KR20240047357A - Compositions and methods for pairwise sequencing - Google Patents

Abstract

The present disclosure provides compositions and methods of using the compositions for performing pairwise sequencing and for generating concatimer template molecules for pairwise sequencing. Concatimers can be produced using a rolling ring amplification reaction carried out on a support or in solution and then distributed on the support. The rolling ring amplification reaction generates concatemers containing tandem copies of the sequence of interest and at least one universal adapter sequence. Increasing the number of tandem copies in a given concatimer increases the number of sites along the concatimer for hybridization to multiple sequencing primers, which serve as multiple initiation sites for the polymerase-catalyzed sequencing reaction. When a sequencing reaction uses detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the nucleotides or nucleotide units that participate in parallel sequencing follow concatemers. The signal emitted by the concatimer yields increased signal intensity for each concatimer.

Description

Compositions and methods for pairwise sequencing

Various publications, patents and/or patent applications are referenced throughout this application. The disclosures of publications, patents, and/or patent applications are incorporated by reference in their entirety into this application to more completely describe the technical field to which the disclosure pertains.

Cross-reference to related applications

This application is related to U.S. Provisional Patent Application No. 63/212,059, filed on June 17, 2021; U.S. Patent Application No. 17/377,284, filed July 15, 2021, now issued as U.S. Patent No. 11,220,707; U.S. Patent Application No. 17/377,285, filed July 15, 2021, now issued as U.S. Patent No. 11,236,388; U.S. Patent Application No. 17/377,279, filed July 15, 2021; U.S. Patent Application No. 17/377,283, filed July 15, 2021; U.S. Patent Application No. 17/521,239, filed November 8, 2021; Claims the benefit of U.S. Patent Application No. 17/554,396, filed on December 17, 2021, the contents of each of which are incorporated herein by reference in their entirety.

Technology field

The present disclosure provides compositions and methods of using the compositions for performing pairwise sequencing and for generating concatemer template molecules for pairwise sequencing.

Polynucleotide sequencing technology is applied in biomedical research and health care settings. Improved processing of polynucleotides requires improved surface chemistry, polynucleotide amplification on supports, and base calling. Currently, these components create barriers to existing sequencing technologies resulting in throughput limitations and poor signal-to-noise ratios, ultimately increasing the costs associated with polynucleotide sequencing.

There is a need for new polynucleotide sequencing methods that utilize improved surface chemistry, amplification on supports, and base calling. The present disclosure provides methods and compositions for improving the sequencing of polynucleotides.

The present disclosure provides: a) a plurality of fixed single-stranded nucleic acid cones each comprising at least one nucleotide having a scissile moiety that can be cleaved to create a non-basic site in the conkatimer template molecule; providing a catimer template molecule, each of the plurality of template molecules being immobilized on a first surface primer immobilized on a support, wherein the immobilized first surface primer lacks a nucleotide having a cleavable moiety; b) Generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules, wherein each immobilized concatimer template molecule includes at least two extended forward sequencing primer strands. This hybridized,step; c) maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward extension strands to the immobilized single-stranded nucleic acid concatimer template molecules. Replacing with; d) creating a non-basic portion of the single-stranded concatimer template molecule anchored at the nucleotide(s) having a cleavable moiety while maintaining a plurality of forward extension strands and maintaining a plurality of immobilized surface primers and removing the retained immobilized concatimer template molecule by creating a gap at the site to generate a plurality of gap-containing single stranded nucleic acid concatimer template molecules; and e) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each maintained forward extension strand is hybridized with two or more extended reverse sequencing primer strands. , and provides a pairwise sequence analysis method, including steps.

In some embodiments, each individual template molecule of the plurality is covalently linked to an immobilized first surface primer. In some embodiments, individual template molecules of the plurality hybridize to an immobilized first surface primer. In some embodiments, the individual immobilized concatimer template molecules of the plurality comprise two or more copies of the sequence of interest, and the individual immobilized concatimer template molecules have (i) universal binding to a soluble forward sequencing primer; two or more copies of the universal binding sequence, (ii) two or more copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) two or more copies of the universal binding sequence for the immobilized first surface primer. , (iv) at least two copies of the universal binding sequence for the immobilized second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) for the second soluble amplification primer. at least two copies of the universal binding sequence, (vii) at least two copies of the universal binding sequence for the soluble compressed oligonucleotide, (viii) at least two copies of the sample barcode sequence, and/or (ix) at least two copies of the unique molecular marker sequence. It further includes any one or any combination of two or more of the two or more copies.

In some embodiments, the sequencing of step (b) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (e) includes hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the non-3'extensible end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

The present disclosure also provides the steps of a) providing a support on which a plurality of first surface primers are immobilized, wherein each of the first surface primers has a 3' extendable end and lacks a nucleotide having a cleavable moiety. ; b) hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers, a plurality of strand displacing polymerases, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and an abasic site. A plurality of fixed single-stranded nucleic acid concatemer template molecules are generated by performing a rolling circle amplification reaction with a nucleotide having a cleavable moiety that can be cleaved to produce a cleavable moiety. generating a plurality of immobilized single-stranded nucleic acid concatimer template molecules having at least one nucleotide with a tee, wherein each single-stranded nucleic acid concatimer template molecule is covalently linked to an immobilized first surface primer, c) Generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules, wherein each immobilized concatimer template molecule includes at least two extended forward sequencing primer strands. This hybridized,step; d) maintaining a plurality of fixed concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward extended strands to the maintained fixed single-stranded nucleic acid concatimer template molecules. Replacing with; e) creating an abasic portion of the fixed single-stranded concatimer template molecule at the nucleotide(s) having a cleavable moiety while maintaining the plurality of forward extension strands and maintaining the plurality of fixed first surface primers; removing the retained immobilized concatimer template molecule by creating a gap at the non-basic site to generate a plurality of gap-containing single-stranded nucleic acid concatimer template molecules; and f) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each forward extension strand is hybridized with two or more extended reverse sequencing primer strands. Provides a pairwise sequence analysis method including.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer. a universal binding sequence, (iii) a universal binding sequence for a first immobilized surface primer, (iv) a universal binding sequence for a second immobilized surface primer, (v) a universal binding sequence for a first soluble amplification primer, (vi ) a universal binding sequence for a second soluble amplification primer, (vii) a universal binding sequence for a soluble compressed oligonucleotide, (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Includes more combinations.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules produced by the rolling ring amplification reaction comprise two or more copies of the sequence of interest, wherein the individual immobilized concatimer template molecules are (i) soluble; (ii) at least two copies of the universal binding sequence for the forward sequencing primer, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) 2 copies of the universal binding sequence for the immobilized first surface primer. at least two copies, (iv) at least two copies of the universal binding sequence for the immobilized second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. at least two copies of the universal binding sequence for the primer, (vii) at least two copies of the universal binding sequence for the soluble compressed oligonucleotide, (viii) at least two copies of the sample barcode sequence, and/or (ix) a unique molecular indicator. It further comprises any one or any combination of two or more copies of the sequence.

In some embodiments, the sequencing of step (c) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (f) includes hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

The present disclosure also provides a) a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable for forming a plurality of library-primer duplexes and performing a rolling ring amplification reaction, comprising a plurality of first soluble amplification primers, a plurality of first soluble amplification primers, A strand transfer polymerase, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety that can be cleaved to create an abasic site, generating a plurality of single-stranded nucleic acid concatemers having at least one nucleotide; b) distributing the rolling ring amplification reaction on a support to which a plurality of first surface primers are immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatimers to the one or more immobilized first surface primers. Wherein each of the first surface primers lacks a nucleotide having a cleavable moiety; c) continuing the rolling ring amplification reaction on the support to generate a plurality of immobilized concatimer template molecules; d) Generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules, wherein each immobilized concatimer template molecule includes at least two extended forward sequencing primer strands. This hybridized,step; e) maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward extension strands to the maintained immobilized single-stranded nucleic acid concatimer template molecules. Replacing with; f) creating an abasic portion of the fixed single-stranded concatimer template molecule at the nucleotide(s) having a cleavable moiety, while maintaining the plurality of forward extension strands and maintaining the plurality of fixed first surface primers; removing the retained immobilized concatimer template molecule by creating a gap at the non-basic site to generate a plurality of gap-containing single-stranded nucleic acid concatimer template molecules; and g) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each forward extension strand is hybridized with two or more extended reverse sequencing primer strands. Provides a pairwise sequence analysis method including.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer. a universal binding sequence, (iii) a universal binding sequence for a first immobilized surface primer, (iv) a universal binding sequence for a second immobilized surface primer, (v) a universal binding sequence for a first soluble amplification primer, (vi ) a universal binding sequence for a second soluble amplification primer, (vii) a universal binding sequence for a soluble compressed oligonucleotide, (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Includes more combinations.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules produced by the rolling ring amplification reaction comprise two or more copies of the sequence of interest, wherein the individual immobilized concatimer template molecules are (i) soluble; (ii) at least two copies of the universal binding sequence for the forward sequencing primer, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) 2 copies of the universal binding sequence for the immobilized first surface primer. at least two copies, (iv) at least two copies of the universal binding sequence for the immobilized second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. at least two copies of the universal binding sequence for the primer, (vii) at least two copies of the universal binding sequence for the soluble compressed oligonucleotide, (viii) at least two copies of the sample barcode sequence, and/or (ix) a unique molecular indicator. It further comprises any one or any combination of two or more copies of the sequence.

In some embodiments, the sequencing of step (d) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (g) includes hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the non-3'extensible end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

The present disclosure includes a) providing a support on which a plurality of first surface primers are fixed, wherein each first surface primer of the plurality has a first part (SP1-A) and a second part (SP1-B). wherein each first surface primer comprises a 3' extendable end at the first surface marker and lacks a nucleotide having a cleavable moiety that can be cleaved to create an abasic site; b) contacting the plurality of first surface primers with a plurality of single-stranded linear nucleic acid library molecules, each library molecule comprising a universal sequence (SP1-) that binds at its 5' end to the first portion of the immobilized first surface primer. A'), and the library molecules each have a universal sequence (SP1-B') that binds to the second part of the immobilized first surface primer at the 3' end, wherein the contacting step is performed with the immobilized first surface primer. performing under conditions suitable for hybridizing the individual library molecules to form a circular library molecule having a gap or crevice between the 5' end and the 3' end of the circular library molecule; c) enzymatically closing the gap or crevice, thereby forming individual covalently closed ring molecules hybridized to the immobilized first surface primer; d) performing a rolling ring amplification reaction with a plurality of strand transfer polymerases and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety that can be cleaved to create an abasic site. thereby generating a plurality of fixed single-stranded nucleic acid conkatimer template molecules, thereby generating a plurality of fixed single-stranded nucleic acid conkatimer template molecules having at least one nucleotide having a cleavable moiety, wherein each single wherein the stranded nucleic acid concatimer template molecules are covalently linked to an immobilized first surface primer; e) generating a plurality of extended forward sequencing primer strands by sequencing the plurality of immobilized concatimer template molecules; , wherein two or more extended forward sequencing primer strands are hybridized to each immobilized concatimer template molecule; f) maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward extension strands to the immobilized single-stranded nucleic acid concatimer template molecules. Replacing with; g) creating an abasic portion of the fixed single-stranded concatimer template molecule at the nucleotide(s) having a cleavable moiety while maintaining the plurality of forward extension strands and maintaining the plurality of fixed first surface primers; removing the retained immobilized concatimer template molecule by creating a gap at the non-basic site to generate a plurality of gap-containing single-stranded nucleic acid concatimer template molecules; and h) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each forward extension strand is hybridized with two or more extended reverse sequencing primer strands. Provides a pairwise sequence analysis method including.

In some embodiments, an individual linear library molecule of the plurality comprises a sequence of interest, and the library molecule comprises: (i) a universal binding sequence to a soluble forward sequencing primer, (ii) a universal binding sequence to a soluble reverse sequencing primer. , (iii) a universal binding sequence for the first part of the immobilized first surface primer (SP1-A), (iv) a universal binding sequence for the second part of the immobilized first surface primer (SP1-B), ( v) a universal binding sequence for an immobilized second surface primer, (vi) a universal binding sequence for a first soluble amplification primer, (vii) a universal binding sequence for a second soluble amplification primer, (viii) a universal binding sequence for a soluble compressed oligonucleotide. It further comprises any one or any combination of two or more of: a universal binding sequence for, (ix) a sample barcode sequence, and/or (x) a unique molecular indicator sequence.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules produced by the rolling ring amplification reaction comprise two or more copies of the sequence of interest, wherein the individual immobilized concatimer template molecules are (i) soluble; at least two copies of the universal binding sequence for the forward sequencing primer, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) universal for the first portion of the immobilized first surface primer. two or more copies of the binding sequence (SP1-A), (iv) two or more copies of the universal binding sequence (SP1-B) for the second portion of the immobilized first surface primer, (v) an immobilized second surface at least two copies of the universal binding sequence for the primer, (vi) at least two copies of the universal binding sequence for the first soluble amplification primer, (vii) at least two copies of the universal binding sequence for the second soluble amplification primer, (viii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide, (ix) two or more copies of the sample barcode sequence, and/or (x) any one or two of the following: two or more copies of the unique molecular indicator sequence. It further includes any combination of the above.

In some embodiments, the sequencing of step (e) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (h) includes hybridizing a plurality of soluble reverse sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the non-3'extensible end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

In some embodiments, closing the gap in the circular library molecule involves performing a polymerase-catalyzed gap fill-in reaction using an immobilized first surface primer as a template molecule, and ligating to form a covalently closed circular molecule, wherein the individual covalently closed circular molecules are hybridized to the immobilized first surface primer. In some embodiments, closing a nick in a circular library molecule includes performing a ligation reaction to form a covalently closed circular molecule, wherein the individual covalently closed circular molecules hybridize to an immobilized first surface primer.

The present disclosure provides a pairwise sequencing method comprising the following steps: a) a plurality of anchored single strands each lacking a cleavable moiety that can be cleaved to create an abasic site in the concatimer template molecule; Providing a nucleic acid concatemer template molecule, wherein each template molecule of the plurality is immobilized on a first surface primer immobilized on a support, wherein the immobilized first surface primer lacks a nucleotide having a cleavable moiety. step; b) Generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules, wherein each immobilized concatimer template molecule includes at least two extended forward sequencing primer strands. This hybridized,step; c) maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-transfer polymerases to extend the plurality of extended forward sequencing primer strands in a plurality of forward directions. strand replacement to generate a plurality of forward extension strands and a plurality of partially translocated forward extension strands hybridized to the immobilized concatimer template molecule to form a plurality of immobilized amplicons, and the primer extension reaction (e.g. , generating a plurality of detached forward extension strands (not hybridized to the immobilized concatimer template molecule); and d) sequencing the plurality of fixed, partially translocated forward extension strands to generate a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of fixed, detached forward extension strands, thereby generating a second plurality of fixed, detached forward extension strands. Generating a plurality of extended reverse sequencing primer strands, wherein at least two extended reverse sequencing primer strands are hybridized to each fixed partially translocated forward extension strand, and each fixed partially translocated forward extension strand is hybridized to the plurality of extended reverse sequencing primer strands. generating a primer strand, wherein two or more extended reverse sequencing primer strands are hybridized.

In some embodiments, each individual template molecule of the plurality is covalently linked to an immobilized first surface primer. In some embodiments, individual template molecules of the plurality hybridize to an immobilized first surface primer. In some embodiments, the individual immobilized concatimer template molecules of the plurality comprise two or more copies of the sequence of interest, and the individual immobilized concatimer template molecules have (i) universal binding to a soluble forward sequencing primer; two or more copies of the sequence, (ii) two or more copies of the universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of the universal binding sequence for a fixed first surface primer, (iv) fixed. at least two copies of the universal binding sequence for the second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) two copies of the universal binding sequence for the second soluble amplification primer. any of the following: (vii) at least two copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) at least two copies of the sample barcode sequence, and/or (ix) at least two copies of the unique molecular indicator sequence. It further includes one or any combination of two or more.

In some embodiments, the sequencing of step (b) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (d) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of fixed partially translocated forward extension strands and a plurality of fixed detached extended forward sequencing primer strands; and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

The present disclosure also provides the steps of a) providing a support on which a plurality of first surface primers are immobilized, wherein each of the first surface primers has a 3' extendable end and lacks a nucleotide having a cleavable moiety. ; b) hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers, a plurality of strand transposition polymerases, and nucleotides having a cleavable moiety that can be cleaved to create an abasic site. A step of generating a plurality of fixed single-stranded nucleic acid concatimer template molecules by performing a rotation amplification reaction with a plurality of nucleotides, wherein each single strand wherein the nucleic acid concatemer template molecule is covalently linked to an immobilized first surface primer; c) Generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules, wherein each immobilized concatimer template molecule includes at least two extended forward sequencing primer strands. This hybridized,step; d) maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-transfer polymerases to extend the plurality of extended forward sequencing primer strands in a plurality of forward directions. strand replacement to generate a plurality of forward extension strands and a plurality of partially translocated forward extension strands hybridized to the immobilized concatimer template molecule to form a plurality of immobilized amplicons, and the primer extension reaction (e.g. , generating a plurality of detached forward extension strands (not hybridized to the immobilized concatimer template molecule); and e) sequencing the plurality of fixed, partially translocated forward extension strands to generate a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of fixed, detached forward extension strands, thereby generating a second plurality of fixed, detached forward extension strands. Generating a plurality of extended reverse sequencing primer strands, wherein at least two extended reverse sequencing primer strands are hybridized to each fixed partially translocated forward extension strand, and each fixed partially translocated forward extension strand is hybridized to the plurality of extended reverse sequencing primer strands. Provides a pairwise sequencing method comprising the step of hybridizing two or more extended reverse sequencing primer strands.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer. a universal binding sequence, (iii) a universal binding sequence for a first immobilized surface primer, (iv) a universal binding sequence for a second immobilized surface primer, (v) a universal binding sequence for a first soluble amplification primer, (vi ) a universal binding sequence for a second soluble amplification primer, (vii) a universal binding sequence for a soluble compressed oligonucleotide, (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Includes more combinations.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules produced by the rolling ring amplification reaction comprise two or more copies of the sequence of interest, wherein the individual immobilized concatimer template molecules are (i) soluble; (ii) at least two copies of the universal binding sequence for the forward sequencing primer, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) 2 copies of the universal binding sequence for the immobilized first surface primer. at least two copies, (iv) at least two copies of the universal binding sequence for the immobilized second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. at least two copies of the universal binding sequence for the primer, (vii) at least two copies of the universal binding sequence for the soluble compressed oligonucleotide, (viii) at least two copies of the sample barcode sequence, and/or (ix) a unique molecular indicator. It further comprises any one or any combination of two or more copies of the sequence.

In some embodiments, the sequencing of step (c) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (e) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of fixed partially translocated forward extension strands and a plurality of fixed detached extended forward sequencing primer strands; and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

The present disclosure also provides a) a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable for forming a plurality of library-primer duplexes and performing a rolling ring amplification reaction, comprising a plurality of first soluble amplification primers, a plurality of first soluble amplification primers, contacting a strand transposition polymerase of and a plurality of nucleotides lacking a nucleotide having a cleavable moiety that can be cleaved to create a non-basic site, thereby producing a plurality of single-stranded nucleic acid concatemers; b) distributing the rolling ring amplification reaction on a support to which a plurality of first surface primers are immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatimers to the one or more immobilized first surface primers. Wherein each of the first surface primers lacks a nucleotide having a cleavable moiety; c) continuing the rolling ring amplification reaction on the support to generate a plurality of immobilized concatimer template molecules; d) Generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules, wherein each immobilized concatimer template molecule includes at least two extended forward sequencing primer strands. This hybridized,step; e) Maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction with a plurality of second soluble amplification primers and a plurality of strand-transfer polymerases to form a plurality of extended forward sequencing primer strands into a plurality of strands. A plurality of forward extension strands and a plurality of partially translocated forward extension strands are hybridized to the immobilized concatimer template molecule by replacing the forward extension strand to form a plurality of immobilized amplicons, and the primer extension reaction is performed (e.g. generating a plurality of detached forward extension strands (e.g., not hybridized to the immobilized concatimer template molecule); and f) sequencing the plurality of fixed, partially translocated forward extension strands to generate a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of fixed, detached forward extension strands, thereby generating a second plurality of extended reverse sequencing primer strands. Generating a plurality of extended reverse sequencing primer strands, wherein at least two extended reverse sequencing primer strands are hybridized to each fixed partially translocated forward extension strand, and each fixed partially translocated forward extension strand is hybridized to the plurality of extended reverse sequencing primer strands. Provides a pairwise sequencing method comprising the step of hybridizing two or more extended reverse sequencing primer strands.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer. a universal binding sequence, (iii) a universal binding sequence for a first immobilized surface primer, (iv) a universal binding sequence for a second immobilized surface primer, (v) a universal binding sequence for a first soluble amplification primer, (vi ) a universal binding sequence for a second soluble amplification primer, (vii) a universal binding sequence for a soluble compressed oligonucleotide, (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Includes more combinations.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules produced by the rolling ring amplification reaction comprise two or more copies of the sequence of interest, wherein the individual immobilized concatimer template molecules are (i) soluble; (ii) at least two copies of the universal binding sequence for the forward sequencing primer, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) 2 copies of the universal binding sequence for the immobilized first surface primer. at least two copies, (iv) at least two copies of the universal binding sequence for the immobilized second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. at least two copies of the universal binding sequence for the primer, (vii) at least two copies of the universal binding sequence for the soluble compressed oligonucleotide, (viii) at least two copies of the sample barcode sequence, and/or (ix) a unique molecular indicator. It further comprises any one or any combination of two or more copies of the sequence.

In some embodiments, the sequencing of step (d) includes hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. In some embodiments, the sequencing of step (f) comprises hybridizing a plurality of soluble reverse sequencing primers to a plurality of fixed partially translocated forward extension strands and a plurality of fixed detached extended forward sequencing primer strands; and performing one or more sequencing reactions.

In some embodiments, the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. In some embodiments, at least one copy of the universal binding sequence for the immobilized second surface primer in an individual concatimer template molecule hybridizes to the immobilized second surface primer. In some embodiments, the plurality of immobilized second surface primers have 3' OH extendable ends. In some embodiments, the plurality of immobilized second surface primers have 3' non-extendable ends. In some embodiments, the non-3'extensible end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group.

The present disclosure also provides a) a plurality of fixed single-stranded nucleic acid conkatimer template molecules each comprising at least one nucleotide having a cleavable moiety that can be cleaved to create a non-basic site in the conkatimer template molecule A step of providing, wherein each of the plurality of template molecules is immobilized on a first surface primer immobilized on a support, the immobilized first surface primer includes a nucleotide having a cleavable moiety, and the support has a cleavable moiety. further comprising a plurality of immobilized second surface primers lacking nucleotides having a t and having 3'OH groups at extendable ends, wherein the immobilized concatimer template molecules have 2 of the universal binding sequences for the immobilized second surface primers. comprising at least one copy, wherein the support includes an excess of immobilized first and second surface primers relative to the number of immobilized concatimer template molecules; b) generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers, wherein each immobilized concatimer template molecule includes wherein two or more extended forward sequencing primer strands are hybridized; c) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule; d) hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized Concatimer template molecule generating a plurality of immobilized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer; e) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution comprising at least one chaotropic agent; f) dissociating at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retaining the immobilized forward extending strand, and forward extending at least one portion of the immobilized concatimer template molecule. rehybridizing to one of the immobilized second surface primers that are not covalently attached to the strand, wherein the dissociation and re-dissociation comprise increasing temperature, temperature plateauing and decreasing temperature, and washing the relaxing solution from the support; g) contacting the rehybridized immobilized Concatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that is rehybridized to a portion of the immobilized Concatimer template molecule to thereby immobilize the Concatimer template molecule. generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of and covalently linked to an immobilized second surface primer; h) repeating steps (e) to (g) at least once; i) a first immobilized single-stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining a plurality of immobilized forward extension strands and a plurality of immobilized second surface primers; creating a non-basic site of the surface primer and removing the retained immobilized concatimer template molecule by creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules; and j) sequencing the plurality of maintained fixed forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands.

In some embodiments, each individual template molecule of the plurality is covalently linked to an immobilized first surface primer. In some embodiments, individual template molecules of the plurality hybridize to an immobilized first surface primer. In some embodiments, the individual immobilized concatimer template molecules of the plurality comprise two or more copies of the sequence of interest, and the individual immobilized concatimer template molecules have (i) universal binding to a soluble forward sequencing primer; two or more copies of the sequence, (ii) two or more copies of the universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of the universal binding sequence for a fixed first surface primer, (iv) fixed. at least two copies of the universal binding sequence for the second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) two copies of the universal binding sequence for the second soluble amplification primer. any of the following: (vii) at least two copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) at least two copies of the sample barcode sequence, and/or (ix) at least two copies of the unique molecular indicator sequence. It further includes one or any combination of two or more.

The present disclosure also provides the steps of a) providing a support to which a plurality of first and second surface primers are immobilized, wherein the first surface primer has a cleavable moiety that can be cleaved to create an abasic site, and providing a two-surface primer lacking a nucleotide having a cleavable moiety, and the second surface primer having a 3'OH group at an extendable end; b) hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers and generating a plurality of strand transposition polymerases, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and abasic sites. Generating a plurality of fixed single-stranded nucleic acid concatimer template molecules by performing a rolling ring amplification reaction with a plurality of nucleotides having a cleavable moiety that can be cleaved, thereby producing at least one nucleotide having a cleavable moiety c) generating a plurality of immobilized single-stranded nucleic acid conkatimer template molecules having a plurality of soluble forward sequences, wherein each single-stranded nucleic acid conkatimer template molecule is covalently linked to an immobilized first surface primer. A step of generating a plurality of extended forward sequencing primer strands by sequencing a plurality of fixed concatimer template molecules using analysis primers, wherein each fixed concatimer template molecule contains two or more extended forward sequences. the assay primer strand is hybridized; d) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule; e) hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized Concatimer template molecule generating a plurality of immobilized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer; f) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution comprising at least one disorder-inducing agent; g) dissociating at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retaining the immobilized forward extending strand, and forward extending at least one portion of the immobilized concatimer template molecule. rehybridizing to one of the immobilized second surface primers that are not covalently attached to the strand, wherein the dissociation and re-dissociation comprise increasing temperature, temperature plateauing and decreasing temperature, and washing the relaxing solution from the support; h) contacting the rehybridized immobilized Concatimer template molecule with the amplification solution and performing a primer extension reaction from the second surface primer that is rehybridized to a portion of the immobilized Concatimer template molecule generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of and covalently linked to an immobilized second surface primer; i) repeating steps (f) to (h) at least once; j) a first immobilized single-stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining a plurality of immobilized forward extension strands and a plurality of immobilized second surface primers; creating a non-basic site of the surface primer and removing the retained immobilized concatimer template molecule by creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules; and k) sequencing the plurality of maintained fixed forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence to a soluble forward sequencing primer, (ii) a universal binding sequence to a soluble reverse sequencing primer. a universal binding sequence, (iii) a universal binding sequence for a first immobilized surface primer, (iv) a universal binding sequence for a second immobilized surface primer, (v) a universal binding sequence for a first soluble amplification primer, (vi ) a universal binding sequence for a second soluble amplification primer, (vii) a universal binding sequence for a soluble compressed oligonucleotide, (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Includes more combinations.

In some embodiments, the individual immobilized concatimer template molecules of the plurality comprise two or more copies of the sequence of interest, and the individual immobilized concatimer template molecules have (i) universal binding to a soluble forward sequencing primer; two or more copies of the sequence, (ii) two or more copies of the universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of the universal binding sequence for a fixed first surface primer, (iv) fixed. at least two copies of the universal binding sequence for the second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) two copies of the universal binding sequence for the second soluble amplification primer. any of the following: (vii) at least two copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) at least two copies of the sample barcode sequence, and/or (ix) at least two copies of the unique molecular indicator sequence. It further includes one or any combination of two or more.

The present disclosure also provides a pairwise sequencing method comprising the following steps: a) a plurality of single-stranded prototypes in solution under conditions suitable to form a plurality of library-primer duplexes and to perform a rolling ring amplification reaction; Nucleic acid library molecules are comprised of a plurality of first soluble amplification primers, a plurality of strand transfer polymerases, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP, and a cleavable moiety that can be cleaved to generate an abasic site. generating a plurality of single-stranded nucleic acid concatemers having at least one nucleotide having a cleavable moiety by contacting a plurality of nucleotides having a cleavable moiety; b) distributing the rolling ring amplification reaction on a support to which a plurality of first surface primers are immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatimers to the one or more immobilized first surface primers. wherein the immobilized first surface primer comprises a nucleotide having a cleavable moiety and the support comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety and having 3'OH groups at extendable ends. further comprising a surface primer; c) continuing the rolling ring amplification reaction on a support in the presence of a plurality of nucleotides comprising a plurality of nucleotides having a cleavable moiety to produce a plurality of immobilized concatimer template molecules; d) generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers, wherein each immobilized concatimer template molecule includes wherein two or more extended forward sequencing primer strands are hybridized; e) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule; f) hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized Concatimer template molecule to form a first primer. generating a plurality of immobilized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer; g) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution comprising at least one disorder-inducing agent; h) dissociating at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retaining the immobilized forward extending strand, and forward extending at least one portion of the immobilized concatimer template molecule. rehybridizing to one of the immobilized second surface primers that are not covalently attached to the strand, wherein the dissociation and re-dissociation comprise increasing temperature, temperature plateauing and decreasing temperature, and washing the relaxing solution from the support; i) contacting the rehybridized immobilized Concatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that is rehybridized to a portion of the immobilized Concatimer template molecule, thereby generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of and covalently linked to an immobilized second surface primer; j) repeating steps (g) to (i) at least once; k) a first immobilized single-stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining a plurality of immobilized forward extension strands and a plurality of immobilized second surface primers; creating a non-basic site of the surface primer and removing the retained immobilized concatimer template molecule by creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules; and l) sequencing the plurality of maintained fixed forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer, (ii) a universal binding sequence for a soluble reverse sequencing primer. a universal binding sequence, (iii) a universal binding sequence for a first immobilized surface primer, (iv) a universal binding sequence for a second immobilized surface primer, (v) a universal binding sequence for a first soluble amplification primer, (vi ) a universal binding sequence for a second soluble amplification primer, (vii) a universal binding sequence for a soluble compressed oligonucleotide, (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Includes more combinations.

In some embodiments, the individual immobilized concatimer template molecules of the plurality comprise two or more copies of the sequence of interest, and the individual immobilized concatimer template molecules have (i) universal binding to a soluble forward sequencing primer; two or more copies of the sequence, (ii) two or more copies of the universal binding sequence for a soluble reverse sequencing primer, (iii) two or more copies of the universal binding sequence for a fixed first surface primer, (iv) fixed. at least two copies of the universal binding sequence for the second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) two copies of the universal binding sequence for the second soluble amplification primer. any of the following: (vii) at least two copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) at least two copies of the sample barcode sequence, and/or (ix) at least two copies of the unique molecular indicator sequence. It further includes one or any combination of two or more.

In any of the foregoing or related embodiments, the support may be made of glass, fused silica, silicone, polymer (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate ( PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof It includes a flat substrate containing a.

In any of the preceding or related embodiments, the support comprises at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less, and at least one of the hydrophilic polymer coatings comprises a branched hydrophilic polymer having at least four branches. do.

In any of the foregoing or related embodiments, the 5' ends of the plurality of first surface primers are secured to the support or to a coating on the support. In any of the foregoing or related embodiments, the plurality of first surface primers comprise modified oligonucleotide molecules having 2 to 10 phosphorothioate linkages at their 5' ends to render them resistant to nuclease degradation. Grant.

In any of the foregoing or related embodiments, the 5' ends of the plurality of second surface primers are secured to the support or to a coating on the support. In some embodiments, the plurality of second surface primers comprise modified oligonucleotide molecules having 2 to 10 phosphorothioate linkages at their 5' ends to confer resistance to nuclease degradation.

In any of the preceding or related embodiments, the immobilized concatimer template molecule has at least one cleavable moiety comprising uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. Contains nucleotides.

In any of the foregoing or related embodiments, the nucleotides having cleavable moieties are located at randomly distributed positions on individual fixed concatimer template molecules of the plurality.

In any of the foregoing or related embodiments, 0.01 to 30% of the thymidine nucleotides in each immobilized concatimer template molecule are replaced with uridine. In any of the foregoing or related embodiments, 0.01 to 30% of the guanosine nucleotides in each immobilized conkatimer template molecule are replaced with 8-oxo-7,8-dihydroguanine or deoxyinosine.

In any of the foregoing or related embodiments, the soluble forward sequencing primer comprises a 3' OH extendable end and lacks a nucleotide having a cleavable moiety. In any of the foregoing or related embodiments, the soluble reverse sequencing primer comprises a 3' OH extendable end and lacks a nucleotide having a cleavable moiety.

In any of the foregoing or related embodiments, the first soluble amplification primer comprises a 3' OH extendable end and lacks a nucleotide having a cleavable moiety. In any of the foregoing or related embodiments, the second soluble amplification primer comprises a 3' OH extendable end and lacks a nucleotide having a cleavable moiety.

In any of the foregoing or related embodiments, the forward sequencing step comprises a) a plurality of sequencing polymerases comprising (i) a plurality of immobilized concatimer template molecules and (ii) a plurality of soluble forward sequencing primers. A contacting step, wherein the contacting step is performed under conditions suitable for forming a plurality of complexed polymerases, each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex is hybridized to a soluble forward sequencing primer. comprising an immobilized concatimer template molecule; b) contacting the plurality of complexed sequencing polymerases with the plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides are labeled with a fluorophore and Comprising at least one nucleotide analog having a removable chain termination moiety at the 3' position; c) incorporating at least one nucleotide into the 3' end of the hybridized forward sequencing primer, thereby generating a plurality of initial extended forward sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

In any of the preceding or related embodiments, the reverse sequencing step comprises a) contacting a plurality of sequencing polymerases with (i) a plurality of maintained forward extension strands and (ii) a plurality of soluble reverse sequencing primers. As a step, the contacting step is performed under conditions suitable to form a plurality of complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex remains hybridized to a soluble reverse sequencing primer. comprising a forward extending strand; b) contacting the plurality of complexed sequencing polymerases with the plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides are labeled with a fluorophore and comprising at least one nucleotide analog having a removable chain termination moiety at the 3' position; c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer to generate a plurality of initial extended reverse sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

In some embodiments, the reverse sequencing of step (a) comprises: (i) a first polar aprotic solvent comprising acetonitrile in 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) hybridizing the plurality of soluble reverse sequencing primers to the plurality of maintained forward extension strands, comprising a crowding agent comprising polyethylene glycol (PEG) in a hybridization buffer at 5 to 35% by volume. Includes.

In some embodiments, the reverse sequencing step comprises a) a plurality of sequencing polymerases comprising: (i) a plurality of fixed partially transposed forward extension strands, (ii) a plurality of fixed detachment extended forward sequencing primer strands. , and (iii) contacting with a plurality of soluble reverse sequencing primers, wherein the contacting is performed under conditions suitable to form a plurality of complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex. wherein the nucleic acid duplex comprises a soluble reverse sequencing primer hybridized to the fixed partially translocated forward extended strand or to the fixed detached extended forward sequencing primer strand; b) contacting the plurality of complexed sequencing polymerases with the plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides are labeled with a fluorophore and comprising at least one nucleotide analog having a removable chain termination moiety at the 3' position; c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer to generate a plurality of initial extended reverse sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

In some embodiments, the reverse sequencing of step a) comprises: (i) a first polar aprotic solvent comprising acetonitrile in 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.

In any of the foregoing or related embodiments, the forward and reverse sequencing steps include: 1) performing a sequencing reaction at a location on the template molecule using a multivalent molecule that binds but does not incorporate; 2) performing a sequencing reaction at the same position on the template molecule using the incorporated nucleotide; and 3) repeating steps a) and b) at the next position on the template molecule.

In any of the preceding or related embodiments, the forward sequencing step and the reverse sequencing step comprise a) a plurality of first sequencing polymerases comprising (i) a plurality of nucleic acid template molecules and (ii) a plurality of soluble sequencing molecules. contacting a primer, wherein the contacting step is performed under conditions suitable for forming a plurality of first complexed polymerases each comprising a first sequencing polymerase linked to a nucleic acid duplex, wherein the nucleic acid duplex is sequenced Comprising a nucleic acid template molecule hybridized to a primer, (1) the plurality of nucleic acid template molecules comprises a plurality of immobilized concatimer template molecules, and the plurality of soluble primers comprise a plurality of soluble forward sequencing primers, or (1) 2) contacting the plurality of nucleic acid template molecules comprising a plurality of maintained forward extension strands and the plurality of soluble sequencing primers comprising a plurality of soluble reverse sequencing primers; b) conditions suitable for linking complementary nucleotide units of the multivalent molecule to at least two of the first plurality of complexed polymerases to form a plurality of multivalent-complexed polymerases, and a plurality of multivalent-complexed polymerases. A plurality of first complexed polymerases are formed by contacting the first plurality of complexed polymerases with a plurality of detectably labeled multivalent molecules under conditions that inhibit the incorporation of complementary nucleotide units into the sequencing primers of the polymerase. forming the polymerase, wherein each multivalent molecule of the plurality of multivalent molecules includes a core attached to multiple nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; c) detecting a plurality of multivalent-complexed polymerases; and d) determining the sequence of the nucleic acid template by identifying the nucleobase of the complementary nucleotide unit bound to the first plurality of complexed polymerases in the plurality of multivalent-complexed polymerases.

In some embodiments, the reverse sequencing step comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.

In some embodiments, the method further comprises the following steps: e) dissociating the plurality of multivalent-complexed polymerases, removing the plurality of first sequencing polymerases and their associated multivalent molecules, and maintaining the nucleic acid duplex; f) contacting the plurality of maintained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein contacting causes the plurality of second sequencing polymerases to bind to the plurality of maintained nucleic acid duplexes. performed under conditions suitable for forming a plurality of second complexed polymerases, each comprising a second sequencing polymerase linked to the retained nucleic acid duplex; g) contacting the plurality of second complexed polymerases with the plurality of nucleotides, wherein the contacting brings complementary nucleotides from the plurality of nucleotides into contact with at least two of the second complexed polymerases of step (f). performed under conditions suitable for linking to form a plurality of nucleotide-complexed polymerases and under conditions suitable for promoting incorporation of complementary nucleotides linked to the sequencing primer of the nucleotide-complexed polymerase; h) detecting complementary nucleotides incorporated into the sequencing primer of the nucleotide-complexed polymerase; and d) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.

In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) a first sequencing primer, a first sequencing polymerase, and a first multivalent linking the molecule to a first portion of a nucleic acid template molecule to form a first binding complex, wherein the first nucleotide unit of the first multivalent molecule binds to a first sequencing polymerase; and b) linking the second sequencing primer, the second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule, thereby forming a second binding complex, wherein the second binding complex of the second multivalent molecule The nucleotide units bind to a second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex.

In some embodiments, (i) the first sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an immobilized concatimer template molecule, and (ii) the second sequencing primer comprises a soluble forward sequencing primer. and (iii) the first sequencing primer and the second sequencing primer have the same sequence.

In some embodiments, (i) the first sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises a maintained forward extension strand, and (ii) the second sequencing primer comprises a soluble reverse sequencing primer. wherein the nucleic acid template molecule comprises the same maintained forward extension strand, and (iii) the first sequencing primer and the second sequencing primer have the same sequence.

In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising a) a plurality of first sequencing polymerases and a plurality of second sequencing primers. contacting different portions of a nucleic acid template molecule to form at least first and second complexed polymerases on the same nucleic acid template molecule; b) contacting the plurality of multivalent molecules with at least the first and second complexed polymerases on the same nucleic acid template molecule under conditions suitable for binding a single multivalent molecule from the plurality to the first and second complexed polymerases. wherein at least the first nucleotide unit of the single multivalent molecule is bound to a first complexed polymerase comprising a first sequencing primer hybridized to a first portion of the nucleic acid template molecule to form a first binding complex, and forming a single At least a second nucleotide unit of the multivalent molecule is bound to a second complexed polymerase comprising a second sequencing primer hybridized to a second portion of the same nucleic acid template molecule to form a second binding complex, wherein contacting comprises: carried out under conditions suitable to inhibit polymerase-catalyzed incorporation of the first and second nucleotide units bound in the first and second binding complexes, wherein the first and second binding complexes bound to the same multivalent molecule have a binding activity. forming a complex; c) detecting the first and second binding complexes on the same nucleic acid template molecule, and d) identifying the first nucleotide unit in the first binding complex to determine the sequence of the first portion of the nucleic acid template molecule and binding the second binding complex. and determining the sequence of a second portion of the same nucleic acid template molecule by identifying the second nucleotide unit in the complex.

In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the nucleic acid template molecules comprise immobilized concatimer template molecules, and (ii) the plurality of first sequencing primers comprise a plurality of first soluble forward sequencing primers, and 2 the sequencing primers comprise a plurality of second soluble forward sequencing primers, the nucleic acid template molecules comprise identical immobilized concatimer template molecules, and (iii) a plurality of first sequencing primers and a second sequencing primer. Primers have identical sequences.

In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecules comprise a maintained forward extension strand, and (ii) the plurality of second sequences The assay primers comprise a plurality of second soluble reverse sequencing primers, the nucleic acid template molecules comprise identical maintained forward extension strands, and (iii) the plurality of first sequencing primers and the second sequencing primers have identical sequences. has

In any of the foregoing or related embodiments, the forward sequencing step and the reverse sequencing step comprise a) a plurality of first sequencing polymerases comprising (i) a plurality of nucleic acid template molecules and (ii) a plurality of soluble sequencing primers. wherein the contacting step is performed under conditions suitable for forming a plurality of first complexed polymerases, each comprising a first complexed polymerase linked to a nucleic acid duplex, wherein the nucleic acid duplex is a soluble sequencing polymerase. Comprising nucleic acid template molecules hybridized to primers, (1) the plurality of nucleic acid template molecules comprises a plurality of immobilized concatimer template molecules, and the plurality of sequencing primers comprise a plurality of soluble forward sequencing primers, or (2) the plurality of nucleic acid template molecules comprise a plurality of fixed, partially translocated forward extension strands, and the plurality of sequencing primers comprise a plurality of soluble reverse sequencing primers, or (3) the plurality of nucleic acid template molecules comprise a plurality of fixed, partially translocated forward extension strands. comprising a plurality of fixed, detachable, extended forward sequencing primer strands, the plurality of sequencing primers comprising a plurality of soluble reverse sequencing primers; b) conditions suitable for binding complementary nucleotide units of the multivalent molecule to at least two of the first plurality of complexed polymerases to form a plurality of multivalent-complexed polymerases, and a plurality of multivalent-complexed polymerases. A plurality of first complexed polymerases are formed by contacting the first plurality of complexed polymerases with a plurality of detectably labeled multivalent molecules under conditions that inhibit the incorporation of complementary nucleotide units into the sequencing primers of the polymerase. forming the polymerase, wherein each multivalent molecule of the plurality of multivalent molecules includes a core attached to multiple nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; c) detecting a plurality of multivalent-complexed polymerases; and d) determining the sequence of the nucleic acid template by identifying the nucleobase of the complementary nucleotide unit bound to the first plurality of complexed polymerases in the plurality of multivalent-complexed polymerases.

In any of the foregoing or related embodiments, the reverse sequencing step includes (i) a first polar aprotic solvent comprising acetonitrile in 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) extending a plurality of fixed, partially transposed forward sequences of a plurality of soluble reverse sequencing primers in the presence of a high-efficiency hybridization buffer comprising a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer. hybridizing to the strand or to a plurality of immobilized, detached, extended forward sequencing primer strands.

In some embodiments, the method comprises e) dissociating the plurality of multivalent-complexed polymerases and removing the plurality of first sequencing polymerases and their associated multivalent molecules, and maintaining the plurality of nucleic acid duplexes; f) contacting the plurality of maintained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein contacting causes the plurality of second sequencing polymerases to bind to the plurality of maintained nucleic acid duplexes. performing under conditions suitable for forming a plurality of second complexed polymerases, each comprising a second sequencing polymerase linked to a retained nucleic acid duplex; g) contacting the plurality of second complexed polymerases with the plurality of nucleotides, wherein the contacting brings complementary nucleotides from the plurality of nucleotides into contact with at least two of the second complexed polymerases of step (f). performed under conditions suitable for linking to form a plurality of nucleotide-complexed polymerases and under conditions suitable for promoting incorporation of complementary nucleotides linked to the sequencing primer of the nucleotide-complexed polymerase; h) detecting complementary nucleotides incorporated into the sequencing primer of the nucleotide-complexed polymerase; and i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.

In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) a first sequencing primer, a first sequencing polymerase, and a first multivalent linking the molecule to a first portion of a nucleic acid template molecule to form a first binding complex, wherein the first nucleotide unit of the first multivalent molecule binds to a first sequencing polymerase; and b) linking the second sequencing primer, the second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule, thereby forming a second binding complex, wherein the second binding complex of the second multivalent molecule The nucleotide units bind to a second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex.

In some embodiments, (i) the first sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an immobilized concatimer template molecule, and (ii) the second sequencing primer comprises a soluble forward sequencing primer. and (iii) the first sequencing primer and the second sequencing primer have the same sequence. In some embodiments, (i) the first sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises a fixed, partially translocated forward extension strand, and (ii) the second sequencing primer comprises a soluble reverse sequencing primer. comprising a reverse sequencing primer, the nucleic acid template molecule comprising an identical fixed partially translocated forward extension strand, and (iii) the first sequencing primer and the second sequencing primer have the same sequence. In some embodiments, (i) the first sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises an immobilized, detached extended forward sequencing primer strand, and (ii) the second sequencing primer comprises and (iii) the first sequencing primer and the second sequencing primer have identical sequences.

In some embodiments, the method further comprises forming at least one avidity complex in step (b), the method comprising: a) a plurality of first sequencing polymerases and a plurality of second sequencing primers contacting a different portion of a nucleic acid template molecule to form at least a first and a second complexed polymerase on the same nucleic acid template molecule; b) contacting the plurality of multivalent molecules with at least the first and second complexed polymerases on the same nucleic acid template molecule under conditions suitable for binding a single multivalent molecule from the plurality to the first and second complexed polymerases. wherein at least the first nucleotide unit of the single multivalent molecule is bound to a first complexed polymerase comprising a first sequencing primer hybridized to a first portion of the nucleic acid template molecule to form a first binding complex, and forming a single At least a second nucleotide unit of the multivalent molecule is bound to a second complexed polymerase comprising a second sequencing primer hybridized to a second portion of the same nucleic acid template molecule to form a second binding complex, wherein contacting comprises: carried out under conditions suitable to inhibit polymerase-catalyzed incorporation of the first and second nucleotide units bound in the first and second binding complexes, wherein the first and second binding complexes bound to the same multivalent molecule have a binding activity. forming a complex; c) detecting the first and second binding complexes on the same nucleic acid template molecule, and d) identifying the first nucleotide unit in the first binding complex to determine the sequence of the first portion of the nucleic acid template molecule and binding the second binding complex. and determining the sequence of a second portion of the same nucleic acid template molecule by identifying the second nucleotide unit in the complex.

In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the nucleic acid template molecules comprise immobilized concatimer template molecules, and (ii) the plurality of first sequencing primers comprise a plurality of first soluble forward sequencing primers, and 2 the sequencing primers comprise a plurality of second soluble forward sequencing primers, the nucleic acid template molecules comprise identical immobilized concatimer template molecules, and (iii) a plurality of first sequencing primers and a second sequencing primer. Primers have identical sequences. In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises a fixed, partially translocated forward extension strand, and (ii) a plurality of first sequencing primers. the second sequencing primer comprises a plurality of second soluble reverse sequencing primers, the nucleic acid template molecule comprises an identical fixed partially translocated forward extension strand, and (iii) a plurality of first sequencing primers and The second sequencing primer has the same sequence. In some embodiments, (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises an immobilized, detached extended forward sequencing primer strand, and (ii) The plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, the nucleic acid template molecules comprise identical fixed detachable extended forward sequencing primer strands, and (iii) the plurality of first sequence The analysis primer and the second sequencing primer have the same sequence.

In any of the foregoing or related embodiments, each nucleotide in the plurality of nucleotides comprises an aromatic base, a 5 carbon sugar and 1 to 10 phosphate groups, wherein the aromatic base of the nucleotide is adenine, guanine, cytosine, thymine or uranium. Contains thread. In some embodiments, the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, and dTTP. In some embodiments, the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, and/or dTTP. In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a fluorescently labeled nucleotide. In some embodiments, at least one of the plurality of nucleotides lacks a fluorophore label.

In any of the foregoing or related embodiments, in the plurality of nucleotides, at least one of the nucleotides comprises a chain termination moiety attached to the 3'-OH sugar position through a cleavable moiety, wherein the chain termination moiety is an alkyl group. , alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group. or includes a silyl group.

In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl, and allyl are grouped together with piperidine by tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4), or 2,3 -cleavable/removable by dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ); (i) chain terminating moieties aryl and benzyl are cleavable/removable by H2 Pd/C; (ii) Chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by thiol reagents including beta-mercaptoethanol or dithiothritol (DTT). do; (iii) Chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) , or cleavable/removable by phosphine reagents including tri(hydroxypropyl)phosphine (THPP); (iv) chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by 4-dimethylaminopyridine (4-DMAP); (v) the chain terminating moiety carbonate is cleavable/removable by potassium carbonate (K2CO3) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH); and (vi) the chain terminating moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride, or by triethylamine trihydrofluoride.

In some embodiments, at least one of the nucleotides in the plurality of nucleotides comprises a chain termination moiety attached to the 3'-OH sugar position through a cleavable moiety, and the chain termination moiety is a 3' O-azido or Contains a 3' O-azidomethyl group. In some embodiments, (i) the chain termination moieties 3' O-azido and 3' O-azidomethyl groups are derivatized tri-alkyl phosphine moieties, derivatized tri-aryl phosphine moieties, tris cleavable/removable by phosphine compounds including (2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or tri(hydroxypropyl)phosphine (THPP); ; and (ii) the chain terminating moieties 3' O-azido and 3' O-azidomethyl groups are cleavable/removable by 4-dimethylaminopyridine (4-DMAP).

In any of the foregoing or related embodiments, each multivalent molecule of the plurality of multivalent molecules comprises (a) a core; and (b) a plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core comprises a core attachment moiety. It is attached to a plurality of nucleotide arms via, a spacer is attached to a linker, and the linker is attached to a nucleotide unit.

In some embodiments, the core comprises an avidin-type moiety and the core attachment moiety comprises biotin. In some embodiments, the linker comprises an aliphatic chain having 2 to 6 subunits or an oligo ethylene glycol chain having 2 to 6 subunits. In some embodiments, the linker further includes an aromatic moiety. In some embodiments, the nucleotide unit includes an aromatic base, a pentose sugar, and 1 to 10 phosphate groups. In some embodiments, the linker is attached to the nucleotide unit via a base.

In some embodiments, the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of multivalent molecules comprises one type of multivalent molecule, wherein each multivalent molecule of the plurality has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and/or dUTP. .

In some embodiments, the plurality of multivalent molecules are fluorescently labeled multivalent molecules. In some embodiments, (i) the core of an individual fluorescently labeled multivalent molecule is attached to a fluorophore corresponding to a nucleotide unit attached to a nucleotide arm; (ii) at least one of the nucleotide arms comprises a linker attached to a fluorophore corresponding to the nucleotide unit attached to the nucleotide arm; and/or (iii) at least one of the nucleotide arms comprises a nucleotide unit attached to a fluorophore that corresponds to the nucleotide unit attached to the nucleotide arm.

In some embodiments, the plurality of multivalent molecules lack a fluorophore.

In some embodiments, at least one of the plurality of multivalent molecules comprises a nucleotide unit having a chain terminating moiety attached to the 3'-OH sugar position through a cleavable moiety, wherein the chain terminating moiety is an alkyl group, Alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or Includes silyl group.

In some embodiments, (i) the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are fused together with piperidine by tetrakis(triphenylphosphine)palladium(0)(Pd(PPh3)4), or Cleavable/removable by 2,3-dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ); (ii) chain terminating moieties aryl and benzyl are cleavable/removable by H2 Pd/C; (iii) chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by thiol reagents including beta-mercaptoethanol or dithiothritol (DTT) do; (iv) Chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) , or cleavable/removable by phosphine reagents including tri(hydroxypropyl)phosphine (THPP); (v) chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by 4-dimethylaminopyridine (4-DMAP); (vi) the chain terminating moiety carbonate is cleavable/removable by potassium carbonate (K2CO3) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH); and (vii) the chain terminating moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride, or by triethylamine trihydrofluoride.

In some embodiments, at least one of the plurality of multivalent molecules comprises a nucleotide unit having a chain terminating moiety attached to the 3'-OH sugar position through a cleavable moiety, and the chain terminating moiety is 3' Contains O-azido or 3' O-azidomethyl group.

In some embodiments, (i) the chain termination moieties 3' O-azido and 3' O-azidomethyl groups are derivatized tri-alkyl phosphine moieties, derivatized tri-aryl phosphine moieties, tris cleavable/removable by phosphine compounds including (2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or tri(hydroxypropyl)phosphine (THPP); ; and (ii) the chain terminating moieties 3' O-azido and 3' O-azidomethyl are cleavable/removable by 4-dimethylaminopyridine (4-DMAP).

In some embodiments, the plurality of sequencing polymerases in step (a) comprises a recombinant wild-type DNA polymerase, and the plurality of nucleotides in step (b) include a fluorescent label having a removable chain termination moiety at the 3' sugar position. contains nucleotides.

In some embodiments, the plurality of sequencing polymerases in step (a) comprises a mutant DNA polymerase, and the plurality of nucleotides in step (b) include a fluorescent label having a removable chain termination moiety at the 3' sugar position. contains nucleotides.

In some embodiments, the plurality of first sequencing polymerases of step (a) comprises a recombinant wild-type DNA polymerase. In some embodiments, the plurality of first sequencing polymerases of step (a) comprises mutant DNA polymerases.

In some embodiments, the plurality of second sequencing polymerases in step (f) comprises a recombinant wild-type DNA polymerase, and the plurality of nucleotides in step (b) have a removable chain termination moiety at the 3′ sugar position. Contains fluorescently labeled nucleotides.

In some embodiments, the plurality of second sequencing polymerases in step (f) comprises a mutant DNA polymerase, and the plurality of nucleotides in step (b) have a removable chain termination moiety at the 3′ sugar position. Contains fluorescently labeled nucleotides.

In any of the foregoing or related embodiments, the plurality of extended forward sequencing primer strands are replaced with a plurality of forward extension strands hybridized to an immobilized single-stranded nucleic acid concatimer template molecule maintained by performing a primer extension reaction. The step of: (i) performing a strand transposition primer extension reaction using at least one extended forward sequencing primer to initiate the primer extension reaction to generate a forward extended strand covalently linked to the extended forward sequencing primer strand. Under conditions suitable for the following, at least one extended forward sequencing primer strand is contacted with a plurality of strand transfer polymerases and a plurality of nucleotides and in the absence of a soluble amplification primer, wherein the forward extension strand is attached to an immobilized concatimer template molecule. Including hybridization.

In any of the preceding or related embodiments, the plurality of extended forward sequencing primer strands are replaced with a plurality of forward extension strands hybridized to an immobilized single-stranded nucleic acid concatimer template molecule maintained by performing a primer extension reaction. The steps include (i) contacting a plurality of extended forward sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease; (ii) contacting the plurality of extended forward sequencing primer strands with a denaturing reagent comprising any combination of formamide, acetonitrile, guanidinium chloride, and/or a pH buffer; or (iii) contacting the plurality of extended forward sequencing primer strands with 100% formamide.

In any of the preceding or related embodiments, the plurality of extended forward sequencing primer strands are replaced with a plurality of forward extension strands hybridized to an immobilized single-stranded nucleic acid concatimer template molecule maintained by performing a primer extension reaction. The step is suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of maintained immobilized concatimer template molecules and performing a polymerase-catalyzed primer extension reaction to generate a plurality of forward extension strands. Under conditions, (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) contacting the plurality of maintained immobilized conkatimer molecules with a second plurality of soluble forward sequencing primers, a plurality of nucleotides, and a plurality of primer extension polymerases, wherein the plurality of nucleotides are dATP, dGTP. , comprising dCTP and dTTP but lacking dUTP, wherein a plurality of primer extension polymerases tolerate the uridine-containing template strand, and the soluble sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized conkatimer molecule. do.

In some embodiments, contacting comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. It includes contacting with a primer.

In any of the foregoing or related embodiments, the plurality of extended forward sequencing primer strands are replaced with a plurality of forward extension strands hybridized to an immobilized single-stranded nucleic acid concatimer template molecule maintained by performing a primer extension reaction. The step of doing so is under conditions suitable for hybridizing a plurality of soluble amplification primers to a plurality of retained immobilized concatimer template molecules and performing a polymerase-catalyzed primer extension reaction to generate a plurality of forward extension strands. , (i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and (ii) contacting the plurality of retained immobilized conkatimer molecules with a plurality of soluble amplification primers, a plurality of nucleotides, and a plurality of primer extension polymerases, wherein the soluble amplification primers are retained immobilized conkatimer molecules. hybridizes to the soluble amplification primer binding sequence, the plurality of nucleotides include dATP, dGTP, dCTP and dTTP but lack dUTP, the plurality of primer extension polymerases tolerate the uridine-containing template strand, and the soluble sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized concatimer molecule.

In some embodiments, contacting comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers in the presence of a high-efficiency hybridization buffer comprising a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. It includes the step of contacting.

In some embodiments, the method further comprises contacting the plurality of retained immobilized concatimer molecules with a plurality of soluble compressed oligonucleotides.

In any of the foregoing or related embodiments, replacing a plurality of extended forward sequencing primer strands includes (i) performing a strand transposition primer extension reaction using at least one extended forward sequencing primer strand to obtain a primer contacting at least one extended forward sequencing primer strand with a plurality of strand transposition polymerases and a plurality of nucleotides in the absence of a soluble amplification primer under conditions suitable to initiate an extension reaction to form a plurality of forward extension strands, a plurality of partially translocating strands; generating an extended forward sequencing strand and a plurality of detached extended forward sequencing primer strands.

In any of the preceding or related embodiments, replacing the plurality of extended forward sequencing primer strands comprises (i) replacing the plurality of extended forward sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease; contacting with an agent; (ii) contacting the plurality of extended forward sequencing primer strands with a denaturing reagent comprising any combination of formamide, acetonitrile, guanidinium chloride, and/or a pH buffer; or (iii) contacting the plurality of extended forward sequencing primer strands with 100% formamide.

In any of the foregoing or related embodiments, the step of replacing the plurality of extended forward sequencing primer strands is suitable for hybridizing the plurality of soluble forward sequencing primers to the plurality of retained immobilized concatimer template molecules; Under conditions suitable for carrying out a polymerase-catalyzed strand transfer reaction, (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) contacting the plurality of retained immobilized concatimer molecules with a second plurality of soluble forward sequencing primers, a plurality of nucleotides, and a plurality of strand transfer polymerases, such that the plurality of immobilized concatimer molecules hybridizes to the immobilized concatimer template molecules. generate a forward extension strand and a plurality of partially translocated extended forward sequencing strands to form a plurality of fixed amplicons, and the primer extension reaction is performed by a plurality of detachable extended forward sequencing primer strands (e.g., fixed amplicons). generating a conkatimer template molecule), wherein the plurality of nucleotides include dATP, dGTP, dCTP, and dTTP, but lack dUTP, and the soluble forward sequencing primer is maintained as a fixed conkatimer. Hybridizes to the forward sequencing primer binding sequence in the mer molecule.

In some embodiments, contacting comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. It includes contacting with a primer.

In any of the foregoing or related embodiments, the step of replacing the plurality of extended forward sequencing primer strands is suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules, and the step of replacing the plurality of extended forward sequencing primer strands is suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and -Under conditions suitable for carrying out a catalytic strand transposition reaction, (i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and (ii) a plurality of forward extension strands that are hybridized to the immobilized concatimer template molecules by contacting the plurality of retained immobilized concatimer molecules with a plurality of soluble amplification primers, a plurality of nucleotides and a plurality of strand transposition polymerases, and A plurality of partially translocated extended forward sequencing strands are generated to form a plurality of immobilized amplicons, and the primer extension reaction is performed using a plurality of detachable extended forward sequencing primer strands (e.g., an immobilized concatimer template). generating an amplification primer binding sequence in an immobilized concatemer molecule, wherein the plurality of nucleotides comprises dATP, dGTP, dCTP and dTTP, but lacks dUTP, and the soluble amplification primer is maintained. is hybridized with

In some embodiments, contacting comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers in the presence of a high-efficiency hybridization buffer comprising a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. It includes the step of contacting.

In any of the foregoing or related embodiments, at least one of the retained anchor conkatimer template molecules comprises one or more nucleotides having a cleavable moiety, wherein the cleavable moiety is uridine or 8-oxo -7,8-dihydroguanine or deoxyinosine. In any of the foregoing or related embodiments, the retained anchored concatimer template molecule comprises one or more uridines, and creating an abasic site at the uridine causes the retained anchored concatimer template molecule to form a uracil and contacting with DNA glycosylase (UDG). In any of the foregoing or related embodiments, the retained anchored concatimer template molecule comprises one or more 8oxoG (8oxoG), and creating a non-basic site at the 8oxoG is a retained anchored concatimer. and contacting the template molecule with the Fpg enzyme (formamidopyrimidine DNA glycosylase). In any of the foregoing or related embodiments, the retained anchored concatimer template molecule comprises one or more deoxyinosines, and creating a non-basic site at the deoxyinosine causes the retained anchored concatimer template molecule to and contacting with AlkA glycosylase enzyme.

In any of the foregoing or related embodiments, the method further comprises creating a gap at a non-basic site to generate at least one gap-containing concatimer template molecule, wherein the method comprises at least one non-basic site. The retained anchor template molecule is endonuclease IV, AP lyase (e.g., DNA-apurinic lyase or DNA-alpyrimidine lyase), FPG glycosylase/AP lyase and/or endo VIII. and contacting with glycosylase/AP lyase.

In any of the foregoing or related embodiments, the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of wild-type sequencing polymerases have an error rate of incorporated dUTP of at least 0.1× compared to the error rate of incorporated dTTP. Obtain an error rate of In any of the foregoing or related embodiments, the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of mutant sequencing polymerases have an incorporation error rate of at least 0.1× compared to the error rate of incorporation dTTP. Obtain the error rate of dUTP. In any of the foregoing or related embodiments, the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of wild-type sequencing polymerases have an error rate of incorporated dUTP of at least 0.1× compared to the error rate of incorporated dTTP. Obtain an error rate of In any of the foregoing or related embodiments, the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of mutant sequencing polymerases have an incorporation error rate of at least 0.1× compared to the error rate of incorporation dTTP. Obtain the error rate of dUTP.

In any of the foregoing or related embodiments, the ratio of the first base fluorescence signal of R2 (e.g., reverse sequencing) to the first base fluorescence signal of R1 (e.g., forward sequencing) is 1, 2. , for sequencing using 3 or 4 dye colors, it is at least 0.7.

In any of the foregoing or related embodiments, the rolling ring amplification step includes a plurality of compressed oligonucleotides and/or hexamine to achieve a more compact size and /or generating an immobilized concatimer template molecule having a shape.

In any of the foregoing or related embodiments, the primer extension reaction of the step comprises a plurality of compressed oligonucleotides and/or hexamine to be more compact in size and compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. /or generate a plurality of forward extending strands having a shape.

In any of the foregoing or related embodiments, the rolling ring amplification step includes a plurality of compressed oligonucleotides and/or hexamine to achieve a more compact size and /or generating a concatimer molecule having a shape.

In any of the foregoing or related embodiments, the primer extension reaction of the step comprises a plurality of compressed oligonucleotides and/or hexamine to be more compact in size and compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. /or generate a plurality of forward extending strands having a shape.

In any of the foregoing or related embodiments, the rolling ring amplification step comprises a plurality of compressed oligonucleotides and/or hexamine to achieve a more compact size and /or generating an immobilized concatimer template molecule having a shape.

In any of the foregoing or related embodiments, the primer extension reaction of the step comprises a plurality of compressed oligonucleotides and/or hexamine to be more compact in size and compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. /or generate a plurality of forward extending strands having a shape.

In any of the foregoing or related embodiments, the primer extension reaction of the step comprises a plurality of compressed oligonucleotides and/or hexamine to be more compact in size and compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. /or generate a plurality of primer extension products having a shape, wherein the plurality of primer extension products include a plurality of forward extension strands, a plurality of partially translocated extended forward sequencing strands, and a plurality of detachment extended forward sequencing primer strands. Includes.

In any of the foregoing or related embodiments, the rolling ring amplification step comprises a plurality of compressed oligonucleotides and/or hexamine to achieve a more compact size and /or generating an immobilized concatimer template molecule having a shape.

In any of the foregoing or related embodiments, the primer extension reaction of the step comprises a plurality of compressed oligonucleotides and/or hexamine to be more compact in size and compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. /or generate a plurality of primer extension products having a shape, wherein the plurality of primer extension products include a plurality of forward extension strands, a plurality of partially translocated extended forward sequencing strands, and a plurality of detachment extended forward sequencing primer strands. Includes.

In any of the foregoing or related embodiments, the rolling ring amplification step comprises a plurality of compressed oligonucleotides and/or hexamine to achieve a more compact size and /or generating a plurality of concatimer molecules having a shape.

In any of the foregoing or related embodiments, the primer extension reaction of the step comprises a plurality of compressed oligonucleotides and/or hexamine to be more compact in size and compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. /or generate a plurality of primer extension products having a shape, wherein the plurality of primer extension products include a plurality of forward extension strands, a plurality of partially transposed extended forward sequencing strands, and a plurality of detachment extended forward sequencing primer strands. Includes.

In any of the foregoing or related embodiments, the plurality of immobilized concatimer template molecules or the plurality of immobilized concatimer molecules have a full width at half maximum (FWHM) of about 5 μm or less. In any of the foregoing or related embodiments, the plurality of forward extending strands have a full width at half maximum (FWHM) of about 5 μm or less. In any of the foregoing or related embodiments, the plurality of primer extension products have a full width at half maximum (FWHM) of about 5 μm or less.

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description, in which the principles of the invention are utilized, presented in exemplary embodiments, and the accompanying drawings:
Figure 1 is Schematic diagram showing an exemplary single-stranded nucleic acid concatimer template molecule immobilized on an immobilized first surface primer. The immobilized concatimer template molecule includes at least one nucleotide with a cleavable moiety that can be cleaved to create an abasic site in the immobilized concatimer template molecule. In some embodiments, immobilized concatimer template molecules can be generated by performing a rolling ring amplification reaction on a support. The arrangement of the various primer binding sequences is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 2 to 12 show flow diagrams of pairwise sequencing of the immobilized concatimer template molecule shown in Figure 1.
Figure 2 is A schematic diagram showing an exemplary forward sequencing reaction performed on the immobilized concatimer template molecule shown in Figure 1. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 3 is a schematic diagram illustrating an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand transfer polymerase in the absence of a soluble primer to generate a forward extended strand.
Figure 4 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer to generate a forward extended strand.
Figure 5 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer to generate a forward extended strand.
Figure 6 shows a method for creating an abasic portion of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 3 or Figure 4.
Figure 7 is Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 6.
Figure 8 shows a method for creating an abasic region of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be generated by the method shown in Figure 5.
Figure 9 is Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 8.
Figure 10 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 7. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 1-10 show exemplary immobilized concatimer molecules with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain two or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 11 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 9. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 1-11 show exemplary immobilized concatimer molecules with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain two or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 12 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 1 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 13 is Schematic diagram showing an exemplary single-stranded nucleic acid concatimer template molecule immobilized on an immobilized first surface primer. The immobilized concatimer template molecule includes at least one nucleotide with a cleavable moiety that can be cleaved to create an abasic site in the immobilized concatimer template molecule. In some embodiments, immobilized concatimer template molecules can be produced by performing a rolling ring amplification reaction in solution and distributing the rolling ring amplification reaction on a support. The arrangement of the various primer binding sequences is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 14-25 show flow diagrams of pairwise sequencing of the immobilized concatimer template molecule shown in Figure 13.
Figure 14 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 13. The forward sequencing reaction may be performed with a plurality of soluble forward sequencing primers. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 15 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with strand transfer polymerase in the absence of a soluble primer.
Figure 16 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer.
Figure 17 is a schematic diagram showing an exemplary method of replacing an extended amplification primer strand by performing a primer extension reaction with a soluble forward sequencing primer.
18 shows a method for creating an abasic portion of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 15 or Figure 16.
Figure 19 is Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 18.
Figure 20 shows a method for creating an abasic portion of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be generated by the method shown in Figure 17.
Figure 21 is Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 20.
Figure 22 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 19. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers. The maintained forward extension strand shown in Figure 22 is a concatemer molecule that can contain two or more tandem copies of the sequence of interest and various primer binding sites. Such concatimer molecules may have two or more extended reverse sequencing primer strands hybridized to them. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 13-23 show exemplary immobilized concatimer molecules with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain two or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 23 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 21. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The maintained forward extension strand shown in Figure 23 is a concatimer molecule containing two or more tandem copies of the sequence of interest and various primer binding sites. Such concatimer molecules may have two or more extended reverse sequencing primer strands hybridized to them. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 13-23 show exemplary immobilized concatimer molecules with two tandem copies containing the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain three or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 24 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 13 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 25 is A schematic diagram depicts an exemplary support to which a first surface primer is immobilized and, in some embodiments, can be used to perform a pairwise sequencing flow diagram on the support.
Figure 26 is a nucleic acid circular library molecule, rotated on an exemplary support using a mixture of nucleotides comprising an immobilized first surface primer shown in Figure 25 and nucleotides with cleavable moieties that can be cleaved to create abasic sites. This is a schematic diagram showing the ring amplification reaction. The rotary ring amplification reaction produces a fixed single-stranded nucleic acid concatimer template molecule having at least one nucleotide with a cleavable moiety that can be cleaved to create a non-basic site in the fixed concatimer template molecule. The arrangement of various primer binding sequences in a circular nucleic acid library molecule is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 26-37 show flow diagrams of pairwise sequencing of the immobilized concatimer template molecule shown in Figure 26.
Figure 27 A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 26. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 28 is a schematic diagram illustrating an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand transposition polymerase in the absence of a soluble primer to generate a forward extended strand.
Figure 29 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer to generate a forward extended strand.
Figure 30 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer to generate a forward extended strand.
Figure 31 shows a nucleotide with a cleavable moiety creating an abasic portion of an anchored single-stranded concatimer template molecule and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 28 or Figure 29.
Figure 32 is Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 31.
33 shows a method for creating an abasic region of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 30.
Figure 34 is Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 33.
Figure 35 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 32. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 26-36 show exemplary immobilized concatimer molecules with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain two or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 36 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 34. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 26-36 show exemplary immobilized concatimer molecules with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain two or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 37 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 26 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 38 shows an exemplary in-solution rotational amplification reaction using a mixture of nucleic acid circular library molecules, soluble first amplification primers, and nucleotides comprising nucleotides with cleavable moieties that can be cleaved to create non-basic sites. This is a schematic diagram. The rolling ring amplification reaction produces a single-stranded nucleic acid concatimer molecule in solution having at least one nucleotide with a cleavable moiety that can be cleaved to create a non-basic site in the concatimer molecule. The arrangement of various primer binding sequences in a circular nucleic acid library molecule is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 38-52 show flow diagrams of pairwise sequencing of the concatimer molecules shown in Figure 38.
Figure 39 is a schematic diagram showing an exemplary method comprising distributing the rolling ring amplification reaction shown in Figure 38 on a support on which a first surface primer is immobilized. The concatimer molecule can hybridize to the immobilized first surface primer.
Figure 40 shows a rotation that continues on a support to generate an immobilized concatimer template molecule comprising at least one nucleotide with a cleavable moiety that can be cleaved to create an abasic site in the immobilized concatimer template molecule. This is a schematic diagram showing an exemplary method of illustrating a ring amplification reaction.
Figure 41 is a schematic diagram showing an exemplary immobilized concatimer template molecule produced by the method shown in Figure 40.
Figure 42 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 41. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 43 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with strand transfer polymerase in the absence of a soluble primer.
Figure 44 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer.
Figure 45 is a schematic diagram showing an exemplary method of replacing an extended amplification primer strand by performing a primer extension reaction with a soluble forward sequencing primer.
46 shows a method for creating an abasic region of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 43 or Figure 44.
Figure 47 Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 46.
48 shows a method for creating an abasic region of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and maintaining a plurality of forward extension strands and a plurality of gaps while maintaining a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 45.
Figure 49 Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 48.
Figure 50 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 47. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers. The maintained forward extension strand shown in Figure 50 is a concatimer molecule that can contain two or more tandem copies of the sequence of interest and various primer binding sites. Such concatimer molecules may have two or more extended reverse sequencing primer strands hybridized to them. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 41-50 show exemplary immobilized concatimer molecules with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain two or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 51 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 49. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The retained forward extension strand shown in Figure 51 is a concatimer molecule containing two or more tandem copies of the sequence of interest and various primer binding sites. Such concatimer molecules may have two or more extended reverse sequencing primer strands hybridized to them. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support. For simplicity, Figures 41-51 show an exemplary immobilized concatimer molecule with two tandem copies containing the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that an immobilized concatimer molecule may contain three or more tandem copies containing the sequence of interest and various universal primer binding sites.
Figure 52 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 41 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 53 is a schematic showing a linear single stranded library molecule (top left schematic) hybridizing with a double stranded splint molecule (bottom left schematic) to generate a circular library molecule with two gaps (right schematic). The Sprint molecule comprises a first Sprint molecule (long strand) hybridized to a second Sprint molecule (short strand). The first sprint molecule includes a left-hand sequence that hybridizes to a sequence on one end of the linear single-stranded library molecule and a right-hand sequence that hybridizes to a sequence on the other end of the linear single-stranded library molecule. The internal portion of the first Sprint molecule hybridizes to the second Sprint molecule.
Figure 54 is Schematic showing the circular library molecule shown in Figure 53 (left schematic) undergoing a ligation reaction to generate a single-stranded covalently closed circular molecule (center schematic) that hybridizes to a first sprint molecule. A single-stranded covalently closed circular molecule is subjected to a rolling ring amplification reaction using the 3' end of the first sprint molecule to initiate the RCA reaction (schematic diagram on the right).
Figure 55 is A schematic diagram depicts an exemplary support on which a first surface primer is immobilized and, in some embodiments, can be used to perform a ligation reaction on the support for a pairwise sequencing flow diagram. Figures 55-72 show flow diagrams of ligation and pairwise sequencing on supports.
Figure 56 is a schematic diagram representing an exemplary single-stranded linear library molecule containing various complementary adapter sequences to the sequence of interest and primer binding sites. The arrangement of the various universal adapter sequences in this schematic is for illustrative purposes. Those skilled in the art will recognize that many different arrangements and combinations of universal adapter sequences are possible.
Figure 57 is a schematic diagram showing an exemplary single-stranded linear library molecule hybridized to an immobilized first surface primer to form a circular library molecule with an asymmetrically positioned gap or crevice.
Figure 58 (left) is a schematic diagram showing an exemplary single-stranded linear library molecule hybridized to an immobilized first surface primer to form a circular library molecule with an asymmetrically positioned gap or crevice. Figure 58 (right) is a schematic diagram showing an exemplary single-stranded linear library molecule hybridized to an immobilized first surface primer to form a circular library molecule with symmetrically positioned gaps or crevices. The schematics shown in Figures 57 and 58 represent several embodiments of circular library molecules comprising single stranded linear library molecules hybridized to immobilized first surface primers.
Figure 59 is a schematic diagram showing an exemplary covalently closed circular library molecule generated by covalently closing a gap or crevice.
Figure 60 (left) is a schematic showing an exemplary covalently closed prototype library molecule generated by covalently closing a gap or crevice. Figure 60 (right) is a schematic showing an exemplary covalently closed prototype library molecule generated by covalently closing a gap or crevice. The schematics shown in Figures 57 and 58 represent several embodiments of covalently closed circular library molecules hybridized to immobilized first surface primers.
Figure 61 is an example using a covalently closed circular library molecule, an immobilized first surface primer shown in Figure 55, and a mixture of nucleotides including nucleotides with cleavable moieties that can be cleaved to create abasic sites. This is a schematic diagram showing the rotational amplification reaction on a suitable support. The rotary ring amplification reaction produces a fixed single-stranded nucleic acid concatimer template molecule having at least one nucleotide with a cleavable moiety that can be cleaved to create a non-basic site in the fixed concatimer template molecule.
Figure 62 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 61. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 63 is a schematic diagram illustrating an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with strand transposition polymerase in the absence of a soluble primer to generate a forward extended strand.
Figure 64 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble forward sequencing primer to generate a forward extended strand.
Figure 65 is a schematic diagram showing an exemplary method of replacing an extended forward sequencing primer strand by performing a primer extension reaction with a soluble amplification primer to generate a forward extended strand.
Figure 66 shows the creation of an abasic portion of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and a plurality of gaps while maintaining a plurality of forward extension strands and a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 63 or Figure 64.
Figure 67 Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 66.
Figure 68 shows the creation of an abasic portion of an anchored single-stranded concatimer template molecule at a nucleotide with a cleavable moiety and a plurality of gaps while maintaining a plurality of forward extension strands and a plurality of anchored first surface primers. -A schematic diagram showing an exemplary method of creating a gap at a non-basic site to create a concatimer template molecule. The forward extension strand can be created by the method shown in Figure 65.
Figure 69 Schematic diagram showing an exemplary retained forward extension strand after removal of the gap-containing concatimer template molecule as shown in Figure 68.
Figure 70 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 67. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support.
Figure 71 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 69. The retained forward extension strand may have two or more extended reverse sequencing primer strands hybridized thereto. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support.
Figure 72 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 61 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 73 is Schematic diagram showing an exemplary single-stranded nucleic acid concatimer template molecule immobilized on an immobilized first surface primer. In some embodiments, immobilized concatimer template molecules can be generated by performing a rolling ring amplification reaction on a support. The arrangement of the various primer binding sequences is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 73-79 show flow diagrams of pairwise sequencing of the immobilized concatimer template molecule shown in Figure 73.
Figure 74 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 73. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 75 shows an ampoule hybridized to an immobilized concatimer template molecule by replacing the extended forward sequencing primer strand by performing a primer extension reaction using a soluble amplification primer and strand transfer polymerase in the presence of compressed oligonucleotides. Schematic diagram showing an exemplary method of forming a forward extending strand and a partially translocated forward extending strand forming a recon.
Figure 76 is a schematic diagram showing a continuation of the exemplary strand transposition method shown in Figure 75, wherein the polymerase-catalyzed strand transposition reaction comprises a forward extending strand and a partially translocated forward extending strand hybridized to an immobilized concatimer template molecule; and generates a detached forward extension strand that does not hybridize to the immobilized concatimer template molecule.
Figure 77 shows an exemplary hybridization complex comprising a forward extension strand and a partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, and an immobilized detached forward extension strand hybridized to the partially translocated forward extension strand. This is a schematic diagram.
Figure 78 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the hybridization complex shown in Figure 77. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers on the partially translocated forward extension strand and the fixed, detached forward extension strand. The reverse sequencing reaction generates an extended reverse sequencing primer strand. For brevity, Figure 78 shows one copy of the extended reverse sequencing primer strand on a partially translocated forward extension strand, and one copy of the extended reverse sequencing primer strand on a fixed, detached forward extension strand. Those skilled in the art will recognize that the partially translocated forward extension strand and the fixed detached forward extension strand include two or more extended reverse sequencing primer strands hybridized thereto.
Figure 79 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 73 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 80 is Schematic diagram showing an exemplary single-stranded nucleic acid concatimer template molecule immobilized on an immobilized first surface primer. In some embodiments, immobilized concatimer template molecules can be produced by performing a rolling ring amplification reaction in solution and distributing the rolling ring amplification reaction on a support. The arrangement of the various primer binding sequences is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 80-86 show flow diagrams of pairwise sequencing of the immobilized concatimer template molecule shown in Figure 80.
Figure 81 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 80. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 82 shows an ampoule hybridized to an immobilized concatimer template molecule by replacing the extended forward sequencing primer strand by performing a primer extension reaction using a soluble amplification primer and strand transfer polymerase in the presence of a compressed oligonucleotide. Schematic diagram showing an exemplary method of forming a forward extending strand and a partially translocated forward extending strand forming a recon.
Figure 83 is a schematic diagram illustrating a continuation of the exemplary strand transposition method shown in Figure 82, wherein the polymerase-catalyzed strand transposition reaction comprises a forward extending strand and a partially translocated forward extending strand hybridized to an immobilized concatimer template molecule; and generates a detached forward extension strand that does not hybridize to the immobilized concatimer template molecule.
Figure 84 shows an exemplary hybridization complex comprising a forward extension strand and a partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, and an immobilized detached forward extension strand hybridized to the partially translocated forward extension strand. This is a schematic diagram.
Figure 85 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the hybridization complex shown in Figure 84. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers on the partially translocated forward extension strand and the fixed, detached forward extension strand. The reverse sequencing reaction generates an extended reverse sequencing primer strand. For brevity, Figure 85 shows one copy of the extended reverse sequencing primer strand on a partially translocated forward extension strand, and one copy of the extended reverse sequencing primer strand on a fixed, detached forward extension strand. Those skilled in the art will recognize that the partially translocated forward extension strand and the fixed detached forward extension strand include two or more extended reverse sequencing primer strands hybridized thereto.
Figure 86 is a schematic diagram showing an exemplary support to which first and second surface primers are immobilized. A portion of the immobilized concatimer template molecule shown in Figure 80 hybridizes to the immobilized second surface primer. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.
Figure 87 A schematic diagram depicts an exemplary support to which a first surface primer is immobilized and, in some embodiments, can be used to perform a pairwise sequencing flow diagram on the support.
Figure 88 is a schematic diagram showing a rolling ring amplification reaction on an exemplary support using a circular nucleic acid library molecule and an immobilized first surface primer shown in Figure 87. The rolling ring amplification reaction generates an immobilized single-stranded nucleic acid concatimer template molecule. The arrangement of various primer binding sequences in a circular nucleic acid library molecule is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 87-94 show flow diagrams of pairwise sequencing of the immobilized concatimer template molecule shown in Figure 87.
Figure 89 Schematic diagram showing an exemplary single-stranded nucleic acid concatimer template molecule immobilized on an immobilized first surface primer.
Figure 90 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 89. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 91 shows an ampoule hybridized to an immobilized concatimer template molecule by replacing the extended forward sequencing primer strand by performing a primer extension reaction using a soluble amplification primer and strand transfer polymerase in the presence of a compressed oligonucleotide. Schematic diagram showing an exemplary method of forming a forward extending strand and a partially translocated forward extending strand forming a recon.
Figure 92 is a schematic diagram illustrating a continuation of the exemplary strand transposition method shown in Figure 91, wherein the polymerase-catalyzed strand transposition reaction comprises a forward extending strand and a partially translocated forward extending strand hybridized to an immobilized concatimer template molecule; and generates a detached forward extension strand that does not hybridize to the immobilized concatimer template molecule.
Figure 93 shows an exemplary hybridization complex comprising a forward extension strand and a partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, and an immobilized detached forward extension strand hybridized to the partially translocated forward extension strand. This is a schematic diagram.
Figure 94 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the hybridization complex shown in Figure 93. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers on the partially translocated forward extension strand and the fixed, detached forward extension strand. The reverse sequencing reaction generates an extended reverse sequencing primer strand. For brevity, Figure 94 shows one copy of the extended reverse sequencing primer strand on a partially translocated forward extension strand, and one copy of the extended reverse sequencing primer strand on a fixed, detached forward extension strand. Those skilled in the art will recognize that the partially translocated forward extension strand and the fixed detached forward extension strand include two or more extended reverse sequencing primer strands hybridized thereto.
Figure 95 is a schematic diagram showing an exemplary in-solution rolling ring amplification reaction using a mixture of nucleic acid circular library molecules, soluble first amplification primers, and nucleotides. Rotational ring amplification reactions produce single-stranded nucleic acid concatemer molecules in solution. The arrangement of various primer binding sequences in a circular nucleic acid library molecule is for illustrative purposes. Those skilled in the art will recognize that many other arrangements are possible. Figures 95-103 show flow diagrams of pairwise sequencing of the concatimer molecules shown in Figure 96.
Figure 96 is a schematic diagram showing an exemplary method comprising distributing the rolling ring amplification reaction shown in Figure 95 on a support on which a first surface primer is immobilized. The concatimer molecule can hybridize to the immobilized first surface primer.
Figure 97 is a schematic diagram illustrating an exemplary method depicting a rolling ring amplification reaction resulting in an immobilized concatimer template molecule by continuing on a support.
Figure 98 is Schematic diagram showing an exemplary single-stranded nucleic acid concatimer template molecule immobilized on an immobilized first surface primer.
Figure 99 is A schematic diagram showing an exemplary forward sequencing reaction performed on an immobilized concatimer template molecule shown in Figure 98. The forward sequencing reaction can be performed with a plurality of soluble forward sequencing primers. The immobilized concatimer template molecule may have two or more extended forward sequencing primer strands hybridized to it.
Figure 100 shows an ampoule hybridized to an immobilized concatimer template molecule by replacing the extended forward sequencing primer strand by performing a primer extension reaction using a soluble amplification primer and strand transfer polymerase in the presence of a compressed oligonucleotide. Schematic diagram showing an exemplary method of forming a forward extending strand and a partially translocated forward extending strand forming a recon.
Figure 101 is a schematic diagram showing a continuation of the exemplary strand transposition method shown in Figure 100, wherein the polymerase-catalyzed strand transposition reaction comprises a forward extending strand and a partially translocated forward extending strand hybridized to an immobilized concatimer template molecule; and generates a detached forward extension strand that does not hybridize to the immobilized concatimer template molecule.
Figure 102 shows an exemplary hybridization complex comprising a forward extension strand and a partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, and an immobilized detached forward extension strand hybridized to the partially translocated forward extension strand. This is a schematic diagram.
Figure 103 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the hybridization complex shown in Figure 102. The reverse sequencing reaction can be performed with a plurality of soluble reverse sequencing primers on the partially translocated forward extension strand and the fixed, detached forward extension strand. The reverse sequencing reaction generates an extended reverse sequencing primer strand. For brevity, Figure 103 shows one copy of the extended reverse sequencing primer strand on a partially translocated forward extension strand, and one copy of the extended reverse sequencing primer strand on a fixed, detached forward extension strand. Those skilled in the art will recognize that the partially translocated forward extension strand and the fixed detached forward extension strand include two or more extended reverse sequencing primer strands hybridized thereto.
Figure 104 is a schematic diagram of various exemplary stereo media of multivalent molecules. Left: Schematic illustration of a multivalent molecule with starburst or helter-skelter configuration. Center: Schematic diagram of a multivalent molecule with dendrimer configuration. Right: Schematic illustration of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. The nucleotide unit is denoted as 'N', biotin is denoted as 'B', and streptavidin is denoted as 'SA'.
Figure 105 Schematic diagram of an exemplary multivalent molecule comprising a general core attached to a plurality of nucleotide-arms.
Figure 106 is Schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.
Figure 107 shows a schematic diagram of an exemplary multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms include biotin, a spacer, a linker, and a nucleotide unit.
Figure 108 is a schematic diagram of an exemplary nucleotide-arm comprising a core attachment moiety, spacer, linker, and nucleotide units.
Figure 109 shows the chemical structures of exemplary spacers and various exemplary linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker.
Figure 110 shows the chemical structures of various exemplary linkers, including linkers 1 through 9.
Figure 111 is The chemical structures of various exemplary linkers bound/attached to nucleotide units are shown.
Figure 112 The chemical structures of various exemplary linkers bound/attached to nucleotide units are shown.
Figure 113 The chemical structures of various exemplary linkers bound/attached to nucleotide units are shown.
Figure 114 shows the chemical structure of an exemplary nucleotide-arm. In this example, the nucleotide units are linked to the linker via a propargyl amine attachment at position 5 of the pyrimidine base or position 7 of the purine base. This nucleotide-arm represents an exemplary biotinylated nucleotide-arm.
115 shows a low binding support comprising a glass substrate and further comprising a chemically reactive functional group that is covalently or non-covalently attached to the glass and serves as an attachment site for oligonucleotide primers (e.g., capture oligonucleotides and cyclic oligonucleotides). This is an exemplary schematic illustration of one embodiment of alternating layers of hydrophilic coating. In alternative embodiments, the support may be made of any material such as glass, plastic or polymeric materials.
Figure 116A is a schematic diagram of a guanine tetrad (e.g., G-tetrad).
Figure 116B is a schematic diagram of the intramolecular G-quadruplex structure.
Figure 117 is an exemplary single cycle showing flow in nucleic acid relaxation buffer at increasing temperature and temperature, washing steps, and flow in flex amplification buffer containing strand-transfer DNA polymerase at increasing temperature and MDA incubation and decreasing temperature. This is a schematic diagram of the cycle. One or more cycles of flow in flex amplification buffer containing strand-translocation DNA polymerase at increasing temperature and MDA amplification and decreasing temperature are performed.
Figure 118 (left) is a graph showing error rates from R1 sequencing reads of template molecules with various levels of uracil. Figure 118 (right) is a graph showing phasing rates from R1 sequencing reads of template molecules with various levels of uracil. The data show that sequencing template molecules with lower levels of incorporated uracil yield lower error rates and phase ratios. The level of uracil in the template molecule also affects the intensity ratio of the R2/R1 readout.
Figure 119 is a sequencing flow chart showing increased ratio of signal intensity for R2/R1 sequencing reads using cleavage reagents comprising compounds that reduce photodamage to nucleic acids. Lanes 1, 3, 5 and 7 show R2/R1 signal intensity using different cleavage reagent formulations without compounds that reduce photodamage. Lanes 2, 4, 6 and 8 show R2/R1 signal intensity using the corresponding cleavage reagent formulation containing a compound that reduces photodamage.

Justice

The headings provided herein are not intended to be limiting of the various aspects of the disclosure, aspects of which may be understood by reference to the specification as a whole.

Unless otherwise defined, technical and scientific terms used herein have meanings commonly understood by those skilled in the art, unless otherwise defined. In general, terms relating to the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization described herein are well known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with this specification and the experimental procedures and techniques described herein are well known and commonly used in the art.

Unless otherwise required by the context of this specification, singular terms shall include pluralities and plural terms shall include the singular. Singular forms and singular uses of any word include plural referents unless otherwise clearly and unambiguously limited to one referent.

It is understood that use of alternative terms (e.g., “or”) is taken to mean one or both of the alternatives or any combination thereof.

As used herein, the term “and/or” shall be taken to mean the specific disclosure of each specified feature or component, regardless of the presence or absence of the other. For example, the term “and/or” as used herein in phrases such as “A and/or B” means “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and/or” as used in phrases such as “A, B and/or C” is intended to encompass each of the following aspects: “A, B and C”; “A, B or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

As used in this specification and the appended claims, the terms “comprising,” “including,” “having,” and “comprising,” and grammatical variations thereof, are intended to be non-limiting; , therefore, one item or multiple items in a list does not exclude one item that replaces or is in addition to the listed item. It is understood that where aspects herein are described with the language "comprising" other similar aspects described in terms of "consisting of" and/or "consisting essentially of" are also provided.

As used herein, the terms "about" and "approximately" refer to the method by which the value or composition is measured or determined, i.e., the allowable error for a particular value or composition as determined by one of ordinary skill in the art depending in part on the limitations of the measurement system. Refers to a value or composition that is within a range. For example, “about” or “approximately” can mean one standard deviation or more per run in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e. ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or processes, the term can mean up to 10 times the value or up to 5 times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, “about” or “approximately” should be assumed to be within an acceptable margin of error for the particular value or composition. Additionally, when ranges and/or subranges of values are provided, the ranges and/or subranges may include endpoints of the ranges and/or subranges.

The term “biological sample” refers to a single cell, multiple cells, tissue, organ, organism, or section of any such biological sample. Biological samples are extracted from an organism (e.g., a biopsy) or obtained from a cell culture grown in liquid or in a culture dish. Biological samples include fresh, frozen, fresh-frozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) samples. Biological samples can be embedded in wax, resin, epoxy, or agar. Biological samples can be fixed in any one or any combination of two or more of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton, or glutaraldehyde. Biological samples may be sectioned or unsectioned. Biological samples may be stained, unstained, or unstained.

Nucleic acids of interest can be extracted from biological samples using a number of techniques known to those skilled in the art. For example, a typical DNA extraction procedure involves (i) collection of cell or tissue samples from which DNA is extracted, (ii) disruption of the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components, and (iii) protein , treatment of the dissolved sample with a concentrated salt solution to precipitate lipids and RNA, followed by centrifugation to separate the precipitated proteins, lipids and RNA, and (iv) detergents, proteins, salts or Involves purification of DNA from the supernatant to remove other reagents. A variety of suitable commercial nucleic acid extraction mill purification kits are consistent with the disclosure herein. Examples include the QIAamp kit (for isolation of genomic DNA from human samples) and the DNAeasy kit (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD) or Promega (Madison, WI). Including, but not limited to, the Maxwell® and ReliaPrep™ series of kits from

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” and other related terms used herein are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids are recombinant and include chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids (PNAs), and non-natural derived nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids may be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, where the nucleotides include natural or unnatural bases and/or sugars. Nucleic acids contain naturally occurring internucleoside linkages, such as phosphodiester linkages. Nucleic acids may lack phosphate groups. Nucleic acids contain non-natural internucleoside linkages, including phosphorothioate, phosphorothioate, or peptide nucleic acid (PNA) linkages. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

The terms “universal sequence,” “universal adapter sequence,” and related terms refer to sequences of nucleic acid molecules that are common among two or more polynucleotide molecules. For example, adapters having the same universal sequence can be linked to a plurality of polynucleotides such that the population of co-assembled molecules has the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or support immobilized capture primers).

As used herein, the terms “operably connected” and “operably coupled” or related terms refer to the juxtaposition of components. Juxtaposed components may be covalently linked together. For example, two nucleic acid components can be enzymatically ligated together, where the linkage that joins the two components together includes a phosphodiester bond. The first nucleic acid and the second nucleic acid component can be linked together, where the first nucleic acid component can confer a function to the second nucleic acid component. For example, linkage between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion capable of binding a primer. In another example, a transgene (e.g., a polypeptide or nucleic acid encoding a sequence of interest) can be ligated to a vector, where the linkage allows expression or functionalization of the transgene sequence contained in the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcription and/or translation initiation sequence, a transcription and/or translation termination sequence, a polypeptide secretion signal sequence, etc. In some embodiments, host cell regulatory sequences control expression of the level, timing and/or location of the transgene.

The terms “linked,” “bound,” “attached,” “suspended,” and their variations refer to any type of fusion between any combination of compounds or molecules that has sufficient stability to withstand use in a particular procedure. , includes bonding, attachment, or association. The procedure involves nucleotide binding; nucleotide incorporation; de-blocking (e.g., removal of a chain terminating moiety); wash; eliminate; flow; detection; It may include, but is not limited to, imaging and/or identification. Such bonds may include, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, etc. In some embodiments, such linking occurs intramolecularly, for example, joining the ends of single- or double-stranded linear nucleic acid molecules together to form a circular molecule. In some embodiments, such linkages include linkages between combinations of different molecules, or between nucleic acid molecules and a solid surface; A link between a protein and a detectable receptor moiety; It can occur between molecules and non-molecules, including but not limited to linkages between nucleotides and detectable receptor moieties. Some examples of combinations include, for example, Hermanson, G., “Bioconjugate Techniques”, Second Edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., "Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences", London: Macmillan (1998).

The term “adapter” and related terms refer to an oligonucleotide that can be operably linked (suspended) to a target polynucleotide, wherein the adapter confers functionality to the co-bound adapter-target molecule. Adapters include DNA, RNA, chimeric DNA/RNA, or analogs thereof. The adapter may include at least one ribonucleoside residue. Adapters may be single-stranded, double-stranded, or may have single-stranded and/or double-stranded portions. Adapters can be arranged in a linear, stem-loop, hairpin or Y-shape. Adapters can be of any length, including from 4 to 100 or more nucleotides. Adapters may have blunt ends, protruding ends, or a combination of both. The overhang ends include the 5' overhang and the 3' overhang ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, may have a 5' phosphate group or lack a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tail adapter), or the adapter may not be a tail. The adapter may include a sequence complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). Adapters may comprise random or degenerate sequences. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothioate and/or phosphoramidate linkage. Adapters may include barcode sequences that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex analysis. Adapters may include a unique identification sequence (e.g., a unique molecular index UMI; or unique molecular tag) that can be used to uniquely identify the nucleic acid molecule on which the adapter is suspended. In some embodiments, unique identification sequences can be used to increase error correction and accuracy, reduce false positive variant call rates, and/or increase sensitivity of variant detection. The adapter may include at least one restriction enzyme recognition sequence comprising any one or any combination of two or more selected from the group consisting of type I, type II, type III, type IV, type Hs or type IIB.

The terms “nucleic acid template”, “template polynucleotide”, “nucleic acid target”, “target polynucleotide”, “template strand” and other variations refer to any of the analytical methods described herein (e.g., primer extension, amplification and/or or sequencing) refers to a nucleic acid strand that serves as the basic nucleic acid molecule for sequencing. The template nucleic acid may be single-stranded or double-stranded, or the template nucleic acid may have single-stranded or double-stranded portions. Template nucleic acids can be obtained from naturally occurring sources, recombinant forms, or can be chemically synthesized to include any type of nucleic acid analog. Template nucleic acids may be linear, circular, or in other forms. The template nucleic acid may include an insertion region with an insertion sequence, also known as a sequence of interest. The template nucleic acid may also include at least one adapter sequence. The template nucleic acid may be a concatemer having two or tandem copies of the sequence of interest and at least one adapter sequence. Insertion regions can be chromosomes, genomes, organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules obtained from fresh frozen paraffin-embedded tissue, needle biopsy, circulating tumor cells, cell-free circulating DNA, or any type of nucleic acid library. , cloned, amplified, cDNA, RNA, such as precursor mRNA or mRNA, oligonucleotides, total genomic DNA. The insertion region may be an organism, such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungi, viral cells, tissues, normal or diseased cells or tissues, blood, urine, serum, lymph, tumors, saliva, Can be isolated from any source, including body fluids including anal and vaginal secretions, amniotic membrane samples, sweat, semen, environmental samples, culture samples, or from synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. there is. Insertion areas include the head, neck, brain, breast, ovaries, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testes, liver, lungs, kidneys, esophagus, pancreas, thyroid, pituitary gland, thymus, and skin. , can be isolated from any organ, including the heart, larynx or other organs. Template nucleic acids can be subjected to nucleic acid analysis, including sequence analysis and composition analysis.

As used herein, the term "polymerase" and variants thereof include enzymes comprising a domain that binds to a nucleotide (or nucleoside), wherein the polymerase is capable of forming a complex with a template nucleic acid and a complementary nucleotide. You can. A polymerase may have one or more activities including, but not limited to, base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand translocation activity, and nucleotide binding and recognition. A polymerase may be any enzyme capable of catalyzing the polymerization of nucleotides (including their analogs) into a nucleic acid strand. Typically, but not necessarily, this nucleotide polymerization may occur in a template-dependent manner. Typically, polymerases contain one or more active sites where nucleotide binding and/or catalysis of nucleotide polymerization can occur. In some embodiments, the polymerase comprises other enzymatic activity, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand translocation activity. Polymerases include naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinants, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and It may include, but is not limited to, any analog, derivative or fragment thereof (e.g., a catalytically active fragment) that retains the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes that contain a nucleotide binding domain. In some embodiments, the polymerase can be isolated from cells or produced using recombinant DNA techniques or chemical synthesis methods. In some embodiments, the polymerase can be expressed in prokaryotic, eukaryotic, viral, or phage organisms. In some embodiments, the polymerase may be a post-translationally modified protein or fragment thereof. Polymerases may be derived from prokaryotes, eukaryotes, viruses, or phages. Polymerases include DNA-forward DNA polymerase and RNA-forward DNA polymerase.

The term “strand translocation” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize new strands in a template-based manner. Strand transposition polymerases translocate the complementary strand from the template strand and catalyze new strand synthesis. Strand transposition polymerases include mesophilic and thermophilic polymerases. Strand transposition polymerases include wild-type enzymes and variants, including exonuclease minus mutants, mutant forms, chimeric enzymes, and truncated enzymes. Examples of strand transposition polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), large fragment of Bca DNA polymerase (exo-), E. coli Includes the Klenow fragment of DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

As used herein, the term “DNA primase-polymerase” and related terms refer to enzymes that have the activities of DNA polymerase and RNA primase. DNA primase-polymerases can utilize deoxyribonucleotide triphosphates and catalytic divalent cations (e.g., magnesium and/or manganese) to synthesize DNA primers on single-stranded DNA templates in a template-sequence dependent manner. Primer strands can be extended through nucleotide polymerization (e.g., primer extension) in the presence of . DNA primase-polymerases include enzymes that are members of the DnaG-like primases (e.g., bacteria) and AEP-like primases (archaea and eukaryotes). An exemplary DNA primase-polymerase is Tth PrimPol from Thermus thermophilus HB27.

As used herein, the term “fidelity” refers to the accuracy of DNA polymerization by template-dependent DNA polymerase. The fidelity of a DNA polymerase is measured by its error rate (the frequency of incorporating incorrect nucleotides, i.e., nucleotides that are not complementary to the template nucleotide). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3'-5' exonuclease activity of DNA polymerase.

As used herein, the term "binding complex" refers to a complex formed by joining together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide units of a multivalent molecule, wherein the nucleic acid duplex is a nucleic acid template molecule hybridized to a nucleic acid primer. Includes. In a binding complex, a free nucleotide or nucleotide unit may or may not be bound to the 3' end of a nucleic acid primer at a position opposite the complementary nucleotide in the nucleic acid template molecule. A “ternary complex” is an example of a binding complex formed by joining together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, wherein the free nucleotide or nucleotide unit is opposite the complementary nucleotide of the nucleic acid template molecule. It is bound to the 3' end of a nucleic acid primer (as part of a nucleic acid duplex) at a position.

The term “duration” and related terms refer to the length of time during which a binding complex remains stable without dissociation of any of its components, wherein the components of the binding complex include nucleic acid templates and nucleic acid primers, polymerases, and multivalent molecules. Includes nucleotide units or free (e.g., unconjugated) nucleotides. Nucleotide units or free nucleotides may be complementary or non-complementary to nucleotide residues in the template molecule. The nucleotide unit or free nucleotide may bind to the 3' end of the nucleic acid primer at a position opposite the complementary nucleotide residue in the nucleic acid template molecule. The duration indicates the stability of the binding complex and the strength of the binding interaction. Duration can be measured by observing the initiation and/or duration of the binding complex, such as by observing signals from labeled components of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in a binding complex, thereby allowing the signal to be detected by the label for the duration of the binding complex. One exemplary label is a fluorescent label. The binding complex (e.g., ternary complex) remains stable until subjected to any polymerase, template molecule, primer, and/or conditions that cause dissociation between nucleotide units or nucleotides. For example, dissociation conditions include contacting the binding complex with any one or any combination of detergent, EDTA, and/or water.

The term “primer” and related terms as used herein refers to an oligonucleotide capable of hybridizing with a DNA and/or RNA polynucleotide template to form a duplex molecule. Primers include natural nucleotides and/or nucleotide analogs. Primers may be recombinant nucleic acid molecules. Primers can be of any length, but typically range from 4 to 50 nucleotides. A typical primer includes a 5' end and a 3' end. The 3' end of the primer may include a 3' OH moiety that acts as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of the primer may lack a 3' OH moiety or may contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer may be labeled with a detectable reporter moiety. Primers may be in solution (e.g., soluble primers) or immobilized on a support (e.g., capture primers).

When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refer to hydrogen bonds between two different nucleic acids forming a duplex nucleic acid. Hybridization also involves hydrogen bonding between two different regions of a single nucleic acid molecule forming a self-hybridizing molecule with a duplex region. Hybridization may involve a duplex double-stranded nucleic acid, or a Watson-Crick or Hoogstein bond that forms a double-stranded region within a nucleic acid molecule. Two different regions of a double-stranded nucleic acid, or a single nucleic acid, may be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other over their entire length. Complementary base pairings may be standard A-T or C-G base pairings, or may be other forms of base-pairing interactions. Duplex nucleic acids may contain mismatched base-paired nucleotides.

When used with reference to nucleic acids, the terms “extend,” “extending,” “extension,” and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides at the terminal 3' OH of a nucleic acid strand (e.g., a nucleic acid primer), resulting in extension of the nucleic acid strand (e.g., an extended primer). Nucleotide incorporation can be accomplished by natural nucleotides and/or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method of extending nucleic acid molecules can be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

In some embodiments, any of the amplification primer sequences, sequencing primer sequences, capture primer sequences (capture oligonucleotides), target capture sequences, circular anchor sequences, sample barcode sequences, spatial barcode sequences, or anchor region sequences are about 3 to 50. nucleotides in length, or about 5 to 40 nucleotides in length, or about 5 to 25 nucleotides in length.

The term “nucleotide” and related terms refer to a molecule comprising an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of the terms above. In some embodiments, the phosphate includes monophosphate, diphosphate, or triphosphate, or corresponding phosphate analogs. The term “nucleoside” refers to a molecule containing an aromatic base and a sugar. Nucleotides and nucleosides may be unlabeled or labeled with a detectable reporter moiety.

Nucleotides (and nucleosides) typically contain a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring commonly found in nucleic acids, including naturally occurring, substituted, modified or engineered variants, or analogs thereof. Contains cyclic bases. The bases of a nucleotide (or nucleoside) can form Watson-Crick and/or Hoogsteen hydrogen bonds with appropriate complementary bases. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N ⁶ -Δ ² -iso Pentenyl adenine (6iA), N ⁶ -Δ ² -isopentenyl-2-methylthioadenine (2ms6iA), N ⁶ -methyladenine, guanine (G), isoguanine, N ² -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O ⁶ -methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); Pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O ⁴ -methylthymine, ura sil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; Pyrrole, such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methycytosine; base (Y); and methylated, glycosylated and acylated base moieties, etc. Additional exemplary bases are described in Fasman, 1989, “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

Nucleotides (and nucleosides) typically contain sugar moieties, such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627) -2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Patent No. 5,558,991). The sugar moiety is ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribyl carbocyclic; Contains acyclic or other modified sugars.

In some embodiments, the nucleotide comprises a chain of 1, 2, or 3 phosphorus atoms, the chain typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atom is linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom of the chain comprises a substituted side group comprising O, S, or BH ₃ . In some embodiments, the chain comprises a phosphate group substituted with analogs including phosphoramidate, phosphorothioate, phosphodithioate, and O-methylphosphoroamidite groups.

The terms “reporter moiety”, “reporter moiety” or related terms refer to a compound that produces or causes to produce a detectable signal. The reporter moiety is sometimes called a “beacon”. Any suitable reporter moiety, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophore, radioisotope, electrochemistry, mass spectrometry, Raman, hapten, affinity tag, atom, or enzyme. can be used. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or proximal events). A proximity event involves two reporter moieties approaching each other, or associating with or binding to each other. It is well known to those skilled in the art to select reporter moieties so that each absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from other reporter moieties to monitor the presence of different reporter moieties in the same reaction or in different reactions. makes possible. Two or more different reporter moieties may be selected that have spectrally distinct emission profiles or have minimally overlapping spectral emission profiles. The reporter moiety may be linked (e.g., operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (e.g., polymerase or reverse transcriptase), or support (e.g., a surface).

The reporter moiety (or label) includes a fluorescent label or fluorophore. Exemplary fluorescent moieties that can act as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, Fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbhydrazinomethylthio. Acetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacet Amides, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS , AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY 530/550 C3 hydrazide, BODIPY 493 /503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, Pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT , Europium chelate, Terbium chelate, Alexa Fluor dye, DyLight dye, Atto dye, LightCycler Red dye, CAL Flour dye, JOE and their derivatives, Oregon Green dye, WellRED dye, IRD dye, phycoerythrin and phycolbilin dye, Malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, OR) 6th Edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. It doesn't work. Cyanine dyes may exist in sulfonated or non-sulfonated forms and consist of two indolenines, benzo-indolium, pyridium, thiozolium and/or separated by a polymethine bridge between the two nitrogen atoms. Or it consists of a quinolinium group. Commercially available cyanine fluorophores include, for example, Cy3(1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1 -[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop p-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]- 2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro -2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5(1-( 6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2 ,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl )-3,3-dimethyl-3H-indole-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-(( 1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5- Sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1, 3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo- 1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate) and , "Cy" stands for 'cyanine', and the first number identifies the number of carbon atoms between the two indolene groups. Cy2, which is an oxazole derivative rather than an indolene, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

In some embodiments, the reporter moiety can be a FRET pair so that multiple fractionations can be performed under a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron-exchange (Dexter) transfer.

As used herein, the term “support” refers to a substrate designed for the deposition of biological molecules or biological samples for assay and/or analysis. Examples of biological molecules to be deposited on the support include nucleic acids (e.g., DNA, RNA), polypeptides, saccharides, lipids, single or multicellular. Examples of biological samples include, but are not limited to, saliva, phlegm, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support may be cylindrical, including, for example, a capillary or an inner surface of a capillary.

In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the scaffold comprising bumps, etchings, pores, three-dimensional scaffolds, or any combination thereof may be regularly or irregularly textured.

In some embodiments, the support includes beads having any shape, including spheres, hemispheres, cylinders, barrels, donuts, discs, rods, cones, triangles, cubes, polygons, tubulars, or wires. .

Supports include glass, fused silica, silicone, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene ( PE), high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. It can be manufactured from materials. Various compositions of both glass and plastic substrates are contemplated.

The support may have a plurality (e.g., two or more) nucleic acid templates immobilized thereon. The plurality of fixed nucleic acid templates may have the same sequence or have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are anchored to different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are anchored to sites on the support.

The term “array” refers to a support comprising a plurality of sites positioned at predetermined locations on the support to form an array of sites. Regions may be separated and may be separated by interstitial regions. In some embodiments, the predetermined portions on the support can be arranged one-dimensionally in rows or columns, or two-dimensionally in rows and columns. In some embodiments, a plurality of predetermined sites are arranged on the support in an organized manner. In some embodiments, the plurality of predetermined portions are arranged in any organized pattern, including straight lines, hexagonal patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, etc. The pitch between different pairs of regions may be the same or different. In some embodiments, the support has at least 10 ² sites, at least 10 ³ sites, at least 10 ⁴ sites, at least 10 ⁵ sites, at least 10 ⁶ sites, at least 10 ⁷ sites, at least 10 ⁸ sites, at least 10 ⁹ sites, at least 10 ¹⁰ sites, at least 10 ¹¹ sites, at least 10 ¹² sites, at least 10 ¹³ sites, at least 10 ¹⁴ sites, at least 10 ¹⁵ sites, where the sites are selected from the dictionary on the support. It is located at a determined location. In some embodiments, a plurality of predetermined sites (eg, 10 ² to 10 ¹⁵ or more sites) on the support are immobilized with a nucleic acid template to form a nucleic acid template array. In some embodiments, the immobilized nucleic acid template is covalently attached to the surface capture primer by hybridization to the immobilized surface capture primer or nucleic acid template at a plurality of predetermined sites. In some embodiments, the nucleic acid template is anchored at a plurality of predetermined sites, for example, 10 ² to 10 ¹⁵ or more sites. In some embodiments, the immobilized nucleic acid template generates immobilized nucleic acid polities at a plurality of predetermined sites. In some embodiments, each fixed nucleic acid polony comprises a single-stranded or double-stranded concatemer.

In some embodiments, a support comprising a plurality of sites positioned at randomly positioned sites on the support is referred to herein as a support having randomly positioned sites thereon. The positions of randomly positioned sites on the support are not predetermined. A plurality of randomly positioned sites are arranged on the support in an irregular and/or unpredictable manner. In some embodiments, the support has at least 10 ² sites, at least 10 ³ sites, at least 10 ⁴ sites, at least 10 ⁵ sites, at least 10 ⁶ sites, at least 10 ⁷ sites, at least 10 ⁸ sites, at least 10 ⁹ sites, at least 10 ¹⁰ sites, at least 10 ¹¹ sites, at least 10 ¹² sites, at least 10 ¹³ sites, at least 10 ¹⁴ sites, at least 10 ¹⁵ sites, where the sites are on the support. are located randomly. In some embodiments, a plurality of randomly positioned sites (eg, 10 ² to 10 ¹⁵ or more sites) on the support are immobilized together with the nucleic acid template to form a support on which the nucleic acid template is immobilized. In some embodiments, the immobilized nucleic acid template is covalently attached to the surface capture primer at a plurality of randomly located sites by hybridization to the immobilized surface capture primer or nucleic acid template. In some embodiments, the nucleic acid template is anchored at a plurality of randomly located sites, for example, at more than 10 ² to 10 ¹⁵ sites. In some embodiments, the fixed nucleic acid template generates fixed nucleic acid polities at a plurality of randomly located sites. In some embodiments, each fixed nucleic acid polony comprises a single-stranded or double-stranded concatemer.

When used in conjunction with low bond surface coatings, one or more layers of the multilayer surface coating may include branched polymers or may be linear. Examples of suitable branched polymers are branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinyl pyridine), branched poly(vinyl pyrrolidone) (branched PVP), branched) , poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched branched poly(2-hydroxylethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched Includes, but is not limited to, branched poly-lysine, branched poly-glucoside, and dextran.

In some embodiments, the branched polymer used to create one or more layers of any of the multilayer surfaces disclosed herein has at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches It may include branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches.

The linear, branched or multi-branched polymer used to create one or more layers of any of the multilayer surfaces disclosed herein may have at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 10,000, It may have a molecular weight of at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000 or at least 50,000 daltons.

In some embodiments, for example, at least one layer of the multilayer surface comprises a branched polymer, and the number of covalent bonds between the branched polymer molecules of the layer being deposited and the molecules of the previous layer is about 1 per molecule. It can range from covalent bonds to about 32 covalent bonds per molecule. In some embodiments, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, There may be at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30 or at least 32 covalent bonds.

Any reactive functional groups remaining after bonding of the quenching layer to the surface can be selectively blocked by coupling small, inert molecules using high-yield bonding chemistries. For example, when using amine bonding chemistry to attach a new layer of material to a previous layer of material, any remaining amine groups may subsequently be acetylated or deacetylated by coupling with a small amino acid such as glycine. You can.

The number of layers of low non-specific binding material, such as a hydrophilic polymeric material, deposited on the surface may range from 1 to about 10. In some embodiments, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some embodiments, the number of layers can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure; for example, in some embodiments, the number of layers may range from about 2 to about 4. In some embodiments, all layers may include the same material. In some embodiments, each layer may include a different material. In some embodiments, the plurality of layers can include multiple materials. In some embodiments, at least one layer can include a branched polymer. In some embodiments, all layers may include branched polymers.

One or more layers of the low non-specific binding material may, in some cases, be deposited and/or bonded onto the substrate surface using a polar protic solvent, a polar or polar aprotic solvent, a non-polar solvent, or any combination thereof. You can. In some embodiments, solvents used for layer deposition and/or bonding include alcohols (e.g., methanol, ethanol, propanol, etc.), other organic solvents (e.g., acetonitrile, dimethyl sulfoxide (DMSO) , dimethyl formamide (DMF), etc.), water, aqueous buffer solutions (e.g., phosphate buffer, phosphate buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any of these. It may include a combination of . In some embodiments, the organic component of the solvent mixture used is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, It may comprise 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, with the remainder comprised of water or an aqueous buffer solution. In some embodiments, the constituents of the solvent mixture used are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, It may contain 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, with the remainder being comprised of organic solvent. The pH of the solvent mixture used may be less than 6, about 6, 6.5, 7, 7.5, 8, 8.5, 9, or greater than pH 9.

The term “branched polymer” and related terms refer to a polymer having a plurality of functional groups that assist in conjugation to biologically active molecules, such as nucleotides, where the functional groups may be on the side chains of the polymer or directly attached to the central core or central backbone of the polymer. You can. Branched polymers may have a linear backbone with one or more functional groups extending beyond the backbone for conjugation. Branched polymers can also be polymers with one or more side chains, where the side chains have sites suitable for conjugation. Examples of functional groups are hydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated sulfone, hydrazide, thiol, alkanoic acid, acid halide, iso. Includes cyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate. It is not limited to these.

When used in reference to immobilized nucleic acids, the term "immobilized" and related terms refer to nucleic acid molecules that are attached to a support through covalent or non-covalent interactions, attached to a coating on a support, or embedded within a matrix formed by a coating on a support. refers to , wherein the nucleic acid molecule includes a surface capture primer, a nucleic acid template molecule, and an extension product of the capture primer. The extension products of the capture primer include nucleic acid concatemers that can form nucleic acid polities.

In some embodiments, one or more nucleic acid templates are immobilized on a support, e.g., anchored to a site on the support. In some embodiments, one or more nucleic acid templates are clonally amplified. In some embodiments, one or more nucleic acid templates are clonally amplified off a support (e.g., in solution) and then precipitated onto the support and immobilized on the support. In some embodiments, a clonal amplification reaction of one or more nucleic acid templates is performed on a support, resulting in immobilization on the support. In some embodiments, one or more nucleic acid templates can be used for polymerase chain reaction (PCR), multiplex translocation amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand translocation amplification (SDA), Any of real-time SDA, bridge amplification, isothermal bridge amplification, rotary ring amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and/or single-stranded binding (SSB) protein-dependent amplification. or clonally amplified using a nucleic acid amplification reaction (e.g., in solution or on a support) including any combination.

The terms “surface primer”, “surface capture primer” and related terms refer to a single-stranded oligonucleotide that is immobilized on a support and includes a sequence capable of hybridizing to at least a portion of a nucleic acid template molecule. Surface primers can be used to immobilize template molecules on the support through hybridization. The surface primer can be immobilized on the support in a manner that resists primer removal during flow, washing, aspiration, and changes in temperature, pH, salt, chemical and/or enzymatic conditions. Typically, but not necessarily, the 5' end of the surface primer may be anchored to the support. Alternatively, the internal portion or 3' end of the surface primer may be anchored to the support.

Surface primers include DNA, RNA, or analogs thereof. Surface primers may include a combination of DNA and RNA. The sequence of the surface primer may be fully or partially complementary along its length to at least a portion of the nucleic acid template molecule (e.g., a linear or circular template molecule). The support may have the same sequence or may include a plurality of immobilized surface primers having two or more different sequences. The surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

The surface primer may include a terminal 3' nucleotide with a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase catalyzed polymerization). Surface primers may include a terminal 3' nucleotide with a moiety that blocks polymerase-catalyzed extension. Surface primers may include a terminal 3' nucleotide with a 3' sugar position linked to a chain-end moiety that inhibits nucleotide polymerization. The 3' chain-termination moiety can be removed (e.g., deblocked) to convert the 3' end to an extendable 3' OH end using a deblocking agent. Examples of chain terminating moieties include alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, and di group. Includes sulfide group, carbonate group, urea group or silyl group. Azide type chain terminating moieties include azide, azido and azidomethyl groups. Examples of deblocking agents include, for chain-terminator azide, azido and azidomethyl groups, phosphine compounds such as tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfo triphenyl phosphine (BS- TPP). Examples of deblocking agents include, for the chain terminators alkyl, alkenyl, alkynyl and allyl, together with piperidine or 2,3-dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ). ) and tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ). Examples of deblocking agents include Pd/C for the chain-terminator aryl and benzyl. Examples of deblocking agents include, for chain-terminator amines, amides, ketos, isocyanates, phosphates, thio and disulfides, phosphine, beta-mercaptoethanol or dithiothritol (DTT). Examples of deblocking agents include, for carbonate chain-terminators, potassium carbonate (K ₂ CO ₃ ) in MeOH, triethylamine in pyridine and Zn in acetic acid (AcOH). Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, along with ammonium fluoride and triethylamine trihydrofluoride, for the chain-terminator urea and silyl.

In some embodiments, a plurality of immobilized surface capture primers on a support comprise a solution of a reagent (e.g., linear or circular nucleic acid template molecule, soluble primer, enzyme, nucleotide, divalent cation, buffer, reagent, etc.) on the support. Multiple immobilized surface capture primers on a support can be reacted simultaneously with reagents in essentially a massively parallel manner and are in fluid communication with each other to enable flow. In some embodiments, fluid communication of a plurality of immobilized surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) essentially simultaneously on a plurality of immobilized surface capture primers. there is.

In some embodiments, a plurality of immobilized single-stranded nucleic acid concatemer template molecules on a support allow a solution of reagents (e.g., soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc.) to flow onto the support. In fluid communication with each other to enable multiple immobilized concatimer template molecules on the support to be reacted simultaneously with the reagents in essentially a massively parallel manner. In some embodiments, fluid communication of a plurality of immobilized single-stranded nucleic acid conkatimer template molecules is used to perform nucleotide binding assays and/or to conduct nucleotide polymerization reactions (e.g., nucleotide polymerization reactions) on a plurality of immobilized single-stranded nucleic acid conkatimer template molecules essentially simultaneously. For example, primer extension or sequencing), and optionally for massively parallel sequencing, detection and imaging.

When used with reference to nucleic acids, the terms "amplify," "amplifying," "amplification," and other related terms include producing multiple copies of an original polynucleotide template molecule, wherein the copies are complementary to the template sequence. or the duplicate comprises a sequence identical to the template sequence. In some embodiments, the replica comprises a sequence that is substantially identical to the template sequence or is substantially identical to a sequence that is complementary to the template sequence.

The present disclosure provides various pH buffering agents. The full names of pH buffering agents are listed herein.

The term “Tris” refers to the pH buffer tris(hydroxymethyl)-aminomethane. The term “Tris-HCl” refers to the pH buffer tris(hydroxymethyl)-aminomethane hydrochloride. The term “tricine” refers to the pH buffer N-[tris(hydroxymethyl)methyl]glycine. The term “bicine” refers to the pH buffer N,N-bis(2-hydroxyethyl)glycine. The term "bis-tris propane" refers to a pH buffering agent. 1,3 refers to bis[tris(hydroxymethyl)methylamino]propane. The term “HEPES” refers to the pH buffer 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid. The term “MES” refers to the pH buffer 2-( N -morpholino)ethanesulfonic acid). The term “MOPS” refers to the pH buffer 3-( N -morpholino)propanesulfonic acid. The term “MOPSO” refers to the pH buffer 3-(N-morpholino)-2-hydroxypropanesulfonic acid. The term “BES” refers to the pH buffer N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid. The term “TES” refers to the pH buffer 2-[(2-hydroxy-1,1bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid). The term “CAPS” refers to the pH buffer 3-(cyclohexylamino)-1-propanesuccinic acid. The term “TAPS” refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-amino propane sulfonic acid. The term “TAPSO” refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid. The term “ACES” refers to the pH buffer N -(2-acetamido)-2-aminoethanesulfonic acid. The term “PIPES” refers to the pH buffer piperazine-1,4-bis(2-ethanesulfonic acid).

introduction

The present disclosure provides compositions and methods of using the compositions for performing pairwise sequencing and for generating concatimer template molecules for pairwise sequencing.

Pairwise sequencing involves obtaining a first sequencing read of a first region of a first nucleic acid strand (e.g., the sense strand) and a first sequencing read of a second nucleic acid strand that is complementary to the first strand (e.g., the antisense strand). Obtaining two regions of second sequencing reads, wherein the first strand and the second strand correspond to two complementary strands of the same double-stranded template molecule. The first sequencing read of the first sequencing region and the second sequencing read of the second sequencing region may have overlapping sequences corresponding to complementary sequences from the first and second strands of the double-stranded template molecule. . Redundant sequencing reads can obtain sequence information of paired regions (e.g., paired regions in a genome) from the original double-stranded nucleic acid source, and the accuracy of the sequence information can be obtained from the first and second sequences with a high level of confidence. The first and second sequencing reads can be aligned so that they can be identified from the analysis reads. The first sequencing read of the first sequencing region and the second sequencing read of the second sequencing region have essentially no overlapping sequences, in which case the sequence information of the paired regions from the original double-stranded nucleic acid source is high-level. The reliability cannot be confirmed. The first and second sequencing reads may begin at one end of their respective template molecules, or may begin at an internal location.

The compositions and methods for pairwise sequencing described herein provide several advantages that improve the quality of sequencing data, including increased signal intensity that improves base calling accuracy. Pairwise sequencing methods also save time by eliminating the need to prepare separate nucleic acid libraries corresponding respectively to the sense and antisense strands of a double-stranded template molecule carrying the sequence of interest. Additionally, pairwise sequencing methods generate and sequence sense and antisense strands directly on the support/substrate used to conduct the sequencing reaction.

The present disclosure provides a pairwise sequencing method using a support on which a plurality of surface primers are immobilized. The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. on the support. In communication, a plurality of immobilized surface primers (and products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

The present disclosure includes the steps of (a) providing a plurality of single-stranded nucleic acid concatimer template molecules immobilized on a support; (b) sequencing a plurality of immobilized concatimer template molecules using a first plurality of sequencing polymerases, a plurality of soluble forward sequencing primers, and a first plurality of multivalent molecules, thereby performing a plurality of extended forward sequencing generating primer strands; (c) maintaining a plurality of fixed Conkatimer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extending strands hybridized to the maintained fixed Conkatimer template molecules by performing a primer extension reaction. steps; (d) removing the retained fixed concatimer template molecule while maintaining the plurality of forward extending strands; and (e) sequencing the plurality of maintained forward extension strands using a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules. provides. In some embodiments, individual template molecules of the plurality are immobilized on a surface primer, wherein the surface primer is immobilized on a support. In some embodiments, individual Concatimer template molecules are covalently linked to a surface primer, or individual Concatimer template molecules are hybridized to a surface primer. In some embodiments, the immobilized surface primer contains or lacks a nucleotide that has a cleavable moiety that can be cleaved to create a non-basic site in the surface primer. In some embodiments, the plurality of conkatimer template molecules comprise at least one nucleotide with a cleavable moiety that can be cleaved to create an abasic site in the conkatimer template molecule. In some embodiments, the plurality of concatimer template molecules lack nucleotides having a cleavable moiety. Exemplary nucleotides with moieties that can cleave (e.g., in a surface primer or concatimer template molecule) include uridine, 8-oxo-7,8-dihydroguanine, and deoxyinosine.

In some embodiments, the pairwise sequencing method includes a rolling ring amplification reaction performed on a support by distributing a plurality of single-stranded circular library molecules on a support on which a plurality of surface primers are immobilized. Individual surface primers are designed to hybridize and capture via single circular library molecules. The rotating ring amplification reaction can be performed on a support. In some embodiments, for RCA reactions on a support, a solution of single-stranded circular library molecules flows onto the support, so that individual circular molecules are captured via hybridization to individual surface primers. Each circular library molecule comprises at least a sequence of interest and a universal surface primer binding site, and optionally includes a universal sequencing primer binding site, a universal amplification primer binding site, additional surface primer binding sites, and a sample barcode and/or molecular indicator. . A single immobilized surface primer will capture a single circular library molecule, and the rolling ring amplification reaction will produce a single-stranded linear concatemer covalently linked to the immobilized surface primer by using the 3' end of the surface primer terminus as the primer extension initiation site. Create a Mer. Accordingly, individual concatimer molecules are immobilized on the support as concatimers covalently linked to the immobilized surface primer. Single-stranded concatemers contain multiple tandem copies of the sequence of interest and universal sequencing primer binding sites. A single surface primer will capture a single circular library molecule and generate a single concatemer molecule.

In some embodiments, the pairwise sequencing method includes a rolling ring amplification reaction performed in solution to generate a plurality of concatimers distributed on a support on which a plurality of surface primers are immobilized. Individual surface primers are designed to capture a single concatemer with the complementary sequence of the original library molecule through hybridization. The rolling ring amplification reaction can continue on the support. In some embodiments, for RCA reactions in solution, a plurality of single-stranded circular library molecules are subjected to a rotational amplification reaction in a reaction vessel. Each circular library molecule comprises at least a sequence of interest and a universal surface primer binding site, and optionally includes a universal sequencing primer binding site, a universal amplification primer binding site, additional surface primer binding sites, and a sample barcode and/or molecular indicator. . The RCA reaction can be carried out over a very short period of time, or generates a plurality of concatimers that hybridize to their respective circular library molecules, which are then distributed on a support to which a plurality of surface primers are immobilized. A solution of concatimer molecules flows onto the support and individual concatimer molecules are captured through hybridization to individual surface primers. Each concatimer molecule comprises at least a sequence of interest, a universal surface primer binding site(s), a universal sequencing primer binding site, and optionally a sample barcode and/or molecular indicator. A single immobilized surface primer will capture a single concatimer molecule, and the rolling ring amplification reaction continues (now on the support) to extend the single stranded concatimer that hybridizes to the immobilized surface primer. Accordingly, individual concatimer molecules are immobilized on the support as concatimers hybridized to the immobilized surface primer. Single-stranded concatemers contain multiple tandem copies of the sequence of interest and universal sequencing primer binding sites. A single surface primer will capture a single concatimer molecule and generate a single extended concatimer molecule.

Rotational ring amplification reactions performed in solution or on a support will produce concatimer immobilized on the support. Immobilized concatimers offer several advantages over non-concatimer molecules. The number of tandem copies in a concatimer can be adjusted by controlling the time, temperature, and concentration of reagents of the rolling ring amplification reaction in solution or on a support. Concatimers can self-disintegrate into compact nucleic acid nanoballs. The inclusion of one or more compression oligonucleotides during the RCA reaction can further compress the size and/or shape of the nanoballs. Increasing the number of tandem copies in a given concatimer increases the number of sites along the concatimer for hybridization to multiple sequencing primers, which serve as multiple initiation sites for the polymerase-catalyzed sequencing reaction. When a sequencing reaction uses detectably labeled nucleotides and/or detectably labeled multivalent molecules (e.g., having nucleotide units), the nucleotides or nucleotide units that participate in parallel sequencing follow concatemers. The signal emitted by the concatimer yields increased signal intensity for each concatimer. Multiple portions of a given concatemer can be sequenced simultaneously. Furthermore, a plurality of binding complexes can be formed along a specific concatimer molecule, each binding complex comprising a sequencing polymerase bound to the multivalent molecule, and the plurality of binding complexes are stably maintained without dissociation, thereby providing a signal. This results in increased duration which increases intensity and reduces imaging time.

The level of sequencing accuracy can be further improved by obtaining partially or fully overlapping sequencing reads from both the sense and antisense strands and aligning the sequencing reads to provide common sequencing data.

Accordingly, the pairwise sequencing compositions and methods described herein provide improved sequencing data quality in a massively parallel manner.

Pairwise sequencing method - generation of non-basic regions

The present disclosure provides a pairwise sequencing method comprising providing a plurality of fixed single-stranded nucleic acid concatemer template molecules, each comprising at least one nucleotide having a cleavable moiety , comprising : Each template molecule of the plurality is immobilized on a first surface primer immobilized on a support, and the immobilized first surface primer lacks a nucleotide having a cleavable moiety. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers.

In some embodiments, individual immobilized concatimer template molecules are covalently linked to an immobilized surface primer (e.g., an immobilized first surface primer) (Figure 1). In an alternative embodiment, individual immobilized concatimer template molecules hybridize to an immobilized surface primer (e.g., an immobilized first surface primer) (Figure 13).

In some embodiments, each individual Concatimer template molecule of the plurality comprises two or more copies of the sequence of interest, and each immobilized Concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer; at least two copies, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) at least two copies of the universal binding sequence for the immobilized first surface primer, (iv) at least two copies of the immobilized agent. 2 at least two copies of the universal binding sequence for the surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. any of the following: duplicates, (vii) two or more copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) two or more copies of the sample barcode sequence, and/or (ix) two or more copies of the unique molecular indicator sequence. Or it further includes any combination of two or more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence for the immobilized first surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, the cleavable moiety in the immobilized concatimer template molecule of step (a) can be converted to a non-basic moiety in the immobilized concatimer template molecule. In some embodiments, moieties that can cleave from the immobilized concatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the concatimer template molecule, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG), and 8oxoG can be converted to a non-basic site using FPG glycosylase. , deoxyinosine can be converted to a non-basic moiety using AlkA glycosylase. In some embodiments, the immobilized concatimer template molecule comprises 1 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 100, or more nucleotides with a cleavable moiety. In some embodiments, about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or a greater percentage of the immobilized concatimer template molecules. dTTP is replaced by a nucleotide with a cleavable moiety. In some embodiments, nucleotides bearing cleavable moieties are distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized first surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized first surface primer comprises a 3' non-extensible moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the immobilized concatimer template molecule further comprises two or more copies of a universal binding sequence (or a complementary sequence thereof) for an immobilized second surface primer having a different sequence than the first immobilized surface primer. do. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. In some embodiments, the end of the 3' end of the immobilized second surface primer is a moiety that blocks primer extension (e.g., the 3' end of the non-extendable end), e.g., a phosphate group, a diade group, etc. Contains an oxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules are bound to or immobilized to a first immobilized surface primer, and at least one portion of the individual concatimer template molecules is hybridized to the immobilized second surface primer. do. The immobilized second surface primer acts to tether the portion of the concatimer template molecule immobilized on the support (Figures 12 and 24).

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

Immobilized surface primers (e.g., first and second surface primers) allow various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. to flow on the support. A plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (b) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (b) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule using a plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 2 and 14). In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, and the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments, the pairwise sequencing method comprises maintaining a plurality of fixed concatimer template molecules and hybridizing a plurality of extended forward sequencing primer strands to the maintained fixed single-stranded nucleic acid concatimer template molecules. It further includes step (c) of replacing with a strand. The plurality of extended forward sequencing primer strands can be removed and replaced with a plurality of forward extension strands by performing a primer extension reaction (Figures 3 to 5 and Figures 15 to 17).

In some embodiments, step (c) performs a strand translocation primer extension reaction using at least one extended forward sequencing primer to initiate the primer extension reaction, such that the forward extension is covalently linked to the extended forward sequencing primer strand. Under conditions suitable to generate strands, at least one extended forward sequencing primer strand is contacted with a plurality of strand transfer polymerases and a plurality of nucleotides and in the absence of soluble amplification primers, wherein the forward extension strand is bound to an immobilized conkatimer. It involves hybridizing to a template molecule. For example, one of the extended forward sequencing primer strands can serve as a primer for strand transposition polymerase. A strand transposition polymerase can extend an extended forward sequencing primer strand, transpose a downstream extended forward sequencing primer strand, and synthesize an extended strand that replaces the downstream extended forward sequencing primer strand ( 3 and 15). The newly extended strand is covalently linked to the extended forward sequencing primer strand. The immobilized concatimer template molecule is maintained.

The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (c) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of soluble forward sequencing primers under conditions suitable for hybridizing to the plurality of retained immobilized concatimer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction. The concatimer molecule is comprised of a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases. contacting each other to generate a plurality of forward extension strands, wherein the soluble sequencing primer hybridizes with the forward sequencing primer binding sequence in the retained immobilized concatimer molecule (Figures 4 and 16). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (c), conditions suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of maintained fixed single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and high efficiency. and hybridizing a fixed concatimer template molecule maintained in the presence of a hybridization buffer with a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, step (c) comprises (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of maintained immobilized cones under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a polymerase-catalyzed primer extension reaction. Contacting the catimer molecule with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases to generate a plurality of forward extension strands, comprising: hybridizes to the soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figures 5 and 17). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (c), conditions suitable for hybridizing a plurality of soluble amplification primers to a plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and a high-throughput hybridization buffer. and hybridizing a fixed concatimer template molecule maintained in the presence of a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (c), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the primer extension polymerase of step (c) comprises a high fidelity polymerase. In some embodiments, the primer extension polymerase of step (c) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). . Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The pairwise method described herein provides increased accuracy in downstream sequencing reactions because step (c) replaces the extended forward sequencing primer strand generated in step (b) with a forward extended strand with reduced base errors. can be provided. The extended forward sequencing primer strand is generated in step (b) and may or may not contain misincorporated nucleotides due to polymerase-catalyzed mispairing bases. When step (c) is performed with a high fidelity DNA polymerase, the resulting forward extended strand may have reduced base errors compared to the extended forward sequencing primer strand. The forward extension strand will be used as a nucleic acid template for downstream sequencing steps (see, eg, step (e) below). Accordingly, step (c) can increase the sequencing accuracy of the downstream step (e), thereby increasing the overall sequencing accuracy of the pairwise sequencing flow diagram.

In some embodiments, the pairwise sequencing method comprises : a single-stranded concatimer template molecule anchored at nucleotide(s) having a cleavable moiety while maintaining a plurality of forward extension strands and a plurality of immobilized surface primers; It further comprises the step (d) of removing the retained fixed concatimer template molecule by creating an abasic site and creating a gap in the abasic site to generate a plurality of gap-containing single stranded nucleic acid concatimer template molecules ( 6 and 18).

Abasic sites are created on the retained concatimer template strand containing nucleotides with cleavable moieties. In some embodiments, moieties that can cleave from the retained conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create multiple single-stranded nucleic acid template molecules with gaps while maintaining multiple forward extension strands. Abasic sites can be created by contacting an immobilized concatimer template molecule with an enzyme that removes a nucleobase from a nucleotide with a cleavable moiety. Uracil in the retained concatimer template strand can be converted to an abasic site using uracil DNA glycosylase (UDG). 8oxoG in the retained concatimer template strand can be converted to an abasic site using FPG glycosylase. Deoxyinosine in the retained concatimer template strand can be converted to an abasic site using AlkA glycosylase.

In some embodiments, in step (d), the gap is formed by an enzyme having lyase activity that releases abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a mixture of enzymes with the non-basic portion of an immobilized concatimer template molecule (Figures 6 and 18). Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (d), the plurality of gap-containing template molecules can be removed using enzymes, chemicals, and/or heat. After the gap-removal procedure, the plurality of retained forward extension strands (e.g., Figures 7 and 9, and Figures 19 and 21) hybridize to the retained immobilized surface primer.

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by . If a 5' to 3' double-stranded DNA exonuclease is used to remove the gap-containing template molecule, the plurality of soluble amplification primers in step (c) will provide soluble amplification primers that are resistant to exonuclease digestion. They may contain at least one phosphorothioate diester bond at their 5' end. In some embodiments, the plurality of soluble amplification primers in step (c) comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of soluble amplification primers in step (c) comprise at least one ribonucleotide and/or at least one 2'-O-methyl nucleotide that can provide a forward sequencing primer that is resistant to exonuclease digestion. or 2'-O-methoxyethyl (MOE) nucleotides.

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5. It can be removed by washing.

In some embodiments, the pairwise sequencing method further comprises step (e) generating a plurality of extended reverse sequencing primer strands by sequencing a plurality of retained forward extension strands. In some embodiments, the sequencing of step (e) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, wherein the forward sequencing reaction generates a plurality of extended reverse sequencing primer strands ( FIGS. 10 and 11 , and 22 and 23). The extended reverse sequencing primer strand hybridizes to the retained forward extended strand. The retained forward extension strand hybridizes to the first surface primer. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support.

For simplicity, Figures 7 and 9 show exemplary maintained forward extension strands each with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that the retained forward extension strand may comprise two or more tandem copies containing the sequence of interest and various universal primer binding sites. Accordingly, a reverse sequencing reaction can generate multiple extended reverse sequencing primer strands that hybridize to the same maintained forward extended strand.

In some embodiments, in step (e), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In an alternative embodiment, the sequencing of step (e) includes using the immobilized surface primer as a sequencing primer and performing a sequencing reaction on the immobilized surface primer to generate a plurality of reverse sequencing strands.

In some embodiments, the reverse sequencing reaction of step (e) comprises a plurality of soluble reverse sequencing primers, a reverse sequencing primer binding sequence of the retained forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides or It includes contacting a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, each maintained forward extension strand may undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site (e.g., Figures 10 and 11 , and Figures 22 and 23). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (e). The washing step can be carried out with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

RCA and pairwise sequencing on supports - generation of abasic sites

The present disclosure provides a pairwise sequencing method comprising step (a) providing a support on which a plurality of surface primers (e.g., a plurality of first surface primers) are immobilized, wherein each of the surface primers has a 3' OH It has an extendable end and lacks nucleotides with a cleavable moiety (Figure 25). For example, the surface primer lacks uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The first surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the nucleic acid library molecules (e.g., linear or circular library molecules). The first surface primer may include a terminal 3' nucleotide with a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase catalyzed polymerization).

The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the support further includes a plurality of second surface primers secured thereto (Figure 37). The second surface primer has a different sequence than the first fixed surface primer. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The second surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the immobilized single-stranded concatimer template molecule. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. The 3' end of the immobilized second surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, an individual immobilized single-stranded nucleic acid concatimer template molecule is covalently linked to an immobilized first surface primer, and at least one portion of the individual concatimer template molecule is hybridized to an immobilized second surface primer. (Figure 37). The immobilized second surface primer serves to tether the portion of the concatimer template molecule immobilized to the support. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable. In some embodiments, the second surface primer has a terminal 3' extendable end.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

Immobilized surface primers (e.g., first and second surface primers) allow various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. to flow on the support. A plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments , a pairwise sequencing method comprises hybridizing a plurality of single stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers, a plurality of strand transfer polymerases, and a plurality of nucleic acid molecules comprising dATP, dCTP, dGTP, dTTP. A rotation ring amplification reaction is performed using nucleotides and nucleotides having a cleavable moiety to generate a plurality of fixed single-stranded nucleic acid concatimer template molecules, thereby producing a plurality of fixed single-stranded nucleic acid concatimer template molecules. generating, further comprising step (b) , wherein the individual single-stranded nucleic acid concatimer template molecules are bound (e.g., covalently linked) to an immobilized surface primer (e.g., an immobilized first surface primer) ( Figure 26). In some embodiments, the rolling ring amplification reaction can be performed with or without a plurality of compressed oligonucleotides.

In some embodiments, single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules. In some embodiments, single-stranded circular nucleic acid library molecules can be removed from the concatimer template molecule using at least one washing step performed under conditions suitable for maintaining the single-stranded nucleic acid concatimer template molecule, wherein the individual concatimer template molecules are The catimer template molecule is operably linked to the immobilized first surface primer.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each immobilized concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer (or its complement); sequence), (ii) a universal binding sequence (or a complementary sequence thereof) to a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer, (iv) an immobilized a universal binding sequence (or a complementary sequence thereof) to a second surface primer, (v) a universal binding sequence (or a complementary sequence thereof) to a first soluble amplification primer, (vi) a universal binding sequence to a second soluble amplification primer. a binding sequence (or a complementary sequence thereof), (vii) a universal binding sequence for soluble compressed oligonucleotides (or a complementary sequence thereof), (viii) a sample barcode sequence and/or (ix) a unique molecular indicator sequence, or It further includes any combination of two or more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, step (b) the rolling ring amplification reaction comprises a plurality of fixed single-stranded nucleic acid cones each comprising a concatemer having at least one nucleotide with a cleavable moiety and two or more copies of the sequence of interest. A concatimer template molecule is generated, and the immobilized concatimer template molecule contains (i) two or more copies of the universal binding sequence (or its complementary sequence) to the soluble forward sequencing primer, (ii) the soluble reverse sequencing primer. two or more copies of the universal binding sequence (or complementary sequence thereof) to the primer, (iii) two or more copies of the universal binding sequence (or complementary sequence thereof) to the immobilized first surface primer, (iv) ) two or more copies of the universal binding sequence (or complementary sequence thereof) to the immobilized second surface primer, (v) two copies of the universal binding sequence (or complementary sequence thereof) to the first soluble amplification primer (vi) two or more copies of the universal binding sequence (or complementary sequence thereof) to the second soluble amplification primer, (vii) the universal binding sequence (or complementary sequence thereof) to the soluble compressed oligonucleotide. (viii) two or more copies of a sample barcode sequence, and/or (ix) two or more copies of a unique molecular indicator sequence, or any combination of two or more.

In some embodiments, the plurality of immobilized single-stranded nucleic acid concatemer template molecules generated by the rolling ring amplification reaction of step (b) have a universal binding sequence (or a complementary sequence thereof) to the immobilized second sequence surface primer. It further contains two or more copies of. In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules are bound (e.g., covalently) to the immobilized first surface primer, and at least one portion of the individual concatimer template molecules is bound to the immobilized first surface primer. Hybridizes to the second surface primer. The immobilized second surface primer serves to tether the portion of the concatimer template molecule immobilized to the support (see Figure 37). In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable.

The rolling ring amplification reaction of step (b) is carried out with a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety to obtain a fixed nucleotide comprising at least one nucleotide having a cleavable moiety. A concatimer template molecule can be generated. Moieties that can be cleaved from the immobilized concatimer template molecule can be converted to non-basic sites. In some embodiments, in the nucleotide mixture, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the immobilized concatimer template molecule, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG), and 8oxoG can be converted to a non-basic site using FPG glycosylase. Deoxyinosine can be converted to a non-basic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture includes an amount of dUTP such that a target percentage of thymidine is replaced by dUTP in the resulting concatimer molecules. For example, when 30% of the dTTP in a conkatimer molecule is replaced with dUTP (e.g., 30% is the target percentage), then the nucleotide mixture will have 7.5% dUTP (e.g., 30/4 = 7.5 %), 17.5% dTTP, and 25% each for dATP, dCTP, and dGTP. The target percentage of dTTP replaced by dUTP is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%, or about 45 to 50%, or a higher percentage of dTTP in the immobilized concatimer template molecule is replaced with a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of deoxyinosine such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by deoxyinosine. For example, when 30% of the dGTP in a conkatimer molecule is replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture has 7.5% deoxyinosine each for dATP, dCTP, and dTTP. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by deoxyinosine is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or about 45 to 50%, or a higher percentage of dGTP in the immobilized concatimer template molecule is replaced by a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of 8oxoG such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by 8oxoG. For example, when 30% of the dGTP in a conkatimer molecule is replaced by 8oxoG (e.g., 30% is the target percentage), the nucleotide mixture contains 7.5% 8oxoG for dATP, dCTP, and dTTP each. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by 8-oxoG is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or about 45 to 50%, or a higher percentage of dGTP in the immobilized concatimer template molecule is replaced by a nucleotide having a cleavable moiety.

In some embodiments, the rolling ring amplification reaction generates fixed concatimer template molecules using incorporated nucleotides with cleavable moieties distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules.

In some embodiments, the pairwise sequencing method further comprises step (c) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (c) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing the plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. It includes performing (Figure 27). In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 27). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments, the pairwise sequencing method comprises maintaining a plurality of fixed concatimer template molecules and hybridizing a plurality of extended forward sequencing primer strands to the maintained fixed single-stranded nucleic acid concatimer template molecules. It further includes step (d) of replacing with a strand. The plurality of extended forward sequencing primer strands can be removed and replaced with a plurality of forward extension strands by performing a primer extension reaction (FIGS. 28 to 30).

In some embodiments, step (d) performs a strand translocation primer extension reaction using at least one extended forward sequencing primer to initiate the primer extension reaction, such that the forward extension is covalently linked to the extended forward sequencing primer strand. Under conditions suitable to generate strands, at least one extended forward sequencing primer strand is contacted with a plurality of strand transfer polymerases and a plurality of nucleotides and in the absence of soluble amplification primers, wherein the forward extension strand is bound to an immobilized conkatimer. and hybridization to the template molecule (Figure 28). For example, one of the extended forward sequencing primer strands can serve as a primer for strand transposition polymerase. A strand transposition polymerase can extend an extended forward sequencing primer strand, transpose a downstream extended forward sequencing primer strand, and synthesize an extended strand that replaces the downstream extended forward sequencing primer strand. The newly extended strand is covalently linked to the extended forward sequencing primer strand. The immobilized concatimer template molecule is maintained. The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (d) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of soluble forward sequencing primers under conditions suitable for hybridizing to the plurality of retained immobilized concatimer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction. The concatimer molecule is comprised of a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases. contacting to generate a plurality of forward extension strands, wherein the soluble sequencing primer hybridizes with the forward sequencing primer binding sequence in the retained immobilized concatimer molecule (Figure 29). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (d), conditions suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of maintained fixed single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and high efficiency. and hybridizing a fixed concatimer template molecule maintained in the presence of a hybridization buffer with a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, step (d) comprises (i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and (ii) a plurality of maintained immobilized cones under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a polymerase-catalyzed primer extension reaction. Contacting the catimer molecule with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases to generate a plurality of forward extension strands, comprising: hybridizes to the soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figure 30). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (d), conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and a high-throughput hybridization buffer. and hybridizing a fixed concatimer template molecule maintained in the presence of a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (c), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the primer extension polymerase of step (d) comprises a high fidelity polymerase. In some embodiments, the primer extension polymerase of step (d) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). . Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The pairwise method described herein provides increased accuracy in downstream sequencing reactions because step (d) replaces the extended forward sequencing primer strand generated in step (c) with a forward extended strand with reduced base errors. can be provided. The extended forward sequencing primer strand is generated in step (c) and may or may not contain misincorporated nucleotides due to polymerase-catalyzed mispairing bases. When step (d) is performed with a high fidelity DNA polymerase, the resulting forward extended strand may have reduced base errors compared to the extended forward sequencing primer strand. The forward extension strand will be used as a nucleic acid template for downstream sequencing steps (see, eg, step (f) below). Accordingly, step (d) can increase the sequencing accuracy of step (f) downstream, thereby increasing the overall sequencing accuracy of the pairwise sequencing flow diagram.

In some embodiments, the pairwise sequencing method comprises : a single stranded concatimer template molecule anchored at nucleotide(s) having a cleavable moiety while maintaining a plurality of forward extension strands and a plurality of immobilized surface primers; It further comprises the step (e) of removing the retained fixed concatimer template molecule by creating an abasic site and creating a gap in the abasic site to generate a plurality of gap-containing single stranded nucleic acid concatimer template molecules ( 31 and 33).

Non-basic sites are created on the retained concatimer template strand containing nucleotides with cleavable moieties. In some embodiments, moieties that can cleave from the retained conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create multiple single-stranded nucleic acid template molecules with gaps while maintaining multiple forward extension strands. Abasic sites can be created by contacting an immobilized concatimer template molecule with an enzyme that removes a nucleobase from a nucleotide with a cleavable moiety. Uracil in the retained concatimer template strand can be converted to an abasic site using uracil DNA glycosylase (UDG). 8oxoG in the retained concatimer template strand can be converted to an abasic site using FPG glycosylase. Deoxyinosine in the retained concatimer template strand can be converted to an abasic site using AlkA glycosylase.

In some embodiments, in step (e), the gap is formed by an enzyme having lyase activity that releases the abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a mixture of enzymes with the non-basic portion of an immobilized concatimer template molecule (Figures 31 and 33). Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (e), the plurality of gap-containing template molecules can be removed using enzymes, chemical compounds, and/or heat. After the gap-removal procedure, the plurality of retained forward extension strands hybridize to the retained immobilized surface primers (Figures 32 and 34).

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by . If a 5' to 3' double-stranded DNA exonuclease is used to remove the gap-containing template molecule, the plurality of soluble amplification primers in step (e) will provide soluble amplification primers that are resistant to exonuclease digestion. They may contain at least one phosphorothioate diester bond at their 5' end. In some embodiments, the plurality of soluble amplification primers in step (d) comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of soluble amplification primers in step (d) comprise at least one ribonucleotide and/or at least one 2'-O-methyl nucleotide that can provide a forward sequencing primer that is resistant to exonuclease digestion. or 2'-O-methoxyethyl (MOE) nucleotides.

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5. It can be removed by washing.

In some embodiments, the pairwise sequencing method further comprises step (f) generating a plurality of extended reverse sequencing primer strands by sequencing a plurality of retained forward extension strands. In some embodiments, the sequencing of step (f) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, thereby producing a plurality of extended reverse sequencing primer strands (FIGS. 35 and 36). The extended reverse sequencing primer strand hybridizes to the retained forward extended strand. The retained forward extension strand hybridizes to the first surface primer. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support.

For simplicity, Figures 32 and 34 show exemplary maintained forward extension strands, each with one copy of the sequence of interest and various universal primer binding sites. Those skilled in the art will recognize that the retained forward extension strand may comprise two or more tandem copies containing the sequence of interest and various universal primer binding sites. Accordingly, a reverse sequencing reaction can generate multiple extended reverse sequencing primer strands that hybridize to the same maintained forward extended strand.

In some embodiments, in step (f), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In an alternative embodiment, the sequencing of step (f) includes using the immobilized surface primer as a sequencing primer and performing a sequencing reaction on the immobilized surface primer to generate a plurality of reverse sequencing strands.

In some embodiments, the reverse sequencing reaction of step (f) comprises a plurality of reverse sequencing primers, a reverse sequencing primer binding sequence of the forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides, and/ or contacting it with a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, an individual maintained forward extension strand may undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site (e.g., Figures 35 and 36 reference). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (f). The washing step can be performed with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the detergent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

RCA and pairwise sequencing in solution - generation of abasic sites

The present disclosure provides a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable to form a plurality of library-primer duplexes and suitable for performing a rotational amplification reaction, a plurality of soluble first amplification primers, a plurality of strand translocations. A concatenation of a plurality of single-stranded nucleic acids having at least one nucleotide having a cleavable moiety by contacting a polymerase and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety. A pairwise sequencing method is provided, including step (a) of generating a mer (FIG. 38). In some embodiments, the soluble first amplification primer comprises a sequence that selectively hybridizes to a universal binding sequence in a circular nucleic acid library molecule, e.g., a universal binding sequence for the first soluble amplification primer (or a complementary sequence thereof). do. Alternatively, the soluble first amplification primer comprises a random sequence that binds non-selectively in the circular nucleic acid library molecule.

In some embodiments, each individual single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence to a soluble forward sequencing primer (or a complementary sequence thereof), (ii) ) a universal binding sequence (or a complementary sequence thereof) to a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer, (iv) a universal binding sequence (or a complementary sequence thereof) to an immobilized second surface primer. a universal binding sequence (or complementary sequence thereof) to a surface primer, (v) a universal binding sequence (or complementary sequence thereof) to a first soluble amplification primer, (vi) a universal binding sequence to a second soluble amplification primer. sequence (or its complementary sequence), (vii) a universal binding sequence for soluble compressed oligonucleotides (or its complementary sequence), (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Or it further includes any combination of two or more. In some embodiments, single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules.

In some embodiments, the rolling ring amplification reaction of step (a) produces a plurality of single-stranded nucleic acid concatimer molecules in solution comprising a concatimer having at least one nucleotide having a cleavable moiety. In some embodiments, each individual Concatimer template molecule of the plurality comprises two or more copies of the sequence of interest, and each immobilized Concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer; at least two copies, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) at least two copies of the universal binding sequence for the immobilized first surface primer, (iv) at least two copies of the immobilized agent. 2 at least two copies of the universal binding sequence for the surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. any of the following: duplicates, (vii) two or more copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) two or more copies of the sample barcode sequence, and/or (ix) two or more copies of the unique molecular indicator sequence. Or it further includes any combination of two or more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence for the immobilized first surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

The rotation ring amplification reaction in solution of step (a) is carried out by a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety, such that it contains at least one nucleotide having a cleavable moiety. A concatimer molecule can be created. Moieties that can be cleaved from the concatimer molecule can be converted to non-basic sites. In some embodiments, in the nucleotide mixture, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the concatimer molecule, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG), and 8oxoG can be converted to a non-basic site using FPG glycosylase. Deoxyinosine can be converted to a basic moiety using AlkA glycosylase.

In some embodiments, the nucleotide mixture includes an amount of dUTP such that a target percentage of thymidine is replaced by dUTP in the resulting concatimer molecules. For example, when 30% of the dTTP in a conkatimer molecule is replaced with dUTP (e.g., 30% is the target percentage), then the nucleotide mixture will have 7.5% dUTP (e.g., 30/4 = 7.5 %), 17.5% dTTP, and 25% each for dATP, dCTP, and dGTP. The target percentage of dTTP replaced by dUTP is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%, or about 45 to 50%, or a higher percentage of dTTP in the concatimer molecule is replaced with a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of deoxyinosine such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by deoxyinosine. For example, when 30% of the dGTP in a conkatimer molecule is replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture has 7.5% deoxyinosine each for dATP, dCTP, and dTTP. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by deoxyinosine is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or may be about 45 to 50%, or a higher percentage of dGTP in the concatimer molecule is replaced by a nucleotide with a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of 8oxoG such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by 8oxoG. For example, when 30% of the dGTP in a conkatimer molecule is replaced by 8oxoG (e.g., 30% is the target percentage), the nucleotide mixture contains 7.5% 8oxoG for dATP, dCTP, and dTTP each. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by 8-oxoG is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or may be about 45 to 50%, or a higher percentage of dGTP in the concatimer molecule is replaced by a nucleotide with a cleavable moiety.

In some embodiments, a rolling ring amplification reaction in solution generates concatimer molecules with incorporated nucleotides having cleavable moieties distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different concatimer molecules.

In some embodiments, the pairwise sequencing method comprises a plurality of first surface primers immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatemers to the one or more immobilized first surface primers. It further comprises a step (b) of distributing the rolling ring amplification reaction from step (a) on a support (Figure 39). In some embodiments, the immobilized first surface primer has a terminal 3' group that is not extensible. In some embodiments, the 3' end of the immobilized first surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT, or amino group. In some embodiments, the immobilized first surface primer has an extendable 3'OH end. In some embodiments, the immobilized first surface primer lacks a nucleotide having a cleavable moiety. The concatimer is immobilized on the support by hybridization to the immobilized first surface primer. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers.

In some embodiments, the pairwise sequencing method further comprises (c) continuing the rolling ring amplification reaction on the support to generate a plurality of extended concatimer template molecules immobilized via hybridization to the immobilized first surface primer. (Figure 40). The RCA reaction on a support involves a plurality of strand transfer polymerases, and dATP, dCTP, dGTP, dTTP, under conditions suitable to generate a plurality of extended concatemers with at least one nucleotide having a cleavable moiety. It can be performed using a plurality of nucleotides and a nucleotide having a cleavable moiety (FIG. 41). In some embodiments, the rolling ring amplification reaction on a support can be performed with or without a plurality of compressed oligonucleotides.

In some embodiments, a rolling ring amplification reaction on a support generates immobilized concatimer template molecules using incorporated nucleotides with cleavable moieties distributed at random positions along the individual immobilized concatimer template molecules. do. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The first surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the concatimer molecule. In some embodiments, the first surface primer may lack a terminal 3' OH extendable end rendering the first surface primer non-extensible. In some embodiments, the first surface primer includes a terminal 3' OH group that is extendable for nucleotide polymerization (e.g., polymerase catalyzed polymerization). The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the plurality of immobilized first surface primers includes a 3' extendable end. In some embodiments, the 3' end of the immobilized first surface primer comprises a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT, or amino group. In some embodiments, the immobilized first surface primer is not extendable in the primer extension reaction. The immobilized first surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the concatimer template molecule.

In some embodiments, the support further includes a plurality of second surface primers secured thereto (Figure 52). The second surface primer has a different sequence than the first fixed surface primer. The immobilized second surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The second surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the concatimer molecule. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. The 3' end of the immobilized second surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprises at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, an individual immobilized single-stranded nucleic acid concatimer template molecule hybridizes to an immobilized first surface primer, and at least one portion of the individual concatimer template molecule hybridizes to an immobilized second surface primer ( Figure 52). The immobilized second surface primer acts to tether the portion of the concatimer template molecule immobilized to the support. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable. In some embodiments, the second surface primer has a terminal 3′ extendable end.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

Immobilized surface primers (e.g., first and second surface primers) allow various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. to flow on the support. Thus, a plurality of immobilized surface primers can be reacted with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (d) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (d) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing a plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. The forward sequencing reaction can generate multiple extended forward sequencing primer strands (Figure 42). In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 42). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments, the pairwise sequencing method comprises maintaining a plurality of fixed concatimer template molecules and hybridizing a plurality of extended forward sequencing primer strands to the maintained fixed single-stranded nucleic acid concatimer template molecules. It further includes step (e) of replacing with a strand. The plurality of extended forward sequencing primer strands can be removed and replaced with a plurality of forward extension strands by performing a primer extension reaction ( FIGS. 43 to 45 ).

In some embodiments, step (e) performs a strand transposition primer extension reaction using at least one extended forward sequencing primer to initiate the primer extension reaction, such that the forward extension is covalently linked to the extended forward sequencing primer strand. Under conditions suitable for generating strands, at least one extended forward sequencing primer strand is contacted with a plurality of strand transfer polymerases and a plurality of nucleotides and in the absence of soluble amplification primers, wherein the forward extension strand is bound to an immobilized conkatimer. and hybridization to the template molecule (Figure 43). For example, one of the extended forward sequencing primer strands can serve as a primer for strand transposition polymerase. A strand transposition polymerase can extend an extended forward sequencing primer strand, transpose a downstream extended forward sequencing primer strand, and synthesize an extended strand that replaces the downstream extended forward sequencing primer strand. The newly extended strand is covalently linked to the extended forward sequencing primer strand. The immobilized concatimer template molecule is maintained. The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (e) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of soluble forward sequencing primers under conditions suitable for hybridizing to the plurality of retained immobilized concatimer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction. The concatimer molecule is comprised of a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases. contacting to generate a plurality of forward extension strands, wherein the soluble sequencing primer hybridizes with the forward sequencing primer binding sequence in the retained immobilized conkatimer molecule (Figure 44). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (e), conditions suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of maintained fixed single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and high efficiency. and hybridizing an immobilized concatimer template molecule maintained in the presence of a hybridization buffer with a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, step (e) comprises (i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and (ii) a plurality of maintained immobilized cones under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a polymerase-catalyzed primer extension reaction. Contacting the catimer molecule with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases to generate a plurality of forward extension strands, comprising: hybridizes to the soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figure 45). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (e), conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and a high-throughput hybridization buffer. and hybridizing a fixed concatimer template molecule maintained in the presence of a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (e), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of buffering agents (e.g. Tris-HCl, MES, HEPES, etc.).

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the primer extension polymerase of step (e) comprises a high fidelity polymerase. In some embodiments, the primer extension polymerase of step (e) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). . Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The pairwise method described herein provides increased accuracy in downstream sequencing reactions because step (e) replaces the extended forward sequencing primer strand generated in step (d) with a forward extended strand with reduced base errors. can be provided. The extended forward sequencing primer strand is generated in step (d) and may or may not contain misincorporated nucleotides due to polymerase-catalyzed mispairing bases. When step (e) is performed with a high fidelity DNA polymerase, the resulting forward extended strand may have reduced base errors compared to the extended forward sequencing primer strand. The forward extension strand will be used as a nucleic acid template for downstream sequencing steps (see, eg, step (f) below). Accordingly, step (e) can increase the sequencing accuracy of step (g) downstream, thereby increasing the overall sequencing accuracy of the pairwise sequencing flow diagram.

In some embodiments, the pairwise sequencing method comprises : a single-stranded concatimer template molecule anchored at nucleotide(s) having a cleavable moiety while maintaining a plurality of forward extension strands and a plurality of immobilized surface primers; It further comprises a step (f) of removing the retained fixed conkatimer template molecule by creating an abasic site and creating a gap in the abasic site to generate a plurality of gap-containing single stranded nucleic acid conkatimer template molecules ( 46 and 48).

Abasic sites are created on the retained concatimer template strand containing nucleotides with cleavable moieties. In some embodiments, moieties that can cleave from the retained conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create multiple single-stranded nucleic acid template molecules with gaps while maintaining multiple forward extension strands. Abasic sites can be created by contacting an immobilized concatimer template molecule with an enzyme that removes a nucleobase from a nucleotide with a cleavable moiety. Uracil in the retained concatimer template strand can be converted to an abasic site using uracil DNA glycosylase (UDG). 8oxoG in the retained concatimer template strand can be converted to an abasic site using FPG glycosylase. Deoxyinosine in the retained concatimer template strand can be converted to an abasic site using AlkA glycosylase.

In some embodiments, in step (f), the gap is formed by an enzyme having lyase activity that releases abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a mixture of enzymes with the non-basic portion of an immobilized concatimer template molecule (Figures 46 and 48). Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (f), the plurality of gap-containing template molecules can be removed using enzymes, chemical compounds, and/or heat. After the gap-removal procedure, the plurality of retained forward extension strands can hybridize to the retained immobilized surface primer (Figures 47 and 49).

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by . If a 5' to 3' double-stranded DNA exonuclease is used to remove the gap-containing template molecule, the plurality of soluble amplification primers in step (e) will provide soluble amplification primers that are resistant to exonuclease digestion. They may contain at least one phosphorothioate diester bond at their 5' end. In some embodiments, the plurality of soluble amplification primers in step (e) comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of soluble amplification primers in step (e) comprise at least one ribonucleotide and/or at least one 2'-O-methyl nucleotide that can provide a forward sequencing primer that is resistant to exonuclease digestion. or 2'-O-methoxyethyl (MOE) nucleotides.

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5. It can be removed by washing.

In some embodiments, the pairwise sequencing method further comprises step (g) generating a plurality of extended reverse sequencing primer strands by sequencing a plurality of retained forward extension strands. In some embodiments, the sequencing of step (g) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, thereby producing a plurality of extended reverse sequencing primer strands (FIGS. 50 and 51). The extended reverse sequencing primer strand hybridizes to the retained forward extended strand. The retained forward extension strand hybridizes to the first surface primer. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support.

For simplicity, Figures 47 and 49 show (i) one copy of the sequence of interest and different universal primer binding sites (Figure 47) or (ii) two tandem copies of the sequence of interest and different universal primer binding sites (Figure 49). Shown are exemplary maintained forward extension strands each having one of the following: Those skilled in the art will recognize that the retained forward extension strand may comprise two, three, four or more tandem copies containing the sequence of interest and various universal primer binding sites. Accordingly, a reverse sequencing reaction can generate multiple extended reverse sequencing primer strands that hybridize to the same maintained forward extended strand.

In some embodiments, in step (g), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In an alternative embodiment, the sequencing of step (g) includes using the immobilized surface primer as a sequencing primer and performing a sequencing reaction on the immobilized surface primer to generate a plurality of reverse sequencing strands.

In some embodiments, the reverse sequencing reaction of step (g) comprises a plurality of reverse sequencing primers, a reverse sequencing primer binding sequence of the forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides and/ or contacting it with a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, an individual maintained forward extension strand may undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer that hybridizes to the reverse sequencing primer binding site (e.g., Figures 50 and 51 ). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (g). The washing step can be performed with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

Ligation and RCA on scaffold and pairwise sequencing

The present disclosure provides a pairwise sequencing method comprising the step (a) of providing a support on which a plurality of surface primers (e.g., a plurality of first surface primers) are immobilized, wherein each first surface primer of the plurality comprises a first portion (SP1-A) and a second portion (SP1-B), each first surface primer comprising a 3' extendable end at the first surface marker and to be cleaved to create a non-basic site. It lacks a nucleotide that has a cleavable moiety. In some embodiments, the immobilized first surface primer lacks a nucleotide having a cleavable moiety (Figure 55). For example, the surface primer lacks uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine. In some embodiments, the first and second portions (SP1-A and SP1-B) of the first surface primer have the same or different lengths. The first portion of the first surface primer (SP1-A) may be about 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. The second portion of the first surface primer (SP1-B) may be about 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the first and second portions (SP1-A and SP1-B) of the immobilized first surface primer have the same or different sequences. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The first surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the nucleic acid library molecules (e.g., linear or circular library molecules). The first surface primer may include a terminal 3' nucleotide with a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase catalyzed polymerization).

The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the support further includes a plurality of second surface primers secured thereto (Figure 72). The second surface primer has a different sequence than the first fixed surface primer. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The second surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the immobilized single-stranded concatimer template molecule. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. The 3' end of the immobilized second surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, an individual immobilized single-stranded nucleic acid concatimer template molecule is covalently linked to an immobilized first surface primer, and at least one portion of the individual concatimer template molecule is hybridized to an immobilized second surface primer. (Figure 72). The immobilized second surface primer serves to tether the portion of the concatimer template molecule immobilized to the support. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable. In some embodiments, the second surface primer has a terminal 3' extendable end.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. on the support. In communication, a plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments , the pairwise sequencing method further comprises the step (b) of contacting a plurality of first surface primers with each library molecule of a plurality of single stranded linear nucleic acid library molecules having 5' and 3' ends. The contacting step is performed under conditions that hybridize the individual library molecules to the immobilized first surface primer to form a circular library molecule with a gap or crevice between the 5' and 3' ends of the circular library molecule ( FIGS. 57 and 58 ).

In some embodiments, the location of the gap or crevice in the circular library molecule may be asymmetric or symmetrical relative to the duplex formed by hybridizing the 5' and 3' ends of the linear library molecule to an immobilized first surface primer. For example, Figure 57 shows an asymmetrically positioned gap or crevice. Figure 58 (left) shows an asymmetrically positioned gap or crevice. Figure 58 (right) shows a symmetrically positioned gap or crevice. Asymmetric or symmetrically positioned gaps/gaps can be created by adjusting the lengths of the first portion (SP1-A) and the second portion (SP1-B) in the fixed first surface primer.

In some embodiments, an individual library molecule of the plurality comprises a sequence of interest, and the library molecule comprises: (i) a universal binding sequence to a soluble forward sequencing primer (or a complementary sequence thereof); (ii) a universal binding sequence (or a complementary sequence thereof) to a soluble reverse sequencing primer; (iii) a universal binding sequence (or a complementary sequence thereof) to the first portion of the immobilized first surface primer (SP1-A); (iv) a universal binding sequence (or a complementary sequence thereof) to the second portion of the immobilized first surface primer (SP1-B); (v) a universal binding sequence (or a complementary sequence thereof) to an immobilized second surface primer; (vi) a universal binding sequence (or a complementary sequence thereof) to the first soluble amplification primer; (vii) a universal binding sequence (or a complementary sequence thereof) to the second soluble amplification primer; (viii) a universal binding sequence for a soluble compressed oligonucleotide (or a complementary sequence thereof); (ix) a sample barcode sequence and/or (x) a unique molecular indicator sequence, or any combination of two or more. An exemplary single stranded linear library molecule is shown in Figure 56.

In some embodiments, the universal binding sequence for the first portion of the immobilized first surface primer (e.g., SP1-A') in the linear library molecule is the first portion of the immobilized first surface primer (e.g., SP1-A'). can be hybridized. In some embodiments, the universal binding sequence for the second portion of the immobilized first surface primer (e.g., SP1-B') in the linear library molecule is the second portion of the immobilized first surface primer (e.g., SP1-B'). can be hybridized. In some embodiments, the immobilized first surface primer comprises a first portion (SP1-A) and a second portion (SP1-B) that hybridize to SP1-A' and SP1-B' in the linear library molecule, and 1 Surface primers act as nucleic acid sprint molecules to circularize linear library molecules.

In some embodiments , the pairwise sequencing method further comprises (c) enzymatically closing the gap or crevice to form an individual single-stranded covalently closed circular molecule that hybridizes to the immobilized first surface primer ( 59, 60 (left) and 60 (right)).

In some embodiments, the gap in the circular library molecule is formed using a polymerase-catalyzed fill reaction using the 3' extendable end of the library molecule as the initiation site for the polymerase-catalyzed filling reaction and an immobilized first surface primer as a template molecule. It is closed by performing a gap-filling reaction to form a circular molecule with a gap. The cleft is closed by performing an enzymatic ligation reaction to form a single-stranded covalently closed circular molecule, with each covalently closed circular molecule hybridizing to the immobilized first surface primer. In some embodiments, the gap filling reaction can be performed using a plurality of nucleotides and a polymerase that lacks 5' to 3' strand translocation activity. Polymerases include E. coli DNA polymerase I, the Klenov fragment of E. coli DNA polymerase I, T7 DNA polymerase, or T4 DNA polymerase. In some embodiments, the ligation reaction may be performed using a DNA ligase including T3, T4, T7, or Taq DNA ligase.

In some embodiments, a cleft in a circular library molecule is closed by performing a ligase-catalyzed ligation reaction to form a single-stranded covalently closed circular molecule, wherein each covalently closed circular molecule hybridizes to an immobilized first surface primer. . In some embodiments, the ligase enzyme includes T3, T4, T7, or Taq DNA ligase.

In some embodiments , a pairwise sequencing method includes a plurality of strand transposition polymerases, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a cleavable moiety that can be cleaved to create an abasic site. A plurality of fixed single-stranded nucleic acid concatimer template molecules are generated by performing a rotation amplification reaction with nucleotides, thereby producing a plurality of fixed single-stranded nucleic acid conkatimer template molecules having at least one nucleotide having a cleavable moiety. Further comprising step (d) of generating molecules, wherein the individual single-stranded nucleic acid concatimer template molecules are covalently linked to the immobilized first surface primer (Figure 61). In some embodiments, the rolling ring amplification reaction can be performed with or without a plurality of compressed oligonucleotides.

In some embodiments, single-stranded circular nucleic acid library molecules can be removed from the concatimer template molecule using at least one washing step performed under conditions suitable for maintaining the single-stranded nucleic acid concatimer template molecule, wherein the individual concatimer template molecules are The catimer template molecule is operably linked to the immobilized first surface primer.

In some embodiments, the individual immobilized concatimer template molecules produced by the rolling ring amplification reaction comprise two or more copies of the sequence of interest, wherein the individual immobilized concatimer template molecules are (i) soluble forward sequencing Two or more copies of the universal binding sequence for the primer; (ii) two or more copies of the universal binding sequence for the soluble reverse sequencing primer; (iii) two or more copies of the universal binding sequence (SP1-A) for the first portion of the immobilized first surface primer; (iv) two or more copies of the universal binding sequence (SP1-B) for the second portion of the immobilized first surface primer; (v) two or more copies of a universal binding sequence for an immobilized second surface primer; (vi) two or more copies of the universal binding sequence for the first soluble amplification primer; (vii) two or more copies of the universal binding sequence for the second soluble amplification primer; (viii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide; (ix) two or more copies of a sample barcode sequence and/or (x) two or more copies of a unique molecular indicator sequence.

In some embodiments, the plurality of immobilized single-stranded nucleic acid concatemer template molecules generated by the rolling ring amplification reaction of step (d) have a universal binding sequence (or a complementary sequence thereof) to the immobilized second sequence surface primer. It further contains two or more copies of. In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules are bound (e.g., covalently) to the immobilized first surface primer, and at least one portion of the individual concatimer template molecules is bound to the immobilized first surface primer. Hybridizes to the second surface primer. The immobilized second surface primer acts to tether the portion of the concatimer template molecule immobilized to the support (see Figure 72). In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable.

The rolling ring amplification reaction of step (d) is carried out with a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety to obtain a fixed nucleotide comprising at least one nucleotide having a cleavable moiety. A concatimer template molecule can be generated. Moieties that can be cleaved from the immobilized concatimer template molecule can be converted to non-basic sites. In some embodiments, in the nucleotide mixture, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the immobilized concatimer template molecule, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG), and 8oxoG can be converted to a non-basic site using FPG glycosylase. Deoxyinosine can be converted to a non-basic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture includes an amount of dUTP such that a target percentage of thymidine is replaced by dUTP in the resulting concatimer molecule. For example, when 30% of the dTTP in a conkatimer molecule is replaced with dUTP (e.g., 30% is the target percentage), then the nucleotide mixture will have 7.5% dUTP (e.g., 30/4 = 7.5 %), 17.5% dTTP, and 25% each for dATP, dCTP, and dGTP. The target percentage of dTTP replaced by dUTP is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%, or about 45 to 50%, or a higher percentage of dTTP in the immobilized concatimer template molecule is replaced with a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of deoxyinosine such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by deoxyinosine. For example, when 30% of the dGTP in a conkatimer molecule is replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture has 7.5% deoxyinosine each for dATP, dCTP, and dTTP. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by deoxyinosine is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or may be about 45 to 50%, or a higher percentage of the dGTP in the immobilized concatimer template molecule is replaced by a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of 8oxoG such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by 8oxoG. For example, when 30% of the dGTP in a conkatimer molecule is replaced by 8oxoG (e.g., 30% is the target percentage), the nucleotide mixture contains 7.5% 8oxoG for dATP, dCTP, and dTTP each. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by 8-oxoG is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or about 45 to 50%, or a higher percentage of dGTP in the immobilized concatimer template molecule is replaced by a nucleotide having a cleavable moiety.

In some embodiments, the rolling ring amplification reaction generates fixed concatimer template molecules using incorporated nucleotides with cleavable moieties distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules.

In some embodiments, the pairwise sequencing method further comprises step (e) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (e) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing the plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes performing (Figure 62). In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 62). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments, the pairwise sequencing method comprises maintaining a plurality of fixed concatimer template molecules and hybridizing a plurality of extended forward sequencing primer strands to the maintained fixed single-stranded nucleic acid concatimer template molecules. It further includes step (f) of replacing with a strand. The plurality of extended forward sequencing primer strands can be removed and replaced with a plurality of forward extension strands by performing a primer extension reaction (Figures 63 to 65).

In some embodiments, step (f) performs a strand translocation primer extension reaction using at least one extended forward sequencing primer to initiate the primer extension reaction, such that the forward extension is covalently linked to the extended forward sequencing primer strand. Under conditions suitable to generate strands, at least one extended forward sequencing primer strand is contacted with a plurality of strand transfer polymerases and a plurality of nucleotides and in the absence of soluble amplification primers, wherein the forward extension strand is bound to an immobilized conkatimer. and hybridization to the template molecule (Figure 63). For example, one of the extended forward sequencing primer strands can serve as a primer for strand transposition polymerase. A strand transposition polymerase can extend an extended forward sequencing primer strand, transpose a downstream extended forward sequencing primer strand, and synthesize an extended strand that replaces the downstream extended forward sequencing primer strand. The newly extended strand is covalently linked to the extended forward sequencing primer strand. The immobilized concatimer template molecule is maintained. The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (f) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of soluble forward sequencing primers under conditions suitable for hybridizing to the plurality of retained immobilized concatimer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction. The concatimer molecule is comprised of a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases. contacting to generate a plurality of forward extension strands, wherein the soluble sequencing primer hybridizes with the forward sequencing primer binding sequence in the retained immobilized conkatimer molecule (Figure 64). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (f), conditions suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of maintained fixed single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and high efficiency. and hybridizing a fixed concatimer template molecule maintained in the presence of a hybridization buffer with a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, step (f) comprises (i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and (ii) a plurality of maintained immobilized cones under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a polymerase-catalyzed primer extension reaction. Contacting the catimer molecule with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases to generate a plurality of forward extension strands, comprising: hybridizes to the soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figure 65). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (f), conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and a high-throughput hybridization buffer. and hybridizing a fixed concatimer template molecule maintained in the presence of a soluble primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (f), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (f), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of buffering agents (e.g. Tris-HCl, MES, HEPES, etc.).

In some embodiments, in step (f), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (f), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65° C. for about 3 minutes and with about 50 mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the primer extension polymerase of step (f) comprises a high fidelity polymerase. In some embodiments, the primer extension polymerase of step (d) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). . Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The pairwise method described herein provides increased accuracy in downstream sequencing reactions because step (f) replaces the extended forward sequencing primer strand generated in step (e) with a forward extended strand with reduced base errors. can be provided. The extended forward sequencing primer strand is generated in step (e) and may or may not contain misincorporated nucleotides due to polymerase-catalyzed mispairing bases. When step (e) is performed with a high fidelity DNA polymerase, the resulting forward extended strand may have reduced base errors compared to the extended forward sequencing primer strand. The forward extension strand will be used as a nucleic acid template for downstream sequencing steps (see, eg, step (h) below). Accordingly, step (f) can increase the sequencing accuracy of the downstream step (h), thereby increasing the overall sequencing accuracy of the pairwise sequencing flow diagram.

In some embodiments, the pairwise sequencing method comprises : a single-stranded concatimer template molecule anchored at nucleotide(s) having a cleavable moiety while maintaining a plurality of forward extension strands and a plurality of immobilized surface primers; It further comprises the step (g) of removing the retained fixed conkatimer template molecule by creating an abasic site and creating a gap in the abasic site to generate a plurality of gap-containing single stranded nucleic acid conkatimer template molecules ( 66 and 67, and 68 and 69).

Abasic sites are created on the retained concatimer template strand containing nucleotides with cleavable moieties. In some embodiments, moieties that can cleave from the retained conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create multiple single-stranded nucleic acid template molecules with gaps while maintaining multiple forward extension strands. Abasic sites can be created by contacting an immobilized concatimer template molecule with an enzyme that removes a nucleobase from a nucleotide with a cleavable moiety. Uracil in the retained concatimer template strand can be converted to an abasic site using uracil DNA glycosylase (UDG). 8oxoG in the retained concatimer template strand can be converted to an abasic site using FPG glycosylase. Deoxyinosine in the retained concatimer template strand can be converted to an abasic site using AlkA glycosylase.

In some embodiments, in step (g), the gap is formed by an enzyme having lyase activity that releases abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a mixture of enzymes with the non-basic portion of an immobilized concatimer template molecule (Figures 66 and 68). Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (g), the plurality of gap-containing template molecules can be removed using enzymes, chemical compounds, and/or heat. After the gap-removal procedure, the plurality of retained forward extension strands hybridize to the retained immobilized surface primers (Figures 67 and 69).

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by . If a 5' to 3' double-stranded DNA exonuclease is used to remove the gap-containing template molecule, the plurality of soluble amplification primers in step (f) will provide soluble amplification primers that are resistant to exonuclease digestion. They may contain at least one phosphorothioate diester bond at their 5' end. In some embodiments, the plurality of soluble amplification primers in step (f) comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of soluble amplification primers in step (f) comprise at least one ribonucleotide and/or at least one 2'-O-methyl nucleotide that can provide a forward sequencing primer that is resistant to exonuclease digestion. or 2'-O-methoxyethyl (MOE) nucleotides.

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5. It can be removed by washing.

In some embodiments, the pairwise sequencing method further comprises step (h) generating a plurality of extended reverse sequencing primer strands by sequencing a plurality of retained forward extension strands. In some embodiments, the sequencing of step (h) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, thereby producing a plurality of extended reverse sequencing primer strands (FIGS. 70 and 71). The extended reverse sequencing primer strand hybridizes to the retained forward extended strand. The retained forward extension strand hybridizes to the first surface primer. The extended reverse sequencing primer strand does not hybridize to the first surface primer or is covalently linked to the first surface primer. Therefore, the extended reverse sequencing primer strand is not anchored to the support.

In some embodiments, in step (h), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In an alternative embodiment, the sequencing of step (h) includes using the immobilized surface primer as a sequencing primer and performing a sequencing reaction on the immobilized surface primer to generate a plurality of reverse sequencing strands.

In some embodiments, the reverse sequencing reaction of step (h) comprises a plurality of reverse sequencing primers, a reverse sequencing primer binding sequence of the forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides and/ or contacting it with a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, an individual maintained forward extension strand may undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer that hybridizes to the reverse sequencing primer binding site (e.g., Figures 70 and 71 reference). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (h). The washing step can be carried out with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

Pairwise sequencing method - lack of non-basic regions

This disclosure Pairwise sequences, comprising the step (a) of providing a plurality of fixed single-stranded nucleic acid concatimer template molecules, each lacking a cleavable moiety that can be cleaved to create an abasic site in the concatimer template molecule. An analysis method is provided, wherein each template molecule of the plurality is immobilized on a first surface primer immobilized on a support, and the immobilized first surface primer lacks a nucleotide having a cleavable moiety. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers. Exemplary nucleotides with cleavable moieties include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine.

In some embodiments, individual immobilized concatimer template molecules are covalently linked to an immobilized surface primer (e.g., an immobilized first surface primer) (Figure 73). In an alternative embodiment, individual immobilized concatimer template molecules hybridize to an immobilized surface primer (e.g., an immobilized first surface primer) (Figure 80).

In some embodiments, each individual Concatimer template molecule of the plurality comprises two or more copies of the sequence of interest, and each immobilized Concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer; at least two copies, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) at least two copies of the universal binding sequence for the immobilized first surface primer, (iv) at least two copies of the immobilized agent. 2 at least two copies of the universal binding sequence for the surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. any of the following: duplicates, (vii) two or more copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) two or more copies of the sample barcode sequence, and/or (ix) two or more copies of the unique molecular indicator sequence. Or it further includes any combination of two or more.

In some embodiments, the individual template molecules of the plurality comprise at least two copies of the sequence of interest and at least two copies of the universal binding sequence for the soluble compressed oligonucleotide, wherein the individual immobilized concatimer template molecules are (i ) two or more copies of the universal binding sequence for the soluble forward sequencing primer, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) the universal binding sequence for the immobilized first surface primer. (iv) at least two copies of the universal binding sequence for the immobilized second surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) a second Any one or any combination of two or more of the following: two or more copies of the universal binding sequence for the soluble amplification primer, (vii) two or more copies of the sample barcode sequence, and/or (viii) two or more copies of the unique molecular indicator sequence. Includes more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized first surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized first surface primer comprises a 3' non-extensible moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the immobilized concatimer template molecule further comprises two or more copies of a universal binding sequence (or a complementary sequence thereof) for an immobilized second surface primer having a different sequence than the first immobilized surface primer. do. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. In some embodiments, the end of the 3' end of the immobilized second surface primer is a moiety that blocks primer extension (e.g., the 3' end of the non-extendable end), e.g., a phosphate group, a diade group, etc. Contains an oxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules are bound to or immobilized to a first immobilized surface primer, and at least one portion of the individual concatimer template molecules is hybridized to the immobilized second surface primer. do. The immobilized second surface primer serves to tether the portion of the concatimer template molecule immobilized to the support (Figures 79 and 86).

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. on the support. In communication, a plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (b) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (b) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing a plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 74 and 81). In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments , the pairwise sequencing method comprises replacing a plurality of extended forward sequencing primer strands with a plurality of forward extension strands by maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction (c ) further includes. The extended forward sequencing primer strand can be removed from the retained fixed concatimer template molecule. The retained immobilized concatimer template molecule can be hybridized to a plurality of soluble amplification or sequencing primers and subjected to a primer extension reaction. The primer extension reaction is performed by a plurality of soluble primers (e.g., soluble amplification primers or soluble forward sequencing primers) and a plurality of strand-transfer polymerases to hybridize the plurality of forward extension strands to the immobilized concatimer template molecule. , and generate a plurality of partially translocated forward extension strands that hybridize to the immobilized concatimer template molecule to form a plurality of immobilized amplicons. The strand translocation primer extension reaction also generates a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. In some embodiments, the strand transposition primer extension reaction can be performed in the presence of a plurality of soluble compression oligonucleotides to anchor the detached forward extension strand to the immobilized amplicon (see Figures 75-77 and Figures 82-84 ).

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (c) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a strand transposition primer extension reaction, the plurality of retained immobilized concatimer molecules. A plurality of partial portions are contacted with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of strand transfer polymerases to hybridize to the plurality of forward extension strands and the immobilized concatimer template molecule. generating a translocated forward extension strand, and a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. The soluble amplification primer hybridizes to the soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figures 75 and 82). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, step (c) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of retained immobilized Concatimer molecules under conditions suitable for hybridizing the plurality of soluble sequencing primers to the plurality of retained immobilized Concatimer template molecules and for performing a strand transposition primer extension reaction. is contacted with a plurality of soluble sequencing primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of strand transfer polymerases, thereby hybridizing the plurality of forward extension strands and the immobilized concatimer template molecules. generating a partially dislocated forward extension strand of and a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. The soluble forward sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized conkatimer molecule. The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (c), conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and a high-throughput hybridization buffer. and hybridizing a fixed concatimer template molecule maintained in the presence of a soluble amplification primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (c), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65° C. for about 3 minutes and with about 50 mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the pairwise sequencing method further comprises step (d) sequencing the plurality of fixed, partially transposed forward extended strands to generate a first plurality of extended reverse sequencing primer strands. In some embodiments, step (d) further comprises sequencing the plurality of fixed, detached forward extended strands to generate a second plurality of extended reverse sequencing primer strands. In some embodiments, each fixed, partially translocated forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto. In some embodiments, each fixed, detached forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto. During sequencing in step (d), the anchored partially translocated forward extension strand remains hybridized to the anchored concatimer template molecule.

In some embodiments, the sequencing of step (d) is performed under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the forward extension strand (e.g., hybridizing to an immobilized concatimer template molecule). contacting the plurality of fixed partially translocated forward extension strands and the plurality of fixed detached forward extension strands with a plurality of soluble reverse sequencing primers (FIGS. 78 and 85). The sequencing in step (d) includes performing a sequencing reaction using hybridized reverse sequencing primers, wherein the forward sequencing reaction generates a plurality of extended reverse sequencing primer strands (Figures 78 and 85). The extended reverse sequencing primer strand hybridizes to a partially translocated forward extension strand that hybridizes to an immobilized concatimer template molecule or to an immobilized detached forward extension strand.

For simplicity, Figures 73-78 and Figures 80-85 do not show immobilized concatimer template molecules with universal binding sequences for soluble compressed oligonucleotides. Those skilled in the art will recognize that the immobilized concatimer template molecule may contain a universal binding sequence for a soluble compacted oligonucleotide.

For simplicity, Figures 78 and 85 show exemplary immobilized partially translocated forward extension strands hybridizing to an immobilized concatimer template molecule and an immobilized detached forward extension strand, each with an extended reverse sequence hybridized thereto. Have one copy of the assay primer strand. Those skilled in the art will recognize that the fixed partially translocated forward extension strand hybridized to the fixed conkatimer template molecule and the fixed detached forward extended strand can have two or more copies of the extended reverse sequencing primer strand hybridized thereto. will recognize Accordingly, the reverse sequencing reaction may generate a plurality of extended reverse sequencing primer strands hybridized to the same immobilized partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, or to an immobilized detached forward extension strand. You can.

In some embodiments, a reverse sequencing reaction may include a plurality of compressed oligonucleotides. The compaction oligonucleotide acts to anchor one or more of the detached forward extension strands (e.g., hybridized to an immobilized concatimer template molecule) via hybridization to an immobilized, partially dislocated forward extension strand.

In some embodiments, in step (d), a reverse sequencing primer is applied to the reverse sequencing primer binding sequence of the immobilized partially translocated forward extension strand and the immobilized detached forward extension strand hybridized to the immobilized concatimer template molecule. Suitable conditions for hybridization include contacting a plurality of soluble reverse sequencing primers and the forward extension strand with a high-throughput hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reaction of step (d) comprises a plurality of soluble reverse sequencing primers of a fixed partially translocated forward extension strand or a fixed detached forward extension strand hybridized to an immobilized concatimer template molecule. contacting the reverse sequencing primer binding sequence with one or more types of sequencing polymerase and a plurality of nucleotides and/or a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given fixed partially translocated forward extension strand hybridized to an immobilized concatimer template molecule or to an immobilized detached forward extension strand can hybridize to a reverse sequencing primer, and the sequencing reaction You may experience Accordingly, each maintained forward extension strand can undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (d). The washing step can be performed with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

RCA and pairwise sequencing on supports - lack of non-basic regions

The present disclosure provides a pairwise sequencing method comprising step (a) providing a support on which a plurality of surface primers (e.g., a plurality of first surface primers) are immobilized, wherein each of the surface primers has a 3' OH It has an extendable end and lacks nucleotides with a cleavable moiety (Figure 87). For example, the surface primer lacks uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The first surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the nucleic acid library molecules (e.g., linear or circular library molecules). The first surface primer may include a terminal 3' nucleotide with a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase catalyzed polymerization).

The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the support further includes a plurality of second surface primers secured thereto (e.g., Figure 79). The second surface primer has a different sequence than the first fixed surface primer. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The second surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the immobilized single-stranded concatimer template molecule. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. The 3' end of the immobilized second surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, an individual immobilized single-stranded nucleic acid concatimer template molecule is covalently linked to an immobilized first surface primer, and at least one portion of the individual concatimer template molecule is hybridized to an immobilized second surface primer. (e.g., Figure 79). The immobilized second surface primer serves to tether the portion of the concatimer template molecule immobilized to the support. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable. In some embodiments, the second surface primer has a terminal 3' extendable end.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc., on the support. In communication, a plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

The present disclosure provides a step (b) of hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers and performing a rolling ring amplification reaction to generate a plurality of immobilized single-stranded nucleic acid concatimer template molecules. Provides a pairwise sequence analysis method including. In some embodiments, the rotary ring amplification reaction can be performed with a plurality of strand transposition polymerases and a plurality of nucleotides lacking nucleotides with a cleavable moiety that can be cleaved to create an abasic site. In some embodiments, the plurality of nucleotides comprises any combination of dATP, dCTP, dGTP, and/or dTTP. Rotational ring amplification reactions involve mixtures of nucleotides that lack cleavable moieties. Exemplary nucleotides with cleavable moieties include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine. In some embodiments, the rolling ring amplification reaction generates a plurality of immobilized single-stranded nucleic acid conkatimer template molecules lacking nucleotides having a cleavable moiety, wherein each single-stranded nucleic acid conkatimer template molecule is an immobilized agent. 1 is covalently linked to the surface primer (Figure 88).

In some embodiments, single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules. In some embodiments, single-stranded circular nucleic acid library molecules can be removed from the concatimer template molecule using at least one washing step performed under conditions suitable for maintaining the single-stranded nucleic acid concatimer template molecule, wherein individual concatimer template molecules are The catimer template molecule is operably linked to the immobilized first surface primer.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each immobilized concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer (or its complement); sequence), (ii) a universal binding sequence (or a complementary sequence thereof) to a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer, (iv) an immobilized a universal binding sequence (or a complementary sequence thereof) to a second surface primer, (v) a universal binding sequence (or a complementary sequence thereof) to a first soluble amplification primer, (vi) a universal binding sequence to a second soluble amplification primer. a binding sequence (or a complementary sequence thereof), (vii) a universal binding sequence for soluble compressed oligonucleotides (or a complementary sequence thereof), (viii) a sample barcode sequence and/or (ix) a unique molecular indicator sequence, or It further includes any combination of two or more.

In some embodiments, each individual single-stranded circular nucleic acid library molecule of the plurality comprises a universal binding sequence (or a complementary sequence thereof) for the sequence of interest and a soluble compressed oligonucleotide, and each immobilized concatimer template molecule is ( i) a universal binding sequence for a soluble forward sequencing primer (or a complementary sequence thereof), (ii) a universal binding sequence for a soluble reverse sequencing primer (or a complementary sequence thereof), (iii) an immobilized first surface primer. (iv) a universal binding sequence (or a complementary sequence thereof) for the immobilized second surface primer, (v) a universal binding sequence for the first soluble amplification primer (or (or its complementary sequence), (vi) a universal binding sequence for the second soluble amplification primer (or its complementary sequence), (vii) a sample barcode sequence, and/or (viii) a unique molecular indicator sequence. It further includes a combination of

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, step (b) the rolling ring amplification reaction comprises a plurality of fixed single-stranded nucleic acid conkatimers each comprising a conkatimer lacking a cleavable moiety and a nucleotide with two or more copies of the sequence of interest. The template molecule is generated, and the immobilized concatimer template molecule contains (i) two or more copies of the universal binding sequence (or its complementary sequence) to the soluble forward sequencing primer, (ii) to the soluble reverse sequencing primer. (iii) two or more copies of the universal binding sequence (or complementary sequence thereof) for the immobilized first surface primer, (iv) immobilization (v) two or more copies of the universal binding sequence (or complementary sequence thereof) to the second surface primer, (v) two or more copies of the universal binding sequence (or complementary sequence thereof) to the first soluble amplification primer. , (vi) two or more copies of the universal binding sequence (or complementary sequence thereof) to the second soluble amplification primer, (vii) two copies of the universal binding sequence (or complementary sequence thereof) to the soluble compressed oligonucleotide. (viii) two or more copies of a sample barcode sequence, and/or (ix) two or more copies of a unique molecular indicator sequence.

In some embodiments, the plurality of immobilized single-stranded nucleic acid concatemer template molecules generated by the rolling ring amplification reaction of step (b) have a universal binding sequence (or a complementary sequence thereof) to the immobilized second sequence surface primer. It further contains two or more copies of. In some embodiments, the individual immobilized single-stranded nucleic acid concatimer template molecules are bound (e.g., covalently) to the immobilized first surface primer, and at least one portion of the individual concatimer template molecules is bound to the immobilized first surface primer. Hybridizes to the second surface primer. The immobilized second surface primer serves to tether portions of the concatimer template molecule immobilized to the support (see, e.g., Figure 79). In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable.

The rolling ring amplification reaction of step (b) can be carried out with a nucleotide mixture containing dATP, dCTP, dGTP, dTTP, and the nucleotide mixture lacking nucleotides having a cleavable moiety. Exemplary nucleotides with cleavable moieties include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine.

In some embodiments, the pairwise sequencing method further comprises step (c) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (c) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing the plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 90). In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments , the pairwise sequencing method comprises replacing a plurality of extended forward sequencing primer strands with a plurality of forward extension strands by maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction (d ) further includes. The extended forward sequencing primer strand can be removed from the retained fixed concatimer template molecule. The retained immobilized concatimer template molecule can be hybridized to a plurality of soluble amplification or sequencing primers and subjected to a primer extension reaction. The primer extension reaction is performed by a plurality of soluble primers (e.g., soluble amplification primers or soluble forward sequencing primers) and a plurality of strand-transfer polymerases to hybridize the plurality of forward extension strands to the immobilized concatimer template molecule. , and generate a plurality of partially translocated forward extension strands that hybridize to the immobilized concatimer template molecule to form a plurality of immobilized amplicons. The strand translocation primer extension reaction also generates a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. In some embodiments, the strand transposition primer extension reaction can be performed in the presence of a plurality of soluble compression oligonucleotides to anchor the detached forward extension strand to the immobilized amplicon (see Figures 91-93).

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (d) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a strand transposition primer extension reaction, the plurality of retained immobilized concatimer molecules. A plurality of partial portions are contacted with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of strand transfer polymerases to hybridize to the plurality of forward extension strands and the immobilized concatimer template molecule. generating a translocated forward extension strand, and a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. The soluble amplification primer hybridizes to the soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figure 91). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, step (d) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of retained immobilized Concatimer molecules under conditions suitable for hybridizing the plurality of soluble sequencing primers to the plurality of retained immobilized Concatimer template molecules and for performing a strand transposition primer extension reaction. is contacted with a plurality of soluble sequencing primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of strand transfer polymerases, thereby hybridizing the plurality of forward extension strands and the immobilized concatimer template molecules. generating a partially dislocated forward extension strand of and a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. The soluble forward sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized conkatimer molecule. The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (d), conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and a high-throughput hybridization buffer. and hybridizing a fixed concatimer template molecule maintained in the presence of a soluble amplification primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (d), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the pairwise sequencing method further comprises step (e) sequencing the plurality of fixed, partially transposed forward extended strands to generate a first plurality of extended reverse sequencing primer strands. In some embodiments, step (e) further comprises sequencing the plurality of fixed, detached forward extended strands to generate a second plurality of extended reverse sequencing primer strands. In some embodiments, each fixed, partially translocated forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto. In some embodiments, each fixed, detached forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto. During sequencing in step (e), the anchored partially translocated forward extension strand remains hybridized to the anchored concatimer template molecule.

In some embodiments, the sequencing of step (e) is performed under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the forward extension strand (e.g., hybridizing to an immobilized concatimer template molecule). (Figure 94). Sequencing in step (e) includes performing a sequencing reaction using hybridized reverse sequencing primers, wherein the reverse sequencing reaction generates a plurality of extended reverse sequencing primer strands (FIG. 94). The extended reverse sequencing primer strand hybridizes to a partially translocated forward extension strand that hybridizes to an immobilized concatimer template molecule or to an immobilized detached forward extension strand.

For simplicity, Figures 88-94 do not show immobilized concatimer template molecules with universal binding sequences for soluble compacted oligonucleotides. Those skilled in the art will recognize that the immobilized concatimer template molecule may contain a universal binding sequence for a soluble compacted oligonucleotide.

For simplicity, Figure 94 shows an exemplary immobilized partially translocated forward extension strand hybridizing to an immobilized concatimer template molecule and an immobilized detached forward extension strand, each with an extended reverse sequencing primer strand hybridized thereto. has one copy of Those skilled in the art will recognize that the fixed partially translocated forward extension strand hybridized to the fixed conkatimer template molecule and the fixed detached forward extended strand can have two or more copies of the extended reverse sequencing primer strand hybridized thereto. will recognize Accordingly, the reverse sequencing reaction may generate a plurality of extended reverse sequencing primer strands hybridized to the same immobilized partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, or to an immobilized detached forward extension strand. You can.

In some embodiments, a reverse sequencing reaction may include a plurality of compressed oligonucleotides. The compaction oligonucleotide acts to anchor one or more of the detached forward extension strands (e.g., hybridized to an immobilized concatimer template molecule) via hybridization to an immobilized, partially dislocated forward extension strand.

In some embodiments, in step (e), a reverse sequencing primer is applied to the reverse sequencing primer binding sequence of the immobilized partially translocated forward extension strand and the immobilized detached forward extension strand hybridized to the immobilized concatimer template molecule. Suitable conditions for hybridization include contacting a plurality of soluble reverse sequencing primers and the forward extension strand with a high-throughput hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reaction of step (e) comprises a plurality of soluble reverse sequencing primers comprising a fixed partially translocated forward extension strand or a fixed detached forward extension strand hybridized to an immobilized concatimer template molecule. contacting the reverse sequencing primer binding sequence with one or more types of sequencing polymerase and a plurality of nucleotides and/or a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given fixed partially translocated forward extension strand hybridized to an immobilized concatimer template molecule or to an immobilized detached forward extension strand can hybridize to a reverse sequencing primer, and the sequencing reaction You may experience Accordingly, each maintained forward extension strand can undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (e). The washing step can be performed with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the detergent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

RCA and pairwise sequencing in solution - lack of non-basic regions

The present disclosure provides a method for combining a plurality of single-stranded circular nucleic acid library molecules in solution with a plurality of soluble first amplification primers, under conditions suitable for forming a plurality of library-primer duplexes and suitable for performing a rotary ring amplification reaction. A pairwise sequence comprising contacting a strand transfer polymerase and a plurality of nucleotides comprising any combination of dATP, dCTP, dGTP and/or dTTP, thereby producing a plurality of single stranded nucleic acid concatemers. Analysis methods are provided (Figure 95). Rotational ring amplification reactions involve a mixture of nucleotides lacking a cleavable moiety that can be cleaved to create a non-basic site. Exemplary nucleotides with cleavable moieties include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine. The rolling ring amplification reaction generates a plurality of single-stranded nucleic acid concatemers that lack nucleotides with cleavable moieties. In some embodiments, single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules. In some embodiments, the soluble first amplification primer comprises a sequence that selectively hybridizes to a universal binding sequence in a circular nucleic acid library molecule, e.g., a universal binding sequence for the first soluble amplification primer (or a complementary sequence thereof). do. Alternatively, the soluble first amplification primer comprises a random sequence that binds non-selectively in the circular nucleic acid library molecule.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each immobilized concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer (or its complement); sequence), (ii) a universal binding sequence (or a complementary sequence thereof) to a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer, (iv) an immobilized a universal binding sequence (or a complementary sequence thereof) to a second surface primer, (v) a universal binding sequence (or a complementary sequence thereof) to a first soluble amplification primer, (vi) a universal binding sequence to a second soluble amplification primer. a binding sequence (or a complementary sequence thereof), (vii) a universal binding sequence for soluble compressed oligonucleotides (or a complementary sequence thereof), (viii) a sample barcode sequence and/or (ix) a unique molecular indicator sequence, or It further includes any combination of two or more.

In some embodiments, each individual single-stranded circular nucleic acid library molecule of the plurality comprises a universal binding sequence (or a complementary sequence thereof) for the sequence of interest and a soluble compressed oligonucleotide, and each immobilized concatimer template molecule is ( i) a universal binding sequence for a soluble forward sequencing primer (or a complementary sequence thereof), (ii) a universal binding sequence for a soluble reverse sequencing primer (or a complementary sequence thereof), (iii) an immobilized first surface primer. (iv) a universal binding sequence (or a complementary sequence thereof) for the immobilized second surface primer, (v) a universal binding sequence for the first soluble amplification primer (or its complementary sequence), (vi) a universal binding sequence for the second soluble amplification primer (or its complementary sequence), (vii) a sample barcode sequence, and/or (viii) a unique molecular indicator sequence. It further includes a combination of

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence for the immobilized first surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, step (a) the rolling ring amplification reaction comprises a plurality of fixed single-stranded nucleic acid conkatimers each comprising a conkatimer lacking a cleavable moiety and a nucleotide with two or more copies of the sequence of interest. The template molecule is generated, and the immobilized concatimer template molecule contains (i) two or more copies of the universal binding sequence (or its complementary sequence) to the soluble forward sequencing primer, (ii) to the soluble reverse sequencing primer. (iii) two or more copies of the universal binding sequence (or complementary sequence thereof) for the immobilized first surface primer, (iv) immobilization (v) two or more copies of the universal binding sequence (or complementary sequence thereof) to the second surface primer, (v) two or more copies of the universal binding sequence (or complementary sequence thereof) to the first soluble amplification primer. , (vi) two or more copies of the universal binding sequence (or complementary sequence thereof) to the second soluble amplification primer, (vii) two copies of the universal binding sequence (or complementary sequence thereof) to the soluble compressed oligonucleotide. (viii) two or more copies of a sample barcode sequence, and/or (ix) two or more copies of a unique molecular indicator sequence.

In some embodiments, the pairwise sequencing method comprises a plurality of first surface primers immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatemers to the one or more immobilized first surface primers. It further includes a step (b) of distributing the rolling ring amplification reaction from step (a) on a support (Figure 96). In some embodiments, the immobilized first surface primer has a terminal 3' group that is not extensible. In some embodiments, the 3' end of the immobilized first surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT, or amino group. In some embodiments, the immobilized first surface primer has an extendable 3'OH end. In some embodiments, the immobilized first surface primer lacks a nucleotide having a cleavable moiety. Exemplary nucleotides with cleavable moieties include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine. The concatimer is immobilized on the support by hybridization to the immobilized first surface primer. In some embodiments, the support includes a plurality of first surface primers. In some embodiments, the support lacks a plurality of second surface primers. In some embodiments, the support includes a plurality of first and second surface primers.

In some embodiments, the pairwise sequencing method further comprises (c) continuing the rolling ring amplification reaction on the support to generate a plurality of extended concatimer template molecules immobilized via hybridization to the immobilized first surface primer. (Figure 97). The RCA reaction on the support can be performed by a plurality of strand transfer polymerases and a plurality of nucleotides comprising any combination of dATP, dCTP, dGTP and/or dTTP. The plurality of nucleotides lacks a nucleotide having a cleavable moiety. In some embodiments, the rolling ring amplification reaction on a support can be performed in the presence of a plurality of compressed oligonucleotides.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The first surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the concatimer molecule. In some embodiments, the first surface primer may lack a terminal 3' OH extendable end rendering the first surface primer non-extensible. In some embodiments, the first surface primer includes a terminal 3' OH group that is extendable for nucleotide polymerization (e.g., polymerase catalyzed polymerization). The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the plurality of immobilized first surface primers includes a 3' extendable end. In some embodiments, the 3' end of the immobilized first surface primer comprises a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT, or amino group. In some embodiments, the immobilized first surface primer is not extendable in the primer extension reaction. The immobilized first surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the concatimer template molecule.

In some embodiments, the support further includes a plurality of second surface primers secured thereto (Figure 86). The second surface primer has a different sequence than the first fixed surface primer. The immobilized second surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The second surface primer comprises a sequence that is fully or partially complementary along its length to at least a portion of the concatimer molecule. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. The 3' end of the immobilized second surface primer includes a moiety that blocks primer extension, such as a phosphate group, dideoxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, an individual immobilized single-stranded nucleic acid concatimer template molecule hybridizes to an immobilized first surface primer, and at least one portion of the individual concatimer template molecule hybridizes to an immobilized second surface primer ( Figure 86). The immobilized second surface primer serves to tether the portion of the concatimer template molecule immobilized to the support. The immobilized concatimer template molecule has at least two copies of the universal binding sequence for the immobilized second surface primer. A portion of the immobilized concatimer template molecule comprising a universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. In some embodiments, the second surface primers include terminal 3' blocking groups that render them non-extendable. In some embodiments, the second surface primer has a terminal 3' extendable end.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc., on the support. In communication, and therefore a plurality of immobilized surface primers can be reacted with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (d) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (d) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing a plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule may undergo more than one sequencing reaction, where each sequencing reaction is initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site (e.g., 99). In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments, the pairwise sequencing method comprises replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands by maintaining a plurality of immobilized concatimer template molecules and performing a primer extension reaction (e) It further includes. The extended forward sequencing primer strand can be removed from the retained fixed concatimer template molecule. The retained fixed concatimer template molecule can be hybridized to a plurality of second soluble amplification primers or a plurality of sequencing primers, and a primer extension reaction can be applied. The primer extension reaction is performed by a plurality of soluble primers (e.g., a second soluble amplification primer or a soluble forward sequencing primer) and a plurality of strand-transfer polymerases to hybridize a plurality of forward direction polymerases to the immobilized concatimer template molecule. An extension strand, and a plurality of partially translocated forward extension strands that hybridize to the immobilized concatimer template molecule are created to form a plurality of immobilized amplicons. The strand translocation primer extension reaction also generates a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. In some embodiments, the strand transposition primer extension reaction can be performed in the presence of a plurality of soluble compression oligonucleotides to anchor the detached forward extension strand to the immobilized amplicon (see Figures 100-102).

Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, step (e) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of maintained immobilized concatimers under conditions suitable for hybridizing the plurality of second soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a strand transposition primer extension reaction. Contacting the molecule with a plurality of second soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of strand transfer polymerases to hybridize to the plurality of forward extension strands and the immobilized concatimer template molecule. generating a plurality of partially dislocated forward extension strands, and a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. The second soluble amplification primer hybridizes to the second soluble amplification primer binding sequence in the retained immobilized concatimer molecule (Figure 100). The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, step (e) includes (i) removing a plurality of extended forward sequencing primer strands while maintaining an immobilized concatimer template molecule; and (ii) a plurality of second soluble sequencing primers, under conditions suitable for hybridizing the plurality of second soluble sequencing primers to the plurality of retained immobilized Concatimer template molecules and for performing a strand transposition primer extension reaction. Contacting the mer molecule with a plurality of second soluble sequencing primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of strand transfer polymerases results in a plurality of forward extension strands and an immobilized concatimer template molecule. generating a plurality of partially dislocated forward extension strands that hybridize to the and a plurality of detached forward extension strands that do not hybridize to the immobilized concatimer template molecule. The soluble forward sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized conkatimer molecule. The primer extension reaction may optionally include a plurality of condensed oligonucleotides and/or hexamine (e.g., cobalt hexamine III) to generate a forward extension strand. Individual forward extension strands collapse into nanoballs having a more compact size and/or shape compared to nanoballs resulting from a primer extension reaction performed without compaction oligonucleotides and/or hexamine (e.g., cobalt hexamine III). It can be. Inclusion of compact oligonucleotides and/or hexamine (e.g., cobalt hexamine III) in the primer extension reaction can improve the full width half maximum (FWHM) of nanoball spot images. Spot images can be represented as Gaussian spots, and their size can be measured as FWHM. A smaller spot size, as indicated by a smaller FWHM, typically correlates with improved imaging of the spot. In some embodiments, the FWHM of a nanoball spot can be about 10 μm or smaller.

In some embodiments, in step (e), conditions suitable for hybridizing the plurality of second soluble amplification primers to the plurality of retained fixed single-stranded nucleic acid concatemer template molecules include primer extension polymerase, a plurality of nucleotides, and high efficiency. and hybridizing an immobilized concatimer template molecule maintained in the presence of a hybridization buffer with a soluble second amplification primer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (e), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the pairwise sequencing method further comprises step (f) sequencing the plurality of fixed, partially transposed forward extended strands to generate a first plurality of extended reverse sequencing primer strands. In some embodiments, step (f) further comprises sequencing the plurality of fixed, detached forward extended strands to generate a second plurality of extended reverse sequencing primer strands. In some embodiments, each fixed, partially translocated forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto. In some embodiments, each fixed, detached forward extension strand has two or more extended reverse sequencing primer strands thereon. During the sequencing of step (f), the fixed partially translocated forward extension strand remains hybridized to the retained fixed concatimer template molecule.

In some embodiments, the sequencing of step (f) is performed under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the forward extension strand (e.g., hybridizing to an immobilized concatimer template molecule). (FIG. 103). The sequencing of step (f) includes performing a sequencing reaction using hybridized reverse sequencing primers, wherein the reverse sequencing reaction generates a plurality of extended reverse sequencing primer strands (FIG. 103). The extended reverse sequencing primer strand hybridizes to a partially translocated forward extension strand that hybridizes to an immobilized concatimer template molecule or to an immobilized detached forward extension strand.

For simplicity, Figures 95-103 do not show immobilized concatimer template molecules with universal binding sequences for soluble compacted oligonucleotides. Those of ordinary skill in the art will recognize that the immobilized concatimer template molecule may contain a universal binding sequence for a soluble compressed oligonucleotide.

For simplicity, Figure 103 shows an exemplary immobilized partially translocated forward extension strand hybridizing to an immobilized concatimer template molecule and an immobilized detached forward extension strand, each with an extended reverse sequencing primer strand hybridized thereto. has one copy of Those skilled in the art will recognize that the fixed partially translocated forward extension strand hybridized to the fixed conkatimer template molecule and the fixed detached forward extended strand can have two or more copies of the extended reverse sequencing primer strand hybridized thereto. will recognize Accordingly, the reverse sequencing reaction may generate a plurality of extended reverse sequencing primer strands hybridized to the same immobilized partially translocated forward extension strand hybridized to an immobilized concatimer template molecule, or to an immobilized detached forward extension strand. You can.

In some embodiments, a reverse sequencing reaction may include a plurality of compressed oligonucleotides. The compaction oligonucleotide acts to anchor one or more of the detached forward extension strands (e.g., hybridized to an immobilized concatimer template molecule) via hybridization to an immobilized, partially dislocated forward extension strand.

In some embodiments, in step (f), a reverse sequencing primer is applied to the reverse sequencing primer binding sequence of the immobilized partially translocated forward extension strand and the immobilized detached forward extension strand hybridized to the immobilized concatimer template molecule. Suitable conditions for hybridization include contacting a plurality of soluble reverse sequencing primers and the forward extension strand with a high-throughput hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reaction of step (f) comprises a plurality of soluble reverse sequencing primers of a fixed partially translocated forward extension strand or a fixed detached forward extension strand hybridized to an immobilized concatimer template molecule. contacting the reverse sequencing primer binding sequence with one or more types of sequencing polymerase and a plurality of nucleotides and/or a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given fixed partially translocated forward extension strand hybridized to an immobilized concatimer template molecule or to an immobilized detached forward extension strand can hybridize to a reverse sequencing primer, and the sequencing reaction You may experience Accordingly, each maintained forward extension strand can undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, at least one washing step may be performed after any of steps (a) through (f). The washing step can be performed with a washing buffer containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the wash buffer is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the wash buffer includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash buffer includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The wash buffer may include a monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the detergent in the wash buffer includes a non-ionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

Introduction - Amplification using plexing amplification cycling

The present disclosure provides a general workflow: (a) providing a plurality of single-stranded concatimer template molecules comprising at least one nucleotide having a cleavable moiety, wherein the concatimer template molecules are immobilized on a support. wherein the support comprises a plurality of immobilized first and second surface primers, the first surface primer comprising a nucleotide having a cleavable moiety, and the second surface primer comprising a cleavable moiety. lacking a nucleotide having a moiety; (b) sequencing at least a portion of the concatimer template molecule and removing extension products of the sequencing reaction; (c) hybridizing individual concatimer template molecules to a portion of the second surface primer and performing a primer extension reaction to generate a plurality of forward extension strands covalently linked to the second surface primer; (d) dissociating the immobilized concatimer template molecules hybridized to the second surface primer and releasing the portions of the individual concatimer template molecules to the second surface primer that are not covalently bound to the forward extending strand by flowing a relaxing solution over the support. rehybridizing; (e) applying temperature dissociation and temperature rehybridization conditions to the immobilized concatimer template molecule and the immobilized forward extension strand; (f) performing an amplification reaction using a concatimer template molecule hybridized to the second surface primer to generate newly synthesized forward extension strands each covalently linked to the second surface primer; (g) repeating steps (d) to (f) at least once, wherein steps (d) to (f) comprise a plexing amplification cycle; (h) creating a non-basic site in the immobilized concatimer template molecule and the immobilized first surface primer, and cleaving the non-basic site to generate a gap-containing nucleic acid molecule, thereby removing the strand from the support while maintaining the immobilized forward extension strand. removing the immobilized concatimer template molecule and the immobilized first surface primer; and (i) sequencing a plurality of maintained fixed forward extension strands.

Method for pairwise sequencing - amplification by plexing amplification cycling

The present disclosure provides a plurality of fixed single-stranded nucleic acid concatimer template molecules , each comprising at least one nucleotide with a cleavable moiety that can be cleaved to create a non-basic site in the concatimer template molecule. Provided is a pairwise sequencing method comprising step (a) , wherein each template molecule among the plurality of molecules is immobilized on a first surface primer immobilized on a support, and the immobilized first surface primer has a cleavable moiety. nucleotides, and the support further comprises a plurality of immobilized second surface primers devoid of nucleotides with cleavable moieties and having 3'OH groups at extendable ends. The immobilized concatimer template molecule contains at least two copies of the sequence of interest, at least two copies of the universal binding sequence for the immobilized first surface primer, and at least two copies of the universal binding sequence for the immobilized second surface primer. Includes. The support may include an excess of immobilized first and second surface primers relative to the number of immobilized concatimer template molecules.

In some embodiments, immobilized concatimer template molecules can self-disintegrate into compact nucleic acid nanoballs. Nanoballs can be imaged and FWHM measurements can be obtained to provide the shape/size of the nanoballs.

In some embodiments, individual immobilized concatimer template molecules are covalently linked to an immobilized surface primer (e.g., an immobilized first surface primer). In an alternative embodiment, individual immobilized concatimer template molecules hybridize to an immobilized surface primer (e.g., an immobilized first surface primer).

In some embodiments, each individual Concatimer template molecule of the plurality comprises two or more copies of the sequence of interest, and each immobilized Concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer; at least two copies, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) at least two copies of the universal binding sequence for the immobilized first surface primer, (iv) at least two copies of the immobilized agent. 2 at least two copies of the universal binding sequence for the surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. any of the following: duplicates, (vii) two or more copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) two or more copies of the sample barcode sequence, and/or (ix) two or more copies of the unique molecular indicator sequence. Or it further includes any combination of two or more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, cleavable moieties in the immobilized concatimer template molecule and immobilized first surface primer of step (a) can be converted to non-basic moieties. In some embodiments, the cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the concatimer template molecule and the immobilized first surface primer, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG). In the concatimer template molecule and the immobilized first surface primer, 8oxoG can be converted to a non-basic site using FPG glycosylase. In the concatimer template molecule and the immobilized first surface primer, deoxyinosine can be converted to a non-basic moiety using AlkA glycosylase.

In some embodiments, the immobilized concatimer template molecule comprises 1 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 100, or more nucleotides with a cleavable moiety. In some embodiments, about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or a greater percentage of the immobilized concatimer template molecules. dTTP is replaced by a nucleotide with a cleavable moiety. In some embodiments, nucleotides bearing cleavable moieties are distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules. In some embodiments, the immobilized first surface primer comprises at least 1 and up to 5 nucleotides having a cleavable moiety.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized first surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized first surface primer comprises a 3' non-extensible moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the immobilized concatimer template molecule further comprises two or more copies of a universal binding sequence (or a complementary sequence thereof) for an immobilized second surface primer having a different sequence than the first immobilized surface primer. do. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. In some embodiments, the end of the 3' end of the immobilized second surface primer is a moiety that blocks primer extension (e.g., the 3' end of the non-extendable end), e.g., a phosphate group, a diade group, etc. Contains an oxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. on the support. In communication, a plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (b) sequencing a plurality of immobilized concatimer template molecules to generate a plurality of extended forward sequencing primer strands. Sequencing in step (b) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing a plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule can undergo more than one sequencing reaction, with each sequencing reaction initiated from a first forward sequencing primer that hybridizes to the forward sequencing primer binding site. In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below.

In some embodiments, the sequencing of step (b) can be performed with or without a plurality of compressed oligonucleotides. Compact oligonucleotides can maintain the compact size and/or shape of nanoballs (e.g., self-collapsed immobilized concatimer template molecules) during the forward sequencing reaction.

In some embodiments, the sequencing of step (b) may be performed by contacting a plurality of immobilized concatimer template molecules with a plurality of forward sequencing primers in the presence of a hybridization solution comprising a pH buffer, a sodium salt, and a disorder inducing agent. You can. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, the sequencing of step (b) may be performed by contacting a plurality of immobilized concatimer template molecules with a plurality of forward sequencing primers in the presence of a high-throughput hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprises step (c) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (c), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (c), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65° C. for about 3 minutes and with about 50 mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments, the pairwise sequencing method hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to the portion of the immobilized Concatimer template molecule to obtain a first plurality of It further includes step (d) of generating a fixed forward extension strand. The primer extension reaction generates a plurality of forward extension strands each having a sequence complementary to at least a portion of the immobilized concatimer template molecule. The primer extension reaction generates a plurality of forward extension strands covalently linked to an immobilized second surface primer.

In some embodiments, the primer extension reaction of step (d) includes a plurality of nucleotides lacking nucleotides having a cleavable moiety. For example, the plurality of nucleotides includes dATP, dGTP, dCTP, and dTTP. The primer extension reaction can be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. there is.

The primer extension reaction of step (d) involves a DNA polymerase capable of catalyzing the primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The primer extension reaction of step (d) involves a polymerase having strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

The immobilized first and second surface primers, immobilized concatimer template molecules, and immobilized forward extension strands are in fluid communication with each other to allow various solutions, such as buffers and reagents, to flow on the support. The immobilized first and second surface primers, the immobilized concatimer template molecule, and the immobilized forward extension strand can react with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (e) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution. The relaxing solution can flow onto the support to enable reaction with the immobilized concatimer template molecule and the immobilized forward extension strand in a massively parallel manner. The relaxing solution may flow onto the support at a temperature of about 20 to 25° C.

The relaxing solution includes at least one nucleic acid relaxant capable of breaking hydrogen bonds between the immobilized concatimer template molecule and the second surface primer. Exemplary relaxants include nucleic acid denaturants, chaotropic compounds, amide compounds, aprotic compounds, primary alcohols, and ethylene glycol derivatives. Chaotropic compounds include urea, guanidine hydrochloride or guanidine thiocyanate. Amide compounds include formamide, acetamide, or NN-dimethylformamide (DMF). Aprotic compounds include acetonitrile, dimethyl sulfoxide (DMSO), 1,4-dioxane, or tetrahydrofuran. Primary alcohols include 1-propanol, ethanol, or methanol. Ethylene glycol derivatives include 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane or 2-methoxyethanol. Other relaxants include sodium iodide, potassium iodide, and polyamines.

The relaxing solution of step (e) may further include an ionic, non-ionic or amphoteric ionic detergent. Exemplary ionic cleaners include SDS (sodium dodecyl sulfate). Exemplary nonionic detergents include Triton X-100, Tween 20, Tween 80 or Nonidet P-40. Exemplary zwitterionic detergents include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio. -Contains 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate.

The relaxing solution of step (e) further comprises a pH buffering compound (e.g., a zwitterionic buffering compound such as or 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES)). can do.

In some embodiments, the relaxing solution of step (e) includes urea, guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide, NN-dimethylformamide (DMF), acetonitrile, dimethyl sulfoxide (DMSO), side), 1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, 2-methoxyethanol, i. It includes any one or a combination of two or more of the group selected from sodium odide, potassium iodide and/or polyamines.

In some embodiments, the relaxing solution of step (e) includes formamide and SSC. In some embodiments, the relaxing solution includes acetonitrile, formamide, and SSC. In some embodiments, the relaxing solution includes acetonitrile, formamide, and MES (2-(4-morpholino)-ethane sulfonic acid). In some embodiments, the relaxing solution includes acetonitrile, formamide, guanidine hydrochloride, and HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid) and a detergent (e.g., an amphoteric ionic detergent). , such as Tween-20 or Tween-80). In some embodiments, the relaxing solution includes acetonitrile, formamide, urea, and HEPES. In some embodiments, the concentration of SSC in the relaxing solution may be 1×, 2×, 3×, or 4×.

In some embodiments, the pairwise sequencing method dissociates at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retains the immobilized forward extension strand, and (f) rehybridizing at least one portion of to one of the immobilized second surface primers that is not covalently linked to the forward extension strand. In some embodiments, nucleic acid dissociation and rehybridization is performed in the presence of a relaxing solution and includes increasing temperature, temperature plateauing, and decreasing temperature (e.g., Figure 117). The temperature increase may begin at about 20 to 25° C. and increases to about 55 to 70° C. The temperature plateau may be maintained at about 50 to 70° C. The temperature decrease may begin at about 50 to 70° C. and then decrease to about 20 to 25° C. The relaxing solution can be removed from the support by performing at least one wash with a washing solution. The wash solution may include SSC (e.g., at any concentration from about 1 to 5x) and a detergent (e.g., Tween-20). Those skilled in the art will recognize that the temperature increase, temperature plateau, and temperature decrease conditions may vary.

In some embodiments, the forward extension strand duplexed by an immobilized concatimer template molecule (e.g., generated in step (d)) is denatured, in the presence of a relaxing solution, an increasing temperature, a temperature stabilizer, and a decreasing temperature. It can be rehybridized with the first surface primer under.

In some embodiments, a pairwise sequencing method involves contacting a rehybridized immobilized Conkatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that rehybridizes to a portion of the immobilized Conkatimer template molecule. and performing a step (g) to generate a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer. Amplification in step (g) is performed after increasing temperature, temperature plateau, decreasing temperature, and washing in step (f) described above.

In some embodiments, the forward extension strand rehybridized with the first surface primer (e.g., generated in step (f)) is contacted with an amplification solution and subjected to a primer extension reaction to produce a plurality of immobilized first surface primers. When comprising a 3' extendable end, multiple newly synthesized concatimer template molecules can be generated. Alternatively, when the immobilized first surface primer contains a non-3'extensible end, the amplification reaction will not produce a newly synthesized concatimer template molecule.

In some embodiments, the amplification solution of step (g) comprises a plurality of nucleotides lacking nucleotides having a cleavable moiety. For example, the plurality of nucleotides includes dATP, dGTP, dCTP, and dTTP. The amplification reaction can be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. .

The amplification solution of step (g) includes a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The amplification solution of step (g) contains a polymerase having strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments , the pairwise sequencing method further comprises step (h) performing a plexing amplification cycle by repeating steps (e) through (g) at least once. Steps (e) to (g) may be repeated 1, 2, 3, 4, 5, 6 or up to 10 times. Each cycle may produce an additional newly synthesized forward extension strand covalently linked to a second surface primer. Each cycle can produce an additional newly synthesized concatimer template molecule covalently linked to the first surface primer. After performing the desired number of plexing amplification cycles, step (i) can be performed as described directly below.

In some embodiments, the pairwise sequencing method comprises: A first surface primer anchored with a single stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining a plurality of anchored forward extension strands and a plurality of anchored second surface primers. and removing the immobilized concatimer template molecule by creating a non-basic site of and creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules. The gap-containing nucleic acid molecule comprises an immobilized concatimer template strand and an immobilized first surface primer.

The non-basic site is created on the concatimer template strand containing nucleotides with a cleavable moiety and on the immobilized first surface primer. In some embodiments, moieties that can cleave from the conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create a first surface primer with a plurality of concatimer template molecules and a gap while maintaining the plurality of forward extending strands. Abasic sites can be created by contacting an immobilized concatimer template molecule and a first surface primer with an enzyme that removes a nucleobase from a nucleotide having a cleavable moiety. Uracil in the concatimer template strand and first surface primer can be converted to a non-basic site using uracil DNA glycosylase (UDG). 8oxoG in the concatimer template strand and first surface primer can be converted to a non-basic site using FPG glycosylase. Deoxyinosine in the concatimer template strand and first surface primer can be converted to a non-basic site using AlkA glycosylase.

In some embodiments, in step (i), the gap is formed by an enzyme having lyase activity that releases abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a non-basic site with a mixture of enzymes. Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (i), the plurality of gap-containing template molecules can be removed using enzymes, chemicals, and/or heat. After the gap-removal procedure, a plurality of anchored forward extension strands remain covalently linked to the second surface primer.

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by .

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5. It can be removed by washing.

In some embodiments , the pairwise sequencing method further comprises step (j) sequencing the plurality of retained forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands. Includes. In some embodiments, the sequencing of step (e) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, whereby the forward sequencing reaction produces a plurality of extended reverse sequencing primer strands.

Each maintained forward extension strand may contain two or more copies of the reverse sequencing primer strand hybridized thereto. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands that hybridize to the same maintained forward extended strand.

In some embodiments, the sequencing of step (j) can be performed with or without a plurality of compressed oligonucleotides. Compact oligonucleotides can maintain the compact size and/or shape of nanoballs (e.g., self-collapsed immobilized concatimer template molecules) during a reverse sequencing reaction.

In some embodiments, in step (j), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand include the plurality of soluble reverse sequencing primers and the retained forward extension strand at a pH and contacting with a hybridization solution comprising a buffer, a sodium salt, and a disorder-inducing agent. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, in step (j), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reaction of step (j) comprises a plurality of soluble reverse sequencing primers, a reverse sequencing primer binding sequence of the retained forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides or It includes contacting a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, each maintained forward extension strand can undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below.

In some embodiments, at least one washing step may be performed after any of steps (a) through (j). The washing step can be performed with a washing solution containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash solution is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash solution at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the cleaning solution is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the washing solution includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash solution includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The washing solution may include the monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the cleaning agent in the cleaning solution includes a non-ionic cleaning agent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the cleaning solution at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

The wash solution may include SSC (e.g., at any concentration from about 1 to 5x) and a detergent (e.g., Tween-20).

RCA and pairwise sequencing on supports - amplification by plexing amplification cycling

The present disclosure provides a pairwise sequencing method comprising step (a) providing a support on which a plurality of first and second surface primers are immobilized. The first surface primer has at least one nucleotide with a cleavable moiety that can be cleaved to create a non-basic site. The second surface primer lacks nucleotides with cleavable moieties and has 3'OH groups at the extensible ends.

In some embodiments, cleavable moieties in the immobilized first surface primer of step (a) can be converted to non-basic moieties. In some embodiments, the cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the immobilized first surface primer, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG). In the immobilized first surface primer, 8oxoG can be converted to a non-basic site using FPG glycosylase. In the immobilized first surface primer, deoxyinosine can be converted to a non-basic moiety using AlkA glycosylase. The immobilized first surface primer contains at least 1 and up to 5 nucleotides with a cleavable moiety.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized first surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized first surface primer comprises a 3' non-extensible moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the immobilized concatimer template molecule further comprises two or more copies of a universal binding sequence (or a complementary sequence thereof) for an immobilized second surface primer having a different sequence than the first immobilized surface primer. do. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. In some embodiments, the end of the 3' end of the immobilized second surface primer is a moiety that blocks primer extension (e.g., the 3' end of the non-extendable end), e.g., a phosphate group, a diade group, etc. Contains an oxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc., on the support. In communication, a plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments, a pairwise sequencing method involves hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers, a plurality of strand transfer polymerases, and a plurality of strand transfer polymerases, and a plurality of strands comprising dATP, dCTP, dGTP, dTTP. A plurality of immobilized single-stranded nucleic acid concatimer template molecules are generated by performing a rolling ring amplification reaction with a plurality of nucleotides having a nucleotide and a cleavable moiety that can be cleaved to create a non-basic site, which can be cleaved. generating a plurality of immobilized single-stranded nucleic acid conkatimer template molecules having at least one nucleotide having a moiety, wherein each single stranded nucleic acid conkatimer template molecule is covalently linked to an immobilized first surface primer. (b) further includes. The rotation amplification reaction may be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. You can. In some embodiments, the rolling ring amplification reaction can be performed with or without a plurality of compressed oligonucleotides. In some embodiments, the support may include an excess of immobilized first and second surface primers relative to the number of immobilized concatimer template molecules.

In some embodiments, single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules. In some embodiments, single-stranded circular nucleic acid library molecules can be removed from the concatimer template molecule using at least one washing step performed under conditions suitable for maintaining the single-stranded nucleic acid concatimer template molecule, wherein individual concatimer template molecules are The catimer template molecule is operably linked to the immobilized first surface primer.

In some embodiments, each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each immobilized concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer (or its complement); sequence), (ii) a universal binding sequence (or a complementary sequence thereof) to a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer, (iv) an immobilized a universal binding sequence (or a complementary sequence thereof) to a second surface primer, (v) a universal binding sequence (or a complementary sequence thereof) to a first soluble amplification primer, (vi) a universal binding sequence to a second soluble amplification primer. a binding sequence (or a complementary sequence thereof), (vii) a universal binding sequence for soluble compressed oligonucleotides (or a complementary sequence thereof), (viii) a sample barcode sequence and/or (ix) a unique molecular indicator sequence, or It further includes any combination of two or more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

In some embodiments, step (b) the rolling ring amplification reaction comprises a plurality of fixed single-stranded nucleic acid cones each comprising a concatemer having at least one nucleotide with a cleavable moiety and two or more copies of the sequence of interest. A concatimer template molecule is generated, and the immobilized concatimer template molecule contains (i) two or more copies of the universal binding sequence (or its complementary sequence) to the soluble forward sequencing primer, (ii) the soluble reverse sequencing primer. two or more copies of the universal binding sequence (or complementary sequence thereof) to the primer, (iii) two or more copies of the universal binding sequence (or complementary sequence thereof) to the immobilized first surface primer, (iv) ) two or more copies of the universal binding sequence (or complementary sequence thereof) to the immobilized second surface primer, (v) two copies of the universal binding sequence (or complementary sequence thereof) to the first soluble amplification primer (vi) two or more copies of the universal binding sequence (or complementary sequence thereof) to the second soluble amplification primer, (vii) the universal binding sequence (or complementary sequence thereof) to the soluble compressed oligonucleotide. (viii) two or more copies of a sample barcode sequence, and/or (ix) two or more copies of a unique molecular indicator sequence, or any combination of two or more.

The rolling ring amplification reaction of step (b) involves a polymerase with strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

The rolling ring amplification reaction of step (b) is carried out with a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety to obtain a fixed nucleotide comprising at least one nucleotide having a cleavable moiety. A concatimer template molecule can be generated. Moieties that can be cleaved from the immobilized concatimer template molecule can be converted to non-basic sites. In some embodiments, in the nucleotide mixture, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the immobilized concatimer template molecule, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG), and 8oxoG can be converted to a non-basic site using FPG glycosylase. Deoxyinosine can be converted to a non-basic site using AlkA glycosylase.

In some embodiments, the nucleotide mixture includes an amount of dUTP such that a target percentage of thymidine is replaced by dUTP in the resulting concatimer molecules. For example, when 30% of the dTTP in a conkatimer molecule is replaced with dUTP (e.g., 30% is the target percentage), then the nucleotide mixture will have 7.5% dUTP (e.g., 30/4 = 7.5 %), 17.5% dTTP, and 25% each for dATP, dCTP, and dGTP. The target percentage of dTTP replaced by dUTP is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%, or about 45 to 50%, or a higher percentage of dTTP in the immobilized concatimer template molecule is replaced with a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of deoxyinosine such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by deoxyinosine. For example, when 30% of the dGTP in a conkatimer molecule is replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture has 7.5% deoxyinosine each for dATP, dCTP, and dTTP. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by deoxyinosine is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or about 45 to 50%, or a higher percentage of dGTP in the immobilized concatimer template molecule is replaced by a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of 8oxoG such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by 8oxoG. For example, when 30% of the dGTP in a conkatimer molecule is replaced by 8oxoG (e.g., 30% is the target percentage), the nucleotide mixture contains 7.5% 8oxoG for dATP, dCTP, and dTTP each. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by 8-oxoG is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or may be about 45 to 50%, or a higher percentage of the dGTP in the immobilized concatimer template molecule is replaced by a nucleotide having a cleavable moiety.

In some embodiments, the rolling ring amplification reaction generates fixed concatimer template molecules using incorporated nucleotides with cleavable moieties distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules.

In some embodiments, immobilized concatimer template molecules can self-disintegrate into compact nucleic acid nanoballs. Nanoballs can be imaged and FWHM measurements can be obtained to provide the shape/size of the nanoballs.

In some embodiments, the pairwise sequencing method comprises sequencing a plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers to generate a plurality of extended forward sequencing primer strands, The method further includes a step (c) of generating primer strands, wherein two or more extended forward sequencing primer strands are hybridized to the immobilized concatimer template molecule.

Sequencing in step (c) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing the plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule can undergo more than one sequencing reaction, with each sequencing reaction initiated from a forward sequencing primer hybridized to the forward sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below.

In some embodiments, the sequencing of step (c) can be performed with or without a plurality of compressed oligonucleotides. Compact oligonucleotides can maintain the compact size and/or shape of nanoballs (e.g., self-collapsed immobilized concatimer template molecules) during the forward sequencing reaction.

In some embodiments, the sequencing of step (c) may be performed by contacting a plurality of immobilized concatimer template molecules with a plurality of forward sequencing primers in the presence of a hybridization solution comprising a pH buffer, a sodium salt, and a disorder inducing agent. You can. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, the sequencing of step (c) may be performed by contacting a plurality of immobilized concatimer template molecules with a plurality of forward sequencing primers in the presence of a high-throughput hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprises step (d) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (d), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (d), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments , the pairwise sequencing method comprises hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and extracting from the second surface primer hybridized to a portion of the immobilized Concatimer template molecule. It further includes step (e) of generating a first plurality of fixed forward extension strands by performing a primer extension reaction of. The primer extension reaction generates a plurality of forward extension strands each having a sequence complementary to at least a portion of the immobilized concatimer template molecule. The primer extension reaction generates a plurality of forward extension strands covalently linked to an immobilized second surface primer.

In some embodiments, the primer extension reaction of step (e) includes a plurality of nucleotides lacking nucleotides having a cleavable moiety. For example, the plurality of nucleotides includes dATP, dGTP, dCTP, and dTTP. The primer extension reaction can be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. there is.

The primer extension reaction of step (e) involves a DNA polymerase capable of catalyzing the primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The primer extension reaction of step (e) involves a polymerase with strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

The immobilized first and second surface primers, immobilized concatimer template molecules, and immobilized forward extension strands are in fluid communication with each other to allow various solutions, such as buffers and reagents, to flow on the support. The immobilized first and second surface primers, the immobilized concatimer template molecule, and the immobilized forward extension strand can react with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (f) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution. The relaxing solution can flow onto the support to enable reaction with the immobilized concatimer template molecule and the immobilized forward extension strand in a massively parallel manner. The relaxing solution may flow onto the support at a temperature of about 20 to 25° C.

The relaxing solution includes at least one nucleic acid relaxant capable of breaking hydrogen bonds between the immobilized concatimer template molecule and the second surface primer. Exemplary relaxants include nucleic acid denaturants, chaotropic compounds, amide compounds, aprotic compounds, primary alcohols, and ethylene glycol derivatives. Chaotropic compounds include urea, guanidine hydrochloride or guanidine thiocyanate. Amide compounds include formamide, acetamide, or NN-dimethylformamide (DMF). Aprotic compounds include acetonitrile, dimethyl sulfoxide (DMSO), 1,4-dioxane, or tetrahydrofuran. Primary alcohols include 1-propanol, ethanol, or methanol. Ethylene glycol derivatives include 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane or 2-methoxyethanol. Other relaxants include sodium iodide, potassium iodide, and polyamines.

The relaxing solution of step (f) may further include an ionic, non-ionic or amphoteric ionic detergent. Exemplary ionic cleaners include SDS (sodium dodecyl sulfate). Exemplary nonionic detergents include Triton X-100, Tween 20, Tween 80 or Nonidet P-40. Exemplary zwitterionic detergents include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio. -Contains 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate.

The relaxing solution of step (f) further comprises a pH buffering compound (e.g., a zwitterionic buffering compound such as or 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES)). can do.

In some embodiments, the relaxing solution of step (f) includes urea, guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide, NN-dimethylformamide (DMF), acetonitrile, dimethyl sulfoxide (DMSO). side), 1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, 2-methoxyethanol, i. It includes any one or a combination of two or more of the group selected from sodium odide, potassium iodide and/or polyamines.

In some embodiments, the relaxing solution of step (f) includes formamide and SSC. In some embodiments, the relaxing solution includes acetonitrile, formamide, and SSC. In some embodiments, the relaxing solution includes acetonitrile, formamide, and MES (2-(4-morpholino)-ethane sulfonic acid). In some embodiments, the relaxing solution includes acetonitrile, formamide, guanidine hydrochloride, and HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid) and a detergent (e.g., an amphoteric ionic detergent). , such as Tween-20 or Tween-80). In some embodiments, the relaxing solution includes acetonitrile, formamide, urea, and HEPES. In some embodiments, the concentration of SSC in the relaxing solution may be 1×, 2×, 3×, or 4×.

In some embodiments, the pairwise sequencing method dissociates at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retains the immobilized forward extension strand, and (g) rehybridizing at least one portion of to one of the immobilized second surface primers that is not covalently linked to the forward extension strand. In some embodiments, nucleic acid dissociation and rehybridization is performed in the presence of a relaxing solution and includes increasing temperature, temperature plateauing, and decreasing temperature (e.g., Figure 117). The temperature increase may begin at about 20 to 25° C. and increases to about 55 to 70° C. The temperature plateau may be maintained at about 50 to 70° C. The temperature decrease may begin at about 50 to 70° C. and then decrease to about 20 to 25° C. The relaxing solution can be removed from the support by performing at least one wash with a washing solution. The wash solution may include SSC (e.g., at any concentration from about 1 to 5x) and a detergent (e.g., Tween-20). Those skilled in the art will recognize that the temperature increase, temperature plateau, and temperature decrease conditions may vary.

In some embodiments, the forward extension strand duplexed by an immobilized concatimer template molecule (e.g., generated in step (e)) is denatured, in the presence of a relaxing solution, an increasing temperature, a temperature stabilizer, and a decreasing temperature. It can be rehybridized with the first surface primer under.

In some embodiments, a pairwise sequencing method involves contacting a rehybridized immobilized Conkatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that rehybridizes to a portion of the immobilized Conkatimer template molecule. and performing a step (h) to generate a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer. The amplification of step (h) is performed after increasing temperature, temperature plateau, decreasing temperature, and washing in step (g) described above.

In some embodiments, the forward extension strand rehybridized with the first surface primer (e.g., generated in step (g)) is contacted with an amplification solution and subjected to a primer extension reaction to produce a plurality of immobilized first surface primers. When comprising a 3' extendable end, multiple newly synthesized concatimer template molecules can be generated. Alternatively, when the immobilized first surface primer includes a non-3'extensible end, the amplification reaction will not produce a newly synthesized concatimer template molecule.

In some embodiments, the amplification solution of step (h) comprises a plurality of nucleotides lacking nucleotides having a cleavable moiety. For example, the plurality of nucleotides includes dATP, dGTP, dCTP, and dTTP. The amplification reaction can be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. .

The amplification solution of step (h) includes a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The amplification solution of step (h) contains a polymerase having strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments , the pairwise sequencing method further comprises step (i) performing a plexing amplification cycle by repeating steps (f) through (h) at least once. Steps (f) to (h) may be repeated 1, 2, 3, 4, 5, 6 or up to 10 times. Each cycle may produce an additional newly synthesized forward extension strand covalently linked to a second surface primer. Each cycle can produce an additional newly synthesized concatimer template molecule covalently linked to the first surface primer. After performing the desired number of plexing amplification cycles, step (j) can be performed as described directly below.

In some embodiments, the pairwise sequencing method comprises: A first surface primer anchored with a single stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining a plurality of anchored forward extension strands and a plurality of anchored second surface primers. and removing the immobilized concatimer template molecule by creating a non-basic site of and creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules. The gap-containing nucleic acid molecule comprises an immobilized concatimer template strand and an immobilized first surface primer.

The non-basic site is created on the concatimer template strand containing nucleotides with a cleavable moiety and on the immobilized first surface primer. In some embodiments, moieties that can cleave from the conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create a first surface primer with a plurality of concatimer template molecules and a gap while maintaining the plurality of forward extending strands. Abasic sites can be created by contacting an immobilized concatimer template molecule and a first surface primer with an enzyme that removes a nucleobase from a nucleotide having a cleavable moiety. Uracil in the concatimer template strand and first surface primer can be converted to a non-basic site using uracil DNA glycosylase (UDG). 8oxoG in the concatimer template strand and first surface primer can be converted to a non-basic site using FPG glycosylase. Deoxyinosine in the concatimer template strand and first surface primer can be converted to a non-basic site using AlkA glycosylase.

In some embodiments, in step (j), the gap is formed by an enzyme having lyase activity that releases the abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a non-basic site with a mixture of enzymes. Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (j), the plurality of gap-containing template molecules can be removed using enzymes, chemicals, and/or heat. After the gap-removal procedure, a plurality of anchored forward extension strands remain covalently linked to the second surface primer.

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by .

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH between about 6.5 and 8.5. It can be removed by washing.

In some embodiments , the pairwise sequencing method further comprises a step (k) of sequencing the plurality of retained forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands. Includes. In some embodiments, the sequencing of step (k) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, whereby the forward sequencing reaction produces a plurality of extended reverse sequencing primer strands.

Each maintained forward extension strand may contain two or more copies of the reverse sequencing primer strand hybridized thereto. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands that hybridize to the same maintained forward extended strand.

In some embodiments, the sequencing of step (k) can be performed with or without a plurality of compressed oligonucleotides. Compact oligonucleotides can maintain the compact size and/or shape of nanoballs (e.g., self-collapsed immobilized concatimer template molecules) during a reverse sequencing reaction.

In some embodiments, in step (k), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand include the plurality of soluble reverse sequencing primers and the maintained forward extension strand at a pH and contacting with a hybridization solution comprising a buffer, a sodium salt, and a disorder-inducing agent. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, in step (k), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reaction of step (k) comprises a plurality of soluble reverse sequencing primers, a reverse sequencing primer binding sequence of the retained forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides or It includes contacting a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, each maintained forward extension strand can undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below.

In some embodiments, at least one washing step may be performed after any of steps (a) through (k). The washing step can be performed with a washing solution containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash solution is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash solution at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the cleaning solution is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the washing solution includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash solution includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The washing solution may include the monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the cleaning agent in the cleaning solution includes a non-ionic cleaning agent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the cleaning solution at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

The wash solution may include SSC (e.g., at any concentration from about 1 to 5x) and a detergent (e.g., Tween-20).

RCA and pairwise sequencing in solution - amplification by plexing amplification cycling

The present disclosure provides a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable to form a plurality of library-primer duplexes and suitable for performing a rotational amplification reaction, a plurality of soluble first amplification primers, a plurality of strand translocations. A concatenation of a plurality of single-stranded nucleic acids having at least one nucleotide having a cleavable moiety by contacting a polymerase and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety. A pairwise sequencing method is provided, including step (a) of generating a mer. The rotation amplification reaction in solution is carried out at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. It can be done.

In some embodiments, the soluble first amplification primer comprises a sequence that selectively hybridizes to a universal binding sequence in a circular nucleic acid library molecule, e.g., a universal binding sequence for the first soluble amplification primer (or a complementary sequence thereof). do. Alternatively, the soluble first amplification primer comprises a random sequence that non-selectively binds to the circular nucleic acid library molecule.

In some embodiments, each individual single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises (i) a universal binding sequence to a soluble forward sequencing primer (or a complementary sequence thereof), (ii) ) a universal binding sequence (or their complementary sequence) for a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) for an immobilized first surface primer, (iv) an immobilized second surface primer. a universal binding sequence (or complementary sequence thereof) to a surface primer, (v) a universal binding sequence (or complementary sequence thereof) to a first soluble amplification primer, (vi) a universal binding sequence to a second soluble amplification primer. sequence (or its complementary sequence), (vii) a universal binding sequence for soluble compressed oligonucleotides (or its complementary sequence), (viii) a sample barcode sequence, and/or (ix) a unique molecular indicator sequence. Or it further includes any combination of two or more. In some embodiments, single-stranded circular nucleic acid library molecules comprise covalently closed circular molecules.

In some embodiments, the rolling ring amplification reaction of step (a) produces a plurality of single-stranded nucleic acid concatimer molecules in solution comprising a concatimer having at least one nucleotide having a cleavable moiety. In some embodiments, each individual Concatimer template molecule of the plurality comprises two or more copies of the sequence of interest, and each immobilized Concatimer template molecule comprises (i) a universal binding sequence for a soluble forward sequencing primer; at least two copies, (ii) at least two copies of the universal binding sequence for the soluble reverse sequencing primer, (iii) at least two copies of the universal binding sequence for the immobilized first surface primer, (iv) at least two copies of the immobilized agent. 2 at least two copies of the universal binding sequence for the surface primer, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer. any of the following: duplicates, (vii) two or more copies of the universal binding sequence for soluble compressed oligonucleotides, (viii) two or more copies of the sample barcode sequence, and/or (ix) two or more copies of the unique molecular indicator sequence. Or it further includes any combination of two or more.

In some embodiments, a universal binding sequence for a forward sequencing primer (or its complementary sequence) can hybridize to at least a portion of the forward sequencing primer. In some embodiments, a universal binding sequence for a reverse sequencing primer (or its complementary sequence) can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, a universal binding sequence (or a complementary sequence thereof) to an immobilized first surface primer can hybridize to at least a portion of the immobilized first surface primer. In some embodiments, the universal binding sequence for the immobilized second surface primer (or its complementary sequence) can hybridize to at least a portion of the immobilized second surface primer. In some embodiments, the universal binding sequence for the first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for the second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compressed oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compressed oligonucleotide.

The rolling ring amplification reaction of step (a) involves a polymerase with strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

The rotation ring amplification reaction in solution of step (a) is carried out by a nucleotide mixture containing dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety, such that it contains at least one nucleotide having a cleavable moiety. Concatimer molecules can be created. Moieties that can be cleaved from the concatimer molecule can be converted to non-basic sites. In some embodiments, in the nucleotide mixture, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. In the concatimer molecule, uridine can be converted to a non-basic site using uracil DNA glycosylase (UDG), and 8oxoG can be converted to a non-basic site using FPG glycosylase. Deoxyinosine can be converted to a basic moiety using AlkA glycosylase.

In some embodiments, the nucleotide mixture includes an amount of dUTP such that a target percentage of thymidine is replaced by dUTP in the resulting concatimer molecules. For example, when 30% of the dTTP in a conkatimer molecule is replaced with dUTP (e.g., 30% is the target percentage), then the nucleotide mixture will have 7.5% dUTP (e.g., 30/4 = 7.5 %), 17.5% dTTP, and 25% each for dATP, dCTP, and dGTP. The target percentage of dTTP replaced by dUTP is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%, or about 45 to 50%, or a higher percentage of dTTP in the concatimer molecule is replaced with a nucleotide having a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of deoxyinosine such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by deoxyinosine. For example, when 30% of the dGTP in a conkatimer molecule is replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture has 7.5% deoxyinosine each for dATP, dCTP, and dTTP. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by deoxyinosine is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or may be about 45 to 50%, or a higher percentage of dGTP in the concatimer molecule is replaced by a nucleotide with a cleavable moiety.

In some embodiments, the nucleotide mixture may include an amount of 8oxoG such that a target percentage of guanosine in the resulting conkatimer molecule is replaced by 8oxoG. For example, when 30% of the dGTP in a conkatimer molecule is replaced by 8oxoG (e.g., 30% is the target percentage), the nucleotide mixture contains 7.5% 8oxoG for dATP, dCTP, and dTTP each. (e.g., 30/4 = 7.5%), may contain 17.5% dGTP and 25%. The target percentage of dGTP replaced by 8-oxoG is about 0.1 to 1%, or about 1 to 5%, or about 5 to 10%, or about 10 to 20%, or about 20 to 30%, or about 30 to 45%. %, or may be about 45 to 50%, or a higher percentage of dGTP in the concatimer molecule is replaced by a nucleotide with a cleavable moiety.

In some embodiments, a rolling ring amplification reaction in solution generates concatimer molecules with incorporated nucleotides having cleavable moieties distributed at random positions along individual fixed concatimer template molecules. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different concatimer molecules.

In some embodiments, pairwise sequencing methods include :

From step (a) on a support to which a plurality of first and second surface primers are immobilized, under conditions suitable for hybridizing one or more portions of the individual single-stranded concatemers to the one or more immobilized first surface primers. It further includes step (b) of distributing the rotation ring amplification reaction. In some embodiments, the immobilized first surface primer includes at least one nucleotide with a cleavable moiety. In some embodiments, the immobilized second surface primer lacks nucleotides having a cleavable moiety and has an extendable 3'OH group.

In some embodiments, the distribution of step (b) can be performed in the presence of a hybridization solution comprising a pH buffer, a sodium salt, and a disorder-inducing agent. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, the distribution of step (b) can be performed in the presence of a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprises (c) continuing the rolling ring amplification reaction on the support to generate a plurality of extended concatimer template molecules immobilized through hybridization to the immobilized first surface primer. do. The RCA reaction on a support involves a plurality of strand transfer polymerases, and dATP, dCTP, dGTP, dTTP, under conditions suitable to generate a plurality of extended concatemers with at least one nucleotide having a cleavable moiety. It can be performed using a plurality of nucleotides and a nucleotide having a cleavable moiety. In some embodiments, the rolling ring amplification reaction on a support can be performed with or without a plurality of compressed oligonucleotides. The rotary ring amplification reaction on the support is carried out at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. It can be done.

The rolling ring amplification reaction of step (c) continues on the support using a polymerase with strand translocation activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, a rolling ring amplification reaction on a support generates immobilized concatimer template molecules using incorporated nucleotides with cleavable moieties distributed at random positions along the individual immobilized concatimer template molecules. do. In some embodiments, nucleotides bearing cleavable moieties are distributed at different positions in different immobilized concatimer template molecules.

In some embodiments, the support may include an excess of immobilized first and second surface primers relative to the number of immobilized concatimer template molecules.

In some embodiments, immobilized concatimer template molecules can self-disintegrate into compact nucleic acid nanoballs. Nanoballs can be imaged and FWHM measurements can be obtained to provide the shape/size of the nanoballs.

In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed first surface primer is fixed to the support or to a coating on the support. The immobilized first surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored first surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored first surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized first surface primers having the same sequence. The immobilized first surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized first surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized first surface primer comprises a 3' non-extensible moiety.

In some embodiments, the plurality of immobilized first surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the first surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized first surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized first surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A first surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the immobilized first surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge bond between the 2' oxygen and 4' carbon of the pentose ring. An immobilized first surface primer comprising at least one LNA may be resistant to nuclease degradation and may exhibit an increased melting temperature when hybridized to the forward extension strand.

In some embodiments, the immobilized concatimer template molecule further comprises two or more copies of a universal binding sequence (or a complementary sequence thereof) for an immobilized second surface primer having a different sequence than the first immobilized surface primer. do. The immobilized second surface primer of step (a) comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed second surface primer is fixed to the support or to a coating on the support. The immobilized second surface primer can be embedded and attached (bonded) to the coating on the support. In some embodiments, the 5' end of the anchored second surface primer is anchored to the support or to a coating on the support. Alternatively, the interior portion or 3' end of the anchored second surface primer may be anchored to the support or to a coating on the support. The support includes a plurality of immobilized second surface primers having the same sequence. The immobilized second surface primer may be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

In some embodiments, the 3' end of the immobilized second surface primer comprises an extendable 3' OH moiety. In some embodiments, the 3' end of the immobilized second surface primer comprises a 3' non-extensible moiety. In some embodiments, the end of the 3' end of the immobilized second surface primer is a moiety that blocks primer extension (e.g., the 3' end of the non-extendable end), e.g., a phosphate group, a diade group, etc. Contains an oxycytidine group, inverted dT or amino group. The immobilized second surface primer is not extendable in the primer extension reaction. The immobilized second surface primer lacks nucleotides having a cleavable moiety.

In some embodiments, the plurality of immobilized second surface primers comprise at least one phosphorothioate diester bond at the 5' end, which can provide the second surface primers resistant to exonuclease digestion. . In some embodiments, the plurality of immobilized second surface primers comprise at least 2 to 5 consecutive phosphorothioate diester linkages at their 5' ends. In some embodiments, the plurality of immobilized second surface primers comprise at least one ribonucleotide and/or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which is an exo A second surface primer that is resistant to nuclease degradation can be provided.

In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized second surface primers per mm 2 . In some embodiments, the support comprises about 10 ² to 10 ¹⁵ immobilized first surface primers and immobilized second surface primers per mm2.

The immobilized surface primers (first and second surface primers) are fluidized together to enable the flow of various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. on the support. In communication, a plurality of immobilized surface primers (and primer extension products resulting from the immobilized surface primers) can be reacted with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method comprises sequencing a plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers to generate a plurality of extended forward sequencing primer strands, It further comprises a step (d) of generating primer strands, wherein two or more extended forward sequencing primer strands are hybridized to the immobilized concatimer template molecule.

Sequencing in step (d) involves hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites/sequences of the immobilized concatimer template molecule under conditions suitable for hybridizing a plurality of immobilized concatimer templates. contacting the molecule with a plurality of soluble forward sequencing primers, and conducting a forward sequencing reaction using a sequencing polymerase, one or more types of a plurality of nucleotides and/or multivalent molecules, and a hybridized first forward sequencing primer. Includes carrying out In some embodiments, the soluble forward sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide having a cleavable moiety. The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, an individual immobilized concatimer template molecule has multiple copies of a forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Individual forward sequencing primer binding sites on a given immobilized conkatimer template molecule can hybridize to the forward sequencing primer and undergo a sequencing reaction. An individual immobilized concatimer template molecule can undergo more than one sequencing reaction, where each sequencing reaction is initiated from a forward sequencing primer that hybridizes to the forward sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having a plurality of nucleotide units attached to a core, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below.

In some embodiments, the sequencing of step (d) can be performed with or without a plurality of compressed oligonucleotides. Compact oligonucleotides can maintain the compact size and/or shape of nanoballs (e.g., self-collapsed immobilized concatimer template molecules) during the forward sequencing reaction.

In some embodiments, the sequencing of step (d) may be performed by contacting a plurality of immobilized concatimer template molecules with a plurality of forward sequencing primers in the presence of a hybridization solution comprising a pH buffer, a sodium salt, and a disorder inducing agent. You can. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, the sequencing of step (d) may be performed by contacting a plurality of immobilized concatimer template molecules with a plurality of forward sequencing primers in the presence of a high-throughput hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the pairwise sequencing method further comprises step (e) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands may be removed using enzymes or chemical reagents. For example, multiple extended forward sequencing primer strands utilize 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, Catalog #M0263S). Therefore, it can be decomposed by enzymes. In some embodiments, multiple extended forward sequencing primer strands can be removed by temperatures that aid in nucleic acid denaturation.

In some embodiments, in step (e), a denaturing reagent may be used to remove a plurality of extended forward sequencing primer strands, wherein the denaturing reagent is a compound such as formamide, acetonitrile, guanidinium chloride, and /or any one or any combination of pH buffering agents (e.g., Tris-HCl, MES, HEPES, MOPS, etc.). Optionally, the denaturing reagent may further include PEG.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands may be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands have a temperature range of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C. It can be treated at a temperature of, or at a higher temperature.

In some embodiments, in step (e), the plurality of extended forward sequencing primer strands are incubated with 100% formamide at a temperature of about 65°C for about 3 minutes and with about 50mM NaCl or equivalent ionic strength; It can be removed by washing with a reagent with a pH of about 6.5 to 8.5.

In some embodiments , the pairwise sequencing method comprises hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and obtaining a hybridization sequence from the second surface primer that hybridizes to a portion of the immobilized Concatimer template molecule. It further includes step (e) of generating a first plurality of fixed forward extension strands by performing a primer extension reaction of. The primer extension reaction generates a plurality of forward extension strands each having a sequence complementary to at least a portion of the immobilized concatimer template molecule. The primer extension reaction generates a plurality of forward extension strands covalently linked to an immobilized second surface primer.

In some embodiments, the primer extension reaction of step (f) includes a plurality of nucleotides lacking nucleotides having a cleavable moiety. For example, the plurality of nucleotides includes dATP, dGTP, dCTP, and dTTP. The primer extension reaction can be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. there is.

The primer extension reaction of step (f) involves a DNA polymerase capable of catalyzing the primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Including, but not limited to, Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The primer extension reaction of step (f) involves a polymerase with strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

The immobilized first and second surface primers, immobilized concatimer template molecules, and immobilized forward extension strands are in fluid communication with each other to allow various solutions, such as buffers and reagents, to flow on the support. The immobilized first and second surface primers, the immobilized concatimer template molecule, and the immobilized forward extension strand can react with the solution in a massively parallel manner.

In some embodiments, the pairwise sequencing method further comprises step (g) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution. The relaxing solution can flow onto the support to enable reaction with the immobilized concatimer template molecule and the immobilized forward extension strand in a massively parallel manner. The relaxing solution may flow onto the support at a temperature of about 20 to 25° C.

The relaxing solution includes at least one nucleic acid relaxant capable of breaking hydrogen bonds between the immobilized concatimer template molecule and the second surface primer. Exemplary relaxants include nucleic acid denaturants, chaotropic compounds, amide compounds, aprotic compounds, primary alcohols, and ethylene glycol derivatives. Chaotropic compounds include urea, guanidine hydrochloride or guanidine thiocyanate. Amide compounds include formamide, acetamide, or NN-dimethylformamide (DMF). Aprotic compounds include acetonitrile, dimethyl sulfoxide (DMSO), 1,4-dioxane, or tetrahydrofuran. Primary alcohols include 1-propanol, ethanol, or methanol. Ethylene glycol derivatives include 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane or 2-methoxyethanol. Other relaxants include sodium iodide, potassium iodide, and polyamines.

The relaxing solution of step (g) may further include an ionic, non-ionic or amphoteric ionic detergent. Exemplary ionic cleaners include SDS (sodium dodecyl sulfate). Exemplary nonionic detergents include Triton X-100, Tween 20, Tween 80 or Nonidet P-40. Exemplary zwitterionic detergents include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio. -Contains 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate.

The relaxing solution of step (g) further comprises a pH buffering compound (e.g., a zwitterionic buffering compound such as or 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid (HEPES)). can do.

In some embodiments, the relaxing solution of step (g) includes urea, guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide, NN-dimethylformamide (DMF), acetonitrile, dimethyl sulfoxide (DMSO). side), 1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, 2-methoxyethanol, i. It includes any one or a combination of two or more of the group selected from sodium odide, potassium iodide and/or polyamines.

In some embodiments, the relaxing solution of step (g) includes formamide and SSC. In some embodiments, the relaxing solution includes acetonitrile, formamide, and SSC. In some embodiments, the relaxing solution includes acetonitrile, formamide, and MES (2-(4-morpholino)-ethane sulfonic acid). In some embodiments, the relaxing solution includes acetonitrile, formamide, guanidine hydrochloride, and HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid) and a detergent (e.g., an amphoteric ionic detergent). , such as Tween-20 or Tween-80). In some embodiments, the relaxing solution includes acetonitrile, formamide, urea, and HEPES. In some embodiments, the concentration of SSC in the relaxing solution may be 1×, 2×, 3×, or 4×.

In some embodiments, the pairwise sequencing method dissociates at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retains the immobilized forward extension strand, and (h) rehybridizing at least one portion of to one of the immobilized second surface primers that is not covalently linked to the forward extension strand. In some embodiments, nucleic acid dissociation and rehybridization is performed in the presence of a relaxing solution and includes increasing temperature, temperature plateauing, and decreasing temperature (e.g., Figure 117). The temperature increase may begin at about 20 to 25°C and increase to about 55 to 70°C. The temperature plateau may be maintained at about 50 to 70° C. The temperature decrease may begin at about 50 to 70° C. and then decrease to about 20 to 25° C. The relaxing solution can be removed from the support by performing at least one wash with a washing solution. The wash solution may include SSC (e.g., at any concentration from about 1 to 5x) and a detergent (e.g., Tween-20). Those skilled in the art will recognize that the temperature increase, temperature plateau, and temperature decrease conditions may vary.

In some embodiments, the forward extension strand duplexed by an immobilized concatimer template molecule (e.g., generated in step (f)) is denatured, in the presence of a relaxing solution, an increasing temperature, a temperature stabilizer, and a decreasing temperature. It can be rehybridized with the first surface primer under.

In some embodiments, a pairwise sequencing method involves contacting a rehybridized immobilized Conkatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that rehybridizes to a portion of the immobilized Conkatimer template molecule. It further comprises step (i) , performing a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer. Amplification in step (i) is performed after increasing temperature, temperature plateau, decreasing temperature, and washing in step (h) described above.

In some embodiments, the forward extension strand rehybridized with the first surface primer (e.g., generated in step (h)) is contacted with an amplification solution and subjected to a primer extension reaction to produce a plurality of immobilized first surface primers. When comprising a 3' extendable end, multiple newly synthesized concatimer template molecules can be generated. Alternatively, when the immobilized first surface primer contains a non-3'extensible end, the amplification reaction will not produce a newly synthesized concatimer template molecule.

In some embodiments, the amplification solution of step (i) comprises a plurality of nucleotides lacking nucleotides having a cleavable moiety. For example, the plurality of nucleotides includes dATP, dGTP, dCTP, and dTTP. The amplification reaction can be performed at an isothermal temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C. .

The amplification solution of step (i) includes a DNA polymerase capable of catalyzing a primer extension reaction using a uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include Q5U Hot Start high-fidelity DNA polymerase (e.g., catalog #M0515S from New England Biolabs), Taq DNA polymerase, One Taq DNA polymerase (e.g., Taq and Deep Vent DNA polymerase) A mixture of enzymes, Catalog #M0480S from New England Biolabs), LongAmp Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs), Epimark Hot Start Taq DNA polymerase (e.g., Catalog #M0323S from New England Biolabs) Catalog #M0490S), Bst DNA polymerase (e.g., large fragment, catalog #M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog #M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., Catalog #M0269S from New England Biolabs), E. coli DNA polymerase (e.g., Catalog #M0209S from New England Biolabs), Therminator DNA polymerase (e.g., New England Biolabs) Catalog #M0261S), Vent DNA Polymerase, and Deep Vent DNA Polymerase.

The amplification solution of step (i) contains a polymerase having strand transposition activity. Examples of strand transposition polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), the large fragment of Bca DNA polymerase (exo-), and the large fragment of E. coli DNA polymerase. knob fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments , the pairwise sequencing method further comprises step (j) performing a plexing amplification cycle by repeating steps (g) through (i) at least once. Steps (g) to (i) may be repeated 1, 2, 3, 4, 5, 6 or up to 10 times. Each cycle may produce an additional newly synthesized forward extension strand covalently linked to a second surface primer. Each cycle can produce an additional newly synthesized concatimer template molecule covalently linked to the first surface primer. After performing the desired number of plexing amplification cycles, step (k) can be performed as described directly below.

In some embodiments, the pairwise sequencing method comprises: A first surface primer anchored with a single stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining a plurality of anchored forward extension strands and a plurality of anchored second surface primers. and removing the immobilized concatimer template molecule by creating a non-basic site of and creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules. The gap-containing nucleic acid molecule comprises an immobilized concatimer template strand and an immobilized first surface primer.

The non-basic site is created on the concatimer template strand containing nucleotides with a cleavable moiety and on the immobilized first surface primer. In some embodiments, moieties that can cleave from the conkatimer template molecule include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Non-basic sites can be removed to create a first surface primer with a plurality of concatimer template molecules and a gap while maintaining the plurality of forward extending strands. Abasic sites can be created by contacting an immobilized concatimer template molecule and a first surface primer with an enzyme that removes a nucleobase from a nucleotide having a cleavable moiety. Uracil in the concatimer template strand and first surface primer can be converted to a non-basic site using uracil DNA glycosylase (UDG). 8oxoG in the concatimer template strand and first surface primer can be converted to a non-basic site using FPG glycosylase. Deoxyinosine in the concatimer template strand and first surface primer can be converted to a non-basic site using AlkA glycosylase.

In some embodiments, in step (k), the gap is formed by an enzyme having lyase activity that releases abasic deoxyribose and breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site creating the gap, or It can be produced by contacting a non-basic site with a mixture of enzymes. Non-basic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase/AP lyase, and Endo VIII glycosylase/AP lyase. In some embodiments, the creation of abasic sites and removal of abasic sites that create gaps can be performed using uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as USER (from New England Biolabs). This can be accomplished using a mixture of Uracil-Specific Excision Reagent Enzyme) or the thermolabile USER (also from New England Biolabs).

In some embodiments, in step (k), the plurality of gap-containing template molecules can be removed using enzymes, chemicals, and/or heat. After the gap-removal procedure, a plurality of anchored forward extension strands are maintained covalently linked to the second surface primer.

For example, a plurality of gap-containing template molecules can be enzymatically digested using 5' to 3' double-stranded DNA exonucleases, including T7 exonuclease (e.g., New England Biolabs, catalog #M0263S). It can be decomposed by .

In some embodiments, multiple gap-containing template molecules can be removed using chemical reagents that favor nucleic acid denaturation. Denaturing reagents may include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and/or buffering agents (e.g., Tris-HCl, MES, HEPES, etc.).

In some embodiments, the plurality of gap-containing template molecules can be removed using elevated temperature (e.g., heating) with or without a nucleic acid denaturing reagent. The gap-containing template molecule is heated to a temperature of about 45 to 50°C, or about 50 to 60°C, or about 60 to 70°C, or about 70 to 80°C, or about 80 to 90°C, or about 90 to 95°C, or Can be processed at higher temperatures.

In some embodiments, the plurality of gap-containing template molecules are prepared using 100% formamide at a temperature of about 65° C. for about 3 minutes and a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5. It can be removed by washing.

In some embodiments , the pairwise sequencing method further comprises the step (l) of sequencing the plurality of retained forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands. Includes. In some embodiments, the sequencing of step (l) is performed in a sequencing reaction using a reverse sequencing primer that has been hybridized under conditions suitable to hybridize the reverse sequencing primer to the reverse sequencing primer binding site of the retained forward extension strand. contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, whereby the forward sequencing reaction produces a plurality of extended reverse sequencing primer strands.

Each maintained forward extension strand may contain two or more copies of the reverse sequencing primer strand hybridized thereto. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands that hybridize to the same maintained forward extended strand.

In some embodiments, the sequencing of step (l) can be performed with or without a plurality of compressed oligonucleotides. Compact oligonucleotides can maintain the compact size and/or shape of nanoballs (e.g., self-collapsed immobilized concatimer template molecules) during a reverse sequencing reaction.

In some embodiments, in step (l), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand include the plurality of soluble reverse sequencing primers and the maintained forward extension strand at a pH. and contacting with a hybridization solution comprising a buffer, a sodium salt, and a disorder-inducing agent. Exemplary disorder-inducing agents include urea, guanidine hydrochloride, and guanidine thiocyanate. In some embodiments, the hybridization solution includes MES buffer, NaCl, and guanidine hydrochloride.

In some embodiments, in step (l), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand are such that the plurality of soluble reverse sequencing primers and the retained forward extension strand can be combined with high efficiency. and contacting with a hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a densifying agent in an amount that enhances or promotes molecular densification. In some embodiments, the high-efficiency hybridization buffer comprises (i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer; (iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

In some embodiments, the reverse sequencing reaction of step (l) comprises a plurality of soluble reverse sequencing primers, a reverse sequencing primer binding sequence of the retained forward extension strand, one or more types of sequencing polymerase, and a plurality of nucleotides or It includes contacting a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primer includes a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer includes a 3' blocking moiety that can be removed to create a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide having a cleavable moiety. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each maintained forward extension strand has multiple copies of the reverse sequencing primer binding sequence/site, such that each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Individual reverse sequencing primer binding sites on a given maintained forward extension strand can hybridize to the reverse sequencing primer and undergo a sequencing reaction. Accordingly, each maintained forward extension strand can undergo more than one sequencing reaction, each sequencing reaction initiated from a reverse sequencing primer hybridized to the reverse sequencing primer binding site. In some embodiments, the sequencing reaction includes a plurality of nucleotides (or analogs thereof) labeled with a detectable reporter moiety. In some embodiments, the sequencing reaction comprises a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. Sequencing reactions using nucleotides and/or multivalent molecules are described in more detail below.

In some embodiments, at least one washing step may be performed after any of steps (a) through (l). The washing step can be performed with a washing solution containing a pH buffer, metal chelating agent, salt and detergent.

In some embodiments, the pH buffering compound in the wash solution is Tris, Tris-HCl, tricine, bicine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES. , ethanolamine (2-amino methanol; also known as MEA), citrate compounds, citrate mixtures NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the wash solution at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein can be adjusted to a pH of about 4 to 9, or to a pH of about 5 to 9, or to a pH of about 5 to 8.

In some embodiments, the metal chelating agent in the cleaning solution is ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), or diethylene triamine pentaacetic acid (DPTA). , NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate or magnesium citrate. In some embodiments, the washing solution includes a chelating agent at a concentration of about 0.01 to 50mM, or about 0.1 to 20mM, or about 0.2 to 10mM.

In some embodiments, the salt of the wash solution includes NaCl, KCl, NH ₂ SO ₄ or potassium glutamate. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). The washing solution may include the monovalent salt at a concentration of about 25 to 500mM, or about 50 to 250mM, or about 100 to 200mM.

In some embodiments, the cleaning agent in the cleaning solution includes a non-ionic cleaning agent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N -dimethyl-3-ammonio- Includes zwitterionic detergents such as 1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the cleaning solution at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

The wash solution may include SSC (e.g., at any concentration from about 1 to 5x) and a detergent (e.g., Tween-20).

Support and low non-specific coating

The present disclosure provides pairwise sequencing compositions and methods using a support comprising a plurality of immobilized oligonucleotide surface primers. In some embodiments, the support is passivated with a low non-specific binding coating. The surface coatings described herein exhibit very low non-specific binding to reagents typically used in nucleic acid capture, amplification and sequencing workflows, such as dyes, nucleotides, enzymes and nucleic acid primers. The surface coatings exhibit low background fluorescence signal or high contrast-to-noise (CNR) ratio compared to conventional surface coatings.

Low non-specific binding coatings include one layer or multiple layers (Figure 115). In some embodiments, a plurality of surface primers are secured to a low non-specific binding coating. In some embodiments, at least one surface primer is incorporated into the low non-specific binding coating. The low non-specific binding coating allows for improved nucleic acid hybridization and amplification performance. Typically, the support includes a substrate (or support structure), one or more covalently or non-covalently attached low-binding layers, a chemical modification layer, such as a silane layer, a polymer film, and a single-stranded nucleic acid library molecule. and one or more covalently or non-covalently attached surface primers that can be used to tether to a support. In some embodiments, the formulation of the coating, e.g., the chemical composition of one or more layers, the support, such that non-specific binding of proteins, nucleic acid molecules and other hybridization and amplification reaction components to the coating is minimized or reduced compared to a similar monolayer. The bonding chemistry used to crosslink one or more layers and/or to each other and the total number of layers may vary. The formulation of the coatings described herein can be varied so that non-specific hybridization on the coating is minimized or reduced compared to a similar monolayer. The formulation of the coating can be varied so that non-specific amplification on the coating is minimized or reduced compared to a similar monolayer. The formulation of the coating can be varied to maximize the specific amplification rate and/or yield on the coating. Suitable amplification levels for detection are no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 30 or more amplification cycles in some cases disclosed herein. achieved.

The support structure comprising one or more chemically modified layers, for example a layer of a low non-specific binding polymer, can be independent or integrated into another structure or assembly. For example, in some embodiments, the support structure may include one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may comprise one or more surfaces within the microplate format, such as the bottom surface of a well in a microplate. In some embodiments, the support structure includes an inner surface of the capillary (eg, a luminal surface). In some embodiments, the support structure includes a capillary inner surface (eg, luminal surface) etched into a planar chip.

The attachment chemistry used to bond the first chemically modified layer to the surface of the support will generally depend on both the chemical nature of the layer and the material from which the surface is fabricated. In some embodiments, the first layer can be covalently attached to the surface. In some embodiments, the first layer can be non-covalently attached, for example, by non-covalent interactions such as electrostatic interactions, hydrogen bonds, or van der Waals interactions between the support and the molecular components of the first layer. It can be adsorbed to the support through action. In either case, the support may be treated prior to attachment or deposition of the first layer. Any of a variety of surface preparation techniques known to those skilled in the art may be used to clean or treat the surface. For example, glass or silicon surfaces can be acid cleaned using a base treatment in Piranha solution (a mixture of sulfuric acid (H ₂ SO ₄ ) and hydrogen peroxide (H ₂ O ₂ )), KOH and NaOH, and/ Or it can be cleaned using an oxygen plasma treatment method.

Silane chemistry involves covalently modifying silane groups on a glass or silicone surface to attach more reactive functional groups (e.g., amines or carboxyl groups), followed by binding linker molecules to the surface (e.g., linear It constitutes a non-limiting approach that can be used on hydrocarbon molecules (e.g., C6, C12, C18 hydrocarbons or linear polyethylene glycol (PEG) molecules) or layered molecules (e.g., branched PEG molecules or other polymers). Examples of suitable silanes that can be used to create any of the disclosed low binding coatings include (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), and any of the various PEGs. -Silanes (e.g., including molecular weights 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, etc., It is not limited to these.

Any of a variety of molecules known to those skilled in the art, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof, can be used to create one or more chemically modified layers on the support. wherein the selection of components used may depend on one or more properties of the layer, such as surface density of functional groups and/or tethered oligonucleotide primers, hydrophilicity/hydrophobicity of the layer, or three-dimensional properties of the layer (i.e. can be changed to change the “thickness”). Examples of polymers that can be used to create one or more layers of low non-specific binding material in any of the disclosed coatings include polyethylene glycol (PEG) of various molecular weights and branched structures, streptavidin, polyacrylamide, polyester, dextran, Including, but not limited to, poly-lysine and poly-lysine copolymers, or any combination thereof. Examples of bonding chemistries that can be used to bond one or more layers of a material (e.g., a polymer layer) to a surface and/or crosslink the layers to each other include biotin-streptavidin interactions (or variations thereof) , his tags - Ni/NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxies, azides, hydrazides, alkynes, isocyanates, and Including, but not limited to, silanes.

The low non-specific binding surface coating can be applied uniformly across the support. Alternatively, the surface coating can be patterned such that the chemical modification layer is confined to one or more discrete areas of the support. For example, the coating can be patterned using photolithographic techniques to create a regular array or random pattern of chemically modified areas on the support. Alternatively or in combination, the coating may be patterned using, for example, contact printing and/or inkjet printing techniques. In some embodiments, the regular array or random pattern of chemically modified regions has at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, It can contain more than 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 separate areas.

In some embodiments, the low non-specific binding coating comprises a hydrophilic polymer non-specifically adsorbed or covalently conjugated to a support. Typically, poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene) or other hydrophilic polymers with different molecular weights and end groups linked to the support using, for example, silane chemistry. Passivation is performed. Terminal groups distal from the surface may include, but are not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide, and bis-silane. In some embodiments, two or more layers of hydrophilic polymer, such as linear polymer, branched polymer, or multi-branched polymer, can be deposited on the surface. In some embodiments, two or more layers can be covalently linked to each other or internally crosslinked to improve the coating of the resulting coating. In some embodiments, surface primers with different nucleotide sequences and/or base modifications (or other biomolecules, such as enzymes or antibodies) can be tethered to the resulting layer at various surface densities. In some embodiments, for example, surface functional group density and surface primer concentration can both be varied to achieve a desired surface primer density range. Additionally, surface primer density can be controlled by diluting the surface primer with other molecules bearing the same functional groups. For example, amine-labeled surface primers can be diluted with amine-labeled polyethylene glycol in reaction with the NHS-ester coated surface reducing the final primer density. Surface primers with different length linkers between the hybridization region and the surface attachment functional group can be applied to control the surface density. Examples of suitable linkers include primers (e.g., 0 to 20 bases), PEG linkers (e.g., 3 to 20 monomer units) and 5' linkers of the carbon chain (e.g., C6, C12, C18, etc.). It contains poly-T and poly-A strands at the ends. To measure primer density, a fluorescently labeled primer can be tethered to a surface and the fluorescence reading is then compared to that for a dye solution of known concentration.

In some embodiments, the low non-specific binding coating comprises a functionalized polymer coating layer covalently linked to at least a portion of the support via chemical groups on the support, a primer conjugated to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. Includes. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM).

To tune primer surface density and add additional dimension to hydrophilic or amphiphilic coatings, supports containing multilayer coatings of PEG and other hydrophilic polymers have been developed. By utilizing hydrophilic and amphiphilic surface layering approaches, including but not limited to the polymer/copolymer materials described below, the primer loading density on the support can be significantly increased. Traditional PEG coating approaches use monolayer primer deposition, which has generally been reported for single molecule approaches but does not yield high copy numbers for nucleic acid amplification applications. “Layering” as described herein can be accomplished using traditional crosslinking approaches with any suitable polymer or monomer subunit such that a surface comprising two or more highly crosslinked layers can be sequentially constructed. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyesters, dextran, poly-lysine, and copolymers of poly-lysine and PEG. In some embodiments, the different layers are biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic reaction between positively charged and negatively charged polymers. They can be attached to each other through any of a variety of conjugation reactions, including but not limited to interactions. In some embodiments, the high primer density material can be built up in solution and subsequently layered on the surface in multiple steps.

Examples of materials from which support structures can be fabricated include glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC). , polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. However, it is not limited to these. Various compositions of both glass and plastic support structures are envisioned.

The support structures may be provided in any of a variety of geometries and dimensions known to those skilled in the art, and may include any of a variety of materials known to those skilled in the art. For example, the support structure may be locally planar (eg, including a microscope slide or the surface of a microscope slide). Overall, the support structure may be cylindrical (e.g., including the inner surface of a capillary or capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., irregularly shaped, including the outer surface of a non-porous bead or particle). ) can be. In some embodiments, the surface of the support structure used for nucleic acid hybridization and amplification may be a solid, non-porous surface. In some embodiments, the surface of the support structure used for nucleic acid hybridization and amplification may be porous such that the coatings described herein penetrate the porous surface and the nucleic acid hybridization and amplification reactions performed thereon occur within the pores. .

The support structure comprising one or more chemically modified layers, for example a layer of a low non-specific binding polymer, can be independent or integrated into another structure or assembly. For example, the support structure can include one or more surfaces within an integrated or assembled microfluidic flow cell. The support structure may comprise one or more surfaces within the microplate format, such as the bottom surface of a well in a microplate. In some embodiments, the support structure includes an inner surface of the capillary (eg, a luminal surface). In some embodiments, the support structure includes a capillary inner surface (eg, luminal surface) etched into a planar chip.

As noted, the low nonspecific binding supports of the present disclosure exhibit reduced nonspecific binding of proteins, nucleic acids, and other components of hybridization and/or amplification formulations used for solid phase nucleic acid amplification. The degree of non-specific binding exhibited by a given support surface can be evaluated quantitatively or qualitatively. For example, fluorescent dyes (e.g., cyanine, such as Cy3, or Cy5, etc., fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein), fluorescently labeled nucleotides, under a standardized set of conditions, Exposure of the surface to fluorescently labeled oligonucleotides and/or fluorescently labeled proteins (e.g., polymerase), followed by a specified rinse protocol and fluorescence imaging allows comparison of non-specific binding on supports containing different surface formulations. It can be used as a quantitative tool for In some embodiments, exposure of the surface to a fluorescent dye, fluorescently labeled nucleotide, fluorescently labeled oligonucleotide, and/or fluorescently labeled protein (e.g., a polymerase) under a standardized set of conditions, followed by a specified rinse protocol. and fluorescence imaging can be used as a quantitative tool for comparison of non-specific binding on supports comprising different surface formulations - provided the fluorescence signal is not present (e.g., signal saturation and/or self-quenching of the fluorophore is not an issue). Care was taken to ensure that fluorescence imaging was performed under conditions that were linearly related (or related in a predictable manner) to the number of fluorophores on the support surface (under conditions) and that appropriate calibration standards were used. In some embodiments, other techniques known to those skilled in the art, such as radioisotope labeling and counting methods, can be used to quantitatively assess the extent to which different support surface formulations of the present disclosure exhibit non-specific binding.

Some surfaces disclosed herein have a ratio of specific to non-specific binding of fluorophores, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, represents a Cy3 of 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value spanning the ranges herein. Some surfaces disclosed herein have a ratio of the specific to non-specific fluorescence of the fluorophore, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, represents a Cy3 of 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value spanning the ranges herein.

The degree of non-specific binding exhibited by the disclosed low-binding scaffolds can be achieved by binding to the surface and labeled proteins (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase) under a standardized set of incubation and rinse conditions. case, single-stranded binding protein (SSB), etc., or any combination thereof), labeled nucleotides, labeled oligonucleotides, etc., and then This can be followed by detection of labeled quantities and comparison of the signals obtained from them with appropriate calibration standards. In some embodiments, the label may include a fluorescent label. In some embodiments, the label may include a radioisotope. In some embodiments, the label may include any other detectable label known to those skilled in the art. In some embodiments, the degree of non-specific binding exhibited by a given support surface formulation can therefore be assessed in terms of the number of protein molecules (or nucleic acid molecules or other molecules) non-specifically bound per unit area. In some embodiments, the low-binding support of the present disclosure has less than 0.001 molecules per μm ² , less than 0.01 molecules per μm ² , less than 0.1 molecules per μm ² , Less than 0.25 molecules per μm ² , less than 0.5 molecules per μm ² , less than 1 molecule per μm ² , less than 10 molecules per μm ² , less than 100 molecules per μm ² or 1,000 molecules per μm ² represents non-specific protein binding of less than a molecule (or non-specific binding of another specified molecule (e.g., cyanine, such as Cy3 or Cy5, etc., fluorescein, coumarin, rhodamine, etc., or other dyes disclosed herein)). You can. Those skilled in the art will recognize that a given support surface of the present disclosure may exhibit non-specific binding of less than 86 molecules per μm ² falling anywhere within this range, for example. For example, some of the modified surfaces disclosed herein were contacted with a 1 μM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS) buffer for 15 minutes and then rinsed three times with deionized water. It exhibits non-specific protein binding of less than 0.5 molecules/㎛ ² . Some of the modified surfaces disclosed herein exhibit non-specific binding of less than 0.25 molecules of Cy3 dye molecules per μm ² . In an independent nonspecific binding assay, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rhol 1 (Jena Biosciences), 10 μM 7-propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propargylamino-7-deaza- dGTP-Cy3 (Jena Biosciences) is incubated on low binding coated supports for 15 minutes at 37°C in 384 well plate format. Each well is rinsed 2-3× with 50 μl deionized RNase/DNase-free water and 2-3× with 25mM ACES buffer pH 7.4. 384 well plates were purified using GE Typhoon using Cy3, AF555, or Cy5 filter sets (depending on the dye test performed) as specified by the manufacturer at a PMT gain setting of 800 and a resolution of 50 to 100 μm. Imaging was performed on the device. For higher resolution imaging, a total internal reflectance fluorescence (TIRF) objective (100×, 1.5 NA, Olympus) and a CCD camera (e.g., Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera) or Olympus DP80 color and monochrome cameras), an illumination source (e.g., an Olympus 100W Hg lamp, an Olympus 75W Images were collected on an inverted fluorescence microscope (Olympus Corp., Center Valley, PA). Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, NY), e.g., 405, 488, 532, or 633 nm dichroic reflectors/beam splitters, and bandpass filters were used at the appropriate excitation wavelength. Therefore, 532 LP or 645 LP trends were selected. Some modified surfaces disclosed herein exhibit non-specific binding of dye molecules of less than 0.25 molecules per μm ² . In some embodiments, the coated support was immersed in a buffer (e.g., 25mM ACES, pH 7.4) while images were acquired.

In some embodiments, some surfaces disclosed herein have a ratio of specific to non-specific binding of fluorophores, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. , 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value spanning the ranges herein. In some embodiments, some surfaces disclosed herein have a ratio of specific to non-specific fluorescence signal for the fluorophore, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Cy3 of greater than 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or 100, or any intermediate value spanning the ranges herein represents.

Consistent with the disclosure herein, a low-background surface has at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1 per non-specifically adsorbed molecule. Specific dye attachment (e.g., Cy3 attachment) versus non-specific dye adsorption (e.g., Cy3 attachment) of 1, 20:1, 30:1, 40:1, 50:1, or more than 50 attached non-specific dye molecules. dye adsorption) ratio can be expressed. Similarly, upon application of excitation energy, consistent with the disclosure herein, a low-background surface to which a fluorophore, e.g., Cy3, is attached produces a specific fluorescence signal (e.g., a Cy3-labeled oligomer attached to the surface). arises from nucleotides) vs. at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, It may represent a ratio of non-specific adsorption dye fluorescence signal of 50:1, or greater than 50:1.

In some embodiments, the degree of hydrophilicity (or "wetability" by an aqueous solution) of a disclosed support surface can be assessed, for example, through measurement of the water contact angle at which a droplet is placed on the surface, where the contact angle between the surface and the surface is: For example, it is measured using an optical tensiometer. In some embodiments, static contact angles can be determined. In some embodiments, advancing or receding contact angles can be determined. In some embodiments, the water contact angle for the hydrophilic, low-binding support surfaces disclosed herein can range from about 0 degrees to about 30 degrees. In some embodiments, the water contact angle for the hydrophilic, low-binding support surfaces disclosed herein is 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees. , may be 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree or less. In many cases the contact angle is less than 40 degrees. Those skilled in the art will recognize that a given hydrophilic, low binding support surface of the present disclosure may exhibit a water contact angle having a value anywhere else within this range.

In some embodiments, the hydrophilic surfaces disclosed herein promote reduced wash times for bioassays, often due to reduced non-specific binding of biomolecules to the low-binding surface. In some embodiments, a suitable cleaning step can be performed in less than 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example, a suitable washing step can be performed in less than 30 seconds.

Some low bond surfaces of the present disclosure exhibit significant improvements in stability or durability of solvents and elevated temperatures, or repeated cycles of solvent exposure or long-term exposure to temperature changes. For example, the stability of the disclosed surfaces can be determined by fluorescently labeling functional groups on the surface or tethered biomolecules on the surface (e.g., oligonucleotide primers), exposure to solvents and elevated temperatures, or repeated cycles of solvent exposure or temperature. Changes can be tested by monitoring the fluorescence signal before, during and after long-term exposure. In some embodiments, the degree of fluorescence change used to assess surface quality is 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours , less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25% over a time period of 40-hour, 45-hour, 50-hour or 100-hour exposure (or such time period) can be any combination of these percentages as measured across). In some embodiments, the degree of change in fluorescence used to assess surface quality is 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles in response to solvent change and/or temperature change. , 1%, 2%, and 3% over repeated exposures of 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 cycles. , may be less than 4%, 5%, 10%, 15%, 20% or 25% (or any combination of these percentages as measured over this period range).

In some embodiments, surfaces disclosed herein may exhibit a high ratio of specific signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces have at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 50,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,0,000,000,0,000,0,000,0,000,0,0,0,0,000,0,0,0,0,000,000,0, were, For, For, For,,, , , , , , , , , , , An amplified signal can be expressed as an excess of 100 times, or any number greater than 100. Similarly, some surfaces have a signal at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100 times or greater than the signal of the adjacent amplified nucleic acid population region of the surface. Indicates an amplified signal in excess of a multiple of the number.

In some embodiments, the low background surface fluorescence disclosed when used in a nucleic acid hybridization or amplification application to generate polities of hybridized or clonally amplified nucleic acid molecules (e.g., directly or indirectly labeled with a fluorophore) Images must have at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240 , 250, or a number greater than 250.

One type of primer can be attached or tethered to the support surface. In some embodiments, one or more types of adapters or primers include a spacer sequence, an adapter sequence for hybridization to an adapter-ligated target library nucleic acid sequence, a forward amplification primer, a reverse amplification primer, a sequencing primer, and/or molecular barcoding. sequences, or any combination thereof. In some embodiments, one primer or adapter sequence may be tethered to at least one layer of the surface. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adapter sequences can be tethered to at least one layer of the surface.

In some embodiments, the tethered adapter and/or primer sequence may range from about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the tethered adapter and/or primer sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides long. . In some embodiments, the tethered adapter and/or primer sequence can be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides long. there is. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure; for example, in some embodiments, the length of the tethered adapter and/or primer sequence is from about 20 nucleotides to It may range from about 80 nucleotides. Those skilled in the art will recognize that the length of the tethered adapter and/or primer sequence can have any value within this range, for example, about 24 nucleotides.

In some embodiments, the resulting surface density of primers (e.g., capture primers) on the low binding support surface of the present disclosure can range from about 100 primer molecules per μm ² to about 100,000 primer molecules per μm ² . In some embodiments, the resulting surface density of primers on the low binding support surface of the present disclosure can range from about 1,000 primer molecules per μm ² to about 1,000,000 primer molecules per μm ² . In some embodiments, the surface density of the primer may be at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000 molecules per μm ² . In some embodiments, the surface density of the primers can be at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000 molecules per μm ² . Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure, for example, in some embodiments the surface density of the primer ranges from about 10,000 molecules per μm ² to about 10,000 molecules per μm ² It can be in the range of 100,000 molecules. Those skilled in the art will recognize that the surface density of primer molecules can have any value within this range, for example about 455,000 molecules per micrometer ² . In some embodiments, the surface density of surface library nucleic acid sequences initially hybridized to adapter or primer sequences on the support surface may be less than or equal to that seen for the surface density of the tethered primers. In some embodiments, the surface density of clonally amplified surface library nucleic acid sequences hybridized to adapter or primer sequences on the support surface may span the same range as shown by the surface density of the tethered primers.

A local density as listed above may be present on the surface such that the surface may, for example, comprise a region having an oligo density of 500,000/μm ² while also comprising at least a second region having a substantially different local density. Fluctuations in density over time are not impossible.

In some embodiments, the performance of nucleic acid hybridization and/or amplification reactions using the disclosed reaction formulations and low-binding supports can be assessed using fluorescence imaging techniques, wherein the contrast-to-noise ratio (CNR) of the image is determined by It provides an important measurement standard in evaluating amplification specificity and non-specific binding. CNR is described in US patent application US 2020/0149095, which is hereby expressly incorporated by reference in its entirety. CNR is usually defined as: CNR=(signal-background)/noise. The background term is typically considered the signal measured for the area between a specified region of interest (ROI) and surrounding a specific feature (diffraction limited spot, DLS). Signal-to-noise ratio (SNR) is often considered the benchmark for overall signal quality, but improved CNR can be beneficial in applications requiring fast image capture (e.g., sequences where cycle time is minimized), as shown in the examples below. In analytical applications, it can be shown that, as a background to signal quality, it can provide significant benefits for SNR. At high CNR, the imaging time required to reach accurate discrimination (and therefore accurate base calling in the case of sequencing applications) can be dramatically reduced even at moderate improvements in CNR. Improved CNR in imaging data relative to imaging integration time provides a more accurate way to detect features such as clonally amplified nucleic acid colonies on the support surface.

In most ensemble-based sequencing approaches, background terms are typically measured as signals associated with 'intervening' regions. In addition to the “between” background (B _inter ), the “between” background (B _intra ) exists within the region occupied by amplified DNA colonies. The combination of these two background signals influences achievable CNR, and subsequently optics requirements, architecture cost, reagent cost, run time, cost/genome, and ultimately accuracy and data quality for cyclic array-based sequencing applications. directly affects. B _inter background signals come from a variety of sources; Some examples include autofluorescence from consumable flowcells, non-specific adsorption of detection molecules yielding false fluorescence signals that can obscure the signal from the ROI, and the presence of non-specific DNA amplification products (e.g., those resulting from primer dimers). ) is included. In a typical next-generation sequencing (NGS) application, this background signal is averaged over time and subtracted from the current field-of-view (FOV). The signal resulting from individual DNA colonies (i.e., (signal)-B(between) in FOV) yields identifiable features that can be classified. In some embodiments, the background background (B(between)) may contribute to a confounding fluorescence signal that is not specific to the target of interest, but is present in the same ROI, thus making averaging and subtraction much more difficult.

Nucleic acid amplification on the low-binding coated supports described herein can reduce B (between) background signal by reducing non-specific binding, resulting in improved specific nucleic acid amplification, resulting from both the between and between regions. This may result in a decrease in non-specific amplification that may affect the background signal. In some embodiments, the disclosed low-binding coated supports, optionally used in combination with the disclosed hybridization and/or amplification reaction formulations, provide a 2, 5-fold higher molecular weight than that achieved using conventional supports and hybridization, amplification and/or sequencing protocols. , can result in an improvement in CNR by as much as 10, 100, 250, 500 or 1000-fold. Although described herein with respect to the use of fluorescence imaging as a readout or detection method, the low-binding coated support and nucleic acid hybridization and amplification formulations are also disclosed for other detection methods, including both optical and non-optical detection methods. The same principles for use apply.

Compositions and methods for making, testing, and using low non-specific binding coatings, including methods for immobilizing any type of primer (e.g., capture oligonucleotide, amplification oligonucleotide, and/or cycling oligonucleotide) to the coating, 2020 U.S. Patent No. 10,768,173, issued September 8; U.S. Patent Application No. 16/363,842, filed March 25, 2019; No. 16/740,355, filed January 10, 2020; No. 16/740,357, filed January 10, 2020; and No. 16,855,877 filed on April 22, 2020. The contents of the foregoing issued patents and patent applications are expressly incorporated herein by reference in their entirety.

library molecule

Pairwise sequencing compositions and methods described herein utilize nucleic acid library molecules, which typically refer to a population of nucleic acid molecules each containing a sequence of interest (e.g., an insertion region) covalently linked to at least one adapter sequence. Individual library molecules in a population may have insert sequences that are the same or different insert sequences from other library molecules in the population. An individual library molecule in a population may have an adapter sequence that is the same (e.g., a universal adapter sequence) or a different (e.g., unique identification sequence) adapter sequence than other library molecules in the population.

Nucleic acid library molecules include DNA, RNA, cDNA, or chimeric DNA/RNA. Nuclear library molecules may be single- or double-stranded, or may contain single- or double-stranded portions. Nucleic acid library molecules may be linear, covalently closed circles, dumbbells, hairpins, or other shapes.

The insertion region of the nucleic acid library molecule contains a sequence of interest extracted from any source containing a biological sample (e.g., a fresh or living sample), such as a single cell, a plurality of cells, or tissue. The insertion area can be isolated from healthy or diseased cells or tissue. Insertion areas can be obtained from archival samples, such as fresh frozen paraffin embedded (FFPE) samples, or from needle biopsies, circulating tumor cells, cell-free circulating DNA. Cells or tissues are typically treated with a lysis buffer to release their DNA and RNA, and the nucleic acids of interest are separated from undesired macromolecules such as proteins.

Insertion regions of nucleic acid library molecules can be isolated in any form, including chromosomes, genomes (e.g., whole genomes), organelles (e.g., mitochondria, chloroplasts, or ribosomes), recombinant molecules, cloning, or amplification. .

The insertion region may be prepared using recombinant nucleic acid techniques, including, but not limited to, any combination of vector cloning, transgenic host cell production, host cell culture, and/or PCR amplification.

The insertion region can be prepared using chemical synthesis procedures using natural nucleotides, with or without nucleotide analogs or modified nucleotide linkages that impart specific properties, including resistance to enzymatic degradation, or increased thermal stability. Examples of nucleotide analogs and modified nucleotide linkages that inhibit nuclease degradation include phosphorothioate, 2'-O-methyl RNA, inverted dT, and 2' 3' dideoxy-dT. Insertion regions containing locked nucleic acids (LNA) have increased thermal stability.

The insertion region can be isolated from any organism, including viruses, fungi, prokaryotes, or eukaryotes. The insertion region can be isolated from any organism, including humans, simians, apes, dogs, cats, cattle, horses, murine, pigs, goats, wolves, toads, fish, plants, insects or bacteria. The insertion area can be isolated from organisms carried in air, water, soil or food.

The insertion area can be isolated from any biological fluid, including blood, urine, serum, lymph, tumor, saliva, anal secretions, vaginal secretions, amniotic membrane samples, sweat, semen, environmental samples, or culture samples. Insertion areas include the head, neck, brain, breast, ovaries, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testes, liver, lungs, kidneys, esophagus, pancreas, thyroid, pituitary gland, thymus, and skin. , can be isolated from any organ, including the heart, larynx or other organs.

The insertion region may be in fragmented or non-fragmented form. Fragmented insertion regions can be obtained by mechanical or enzymatic fragmentation methods. Fragmented insertion regions can be generated using procedures that yield populations of fragments with overlapping or non-overlapping sequences.

Mechanical fragmentation typically produces randomly fragmented nucleic acid molecules. Mechanical fragmentation methods include mechanical shear, such as fluid shear, constant shear, and pulsatile shear. Mechanical fragmentation methods also include mechanical stress, including sonication, atomization and acoustic cavitation.

Enzymatic fragmentation procedures can be performed under conditions suitable to produce randomly or non-randomly fragmented nucleic acid molecules. For example, restriction enzyme digestion can be performed to completion to generate non-randomly fragmented nucleic acid molecules. Alternatively, partial or incomplete restriction enzyme digestion can be performed to generate randomly fragmented nucleic acid molecules. Enzyme fragmentation using restriction enzymes includes any one or any combination of two or more restriction enzymes selected from the group consisting of type I, type II, type IIs, type IIB, type III, or type IV restriction enzymes. Enzymatic fragmentation involves the use of any combination of nicking restriction endonucleases, endonucleases, and/or exonucleases.

Fragments of the insertion region can be generated by PCR using sequence-specific primers that hybridize to the target region in the genomic DNA sample to generate an insertion region of known fragment length and sequence.

Targeted genome fragmentation methods using CRISPR/Cas9 can be used to generate fragmented insertion regions.

Fragments of the insertion region can also be generated using transposase-based tagging using NEXTERA (Epicentre).

The insertion region may be single-stranded or double-stranded. The ends of the double-stranded insertion region may be blunt-ended, or may have 5' overhangs or 3' overhangs, or any combination thereof. One or both ends of the insertion region can be subjected to an enzymatic tail reaction to generate a non-template poly-A tail by using a terminal transferase reaction. An end of the insertion region may be suitable for coupling to at least one adapter.

The insertion region may be coupled at one or both ends to at least one adapter. Covalent linkage between the insertion region and the adapter(s) can be achieved by DNA or RNA ligase. Exemplary DNA ligases capable of ligating double-stranded DNA molecules include T4 DNA ligase and T7 DNA ligase. The adapter sequence can be suspended from the insert sequence by PCR using a tail primer having a 5' region carrying the adapter sequence and a 3' region complementary to a portion of the insert sequence. Adapter sequences are flanked by PCR using tail primers having a 5' region carrying a third adapter sequence and a 3' region complementary to a portion of the first or second adapter sequence. It may be suspended from an insertion sequence flanking the end.

Adapters include DNA or RNA or analogs thereof, or chimeric DNA/RNA. The adapter may include at least one ribonucleoside residue. Adapters may be single-stranded, double-stranded, or may have single-stranded and/or double-stranded portions.

Adapters can be arranged in a linear, stem-loop, hairpin or Y-shape. Adapters can be of any length, including from 4 to 100 or more nucleotides.

Adapters may have blunt ends, protruding ends, or a combination of both. The overhang ends include the 5' overhang and the 3' overhang ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, may have a 5' phosphate group or lack a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tail adapter), or the adapter may not be a tail.

The adapter may comprise a sequence complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, a fixed surface capture primer, or a soluble primer (e.g., a universal adapter sequence).

Adapters may comprise random or degenerate sequences. The adapter may include at least one inosine residue.

Adapters can be synthesized to contain nucleotide analogs or modified nucleotide linkages that impart specific properties, including resistance to enzymatic degradation, or increased thermal stability. Examples of nucleotide analogs and modified nucleotide linkages that inhibit nuclease degradation include phosphorothioate, 2'-O-methyl RNA, inverted dT, or 2' 3' dideoxy-dT. Adapters containing locked nucleic acids (LNA) have increased thermal stability. The adapter may include at least one phosphorothioate, phosphorothioate and/or phosphoramidate linkage.

The adapter may include at least one restriction enzyme recognition sequence comprising any one or any combination of two or more selected from the group consisting of type I, type II, type III, type IV, type Hs or type IIB.

Adapters may include sample barcode sequences used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex analysis. An adapter is a unique identifying sequence (e.g., molecule) that is used to uniquely identify an individual nucleic acid molecule (e.g., an insertion sequence) suspended from a population of other nucleic acid molecules to which the adapter is bound to another distinct, uniquely identifying sequence adapter. tags, unique molecular indicators, UMI).

In some embodiments, single-stranded nucleic acid library molecules can serve as template molecules for sequencing. In some embodiments, complementary strands of nucleic acid library molecules can be synthesized, and the complementary strands can serve as template molecules for sequencing. In some embodiments, single-stranded nucleic acid library molecules can be amplified, and the resulting amplicon strands can serve as template molecules for sequencing. In some embodiments, double-stranded library molecules can be denatured or subjected to enzymatic treatment to produce single-stranded library molecules that can be linked and/or sequenced.

In some embodiments, a single-stranded circular library molecule can be hybridized to an amplification primer (e.g., a surface primer) and subjected to a primer extension reaction to generate a complementary conkatimer molecule, wherein the conkatimer molecule is It acts as a template molecule for sequence analysis. Sequencing of concatimer molecules can generate complementary strands, which in turn can serve as template molecules for sequencing. In some embodiments, a copy of the complementary strand may serve as a template molecule for sequencing.

Circulation of library molecules

Each linear library molecule contains insert sequences flanked on both ends by at least one adapter sequence. A population of single-stranded linear library molecules can be circularized to create a covalently closed circular library molecule. For example, the ends of single-stranded linear library molecules can undergo intramolecular ligation using a single-strand ligase (e.g., CircLigase from Epicentre or Lucigen).

In some embodiments, circular DNA molecules can be produced using protelomerase instead of nucleic acid ligase. Protelomerase enzymes identify an enzyme recognition sequence within a nucleic acid molecule and cleave the enzyme recognition sequence to produce ends with exposed 5' and 3' exposed cleaved ends, with a single exposed 5' end at the enzyme recognition site. and recombines at the 3' cleaved end to form a single linear molecule from the cleaved 5' and 3' ends. When this reaction is performed on both ends of a double-stranded nucleic acid molecule with an enzyme recognition sequence at each end, the result is a circular nucleic acid molecule. An adapter having an enzyme recognition sequence can be bound to both ends of a double-stranded DNA molecule through ligation or PCR using a tail PCR primer. A number of enzymes or combinations of enzymes, including proteinomerase, are suitable for this reaction. One type of proteinomerase is TelN proteinase, such as the proteinase from E. coli phage Nl.

In some embodiments, a population of double-stranded linear library molecules can be circularized to create circular library molecules. Each linear library molecule contains insert sequences flanked on both ends by at least one adapter sequence. In some embodiments, a population of linear library molecules can be contacted with an enzyme that catalyzes 5' phosphorylation of the ends of linear molecules, such as T4 polynucleotide kinase. In some embodiments, a population of linear molecules with blunt ends can be contacted with a ligase enzyme for intramolecular ligation, where the ligase enzyme comprises a T3 or T4 DNA ligase. In some embodiments, a population of linear molecules has protruding ends (e.g., sticky ends) and can be contacted with T7 DNA ligase to generate circular molecules. In some embodiments, linear library molecules can be reacted sequentially or simultaneously with T4 polynucleotide kinase enzyme and ligase enzyme to generate circular molecules. Non-circular molecules can be cleaved using at least one exonuclease enzyme, such as T7 exonuclease and/or exonuclease I (e.g., thermolabile exonuclease I). .

In some embodiments, a padlock probe workflow can be used to generate single-stranded circular molecules. Typically, in paddlelock probes, the arrangement of the insertion sequence (sequence of interest) and adapters is different from standard linear library molecules. In some embodiments, the paddlelock probe comprises a single-stranded linear oligonucleotide having a 5' portion, an internal linker portion, an internal linker portion, and a 3' portion. The 5' and 3' portions each contain portions of the target sequence of interest. The 5' and 3' portions are individually complementary to the target sequence of interest (e.g., an adjacent target sequence of interest), while the linker portion is designed to have little or no complementarity to the target sequence. The 5' and 3' ends of the paddlelock probe can hybridize to adjacent sites on the target nucleic acid molecule to form an open circular molecule with a gap between the hybridized 5' and 3' ends. The gap can be ligated to create a covalently closed circular molecule. Alternatively, the 5' and 3' ends of the paddlelock probe can hybridize to adjacent positions on the target nucleic acid molecule to form an open circular molecule with a gap between the hybridized 5' and 3' ends. The gap is subjected to a polymerase-mediated filling reaction to form a gap, which can then be ligated to produce a covalently closed circular molecule. Covalently closed circular molecules can be subjected to a rolling ring amplification reaction to generate concatemers with tandem repeat regions containing the sequence of interest. The internal linker portion can be engineered to include one or more universal adapter sequences, barcode adapters, or unique identifier adapters.

In some embodiments, a population of single-stranded linear library molecules can be circularized to generate single-stranded circular library molecules. Each single-stranded linear library molecule contains insert sequences flanked on both ends by at least one adapter sequence. For example, a single-stranded linear library molecule contains the following sequence: 5'-first surface primer binding sequence (SP1); A first sequencing primer binding sequence (Seq1); Insertion sequence (sequence of interest); a second sequencing primer binding sequence (Seq2); Second surface primer binding sequence (SP2)-3' (e.g., see schematic top left of Figure 53). A population of double-stranded linear library molecules can be denatured to produce single-stranded linear library molecules. Single-stranded linear library molecules can be hybridized to partially double-stranded sprint molecules to generate circular library molecules with gaps. A partially double-stranded splint molecule comprises a double-stranded oligonucleotide with a first splint molecule (long strand) and a second splint molecule (short strand) (see, e.g., Figure 53, bottom left schematic). In some embodiments, the first sprint molecule comprises (i) a left sequence that hybridizes to the first surface primer binding sequence (SP1'); (ii) an internal portion that hybridizes to the second Sprint molecule; and (iii) a right-hand sequence (see, e.g., Figure 53) that hybridizes to the second surface primer binding sequence (SP2'). In some embodiments, the internal portion of the first sprint molecule comprises a complementary sequence of the sample barcode and/or unique identification sequence (N'N'N'). In some embodiments, the second Sprint molecule comprises a sequence that hybridizes to an internal portion of the first Sprint molecule. The second Sprint molecule may include a sample barcode and/or unique identification sequence (NNN). The insert sequence of interest does not hybridize to the first or second Sprint molecule (Figure 53).

Single-stranded library molecules hybridize to double-stranded splint molecules to create a library-splint complex with two gaps (Figure 54). The first gap is located between the 5' end of the library molecule and the 3' end of the second sprint molecule. The second gap is located between the 3' end of the library molecule and the 5' end of the second sprint molecule (Figure 54, left schematic).

The library-splint complex is reacted sequentially or simultaneously with a T4 polynucleotide kinase and a ligase (e.g., T7 DNA ligase) to bind (i) the 5' end of the library molecule, the 5' end of the first splint molecule, and the first splint molecule. 2 phosphorylate the 5' end of the Sprint molecule, and (ii) close the first and second clefts by enzymatic ligation, thereby generating a single-stranded covalently closed circular library molecule hybridized to the second Sprint molecule. (Figure 54, central schematic). The non-circular molecule and the second splint strand utilize at least one exonuclease enzyme, such as T7 exonuclease and/or exonuclease I (e.g., thermolabile exonuclease I). It can be decomposed. The remaining single-stranded circular library molecules can be subjected to a rotation amplification reaction in solution or on a support using the 3' end of the linear first sprint molecule (Figure 54, right schematic). Alternatively, the first sprint molecule can be removed and the closed circular molecule can be hybridized with a soluble amplification primer that can be used to initiate the rolling ring amplification reaction. In another example, the first sprint molecule can be removed and the closed circular molecule can be hybridized with a surface primer immobilized on the support, followed by a spin ring amplification reaction. The surface primer immobilized on the support lacks nucleotides having a cleavable moiety. For example, the surface primer lacks uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), and deoxyinosine.

compressed oligonucleotide

The rolling ring amplification reaction on any of the supports or in solution described herein may include a plurality of compressed oligonucleotides capable of compressing the size and/or shape of the concatemer into nanoballs. Condensed oligonucleotides are single-stranded nucleic acid molecules containing DNA or their analogues. The compressed oligonucleotide is capable of cross-hybridizing to portions of the concatimer molecule. The condensed oligonucleotide comprises two identical sequences separated by a short linker sequence, where the two identical sequences are reverse complementary to portions of the concatemer. Compact oligonucleotides can be of any length, for example 20 to 100 nucleotides. Two identical sequence regions of the compacted oligonucleotide can hybridize to the concatimer, pulling the distal portions of the concatimer together, causing compaction of the concatimer and forming a nanoball. In some embodiments, the compressed oligonucleotide is resistant to 3' exonuclease digestion and/or single strand endonuclease digestion. In some embodiments, the compacted oligonucleotide comprises any one or any combination of two or more of the following: end phosphorylation of the 3' end; at least two terminal nucleotides at the 3' end with a phosphorothioate bond therebetween; at least one terminal nucleotide at the 3' end having a 2'-O-methyl moiety; and/or at least one 3' end nucleotide with a 2' fluoro base.

The condensed oligonucleotide may include at least one region with consecutive guanines. For example, the condensed oligonucleotide may include at least one region with 2, 3, 4, 5, 6 or more consecutive guanines. In some embodiments, the condensed oligonucleotide includes four consecutive guanines capable of forming a guanine tetramolecular structure (see Figure 116A). The guanine tetramolecular structure can be stabilized through Hoogsteen hydrogen bonds. Guanine tetramolecular structures can be stabilized by central cations including potassium, sodium, lithium, rubidium, or cesium.

In some embodiments, the rolling ring amplification reaction comprises the sequence of interest for the compressed oligonucleotide and the universal binding sequence, and further comprises (i) two or more copies of the universal binding sequence for the soluble forward sequencing primer, (ii) the soluble at least two copies of the universal binding sequence for the reverse sequencing primer, (iii) at least two copies of the universal binding sequence for the immobilized first surface primer, (iv) of the universal binding sequence for the immobilized second surface primer. at least two copies, (v) at least two copies of the universal binding sequence for the first soluble amplification primer, (vi) at least two copies of the universal binding sequence for the second soluble amplification primer, (vii) of the sample barcode sequence. This may be performed by circular library molecules comprising any one or any combination of two or more copies of two or more copies and/or (viii) two or more copies of a unique molecular indicator sequence.

The rolling ring amplification reaction can be performed in the presence of a plurality of compressed oligonucleotides having at least four consecutive guanines. The resulting concatemer contains repeated copies of the universal binding sequence for the compressed oligonucleotide. At least one compressed oligonucleotide can form a guanine tetramolecule (Figure 116A) and hybridize to the universal binding sequence for the compressed oligonucleotide, and the resulting concatemer can be folded to form an intramolecular G-quadruple structure (Figure 116A). Figure 116B). Concatimer can self-collapse to form dense nanoballs. The formation of guanine tetramers and G-quadruplexes in the nanoballs increases the stability of the nanoballs, making their compact structure capable of withstanding repeated flows of reagents to perform any of the pairwise sequencing workflows described herein. It can maintain its size and shape.

Rotary ring amplification on supports

The present disclosure provides pairwise sequencing compositions and methods for generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules using rolling ring amplification on a support. In some embodiments, the rolling ring amplification reaction on a support involves (1) hybridizing a plurality of single-stranded covalently closed circular nucleic acid library molecules to a plurality of immobilized surface primers to form a plurality of immobilized covalently closed circular nucleic acid library molecules; generating nucleic acid library molecules; (2) a plurality of immobilized, covalently closed, circular nucleic acid library molecules comprising (i) a plurality of polymerases having strand transposition activity, (ii) nucleotides having dATP, dGTP, dCTP, dTTP, and a cleavable moiety; By contacting the plurality of nucleotides comprising the mixture, and (iii) a catalytic divalent cation (e.g., magnesium and/or manganese) that mediates nucleotide binding to the nucleotide-polymerase complex and promotes nucleotide incorporation, forming a nucleotide-polymerase complex, wherein contacting is performed under conditions suitable for binding the individual polymerase to the immobilized, covalently closed circular nucleic acid library molecule to form a complexed polymerase, and conditions. is suitable for forming a nucleotide-polymerase complex by linking the individually complexed polymerase to a nucleotide or nucleoside analog. In some embodiments, in a nucleotide mixture of a plurality of nucleotides, the nucleotide having a cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Contains; (3) A nucleotide polymerization reaction (e.g., rolling ring amplification reaction) is performed under isothermal temperature conditions to produce a plurality of fixed single-stranded concatimer template molecules.

In some embodiments, the rolling ring amplification reaction lacks a plurality of compacted oligonucleotides, or the rolling ring amplification reaction lacks a plurality of compacted oligonucleotides that can hybridize to a portion of the concatimer and collapse the concatimer into a more compact shape and size. It further contains oligonucleotides. A condensed oligonucleotide is a single-stranded nucleic acid molecule that has two identical sequences separated by a short linker sequence, and the two identical sequences are reverse complementary to portions of the concatemer. Compact oligonucleotides can be of any length, for example 20 to 100 nucleotides. Two identical sequence regions of the condensation oligonucleotide can hybridize to the concatimer, pulling the distal portions of the concatimer together, resulting in compaction of the concatimer. In some embodiments, the compressed oligonucleotide is resistant to 3' exonuclease digestion and/or single strand endonuclease digestion. In some embodiments, the compacted oligonucleotide comprises any one or any combination of two or more of the following: end phosphorylation of the 3' end; at least two terminal nucleotides at the 3' end with a phosphorothioate bond therebetween; at least one terminal nucleotide at the 3' end having a 2'-O-methyl moiety; and/or at least one 3' end nucleotide with a 2' fluoro base. In some embodiments, compressed oligonucleotides may be included in step (2) or (3).

In some embodiments, the plurality of polymerases having strand transposition activity include phi29 DNA polymerase, large fragment of Bst DNA polymerase, Bsu DNA polymerase and large fragment of Bca(exo-) DNA polymerase, E. coli DNA polymerase. Klenov fragment, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, the rolling ring amplification reaction involves a helicase, a single-stranded binding (SSB) protein, or a recombinase (e.g., T4 uvsX) and/or a recombinase accessory factor (e.g., T4 uvsY or T4 gp32). It further includes at least one accessory protein or enzyme.

In some embodiments, the rolling ring amplification reaction can be performed at an isothermal temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

In some embodiments, a conkatimer is a group of repeat units comprising the sequence of interest (or its complementary sequence) and any adapter sequences (or its complementary sequence) present in the original covalently closed circular nucleic acid library molecule. It may contain at least 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more tandem copies.

In some embodiments, a rotating ring amplification (RCA) reaction may be followed by a multiple dislocation amplification (MDA) reaction or a plexing amplification reaction. In some embodiments, the RCA reaction is not followed by a multiple transposition amplification reaction or a plexing amplification reaction. Exemplary MDA and plexing reactions are described below.

Two-step rotational amplification on support

Trapped nucleotide-polymerase complex

The present disclosure provides pairwise sequencing compositions and methods for generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules using a two-step rolling ring amplification reaction on a support. The two-step rotary ring amplification method described herein uses non-catalytic, then catalytic divalent cations to synchronize rotary ring amplification events on a surface/support and generate concatemers. The two-step rotating ring amplification reaction is followed by relaxation conditions and flexing amplification reaction, so that new concatimers can be generated from existing concatimers. Taken together, this amplification method produces highly dense nanoballs containing high copy numbers of target sequence, improving sequencing signal intensity.

In some embodiments, a two-step rolling ring amplification reaction on a support involves (1) hybridizing a plurality of single-stranded covalently closed circular nucleic acid library molecules to a plurality of immobilized surface primers to form a plurality of immobilized covalently closed nucleic acid library molecules; generating circular nucleic acid library molecules; (2) a plurality of immobilized, covalently closed, circular nucleic acid library molecules comprising (i) a plurality of polymerases having strand transposition activity, (ii) nucleotides having dATP, dGTP, dCTP, dTTP, and a cleavable moiety; a first plurality of nucleotides comprising the mixture, and (iii) a non-catalytic divalent cation (e.g., strontium and/or barium) to form a plurality of trapped nucleotide-polymerase complexes, wherein the contacting is performed under conditions suitable for binding the individual polymerases to an immobilized covalently closed circular nucleic acid library molecule. To form a complexed polymerase, conditions are suitable to bind the individual complexed polymerase to a nucleotide or nucleoside analog to form a trapped nucleotide-polymerase complex. In some embodiments, in the nucleotide mixture of the first plurality of nucleotides, the nucleotide having a cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxy Contains inosine. The rolling ring amplification reaction further comprises: (3) a plurality of trapped nucleotide-polymerase complexes with (i) at least one divalent cation (e.g., magnesium) that mediates nucleotide binding and mediates nucleotide incorporation; and/or manganese), and (ii) a mixture of nucleotide analogs having a cleavable moiety under conditions suitable for carrying out an isothermal rolling ring amplification reaction producing a plurality of fixed single-stranded concatimer template molecules. Performing nucleotide polymerization by contacting with a second plurality of nucleotides. In some embodiments, in the nucleotide mixture of the second plurality of nucleotides, the nucleotide analog having a cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or Contains oxyinosine.

In some embodiments, the two-step rotary ring amplification reaction lacks a plurality of condensed oligonucleotides, or the rotary ring amplification reaction can hybridize to a portion of the concatimer and collapse the concatimer into a more compact shape and size. It further comprises a compressed oligonucleotide. A condensed oligonucleotide is a single-stranded nucleic acid molecule that has two identical sequences separated by a short linker sequence, and the two identical sequences are reverse complementary to portions of the concatemer. Compact oligonucleotides can be of any length, for example 20 to 100 nucleotides. Two identical sequence regions of the condensation oligonucleotide can hybridize to the concatimer, pulling the distal portions of the concatimer together, resulting in compaction of the concatimer. In some embodiments, the compressed oligonucleotide is resistant to 3' exonuclease digestion and/or single strand endonuclease digestion. In some embodiments, the compacted oligonucleotide comprises any one or any combination of two or more of the following: end phosphorylation of the 3' end; at least two terminal nucleotides at the 3' end with a phosphorothioate bond therebetween; at least one terminal nucleotide at the 3' end having a 2'-O-methyl moiety; and/or at least one 3' end nucleotide with a 2' fluoro base. In some embodiments, compressed oligonucleotides may be included in step (2) or (3).

In some embodiments, in the trapped nucleotide-polymerase mixture of step (2), the plurality of polymerases with strand translocation activity include phi29 DNA polymerase, large fragment of Bst DNA polymerase, Bsu DNA polymerase, and Bca ( Exo-) large fragment of DNA polymerase, Klenov fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, the two-step rolling ring amplification reaction on the support involves a helicase, a single-stranded binding (SSB) protein or a recombinase (e.g., T4 uvsX) and/or a recombinase accessory factor (e.g., T4 uvsY or It further comprises at least one accessory protein or enzyme including T4 gp32).

In some embodiments, the two-step rolling ring amplification reaction on the support can be performed at an isothermal temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

In some embodiments, a conkatimer is a group of repeat units comprising the sequence of interest (or its complementary sequence) and any adapter sequences (or its complementary sequence) present in the original covalently closed circular nucleic acid library molecule. It may contain at least 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more tandem copies.

In some embodiments, a two-step rolling ring amplification (RCA) reaction on the support by a trapped nucleotide-polymerase complex may be followed by a multiple translocation amplification (MDA) reaction or a plexing amplification reaction. In some embodiments, the RCA reaction with a trapped nucleotide-polymerase complex is not followed by a multiple transposition amplification reaction or a plexing amplification reaction. Exemplary MDA and plexing reactions are described below.

Two-step rotational amplification initiated in solution

Trapped nucleotide-polymerase complex

The present disclosure provides pairwise sequencing compositions and methods for generating a plurality of single-stranded nucleic acid concatemer template molecules using a two-step rolling ring amplification reaction in solution. The two-step rolling ring amplification method described herein uses non-catalytic, then catalytic divalent cations to synchronize rolling ring amplification events in solution and generate concatimers. Concatimer produced in solution using a two-step rolling ring amplification reaction can be distributed on a support on which a surface primer is immobilized. The concatimer can be hybridized to an immobilized surface primer and the rolling ring amplification reaction can continue on the support. This amplification method produces highly dense nanoballs containing high copy numbers of target sequence, which improves sequencing signal intensity.

In some embodiments, a two-step rolling ring amplification reaction in solution comprises (1) hybridizing a circular nucleic acid library molecule in solution to a soluble first amplification primer to form a template-primer duplex; (2) a template-primer duplex comprising (i) a plurality of polymerases having strand translocation activity, (ii) a first plurality comprising a mixture of dATP, dGTP, dCTP, dTTP, and a nucleotide analog having a cleavable moiety. a nucleotide, and (iii) a plurality of trapped nucleotides by contacting them with a non-catalytic divalent cation (e.g., strontium or barium) that mediates nucleotide binding but does not mediate nucleotide incorporation into the trapped nucleotide-polymerase complex. -forming a polymerase complex, wherein contacting is performed under conditions suitable for binding the individual polymerase to an immobilized, covalently closed circular nucleic acid library molecule to form a complexed polymerase and the individual complexed polymerase. is carried out under conditions suitable for binding to a nucleotide or nucleotide analog to form a trapped nucleotide-polymerase complex. In some embodiments, in the nucleotide mixture of the first plurality of nucleotides, the nucleotide having a cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxy Contains inosine. The rolling ring amplification reaction further comprises: (3) a plurality of trapped nucleotide-polymerase complexes with (i) at least one divalent cation (e.g., magnesium) that mediates nucleotide binding and mediates nucleotide incorporation; and/or manganese), and (ii) a mixture of nucleotide analogs having a cleavable moiety under conditions suitable for carrying out an isothermal rolling ring amplification reaction producing a plurality of fixed single-stranded concatimer template molecules. Performing nucleotide polymerization by contacting with a second plurality of nucleotides. In some embodiments, in the nucleotide mixture of the second plurality of nucleotides, the nucleotide analog having a cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or Contains oxyinosine. The RCA reaction in solution can be performed for an amount of time suitable to generate a plurality of concatimers that hybridize to each circular library molecule and then are distributed on a support to which a plurality of surface primers are immobilized. The RCA reaction in solution can be carried out for a very short period of time, a moderate period of time, or a longer period of time before distribution on the support.

In some embodiments, the two-step rotary ring amplification reaction lacks a plurality of condensed oligonucleotides, or the rotary ring amplification reaction can hybridize to a portion of the concatimer and collapse the concatimer into a more compact shape and size. It further comprises a compressed oligonucleotide. A condensed oligonucleotide is a single-stranded nucleic acid molecule that has two identical sequences separated by a short linker sequence, and the two identical sequences are reverse complementary to portions of the concatemer. Compact oligonucleotides can be of any length, for example 20 to 100 nucleotides. Two identical sequence regions of the condensation oligonucleotide can hybridize to the concatimer, pulling the distal portions of the concatimer together, resulting in compaction of the concatimer. In some embodiments, the compressed oligonucleotide is resistant to 3' exonuclease digestion and/or single strand endonuclease digestion. In some embodiments, the compacted oligonucleotide comprises any one or any combination of two or more of the following: end phosphorylation of the 3' end; at least two terminal nucleotides at the 3' end with a phosphorothioate bond therebetween; at least one terminal nucleotide at the 3' end having a 2'-O-methyl moiety; and/or at least one 3' end nucleotide with a 2' fluoro base. In some embodiments, compressed oligonucleotides may be included in step (2) or (3).

In some embodiments, in the trapped nucleotide-polymerase mixture of step (2), the plurality of polymerases with strand translocation activity include phi29 DNA polymerase, large fragment of Bst DNA polymerase, Bsu DNA polymerase, and Bca ( Exo-) large fragment of DNA polymerase, Klenov fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, the two-step rolling ring amplification reaction in solution involves a helicase, a single-stranded binding (SSB) protein, or a recombinase (e.g., T4 uvsX) and/or a recombinase accessory factor (e.g., T4 uvsY or It further comprises at least one accessory protein or enzyme including T4 gp32).

In some embodiments, the two-step spinning ring amplification reaction in solution can be performed at an isothermal temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

In some embodiments, a conkatimer is a group of repeat units comprising the sequence of interest (or its complementary sequence) and any adapter sequences (or its complementary sequence) present in the original covalently closed circular nucleic acid library molecule. It may contain at least 2, 5, 10, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more tandem copies.

In some embodiments, a two-step rolling ring amplification (RCA) reaction in solution by a trapped nucleotide-polymerase complex may be followed by a multiple dislocation amplification (MDA) reaction in solution or a plexing amplification reaction on a support. In some embodiments, the RCA reaction in solution with a trapped nucleotide-polymerase complex is not followed by a multiplex transposition amplification reaction or a plexing amplification reaction. Exemplary MDA and plexing reactions are described below.

Multiplex transposition amplification reaction with random-sequence primers

In some embodiments, the two-step rolling ring amplification method on a support or in solution can optionally be followed by a multiplex transposition amplification reaction using random-sequence soluble primers. The multiple translocation amplification reaction involves (1) a plurality of immobilized single-stranded concatimer template molecules as (i) a second plurality of polymerases having strand transposition activity, and (ii) a plurality of soluble amplification primers, each of which a plurality of soluble amplification primers, wherein the amplification primers are exonuclease-resistant, have 3' OH extendable ends, and comprise a random sequence capable of hybridizing to a portion of the concatimer template molecule, (iii) dATP , a third plurality of nucleotides comprising a mixture of dGTP, dCTP, dTTP and nucleotide analogs having a selectively cleavable moiety, and (iv) at least one divalent cation that mediates nucleotide binding and mediates nucleotide incorporation ( For example, magnesium and/or manganese) to form a multiple dislocation amplification (MDA) reaction mixture; and (2) performing an isothermal multiple dislocation amplification (MDA) reaction to generate a plurality of immobilized branched concatimers. In some embodiments, the nucleotide analog having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine.

In some embodiments, in the multiple translocation amplification (MDA) reaction mixture, the second plurality of polymerases having strand translocation activity is phi29 DNA polymerase, large fragment of Bst DNA polymerase, Bsu DNA polymerase, and Bca(exo-) Includes the large fragment of DNA polymerase, the Klenov fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, in a multiplex translocation amplification (MDA) reaction mixture, the plurality of amplification primers comprise single-stranded nucleic acid primers that are about 5 to 25 nucleotides in length. In some embodiments, the plurality of soluble amplification primers comprise unprotected single-stranded nucleic acid primers. In some embodiments, the plurality of soluble amplification primers comprise protected single-stranded nucleic acid primers that are resistant to 3' exonuclease digestion and/or single-strand endonuclease digestion. In some embodiments, the plurality of soluble amplification primers include terminal phosphorylation at the 3'end; at least two terminal nucleotides at the 3' end with a phosphorothioate bond therebetween; at least one terminal nucleotide at the 3' end having a 2'-O-methyl moiety; and/or at least one 3' end nucleotide with a 2' fluoro base. In some embodiments, the plurality of soluble amplification primers comprise a population of primers of the same length, for example, 6 or 9 nucleotides in length. In some embodiments, the plurality of soluble amplification primers comprise a population of primers having a mixture of different lengths, for example, a mixture comprising hexamer and nine-mer primers. In some embodiments, the plurality of soluble amplification primers are random sequences comprising up to 4 ⁶ different sequences (e.g., for hexamers) or ⁴⁹ different sequences (e.g., for 9-mers). It includes a mixture of primers having.

In some embodiments, the multiple translocation amplification (MDA) reaction mixture comprises a helicase, a single strand binding (SSB) protein or a recombinase (e.g., T4 uvsX) and/or a recombinase accessory factor (e.g., T4 uvsY or It may further include at least one accessory protein or enzyme including T4 gp32).

In some embodiments, the multiple potential amplification (MDA) reaction is conducted at an isothermal temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C. It can be done.

Multiple translocation amplification reaction using primase polymerase

In some embodiments, the two-step rotary ring amplification method on a support or in solution can optionally be followed by a multiplex translocation amplification reaction using primase-polymerase. The multiple translocation amplification reaction involves (1) a plurality of immobilized single-stranded concatimer template molecules , (i) a second plurality of polymerases having strand transposition activity, (ii) a plurality of DNA primase-polymerase enzymes, ( iii) a third plurality of nucleotides comprising a mixture of dATP, dGTP, dCTP, dTTP and nucleotide analogs with optionally cleavable moieties, and (iv) at least one 2 that mediates nucleotide binding and mediates nucleotide incorporation. contacting a cation (e.g., magnesium and/or manganese) to form a multiple dislocation amplification (MDA) reaction mixture; and (2) performing an isothermal multiple dislocation amplification (MDA) reaction to generate a plurality of immobilized branched concatimers. In some embodiments, multiple transposition amplification reactions are performed without added amplification primers (e.g., primer-free reactions). In some embodiments, the nucleotide analog having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine.

In some embodiments, in the multiple translocation amplification (MDA) reaction mixture, the second plurality of polymerases having strand translocation activity is phi29 DNA polymerase, large fragment of Bst DNA polymerase, Bsu DNA polymerase, and Bca(exo-) Includes the large fragment of DNA polymerase, the Klenov fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, the plurality of DNA primase-polymerases comprise an enzyme from Thermus thermophilus HB27 (e.g., a Tth PrimPol enzyme).

In some embodiments, the multiple translocation amplification (MDA) reaction mixture comprises a helicase, a single strand binding (SSB) protein or a recombinase (e.g., T4 uvsX) and/or a recombinase accessory factor (e.g., T4 uvsY or It further comprises at least one accessory protein or enzyme including T4 gp32).

In some embodiments, the multiple potential amplification (MDA) reaction is conducted at an isothermal temperature of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45°C. It can be done.

Flexing Amplification

In some embodiments, an immobilized single-stranded concatimer template molecule produced by a two-step rotating ring amplification method on a support or in solution can optionally be subjected to a two-step plexing amplification method on a support, which method Contacting a single-stranded concatimer template molecule with a nucleic acid relaxant (first step) and then performing a plexing amplification reaction during the second step. Without being bound by theory, the nucleic acid relaxant(s) cause the structure of the nucleic acid concatemer to relax and increase the number of new duplex formations between the anchored surface primer and the portion of the nucleic acid concatemer, thereby creating a duplexed anchored surface. It is postulated that it may be possible to disrupt hydrogen bonds (e.g., denaturation) in multiple immobilized concatimer template molecules, which increases the chance of generating new concatimers from the primers. New concatimers can be generated during the plexing amplification reaction. The inclusion of a relaxant can cause nucleic acid denaturation without the denaturation temperature or use of denaturation chemicals.

Generally, the amplification method includes the steps of: (1) performing a rolling ring amplification (e.g., two-step RCA) on a support to generate a plurality of immobilized single-stranded concatimer template molecules; (2) forming a relaxant reaction mixture by increasing and decreasing temperature, followed by washing; (3) forming a plexing amplification reaction mixture; (4) performing a plexing amplification reaction (e.g., without added soluble primer) on the support to generate a plurality of double-stranded concatemers; (5) washing; and (6) repeating steps (2) to (5) at least once.

In some embodiments, the plexing amplification method includes the steps of: (1) performing a rolling ring amplification on a support to generate a plurality of immobilized single-stranded concatimer template molecules; (2) Surface primers (e.g., covalently closed circular nucleic acid library molecules) immobilized by contacting a single-stranded concatimer template molecule with at least one nucleic acid relaxant under conditions of increasing temperature, relaxer incubation, and decreasing temperature. After forming the relaxer reaction mixture under conditions suitable to increase the number of duplex formations between portions of the single-stranded concatimer template molecule (not yet hybridized to or covalently linked to the single-stranded concatimer template molecule) , washing; (3) a single-stranded concatemer template molecule comprising (i) a plurality of polymerases having strand transposition activity, (ii) a plurality of nucleotides, wherein the concentration of the plurality of nucleotides is at least a threshold concentration or exceeds the threshold concentration, a plurality of nucleotides that promote polymerization, (iii) mediating nucleotide binding and mediating nucleotide polymerization (e.g., magnesium and/or manganese), but the plexing reaction mixture does not contain an added soluble amplification primer, 2 forming a plexing amplification reaction mixture by contacting it with a positive ion; (4) A step of performing a plexing amplification reaction under conditions suitable for generating a plurality of fixed double-stranded concatemers by carrying out temperature increasing conditions, amplification incubation conditions, and temperature decreasing conditions, wherein the plurality of double-stranded nucleic acid concatemers Each conkatimer in a mer consists of two or more tandems of repeat units comprising the sequence of interest (or its complementary sequence) and any adapter sequences (or its complementary sequence) present on the immobilized single-stranded conkatimer template molecule. carrying out the amplification reaction with duplicates; (5) washing the immobilized double-stranded concatimer to remove strand-transfer polymerase under conditions suitable for maintaining the immobilized double-stranded concatimer; and (6) repeating steps (2) to (5) at least once (e.g., at least one cycle).

In some embodiments, the relaxant reaction mixture of step (2) may be formed by at least one nucleic acid relaxant capable of disrupting hydrogen bonds in the immobilized nucleic acid concatemer. Exemplary relaxants include nucleic acid denaturants, chaotropic compounds, amide compounds, aprotic compounds, primary alcohols, and ethylene glycol derivatives. Chaotropic compounds include urea, guanidine hydrochloride or guanidine thiocyanate. Amide compounds include formamide, acetamide, or NN-dimethylformamide (DMF). Aprotic compounds include acetonitrile, dimethyl sulfoxide (DMSO), 1,4-dioxane, or tetrahydrofuran. Primary alcohols include 1-propanol, ethanol, or methanol. Ethylene glycol derivatives include 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane or 2-methoxyethanol. Other relaxants include sodium iodide, potassium iodide, and polyamines.

In some embodiments, the relaxant reaction mixture of step (2) includes urea, guanidine hydrochloride, guanidine thiocyanate, formamide, acetamide, NN-dimethylformamide (DMF), acetonitrile, dimethyl SO (DMSO), sulfoxide), 1,4-dioxane, tetrahydrofuran, 1-propanol, ethanol, methanol, 1,3-propanediol, ethylene glycol, glycerol, 1,2-dimethoxyethane, 2-methoxyethanol, It includes any one or a combination of two or more of the group selected from sodium iodide, potassium iodide and/or polyamines.

In some embodiments, the relaxant reaction mixture of step (2) includes formamide and SSC. In some embodiments, the relaxant reaction mixture includes acetonitrile, formamide, and SSC. In some embodiments, the relaxant reaction mixture includes acetonitrile, formamide, and MES (2-(4-morpholino)-ethane sulfonic acid). In some embodiments, the relaxant reaction mixture includes acetonitrile, formamide, guanidium hydrochloride, and HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid). In some embodiments, the relaxant reaction mixture includes acetonitrile, formamide, urea, and HEPES. In some embodiments, the SSC in the relaxant reaction mixture may be 1×, 2×, 3×, or 4×.

In some embodiments, in forming the relaxant reaction mixture of step (2), the temperature increase conditions may be carried out at a temperature of about 20°C to about 70°C and the relaxant incubation conditions may be carried out at a temperature of about 40°C to 70°C. and the temperature reduction condition may be performed at about 70°C to about 20°C. Those skilled in the art will recognize that the temperature increase, relaxant incubation temperature, and temperature decrease conditions may be modified.

In some embodiments, in the plexing amplification reaction mixture of step (3), the second plurality of polymerases having strand transposition activity is a large fragment of Bst DNA polymerase (e.g., exonuclease minus), phi29 DNA polymerase, the large fragment of Bsu DNA polymerase and Bca(exo-) DNA polymerase, the Klenov fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. Includes. phi29 DNA polymerase is wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific) or chimeric QualiPhi DNA polymerase (e.g., from 4basebio). ) can be.

In some embodiments, in the plexing amplification reaction mixture of step (3), the concentration (e.g., total concentration) of the third plurality of nucleotides can promote a nucleotide polymerization reaction. For example, the concentration (e.g., total concentration) of the third plurality of nucleotides is about 0.1 to 10 mM.

In some embodiments, the plurality of nucleotides in the plexing amplification reaction mixture of step (3) comprises a mixture of nucleotides comprising dATP, dGTP, dCTP, and a nucleotide analog having a cleavable moiety. In some embodiments, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine.

In some embodiments, in the plexing amplification reaction mixture of step (3), at least one divalent cation that mediates nucleotide binding and mediates nucleotide polymerization comprises a catalytic divalent cation. In some embodiments, the catalytic divalent cation includes magnesium and/or manganese. The concentration of catalytic divalent cations in the amplification reaction mixture may be about 1 to 20mM.

In one embodiment, the plexing amplification reaction mixture of step (3) contains a helicase, a single-stranded binding (SSB) protein or a recombinase (e.g., T4 uvsX) and/or a recombinase accessory factor (e.g., T4 uvsY). or at least one accessory protein or enzyme including T4 gp32). In some embodiments, these accessory proteins may be omitted.

In some embodiments, in the plexing amplification reaction of step (4), the temperature increase conditions may be performed at about 20°C to about 90°C.

In some embodiments, in the plexing amplification reaction of step (4), the temperature increase conditions are for about 5 to 15 seconds, or about 15 to 30 seconds, or about 30 to 45 seconds, or about 45 to 60 seconds, or longer. It can be done.

In some embodiments, in the plexing amplification reaction of step (4), the amplification incubation conditions are about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64. , 65, 66, 67, 68, 69 or 70° C., or higher.

In some embodiments, in the plexing amplification reaction of step (4), the amplification incubation conditions are about 30 to 45 seconds, or about 45 to 60 seconds, or about 60 to 75 seconds, or about 75 to 90 seconds, or longer. It can be.

In some embodiments, in the plexing amplification reaction of step (4), the temperature reducing conditions may be performed at about 90°C to about 20°C.

In some embodiments, in the plexing amplification reaction of step (4), the temperature reduction conditions are for about 5 to 15 seconds, or about 15 to 30 seconds, or about 30 to 45 seconds, or about 45 to 60 seconds, or longer. It can be done.

In some embodiments, in the wash of step (5), the wash buffer includes 1x SSC, or 1xSSC with cobalt hexamine.

In some embodiments, steps (2) through (5) can be repeated at least once, up to 10 times, up to 15 times, up to 20 times, or up to 30 or more times.

Sequencing method

The present disclosure provides a method of performing pairwise sequencing using a sequencing polymerase and labeled or unlabeled chain terminating nucleotides, wherein the chain terminating nucleotides contain a 3'-O-azido group (or 3'-O-methyl azido group) or any other type of bulky blocking group at the 3' position. For example, the sequencing-by-synthesis (SBS) method uses labeled chain-ending nucleotides, and the sequencing-by-binding method (SBB) uses unlabeled chains. -Use a termination nucleotide.

The present disclosure also provides a method for performing pairwise sequencing using sequencing-by-avidity method (SBA) using sequencing polymerase and labeled multivalent molecules and unlabeled chain termination nucleotides. Provides a method.

Sequencing by the binding activity of a template molecule (e.g., a DNA template molecule) ideally requires detection of (a) n+1 bases, two or more copies of the target nucleic acid sequence, and Two or more primer nucleic acid molecules complementary to one or more regions, and a polyvalent molecule ( (e.g., polymer-nucleotide conjugate), wherein the polymer-nucleotide conjugate comprises two or more nucleotide moieties; The detection substrate is subsequently washed, and (b) to ensure that only single incorporation occurs, structural modifications ('blockers') of the unlabeled nucleotide are required to ensure single nucleotide incorporation, which is then Prevents the incorporation of any additional nucleotides into the chain. The blocking group must then be removable under reaction conditions that do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then be continued by N+1 detection of the next multivalent polymerase-conjugate-DNA complex, etc. For practical use, the avidity step requires both (a) a stable substrate that lasts long enough to be imaged for >30 seconds and (b) a stepping step, so that the overall process is high yield, fairly specific. It should be comprised of a number of chemical and enzymatic steps that facilitate multiple cycles of sequencing.

Sequencing by synthesis (SBS) of template molecules (e.g., DNA template molecules) ideally requires controlled (i.e., one-by-one) incorporation of the correct complementary nucleotides facing the oligonucleotide being sequenced. . This allows accurate sequencing by adding nucleotides in multiple cycles, as each nucleotide residue is sequenced one by one, preventing an uncontrolled series of incorporations. Incorporated nucleotides are read using an appropriate label attached to them prior to removal of the label moiety and subsequent next round of sequencing. To ensure that only single incorporations occur, structural modifications ('blockers') of the sequenced nucleotide are necessary to ensure single nucleotide incorporation, but then prevent the incorporation of any additional nucleotides into the polynucleotide chain. The blocking group must then be removable under reaction conditions that do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then be continued by incorporation of the next blocking, marker nucleotide. To be of practical use, the entire process must consist of high yield, highly specific chemicals, and enzymatic steps to facilitate multiple cycles of sequencing.

Sequencing by binding of a template molecule (e.g., a DNA template molecule) (SBB) involves (a) sequentially contacting a primed template nucleic acid under ternary complex stabilizing conditions with at least two distinct mixtures, comprising: sequentially contacting the primed template nucleic acid in contact, each of the separate mixtures comprising a polymerase and a nucleotide, with the nucleotide cognate for the first, second and third base types in the template, under ternary complex stabilizing conditions. ; (b) testing at least two separate mixtures to determine whether a ternary complex is formed; and (c) identifying the next exact nucleotide for the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next exact nucleotide is a cognate of the first, second and third base types. identifying, wherein the correct nucleotide is believed to be a nucleotide cognate of the fourth base type based on the absence of a ternary complex in step (b); (d) adding the following purified nucleotides to the primers of the template nucleic acid primed after step (b) to generate extended primers; and (e) repeating steps (a) to (d) for the primed template nucleic acid comprising the extended primer.

How to analyze sequences with nucleotides

The present disclosure provides pairwise sequencing compositions and methods using methods for sequencing a plurality of immobilized concatimer template molecules and methods for sequencing a plurality of maintained forward extension strands. The sequencing method includes (a) a plurality of sequencing polymerases (i) a plurality of nucleic acid template molecules (e.g., a plurality of immobilized concatimer template molecules or a plurality of maintained forward extension strands) and (ii) A step of contacting a plurality of nucleic acid sequencing primers (e.g., a plurality of soluble forward sequencing primers or a plurality of soluble reverse sequencing primers), wherein the contacting step involves each sequencing polymerase bound to the nucleic acid duplex. The nucleic acid duplex comprises a nucleic acid template molecule that hybridizes to a nucleic acid sequencing primer. In some embodiments, the plurality of sequencing primers include a 3' OH extendable end. In some embodiments, soluble sequencing primers lack nucleotides with a cleavable moiety comprising uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. In some embodiments, the plurality of sequencing polymerases comprise recombinant sequencing polymerases. In some embodiments, the plurality of sequencing polymerases include an exonuclease-minus sequencing polymerase.

In some embodiments, the sequencing method further comprises (b) contacting the plurality of complexed sequencing polymerases with the plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase. Includes. In some embodiments, the complexed sequencing polymerase is contacted with a plurality of nucleotides in the presence of at least one catalytic cation that catalyzes polymerase-catalyzed nucleotide incorporation, wherein the catalytic cation comprises magnesium and/or manganese. do. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain terminating moiety at the 2' or 3' position of the sugar, wherein the chain terminating moiety may or may not be removable. In some embodiments, the plurality of nucleotides includes at least one nucleotide lacking a chain terminating moiety. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base by a linker that is cleavable/removable from the base, or is not removed from the base. In some embodiments, at least one nucleotide of the plurality is not labeled with a detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to detect and identify the nucleotide base. can make it possible.

In some embodiments, the sequencing method further comprises the step of (c) incorporating at least one nucleotide into the 3' end of the extendable primer. In some embodiments, at least one nucleotide binds to the complexed sequencing polymerase and is incorporated into the 3' end of the extendable primer. In some embodiments, the incorporation of a nucleotide into the 3' end of the primer in step ( c) comprises a primer extension reaction.

In some embodiments, the sequencing method further comprises the step of (d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

In some embodiments, the sequencing method further comprises (e) repeating steps (b), (c), and (d) at least once.

The present disclosure provides pairwise sequencing compositions and methods using the method to sequence a plurality of immobilized concatimer template molecules. The sequencing method includes the steps of (a) contacting a plurality of sequencing polymerases with (i) a plurality of immobilized concatimer template molecules and (ii) a plurality of soluble forward sequencing primers, which bind to a nucleic acid duplex. performing under conditions suitable for forming a plurality of complexed polymerases, each comprising a sequenced polymerase, wherein the nucleic acid duplex comprises an immobilized concatimer template molecule hybridized to a soluble forward sequencing primer; (b) in the presence of at least one catalytic cation (e.g., magnesium and/or manganese) that promotes polymerase-catalyzed nucleotide incorporation and for binding at least one nucleotide to the complexed sequencing polymerase. Contacting a plurality of complexed sequencing polymerases under suitable conditions with a plurality of nucleotides, wherein the plurality of nucleotides has a removable chain termination moiety at the sugar 3' position and has at least one nucleotide analog labeled with a fluorophore. comprising: steps; (c) incorporating at least one nucleotide into the 3' end of the hybridized forward sequencing primer to generate a plurality of initial extended forward sequencing primers; and (d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide. In some embodiments, the sequencing method includes (e) removing the chain termination moiety from the incorporated nucleotide while maintaining a plurality of complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, generating an extendable 3'OH group for the moiety, wherein the nucleic acid duplex comprises an immobilized concatimer template molecule hybridized to an initial extended forward sequencing primer; (f) the complexed polymerase is maintained in the presence of at least one catalytic cation that promotes polymerase-catalyzed nucleotide incorporation under conditions suitable for binding at least one nucleotide to the complexed sequencing polymerase. contacting a plurality of nucleotides, the plurality of nucleotides comprising at least one nucleotide analog that has a removable chain termination moiety at the sugar 3' position and is labeled with a fluorophore; (g) incorporating at least one nucleotide into the 3' end of the initial extended forward sequencing primer; (h) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide; and (i) repeating steps (e) to (g) at least once. In some embodiments, the removing step of step (e) can be performed using a cleavage reagent. Cleavage reagents may contain or lack one or more compounds that reduce photodamage, including antioxidants, triplet state quenchers, singlet oxygen quenchers, oxygen scavengers, electron scavengers, anti-fade formulations.

The present disclosure provides pairwise sequencing compositions and methods using the method for multiple maintained forward extension strands. The sequencing method comprises the steps of (a) contacting a plurality of sequencing polymerases with (i) a plurality of maintained forward extension strands and (ii) a plurality of soluble reverse sequencing primers, wherein the contacting is performed on the sequence bound to the nucleic acid duplex. Performed under conditions suitable to form a plurality of complexed polymerases, each comprising an assay polymerase, wherein the nucleic acid duplex comprises a retained forward extension strand hybridized to a soluble reverse sequencing primer; (b) in the presence of at least one catalytic cation (e.g., magnesium and/or manganese) that promotes polymerase-catalyzed nucleotide incorporation and for binding at least one nucleotide to the complexed sequencing polymerase. Contacting a plurality of complexed sequencing polymerases under suitable conditions with a plurality of nucleotides, wherein the plurality of nucleotides has a removable chain termination moiety at the sugar 3' position and has at least one nucleotide analog labeled with a fluorophore. comprising: steps; (c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer to generate a plurality of initial extended reverse sequencing primers; and (d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide. In some embodiments, the sequencing method includes (e) removing a chain termination moiety from an incorporated nucleotide while maintaining a plurality of complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex, generating an extendable 3'OH group for the moiety, wherein the nucleic acid duplex comprises a retained forward extension strand hybridized to the initial extended reverse sequencing primer; (f) the complexed polymerase is maintained in the presence of at least one catalytic cation that promotes polymerase-catalyzed nucleotide incorporation under conditions suitable for binding at least one nucleotide to the complexed sequencing polymerase. contacting a plurality of nucleotides, the plurality of nucleotides comprising at least one nucleotide analog that has a removable chain termination moiety at the sugar 3' position and is labeled with a fluorophore; (g) incorporating at least one nucleotide into the 3' end of the initial extended reverse sequencing primer; (h) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide; and (i) repeating steps (e) to (g) at least once. In some embodiments, the removing step of step (e) can be performed using a cleavage reagent. Cleavage reagents may contain or lack one or more compounds that reduce photodamage, including antioxidants, triplet state quenchers, singlet oxygen quenchers, oxygen scavengers, electron scavengers, and anti-fade formulations.

Sequencing method using multivalent molecules and nucleotides

The present disclosure provides a method for sequencing a plurality of immobilized conkatimer template molecules (or a plurality of immobilized non-conkatimer template molecules) and pairwise sequencing of a plurality of maintained forward extension strands using the sequencing method. Compositions and methods are provided. Sequencing methods generally include the following steps: (1) performing a sequencing reaction at a position on a template molecule using a multivalent molecule that binds but does not incorporate; and (2) performing a sequencing reaction at the same position on the template molecule using nucleotides by incorporation; and (3) repeating steps (1) and (2) at the next position on the template molecule. Nucleotide incorporation in step (2) generates an extended forward sequencing primer strand or an extended reverse sequencing primer strand. Repeating steps (1) and (2) produces an extended forward sequencing primer strand or an extended reverse sequencing primer strand.

A sequencing method involves (a) a plurality of first sequencing polymerases comprising (i) a plurality of nucleic acid template molecules (e.g., a plurality of immobilized concatimer template molecules or a plurality of maintained forward extension strands) and (ii) a plurality of first sequencing polymerases. ) a step of contacting with a plurality of soluble nucleic acid sequencing primers (e.g., a plurality of soluble forward sequencing primers or a plurality of soluble reverse sequencing primers), wherein the contacting step includes the first sequencing coupled to the nucleic acid duplex. The nucleic acid duplex comprises a nucleic acid template molecule that hybridizes to a soluble nucleic acid sequencing primer. In some embodiments, the plurality of sequencing primers include a 3' OH extendable end. In some embodiments, the plurality of nucleic acid template molecules comprises a plurality of immobilized concatimer template molecules and the plurality of nucleic acid sequencing primers comprise a plurality of soluble forward sequencing primers. In some embodiments, the plurality of nucleic acid template molecules comprise a plurality of maintained forward extension strands and the plurality of nucleic acid sequencing primers comprise a plurality of soluble reverse sequencing primers. In an alternative embodiment, the first and second template molecules are clonally amplified, non-concatimer template molecules that are positioned proximally and in close proximity to each other. For example, the first and second clonally amplified template molecules include linear template molecules generated through bridge amplification and anchored to the same position or feature on the support. The first and second non-conkatimeric template molecules comprise the sequence of interest and at least one universal sequencing primer binding site. The first and second soluble sequencing primers can bind to sequencing primer binding sites on the first and second non-concatimer template molecules, respectively.

In some embodiments, the sequencing method further comprises step (b) contacting the plurality of first complexed polymerases with a plurality of multivalent molecules to form a plurality of multivalent-complexed polymerases. In some embodiments, individual multivalent molecules of the plurality of multivalent molecules comprise a core attached to multiple nucleotide arms, each nucleotide arm attached to a nucleotide (e.g., a nucleotide unit) (see Figures 104-114 ). In some embodiments, the contacting step of step (b) is performed with a mixture of any combination of two or more types of multivalent molecules, wherein the individual multivalent molecules in the mixture are dATP, dGTP, dCTP, dTTP and/or dUTP. It contains a nucleotide unit selected from the group consisting of. In some embodiments, the contacting step of step (b) is suitable for linking complementary nucleotides of a multivalent molecule to at least two of the plurality of first complexed polymerases to form a plurality of multivalent-complexed polymerases. carried out under the following conditions: The nucleotide unit of the multivalent molecule binds to the 3' end of the sequencing primer (or the 3' end of the initial extended sequencing primer) at a position opposite the complementary nucleotide in the template molecule. In some embodiments, conditions are suitable to inhibit incorporation of complementary nucleotide units into the sequencing primer (or into the initial extended sequence primer) of the plurality of multivalent-complexed polymerases. In some embodiments, the contacting step of step (b) allows binding of the nucleotide units to the complexed polymerase but does not allow binding of the nucleotide units to the sequencing primer of the complexed polymerase (or to the initial extended sequencing primer). It is performed in the presence of non-catalytic divalent cations that inhibit polymerase-catalyzed incorporation, the non-catalytic divalent cations including strontium and/or barium. In some embodiments, the plurality of multivalent molecules comprise at least one multivalent molecule having multiple nucleotide arms each attached to a nucleotide analog (e.g., nucleotide analog unit), wherein the nucleotide analog has a sugar 2' and/or It contains a removable chain terminating moiety at the 3' position. In some embodiments, the plurality of multivalent molecules comprise at least one multivalent molecule comprising multiple nucleotide arms each attached to a nucleotide unit lacking a chain termination moiety. In some embodiments, at least one multivalent molecule of the plurality of multivalent molecules is labeled with a detectable reporter moiety. In each multivalent molecule, at least one detectable moiety is attached to the core, or the detectable moiety is attached to at least one nucleotide unit, or the detectable moiety is attached to at least one linker. In some embodiments, the detectable reporter moiety comprises a fluorophore.

In some embodiments, the sequencing method further comprises step (c) detecting a plurality of multivalent-complexed polymerases. In some embodiments, the detecting step includes detecting a multivalent molecule bound to a complexed polymerase, wherein complementary nucleotide units of the multivalent molecule are bound to the sequencing primer, but incorporation of the complementary nucleotide units is inhibited. . In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety to enable detection. In some embodiments, the core is labeled with a detectable reporter moiety. In some embodiments, at least one linker and/or at least one nucleotide unit of the nucleotide arm is labeled with a detectable reporter moiety.

In some embodiments, the sequencing method determines the sequence of a nucleic acid template by identifying the nucleobases of complementary nucleotide units bound to a first plurality of complexed polymerases (in a plurality of multivalent-complex polymerases). Further comprising step (d) . In some embodiments, the multivalent molecule comprises nucleotides to enable identification of complementary nucleotide units (e.g., nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to a plurality of first complexed polymerases. The arm is labeled with a detectable reporter moiety corresponding to the specific nucleotide unit attached. In some embodiments, a given multivalent molecule is labeled with a known fluorophore to enable differentiation and identification of the nucleobases of the given multivalent molecule.

In some embodiments, in a sequencing method, association of the plurality of first complexed polymerases with the plurality of multivalent molecules forms at least one avidity complex, the method comprising the following steps: (1) 1 A soluble sequencing primer (e.g., a soluble forward sequencing primer or a soluble reverse sequencing primer), a first sequencing polymerase, and a first multivalent molecule are coupled to a first portion of a nucleic acid concatemer template molecule (e.g., binding to an immobilized concatimer template molecule or a maintained forward extension strand to form a first binding complex (e.g., a first multivalent-complexed polymerase), wherein the first nucleotide of the first multivalent molecule wherein the unit binds to a first sequencing polymerase; and (2) a second soluble sequencing primer (e.g., a soluble forward sequencing primer or a soluble reverse sequencing primer), a second sequencing polymerase, and the first multivalent molecule of the same nucleic acid concatemer template molecule. linking the moiety to form a second binding complex (e.g., a second multivalent-complexed polymerase), wherein the second nucleotide unit of the second multivalent molecule binds to the second sequencing polymerase, but is identical to the second binding complex. Forming the second binding complex, wherein the first and second binding complexes comprising the multivalent molecule form an avidity complex.

In some embodiments, the first sequencing primer comprises a soluble forward sequencing primer and the nucleic acid template molecule comprises an immobilized concatimer template molecule. In some embodiments, the second sequencing primer comprises a soluble forward sequencing primer and the nucleic acid template molecule comprises the same immobilized concatimer template molecule. The first sequencing primer and the second sequencing primer have the same sequence.

In some embodiments, the first sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises a retained forward extension strand. In some embodiments, the second sequencing primer comprises a soluble reverse sequencing primer and the nucleic acid template molecule comprises the same maintained forward extension strand. The first sequencing primer and the second sequencing primer have the same sequence.

In some embodiments, the first sequencing polymerase comprises any wild type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild type or mutant polymerase described herein. The concatimer template molecule comprises tandem repeat sequences of the sequence of interest and at least one universal sequencing primer binding site. The first and second soluble sequencing primers can bind to the sequencing primer binding site along the concatimer template molecule.

In some embodiments, the sequencing method includes combining a plurality of first complexed polymerases with a plurality of multivalent molecules to form at least one avidity complex, the method comprising the following steps: (1 ) A plurality of first sequencing polymerases and a plurality of second sequencing primers are contacted with different portions of a nucleic acid concatimer template molecule (e.g., an immobilized concatimer template molecule or a maintained forward extension strand) to obtain the same forming at least first and second complexed polymerases on a nucleic acid concatemer template molecule; (2) at least first and second complexed polymerization of a plurality of multivalent molecules on the same nucleic acid concatemer template molecule under conditions suitable for binding a single multivalent molecule from the plurality to the first and second complexed polymerases. contacting the enzyme, wherein at least the first nucleotide unit of the single multivalent molecule is bound to a first complexed polymerase comprising a first sequencing primer hybridized to a first portion of the nucleic acid concatemer template molecule to form a first complexed polymerase. A second sequencing primer that forms a binding complex (e.g., a first multivalent-complexed polymerase) and hybridizes to a second portion of the nucleic acid concatemer template molecule, wherein at least the second nucleotide unit of the single multivalent molecule is the same. is bound to a second complexed polymerase comprising to form a second binding complex (e.g., a second multivalent-complexed polymerase), and the contacting step includes binding in the first and second binding complexes. performed under conditions suitable to inhibit polymerase-catalyzed incorporation of the first and second nucleotide units, wherein the first and second binding complexes bound to the same multivalent molecule form an avidity complex; (3) detecting the first and second binding complexes on the same nucleic acid concatemer template molecule, and (4) identifying the first nucleotide unit in the first binding complex to determine the sequence of the first portion of the nucleic acid template molecule. and determining the sequence of the second portion of the same nucleic acid template molecule by identifying the second nucleotide unit in the second binding complex.

In some embodiments, the plurality of DNA polymerases include any of the wild-type or mutant polymerases described herein. The concatimer template molecule comprises tandem repeat sequences of the sequence of interest and at least one universal sequencing primer binding site. A plurality of first and second sequencing primers may bind to sequencing primer binding sites along the concatimer template molecule.

In some embodiments, the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the nucleic acid concatimer template molecules comprise immobilized concatimer template molecules. In some embodiments, the plurality of second sequencing primers comprises a plurality of second soluble forward sequencing primers, and the nucleic acid template molecules comprise identical immobilized concatimer template molecules. The plurality of first sequencing primers and second sequencing primers have the same sequence.

In some embodiments, the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises a maintained forward extension strand. In some embodiments, the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, and the nucleic acid template molecules comprise identical maintained forward extension strands. The plurality of first sequencing primers and second sequencing primers have the same sequence.

In some embodiments, in a sequencing method, association of the plurality of first complexed polymerases with the plurality of multivalent molecules forms at least one avidity complex, the method comprising the following steps: (1) 1 A soluble sequencing primer (e.g., a soluble forward sequencing primer or a soluble reverse sequencing primer), a first sequencing polymerase, and a first multivalent molecule are combined with a first template molecule (e.g., an immobilized template molecule or retainer). forming a first binding complex (e.g., a first multivalent-complexed polymerase), wherein the first nucleotide unit of the first multivalent molecule is attached to the first sequencing polymerase. combining, step; and (2) linking a second soluble sequencing primer (e.g., a soluble forward sequencing primer or a soluble reverse sequencing primer), a second sequencing polymerase, and the first multivalent molecule to a second template molecule, thereby forming a second linkage. Forming a complex (e.g., a second multivalent-complexed polymerase), wherein the second nucleotide unit of the second multivalent molecule binds to the second sequencing polymerase, but comprises the first multivalent molecule. and forming the second binding complex, wherein the second binding complex forms an avidity complex.

In some embodiments, the first sequencing polymerase comprises any wild type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild type or mutant polymerase described herein. In some embodiments, the first and second template molecules are clonally amplified template molecules. In some embodiments, the first and second template molecules are positioned close together and proximal. For example, the first and second clonally amplified template molecules include linear template molecules generated through bridge amplification and anchored to the same position or feature on the support. The first and second template molecules include the sequence of interest and at least one universal sequencing primer binding site. The first and second soluble sequencing primers can bind to sequencing primer binding sites on the first and second template molecules, respectively.

In some embodiments, the first soluble sequencing primer comprises a soluble forward sequencing primer and the first template molecule comprises an immobilized first template molecule. In some embodiments, the second soluble sequencing primer comprises a soluble forward sequencing primer and the first template molecule comprises the same immobilized first template molecule. The first and second soluble sequencing primers have identical sequences.

In some embodiments, the first soluble sequencing primer comprises a soluble reverse sequencing primer and the first template molecule comprises a retained forward extension strand. In some embodiments, the second soluble sequencing primer comprises a soluble reverse sequencing primer and the second template molecule comprises an identical maintained forward extension strand. The first and second soluble sequencing primers have identical sequences.

In some embodiments, the sequencing method includes combining a plurality of first complexed polymerases with a plurality of multivalent molecules to form at least one avidity complex, the method comprising the following steps: (1 ) a plurality of sequencing polymerases and a plurality of soluble sequencing primers (including first and second soluble sequencing primers), respectively, into first and second template molecules (e.g., immobilized first or second templates) forming at least a first and a second complexed polymerase on the first and second template molecules, respectively; (2) contacting the plurality of multivalent molecules with at least the first and second complexed polymerases under conditions suitable to bind the single multivalent molecules from the plurality to the first and second complexed polymerases, At least the first nucleotide unit of the multivalent molecule is bound to a first complexed polymerase comprising a first sequencing primer hybridized to a first template molecule to form a first binding complex (e.g., a first multivalent-complexed polymerase), and at least the second nucleotide unit of the single multivalent molecule is bound to a second complexed polymerase comprising a second sequencing primer hybridized to a second template molecule to form a second binding complex (e.g. , a second multivalent-complexed polymerase), and the step of contacting is under conditions suitable to inhibit polymerase-catalyzed incorporation of the first and second nucleotide units bound in the first and second binding complexes. wherein the first and second binding complexes bound to the same multivalent molecule form an avidity complex; (3) detecting the first and second binding complexes on the first and second template molecules, and (4) identifying the first nucleotide unit in the first binding complex to determine the sequence of the first template molecule, 2. Determining the sequence of the second template molecule by identifying the second nucleotide unit in the binding complex.

In some embodiments, the plurality of DNA polymerases include any of the wild-type or mutant polymerases described herein. The first and second template molecules are clonally amplified template molecules. In some embodiments, the first and second template molecules are positioned close together and proximal. For example, the first and second clonally amplified template molecules include linear template molecules generated through bridge amplification and anchored to the same position or feature on the support. The first and second template molecules include the sequence of interest and at least one universal sequencing primer binding site. The first and second soluble primers can bind to sequencing primer binding sites on the first and second template molecules, respectively.

In some embodiments, the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the first template molecules comprise immobilized first template molecules. In some embodiments, the plurality of second sequencing primers comprises a plurality of second soluble forward sequencing primers, and the second template molecules comprise the same immobilized second template molecule. The plurality of first sequencing primers and second sequencing primers have the same sequence.

In some embodiments, the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the first template molecule comprises a first maintained forward extension strand. In some embodiments, the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, and the second template molecule comprises a second retained forward extension strand. The plurality of first sequencing primers and second sequencing primers have the same sequence.

In some embodiments, the sequencing method comprises the steps of dissociating the plurality of multivalent-complexed polymerases, removing the plurality of first sequencing polymerases and their associated multivalent molecules, and maintaining the plurality of nucleic acid duplexes (e ) further includes.

In some embodiments, the sequencing method comprises contacting the plurality of maintained nucleic acid duplexes of (e) with a plurality of second sequencing polymerases, wherein contacting comprises contacting the plurality of second sequencing polymerases with the plurality of maintained nucleic acid duplexes. further comprising step (f) , performed under conditions suitable for binding to the retained nucleic acid duplex, to form a plurality of second complexed polymerases each comprising a second sequencing polymerase linked to the retained nucleic acid duplex. do.

In some embodiments, the first plurality of sequencing polymerases of step (a) has an amino acid sequence that is 100% identical to the amino acid sequence of the second plurality of sequencing polymerases of step (f). In some embodiments, the first plurality of sequencing polymerases of step (a) has an amino acid sequence that is different from the amino acid sequence of the second plurality of sequencing polymerases of step (f).

In some embodiments, the sequencing method further comprises (g) contacting the plurality of second complexed polymerases with a plurality of nucleotides (e.g., free nucleotides), wherein contacting comprises contacting the plurality of second complexed polymerases with a plurality of nucleotides (e.g., free nucleotides). is carried out under conditions suitable for linking the complementary nucleotides of to at least two of the second complexed polymerases of step (f) to form a plurality of nucleotide-complexed polymerases. In some embodiments, the contacting step of step (g) promotes incorporation of the bound complementary nucleotide into the sequencing primer (or initial extended sequencing primer) of the nucleotide-complexed polymerase, thereby forming a plurality of nucleotide- This is carried out under conditions suitable to form complexed polymerase. In some embodiments, the incorporation of nucleotides into the 3' end of the primer in step (g) comprises a primer extension reaction. In some embodiments, the contacting step of step (g) is performed in the presence of a catalytic divalent cation that promotes polymerase-catalyzed nucleotide incorporation. In some embodiments, the catalytic divalent cation includes magnesium and/or manganese. In some embodiments, the plurality of nucleotides comprise natural nucleotides (e.g., non-analog nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides comprise removable or non-removable 2' and/or 3' chain termination moieties. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base by a linker that is cleavable/removable from the base, or is not removed from the base. In some embodiments, at least one nucleotide of the plurality is not labeled with a detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to detect and identify the nucleotide base. can make it possible.

In some embodiments, the sequencing method further comprises step (h) detecting complementary nucleotides incorporated into the sequencing primer (or initial extended sequencing primer) of the nucleotide-complexed polymerase. In some embodiments, the plurality of nucleotides are labeled with a detectable reporter moiety to enable detection. In some embodiments, step (h) of detecting is omitted.

In some embodiments, the sequencing method further comprises step (i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer (or initial extended sequencing primer) of the nucleotide-complexed polymerase. In some embodiments, the identification of complementary nucleotides incorporated in step (i) can be used to identify the identity of the complementary nucleotide units of the multivalent molecule bound to the plurality of first complexed polymerases in step (d). there is. In some embodiments, identifying step (i) can be used to determine the sequence of a nucleic acid template molecule. In some embodiments, identifying step (i) is omitted.

In some embodiments, the sequencing method comprises step (g) contacting a plurality of second complexed polymerases with a plurality of nucleotides comprising at least one nucleotide having a 2' and/or 3' chain termination moiety. It further comprises step (j), when performed, removing the chain termination moiety from the incorporated nucleotide. In some embodiments, the removing step of step (j) can be performed using a cleavage reagent. Cleavage reagents may contain or lack one or more compounds that reduce photodamage, including antioxidants, triplet state quenchers, singlet oxygen quenchers, oxygen scavengers, electron scavengers, anti-fade formulations.

In some embodiments, the sequencing method further comprises step (k) repeating steps (a) through (j) at least once by using the same nucleic acid sequencing primers of step (a). In some embodiments, the sequence of a nucleic acid template molecule can be determined by detecting and identifying multivalent molecules that bind to the sequencing polymerase but are not incorporated into the 3' ends of the primers of steps (c) and (d). In some embodiments, the sequence of a nucleic acid template molecule can be determined (or confirmed) by detecting and identifying nucleotides incorporated into the 3' end of the primer in steps (h) and (i).

In an alternative sequencing method, forming the plurality of complexed polymerases comprises the step (a) under conditions suitable for linking complementary nucleotides from the plurality of nucleotides to the complexed polymerase from the plurality of complexed polymerases ) contacting the plurality of complexed polymerases with a plurality of nucleotides to form a nucleotide-complexed polymerase (b2) . In some embodiments, the contacting step of step (b2) is performed under conditions suitable to promote nucleotide binding but inhibit incorporation of the bound complementary nucleotide into the 3' end of the primer of the nucleotide-complexed polymerase. . In some embodiments, the contacting step of step (b2) is performed in the presence of at least one cation selected from the group consisting of strontium, barium, sodium, magnesium, potassium, manganese, calcium, lithium, nickel, and cobalt. A plurality of complexed polymerases may be sequentially contacted with at least two separate mixtures, each mixture comprising an engineered polymerase and nucleotides. The contacting step is carried out under conditions suitable to form a stable ternary complex with the cognate for the first, second and third base types in the template. The method further comprises step (c3) testing at least two separate mixtures to determine whether a ternary complex is formed. The method includes identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (c3) the next correct nucleotide is identified as a cognate of the first, second and third base types. and further comprising a step (d3) of identifying, wherein the next correct nucleotide is believed to be a nucleotide cognate of the fourth base type based on the absence of a ternary complex in step (c3). The method includes the steps of step (e3) adding the following correct nucleotides to the primer of the primed template nucleic acid after step (c3) to generate an extended primer; and step (f3 ) repeating steps (a) to (e3) for the primed template nucleic acid comprising the extended primer.

nucleotide

In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides comprises a base, a sugar, and at least one phosphate group. In some embodiments, at least one nucleotide in the plurality comprises an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of nucleotides may include at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides may be comprised in a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP. In some embodiments, at least one nucleotide in the plurality is not a nucleotide analog. In some embodiments, at least one nucleotide in the plurality comprises a nucleotide analog.

In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides comprises a chain of 1, 2, or 3 phosphorus atoms, the chain typically comprising ester or phosphoramide linkages. It is attached to the 5' carbon of the sugar moiety. In some embodiments, at least one nucleotide in the plurality is an analog having a phosphorus chain in which the phosphorus atom is linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom of the chain comprises a substituted side group comprising O, S, or BH ₃ . In some embodiments, the chain comprises a phosphate group substituted with analogs including phosphoramidate, phosphorothioate, phosphodithioate, and O-methylphosphoroamidite groups.

In some embodiments, in any of the sequencing methods described herein, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. Detectable reporter moieties include fluorophores. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base by a linker that is cleavable/removable from the base. In some embodiments, at least one nucleotide of the plurality is not labeled with a detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to detect and identify the nucleotide base. can make it possible. In some embodiments, the method further comprises detecting at least one incorporated nucleotide in steps (c) and/or (d). In some embodiments, the method further comprises identifying at least one incorporated nucleotide in steps (c) and/or (d). In some embodiments, the sequence of a nucleic acid template molecule can be determined by detecting and identifying nucleotides (nucleobases) that bind to a sequencing polymerase, thereby determining the sequence of the nucleic acid template. In some embodiments, the sequence of a nucleic acid template molecule can be determined by detecting and identifying nucleotides (nucleobases) incorporated into the 3' end of a primer, thereby determining the sequence of the nucleic acid template. In some embodiments, detection and identification of fluorescently labeled nucleotides can be achieved by imaging the support on which the sequencing reaction is performed.

In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides has a chain termination moiety at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. (e.g., a blocking moiety). In some embodiments, the chain termination moiety may inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent strand during the primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3' sugar hydroxyl position and the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain termination moiety is removable/cleavable from the 3' sugar hydroxyl position, creating a nucleotide with a 3'OH sugar group that is extendable by subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety is an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, t It includes radical groups, disulfide groups, carbonate groups, urea groups, or silyl groups. In some embodiments, the chain terminating moiety is cleavable/removable from the nucleotide, for example, by reacting the chain terminating moiety with a chemical agent, pH change, light, or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are terminated by tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ), by piperidine, or 2, It can be cleaved by 3-dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the chain terminating moieties aryl and benzyl are cleavable by H2 Pd/C. In some embodiments, the chain terminating moiety amine, amide, keto, isocyanate, phosphate, thio, disulfide is conjugated with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). It can be cut by In some embodiments, the chain terminating moiety carbonate is cleavable by potassium carbonate (K ₂ CO ₃ ) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride, or by triethylamine trihydrofluoride.

In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides has a chain termination moiety at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. (e.g., a blocking moiety). In some embodiments, the chain terminating moiety comprises an azide, azido, or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3'-O-azido or 3'-O-azidomethyl group. In some embodiments, the chain terminating moieties azide, azido and azidomethyl groups are cleavable/removable by phosphine compounds. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound is tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or tris(hydroxypropyl)phosphine (THPP) or tris(hydroxypropyl)phosphine (THPP). Includes oxymethyl)phosphine (THMP). In some embodiments, the cutting agent includes 4-dimethylaminopyridine (4-DMAP).

In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides has a chain termination moiety (e.g., a blocking moiety) at the 2' and/or 3' positions. and a terminator nucleotide, wherein the incorporated chain terminating nucleotide is removed/cleaved by contacting the incorporated chain terminating nucleotide with a cleavage agent. Cleavage reagents may contain or lack one or more compounds that reduce photodamage, including antioxidants, triplet state quenchers, singlet oxygen quenchers, oxygen scavengers, electron scavengers, anti-fade formulations.

In some embodiments, in any of the sequencing methods described herein in a plurality of nucleotides, at least one nucleotide is 3'-deoxy nucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3' -azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoro Methyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3 '-aminomethyl, 3'-ethyl, 3'butyl, 3'- tert butyl, 3'-fluorenylmethyloxycarbonyl, 3'tert-butyloxycarbonyl , 3'-O-alkyl hydroxylamino group, and a chain terminating moiety selected from the group consisting of 3'-phosphorothioate and 3-O-benzyl, or derivatives thereof.

In some embodiments, in any of the sequencing methods described herein, at least one nucleotide of the plurality of nucleotides is labeled with a detectable reporter moiety. Detectable reporter moieties include fluorophores. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base by a linker that is cleavable/removable from the base. In some embodiments, at least one nucleotide of the plurality is not labeled with a detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to detect and identify the nucleotide base. can make it possible.

In some embodiments, in any of the sequencing methods described herein, a linker cleavable on a base may include an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, It includes cleavable moieties including an earthenware group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, a linker cleavable on a base is cleavable/removable from the base by reacting the cleavable moiety with a chemical agent, pH change, light, or heat. In some embodiments, the cleavable moieties alkyl, alkenyl, alkynyl, and allyl are cleavable with tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ), with piperidine, or 2, It can be cleaved by 3-dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the cleavable moieties aryl and benzyl are cleavable by H2 Pd/C. In some embodiments, the cleavable moiety amine, amide, keto, isocyanate, phosphate, thio, disulfide is conjugated with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). It can be cut by In some embodiments, the cleavable moiety carbonate is cleavable by potassium carbonate (K ₂ CO ₃ ) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH). In some embodiments, the cleavable moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride, or by triethylamine trihydrofluoride.

In some embodiments, in any of the sequencing methods described herein, the cleavable linker on a base comprises a cleavable moiety comprising an azide, azido, or azidomethyl group. In some embodiments, the cleavable moieties azide, azido and azidomethyl groups are cleavable/removable by phosphine compounds. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or tri(hydroxypropyl)phosphine (THPP). In some embodiments, the cutting agent includes 4-dimethylaminopyridine (4-DMAP).

In some embodiments, in any of the sequencing methods described herein, the chain termination moiety on the base (e.g., at the sugar 2' and/or sugar 3' positions) and the removable linker are identical or different removable moieties. have a tee In some embodiments, the chain termination moiety linked to the base (e.g., at the sugar 2' and/or sugar 3' positions) and the detectable reporter moiety are chemically cleavable/removable by the same chemical agent. In some embodiments, the chain termination moiety linked to the base (e.g., at the sugar 2' and/or sugar 3' positions) and the detectable reporter moiety are chemically cleavable/removable by different chemical agents.

multivalent molecule

The present disclosure provides compositions and methods for pairwise sequencing, including a method for sequencing a plurality of immobilized concatimer template molecules and a method for sequencing a plurality of maintained forward extension strands, wherein the sequencing method includes a method for sequencing a multivalent molecule. Use . In some embodiments, in the sequencing method, at least one multivalent molecule of the plurality of multivalent molecules of step (b) comprises (1) a core; and (2) a plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core comprises a plurality of nucleotides. Attached to the arm, the spacer is attached to the linker, and the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit includes a base, a sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the linker comprises an aliphatic chain or an oligo ethylene glycol chain, both linker chains having 2 to 6 subunits. In some embodiments, the linker also includes an aromatic moiety. Exemplary multivalent molecules are shown in Figures 104-107. Exemplary nucleotide arms are shown in Figures 108 and 114. An exemplary spacer is shown in Figure 109 (top) and an exemplary linker is shown in Figure 109 (bottom) and Figures 110-113.

In some embodiments, in the sequencing method, each multivalent molecule of the plurality of multivalent molecules of step (b) comprises a core attached to a multiple nucleotide arm, and the multiple nucleotide arms are comprised of dATP, dGTP, dCTP, dTTP, and dUTP. It has nucleotide units of the same type selected from the group consisting of Alternatively, a multivalent molecule comprises a core attached to multiple nucleotide arms, with the different nucleotide arms attached to different types of nucleotide units (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), and the multivalent molecule has 2 Contains any combination of more than two different nucleotide units.

In some embodiments, in the sequencing method, the nucleotide units of at least one multivalent molecule of step (b) include an aromatic base, a pentose sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1 to 10 phosphate groups). The plurality of multivalent molecules may include one type of multivalent molecule having one type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP. The plurality of multivalent molecules may comprise a mixture of any combination of two or more types of multivalent molecules, wherein each multivalent molecule in the mixture contains nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP. Includes.

In some embodiments, in the sequencing method, at least one of the plurality of multivalent molecules of step (b) comprises a nucleotide unit having a chain of 1, 2, or 3 phosphorus atoms, the chain typically being an ester or It is attached to the 5' carbon of the sugar moiety via a phosphoramide linkage. In some embodiments, at least one nucleotide unit is a nucleotide analog having a phosphorus chain in which the phosphorus atom is linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atom of the chain comprises a substituted side group comprising O, S, or BH ₃ . In some embodiments, the chain comprises a phosphate group substituted with analogs including phosphoramidate, phosphorothioate, phosphodithioate, and O-methylphosphoroamidite groups.

In some embodiments, in the sequencing method, each multivalent molecule of the plurality of multivalent molecules of step (b) comprises a core attached to multiple nucleotide arms, wherein each nucleotide arm is at the sugar 2' position and at the sugar 3' position. or at the 2' and 3' positions. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In some embodiments, in the sequencing method, at least one of the plurality of multivalent molecules of step (b) has a chain termination moiety at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. It comprises a nucleotide unit comprising a terminator nucleotide analog having a group (e.g., a blocking moiety). In some embodiments, the chain termination moiety may inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent strand during the primer extension reaction. In some embodiments, the chain terminating moiety is attached to the 3' sugar hydroxyl position and the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain termination moiety is removable/cleavable from the 3' sugar hydroxyl position, creating a nucleotide with a 3'OH sugar group that is extendable by subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain terminating moiety is an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, t It includes radical groups, disulfide groups, carbonate groups, urea groups, or silyl groups. In some embodiments, the chain termination moiety is cleavable/removable from a nucleotide unit, for example, by reacting the chain termination moiety with a chemical agent, pH change, light, or heat. In some embodiments, the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are terminated by tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ), by piperidine, or 2, It can be cleaved by 3-dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ). In some embodiments, the chain terminating moieties aryl and benzyl are cleavable by H2 Pd/C. In some embodiments, the chain terminating moiety amine, amide, keto, isocyanate, phosphate, thio, disulfide is conjugated with phosphine or with a thiol group including beta-mercaptoethanol or dithiothritol (DTT). It can be cut by In some embodiments, the chain terminating moiety carbonate is cleavable by potassium carbonate (K ₂ CO ₃ ) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH). In some embodiments, the chain terminating moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride, or by triethylamine trihydrofluoride.

In some embodiments, in the sequencing method, at least one of the plurality of multivalent molecules of step (b) has a chain termination moiety at the sugar 2' position, at the sugar 3' position, or at the sugar 2' and 3' positions. It comprises a nucleotide unit comprising a terminator nucleotide analog having a group (e.g., a blocking moiety). In some embodiments, the chain terminating moiety comprises an azide, azido, or azidomethyl group. In some embodiments, the chain terminating moiety comprises a 3'-O-azido or 3'-O-azidomethyl group. In some embodiments, the chain terminating moieties azide, azido and azidomethyl groups are cleavable/removable by phosphine compounds. In some embodiments, the phosphine compound comprises a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine compound includes tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP) or tri(hydroxypropyl)phosphine (THPP). In some embodiments, the cutting agent includes 4-dimethylaminopyridine (4-DMAP).

In some embodiments, in the sequencing method, at least one of the plurality of multivalent molecules of step (b) is 3'-deoxy nucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3 '-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoro Romethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydral, 3'-aminomethyl, 3'-ethyl, 3'butyl, 3'- tert butyl, 3'-fluorenylmethyloxycarbonyl, 3' tert -butyloxycarbonyl, 3'-O-alkyl hydroxylamino group , 3'-phosphorothioate and 3-O-benzyl, or derivatives thereof.

In some embodiments, in a sequencing method, at least one of the plurality of multivalent molecules of step (b) comprises a core attached to a multi-nucleotide arm, wherein the core is labeled with a detectable reporter moiety, or has at least one One nucleotide unit is labeled with a detectable reporter moiety, or at least one linker is labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to the multivalent molecule corresponds to a base in a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), It can enable detection and identification.

In some embodiments, in a sequencing method, at least one nucleotide arm of one of the plurality of multivalent molecules of step (b) has a nucleotide unit attached to a detectable reporter moiety. In some embodiments, the detectable reporter moiety is attached to a nucleotide base. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a multivalent molecule corresponds to a base in a nucleotide unit (e.g., dATP, dGTP, dCTP, dTTP, or dUTP), It can enable detection and identification.

In some embodiments of the sequencing method, the core of the multivalent molecule can be labeled with a detectable reporter moiety (e.g., a fluorophore) in a manner that allows for differentiation between different multivalent molecules carrying different types of nucleotide units. . For example, the core of the first multivalent molecule is labeled with a first fluorophore, and the first multivalent molecule includes multiple nucleotide-arms with dGTP nucleotide units. The core of the second multivalent molecule is labeled with a second fluorophore (different from the first fluorophore), and the second multivalent molecule comprises multiple nucleotide-arms with dATP nucleotide units. Binding and incorporation events of nucleotide units can be detected, and specific bases of nucleotide units (as part of a multivalent molecule) can be identified based on detection and identification of detectable reporter moieties on the first and second cores.

In some embodiments of the sequencing method, at least one linker of the nucleotide-arm of the multivalent molecule may contain a detectable reporter moiety (e.g., a fluorescent reporter moiety) in a manner that allows differentiation between different multivalent molecules carrying different types of nucleotide units. However, it can be labeled as). For example, at least one linker of the first multivalent molecule is labeled with a first fluorophore, and the first multivalent molecule comprises multiple nucleotide-arms having dGTP nucleotide units. At least one linker of the second multivalent molecule is labeled with a second fluorophore (different from the first fluorophore), and the second multivalent molecule comprises multiple nucleotide-arms with dATP nucleotide units. Binding and incorporation events of nucleotide units can be detected, and specific bases of nucleotide units (as part of a multivalent molecule) can be identified based on detection and identification of detectable reporter moieties on the first and second linkers.

In some embodiments of the sequencing method, at least one nucleotide unit (e.g., a nucleobase) of the nucleotide-arm of the multivalent molecule is detectable in a manner that allows for differences between different multivalent molecules carrying different types of nucleotide units. Can be labeled with a reporter moiety (eg, a fluorophore). For example, at least one nucleotide unit of the first multivalent molecule is labeled with a first fluorophore, and the first multivalent molecule includes multiple nucleotide-arms having dGTP nucleotide units. At least one nucleotide unit of the second multivalent molecule is labeled with a second fluorophore (different from the first fluorophore), and the second multivalent molecule comprises multiple nucleotide-arms with dATP nucleotide units. Binding and incorporation events of nucleotide units can be detected, and specific bases of a nucleotide unit (as part of a multivalent molecule) can be identified based on detection and identification of detectable reporter moieties on the first and second nucleotide units. .

In some embodiments, in the sequencing method, the core of the multivalent molecule of step (b) comprises a streptavidin-type or avidin-type moiety and the core attachment moiety comprises biotin. In some embodiments, the core is a streptavidin-type or avidin-type moiety comprising streptavidin or an avidin protein, as well as any derivatives, analogs, and other non-natural forms of avidin that can bind to at least one biotin moiety. Includes shape. Other forms of streptavidin or avidin moiety include natural and recombinant avidin and streptavidin as well as derivatized molecules, such as aglycosylated avidin and truncated streptavidin. . For example, the avidin moiety may include the deglycosylated form of avidin, the bacterial streptavidin produced by Streptomyces (e.g., Streptomyces avidinii ), as well as the derivatized Forms, for example, of N-acyl avidins, such as N-acetyl, N-phthalyl and N-succinyl avidin, and the commercially available products ExtrAvidin™, Captavidin™, Neutravidin™ and Neutralite Avidin™.

In some embodiments, in the sequencing method, the core of the multivalent group of step (b) is comprised of at least one detectable reporter moiety, e.g., a streptavidin-type or avidin-type moiety labeled with a fluorophore. Includes. The streptavidin-type or avidin-type core may be labeled with 1, 2, 3, 4 or more detectable moieties. The streptavidin-type or avidin-type core may be labeled with one or more of the same type of detectable reporter moiety. In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a streptavidin-type or avidin-type core is a base (e.g., a base of a nucleotide unit) that allows detection and identification of the multivalent molecule. For example, dATP, dGTP, dCTP, dTTP or dUTP).

It is postulated that increased binding of a nucleotide to an enzyme (e.g., a polymerase) or enzyme complex can be achieved by increasing the effective concentration of the nucleotide. The increase can be achieved by increasing the concentration of the nucleotide in free solution, or by increasing the amount of nucleotide proximate to the relevant binding site. Increases can also be achieved by physically confining a large number of nucleotides to a limited volume, thus resulting in a local increase in concentration, e.g., thus resulting in a structure that is larger than that observed with individual nucleotides that are unconjugated, untethered, or otherwise unconstrained. It can bind to the binding site with higher apparent binding activity. One exemplary means of achieving this limitation is that the multivalent nucleotides are incorporated into particles (or cores), such as polymers, branched polymers, dendrimers, micelles, liposomes, microparticles, nanoparticles, quantum dots, or other suitable particles known in the art. By providing a multivalent molecule bound to the particle.

Multivalent molecules can increase the effective concentration of nucleotides in free solution or can increase the amount of nucleotides proximal to the nucleotide binding site or nucleotide incorporation site of the polymerase. Increases in effective concentration can also be achieved by physically limiting the number of nucleotides to a limited volume and thereby increasing the local nucleotide concentration. Multivalent molecules can bind polymerase with increased apparent binding activity compared to unconjugated nucleotides. When a nucleotide unit is complementary to the interrogated position of the template molecule, the multivalent molecule forms a binding complex with the polymerase and the template molecule, and the binding complex is formed using a single unconjugated or untethered nucleotide (e.g., a free nucleotide). It exhibits increased stability and longer duration than the bound complex. When bound to polymerase, the multivalent molecule can increase the local concentration of nucleotides increasing multiple fold, ultimately improving signal intensity. When bound to polymerase, multivalent molecules can exhibit longer durations that can shorten the fluorescence signal imaging for detection and identification steps. When combined with a polymerase, the multivalent molecule forms a stable complex with the polymerase. The stable complex can survive the washing steps, so the signal intensity remains high throughout the imaging and washing steps. The stable complex can dissociate (e.g., by changing the buffer composition) and thus the primed target nucleic acid can undergo extension in subsequent reactions with free nucleotides.

Multivalent molecules can be used to localize a detectable signal to the active site of a biochemical interaction, such as a protein-nucleic acid interaction, a nucleic acid hybridization reaction, or an enzymatic reaction, such as a polymerase reaction. For example, the multivalent molecules described herein can be used to elongate nucleic acid chains during polymerase-catalyzed reactions, to identify base binding sites for nucleic acid template molecules or base incorporation, and to distinguish bases for sequencing and array-based applications. It can be used to provide When the nucleotides are complementary to the target nucleic acid, the increased binding between the nucleic acid template molecules and nucleotide units of the multivalent binding composition provides an enhanced signal that greatly improves base calling accuracy and reduces imaging time.

Additionally, the use of multivalent molecules allows the sequencing signal from a given template molecule to be derived from within the polony region containing multiple copies of the template sequence. Sequencing methods involving multiple copies of a target sequence (e.g., concatimer molecules) have the advantage that the signal can be amplified due to the presence of multiple simultaneous sequencing reactions within a defined region, each providing its own signal. have The presence of multiple signals within a given region can also result in any single skipped cycle due to the fact that signals from a very large number of correct base calls can overwhelm signals from a smaller number of skipped or incorrect base calls. A method is provided to reduce the impact of and thus reduce phase errors and/or improve read length of a sequencing reaction.

The multivalent molecules disclosed herein and their uses include (i) stronger signals for better base-calling accuracy compared to conventional nucleic acid amplification and sequencing methods; ii) allows greater differentiation of sequence-specific and background signals; (iii) reduced requirements for the amount of starting material required, (iv) increased sequencing rates and shortened sequencing times; (v) reducing phase error and (vi) improving read length of the sequencing reaction.

Those skilled in the art will recognize that, in a series of iterative sequencing reactions, sometimes one or more sites will fail to incorporate nucleotides during a given cycle, thus rendering one or more sites out of synchronization with the bulk of the extended nucleic acid chain. Under conditions where the sequencing signal is derived from a reaction occurring on a single copy of the target nucleic acid, this failure to incorporate will yield extra errors in the output sequence. The use of multivalent molecules for sequencing can reduce these types of errors in sequencing reactions. For example, the use of a multivalent substrate that can bind to the polymerase-template-primer complex or be incorporated into the extended strand provides an increased likelihood of recombination upon premature dissociation of the ternary polymerase complex, thereby allowing bases to be incorporated. It can reduce the frequency of “skipped” cycles that are not completed. Accordingly, in some embodiments, the present disclosure contemplates the use of multivalent molecules as disclosed herein comprising nucleotides having a 5' phosphate, diphosphate, or triphosphate moiety, either free or reversibly modified. , the nucleotides are linked to the particle or polymer as disclosed herein through labile or cleavable linkages. In some embodiments, the present disclosure contemplates a reduction in the inherent error rate due to skipped incorporation as a result of using the multivalent substrates disclosed herein.

When each sequencing cycle runs completely, each reaction within a given region will give the same signal. However, as noted elsewhere herein, in a series of iterative sequencing reactions, sometimes one or more sites will fail to incorporate nucleotides during a given cycle, and thus one or more sites will become out of sync with the bulk of the extended nucleic acid chain. do. This problem, referred to as “topology,” causes degradation of the sequencing signal because the signal is contaminated with spurious signals from regions that have one or more skipped cycles. This ultimately creates the possibility for errors in base identification. The gradual accumulation of skipped cycles over multiple cycles also reduces the effective read length due to gradual degradation of the sequencing signal with each cycle. A further object of the present disclosure is to provide a method to reduce phase errors and/or improve read length of a sequencing reaction.

Sequencing methods using multivalent molecules can result in reduced error rates manifested by fewer base misidentifications, reporting of non-existent bases, or failure to report correct bases. In some embodiments, the reduction in error rate is about 5% compared to the error rate observed using a monovalent ligand comprising a free nucleotide, a labeled free nucleotide, a protein or peptide linked nucleotide, or a labeled protein or peptide linked nucleotide. , it can be a reduction of 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200% or more.

Sequencing methods using multivalent molecules result in approximately 5 read lengths compared to the average read length observed using monovalent ligands comprising free nucleotides, labeled free nucleotides, protein- or peptide-linked nucleotides, or labeled protein- or peptide-linked nucleotides. %, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300% or more.

Sequencing methods using multivalent molecules result in approximately 10 read lengths compared to the average read length observed using monovalent ligands comprising free nucleotides, labeled free nucleotides, protein- or peptide-linked nucleotides, or labeled protein- or peptide-linked nucleotides. , may result in increased average read lengths of more than 20, 25, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 500 nucleotides.

The use of multivalent molecules for sequencing can effectively shorten the sequencing time. The sequencing reaction cycle including contact, detection and incorporation steps is performed in a total time ranging from about 5 minutes to about 60 minutes. In some embodiments, the sequencing reaction cycle lasts at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes. In some embodiments, the sequencing reaction cycle is performed for at most 60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 10 minutes, or at most 5 minutes. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure, for example, in some embodiments, the sequencing reaction cycle may have a total time ranging from about 10 minutes to about 30 minutes. can be performed. Those skilled in the art will recognize that the sequencing cycle time can have any value within this range, for example about 16 minutes.

The use of multivalent molecules for sequencing can provide improved base read accuracy. Improved compositions and methods for nucleic acid sequencing will provide average Q-scores for base-call accuracy over sequencing runs ranging from about 20 to about 50. In some embodiments, the average Q-score is at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. Those skilled in the art will recognize that the average Q-score can have any value within this range, for example about 32. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, It will provide a Q-score greater than 30 for at least 95%, at least 98%, or at least 99%. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, It will provide a Q-score greater than 35 for at least 95%, at least 98%, or at least 99% of the time. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, It will provide a Q-score greater than 40 for at least 95%, at least 98%, or at least 99%. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, It will provide a Q-score greater than 45 for at least 95%, at least 98%, or at least 99%. In some embodiments, the disclosed compositions and methods for nucleic acid sequencing comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, It will provide a Q-score greater than 50 for at least 95%, at least 98%, or at least 99%.

In some embodiments, the multivalent molecule has a core comprising a branched polymer known in the art; dendrimer; crosslinked polymer particles such as agarose, polyacrylamide, acrylates, methacrylates, cyanoacrylates, methyl methacrylate particles; glass particles; ceramic particles; metal particles; Quantum dot; liposome; Emulsion particles or any other particles (e.g., nanoparticles, microparticles, etc.).

The particles or cores may also have binding moieties. In some embodiments, the particles or cores can self-associate without the use of separate interacting moieties. In some embodiments, the particles or cores are subjected to buffer conditions or salt conditions, e.g., lipid- or polymer-mediated interactions of micelles or liposomes, as in the case of calcium-mediated interactions of hydroxyapatite particles, or metals (e.g., iron). or gold) nanoparticles may self-associate due to salt-mediated aggregation.

A multivalent molecule may have one or more labels (e.g., a detectable reporter moiety). Examples of labels include fluorophores, spin labels, metals or metal ions, color labels, nanoparticles, PET labels, radiolabels, or other methods known in the art to provide the composition detectable by macromolecular or molecular interaction detection methods. Other such signs may include, but are not limited to. The label can be attached to the nucleotide (e.g., by attachment to the base or 5' phosphate moiety of the nucleotide), to the particle itself (e.g., to the PEG subunit), or to the core (e.g., streptavidin or avidin core). ), at the ends of the polymer, at the central moiety, or at any other within the multivalent molecule recognized by those skilled in the art sufficient to provide such compositions as particles detectable by such methods known in the art or described elsewhere herein. Can be attached to any location. In some embodiments, a given multivalent molecule is labeled with a known fluorophore to enable differentiation and identification of the nucleobases of the given multivalent molecule.

One example of a multivalent molecule is a polymer-nucleotide conjugate. Examples of branched polymers include polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyglycine, polyvinyl acetate, dextran, or other such polymers. In one embodiment, the polymer is PEG. In other embodiments, the polymer may have PEG branches.

Suitable polymers can be characterized by repeating units bearing functional groups suitable for derivatization, such as amine, hydroxyl, carbonyl or allyl groups. The polymer may also have one or more pre-derivatized substituents such that one or more particular subunits contain derivatization or branching sites, whether or not the other subunits contain the same sites, substituents or moieties. Prederivatized substituents include, for example, nucleotides, nucleosides, nucleotide analogs, labels, such as fluorescent labels, radioactive labels, or spin labels, interaction moieties, additional polymeric moieties, etc., or any of the foregoing. It may include any combination, or may include additionally.

In multivalent molecules (e.g., polymer-nucleotide conjugates), the polymer may have multiple branches. Branched polymers can have a variety of configurations, including but not limited to star-like (“starburst”) forms, aggregated star-like (“helter skelter”) forms, bottle brushes, or dendrimers. there is. The branched polymer may radiate from a central point of attachment or central moiety, or may contain multiple branching points, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more branching points. there is. In some embodiments, each subunit of the polymer may optionally constitute a separate branching point. Exemplary nucleotide arms are shown in Figure 108 and exemplary multivalent molecules are shown in Figures 104-107.

In multivalent molecules, the length and size of the branches can vary based on the type of polymer. In some branched polymers, the branches are 1 to 1,000 nm, 1 to 100 nm, 1 to 200 nm, 1 to 300 nm, 1 to 400 nm, 1 to 500 nm, 1 to 600 nm, 1 to 700 nm, 1 to 700 nm. It may have a length of greater than 800 nm, or 1 to 900 nm, or within or between any of the values disclosed herein. In some branched polymers, the branches are 1K, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 30K, 50K, 80K, 100K, or any number within the range defined by any two of the foregoing. The value may have a size corresponding to the apparent molecular weight. The apparent molecular weight of the polymer is the known molecular weight of a representative number of subunits, as determined by size exclusion chromatography, as determined by mass spectra, or by any other method known in the art. It can be calculated from The polymer may have multiple branches. The number of polymer branches can be 2, 3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number within a range determined by any two of these values.

For multivalent molecules (e.g., polymer-nucleotide conjugates), the branched polymer may have 4, 8, 16, 32, or 64 branches, with 0, 1, 2, 3, 4, 5, or 6 branches at each end. The PEG molecule may have a nucleotide attached to the end, such that more than one nucleotide is attached. In one non-limiting example, the branched polymer of 3 to 128 PEG arms attached to a polymer branch is such that 0, 1, 2, 3, 4, 5, 6 or more nucleotides or nucleotide analogs are attached to each end. , ends with one or more nucleotides. In some embodiments, the branched polymer or dendrimer has an even number of arms. In some embodiments, the branched polymer or dendrimer has an odd number of arms.

In multivalent molecules (e.g., polymer-nucleotide conjugates), each branch or subset of branches of the polymer contains nucleotides (e.g., adenine, thymine, uracil, cytosine, or guanine residues or derivatives or mimetics thereof). A moiety comprising a can be attached, and the moiety can bind to a polymerase, reverse transcriptase, or other nucleotide binding domain. Optionally, the nucleotide moiety may bind but not be incorporated into the polymerase-template-primer complex, or may be incorporated into the extending nucleic acid chain during the polymerase reaction. In some embodiments, the nucleotide moiety includes a chain termination moiety that blocks incorporation of subsequent nucleotides during a polymerase-mediated reaction. In some embodiments, a nucleotide moiety may be unblocked (reversibly blocked) such that subsequent nucleotides cannot be incorporated into the extending nucleic acid chain during the polymerase reaction until such blocking is removed, and then subsequently Nucleotides can be incorporated into an extended nucleic acid chain during a polymerase reaction.

Multivalent molecules may additionally have binding moieties on each branch or subset of branches. Some examples of binding moieties include biotin, avidin, streptavidin, etc., polyhistidine domains, complementary paired nucleic acid domains, G-quadruplet forming nucleic acid domains, calmodulin, maltose-binding protein, cellulase, mal Contains methylcellulose, sucrose, glutathione-S-transferase, glutathione, O-6-methylguanine-DNA methyltransferase, benzylguanine and its derivatives, benzylcysteine and its derivatives, antibodies, epitopes, protein A, protein G. However, it is not limited to these. The binding moiety may be any interaction molecule or fragment or small molecule interaction domain or moiety known in the art that binds to or promotes the interaction between proteins, between proteins and ligands, between proteins and nucleic acids, or between nucleic acids. You can.

In some embodiments, a multivalent molecule may include one or more components of a complementary interaction moiety. Exemplary complementary interaction moieties include, for example, biotin and avidin; SNAP-benzylguanosine; Antibodies or FABs and epitopes; IgG FC and protein A, protein G, protein A/G, or protein L; maltose binding protein and maltose; lectins and cognate polysaccharides; Nucleic acids capable of forming ionic chelating moieties, complementary nucleic acids, triple strands or triple helix interactions; Includes nucleic acids that can form a G-quadruplet. Those skilled in the art will recognize that multiple moiety pairs exist and are commonly used for their ability to interact strongly and specifically with each other; Accordingly, it will be readily appreciated that any such complementary pair or set is considered suitable for this purpose in constituting or contemplating the compositions of the present disclosure. In some embodiments, compositions as disclosed herein are such that one component of the complementary interaction moiety is attached to one molecule or multivalent ligand and the other component of the complementary interaction moiety is attached to a separate molecule or It may include a composition that attaches to a multivalent ligand. In some embodiments, compositions as disclosed herein may include compositions in which both or both components of a complementary interaction moiety are attached to a single molecule or multivalent ligand. In some embodiments, compositions as disclosed herein may include compositions in which both or both components of a complementary interaction moiety are attached to separate arms or positions of a single molecule or multivalent ligand. In some embodiments, compositions as disclosed herein may include compositions in which both or both components of a complementary interaction moiety are attached to the same arm or position of a single molecule or multivalent ligand. In some embodiments, a composition comprising one component of a complementary interaction moiety and a composition comprising another component of a complementary interaction moiety may be mixed simultaneously or sequentially. In some embodiments, interactions between molecules or particles as disclosed herein enable, for example, the association or aggregation of multiple molecules or particles such that a detectable signal is increased. In some embodiments, fluorescent, chromatic, or radioactive signals are enhanced. In other embodiments, other interaction moieties as disclosed herein or known to those skilled in the art are contemplated. In some embodiments, one or more molecules comprising a first interaction moiety, e.g., one or more imidazole or pyridine moieties, and one or more additional molecules comprising a second interaction moiety, e.g. For example, compositions as provided herein may be provided such that histidine residues are mixed simultaneously or sequentially. In some embodiments, the composition includes 1, 2, 3, 4, 5, 6 or more imidazole or pyridine moieties. In some embodiments, the composition includes 1, 2, 3, 4, 5, 6 or more histidine residues. In this embodiment, interactions between molecules or particles as provided may be promoted by the presence of divalent cations such as nickel, manganese, magnesium, calcium, strontium, etc. In some embodiments, for example, a (His)3 group may interact with a (His)3 group on another molecule or phase through coordination of nickel or manganese ions.

The multivalent binding composition may include one or more buffers, salts, ions, or additives. In some embodiments, representative additives include betaine, spermidine, detergents such as Triton Cellulose, heparin, heparan sulfate, glycerol, sucrose, 1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol, propylene glycol, polypropylene glycol, block copolymer, For example, the Pluronic (r) series polymers, arginine, histidine, imidazole, or any combination thereof, or any substance known in the art to be a DNA "relaxer" (which increases the accessibility of a site within a strand to the DNA binding moiety). Preferably, compounds having the effect of altering the persistence length of DNA, altering the number of bonds or cross-links in the polymer, or altering the conformational dynamics of the DNA molecule) may include, but are not limited to, these.

Multivalent molecules may include zwitterionic compounds as additives. Additional exemplary additives include those described in Lorenz, T.C., incorporated herein by reference for a disclosure of additives for promoting nucleic acid binding or kinetics, or for promoting processes involving the manipulation, use or storage of nucleic acids. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012)].

In some embodiments, the sequencing reaction involves polymerization of multivalent molecules known in the art to promote nucleic acid interactions, such as self-association, secondary or tertiary structure formation, base pairing, surface association, peptide association, protein binding, etc. It includes an enzyme, a primed template molecule, and at least one cation. Examples of cations include, but are not limited to, sodium, magnesium, strontium, barium, potassium, manganese, calcium, lithium, nickel, cobalt, or other such cations.

When multivalent molecules are used to replace unconjugated or untethered nucleotides to form complexes with polymerase and nucleic acid template molecules, the local concentration of nucleotides is increased severalfold, which ultimately improves signal intensity, especially mismatch versus correction signal. I order it. The present disclosure contemplates contacting a multivalent binding composition with a polymerase and a primed template molecule to determine the formation of a binding complex.

Because of the increased local concentration of nucleotides for the polymer-nucleotide conjugate, binding between the polymerase, the primed template strand and the nucleotide becomes more favorable when the nucleotide is complementary to the next base on the template strand. The binding complex formed has a longer duration, ultimately shortening the imaging step. The high signal intensity resulting from the use of polymer nucleotide conjugates remains for the entire binding and imaging steps. The strong bond between the polymerase, the primed template strand and the nucleotide or nucleotide analog also ensures that the formed binding complex will remain stabilized during the washing steps and that the signal will remain high intensity when other reaction mixtures and unmatched nucleotide analogs are washed away. It means there will be. After the imaging step, the binding complex can be destabilized and the primed template strand can then be extended for one base. After extension, the binding and imaging steps can be repeated again by the use of polymer nucleotide conjugates to determine the identity of the next base.

The compositions and methods of the present disclosure not only provide a robust and controllable means of establishing and maintaining a three-way enzyme complex (e.g., during sequencing), but also provide a greatly improved means by which the presence of the complex can be identified and/or measured. Means are provided, and means by which the persistence of the complex can be controlled. This provides an important solution to problems such as determining the identity of N+1 bases in nucleic acid sequencing applications.

While not intending to be bound by any particular theory, the multivalent binding compositions disclosed herein form ternary complexes at a time-dependent rate that is substantially slower than the association rate known to be achieved with nucleotides in free solution. It was observed to associate with the polymerase nucleotide complex. Accordingly, the on-rate (Kon) is substantially and surprisingly slower than the on-rate for a single nucleotide or a nucleotide not attached to a multivalent ligand complex. However, importantly, the off rate (Koff) of the multivalent ligand complex is substantially slower than that observed for the nucleotide in free solution. Accordingly, the multivalent ligand complexes of the present disclosure provide a surprising and advantageous improvement in the persistence of ternary polymerase-polynucleotide-nucleotide complexes (particularly over complexes formed by free nucleotides), compared to, for example, currently available methods and reagents. It enables significant improvements in imaging quality for even more nucleic acid sequencing applications. Importantly, it is not inherently involved in the dissociation of the complex and allows it to undergo subsequent visualization, transformation or processing steps - i.e., the complex can be formed, imaged, modified or used in other ways if required, and the user can use a dissociation buffer This property of the multivalent substrates disclosed herein provides for controllable formation of visible ternary complexes, such that they remain stable until a positive dissociation step is performed, such as exposing the complex to .

Compositions and methods for making and using multivalent molecules (also called polymer-nucleotide conjugates) are described in U.S. Patent No. 16/579,794, filed September 23, 2019, the contents of which are herein expressly incorporated in their entirety. INCORPORATED BY REFERENCE.

sequencing polymerase

The present disclosure provides compositions and methods for pairwise sequencing, including methods for sequencing a plurality of immobilized concatimer template molecules and methods for sequencing a plurality of maintained forward extension strands, including any of the methods described herein. The sequencing method uses at least one type of sequencing polymerase and a plurality of nucleotides. In some embodiments, the sequencing polymerase(s) may incorporate complementary nucleotides opposite the nucleotides bearing the cleavable moiety in the immobilized concatimer template molecule. In some embodiments, the plurality of sequencing polymerases include recombinant sequencing polymerases.

Examples of suitable polymerases for use in sequencing utilizing nucleotides include, but are not limited to: Klenow DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaea; Candidatus Hadarchaeum Yellowstonense ; Hadesarchaea archaea; Euryarchaeota archaea; Thermoplasmata archaea; Thermococcus polymerase, such as Thermococcus litoralis , bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; Bacteriophage polymerases, such as T4, RB69 and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alpha and epsilon; 9° N polymerase; Reverse transcriptase, such as HIV type M or O reverse transcriptase; avian myeloblastosis virus reverse transcriptase; Moloney Murine Leukemia Virus (MMLV) reverse transcriptase; Or telomerase. Additional non-limiting examples of DNA polymerases include those from various archaeal genera, such as Aeropyrum , Archaeglobus , Desulfurococcus , Pyrobaculum , Pyrococcus ( Polymerization from Pyrococcus , Pyrolobus , Pyrodictium , Staphylothermus , Steteria, Sulfolobus , Thermococcus and Vulcanisaeta, etc. or variants thereof Enzymes include polymerases such as those known in the art such as 9° N, VENT, DEEP VENT, THERMINATOR, Pfu, KOD, Pfx, Tgo and RB69 polymerase.

The present disclosure provides compositions and methods for pairwise sequencing, including methods for sequencing a plurality of immobilized concatimer template molecules and methods for sequencing a plurality of maintained forward extension strands, including any of the methods described herein. The sequencing method uses a first type of sequencing polymerase and a plurality of multivalent molecules, and a second type of sequencing polymerase and a plurality of nucleotides. In some embodiments, the first sequencing polymerase is a multivalent molecule facing a nucleotide (e.g., uridine, 8-oxoG, or deoxyinosine) having a moiety that can cleave from the immobilized conkatimer template molecule. It can bind to the complementary nucleotide unit of. In some embodiments, the second sequencing polymerase binds a complementary nucleotide opposite a nucleotide (e.g., uridine, 8-oxoG, or deoxyinosine) that has a moiety that can cleave from the immobilized concatimer template molecule. Can be incorporated into nucleotides. In some embodiments, the first and second plurality of sequencing polymerases comprise recombinant sequencing polymerases. In some embodiments, the plurality of first and second sequencing polymerases comprise one, two or more substitution mutations that confer exonuclease minus properties.

Examples of suitable first and/or second sequencing polymerases for use in sequencing by multivalent molecules and nucleotides include, but are not limited to: Klenow DNA polymerase; Dermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaea; Candidatus hadarkum yellowstonens; Hadesarchea archaea; Eurycarcheota archaea; Thermoplasmata archaea; Thermococcus polymerase, such as Thermococcus littoralis, bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; Bacteriophage polymerases, such as T4, RB69 and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alpha and epsilon; 9° N polymerase; Reverse transcriptase, such as HIV type M or O reverse transcriptase; avian myeloblastosis virus reverse transcriptase; Moloney murine leukemia virus (MMLV) reverse transcriptase; Or telomerase. Additional non-limiting examples of DNA polymerases include those from various archaeal genera, such as Erophirum, Acaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Includes polymerases from Stetheria, Sulfolobus, Thermococcus and Vulcanisaeta, etc. or variants thereof, including 9° N, VENT, DEEP VENT, THERMINATOR, Pfu, KOD, Pfx, Tgo and RB69 polymerases It includes polymerases such as those known in the art.

In some embodiments, the plurality of first and/or second sequencing polymerases comprise mutant polymerases that exhibit increased fidelity and/or increased incorporation rates of nucleotide analogs compared to wild-type polymerases. In some embodiments, the nucleotide analog includes a chain terminating moiety (e.g., a blocking moiety) at the sugar 2' position and/or the 3' sugar position. In some embodiments, the plurality of first and/or second sequencing polymerases exhibit increased thermal stability compared to the wild-type polymerase. In some embodiments, the plurality of first and/or second sequencing polymerases exhibit increased ability to incorporate nucleotides into the 3' end of a primer or the template molecule contains at least one uridine (e.g., uracil). -resistant sequencing polymerase) incorporates nucleotides into the nascent strand in the primer extension reaction. In some embodiments, the first polymerase has the same or different amino acid sequence as the second polymerase.

In some embodiments, a polymerase suitable for performing a sequencing reaction includes a polymerase capable of binding a nucleic acid duplex (e.g., a template molecule hybridized to a primer) to form a complexed polymerase, the complex The converted polymerase binds to nucleotides to form a binding complex. The binding complex can be converted to a ternary complex.

In some embodiments, a polymerase suitable for performing a sequencing reaction (e.g., a first polymerase) binds to a nucleic acid duplex (e.g., a template molecule hybridized to a primer) to form a complexed polymerase. It contains a polymerase capable of forming a binding complex by binding to the nucleotide units of the multivalent molecule. The binding complex can be converted to a ternary complex.

In some embodiments, a polymerase suitable for performing a sequencing reaction (e.g., a second polymerase) binds to a nucleic acid duplex (e.g., a template molecule hybridized to a primer) to form a complexed polymerase. It contains a polymerase capable of forming a binding complex by binding to a nucleotide. The binding complex can be converted to a ternary complex.

Any suitable sequencing polymerase described herein, when a free nucleotide or a nucleotide unit of a multivalent molecule binds to the 3' end of a nucleic acid primer (as part of a nucleic acid duplex) at a position opposite the complementary nucleotide in the nucleic acid template molecule. Can form a binding complex that can be converted into a ternary complex.

When the nucleotide units of the multivalent molecule are complementary to the target nucleic acid compared to the duration of the non-complementary nucleotide units, the ternary complex has a longer duration. Ternary complexes also have a longer duration when the nucleotide units of the multivalent molecule are complementary to the target nucleic acid compared to the complementary free nucleotides. In some embodiments, the ternary complex has a duration of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 second, or greater than 30 seconds. For example, in some embodiments, the ternary complex may last for about 0.1 to 0.25 seconds, or about 0.25 to 0.5 seconds, or about 0.5 to 0.75 seconds, or about 0.75 to 1 second, or about 1 to 2 seconds, or about 2 to 2 seconds. has a duration of at least 3 seconds, or about 3 to 4 seconds, or about 4 to 5 seconds, or about 5 to 30 seconds, or greater than 30 seconds, and/or the method , 35°C or higher, 37°C or higher, 42°C or higher, 55°C or higher, 60°C or higher, or 72°C or higher, or any of the foregoing. In some embodiments, the ternary complex is less than 1 s, greater than 1 s, greater than 2 s, greater than 3 s, greater than 5 s, greater than 10 s, greater than 15 s, greater than 20 s, greater than 30 s, greater than 60 s, greater than 120 s, greater than 360 s, greater than 3600 s, or any of these values. It can have a duration of time lying within a range defined by any two or more factors. The ternary complex (e.g., binding complex) remains stable until subjected to any polymerase, template molecule, primer, and/or conditions that cause dissociation between nucleotide units or nucleotides. For example, dissociation conditions include contacting the binding complex with any one or any combination of detergent, EDTA, and/or water.

Duration can be measured, for example, by observing the onset and/or duration of the ternary complex, for example, by observing the signal from a labeled component of the ternary complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in a ternary complex, thereby allowing the signal to be detected by the label for the duration of the ternary complex.

It has been observed that different ranges of durations are achievable with different salts or ions, indicating that complexes formed in the presence of, for example, forms of magnesium or manganese form more rapidly than complexes formed by other ions. was observed. Additionally, for example, it has been observed that complexes formed in the presence of strontium or barium readily form and dissociate completely or substantially completely upon recovery of the strontium or barium or upon washing with a buffer lacking strontium or barium.

Dissociation of the ternary complex can be controlled by varying the buffer conditions. During a sequencing method, an imaging step can be used to detect and/or identify nucleotide units bound to a nucleic acid duplex in a ternary complex. After the imaging step, buffers of increasing salt content are used to dissociate the ternary complex so that the labeled multivalent molecules are washed away, providing a means by which the signal can be attenuated or terminated, such as in the transition between one sequencing cycle and the next. You can. This dissociation may, in some embodiments, be accomplished by washing the complex with a buffer lacking the required metal or cofactor. In some embodiments, the wash buffer may include one or more reagents for the purpose of maintaining pH control. In some embodiments, the wash buffer may include one or more monovalent cations, such as sodium. In some embodiments, the wash buffer is free or substantially free of divalent cations, for example, free or substantially free of strontium, barium, calcium, magnesium or manganese. In some embodiments, the wash buffer further includes a chelating agent, such as, for example, EDTA, EGTA, nitrilotriacetic acid, polyhistidine, imidazole, etc. In some embodiments, the wash buffer can maintain the pH of the environment at the same level as the ternary complex. In some embodiments, the wash buffer may raise or lower the pH of the environment relative to the level seen for the ternary complex. In some embodiments, the pH is within the range of 2 to 4, 2 to 7, 5 to 8, 7 to 9, 7 to 10, lower than 2, higher than 10, or any two of the values provided herein. It may be a range determined by .

The addition of specific ions can affect the binding of the polymerase to the primed target nucleic acid, the formation of a ternary complex, the dissociation of the ternary complex, or the incorporation of one or more nucleotides into the extended nucleic acid, e.g., during the polymerase reaction. In some embodiments, relevant anions may include chloride, acetate, gluconate, sulfate, phosphate, etc. In some embodiments, the ion is a binding buffer comprising one or more acids, bases, or salts, such as NiCl ₂ , CoCl ₂ , MgCl ₂ , MnCl ₂ , SrCl ₂ , CaCl ₂ , CaSO ₄ , SrCO ₃ , BaCl ₂ , etc. may be included in Representative salts, ions, solutions and conditions are described in Remington: The Science and Practice of Pharmacy, 20th., which is incorporated herein by reference in its entirety. Edition, Gennaro, AR, Ed. (2000), especially Chapter 17 and the related disclosure of salts, ions, salt solutions and ionic solutions.

The linkage between the nucleic acid template molecule and the multivalent molecule can be performed in the presence of a sequencing polymerase provided that it is catalytically inactive. In some embodiments, the sequencing polymerase has been rendered catalytically inactive by mutation. In some embodiments, the sequencing polymerase is rendered catalytically inactive by chemical modification. In some embodiments, the sequencing polymerase is rendered catalytically inactive by the absence of essential substrates, ions, or cofactors. In some embodiments, the sequencing polymerase is rendered catalytically inactive by the absence of magnesium or manganese ions.

Binding between a nucleic acid template molecule and a multivalent molecule can occur in the presence of a sequencing polymerase, provided that it is catalytically inactive, but the binding solution lacks catalytic ions such as magnesium or manganese. Catalytic ions can catalyze polymerase-catalyzed incorporation of nucleotides or nucleotide units. Alternatively, binding between the nucleic acid template molecule and the multivalent molecule can occur in the presence of a sequencing polymerase provided that it is catalytically inert, but the binding solution contains non-catalytic ions such as strontium, barium or calcium. Non-catalytic ions can inhibit polymerase-catalyzed incorporation of nucleotides or nucleotide units.

A non-catalytic divalent cation (e.g., strontium or barium) will promote the formation of a stable ternary complex with the polymerase bound to a primed nucleic acid template molecule and a multivalent molecule (e.g., a fluorescently labeled multivalent molecule). can inhibit polymerase-catalyzed incorporation of nucleotide units, and the stable ternary complex has a duration that allows detection and imaging by fluorescence detection or other methods known in the art. Unbound polymer-nucleotide conjugates can optionally be washed prior to detection of the ternary complex.

A catalytic divalent cation (e.g., magnesium or manganese) can promote the formation of a stable ternary complex with the polymerase bound to a primed nucleic acid template molecule and a free nucleotide (e.g., a fluorescently labeled nucleotide) , promotes polymerase-catalyzed incorporation of nucleotides, and the stable ternary complex has a duration that allows detection and imaging by fluorescence detection or other methods known in the art.

cutting reagent

The present disclosure provides compositions and methods for pairwise sequencing, including methods for cleaving/removing chain terminator moieties from incorporated terminal nucleotides on the nascent strand during a sequencing workflow. For example, the terminal nucleotide incorporated includes a chain terminator moiety linked to the 3' sugar position by a cleavable linker. In some embodiments, cleavable linkers can be cleaved using cleavage reagents.

The present disclosure also provides compositions and methods for pairwise sequencing, including methods for cleaving/removing detectable reporter moieties (e.g., fluorophores) from incorporated nucleotides on the nascent strand during the sequencing workflow. . For example, the incorporated nucleotide includes a fluorophore linked to a nucleobase. In some embodiments, a linker attaching a fluorophore to a nucleobase is cleavable under the same conditions that would cleave a cleavable linker at the 3' sugar position.

The present disclosure also provides the cleavage of a chain terminator from a multivalent molecule bound and/or unbound to a complexed polymerase using a cleavage reagent under conditions suitable for cleaving the chain terminator moiety from the nucleotide unit of the multivalent molecule. Provided are compositions and methods for pairwise sequencing, including cleavage/removal methods.

The present disclosure also provides a method for cleaving a detectable reporter moiety from a multivalent molecule bound and/or unbound to a polymerase complexed using a cleavage reagent under conditions suitable for cleaving the detectable reporter moiety from the nucleotide units of the multivalent molecule. Provided are compositions and methods for pairwise sequencing, including methods for cleaving/removing tags (e.g., fluorophores).

In some embodiments, the cleavage reagent includes at least one cleavage compound that cleaves the linker that attaches the fluorophore to the nucleotide or nucleotide unit of the multivalent molecule.

In some embodiments, the cleavage reagent includes at least one cleavage compound that cleaves the linker attaching the chain terminator moiety to a nucleotide or nucleotide unit.

In some embodiments, the cleavage compound is piperidine with piperidine, 2,3-dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ), or tetrakis(triphenylphosphine) ) Contains palladium(0)(Pd(PPh ₃ ) ₄ ).

In some embodiments, the cleavage compound includes a palladium catalyst, such as palladium on carbon (Pd/C).

In some embodiments, the cleavage compound includes beta-mercaptoethanol or dithiothritol (DTT).

In some embodiments, the cleavage compound includes potassium carbonate (K ₂ CO ₃ ) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH).

In some embodiments, the cleavage compound includes tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

In some embodiments, the cleavage compound comprises a phosphine compound, e.g., a phosphine with a derivatized tri-alkyl phosphine moiety or a derivatized tri-aryl phosphine moiety. In some embodiments, the phosphine cleavage compound includes tris(2-carboxyethyl)phosphine (TCEP) or bis-sulfo triphenyl phosphine (BS-TPP). In some embodiments, the phosphine cleavage compound includes tri(hydroxypropyl)phosphine (THPP) or tris(hydroxymethyl)phosphine (THMP) or 4-dimethylaminopyridine (4-DMAP).

In some embodiments, the cleavage catalyst includes 4-dimethylaminopyridine (4-DMAP).

The cleavage reagent may include a cleavage compound and/or cleavage catalyst at a concentration of about 1 to 10mM, or about 10 to 25mM, or about 25 to 50mM, or about 50 to 75mM, or about 75 to 100mM.

In some embodiments, the cleavage reagent may include a pH buffer. pH buffers include Tris, Tris-HCl, trisine, bisine, bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, and ethanolamine (also called 2-amino methanol). MEA), citrate compounds, citrate mixtures, NaOH and/or KOH, or any combination of two or more. In some embodiments, the pH buffering agent may be present in the cleavage reagent at a concentration of about 1 to 100mM, or about 10 to 50mM, or about 10 to 25mM. In some embodiments, the pH of the pH buffer present in the cleavage reagent can be adjusted to a pH of about 4 to 9.5, or a pH of about 5 to 9.5, or a pH of about 6 to 8.5.

In some embodiments, the cleavage reagent may include a monovalent cation including sodium or potassium. In some embodiments, the monovalent cation is in the form of MgCl ₂ or NaCl. In some embodiments, the cleavage reagent includes MgCl ₂ at a concentration of about 1 to 10mM, or about 10 to 20mM, or about 10 to 50mM. In some embodiments, the cleavage reagent includes NaCl at a concentration of about 25 to 200mM, or about 50 to 150mM.

In some embodiments, the cleavage reagent may include a detergent. In some embodiments, the cleaning agent includes an ionic cleaning agent such as sodium dodecyl sulfate (SDS). In some embodiments, the cleaning agent includes a non-ionic cleaning agent, such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent is an amphoteric ionic detergent, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N -dodecyl- N,N- Contains dimethyl-3-ammonio-1-propane sulfate (DetX). In some embodiments, the cleaning agent includes LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycolate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the cleavage reagent at a concentration of about 0.01 to 0.05%, or about 0.05 to 0.1%, or about 0.1 to 0.15%, or about 0.15 to 0.2%, or about 0.2 to 0.25%.

Cleavage reagents by compounds that reduce photodamage

The various pairwise sequencing workflows described herein generally include performing a forward sequencing reaction to generate a plurality of extended forward sequencing primer strands, and reverse sequencing to generate a plurality of extended reverse sequencing primer strands. Including carrying out a reaction. Forward and reverse sequencing reactions can be performed using fluorescently labeled nucleotides, or using fluorescently labeled multivalent molecules and nucleotides (labeled and/or unlabeled nucleotides). Detection and identification of fluorescently labeled nucleotides and fluorescently labeled multivalent molecules during forward and reverse sequencing reactions yields forward reads (e.g., R1 reads) and reverse reads (e.g., R2 reads), respectively. The detection step involves excitation of the fluorophore by radiation energy (e.g., light) and emission of a fluorescence signal from the fluorophore. Typically, high intensity fluorescent signals during forward and reverse reads increase sequencing accuracy, whereas low intensity signals affect the accuracy of forward and reverse reads. Ideally, the intensities of the forward and reverse reads are similar to each other and are not affected by the read length (e.g., number of sequencing cycles) of the forward sequencing reads. In non-ideal situations, the intensity of reverse sequencing reads (R2 reads) decreases as the number of forward sequencing reads increases. Without being bound by theory, when the forward sequencing reads are long (e.g., longer than 5 to 10 sequencing cycles), the decrease in signal intensity of the reverse sequencing reads is due to the immobilized concatimer template molecule and/or It is postulated that this is caused by photodamage to the retained forward extension strands. When the cleavage reagent includes at least one compound that reduces photodamage to nucleic acids, the signal intensity of reverse sequencing reads is increased even for long forward sequencing reads (see, e.g., Figure 119).

The present disclosure relates to at least one solvent; pH buffering agent; at least one monovalent cation; detergent; Cleavage compounds and/or cleavage catalysts; and at least one or a combination of two or more compounds capable of reducing light damage, including antioxidants, triplet state quenchers, singlet oxygen quenchers, oxygen scavengers, electron scavengers, and anti-fade formulations. Provide cutting reagents. Those skilled in the art will recognize that some of these compounds may be classified as more than one type of photodamage reduction compound.

In some embodiments, the cleavage reagent is ascorbic acid, ascorbyl palmitate, D-isoascorbic acid (erythorbic acid), sodium ascorbate, butylated hydroxytoluene (BTH), butylated hydroxytoluene (BHT). , polyphenol antioxidants, polyvinyl alcohol, butylated hydroxyanisole (BHA) or Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or nitrated Trolox derivatives. and at least one antioxidant, including other vitamin E analogs, including (US Pat. No. 9,994,541, the entirety of which is hereby expressly incorporated by reference in its entirety).

In some embodiments, the cleavage reagent is ascorbic acid, 1,4-diazobicyclo[2.2.2]octane (DABCO), cyclo-octatetraene (COT), dithiothreitol (DTT), mercaptoethylamine. (MEA), β-mercaptoethanol (BME), n-propyl gallate, p-phenylenediamene (PPD), hydroquinone and sodium azide (NaN ₃ ), TEMPO (2,2,6,6- At least one triplet state quencher comprising tetramethyl1-1-piperidinyloxyl), HTEMPO (4-hydroxy derivative of TEMPO) and/or DTBN (di-t-butylnitroxide) It can be included.

In some embodiments, the cleavage compound is a thiol-based quencher, such as glutathione, dithiothreitol, ergothione, methionine, cysteine, beta-dimethyl cysteine (penicylamine), mercaptopropionylglycine, MESNA, It may include at least one singlet oxygen quenching agent, including dazole and/or N-acetyl cysteine and captopril.

In some embodiments, the cleavage compound contains glutathione and at least one oxygen scavenger including N-acetylcysteine, histidine, tryptophan, hydrazine (N ₂ H ₄ ), sodium sulfate (Na ₂ SO ₃ ), and/or hydroxylamine. It can be included.

In some embodiments, the cleavage compound may include at least one electron scavenger including methyl viologen (e.g., 1,1'-dimethyl-4,4'-bipyridinium dichloride). You can.

In some embodiments, the cleavage compound is Fluoroguard anti-fade reagent (e.g., from BioRad), SlowFade anti-fade kit (e.g., including DABCO from Molecular Probes-Invitrogen), ProLong Gold anti-fade reagent (e.g., It may include at least one anti-fade formulation, including commercially available products including Invitrogen) and/or CitiFluor (e.g., CitiFluor).

The cleavage reagent is about 0.1 to 1mM, or about 1 to 10mM, or about 10 to 25mM, or about 25 to 50mM, or about 50 to 75mM, or about 75 to 100mM of at least one of the compounds that reduce photodamage. It can be included in concentration.

High-efficiency hybridization buffer

The present disclosure provides pairwise sequencing compositions and methods using high-throughput hybridization buffers. A high-throughput hybridization buffer can be used to hybridize a soluble forward sequencing primer (e.g., a second plurality of forward sequencing primers) to the retained immobilized concatimer template molecule. High-throughput hybridization buffers can be used to hybridize soluble reverse sequencing primers to the retained forward extension strand.

The high-throughput hybridization buffers described herein promote high stringency (e.g., specificity), speed, and efficacy of nucleic acid hybridization reactions and increase the efficiency of subsequent amplification and sequencing steps. High-efficiency hybridization buffers can significantly shorten nucleic acid hybridization times and reduce sample input requirements. High-efficiency hybridization buffers can be used for nucleic acid annealing workflows under isothermal conditions, eliminating the requirement of a cooling step for annealing.

In some embodiments, the high-efficiency hybridization buffer includes first and second polar aprotic solvents, a pH buffer system, and a densifying agent. A polar solvent may be a solvent or solvent system comprising one or more molecules characterized by the presence of molecules with a permanent dipole moment, i.e. a spatially unequal distribution of charge density. Polar solvents may be characterized by a dielectric constant of 20, 25, 30, 35, 40, 45, 50, 55, 60 or any of the previously mentioned values or ranges. Polar solvents may include polar aprotic solvents. A polar aprotic solvent will contain no additional ionizable hydrogen in the molecule. Additionally, the polar solvent or polar aprotic solvent preferably has strongly polarizing functional groups such as nitrile, carbonyl, thiol, lactone, sulfone, sulfite and carbonate groups as disclosed herein such that the base solvent molecules have a dipole moment. Substitutions may be made with respect to the composition. Polar solvents and polar aprotic solvents can exist in both aliphatic and aromatic or cyclic forms. In some embodiments, the polar solvent is acetonitrile.

In some embodiments, the polar or polar aprotic solvent may have a dielectric constant equal to or close to that of acetonitrile. The dielectric constant of the polar or polar aprotic solvent is about 20 to 60, about 25 to 55, about 25 to 50, about 25 to 45, about 25 to 40, about 30 to 50, about 30 to 45, or about 30 to 40. It may be in the range of . The dielectric constant of the polar or polar aprotic solvent may be greater than 20, 25, 30, 35 or 40. The dielectric constant of the polar or polar aprotic solvent may be lower than 30, 40, 45, 50, 55 or 60. The dielectric constant of the polar or polar aprotic solvent may be about 35, 36, 37, 38 or 39.

In some embodiments, the polar or polar aprotic solvents described herein may have a polarity index equal to or close to acetonitrile. The polarity index of a polar or polar aprotic solvent is about 2 to 9, 2 to 8, 2 to 7, 2 to 6, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 4 to 9, 4 to 4. It may range from 8, 4 to 7 or 4 to 6. The polarity index of the polar or polar aprotic solvent may be greater than about 2, 3, 4, 4.5, 5, 5.5 or 6. The polarity index of the polar or polar aprotic solvent may be less than about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 10. The polarity index of the polar or polar aprotic solvent may be greater than about 5.5, 5.6, 5.7 or 5.8.

Some examples of polar or polar aprotic solvents include acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetanilide, N-acetyl pyrrolidone, 4-amino pyridine, benzamide, Benzimidazole, 1,2,3-benzotriazole, butadiene dioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride, 2-chlorocyclohexanone , chloroethylene carbonate, chloronitromethane, citric anhydride, crotonactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, l,3-dimethyl- 5-tetrazole, 1,5-dimethyl tetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, diphenylsulfone, 1,2-dinitrobenzene, 2,4-dinitrobenzene Nitrotoluene, diphenylsulfone, epsilon-caprolactam, ethanesulfonyl chloride, ethyl ethyl phosphinate, N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate, ethylene glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile, l-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1 -Methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methyl morpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone, methyl sulfolane, methyl-4-toluenesulfo Nate, 3-nitroaniline, nitrobenzimidazole, 2-nitrofuran, l-nitroso-2-pyrrolidinone, 2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrene Quinone, N-phenyl sidone, phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone, propylene carbonate, 4H-pyran-4-thione, 4H-pyran -4-one (γ-pyrone), pyridazine, 2-pyrrolidone, saccharin, succinonitrile, sulfanylamide, sulfolane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothia Pyran oxide, tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil, 3,3,3-trichloropropene, 1,1,2-trichloropropene, 1,2,3-trichloropropene Including, but not limited to, propene, trimethylene sulfide-dioxide, and trimethylene sulfite.

In some embodiments, the high-throughput hybridization buffer includes a polar solvent or a polar aprotic solvent in an amount effective to denature double-stranded nucleic acids. In some embodiments, the amount of polar or polar aprotic solvent is greater than about 10% by volume based on the total volume of the formulation. The amount of polar or polar aprotic solvent is about 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, 35% by volume, 40% by volume, 50% by volume, based on the total volume of the formulation. Greater than volume%, 60 volume%, 70 volume%, 80 volume%, 90 volume% or a larger number. The amount of polar or polar aprotic solvent is about 15% by volume, 20% by volume, 25% by volume, 30% by volume, 35% by volume, 40% by volume, 50% by volume, 60% by volume, 70% by volume, based on the total volume of the formulation. lower than volume%, 80 volume%, 90 volume% or the greater number. In some embodiments, the amount of polar or polar aprotic solvent ranges from about 10% to 90% by volume based on the total volume of the formulation. In some embodiments, the amount of polar or polar aprotic solvent ranges from about 25% to 75% by volume based on the total volume of the formulation. In some embodiments, the amount of polar or polar aprotic solvent is about 10% to 95% by volume, 10% to 85% by volume, 20% to 90% by volume, or 20% to 20% by volume, based on the total volume of the formulation. It ranges from 80 volume%, 20 volume% to 75 volume%, or 30 volume% to 60 volume%.

In some embodiments, the high-efficiency hybridization buffer can include an organic solvent. Examples of suitable solvents include, but are not limited to, acetonitrile, ethanol, DMF, and methanol in various percentages (typically greater than 5%), or any combination thereof. In some embodiments, the (volume) percentage of organic solvent included in the high-throughput hybridization buffer may range from about 1% to about 20%. In some embodiments, the volume percentage of organic solvent is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%. , may be at least 15%, or may be at least 20%. In some embodiments, the volume percentage of organic solvent is up to 20%, up to 15%, up to 10%, up to 9%, up to 8%, up to 7%, up to 6%, up to 5%, up to 4%, up to 3%. , may be up to 2%, or may be up to 1%. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure; for example, the volume percentage of organic solvent may range from about 4% to about 15%. Those skilled in the art will recognize that the volume percentage of organic solvent can have any value within this range, for example about 7.5%.

In some embodiments, use of a high-efficiency hybridization buffer can improve nucleic acid hybridization rates. When a high-efficiency hybridization buffer is used in combination with a support with a low non-specific binding coating, the relative hybridization ratio can be improved, approximately 2% higher than that for a conventional hybridization protocol using a conventional hybridization buffer with a support with a conventional coating. × to about 20× faster. In some embodiments, the use of a high-throughput hybridization buffer is at least 2×, at least 3×, at least 4×, at least 5×, at least 6×, at least 7×, at least 8×, or at least 9× compared to conventional hybridization buffer protocols. ×, at least 10×, at least 12×, at least 14×, at least 16×, at least 18×, or at least 20×.

Improvement in hybridization efficiency (or yield) is generally a measure of the percentage of total available tethered adapter sequences (e.g., surface primers), primer sequences, or oligonucleotide sequences on the solid surface that have hybridized to complementary sequences. In some embodiments, the use of a high-efficiency hybridization buffer (optionally in combination with a low non-specific binding surface) results in improved hybridization efficiency compared to conventional hybridization protocols. In some embodiments, the hybridization efficiency that can be achieved is better than 80%, 85%, 90%, 95%, 98%, or 99% at any of the hybridization reaction times specified above.

Improvement in hybridization specificity is generally a measure of the ability of a tethered adapter sequence (e.g., surface primer), primer sequence, or oligonucleotide sequence to selectively and accurately hybridize to a complementary sequence. In some embodiments, the use of a high-efficiency hybridization buffer (optionally in combination with a low non-specific binding surface) results in improved hybridization specificity compared to conventional hybridization protocols. In some embodiments, the hybridization specificity that can be achieved is 1 base mismatch in 10 hybridization events, 1 base mismatch in 100 hybridization events, 1 base mismatch in 1,000 hybridization events, or 1 base mismatch in 10,000 hybridization events. Better than one base mismatch.

The term “densifying agent” and related terms refer to compounds that alter the properties of other molecules in solution. Densifying agents typically have high molecular weight and/or bulky structure. Densifying agents in solution can increase the concentration of other molecules in solution. A crowding agent can reduce the volume of solvent available to other molecules in solution, which can create a molecular crowding environment. A crowding agent in solution can create a crowded environment for molecules in solution. Densifying agents can change the rate or equilibrium constant of a reaction. Examples of milling agents include polyethylene glycol (e.g., PEG), picol, dextran, glycogen, polyvinyl alcohol, triblock polymers (e.g., Pluronics), polystyrene, polyvinylpyrrolidone (PVP), hydroxyl Includes propyl methyl cellulose (HPMC), hydroxyethyl methyl cellulose (HEMC), hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methicellulose and hydroxyl methyl cellulose. In some embodiments, the densifying agent includes linear or branched polyethylene glycol (PEG). In some embodiments, the densifying agent includes PEG 400, PEG 1500, PEG 2000, PEG 3400, PEG 3350, PEG 4000, PEG 6000, or PEG 8000. In some embodiments, the high-efficiency hybridization buffer contains at least one crowding agent in an amount of about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, by volume of the solution. You can include 35%, 40%, 50%, 60% or higher percentages. In some embodiments, high-throughput hybridization buffers can be used for nucleic acid sequencing reactions, or for nucleic acid amplifications including rotary ring amplification and/or multiple transposition amplification reactions.

High-efficiency hybridization buffers include amounts of crowding agents that will enable, enhance, or promote molecular crowding. The amount of crowding agent is approximately 1% by volume, 2% by volume, 3% by volume, 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, and 30% by volume based on the total volume of the high-efficiency hybridization buffer formulation. , 35 volume%, 40 volume%, 50 volume%, 60 volume%, or more. In some embodiments, the amount of molecular crowding agent is greater than 5% by volume based on the total volume of the high-throughput hybridization buffer formulation. The amount of densifying agent is about 3% by volume, 5% by volume, 10% by volume, 12.5% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, 35% by volume based on the total volume of the high efficiency hybridization buffer formulation. , lower than 40 volume%, 50 volume%, 60 volume%, 70 volume%, 80 volume%, 90 volume%, or greater than 90 volume%. In some embodiments, the amount of molecular crowding agent may be less than about 30% by volume based on the total volume of the formulation. In some embodiments, the amount of polar or polar aprotic solvent ranges from about 25% to 75% by volume based on the total volume of the formulation. In some embodiments, the amount of polar or polar aprotic solvent is about 1% to 40% by volume, 1% to 35% by volume, 2% to 50% by volume, or 2% to 2% by volume, based on the total volume of the formulation. 40 volume%, 2 volume% to 35 volume%, 2 volume% to 30 volume%, 2 volume% to 25 volume%, 2 volume% to 20 volume%, 2 volume% to 10 volume%, 5 volume% to 50 volume %, ranging from 5 volume% to 40% by volume, 5% to 35% by volume, 5% to 30% by volume, 5% to 25% by volume, and 5% to 20% by volume. In some cases, the amount of molecular crowding agent may range from about 5% to about 20% by volume based on the total volume of the formulation. In some embodiments, the amount of compacting agent ranges from about 1% to 30% by volume based on the total volume of the formulation.

In some embodiments, high-throughput hybridization buffer formulations may include the addition of molecular crowding agents or exclusion agents. Molecular crowding agents or volume exclusion agents are typically macromolecules (e.g., proteins) that, when added to a solution at high concentrations, can alter the properties of other molecules in solution by reducing the volume of solvent available to them. You can. In some embodiments, the volume percentage of molecular crowding agent or volume exclusion agent in the high-throughput hybridization buffer formulation may range from about 1% to about 50%. In some embodiments, the volume percentage of molecular crowding agent or volume exclusion agent is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%. %, at least 45%, or at least 50%. In some embodiments, the volume percentage of molecular crowding agent or volume exclusion agent is at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10. %, up to 5%, or up to 1%. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure, for example, the volume percentage of molecular crowding agent or volume exclusion agent may range from about 5% to about 35%. You can. Those skilled in the art will recognize that the volume percentage of molecular crowding agent or volume exclusion agent can have any value within this range, for example about 12.5%.

In some embodiments, the high-efficiency hybridization buffer includes a pH buffering system that maintains the pH of the composition in a range suitable for the hybridization process. The pH buffering system may include one or more buffering agents selected from the group consisting of Tris, HEPES, TAPS, Trisine, Bisine, Bis-Tris, NaOH, KOH, TES, EPPS, MES and MOPS. The pH buffering system may additionally include a solvent. Preferred pH buffering systems include MOPS, MES, TAPS, phosphate buffers in combination with methanol, acetonitrile, ethanol, isopropanol, butanol, t-butyl alcohol, DMF, DMSO, or any combination thereof.

In some embodiments, the high-efficiency hybridization buffer comprises an amount of pH buffering system effective to maintain the pH of the formulation in a range suitable for hybridization. In some embodiments, the pH can be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some embodiments, the pH can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, or at most 3. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure; for example, the pH of the hybridization buffer may range from about 4 to about 8. Those skilled in the art will recognize that the pH of the hybridization buffer can have any value within this range, for example about pH 7.8. In some cases, the pH range is from about 3 to about 10. In some embodiments, the disclosed hybridization buffer formulations may include adjustment of pH over a range of about pH 3 to pH 10, with preferred buffers ranging from 5 to 9.

In some embodiments, the high-throughput hybridization buffer includes additives (e.g., polar aprotic solvents) to control the melting temperature of the nucleic acids, which may vary depending on the other agents used in the composition. The amount of additives to control the melting temperature of nucleic acids is about 1% by volume, 2% by volume, 3% by volume, 5% by volume, 10% by volume, 15% by volume, 20% by volume, and 25% by volume based on the total volume of the formulation. , 30 volume%, 35 volume%, 40 volume%, 50 volume%, 60 volume%, or a greater number. In some cases, the amount of additive to control the melting temperature of the nucleic acid is greater than about 2% by volume based on the total volume of the formulation. In some cases, the amount of additive to control the melting temperature of the nucleic acid is greater than 5% by volume based on the total volume of the formulation. In some cases, the amount of additive to control the melting temperature of the nucleic acid is about 3% by volume, 5% by volume, 10% by volume, 12.5% by volume, 15% by volume, 20% by volume, 25% by volume, based on the total volume of the formulation. , 30 volume%, 35 volume%, 40 volume%, 50 volume%, 60 volume%, 70 volume%, 80 volume%, 90 volume%, or a greater number. In some embodiments, the amount of additive to control the melting temperature of the nucleic acid is about 1% to 40% by volume, 1% to 35% by volume, 2% to 50% by volume, 2% by volume, based on the total volume of the formulation. % to 40% by volume, 2% to 35% by volume, 2% to 30% by volume, 2% to 25% by volume, 2% to 20% by volume, 2% to 10% by volume, 5% to 5% by volume The range is 50 volume%, 5 volume% to 40 volume%, 5 volume% to 35 volume%, 5 volume% to 30 volume%, 5 volume% to 25 volume%, and 5 volume% to 20 volume%. In some embodiments, the amount of additive to control the melting temperature of the nucleic acid ranges from about 2% to 20% by volume based on the total volume of the formulation. In some cases, the amount of additive to control the melting temperature of the nucleic acid ranges from about 5% to 10% by volume based on the total volume of the formulation.

In some embodiments, high-throughput hybridization buffer formulations may include the addition of additives that alter the nucleic acid duplex melting temperature. Examples of suitable additives that can be used to alter the nucleic acid melting temperature include, but are not limited to, formamide. In some embodiments, the volume percentage of melting temperature additives included in the high-throughput hybridization buffer formulation may range from about 1% to about 50%. In some embodiments, the volume percentage of melt temperature additive is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%. % or at least 50%. In some embodiments, the volume percentage of melt temperature additive is up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 5. % or up to 1%. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within this disclosure; for example, the volume percentage of melt temperature additive may range from about 10% to about 25%. Those skilled in the art will recognize that the volume percentage of melt temperature additive can have any value within this range, for example about 22.5%.

In some embodiments, the hybridization buffers described herein include additives that affect DNA hydration: In some embodiments, the hybridization buffer formulations disclosed can include the addition of additives that affect nucleic acid hydration. Examples include, but are not limited to, betaine, urea, glycine betaine, or any combination thereof. In some embodiments, the volume percentage of hydration additive included in the hybridization buffer formulation may range from about 1% to about 50%. In some embodiments, the volume percentage of hydration additive is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%. Or it could be at least 50%. In some embodiments, the volume percentage of hydration additive is up to 50%, up to 45%, up to 40%, up to 35%, up to 30%, up to 25%, up to 20%, up to 15%, up to 10%, up to 5%. Or it could be up to 1%. Any of the lower and upper limits set forth in this paragraph may be combined to form ranges encompassed within the present disclosure; for example, the volume percentage of hydration additive may range from about 1% to about 30%. Those skilled in the art will recognize that the volume percentage of melt temperature additive can have any value within this range, for example about 6.5%.

Compositions and methods for making and using high-efficiency hybridization buffers are described in U.S. Patent Application No. 16/543,351, filed August 16, 2019, the contents of which are hereby expressly incorporated by reference in their entirety.

bubble fluidics

The present disclosure provides systems for controlling fluidics that can be used to perform any of the pairwise sequencing methods described herein, including any of the rolling ring amplification reactions and/or any of the sequencing reactions, in any solution or on a support, and Provides a method. Pairwise sequencing workflows performed in flow cells in a massively parallel manner require numerous reagent changes to perform different biochemical reactions throughout the workflow. A fluid dispensing system can be used to deliver reagents to the flow cell. Fluid dispensing systems are required to control very small fluid volumes and reduce mixing of different reagents flowing sequentially onto the flow cell. In some embodiments, bubbles can be introduced into the fluidics system to separate different reagents from flowing onto the flow cell and reduce mixing.

The system includes a flow cell fluidly coupled to a reagent reservoir and an injector pump. The flow cell includes an upstream inlet and a downstream outlet. A reagent reservoir is connected to the inlet of the flow cell, and an injector pump is connected to the flow cell outlet in such a way as to establish a fluid flow from the reagent reservoir, through the flow cell, and toward the injector pump. The system further includes a bubble injector fluidly coupled to the fluid flow path and configured to inject bubbles between different reagents flowing through the fluid path. Fluidic lines may be used to fluidly connect the reagent reservoir, bubble injector, flow cell, and injector pump.

The system can be used to allow very small volumes of different reagents to flow in the fluid flow path and reduce mixing of different reagents. For example, a first fluid from a reagent reservoir enters the flow path through a fluid line connecting the reagent reservoir and the inlet of the flow cell. Bubbles are injected into the flow path behind the first reagent in the fluid line. A second fluid from the reagent reservoir enters the flow path through a fluid line connecting the reagent reservoir to the flow cell inlet, with the second reagent behind the bubble. The bubbles prevent mixing of the first and second reagents in the fluid line before the reagents reach the flow cell. Subsequent bubbles and reagents may flow through the flow cell. The system further includes a bubble aspirator to remove air bubbles between the first and second reagents, so that the first and second reagents flow sequentially onto the flow cell without bubbles. To prevent air bubbles from flowing onto the flow cell, a bubble aspirator is configured to introduce air bubbles between different reagents in the fluid line before entering the flow cell. The injector pump is configured to pull the first and second fluids and air bubbles from the reagent reservoir along the flow path toward the flow cell outlet. In this way, different reagents can flow sequentially onto the flow cell without bubbles.

The system includes at least one reagent reservoir, each reservoir having a plurality of separate compartments, each compartment holding reagents for carrying out a biochemical or biological reaction. The reservoir(s) contain reagents for performing nucleic acid amplification and/or nucleic acid sequencing reactions on the flow cell. Each compartment may contain different reagents or washing reagents in fluid form for performing nucleic acid amplification reactions, nucleic acid sequencing reactions. For example, the first storage compartment may be cleaved with a plurality of amplification polymerases, a plurality of amplification primers, a mixture of four different nucleotide triphosphates or analogs thereof (e.g., dATP, dGTP, dCTP or dTTP). May contain nucleotides containing moieties. The second storage compartment may contain a plurality of sequencing polymerases, a plurality of soluble sequencing primers, and a mixture of nucleotide triphosphates or analogs thereof (e.g., dATP, dGTP, dCTP or dTTP). The third storage compartment may contain a plurality of sequencing polymerases, a plurality of soluble sequencing primers, and a mixture of multivalent molecules, each multivalent molecule having a nucleotide arm having a nucleotide unit of dATP, dGTP, dCTP, or dTTP. Includes. The fourth storage compartment may contain a plurality of linear or circular library molecules. The fifth storage compartment may contain wash buffer.

The system includes at least one flow cell with at least one passageway. In some embodiments, the flow cell contains 1, 2, 3, 4 or more passages. In some embodiments, the system includes two or more flow cells. Each flow cell may be composed of a plurality of passages arranged parallel to each other. Each passage has an inlet on the upstream side and an outlet on the downstream side. The flow cell(s) are secured to the fluidic system such that the inlet side of each passageway is fluidly connected to a reagent reservoir and the outlet side of each passageway is fluidly connected to an injector pump.

The flow cell passageway has a surface that can be coated with at least one hydrophilic polymer layer or at least one functionalized polymer coating. The passageway surface may include at least one surface capture primer fixed on or embedded in the coating. The passageway surface may comprise at least 1000 immobilized surface capture primers per mm2. Surface capture primers immobilized on the passageway surface are in fluid communication with each other during fluid flow through the passageway and can react essentially simultaneously with the reagents in a massively parallel manner. Immobilized surface capture primers can hybridize to linear or circular nucleic acid library molecules, each carrying the sequence of interest. Hybridized linear or circular nucleic acid molecules can be subjected to an amplification reaction (e.g., rolling ring amplification reaction) in a passage using a fluidic system to generate a plurality of concatemers. Multiple concatemers in a passage can be subjected to sequential sequencing reactions using a fluidic system.

Example

The following examples are intended to be illustrative and may be used to further understand embodiments of the present disclosure and should not be construed to limit the scope of the present teachings in any way.

Example 1: Rotational ring amplification on a support

Rotary ring amplification on a support:

A support was prepared with a low non-specific binding coating to which a plurality of one type of surface primer (e.g., a first surface primer) was immobilized. The first surface primer included an extendable 3'OH end and lacked any nucleotides with a cleavable moiety (e.g., lacked uracil, 8oxoG, and deoxyinosine). Optionally, the coating also included a plurality of a second type of surface primer (e.g., a second surface primer) lacking any nucleotides having a cleavable moiety and comprising a non-extensible 3' phosphate group. .

The circular library molecules included the sequence of interest (e.g., insert size ranges from 150 to 400 bases) and primer binding sequences for each of the following: a first surface primer; Forward sequencing primer; and reverse sequencing primer. The circular library molecule optionally contained a primer binding sequence for a second surface primer.

A preparation of a single-stranded circular nucleic acid library is distributed on a support and incubated under conditions suitable for hybridizing the circular library molecules to the first surface primer to form a library-primer duplex. For example, hybridization was performed at 70°C for approximately 3 minutes, at 55°C for 3 minutes, at 37°C for 3 minutes, and at room temperature for 3 minutes. The support was washed three times with buffer containing Tris (pH 8), NaCl, EDTA, and Tween-20.

A two-step rolling ring amplification reaction was performed. In a first step, the library-primer duplex is incubated with a strand transfer polymerase (e.g., phi29, an amplification polymerase) in the presence of a binding buffer lacking magnesium and nucleotides under conditions suitable for the polymerase to bind the library-primer duplex. Forms complexed polymerase but inhibits polymerase-catalyzed nucleotide incorporation. For example, polymerase was incubated with the library-primer duplex for about 15 minutes at room temperature. Binding buffer was removed. In a second step, amplification of the rolling ring by adding extension buffer containing MgCl ₂ to a mixture of nucleotides (e.g., about 1 mM each of dATP, dGTP, dCTP, and dTTP) and dUTP (e.g., 1 μM or 10 μM dUTP). The reaction (primer extension) was initiated. Rotational ring amplification reactions contained varying amounts of dUTP (e.g., about 1%, about 2.5%, about 5%, about 10%, or about 15%). Optionally, a plurality of condenser oligonucleotides were added (eg, 25-200 nM). The rotating ring reaction was incubated at 45° C. (isothermal conditions) for about 60 minutes (or less than 60 minutes) to produce a plurality of immobilized concatimers. The reaction was cooled to room temperature and washed several times.

Read 1 Sequencing:

A two-step sequencing reaction was performed. The first step sequencing reaction is performed by hybridizing a plurality of soluble forward sequencing primers (e.g., 5' exonuclease-resistant primers) to the immobilized concatimer to form a primer-concatimer duplex. A plurality of first sequencing polymerases are added to the duplex and incubated under conditions suitable for binding the sequencing polymerase to the duplex to form a complexed polymerase. A mixture of fluorescently labeled multivalent molecules (e.g., about 40 to 100 nM) was added to the complexed polymerase in the presence of a buffer containing a non-catalytic cation (e.g., strontium or barium), and polymerization of nucleotide units occurred. The complementary nucleotide units of the multivalent molecule were incubated under conditions suitable for binding to the complexed polymerase without enzyme-catalyzed incorporation. Complexed polymerase was washed. Images of the fluorescently labeled multivalent molecule remaining bound to the complexed polymerase were obtained. The first sequencing polymerase and multivalent molecules were removed, while the sequencing primers hybridized to the concatemer (retaining the duplex) were maintained by washing with a buffer containing detergent.

A second step sequencing reaction was performed by contacting the retained duplex with a plurality of second sequencing polymerases to form a complexed polymerase. A mixture (e.g., about 1 to 5 μM) and incubated under conditions suitable for binding the complementary nucleotides to the complexed polymerase and promoting polymerase-catalyzed incorporation of the nucleotides to generate the initial extended sequencing primers. Complexed polymerase was washed. Images of fluorescently labeled nucleotide analogs incorporated as part of the complexed polymerase were obtained. The incorporated fluorescently labeled nucleotide analog was reacted with a cleavage reagent to remove the 3' O-methylazido group and generate an extendable 3'OH group. The initial extended sequencing primer hybridized to the concatemer (retaining the duplex) was maintained by washing with a buffer containing detergent while the secondary sequencing polymerase was removed. Repeat sequencing reactions were performed by performing multiple cycles of the first and second step sequencing reactions to generate extended forward sequencing primer strands. Figure 118 (left) shows a comparison of error rates determined for concatimers produced by varying amounts of dUTP (e.g., about 1%, about 2.5%, about 5%, about 10%, or about 15%) . Figure 118 (right) shows a comparison of phase ratios determined for concatimers produced by varying amounts of dUTP (e.g., about 1%, about 2.5%, about 5%, about 10%, or about 15%). indicates.

Replacement of the extended forward sequencing primer strand:

In the absence of newly added soluble primers (e.g., no soluble sequencing primers and no soluble amplification primers), the extended forward sequencing primer strand is digested with a plurality of strand transposition polymerases (e.g., Phi29 polymerase) and a sequence of nucleotides. brought into contact with the mixture. The mixture of nucleotides included dATP, dGTP, dCTP and dTTP. Optionally, compacted oligonucleotides were added (e.g., approximately 100 μM). The reaction was incubated at 45°C for approximately 30 minutes to generate multiple forward extended strands hybridized to the immobilized concatimer molecules. The scaffold was washed several times with washing buffer containing Tris-HCl, NaCl, EDTA, and Tween-20.

Creation of non-basic sites and gaps:

Multiple forward extension strands were digested with Thermolabile USER (II) enzyme (0.02 U/μl) (e.g., from New England Biolabs, catalog #M5508S) and T7 exonuclease (02 U/μl) (e.g., New England Biolabs). were contacted with England Biolabs, catalog #M0263S) and 1× CutSmart buffer (e.g., from New England Biolabs, catalog #B7204S) and incubated at 37°C for approximately 15 minutes, followed by incubation at 25°C for approximately 15 minutes. A plurality of non-basic sites were created in the immobilized concatimer molecule and a gap was created in the non-basic site. The forward extension strand was maintained as an intact molecule. The support was washed several times with a wash buffer containing Tris-HCl, NaCl, EDTA, and Tween-20 to remove gap-containing concatimer molecules.

Read 2 Sequencing:

Except that the first step sequencing reaction is performed by hybridizing a plurality of soluble reverse sequencing primers (e.g., 5' exonuclease-resistant primers) to the retained forward extension strand to form a primer-extension strand duplex. And, a two-step sequencing reaction was performed as described in ‘Read 1 Sequencing’ above.

Example 2: Initiated Rotary Ring Amplification in Solution

Rotary Ring Amplification Initiated in Solution:

A support was prepared with a low non-specific binding coating to which a plurality of one type of surface primer (e.g., a first surface primer) was immobilized. The first surface primer included an extendable 3'OH end and lacked any nucleotides with a cleavable moiety (e.g., lacked uracil, 8oxoG, and deoxyinosine). Optionally, the coating also included a plurality of a second type of surface primer (e.g., a second surface primer) lacking any nucleotides having a cleavable moiety and comprising a non-extensible 3' phosphate group. .

The circular library molecules included the sequence of interest (e.g., insert size ranges from 150 to 400 bases) and primer binding sequences for each of the following: soluble amplification primers; a first surface primer; Forward sequencing primer; and reverse sequencing primer. The circular library molecule optionally contained a primer binding sequence for a second surface primer.

Prototype library molecules (1 to 8 nM) were incubated with soluble amplification primers (2 to 80 nM) in buffer containing 10mM ACES pH 7.4, 10 μM dNTPs (total), 1mM strontium acetate, 0.01% Tween-20, (and optionally 50mM ammonium sulfate). ) (e.g., about 2 to 10 equivalents). The hybridization mixture was heated to 85°C in 5°C steps and cooled to 40°C, with a residence time of 30 seconds at each step and an increase rate of 0.2°C/sec between steps for a total of 10 minutes. The mixture was incubated at a temperature of about 1 to 10° C. below the Tm of the primers for about 10 minutes.

To the hybridization mixture described above, DTT was added at a final concentration of 1 to 50mM, Phi29 polymerase was added at a final concentration of 2 to 10 equivalents relative to the soluble amplification primer, and the final concentrations of ACES, dNTP, strontium acetate, and Tween-20 were the same as the hybridization mixture. A trapped nucleotide-polymerase mixture was prepared by adding preferably 5× trap buffer. The trapped nucleotide-polymerase mixture was incubated for 15 minutes at room temperature or different temperatures up to 35°C.

The nucleotide polymerization reaction mixture was prepared by diluting the trapped nucleotide-polymerase mixture (about 40 to 1000× dilution) with extension buffer, so that the final concentrations were 50mM ACES pH 74, 100mM potassium acetate, 10mM magnesium sulfate, 2mM dNTPs, It contained 10mM DTT, 0.01% Tween-20, and 50mM ammonium sulfate, and the concentration of the original library molecule was 5 to 100pM.

The nucleotide polymerization reaction mixture was distributed on a support immobilized with two different types of surface capture primers and incubated at 30-45°C for 5 minutes for the rolling ring amplification reaction (but time ranges of up to 2 hours were tested). . Alternatively, the nucleotide polymerization reaction mixture was incubated at 30-45° C. for 30 minutes, then distributed on the support, and the rolling ring amplification reaction continued on the support for 5 minutes or up to 2 hours.

The scaffold was washed with wash buffer containing 50mM Tris-HCl pH 8, 750mM NaCl, 0.1mM EDTA, and 0.02% Tween-20. The reaction was cooled to room temperature and washed several times.

Read 1 Sequencing:

The two-step sequencing reaction was performed as described above in Example 1.

Replacement of extended forward sequencing primer strands

The extended forward sequencing primer strand was replaced with the forward extended strand as described above in Example 1.

Creation of non-basic sites and gaps

Non-basic sites and gaps were created in the forward extending strand as described above in Example 1.

Read 2 Sequencing:

The two-step sequencing reaction was performed as described above in Example 1.

Claims (309)

As a pairwise sequencing method,
a) providing a plurality of immobilized single stranded nucleic acid concatemer template molecules, each of which creates a non-basic site in the concatemer template molecule Comprising at least one nucleotide having a scissile moiety that can be cleaved so as to wherein the first surface primer lacks a nucleotide having a cleavable moiety;
b) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
c) maintaining the plurality of immobilized concatimer template molecules, and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of immobilized single-stranded nucleic acid concatimer template molecules to the maintained concatimer template molecules. replacing with a forward extending strand;
d) an abasic portion of the fixed single-stranded concatimer template molecule at the nucleotide(s) having the cleavable moiety, while maintaining the plurality of forward extending strands and maintaining the plurality of fixed surface primers. generating a gap at the non-basic site to generate a plurality of gap-containing single-stranded nucleic acid concatimer template molecules, thereby removing the retained immobilized concatimer template molecule; and
e) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each maintained forward extension strand is hybridized with two or more extended reverse sequencing primer strands. , step
Including, pairwise sequencing method. The method of claim 1, wherein each template molecule of the plurality is covalently linked to an immobilized first surface primer. The method of claim 1, wherein each template molecule of the plurality is hybridized to an immobilized first surface primer. The method of claim 1, wherein each of the plurality of immobilized concatimer template molecules comprises two or more copies of the sequence of interest, and wherein the individual immobilized concatimer template molecules are one or more of the following: Methods, further comprising combinations:
(i) two or more copies of the universal binding sequence for the soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) Two or more copies of a unique molecular indicator sequence. The method of claim 1, wherein the sequencing in step (b) comprises hybridizing a plurality of soluble forward sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. The method of claim 1, wherein the sequencing in step (e) comprises hybridizing a plurality of soluble reverse sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 5. The method of claim 4, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 8. The method of claim 7, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 8. The method of claim 7, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 8. The method of claim 7, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 11. The method of claim 10, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. As a pairwise sequencing method,
a) providing a support on which a plurality of first surface primers are immobilized, each of the first surface primers having a 3' extendable end and lacking a nucleotide having a cleavable moiety;
b) hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized first surface primers, a plurality of strand displacing polymerases, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP, and a basic base A plurality of immobilized single-stranded nucleic acid concatemer template molecules are generated by performing a rolling circle amplification reaction with nucleotides having a cleavable moiety that can be cleaved to create a cleavable moiety. Generating a plurality of immobilized single-stranded nucleic acid conkatimer template molecules having at least one nucleotide having a moiety, wherein each single-stranded nucleic acid conkatimer template molecule is covalently linked to an immobilized first surface primer. ,
c) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
d) maintaining the plurality of immobilized concatimer template molecules, and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of immobilized single-stranded nucleic acid concatimer template molecules to the maintained concatimer template molecules. replacing with a forward extending strand;
e) a non-salt of the fixed single stranded concatimer template molecule at the nucleotide(s) having the cleavable moiety, while maintaining the plurality of forward extending strands and maintaining the plurality of fixed first surface primers. removing the retained immobilized conkatimer template molecules by creating a basic site and creating a gap in the non-basic site to generate a plurality of gap-containing single stranded nucleic acid conkatimer template molecules; and
f) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each forward extension strand is hybridized with two or more extended reverse sequencing primer strands.
Method, including. 13. The method of claim 12, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for the first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for soluble compressed oligonucleotides,
(viii) sample barcode sequence and/or
(ix) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 13. The method of claim 12, wherein the individual fixed single-stranded nucleic acid concatimer template molecule produced by the rolling ring amplification reaction comprises two or more copies of the sequence of interest, and the individual fixed concatimer template molecule is:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 13. The method of claim 12, wherein the sequencing in step (c) comprises hybridizing a plurality of soluble forward sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 13. The method of claim 12, wherein the sequencing in step (f) comprises hybridizing a plurality of soluble reverse sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 15. The method of claim 14, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 18. The method of claim 17, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 18. The method of claim 17, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 18. The method of claim 17, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 21. The method of claim 20, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. As a pairwise sequencing method,
a) a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable for forming a plurality of library-primer duplexes and suitable for performing a rolling ring amplification reaction, a plurality of first soluble amplification primers, a plurality of strand transposition polymerases. , and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and at least one nucleotide having a cleavable moiety by contacting the nucleotide with a cleavable moiety that can be cleaved to create an abasic site. generating a plurality of single-stranded nucleic acid concatemers having;
b) distributing the rolling ring amplification reaction on a support to which a plurality of first surface primers are immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatimers to the one or more immobilized first surface primers. cleaving, wherein each of the first surface primers lacks a nucleotide having a cleavable moiety;
c) continuing the rolling ring amplification reaction on the support to generate a plurality of immobilized concatimer template molecules;
d) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
e) maintaining the plurality of immobilized concatimer template molecules, and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of immobilized single-stranded nucleic acid concatimer template molecules to the maintained concatimer template molecules. replacing with a forward extending strand;
f) the non-basic nature of the fixed single stranded concatimer template molecule at the nucleotide(s) having the cleavable moiety while maintaining the plurality of forward extending strands and maintaining the plurality of fixed first surface primers. removing the retained immobilized concatimer template molecules by creating a site and creating a gap at the non-basic site to generate a plurality of gap-containing single stranded nucleic acid concatimer template molecules; and
g) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each forward extension strand has two or more extended reverse sequencing primer strands hybridized thereto. Steps to create
Method, including. 23. The method of claim 22, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for the first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for soluble compressed oligonucleotides,
(viii) sample barcode sequence and/or
(ix) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 23. The method of claim 22, wherein the individual fixed single-stranded nucleic acid concatimer template molecule produced by the rolling ring amplification reaction comprises two or more copies of the sequence of interest, and the individual fixed concatimer template molecule is:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 23. The method of claim 22, wherein the sequencing in step (d) comprises hybridizing a plurality of soluble forward sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 23. The method of claim 22, wherein the sequencing in step (g) comprises hybridizing the plurality of soluble reverse sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. How to. 25. The method of claim 24, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 28. The method of claim 27, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 28. The method of claim 27, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 28. The method of claim 27, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 31. The method of claim 30, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. As a pairwise sequencing method,
a) providing a support on which a plurality of first surface primers are immobilized, wherein each first surface primer of the plurality includes a first portion (SP1-A) and a second portion (SP1-B), wherein each first surface primer comprises a 3' extendable end at the first surface marker and lacks a nucleotide having a cleavable moiety that can be cleaved to create an abasic site;
b) contacting the plurality of first surface primers with a plurality of single stranded linear nucleic acid library molecules, each library molecule binding to the first portion of the immobilized first surface primer at the 5' end. The library molecules each have a universal sequence (SP1-B') that binds to the second portion of the immobilized first surface primer at the 3' end, wherein the library molecules each have a universal sequence (SP1-B') that binds to the second portion of the immobilized first surface primer. The step is performed under conditions suitable for hybridizing the individual library molecules to the immobilized first surface primer, forming a circular library molecule having a gap or crevice between the 5' end and the 3' end of the circular library molecule;
c) closing the gap or crevice with an enzyme, thereby forming individual covalently closed ring molecules hybridized to the immobilized first surface primer;
d) performing a rolling ring amplification reaction with a plurality of strand transfer polymerases and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable moiety that can be cleaved to create an abasic site. generating a plurality of fixed single-stranded nucleic acid conkatimer template molecules, thereby generating a plurality of fixed single-stranded nucleic acid conkatimer template molecules having at least one nucleotide having a cleavable moiety, wherein each of the A single-stranded nucleic acid concatimer template molecule is covalently linked to an immobilized first surface primer, comprising:
e) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
f) maintaining the plurality of fixed concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward extensions to the maintained fixed single-stranded nucleic acid concatimer template molecules. replacing with a strand;
g) the non-basic nature of the fixed single stranded concatimer template molecule at the nucleotide(s) having the cleavable moiety while maintaining the plurality of forward extending strands and maintaining the plurality of fixed first surface primers. removing the retained immobilized concatimer template molecules by creating a site and creating a gap at the non-basic site to generate a plurality of gap-containing single stranded nucleic acid concatimer template molecules; and
h) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of maintained forward extension strands, wherein each forward extension strand is hybridized with two or more extended reverse sequencing primer strands.
Method, including. 33. The method of claim 32, wherein each linear library molecule of the plurality comprises a sequence of interest, and the library molecule is:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for the first portion of the immobilized first surface primer (SP1-A),
(iv) a universal binding sequence for the second portion of the immobilized first surface primer (SP1-B),
(v) a universal binding sequence for an immobilized second surface primer,
(vi) a universal binding sequence for the first soluble amplification primer,
(vii) a universal binding sequence for a second soluble amplification primer,
(viii) a universal binding sequence for soluble compressed oligonucleotides,
(ix) sample barcode sequence and/or
(x) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 33. The method of claim 32, wherein the individual fixed single-stranded nucleic acid concatimer template molecule produced by the rolling ring amplification reaction comprises two or more copies of the sequence of interest, and the individual fixed concatimer template molecule is:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of the universal binding sequence (SP1-A) for the first portion of the immobilized first surface primer,
(iv) two or more copies of the universal binding sequence (SP1-B) for the second portion of the immobilized first surface primer,
(v) two or more copies of a universal binding sequence for an immobilized second surface primer,
(vi) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the second soluble amplification primer,
(viii) two or more copies of the universal binding sequence for the soluble compacted oligonucleotide,
(ix) two or more copies of the sample barcode sequence and/or
(x) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 33. The method of claim 32, wherein the sequencing of step (e) comprises hybridizing a plurality of soluble forward sequencing primers to the plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 33. The method of claim 32, wherein said sequencing in step (h) comprises hybridizing a plurality of soluble reverse sequencing primers to said plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 33. The method of claim 32, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 35. The method of claim 34, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 35. The method of claim 34, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 35. The method of claim 34, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 41. The method of claim 40, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. The method of claim 32, wherein closing the gap in the circular library molecule is performed by performing a polymerase-catalyzed gap fill-in reaction using a first surface primer immobilized as a template molecule; and ligating the gap to form a covalently closed circular molecule, wherein the individual covalently closed circular molecules are hybridized to the immobilized first surface primer. 33. The method of claim 32, wherein closing the cleft in the circular library molecule comprises performing a ligation reaction to form a covalently closed circular molecule, wherein each covalently closed circular molecule is hybridized to an immobilized first surface primer. done, how. As a pairwise sequencing method,
a) providing a plurality of immobilized single-stranded nucleic acid concatimer template molecules, each lacking a cleavable moiety that can be cleaved to create an abasic site in the concatimer template molecule, wherein each of the plurality A template molecule is immobilized on a first surface primer immobilized on a support, the immobilized first surface primer lacking a nucleotide having a cleavable moiety;
b) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
c) Maintaining the plurality of immobilized concatimer template molecules, performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-transfer polymerases, thereby converting the plurality of extended forward sequencing primer strands into a plurality of strands. Replacing with the forward extension strand, generating a plurality of forward extension strands and a plurality of partially transposed forward extension strands hybridized to the immobilized concatimer template molecule to form a plurality of immobilized amplicons, and the primer extension reaction generating a plurality of detached forward extension strands (e.g., not hybridized to the immobilized concatimer template molecule); and
d) sequencing the plurality of fixed partially translocated forward extension strands to generate a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of fixed detached forward extension strands, 2 Generating a plurality of extended reverse sequencing primer strands, wherein each fixed partially translocated forward extension strand is hybridized with two or more extended reverse sequencing primer strands, and each fixed partially translocated forward extension strand is hybridized to each other. The strand is hybridized with two or more extended reverse sequencing primer strands,
Method, including. 45. The method of claim 44, wherein each template molecule of the plurality is covalently linked to an immobilized first surface primer. 45. The method of claim 44, wherein each template molecule of the plurality is hybridized to an immobilized first surface primer. 45. The method of claim 44, wherein each of the plurality of immobilized concatimer template molecules comprises two or more copies of a sequence of interest, and wherein the individual immobilized concatimer template molecules comprises:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 45. The method of claim 44, wherein the sequencing of step (b) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. . 45. The method of claim 44, wherein said sequencing in step (d) hybridizes a plurality of soluble reverse sequencing primers to said plurality of fixed partially transposed forward extension strands and a plurality of fixed detached extended forward sequencing primer strands. A method comprising performing one or more sequencing reactions. 48. The method of claim 47, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 51. The method of claim 50, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 51. The method of claim 50, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 51. The method of claim 50, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 54. The method of claim 53, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. As a pairwise sequencing method,
a) providing a support on which a plurality of first surface primers are immobilized, each of the first surface primers having a 3' extendable end and lacking a nucleotide having a cleavable moiety;
b) a plurality of single stranded circular nucleic acid library molecules hybridized to the plurality of immobilized first surface primers and a plurality of strand transfer polymerases, and a cleavable moiety that can be cleaved to generate a plurality of nucleotides and non-basic sites. A step of producing a plurality of fixed single-stranded nucleic acid concatimer template molecules by performing a rotation amplification reaction with a nucleotide having, wherein each single-stranded nucleic acid wherein the concatimer template molecule is covalently linked to the immobilized first surface primer;
c) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
d) Maintaining the plurality of immobilized concatimer template molecules, performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-transfer polymerases, thereby converting the plurality of extended forward sequencing primer strands into the plurality of forward sequencing primer strands. Replacing with the extension strands generates a plurality of forward extension strands and a plurality of partially translocated forward extension strands hybridized to the immobilized concatimer template molecule to form the plurality of immobilized amplicons, and the primer extension reaction is ( generating a plurality of detached forward extension strands (e.g., not hybridized to the immobilized concatimer template molecule); and
e) sequencing the plurality of fixed, partially translocated forward extension strands to generate a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of fixed, detached forward extension strands, thereby generating a second plurality of extended reverse sequencing primer strands. A step of generating an extended reverse sequencing primer strand, wherein at least two extended reverse sequencing primer strands are hybridized to each fixed partially translocated forward extension strand, and to each fixed partially translocated forward extension strand: wherein two or more extended reverse sequencing primer strands are hybridized.
Method, including. 56. The method of claim 55, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and wherein the individual library molecule is:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for the first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for soluble compressed oligonucleotides,
(viii) sample barcode sequence and/or
(ix) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 56. The method of claim 55, wherein the individual fixed single-stranded nucleic acid concatimer template molecule produced by the rolling ring amplification reaction comprises two or more copies of the sequence of interest, and the individual fixed concatimer template molecule is:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 56. The method of claim 55, wherein the sequencing in step (c) comprises hybridizing a plurality of soluble forward sequencing primers to a plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. method. 56. The method of claim 55, wherein the sequencing of step (e) combines the plurality of soluble reverse sequencing primers with the plurality of fixed partially translocated forward extension strands and the plurality of fixed detachable extended forward sequencing primer strands. Hybridizing to and performing one or more sequencing reactions. 58. The method of claim 57, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 61. The method of claim 60, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 61. The method of claim 60, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 61. The method of claim 60, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 64. The method of claim 63, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. As a pairwise sequencing method,
a) a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable for forming a plurality of library-primer duplexes and suitable for performing a rolling ring amplification reaction, a plurality of first soluble amplification primers, a plurality of strand transposition polymerases. , and a nucleotide that has a cleavable moiety that can be cleaved to create a non-basic site;
b) distributing the rolling ring amplification reaction on a support to which a plurality of first surface primers are immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatimers to the one or more immobilized first surface primers. wherein each of the first surface primers lacks a nucleotide having a cleavable moiety;
c) continuing a rolling ring amplification reaction on the support to generate a plurality of immobilized concatimer template molecules;
d) Generating a plurality of extended forward sequencing primer strands by sequencing the plurality of fixed Concatimer template molecules, wherein each fixed Concatimer template molecule contains two or more extended forward sequencing primers. strands are hybridized;
e) Maintaining the plurality of immobilized concatimer template molecules and performing a primer extension reaction with a plurality of second soluble amplification primers and a plurality of strand-transfer polymerases to produce the plurality of extended forward sequencing primer strands. replacing the plurality of forward extension strands, thereby generating a plurality of forward extension strands and a plurality of partially translocated forward extension strands hybridized to the immobilized concatimer template molecule to form the plurality of immobilized amplicons, The primer extension reaction generates a plurality of detached forward extension strands (e.g., that do not hybridize to the immobilized concatimer template molecule); and
f) sequencing the plurality of fixed, partially translocated forward extension strands to generate a first plurality of extended reverse sequencing primer strands, and sequencing the plurality of fixed, detached forward extension strands, 2 Generating a plurality of extended reverse sequencing primer strands, wherein each fixed partially translocated forward extension strand is hybridized with two or more extended reverse sequencing primer strands, and each fixed partially translocated forward extension strand is hybridized to each other. generating a primer strand to which two or more extended reverse sequencing primer strands are hybridized;
Method, including. 66. The method of claim 65, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and wherein the individual library molecule is:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for the first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for soluble compressed oligonucleotides,
(viii) sample barcode sequence and/or
(ix) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 66. The method of claim 65, wherein the individual immobilized single-stranded nucleic acid concatimer template molecule produced by the rolling ring amplification reaction comprises two or more copies of the sequence of interest, and the individual immobilized concatimer template molecule is:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 66. The method of claim 65, wherein said sequencing in step (d) comprises hybridizing a plurality of soluble forward sequencing primers to said plurality of immobilized concatimer template molecules and performing one or more sequencing reactions. , method. 66. The method of claim 65, wherein said sequencing of step (f) comprises combining a plurality of soluble reverse sequencing primers to said plurality of fixed partially translocated forward extension strands and said plurality of fixed detachable extended forward sequencing primer strands. A method comprising hybridizing and performing one or more sequencing reactions. 66. The method of claim 65, wherein the support further comprises a plurality of immobilized second surface primers lacking nucleotides having a cleavable moiety. 71. The method of claim 70, wherein at least one copy of the universal binding sequence for the immobilized second surface primer in the individual concatimer template molecule is hybridized to the immobilized second surface primer. 71. The method of claim 70, wherein the plurality of immobilized second surface primers have 3' OH extendable ends. 71. The method of claim 70, wherein the plurality of immobilized second surface primers have 3' non-extendable ends. 74. The method of claim 73, wherein the 3' non-extendable end comprises a phosphate group, dideoxycytidine group, inverted dT, or amino group. As a pairwise sequencing method,
a) providing a plurality of fixed single-stranded nucleic acid conkatimer template molecules each comprising at least one nucleotide having a cleavable moiety that can be cleaved to create a non-basic site in the conkatimer template molecule, , each template molecule among the plurality is fixed to a first surface primer fixed to a support, and the fixed first surface primer includes a nucleotide having a cleavable moiety, and the support has a cleavable moiety. and a plurality of immobilized second surface primers lacking nucleotides and having 3'OH groups at extendable ends, wherein the immobilized concatimer template molecules comprise two of the universal binding sequences for the immobilized second surface primers. comprising at least two copies, wherein the support includes an excess of immobilized first and second surface primers relative to the number of immobilized concatimer template molecules;
b) generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers, wherein each immobilized concatimer template molecule includes wherein two or more extended forward sequencing primer strands are hybridized;
c) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule;
d) hybridizing at least one portion of an individual immobilized Concatimer template molecule to the second surface primer and performing a primer extension reaction from the second surface primer hybridized to the portion of the immobilized Concatimer template molecule. 1 generating a plurality of immobilized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to an immobilized second surface primer;
e) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution containing at least one chaotropic agent;
f) dissociating at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retaining the immobilized forward extension strand, and at least one portion of the immobilized concatimer template molecule The portion is rehybridized to one of the immobilized second surface primers that is not covalently attached to the forward extension strand, wherein the dissociation and re-dissociation comprise a temperature increase, a temperature plateau, and a temperature decrease, and the relaxing solution is washed from the support. step;
g) contacting the rehybridized immobilized Concatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that rehybridizes to a portion of the immobilized Concatimer template molecule to generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the mer template molecule and covalently linked to an immobilized second surface primer;
h) repeating steps (e) to (g) at least once;
i) said fixed single-stranded concatimer template molecule at nucleotide(s) having a cleavable moiety, while maintaining said plurality of fixed forward extension strands and maintaining said plurality of fixed second surface primers; creating a non-basic site of an immobilized first surface primer and removing the retained immobilized concatimer template molecule by creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules; and
j) sequencing the plurality of maintained fixed forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands.
Method, including. 76. The method of claim 75, wherein each template molecule of the plurality is covalently linked to an immobilized first surface primer. 76. The method of claim 75, wherein each template molecule of the plurality is hybridized to an immobilized first surface primer. 76. The method of claim 75, wherein each of the plurality of immobilized concatimer template molecules comprises two or more copies of the sequence of interest, and wherein the individual immobilized concatimer template molecules are one or more of the following: Methods, further comprising combinations:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) Two or more copies of a unique molecular indicator sequence. As a pairwise sequencing method,
a) providing a support on which a plurality of first and second surface primers are immobilized, wherein the first surface primer has a cleavable moiety that can be cleaved to create an abasic site, and the second surface primer lacks a nucleotide having a cleavable moiety, and the second surface primer has a 3'OH group at an extendable end;
b) hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first surface primers and generating a plurality of strand transposition polymerases, and a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and abasic sites. Generating a plurality of fixed single-stranded nucleic acid concatimer template molecules by performing a rolling ring amplification reaction with a plurality of nucleotides having a cleavable moiety that can be cleaved, thereby producing at least one nucleotide having a cleavable moiety generating a plurality of immobilized single-stranded nucleic acid conkatimer template molecules having, wherein each single-stranded nucleic acid conkatimer template molecule is covalently linked to an immobilized first surface primer,
c) generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers, wherein each immobilized concatimer template molecule includes wherein two or more extended forward sequencing primer strands are hybridized;
d) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule;
e) hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and performing a primer extension reaction from said second surface primer hybridized to a portion of the immobilized Concatimer template molecule. 1 generating a plurality of immobilized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to the immobilized second surface primer;
f) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution containing at least one disorder-inducing agent;
g) dissociating the at least one portion of the immobilized concatimer template molecule from the immobilized second surface primer, retaining the immobilized forward extension strand, and at least one portion of the immobilized concatimer template molecule rehybridize a portion of steps;
h) contacting the rehybridized immobilized Concatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that rehybridizes to a portion of the immobilized Concatimer template molecule to generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the mer template molecule and covalently linked to an immobilized second surface primer;
i) repeating steps (f) to (h) at least once;
j) an anchored single-stranded concatimer template molecule and an anchored agent at the nucleotide(s) having said cleavable moiety, while maintaining a plurality of anchored forward extension strands and maintaining a plurality of anchored second surface primers. 1 removing the retained immobilized concatimer template molecule by creating a non-basic site of a surface primer and creating a gap in the non-basic site, thereby generating the plurality of gap-containing nucleic acid molecules; and
k) sequencing the plurality of maintained fixed forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands.
Steps containing. 80. The method of claim 79, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and each library molecule comprises:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for the first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for soluble compressed oligonucleotides,
(viii) sample barcode sequence and/or
(ix) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 80. The method of claim 79, wherein each of the plurality of immobilized concatimer template molecules comprises two or more copies of the sequence of interest, and wherein the individual immobilized concatimer template molecules comprises:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. As a pairwise sequencing method,
a) a plurality of single-stranded circular nucleic acid library molecules in solution under conditions suitable for forming a plurality of library-primer duplexes and suitable for performing a rolling ring amplification reaction, a plurality of first soluble amplification primers, a plurality of strand transposition polymerases. , and at least one having a cleavable moiety by contacting a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP and a plurality of nucleotides having a cleavable moiety that can be cleaved to create an abasic site. generating a plurality of single-stranded nucleic acid concatemers having nucleotides;
b) distributing the rolling ring amplification reaction on a support to which a plurality of first surface primers are immobilized under conditions suitable for hybridizing one or more portions of the individual single-stranded concatimers to the one or more immobilized first surface primers. wherein the immobilized first surface primer comprises a nucleotide having a cleavable moiety, and the support lacks a nucleotide having a cleavable moiety and has a plurality of immobilized primers having a 3'OH group at an extendable end. further comprising a second surface primer;
c) continuing a rolling ring amplification reaction on the support in the presence of a plurality of nucleotides comprising a plurality of nucleotides having a cleavable moiety to generate a plurality of immobilized concatimer template molecules;
d) generating a plurality of extended forward sequencing primer strands by sequencing a plurality of immobilized concatimer template molecules using the plurality of soluble forward sequencing primers, wherein each immobilized concatimer template molecule wherein two or more extended forward sequencing primer strands are hybridized;
e) removing the extended forward sequencing primer strand and maintaining the immobilized concatimer template molecule;
f) hybridizing at least one portion of an individual immobilized Concatimer template molecule to a second surface primer and performing a primer extension reaction from the second surface primer hybridized to a portion of the immobilized Concatimer template molecule to form a first primer. generating a plurality of immobilized forward extension strands having a sequence complementary to at least a portion of the immobilized concatimer template molecule and covalently linked to an immobilized second surface primer;
g) contacting the plurality of immobilized concatimer template molecules and the plurality of immobilized forward extension strands with a relaxing solution containing at least one disorder-inducing agent;
h) dissociating at least one portion of the immobilized Concatimer template molecule from the immobilized second surface primer, retaining the immobilized forward extension strand, and releasing at least one portion of the immobilized Concatimer template molecule from the immobilized second surface primer. rehybridizing to one of the immobilized second surface primers that is not covalently attached to the forward extension strand, wherein the dissociation and re-dissociation comprise a temperature increase, a temperature plateau and a temperature decrease, and washing the relaxing solution from the support;
i) contacting the rehybridized immobilized Concatimer template molecule with an amplification solution and performing a primer extension reaction from a second surface primer that rehybridizes to a portion of the immobilized Concatimer template molecule to generating a plurality of newly synthesized forward extension strands having a sequence complementary to at least a portion of the mer template molecule and covalently linked to an immobilized second surface primer;
j) repeating steps (g) to (i) at least once;
k) said fixed single stranded concatimer template molecule at said nucleotide(s) having said cleavable moiety, while maintaining said plurality of fixed forward extension strands and maintaining said plurality of fixed second surface primers; removing the retained immobilized concatimer template molecule by creating a non-basic site of the immobilized first surface primer and creating a gap in the non-basic site, thereby generating a plurality of gap-containing nucleic acid molecules; and
l) sequencing the plurality of maintained fixed forward extension strands using a plurality of soluble reverse sequencing primers to generate a plurality of extended reverse sequencing primer strands.
Method, including. 83. The method of claim 82, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and wherein the individual library molecule further comprises any one or any combination of two or more of the following:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized first surface primer,
(iv) a universal binding sequence for an immobilized second surface primer,
(v) a universal binding sequence for the first soluble amplification primer,
(vi) a universal binding sequence for a second soluble amplification primer,
(vii) a universal binding sequence for soluble compressed oligonucleotides,
(viii) sample barcode sequence and/or
(ix) Unique molecular indicator sequence. 83. The method of claim 82, wherein each of the plurality of immobilized concatimer template molecules comprises two or more copies of a sequence of interest, and wherein the individual immobilized concatimer template molecules comprises:
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of a universal binding sequence for an immobilized first surface primer,
(iv) two or more copies of a universal binding sequence for an immobilized second surface primer,
(v) two or more copies of the universal binding sequence for the first soluble amplification primer,
(vi) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vii) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(viii) two or more copies of the sample barcode sequence and/or
(ix) two or more copies of a unique molecular indicator sequence
A method further comprising any one or any combination of two or more. The method according to any one of claims 1, 12, 22, 32, 44, 55, 65, 75, 79 and 82, wherein the support is free. , fused silica, silicone, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), A method comprising a planar substrate comprising high density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. The method according to any one of claims 1, 12, 22, 32, 44, 55, 65, 75, 79, and 82, wherein the support is water. A method comprising at least one hydrophilic polymer coating having a contact angle of less than or equal to 45 degrees, wherein at least one of the hydrophilic polymer coatings comprises a branched hydrophilic polymer having at least four branches. According to any one of claims 1, 12, 22, 32, 44, 55, 65, 75, 79, and 82, 1 The 5' end of the surface primer is secured to the support or to a coating on the support. According to any one of claims 1, 12, 22, 32, 44, 55, 65, 75, 79, and 82, 1 The method of claim 1, wherein the surface primer comprises a modified oligonucleotide molecule having 2 to 10 phosphorothioate linkages at the 5' end to confer resistance to nuclease degradation. 71. The method of any one of claims 7, 17, 27, 37, 50, 60, and 70, wherein the 5' end of the plurality of second surface primers is attached to the support. Fixed or fixed to a coating on the support. 71. The method of any one of claims 7, 17, 27, 37, 50, 60, and 70, wherein the plurality of second surface primers comprises 2 to 10 primers at the 5' end. A method comprising a modified oligonucleotide molecule having a phosphorothioate linkage, thereby conferring resistance to nuclease degradation. 83. The method of any one of claims 1, 12, 22, 32, 75, 79 and 82, wherein the immobilized concatimer template molecule is uridine, 8-oxo- A method comprising at least one nucleotide having a cleavable moiety comprising 7,8-dihydroguanine or deoxyinosine. The method of any one of claims 1, 12, 22, 32, 75, 79, and 82, wherein the nucleotide having the cleavable moiety is an individual of the plurality. Positioned at randomly distributed positions on an immobilized concatimer template molecule, the method. 83. The method according to any one of claims 1, 12, 22, 32, 75, 79 and 82, wherein in said individual immobilized concatimer template molecule, 0.01 of the thymidine nucleotides. to 30% replaced with uridine. 83. The method according to any one of claims 1, 12, 22, 32, 75, 79, and 82, wherein in said individual fixed conkatimer template molecule, said guanosine nucleotide is 0.01 to 30% replaced with 8-oxo-7,8-dihydroguanine or deoxyinosine. 83. The soluble forward sequence of any one of claims 5, 15, 25, 35, 48, 58, 68, 75, 79 and 82. The assay primer comprises a 3' OH extendable end and lacks a nucleotide having a cleavable moiety. 83. The soluble reverse sequence of any one of claims 6, 16, 26, 36, 49, 59, 69, 75, 79 and 82. The assay primer comprises a 3' OH extendable end and lacks a nucleotide having a cleavable moiety. The method of any one of claims 4, 13, 22, 23, 33, 47, 56, 65 and 66, wherein the first soluble amplification primer is 3 ' A method comprising an OH extendable end and lacking a nucleotide having a cleavable moiety. 67. The method of any one of claims 4, 13, 23, 33, 47, 56 and 66, wherein the second soluble amplification primer comprises a 3' OH extendable end. , a method lacking a nucleotide having a cleavable moiety. The method of claim 1, wherein said forward sequencing of step (b), or according to claim 12, said forward sequencing of step (c), or according to claim 22, said forward sequencing of step (d), or 33. The method of claim 32, wherein said forward sequencing of step (e), or according to claim 44, said forward sequencing of step (b), or according to claim 55, said forward sequencing of step (c), or The method of claim 65, wherein the forward sequencing of step (d), or the forward sequencing of step (b), or the forward sequencing of step (c) of claim 79, or the forward sequencing of step (c). 82. The method of claim 82, wherein the forward sequencing of step (d) comprises:
a) contacting a plurality of sequencing polymerases with (i) a plurality of immobilized concatimer template molecules and (ii) a plurality of soluble forward sequencing primers, wherein the contacting step involves sequencing bound to a nucleic acid duplex. performing under conditions suitable to form a plurality of complexed polymerases, each comprising a polymerase, wherein the nucleic acid duplex comprises an immobilized concatimer template molecule hybridized to a soluble forward sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides are labeled with a fluorophore. and comprising at least one nucleotide analog having a removable chain termination moiety at the 3' position of the sugar;
c) incorporating at least one nucleotide into the 3' end of the hybridized forward sequencing primer to generate a plurality of initial extended forward sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.
Method, including. 23. The reverse sequencing of step (e), or the reverse sequencing of step (f), or the reverse sequencing of step (g) of claim 22, or according to claim 32, wherein said reverse sequencing of step (h), or according to claim 75, wherein said reverse sequencing of step (j), or according to claim 79, wherein said reverse sequencing of step (k). , or the method of claim 82, wherein the reverse sequencing of step (l) comprises:
a) contacting a plurality of sequencing polymerases with (i) a plurality of said maintained forward extension strands and (ii) a plurality of said soluble reverse sequencing primers, wherein said contacting step involves sequencing linked to a nucleic acid duplex. performing under conditions suitable to form a plurality of complexed polymerases, each comprising a polymerase, wherein the nucleic acid duplex comprises a retained forward extension strand hybridized to a soluble reverse sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with the plurality of nucleotides under conditions suitable for binding at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides is a fluorophore. comprising at least one nucleotide analog that is labeled and has a removable chain termination moiety at the 3' position of the sugar;
c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer to generate a plurality of initial extended reverse sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.
Method, including. 101. The method of claim 100, wherein the reverse sequencing of step (a) comprises the plurality of soluble reverse sequencing primers,
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a crowding agent comprising polyethylene glycol (PEG) in hybridization buffer at 5 to 35% by volume.
Hybridizing to said plurality of said maintained forward extension strands in the presence of a high efficiency hybridization buffer comprising. The reverse sequencing of step (d) of claim 44, or the reverse sequencing of step (e) of claim 55, or the reverse sequencing of step (f) of claim 65,
a) a plurality of sequencing polymerases comprising (i) a plurality of fixed partially translocated forward extension strands, (ii) a plurality of fixed detachment extended forward sequencing primer strands, and (iii) a plurality of said soluble reverse sequences. A step of contacting an analytical primer, wherein the contacting step is performed under conditions suitable for forming a plurality of complexed polymerases, each comprising a sequencing polymerase linked to a nucleic acid duplex, wherein the nucleic acid duplex is a fixed partial comprising a soluble reverse sequencing primer hybridized to the translocated forward extension strand or to the immobilized detached extended forward sequencing primer strand;
b) contacting the plurality of complexed sequencing polymerases with the plurality of nucleotides under conditions suitable for binding at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides is a fluorophore. comprising at least one nucleotide analog that is labeled and has a removable chain termination moiety at the 3' position of the sugar;
c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer to generate a plurality of initial extended reverse sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.
Method, including. 103. The method of claim 102, wherein the reverse sequencing of step (a) comprises the plurality of soluble reverse sequencing primers,
(i) 25 to 50% by volume of a first polar aprotic solvent comprising acetonitrile in the hybridization buffer;
(ii) 5 to 10% by volume of a second polar aprotic solvent comprising formamide in the hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) 5 to 35% by volume of a densifying agent comprising polyethylene glycol (PEG) in the hybridization buffer.
Hybridizing to said plurality of said maintained forward extension strands in the presence of a high efficiency hybridization buffer comprising. 13. The method of claim 1, wherein the forward sequencing of step (b) and the reverse sequencing of step (e), or the forward sequencing of step (c) and the reverse sequencing of step (f). Sequencing, or according to claim 22, said forward sequencing of step (d) and said reverse sequencing of step (g), or according to claim 32, said forward sequencing of step (e) and step (h). the reverse sequencing forward sequencing, or according to claim 44, wherein the forward sequencing of step (b) and the reverse sequencing forward sequencing of step (d), or the method of claim 55, wherein step (c) The forward sequencing and the reverse sequencing of step (e), or the forward sequencing of step (d) and the reverse sequencing of step (f), or the method of claim 75, The forward sequencing of step (b) and the reverse sequencing of step (j), or the forward sequencing of step (c) and the reverse sequencing of step (k), or the method of claim 82 The method of claim 1, wherein the forward sequencing in step (d) and the reverse sequencing in step (l) are:
1) performing a sequencing reaction at a position on the template molecule using a multivalent molecule that binds but does not incorporate;
2) performing a sequencing reaction at the same position on the template molecule using the incorporated nucleotide; and
3) repeating steps a) and b) at the next position on the template molecule.
Method, including. 13. The method of claim 1, wherein the forward sequencing of step (b) and the reverse sequencing of step (e), or the forward sequencing of step (c) and the reverse sequencing of step (f). sequencing or according to claim 22, said forward sequencing of step (d) and said reverse sequencing of step (g), or according to claim 32, said forward sequencing of step (e) and step (h). said reverse sequencing forward sequencing, or according to claim 75, said forward sequencing of step (b) and said reverse sequencing of step (j), or according to claim 79, said forward sequencing of step (c). The sequencing and the reverse sequencing of step (k), or the forward sequencing of step (d) and the reverse sequencing of step (l), are
a) contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of soluble sequencing primers, wherein the contacting comprises contacting the first sequencing polymerase linked to the nucleic acid duplex. Under conditions suitable for forming a first plurality of complexed polymerases, each comprising an enzyme, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to the sequencing primer, and (1) the plurality of nucleic acid templates. the molecule comprises a plurality of said fixed concatimer template molecules, said plurality of soluble primers comprises a plurality of said soluble forward sequencing primers, or (2) said plurality of nucleic acid template molecules comprises a plurality of maintained forward extensions. strands, wherein the plurality of soluble sequencing primers comprise a plurality of soluble reverse sequencing primers;
b) conditions suitable for linking complementary nucleotide units of said multivalent molecule to at least two of said plurality of first complexed polymerases to form said plurality of multivalent-complexed polymerases, and said plurality of multivalent polymerases. -by contacting the first plurality of complexed polymerases with the plurality of detectably labeled multivalent molecules under conditions that inhibit the incorporation of complementary nucleotide units into the sequencing primers of the complexed polymerase to form a plurality of multivalent- Forming a complexed polymerase, wherein each multivalent molecule of the plurality of multivalent molecules comprises a core attached to multiple nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. ;
c) detecting the plurality of multivalent-complexed polymerases; and
d) determining the sequence of the nucleic acid template by identifying the nucleobase of the complementary nucleotide unit bound to the plurality of first complexed polymerases in the plurality of multivalent-complexed polymerases.
Method, including. 105. The method of claim 105, wherein said reverse sequencing of step (e) of claim 1 or said reverse sequencing of step (f) of claim 12, or said reverse sequencing of step (g) of claim 22, or said reverse sequencing of step (g) of claim 32. The reverse sequencing of step (h), or the reverse sequencing of step (j) of claim 75, or the reverse sequencing of step (k) of claim 79, or the reverse sequencing of step (l) of claim 82,
The plurality of soluble reverse sequencing primers,
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.
Hybridizing to said plurality of said maintained forward extension strands in the presence of a high efficiency hybridization buffer comprising. Paragraph 105:
e) dissociating the plurality of multivalent-complexed polymerases and removing the plurality of first sequencing polymerases and their associated multivalent molecules, and maintaining the plurality of nucleic acid duplexes;
f) contacting the plurality of maintained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein contacting comprises contacting the plurality of second sequencing polymerases with the plurality of maintained nucleic acid duplexes. performing under conditions suitable for binding to a duplex, forming a plurality of second complexed polymerases each comprising a second sequencing polymerase bound to a retained nucleic acid duplex;
g) contacting the plurality of second complexed polymerases with a plurality of nucleotides, wherein the contacting comprises combining complementary nucleotides from the plurality of nucleotides with the second complexed polymerase of step (f). Performed under conditions suitable for forming a plurality of nucleotide-complexed polymerases by binding to at least two of them and under conditions suitable for promoting the incorporation of complementary nucleotides bound to the sequencing primer of the nucleotide-complexed polymerase. becoming, stage;
h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase; and
i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.
A method further comprising: 106. The method of claim 105, further comprising forming at least one avidity complex of step (b),
a) linking a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a nucleic acid template molecule to form a first binding complex, wherein the first nucleotide unit of the first multivalent molecule binding to the first sequencing polymerase; and
b) linking a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule to form a second binding complex, wherein the first multivalent molecule of the second multivalent molecule 2 nucleotide units bind to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex.
Method, including. 109. The method of claim 108, wherein (i) the first sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an immobilized concatimer template molecule, and (ii) the second sequencing primer. comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises the same fixed concatimer template molecule, and (iii) the first sequencing primer and the second sequencing primer have the same sequence, method. 109. The method of claim 108, wherein (i) the first sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises a maintained forward extension strand, and (ii) the second sequencing primer comprises a soluble reverse sequencing primer. A method comprising a reverse sequencing primer, wherein the nucleic acid template molecule comprises the identical maintained forward extension strand, and (iii) the first sequencing primer and the second sequencing primer have identical sequences. 109. The method of claim 108, further comprising forming at least one avidity complex of step (b), wherein:
a) contacting a plurality of first sequencing polymerases and a plurality of second sequencing primers with different portions of a nucleic acid template molecule to form at least first and second complexed polymerases on the same nucleic acid template molecule. ;
b) binding a plurality of multivalent molecules to said at least first and second complexed polymerases on the same nucleic acid template molecule under conditions suitable for binding a single multivalent molecule from said plurality to said first and second complexed polymerases. wherein at least the first nucleotide unit of the single multivalent molecule is bound to a first complexed polymerase comprising a first sequencing primer hybridized to a first portion of the nucleic acid template molecule to form a first binding complex. wherein at least a second nucleotide unit of the single multivalent molecule is bound to a second complexed polymerase comprising a second sequencing primer hybridized to a second portion of the same nucleic acid template molecule to form a second binding complex. wherein the contacting step is performed under conditions suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes, and binding to the same multivalent molecule. The first and second binding complexes form a binding active complex;
c) detecting the first and second binding complexes on the same nucleic acid template molecule, and
d) determining the sequence of the first portion of the nucleic acid template molecule by identifying the first nucleotide unit in the first binding complex, and identifying the second nucleotide unit in the second binding complex to produce the same nucleic acid template molecule. determining the sequence of the second portion of
Method, including. 112. The method of claim 111, wherein (i) the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the nucleic acid template molecules comprise immobilized concatimer template molecules, and (ii) the plurality of second sequencing primers comprises a plurality of second soluble forward sequencing primers, the nucleic acid template molecules comprise the identical immobilized concatimer template molecules, and (iii) the plurality of first sequences Wherein the analysis primer and the second sequencing primer have the same sequence. 112. The method of claim 111, wherein (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecules comprise a retained forward extension strand, and (ii) the plurality of first sequencing primers comprise a plurality of first soluble reverse sequencing primers. the second sequencing primer comprises a plurality of second soluble reverse sequencing primers, the nucleic acid template molecule comprises the identical maintained forward extension strand, and (iii) the plurality of first sequencing primers and 2 sequencing primers have the same sequence. 45. The method of claim 44, wherein the forward sequencing of step (b) and the reverse sequencing of step (d), or the forward sequencing of step (c) and the reverse sequencing of step (e). Analysis, according to claim 65, wherein said forward sequencing of step (d) and reverse sequencing of step (f) comprises:
a) contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of soluble sequencing primers, wherein the contacting comprises contacting the first sequencing polymerase linked to the nucleic acid duplex. Carried out under conditions suitable for forming a first plurality of complexed polymerases, each comprising an enzyme, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a soluble sequencing primer, and (1) the plurality of nucleic acid template molecules comprises a plurality of fixed concatimer template molecules, and the plurality of sequencing primers comprises a plurality of the soluble forward sequencing primers, or (2) the plurality of nucleic acid template molecules comprise a plurality of fixed partially translocated molecules. (3) the plurality of nucleic acid template molecules comprise a plurality of fixed, detached extended forward sequencing primer strands, and , wherein the plurality of sequencing primers comprises a plurality of the soluble reverse sequencing primers;
b) conditions suitable for linking complementary nucleotide units of a multivalent molecule to at least two of said plurality of first complexed polymerases to form a plurality of multivalent-complexed polymerases, and said plurality of multivalent-complexes. The plurality of first complexed polymerases are contacted with a plurality of detectably labeled multivalent molecules under conditions that inhibit the incorporation of complementary nucleotide units into the sequencing primers of the complexed polymerase to form the plurality of multivalent-complexed polymerases. forming a polymerase, wherein each multivalent molecule of the plurality of multivalent molecules comprises a core attached to multiple nucleotide arms, each nucleotide arm being attached to a nucleotide unit;
c) detecting the plurality of multivalent-complexed polymerases; and
d) determining the sequence of the nucleic acid template by identifying the nucleobase of the complementary nucleotide unit bound to the first plurality of complexed polymerases in the plurality of multivalent-complexed polymerases.
Method, including. 115. The method of claim 114, wherein said reverse sequencing of step (d) of claim 44 or said reverse sequencing of step (e) of claim 55 or said reverse sequencing of step (f) of claim 65,
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.
Hybridizing the plurality of soluble reverse sequencing primers to the plurality of fixed partially translocated forward extension strands or to the plurality of fixed detachable extended forward sequencing primer strands in the presence of a high-efficiency hybridization buffer comprising , method. According to clause 114,
e) dissociating the plurality of multivalent-complexed polymerases and removing the plurality of first sequencing polymerases and their associated multivalent molecules, and maintaining the plurality of nucleic acid duplexes;
f) contacting the plurality of maintained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein contacting comprises contacting the plurality of second sequencing polymerases with the plurality of maintained nucleic acid duplexes. performing under conditions suitable for binding to a duplex, forming said plurality of second complexed polymerases each comprising a second sequencing polymerase bound to said retained nucleic acid duplex;
g) contacting the plurality of second complexed polymerases with a plurality of nucleotides, wherein the contacting step causes complementary nucleotides from the plurality of nucleotides to be brought into contact with at least one of the second complexed polymerases of step (f). carried out under conditions suitable for combining the two to form a plurality of nucleotide-complexed polymerases and under conditions suitable for promoting incorporation of complementary nucleotides linked to the sequencing primer of the nucleotide-complexed polymerase. step;
h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase; and
i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.
A method further comprising: 115. The method of claim 114, further comprising forming at least one avidity complex of step (b), wherein the method comprises:
a) linking a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a nucleic acid template molecule to form a first binding complex, wherein the first nucleotide of the first multivalent molecule wherein the unit binds to the first sequencing polymerase; and
b) linking the second sequencing primer, the second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule to form a second binding complex, wherein the second nucleotide of the second multivalent molecule forming the second binding complex, wherein the unit binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex.
Method, including. 118. The method of claim 117, wherein (i) the first sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an immobilized concatimer template molecule, and (ii) the second sequencing primer comprises A method comprising a soluble forward sequencing primer, wherein the nucleic acid template molecules comprise identical immobilized concatimer template molecules, and (iii) the first sequencing primer and the second sequencing primer have identical sequences. 118. The method of claim 117, wherein (i) the first sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises a fixed, partially translocated forward extension strand, and (ii) the second sequence The assay primers comprise a soluble reverse sequencing primer, the nucleic acid template molecules comprise identical fixed partially translocated forward extension strands, and (iii) the first sequencing primer and the second sequencing primer have identical sequences. Having, way. 118. The method of claim 117, wherein (i) the first sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises an immobilized, detached extended forward sequencing primer strand, and (ii) the second sequencing primer. The primers comprise a soluble reverse sequencing primer, the nucleic acid template molecules comprise identical immobilized, detached extended forward sequencing primer strands, and (iii) the first sequencing primer and the second sequencing primer have identical sequences. , method. 118. The method of claim 117, further comprising forming at least one avidity complex of step (b), wherein:
a) contacting a plurality of first sequencing polymerases and a plurality of second sequencing primers with different portions of a nucleic acid template molecule to form at least first and second complexed polymerases on the same nucleic acid template molecule. ;
b) contacting the plurality of multivalent molecules with at least the first and second complexed polymerases on the same nucleic acid template molecule under conditions suitable for binding a single multivalent molecule from the plurality to the first and second complexed polymerases. wherein at least the first nucleotide unit of the single multivalent molecule is linked to a first complexed polymerase comprising a first sequencing primer hybridized to a first portion of the nucleic acid template molecule to form a first binding complex; , wherein at least a second nucleotide unit of the single multivalent molecule is bound to a second complexed polymerase comprising a second sequencing primer hybridized to a second portion of the same nucleic acid template molecule to form a second binding complex, The contacting step is performed under conditions suitable to inhibit polymerase-catalyzed incorporation of the first and second nucleotide units bound in the first and second binding complexes, wherein the first and second nucleotide units bound to the same multivalent molecule 2. The binding complex forms an avidity complex;
c) detecting the first and second binding complexes on the same nucleic acid template molecule, and
d) determining the sequence of the first portion of the nucleic acid template molecule by identifying the first nucleotide unit in the first binding complex, and identifying the second nucleotide unit in the second binding complex to produce the same nucleic acid template molecule determining the sequence of the second portion of
Method, including. 122. The method of claim 121, wherein (i) the plurality of first sequencing primers comprises a plurality of first soluble forward sequencing primers, and the nucleic acid template molecules comprise immobilized concatimer template molecules, and (ii) the plurality of second sequencing primers comprises a plurality of second soluble forward sequencing primers, the nucleic acid template molecules comprise identical immobilized concatimer template molecules, and (iii) the plurality of first sequencing primers. and the second sequencing primer has the same sequence. 122. The method of claim 121, wherein (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises a fixed, partially translocated forward extension strand, and (ii) the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, the nucleic acid template molecules comprise identical fixed partially translocated forward extension strands, and (iii) the plurality of second sequencing primers comprise a plurality of second soluble reverse sequencing primers, and The first sequencing primer and the second sequencing primer have the same sequence. 122. The method of claim 121, wherein (i) the plurality of first sequencing primers comprises a plurality of first soluble reverse sequencing primers, and the nucleic acid template molecule comprises an immobilized, detached extended forward sequencing primer strand, and (ii) the plurality of second sequencing primers comprises a plurality of second soluble reverse sequencing primers, and the nucleic acid template molecules comprise identical fixed, detachable, extended forward sequencing primer strands, and (iii) the plurality of second soluble reverse sequencing primers, The method of claim 1, wherein the plurality of first sequencing primers and second sequencing primers have the same sequence. The method of any one of claims 99, 100, 102, 107, and 116, wherein in said plurality of nucleotides, each nucleotide comprises an aromatic base, a pentose sugar, and 1 to 10 phosphate groups, The method of claim 1, wherein the aromatic base includes adenine, guanine, cytosine, thymine, or uracil. 126. The method of claim 125, wherein the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, and dTTP. 126. The method of claim 125, wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, and/or dTTP. 126. The method of claim 125, wherein at least one of the nucleotides in the plurality of nucleotides comprises a fluorescently labeled nucleotide. 126. The method of claim 125, wherein at least one of the plurality of nucleotides lacks a fluorophore label. 117. The method according to any one of claims 99, 100, 102, 107, and 116, wherein in said plurality of nucleotides at least one of said nucleotides has a 3′-OH sugar position via a cleavable moiety. Comprising a chain termination moiety attached to, wherein the chain termination moiety is an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group. A method comprising a nate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. According to clause 130,
(i) the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are terminated by tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ), by piperidine or by 2,3 -cleavable/removable by dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ);
(ii) the chain terminating moieties aryl and benzyl are cleavable/removable by H2 Pd/C;
(iii) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by thiol reagents including beta-mercaptoethanol or dithiothritol (DTT) possible;
(iv) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) ) or cleavable/removable by phosphine reagents including tri(hydroxypropyl)phosphine (THPP);
(v) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by 4-dimethylaminopyridine (4-DMAP);
(vi) the chain terminating moiety carbonate is cleavable/removable by potassium carbonate (K ₂ CO ₃ ) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH); and
(vii) the chain terminating moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride or by triethylamine trihydrofluoride. 131. The method of claim 130, wherein at least one of the nucleotides in the plurality of nucleotides comprises a chain terminating moiety attached to the 3'-OH sugar position through a cleavable moiety, and wherein the chain terminating moiety is 3' O- A method comprising an azido or 3' O-azidomethyl group. According to clause 130,
(i) the chain termination moieties 3' O-azido and 3' O-azidomethyl groups are derivatized tri-alkyl phosphine moieties, derivatized tri-aryl phosphine moieties, tris(2-carboxylic cleavable/removable by phosphine compounds including ethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or tri(hydroxypropyl)phosphine (THPP); and
(ii) the chain terminating moieties 3' O-azido and 3' O-azidomethyl groups are cleavable/removable by 4-dimethylaminopyridine (4-DMAP). The method according to any one of claims 104, 105, 108, 111, 114, 117, and 121, wherein each multivalent molecule of the plurality of multivalent molecules is (a) a core ; and (b) a plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core comprises a core attachment moiety. A method of claim 1, wherein the spacer is attached to a plurality of nucleotide arms via a tee, the spacer is attached to a linker, and the linker is attached to a nucleotide unit. 135. The method of claim 134, wherein the core comprises an avidin-type moiety and the core attachment moiety comprises biotin. 135. The method of claim 134, wherein the linker comprises an aliphatic chain having 2 to 6 subunits or an oligo ethylene glycol chain having 2 to 6 subunits. 135. The method of claim 134, wherein the linker further comprises an aromatic moiety. 135. The method of claim 134, wherein the nucleotide units comprise an aromatic base, a pentose sugar, and 1 to 10 phosphate groups. 135. The method of claim 134, wherein the linker is attached to the nucleotide unit via a base. 135. The method of claim 134, wherein the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. 135. The method of claim 134, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, wherein each multivalent molecule of the plurality has the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. , method. 135. The method of claim 134, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. , method. 135. The method of claim 134, wherein the plurality of multivalent molecules are fluorescently labeled multivalent molecules. According to clause 134,
(i) the core of the individual fluorescently labeled multivalent molecule is attached to a fluorophore corresponding to the nucleotide unit attached to the nucleotide arm;
(ii) at least one of said nucleotide arms comprises a linker attached to a fluorophore corresponding to a nucleotide unit attached to said nucleotide arm; and/or
(iii) at least one of the nucleotide arms comprises a nucleotide unit attached to a fluorophore corresponding to the nucleotide unit attached to the nucleotide arm. 135. The method of claim 134, wherein the plurality of multivalent molecules lack a fluorophore. 135. The method of claim 134, wherein at least one of the plurality of multivalent molecules comprises a nucleotide unit having a chain termination moiety attached to the 3'-OH sugar position through a cleavable moiety, and the chain termination moiety is Alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea A method comprising a group or a silyl group. Paragraph 146:
(i) the chain terminating moieties alkyl, alkenyl, alkynyl and allyl are terminated by tetrakis(triphenylphosphine)palladium(0)(Pd(PPh ₃ ) ₄ ), by piperidine or by 2,3 -cleavable/removable by dichlor-5,6-dicyano-1,4-benzo-quinone (DDQ);
(ii) the chain terminating moieties aryl and benzyl are cleavable/removable by H2 Pd/C;
(iii) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by thiol reagents including beta-mercaptoethanol or dithiothritol (DTT) possible;
(iv) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are tris(2-carboxyethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) ) or cleavable/removable by phosphine reagents including tri(hydroxypropyl)phosphine (THPP);
(v) the chain terminating moieties amine, amide, keto, isocyanate, phosphate, thio, disulfide are cleavable/removable by 4-dimethylaminopyridine (4-DMAP);
(vi) the chain terminating moiety carbonate is cleavable/removable by potassium carbonate (K ₂ CO ₃ ) in MeOH, by triethylamine in pyridine, or by Zn in acetic acid (AcOH); and
(vii) the chain terminating moieties urea and silyl are cleavable by tetrabutylammonium fluoride, pyridine-HF, by ammonium fluoride or by triethylamine trihydrofluoride. 147. The method of claim 146, wherein at least one of the plurality of multivalent molecules comprises a nucleotide unit having a chain termination moiety attached to the 3'-OH sugar position through a cleavable moiety, and the chain termination moiety is A method comprising a 3' O-azido or 3' O-azidomethyl group. Paragraph 146:
(i) the chain termination moieties 3' O-azido and 3' O-azidomethyl groups are derivatized tri-alkyl phosphine moieties, derivatized tri-aryl phosphine moieties, tris(2-carboxylic cleavable/removable by phosphine compounds including ethyl)phosphine (TCEP), bis-sulfo triphenyl phosphine (BS-TPP) or tri(hydroxypropyl)phosphine (THPP); and
(ii) the chain terminating moieties 3' O-azido and 3' O-azidomethyl are cleavable/removable by 4-dimethylaminopyridine (4-DMAP). 103. The method of any one of claims 99, 100, and 102, wherein the plurality of sequencing polymerases in step (a) comprises a recombinant wild-type DNA polymerase, and the plurality of nucleotides in step (b) are 3. ' A method comprising a fluorescently labeled nucleotide having a removable chain termination moiety at the sugar position. 103. The method of any one of claims 99, 100, and 102, wherein the plurality of sequencing polymerases in step (a) comprises a mutant DNA polymerase, and the plurality of nucleotides in step (b) are 3 ' A method comprising a fluorescently labeled nucleotide having a removable chain termination moiety at the sugar position. 115. The method of claim 105 or 114, wherein the first plurality of sequencing polymerases in step (a) comprises a recombinant wild-type DNA polymerase. 115. The method of claim 105 or 114, wherein the first plurality of sequencing polymerases of step (a) comprises a mutant DNA polymerase. 117. The method of claim 107 or 116, wherein the plurality of second sequencing polymerases in step (f) comprises a recombinant wild-type DNA polymerase, and in step (b) the plurality of nucleotides are at the 3′ sugar position. A method comprising a fluorescently labeled nucleotide having a removable chain termination moiety. 117. The method of claim 107 or 116, wherein the plurality of second sequencing polymerases in step (f) comprises a mutant DNA polymerase, and in step (b) the plurality of nucleotides are at the 3′ sugar position. A method comprising a fluorescently labeled nucleotide having a removable chain termination moiety. In step (c) of claim 1, or step (d) of claim 12, or step (e) of claim 22, or step (f) of claim 32, the plurality of extended forward sequences are performed by performing a primer extension reaction. Replacing the assay primer strand with a plurality of forward extension strands that hybridize to the retained fixed single-stranded nucleic acid concatimer template molecule comprises
(i) under conditions suitable for performing a strand transposition primer extension reaction using at least one extended forward sequencing primer to initiate a primer extension reaction, a plurality of strand transposes the at least one extended forward sequencing primer strand; contact with a polymerase and a plurality of nucleotides and in the absence of a soluble amplification primer to generate a forward extension strand covalently linked to the extended forward sequencing primer strand, wherein the forward extension strand hybridizes to the immobilized concatimer template molecule. stages of becoming
Method, including. In step (c) of claim 1, or step (d) of claim 12, or step (e) of claim 22, or step (f) of claim 32, the plurality of extended forward sequences are performed by performing a primer extension reaction. Replacing the assay primer strand with a plurality of forward extension strands that hybridize to the retained fixed single-stranded nucleic acid concatimer template molecule comprising:
(i) contacting the plurality of extended forward sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease;
(ii) contacting the plurality of extended forward sequencing primer strands with a denaturing reagent comprising any combination of formamide, acetonitrile, guanidinium chloride, and/or a pH buffer; or
(iii) contacting the plurality of extended forward sequencing primer strands with 100% formamide.
A method comprising removing a plurality of extended forward sequencing primer strands by . In step (c) of claim 1, or step (d) of claim 12, or step (e) of claim 22, or step (f) of claim 32, the plurality of extended forward sequences are performed by performing a primer extension reaction. Replacing the assay primer strand with a plurality of forward extension strands that hybridize to the retained fixed single-stranded nucleic acid concatimer template molecule comprising:
(i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and
(ii) said plurality of soluble forward sequencing primers are combined with said plurality of soluble forward sequencing primers under conditions suitable for hybridization to a plurality of retained immobilized conkatimer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction. Contacting the concatimer molecule with a second plurality of soluble forward sequencing primers, a plurality of nucleotides, and a plurality of primer extension polymerases to generate a plurality of forward extension strands.
Including,
wherein the plurality of nucleotides includes dATP, dGTP, dCTP and dTTP, but lacks dUTP,
the plurality of primer extension polymerases are resistant to uridine-containing template strands,
The method of claim 1 , wherein the soluble sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized concatimer molecule. 159. The method of claim 158, wherein the contacting step comprises:
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.
A method comprising contacting the plurality of retained immobilized concatimer molecules with the plurality of soluble forward sequencing primers in the presence of a high-throughput hybridization buffer comprising. In step (c) of claim 1, or step (d) of claim 12, or step (e) of claim 22, or step (f) of claim 32, the plurality of extended forward sequences are performed by performing a primer extension reaction. Replacing the assay primer strand with a plurality of forward extension strands that hybridize to the retained fixed single-stranded nucleic acid concatimer template molecule comprising:
(i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and
(ii) under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatimer template molecules and for performing a polymerase-catalyzed primer extension reaction, Contacting the concatimer molecule with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides) and a plurality of primer extension polymerases to generate a plurality of forward extension strands, wherein the soluble amplification primers are Hybridizing the soluble amplification primer binding sequence in the maintained immobilized concatimer molecule.
Including,
wherein the plurality of nucleotides includes dATP, dGTP, dCTP and dTTP, but lacks dUTP,
the plurality of primer extension polymerases are resistant to uridine-containing template strands,
The method of claim 1 , wherein the soluble sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized concatimer molecule. 161. The method of claim 160, wherein the contacting step comprises:
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.
A method comprising contacting the plurality of retained immobilized concatimer molecules with the plurality of soluble amplification primers in the presence of a high-efficiency hybridization buffer comprising. 161. The method of any one of claims 156, 158, and 160, further comprising contacting the plurality of retained immobilized concatimer molecules with a plurality of soluble compacted oligonucleotides. The method of step (c) of claim 44, or step (d) of claim 55, or step (e) of claim 65, wherein the plurality of extended forward sequencing primer strands comprises:
(i) at least one extended forward sequencing primer in the absence of a soluble amplification primer under conditions suitable for performing a strand transposition primer extension reaction using at least one extended forward sequencing primer strand to initiate the primer extension reaction. Contacting the strand with a plurality of strand transposition polymerases and a plurality of nucleotides to generate a plurality of forward extension strands, a plurality of partially transposed extended forward sequencing strands, and a plurality of detachment extended forward sequencing primer strands.
Method, including. The method of step (c) of claim 44, or step (d) of claim 55, or step (e) of claim 65, wherein the plurality of extended forward sequencing primer strands comprises:
(i) contacting the plurality of extended forward sequencing primer strands with a 5' to 3' double-stranded DNA exonuclease;
(ii) contacting the plurality of extended forward sequencing primer strands with a denaturing reagent comprising any combination of formamide, acetonitrile, guanidinium chloride, and/or a pH buffer; or
(iii) contacting the plurality of extended forward sequencing primer strands with 100% formamide.
A method comprising removing the plurality of extended forward sequencing primer strands by . The method of step (c) of claim 44, or step (d) of claim 55, or step (e) of claim 65, wherein the plurality of extended forward sequencing primer strands comprises:
(i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and
(ii) under conditions suitable for hybridizing the plurality of soluble forward sequencing primers to the plurality of retained immobilized concatimer template molecules and for performing a polymerase-catalyzed strand transfer reaction, Contacting an immobilized concatimer molecule with a second plurality of soluble forward sequencing primers, a plurality of nucleotides, and a plurality of strand transfer polymerases to produce a plurality of forward extension strands and a plurality of partial fragments hybridized to the immobilized concatimer template molecule. to generate a translocated extended forward sequencing strand to form a plurality of immobilized amplicons, and the primer extension reaction is performed by attaching a plurality of detached extended forward sequencing primer strands (e.g., to an immobilized concatimer template molecule). Steps to produce (not hybridized)
Including,
wherein the plurality of nucleotides includes dATP, dGTP, dCTP and dTTP, but lacks dUTP,
The method of claim 1 , wherein the soluble forward sequencing primer hybridizes to the forward sequencing primer binding sequence in the retained immobilized concatimer molecule. 166. The method of claim 165, wherein the contacting step comprises contacting the plurality of retained immobilized concatimer molecules with the plurality of soluble forward sequencing primers in the presence of a high-throughput hybridization buffer comprising: :
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a densifying agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of hybridization buffer. The method of step (c) of claim 44, or step (d) of claim 55, or step (e) of claim 65, wherein the plurality of extended forward sequencing primer strands comprises:
(i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and
(ii) a plurality of soluble amplification sequencing primers suitable for hybridizing a plurality of soluble amplification sequencing primers to a plurality of retained immobilized concatimer template molecules and hybridizing to the immobilized concatimer template molecules by performing a polymerase-catalyzed strand transfer reaction. Under conditions suitable for generating a forward extension strand and a plurality of partially transposed extended forward sequencing strands to form a plurality of fixed amplicons, the plurality of retained fixed concatimer molecules is combined with a plurality of soluble amplification primers; contacting a plurality of nucleotides and a plurality of strand transposition polymerases, wherein the primer extension reaction generates a plurality of detached extended amplification primer strands (e.g., that do not hybridize to the immobilized concatimer template molecule).
Including,
wherein the plurality of nucleotides includes dATP, dGTP, dCTP and dTTP, but lacks dUTP,
The method of claim 1 , wherein the soluble amplification primer is hybridized with a soluble amplification primer binding sequence in the retained immobilized conkatimer molecule. 167. The method of claim 167, wherein the contacting step comprises:
(i) a first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of hybridization buffer;
(ii) a second polar aprotic solvent comprising formamide at 5 to 10% by volume of hybridization buffer;
(iii) a pH buffering system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and
(iv) a densifying agent comprising polyethylene glycol (PEG) in 5 to 35% by volume of hybridization buffer.
A method comprising contacting the plurality of retained immobilized concatimer molecules with the plurality of soluble amplification primers in the presence of a high-efficiency hybridization buffer comprising. 83. The method according to any one of claims 1, 12, 22, 32, 75, 79, and 82, wherein at least one of the retained fixed concatimer template molecules is capable of cleavage. A method comprising one or more nucleotides having a moiety, wherein the cleavable moiety comprises uridine or 8-oxo-7,8-dihydroguanine or deoxyinosine. 169. The method of claim 169, wherein the retained fixed conkatimer template molecule comprises one or more uridines, and creating the non-basic site at the uridine comprises uracil DNA glycolysis of the retained fixed conkatimer template molecule. A method comprising contacting with silase (UDG). 169. The method of claim 169, wherein the retained fixed concatimer template molecule comprises one or more 8oxoG (8oxoG), and generating the non-basic site at the 8oxoG is a retained fixed conkatimer template molecule. A method comprising contacting with Fpg enzyme (formamidopyrimidine DNA glycosylase). 169. The method of claim 169, wherein the retained fixed conkatimer template molecule comprises one or more deoxyinosines, and creating an abasic site at the deoxyinosine causes the retained fixed conkatimer template molecule to be activated by AlkA glycolysis. A method comprising contacting with a silase enzyme. 173. The method of any one of claims 170-172, further comprising creating a gap at an abasic site to generate at least one gap-containing concatimer template molecule, comprising at least one abasic site. The retained anchor template molecule is endonuclease IV, AP lyase (e.g., DNA-apurinic lyase or DNA-alpyrimidine lyase), FPG glycosylase/AP lyase and/or endo VIII. A method comprising contacting with glycosylase/AP lyase. 151. The method of claim 150, wherein the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of wild-type sequencing polymerases have an error rate of incorporated dUTP of at least 0.1× compared to the error rate of incorporated dTTP. How to obtain. 152. The method of claim 151, wherein the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of mutant sequencing polymerases has an error rate of incorporated dUTP of at least 0.1× compared to the error rate of incorporated dTTP. How to obtain. 155. The method of claim 154, wherein the immobilized conkatimer template molecule comprises 0.1 to 30% uridine, and the plurality of wild-type sequencing polymerases yield an error rate of incorporated dUTP of at least 0.1× compared to the error rate of incorporated dTTP. How to. 156. The method of claim 155, wherein the immobilized concatimer template molecule comprises 0.1 to 30% uridine, and the plurality of mutant sequencing polymerases have an error rate of incorporated dUTP of at least 0.1× compared to the error rate of incorporated dTTP. How to obtain. 1 of R2 according to any one of claims 1, 12, 22, 32, 44, 55, 65, 75, 79 and 82. The ratio of the base fluorescence signal (e.g., reverse sequencing) to the first base fluorescence signal of R1 (e.g., forward sequencing) is at least 0.7 for sequencing using 1, 2, 3, or 4 dye colors. In,method. 13. The method of claim 12, wherein the rolling ring amplification of step (b) comprises a plurality of compressed oligonucleotides and/or hexamine and is more compact in size and compared to a rolling ring amplification reaction in the absence of compressed oligonucleotides and/or hexamine. /or generating an immobilized concatimer template molecule having a shape. 13. The method of claim 12, wherein the primer extension reaction of step (d) comprises the plurality of compressed oligonucleotides and/or hexamine, and is more compact in size compared to the primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. and/or generating a plurality of forward extending strands having a shape. 23. The method of claim 22, wherein the rolling ring amplification of steps (a), (b) and/or (c) comprises a plurality of compressed oligonucleotides and/or hexamine, thereby reducing the rotation in the absence of compressed oligonucleotides and/or hexamine. A method comprising producing concatimer molecules having a more compact size and/or shape compared to a ring amplification reaction. 23. The method of claim 22, wherein the primer extension reaction of step (e) includes the plurality of compressed oligonucleotides and/or hexamine and is more compact in size and/or compared to the primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. or generating a plurality of forward extending strands having a shape. 33. The method of claim 32, wherein the rolling ring amplification of step (d) comprises a plurality of compressed oligonucleotides and/or hexamine and is more compact in size and compared to a rolling ring amplification reaction in the absence of compressed oligonucleotides and/or hexamine. /or generating an immobilized concatimer template molecule having a shape. 33. The method of claim 32, wherein the primer extension reaction of step (f) comprises the plurality of compressed oligonucleotides and/or hexamine and is more compact in size and /or generating a plurality of forward extending strands having a shape. 45. The method of claim 44, wherein the primer extension reaction of step (c) comprises a plurality of compressed oligonucleotides and/or hexamine and is more compact in size and/or compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. or generate a plurality of primer extension products having a shape, wherein the plurality of primer extension products include a plurality of forward extension strands, a plurality of partially transposed extended forward sequencing strands, and a plurality of detachable extended forward sequencing primer strands. Including, method. 56. The method of claim 55, wherein the rolling ring amplification of step (b) comprises a plurality of compressed oligonucleotides and/or hexamine and is more compact in size and compared to a rolling ring amplification reaction in the absence of compressed oligonucleotides and/or hexamine. /or generating an immobilized concatimer template molecule having a shape. 56. The method of claim 55, wherein the primer extension reaction of step (d) comprises a plurality of compressed oligonucleotides and/or hexamine and is more compact in size and/or compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. or generate a plurality of primer extension products having a shape, wherein the plurality of primer extension products include a plurality of forward extension strands, a plurality of partially transposed extended forward sequencing strands, and a plurality of detachable extended forward sequencing primer strands. Including, method. 66. The method of claim 65, wherein said rolling ring amplification of steps (a), (b) and/or (c) comprises a plurality of compressed oligonucleotides and/or hexamine and rotates in the absence of compressed oligonucleotides and/or hexamine. A method comprising producing a plurality of concatimer molecules having a more compact size and/or shape compared to a ring amplification reaction. 66. The method of claim 65, wherein the primer extension reaction of step (e) comprises a plurality of compressed oligonucleotides and/or hexamine and is more compact in size and/or compared to a primer extension reaction in the absence of compressed oligonucleotides and/or hexamine. or generate a plurality of primer extension products having a shape, wherein the plurality of primer extension products include a plurality of forward extension strands, a plurality of partially transposed extended forward sequencing strands, and a plurality of detachable extended forward sequencing primer strands. Including, method. 188. The method of any one of claims 179, 181, 183, 186, and 188, wherein the plurality of immobilized concatimer template molecules or the plurality of immobilized concatimer molecules have a FWHM (full width half maximum) is less than or equal to about 5 μm. 185. The method of any one of claims 180, 182, and 184, wherein the plurality of forward extending strands have a full width at half maximum (FWHM) of about 5 μm or less. 189. The method of any one of claims 185, 187, and 189, wherein the plurality of primer extension products have a full width at half maximum (FWHM) of about 5 μm or less. As a pairwise sequencing method,
a) providing a plurality of single-stranded nucleic acid concatimer template molecules immobilized on a support;
b) sequencing the plurality of immobilized concatimer template molecules with a first plurality of sequencing polymerase, a plurality of soluble forward sequencing primers, and a first plurality of multivalent molecules, thereby forming a plurality of extended forward sequencing primer strands. generating a;
c) Retaining the plurality of immobilized Concatimer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the maintained immobilized Concatimer template molecules by performing a primer extension reaction. steps;
d) removing the retained immobilized concatimer template molecules while retaining the plurality of forward extending strands and retaining the plurality of immobilized surface primers; and
e) sequencing the plurality of maintained forward extension strands using a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules.
Including,
Each of the first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprises: (i) a core; and (ii) a plurality of nucleotide arms comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via a core attachment moiety, and the spacer is attached to the linker. and wherein the linker is attached to the nucleotide unit. 194. The method of claim 193, wherein the nucleotide units of the individual multivalent molecules of step (b) bind to the first polymerase linked to a nucleic acid duplex comprising an immobilized concatimer template molecule hybridized to a forward sequencing primer. . 194. The method of claim 193, wherein the nucleotide units of the individual multivalent molecules of step (e) bind to a second polymerase linked to a nucleic acid duplex comprising a retained forward extension strand hybridized to a reverse sequencing primer. 194. The method of claim 193, wherein the core comprises streptavidin and the core attachment moiety comprises biotin. 194. The method of claim 193, wherein the spacer comprises a polyethylene glycol (PEG) moiety. 194. The method of claim 193, wherein the linker comprises an aliphatic chain having 2 to 6 subunits or an oligo ethylene glycol chain having 2 to 6 subunits. 194. The method of claim 193, wherein the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. 194. The method of claim 193, wherein said first plurality of multivalent molecules of step (b) and said second plurality of multivalent molecules of step (e) comprise one type of multivalent molecule, wherein each multivalent molecule of said plurality is having nucleotide units of the same type selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP. 194. The method of claim 193, wherein the first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprise a mixture of any combination of two or more types of multivalent molecules, each wherein the type has nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP. 194. The method of claim 193, wherein at least one of said first plurality of multivalent molecules of step (b) is labeled with a multivalent molecule fluorophore, and wherein at least one multivalent molecule of said second plurality of multivalent molecules of step (e) is fluorescent. Labeled as a hem, method. 194. The method of claim 193, wherein each multivalent molecule is attached to a fluorophore corresponding to a nucleotide unit attached to the nucleotide arm of a given multivalent molecule. 193. The method of claim 193, wherein said sequencing in step (b) comprises:
a) contacting the first plurality of sequencing polymerases with (i) the plurality of immobilized concatimer template molecules and (ii) the plurality of soluble forward sequencing primers, wherein the contacting step includes the soluble forward sequencing primers. performed under conditions suitable for forming a plurality of first complexed polymerases, each comprising a first sequencing polymerase linked to a nucleic acid duplex comprising an immobilized conkatimer template molecule hybridized to a sequencing primer, step;
b) contacting the plurality of first complexed polymerases with a plurality of fluorophore-labeled multivalent molecules to form a plurality of binding complexes, wherein the contacting step causes the complementary nucleotide units of the multivalent molecule to form a plurality of binding complexes. performing conditions suitable for binding to at least two of the plurality of first complexed polymerases to form a plurality of binding complexes, and conditions that inhibit incorporation of the complementary nucleotide units into the forward sequencing primer. ;
c) detecting the plurality of binding complexes; and
d) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first complexed polymerases to determine the sequence of the immobilized concatimer template molecule.
Method, including. 205. The method of claim 204, wherein each of the plurality of binding complexes comprises a first sequencing polymerase bound to a multivalent molecule, and wherein the binding complex exhibits a duration of greater than 0.5 seconds. 194. The method of claim 193, further comprising forming at least one avidity complex of step (b), wherein:
a) binding a first soluble forward sequencing primer, a first forward sequencing polymerase, and a first multivalent molecule to a first portion of a first concatimer template molecule to form a first binding complex, wherein wherein the first nucleotide unit of the multivalent molecule binds to a first sequencing polymerase; and
b) linking a second forward sequencing primer, a second forward sequencing polymerase, and a first multivalent molecule to a second portion of the same first concatimer template molecule to form a second binding complex, A second nucleotide unit of the second multivalent molecule binds to a second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex. step
Method, including. 194. The method of claim 193, wherein the plurality of fixed concatimer template molecules comprise at least one nucleotide having a cleavable moiety that can be cleaved to create an abasic site in the concatimer template molecule, wherein one nucleotide has a moiety capable of cleavage from the immobilized concatimer template molecule comprising uridine, 8-oxo-7,8-dihydroguanine or deoxyinosine. 194. The method of claim 193, wherein the plurality of immobilized concatimer template molecules lack nucleotides having a cleavable moiety that can be cleaved to create an abasic site in the concatimer template molecule, and wherein the immobilized concatimer template molecules The method of claim 1, wherein the template molecule lacks a nucleotide having a cleavable moiety comprising uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. 194. The method of claim 193, wherein each template molecule of the plurality is covalently linked to the immobilized surface primer. 194. The method of claim 193, wherein each template molecule of the plurality is hybridized to the immobilized surface primer. 194. The method of claim 193, wherein each molecule of the plurality is immobilized on a surface primer immobilized on the support, wherein the immobilized surface primer lacks a nucleotide having a cleavable moiety and the nucleotide having a cleavable moiety is The method of claim 1, wherein the nucleotides comprise uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. As a pairwise sequencing method,
a) providing a plurality of fixed single-stranded nucleic acid conkatimer template molecules each comprising at least one nucleotide having a cleavable moiety that can be cleaved to create a non-basic site in the conkatimer template molecule, , each template molecule of the plurality is immobilized on a surface primer immobilized on a support, the immobilized surface primer lacks a nucleotide having a cleavable moiety, and the nucleotide having a cleavable moiety contains uridine. to do, step;
b) sequencing the plurality of immobilized concatimer template molecules using a first plurality of sequencing polymerases, a plurality of soluble forward sequencing primers, and a first plurality of multivalent molecules, thereby performing a plurality of extended forward sequencing Generating primer strands, wherein each immobilized concatimer template molecule has at least two extended forward sequencing primer strands hybridized thereto;
c) maintaining the plurality of fixed Concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands into a plurality of forward extension strands hybridized to the maintained fixed Concatimer template molecules. replacing;
d) creating an abasic site of the fixed concatimer template molecule at the nucleotide(s) having the cleavable moiety, while maintaining the plurality of forward extension strands and maintaining the plurality of fixed surface primers. and removing the retained immobilized concatimer template molecule by creating a gap at the non-basic site to generate a plurality of gap-containing concatimer template molecules; and
e) sequencing the plurality of maintained forward extension strands using a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules, thereby forming a plurality of extended reverse sequencing primer strands. A step of generating a primer strand, wherein each maintained forward extension strand is hybridized with two or more extended reverse sequencing primer strands.
Including,
Each of the first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprises: (i) a core; and (ii) a plurality of nucleotide arms comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms via a core attachment moiety, and the spacer is attached to the linker. and wherein the linker is attached to the nucleotide unit. 213. The method of claim 212, wherein the nucleotide units of the individual multivalent molecules of step (b) bind to a first polymerase linked to a nucleic acid duplex comprising an immobilized concatimer template molecule hybridized to a forward sequencing primer. . 213. The method of claim 212, wherein the nucleotide units of the individual multivalent molecules of step (e) bind to a second polymerase linked to a nucleic acid duplex comprising a retained forward extension strand hybridized to a reverse sequencing primer. 213. The method of claim 212, wherein the core comprises streptavidin and the core attachment moiety comprises biotin. 213. The method of claim 212, wherein the spacer comprises a polyethylene glycol (PEG) moiety and the linker comprises an aliphatic chain having 2 to 6 subunits or an oligo ethylene glycol chain having 2 to 6 subunits. method. 213. The method of claim 212, wherein the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. 213. The method of claim 212, wherein said first plurality of multivalent molecules of step (b) and said second plurality of multivalent molecules of step (e) comprise one type of multivalent molecule, wherein each multivalent molecule of said plurality is having nucleotide units of the same type selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP. 213. The method of claim 212, wherein the first plurality of multivalent molecules of step (b) and the second plurality of multivalent molecules of step (e) comprise a mixture of any combination of two or more types of multivalent molecules, each wherein the type has nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP. 213. The method of claim 212, wherein at least one of said first plurality of multivalent molecules of step (b) is labeled with a multivalent molecule fluorophore, and wherein at least one multivalent molecule of said second plurality of multivalent molecules of step (e) is fluorescent. Labeled as a hem, method. 213. The method of claim 212, wherein creating the non-basic site in the uridine in step (d) comprises contacting the immobilized concatimer template molecule with uracil DNA glycosylase (UDG). 213. The method of claim 212, wherein generating the plurality of gap-containing single stranded nucleic acid concatimer template molecules of step (d) comprises an endonuclease IV, a method comprising contacting with an AP lyase (e.g., DNA-depurine lyase or DNA-depyrimidine lyase), FPG glycosylase/AP lyase and/or endo VIII glycosylase/AP lyase. As a pairwise sequencing method,
a) providing a plurality of single-stranded nucleic acid concatemer template molecules immobilized on a support, wherein each template molecule of the plurality is immobilized on a surface primer, and the surface primer is immobilized on the support;
b) sequencing the plurality of immobilized concatimer template molecules with a first plurality of sequencing polymerase, a plurality of soluble forward sequencing primers and a first plurality of multivalent molecules, thereby forming a plurality of extended forward sequencing primer strands. generating a;
c) maintaining the plurality of fixed concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward extensions to the maintained fixed single-stranded nucleic acid concatimer template molecules. replacing with a strand;
d) removing the retained immobilized concatimer template molecules while retaining the plurality of forward extending strands and retaining the plurality of immobilized surface primers; and
e) sequencing the plurality of maintained forward extension strands using a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules.
Including,
The method of claim 1 , wherein the support includes at least one hydrophilic polymer coating layer and a plurality of surface primers fixed to the at least one hydrophilic polymer coating layer, wherein the at least one hydrophilic polymer coating layer has a water contact angle of 45 degrees or less. 223. The method of claim 223, wherein the at least one hydrophilic polymer coating layer is polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid). (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo A method comprising a molecule selected from the group consisting of (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran. 223. The method of claim 223, wherein the at least one hydrophilic polymer coating layer comprises polyethylene glycol (PEG). 223. The method of claim 223, wherein the at least one hydrophilic polymer coating layer comprises polymer molecules having a molecular weight of at least 1000 daltons. 224. The method of claim 223, wherein the at least one hydrophilic polymer coating layer comprises branched polymer molecules having 4 to 8 branches. 224. The method of claim 223, wherein the support comprises one hydrophilic polymer coating layer and a plurality of surface primers with a surface density of at least 1000/μm ² . The method of claim 223, wherein the support is
a) a first coating layer comprising a first monolayer of hydrophilic polymer molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first, second or third layer form a branched polymer layer. Comprising, the third coating layer
Method, including. 223. The method of claim 223, wherein the surface primer is fixed to the hydrophilic polymer molecules of the second or third monolayer, and the surface primer is distributed to a plurality of depths throughout the second or third layer. method. 224. The method of claim 223, wherein one or more of the at least one hydrophilic polymer coating layer comprises a plurality of surface primers with a surface density of at least 1000/μm ² . 224. The method of claim 223, wherein one or more of the at least one hydrophilic polymer coating layer comprises a plurality of surface primers at a surface density of at least 1000 to 15,000 per μm ² . 223. The method of claim 223, wherein the hydrophilic polymer coating layer on the support is formed by contacting the support with a fluorescently labeled nucleotide (Cy3-labeled nucleotide) and acquiring a fluorescence image using an inverted fluorescence microscope and camera under non-signal saturating conditions. A method according to claim 1, wherein a fluorescence image of the support is obtained and exhibits a contrast-to-noise (CNR) ratio of at least 20, while the support is immersed in a buffer. 223. The method of claim 223, wherein the support comprises glass or plastic. 224. The method of claim 223, wherein the support is configured on the inner surface of a flow cell or capillary lumen. 223. The method of claim 223, wherein the plurality of fixed single stranded concatimer template molecules comprise at least one nucleotide having a cleavable moiety that can be cleaved to create an abasic site in the concatimer template molecule, , wherein the at least one nucleotide has a cleavable moiety in the immobilized concatimer template molecule comprising uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. 223. The method of claim 223, wherein the plurality of anchored single stranded conkatimer template molecules lack nucleotides having a cleavable moiety that can be cleaved to create an abasic site in the conkatimer template molecule, and The method of claim 1, wherein the concatimer template molecule lacks a nucleotide with a cleavable moiety comprising uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. 224. The method of claim 223, wherein each template molecule of the plurality is covalently linked to the immobilized surface primer. 223. The method of claim 223, wherein each template molecule of the plurality is hybridized to the immobilized surface primer. 223. The method of claim 223, wherein the concatimer template molecule comprises a clonally amplified nucleic acid molecule. 223. The method of claim 223, wherein the immobilized surface primer lacks a nucleotide having a cleavable moiety, and the nucleotide having a cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine, or A method comprising oxyinosine. As a pairwise sequencing method,
a) providing a plurality of fixed single-stranded nucleic acid conkatimer template molecules each comprising at least one nucleotide having a cleavable moiety that can be cleaved to create a non-basic site in the conkatimer template molecule, , each template molecule of the plurality is immobilized on a surface primer immobilized on a support, the immobilized surface primer lacks a nucleotide having a cleavable moiety, and the concatimer template molecule contains a cleavable moiety. wherein the nucleotide comprising uridine;
b) sequencing the plurality of immobilized concatimer template molecules using a first plurality of sequencing polymerases, a plurality of soluble forward sequencing primers, and a first plurality of multivalent molecules, thereby performing a plurality of extended forward sequencing Generating primer strands, wherein each immobilized concatimer template molecule has at least two extended forward sequencing primer strands hybridized thereto;
c) maintaining the plurality of fixed concatimer template molecules and performing a primer extension reaction with the plurality of extended forward sequencing primer strands, thereby hybridizing the plurality of forward sequencing primer molecules to the maintained fixed single-stranded nucleic acid concatimer template molecules. replacing with an extended strand;
d) creating abasic sites in the anchored single-stranded conkatimer template molecule by contacting the retained fixed conkatimer template molecule with uracil DNA glycosylase (UDG), and binding the nonbasic sites to endonuclease IV, at the non-basic site by contacting with an AP lyase (e.g., DNA-depurine lyase or DNA-depyrimidine lyase), FPG glycosylase/AP lyase and/or endo VIII glycosylase/AP lyase. Removing the retained fixed conkatimer template molecules by creating a gap to create a plurality of gap-containing single stranded nucleic acid concatimer template molecules, while maintaining the plurality of forward extension strands and generating a plurality of fixed surface primers maintaining; and
e) sequencing the plurality of maintained forward extension strands using a second plurality of sequencing polymerases, a plurality of soluble reverse sequencing primers, and a second plurality of multivalent molecules, thereby forming a plurality of extended reverse sequencing primer strands. A step of generating a primer strand, wherein each maintained forward extension strand is hybridized with two or more extended reverse sequencing primer strands.
Including,
The method of claim 1 , wherein the support includes at least one hydrophilic polymer coating layer and a plurality of surface primers fixed to the at least one hydrophilic polymer coating layer, wherein the at least one hydrophilic polymer coating layer has a water contact angle of 45 degrees or less. 243. The method of claim 242, wherein the at least one hydrophilic polymer coating layer is polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid). (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo A method comprising a molecule selected from the group consisting of (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran. 243. The method of claim 242, wherein the at least one hydrophilic polymer coating layer comprises polymer molecules having a molecular weight of at least 1000 daltons. 243. The method of claim 242, wherein the at least one hydrophilic polymer coating layer comprises branched polymer molecules having 4 to 8 branches. 243. The method of claim 242, wherein the support comprises one hydrophilic polymer coating layer and a plurality of surface primers with a surface density of at least 1000/μm ² . The method of claim 242, wherein the support is
a) a first coating layer comprising a first monolayer of hydrophilic polymer molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first, second or third layer form a branched polymer layer. Comprising, the third coating layer
Method, including. 243. The method of claim 242, wherein one or more of the at least one hydrophilic polymer coating layer comprises a plurality of surface primers with a surface density of at least 1000/μm ² . 243. The method of claim 242, wherein the fluorescence image of the support is obtained by contacting the support with a fluorescently labeled nucleotide (Cy3-labeled nucleotide) and acquiring the fluorescence image using an inverted fluorescence microscope and camera under non-signal saturating conditions. The method of claim 1, wherein the support exhibits a contrast-to-noise (CNR) ratio of at least 20 while the support is immersed in a buffer. 243. The method of claim 242, wherein the support comprises glass or plastic. 243. The method of claim 242, wherein the individual template molecules in the plurality are covalently bound to an immobilized surface primer, or the individual concatimer template molecules in the plurality are hybridized to an immobilized surface primer. As a pairwise sequencing method,
a) providing a plurality of immobilized single-stranded nucleic acid concatemer template molecules, wherein each template molecule of the plurality is immobilized on a first surface primer immobilized on a support, wherein the immobilized first surface primer is uridine and wherein at least one of the plurality of immobilized concatimer template molecules comprises a uridine-containing concatimer template molecule having at most 30% of the thymidine replaced with uridine;
b) the plurality of soluble forward sequencing primers and (i) the plurality of first sequencing polymerases and the plurality of multivalent molecules and (ii) the plurality of second sequencing polymerases and the plurality of nucleotide analogs. A step of generating a plurality of extended forward sequencing primer strands by sequencing an immobilized concatimer template molecule, wherein each immobilized concatimer template molecule is hybridized with two or more extended forward sequencing primer strands. , step;
c) remove the plurality of extended forward sequencing primer strands while maintaining the fixed conkatimer template molecule, and perform a primer extension reaction using a plurality of soluble extension primers, a plurality of nucleotides, and a plurality of primer extension polymerases. performing a step of generating a plurality of forward extension strands hybridized to the maintained fixed concatimer template molecule;
d) while maintaining the plurality of forward extension strands and maintaining the plurality of immobilized surface primers, creating a non-basic portion of the immobilized concatimer template molecule at the nucleotide(s) having a cleavable moiety and a plurality of immobilized surface primers. removing the retained immobilized concatimer template molecule by creating a gap at an abasic site creating a gap-containing concatimer template molecule; and
e) maintaining the plurality using the plurality of soluble reverse sequencing primers and (i) a plurality of first sequencing polymerases and a plurality of multivalent molecules and (ii) a plurality of second sequencing polymerases and a plurality of nucleotide analogs. Generating a plurality of extended reverse sequencing primer strands by sequencing the maintained forward extension strand, wherein each maintained forward extension strand is hybridized with two or more extended reverse sequencing primer strands.
Including,
The individual multivalent molecules of steps (b) and (e) include (1) a core; and (2) a plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of arms via the core attachment moiety. Attached to a nucleotide arm, the spacer is attached to the linker, and the linker is attached to the nucleotide unit. 253. The method of claim 252, wherein each template molecule of the plurality is covalently linked to an immobilized first surface primer. 253. The method of claim 252, wherein each template molecule of the plurality is hybridized to an immobilized first surface primer. 253. The method of claim 252, wherein at least one of the plurality of immobilized concatimer template molecules lacks uridine. 253. The method of claim 252, wherein the immobilized concatimer template molecule comprises at least two copies of the sequence of interest and at least two copies of a universal binding sequence for a soluble amplification primer, and wherein the plurality of soluble extensions of step (c) The method of claim 1 , wherein the primers comprise a plurality of soluble amplification primers hybridized to the universal binding sequence for the soluble amplification primer. 253. The method of claim 252, wherein the immobilized concatimer template molecule comprises two or more copies of the sequence of interest and two or more copies of a universal binding sequence for a soluble forward sequencing primer, and The method of claim 1 , wherein the soluble extension primer comprises a plurality of soluble forward sequencing primers hybridized to the universal binding sequence for the soluble forward sequencing primer. 253. The method of claim 252, wherein the plurality of first sequencing polymerases of steps (b) and (e) bind to the concatimer template molecule, the soluble sequencing primer, and the multivalent molecule with a binding duration of greater than 0.5 seconds. A method of forming a complex, wherein the nucleic acid template molecule comprises an immobilized concatimer template molecule or a maintained forward extension strand, and the soluble sequencing primer comprises a soluble forward sequencing primer or a soluble reverse sequencing primer. 253. The method of claim 252, wherein the core of the multivalent molecule comprises streptavidin and the core attachment moiety comprises biotin. 253. The method of claim 252, wherein the spacer of the multivalent molecule comprises a polyethylene glycol (PEG) moiety. 253. The method of claim 252, wherein the linker of the multivalent molecule comprises an aliphatic chain with 2 to 6 subunits or an oligo ethylene glycol chain with 2 to 6 subunits. 253. The method of claim 252, wherein the plurality of nucleotide arms attached to the core of the multivalent molecule have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. 253. The method of claim 252, wherein the plurality of multivalent molecules of steps (b) and (e) comprises one type of multivalent molecule, wherein each multivalent molecule of the plurality consists of dATP, dGTP, dCTP, dTTP, and dUTP. A method having nucleotide units of the same type selected from the group. 253. The method of claim 252, wherein the plurality of multivalent molecules of steps (b) and (e) comprises a mixture of any combination of two or more types of multivalent molecules, each type being dATP, dGTP, dCTP, dTTP, and dUTP. A method having a nucleotide unit selected from the group consisting of 253. The method of claim 252, wherein the plurality of multivalent molecules of steps (b) and (e) comprises fluorophore-labeled multivalent molecules. 253. The method of claim 252, wherein the nucleotide analog of step (b) comprises a chain termination moiety removable from the 3' sugar group, and the nucleotide analog of step (e) comprises a chain termination moiety removable from the 3' sugar group. moieties, wherein the removable chain terminating moiety includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, It includes a keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and the removable chain terminating moiety is cleavable by a chemical compound to form the sugar group. Method for generating an extendable 3'OH moiety. 253. The method of claim 252, wherein the plurality of nucleotide analogs of steps (b) and (e) comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. 253. The method of claim 252, wherein the plurality of nucleotide analogs of steps (b) and (e) comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. , method. 253. The method of claim 252, wherein the plurality of nucleotide analogs of steps (b) and (e) comprises at least one fluorophore-labeled nucleotide analog. 253. The method of claim 252, wherein said sequencing in step (b)
a) a plurality of first sequencing polymerases comprising (i) a plurality of immobilized concatimer template molecules or a plurality of nucleic acid template molecules comprising a plurality of maintained forward extension strands, and (ii) a plurality of soluble forward extension strands. contacting the plurality of soluble sequencing primers comprising a sequencing primer or a plurality of soluble reverse sequencing primers, wherein the contacting step comprises a plurality of sequencing polymerases each comprising a first sequencing polymerase linked to a nucleic acid duplex. 1, performed under conditions suitable to form a complexed polymerase, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a soluble sequencing primer;
b) contacting the plurality of first complexed polymerases with a plurality of fluorophore-labeled multivalent molecules to form a plurality of multivalent-complexed polymerases, wherein the contacting step is complementary to the multivalent molecules. carrying out conditions suitable for forming a plurality of multivalent-complexed polymerases by linking red nucleotide units to at least two of the plurality of first complexed polymerases, wherein the conditions are such that the plurality of multivalent-complexed polymerases Inhibiting the incorporation of the complementary nucleotide unit into the hybridized sequencing primer by the polymerase, the forming step;
c) detecting a plurality of multivalent-complexed polymerases; and
d) determining the sequence of the nucleic acid template by identifying the nucleobase of the complementary nucleotide unit bound to the first plurality of complexed polymerases in the plurality of multivalent-complexed polymerases.
Method, including. According to Article 270,
e) dissociating the plurality of multivalent-complexed polymerases and maintaining the plurality of nucleic acid duplexes by removing the plurality of first sequencing polymerases and their associated multivalent molecules;
f) contacting the plurality of maintained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein contacting causes the plurality of second sequencing polymerases to bind to the plurality of maintained nucleic acid duplexes. performing under conditions suitable for forming a plurality of second complexed polymerases, each comprising a second sequencing polymerase linked to a retained nucleic acid duplex;
g) contacting the plurality of second complexed polymerases with a plurality of nucleotides, wherein the contacting causes complementary nucleotides to bind to at least two of the second complexed polymerases of step (f). carried out under conditions suitable for forming a plurality of nucleotide-complexed polymerases, wherein the conditions promote incorporation of the linked complementary nucleotides of the nucleotide-complexed polymerase into the hybridized sequencing primer. suitable for generating a plurality of extended sequencing primer strands, wherein the plurality of extended sequencing primer strands comprises a plurality of extended forward sequencing primer strands or a plurality of extended reverse sequencing primer strands.
A method further comprising: 253. The method of claim 252, wherein the step of creating the non-basic site at the uridine of the immobilized conkatimer template molecule in step (d) comprises the immobilized conkatimer template molecule using uracil DNA glycosylase (UDG). ), a method comprising contacting with. 253. The method of claim 252, wherein generating the plurality of gap-containing concatimer template molecules of step (d) comprises: an endonuclease IV, A method comprising contacting with an AP lyase (e.g., DNA-talpurine lyase or DNA-talpyrimidine lyase), FPG glycosylase/AP lyase and/or endo VIII glycosylase/AP lyase. 253. The method of claim 252, wherein the support includes at least one hydrophilic polymer coating layer and a plurality of surface primers secured to the at least one hydrophilic polymer coating layer, wherein the at least one hydrophilic polymer coating layer has a water contact angle of 45 degrees or less. . 275. The method of claim 274, wherein the at least one hydrophilic polymer coating layer is polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid). (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo A method comprising a molecule selected from the group consisting of (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran. 276. The method of claim 275, wherein the at least one hydrophilic polymer coating layer comprises polymer molecules having a molecular weight of at least 1000 daltons. 276. The method of claim 275, wherein the at least one hydrophilic polymer coating layer comprises branched polymer molecules having 4 to 8 branches. The method of claim 275, wherein the support is:
a) a first coating layer comprising a first monolayer of hydrophilic polymer molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first, second or third layer form a branched polymer layer. Comprising, the third coating layer
Method, including. 279. The method of claim 278, wherein the surface primer is anchored to the hydrophilic polymer molecules of the second or third monolayer, and the surface primer is distributed to a plurality of depths throughout the second or third layer. method. 276. The method of claim 275, wherein one or more of the at least one hydrophilic polymer coating layer comprises a plurality of surface primers with a surface density of at least 1000/μm ² . As a pairwise sequencing method,
a) providing a support on which a plurality of surface primers are immobilized, wherein the surface primers include a 3' extendable end and a nucleotide having a cleavable moiety that can be cleaved to create an abasic site in the surface primer. step, lacking;
b) hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized surface primers and performing a rotation amplification reaction using a plurality of strand transposition polymerases and a plurality of nucleotides to produce a plurality of immobilized single-stranded nucleic acid concatenations. generating a primer template molecule to produce a plurality of immobilized single-stranded nucleic acid concatimer template molecules, wherein each single-stranded nucleic acid concatimer template molecule is covalently linked to an immobilized surface primer, and the plurality of nucleotides are lacking a nucleotide having a cleavable moiety that can be cleaved to create a non-basic site in the concatimer template molecule;
c) generating a plurality of extended forward sequencing primer strands by sequencing the plurality of immobilized concatimer template molecules using a plurality of soluble forward sequencing primers, wherein each immobilized concatimer template molecule wherein two or more extended forward sequencing primer strands are hybridized;
d) Maintaining the plurality of immobilized concatimer template molecules, performing a primer extension reaction with a plurality of soluble amplification primers and a plurality of strand-transfer polymerases, thereby converting the plurality of extended forward sequencing primer strands into the plurality of forward sequencing primer strands. replacing the extension strands, thereby generating a plurality of forward extension strands and a plurality of partially translocated forward extension strands hybridized to an immobilized concatimer template molecule to form the plurality of immobilized amplicons, wherein the forward extension hybridizing the strand and the partially translocated forward extension strand to the anchored concatimer template molecule to form a plurality of anchored amplicons; and
e) generating a plurality of extended reverse sequencing primer strands by sequencing the plurality of fixed partially transposed forward extension strands using a plurality of soluble reverse sequencing primers, wherein each fixed partial translocated forward extension strand is sequenced using a plurality of soluble reverse sequencing primers. The transposed forward extension strand is hybridized with two or more extended reverse sequencing primer strands, and the fixed partially translocated forward extension strand is hybridized to the fixed concatimer template molecule maintained during the sequencing of step (e). stage that remains
Method, including. 282. The method of claim 281, wherein each single-stranded circular nucleic acid library molecule of the plurality comprises a sequence of interest, and wherein the individual library molecule comprises:
(i) universal binding sequence for soluble forward sequencing primers,
(ii) a universal binding sequence for soluble reverse sequencing primers,
(iii) a universal binding sequence for an immobilized surface primer;
(iv) a universal binding sequence for the first soluble amplification primer,
(v) a universal binding sequence for a second soluble amplification primer,
(vi) a universal binding sequence for soluble compressed oligonucleotides,
(vii) sample barcode sequence and/or
(viii) unique molecular indicator sequence
A method further comprising any one or any combination of two or more. 282. The method of claim 281, wherein the individual immobilized single-stranded nucleic acid concatimer template molecule produced by the spin ring amplification reaction comprises two or more copies of the sequence of interest, and the individual immobilized concatimer template molecule is
(i) two or more copies of a universal binding sequence for a soluble forward sequencing primer,
(ii) two or more copies of the universal binding sequence to the soluble reverse sequencing primer,
(iii) two or more copies of the universal binding sequence for the immobilized surface primer;
(iv) two or more copies of the universal binding sequence for the first soluble amplification primer,
(v) two or more copies of a universal binding sequence for a second soluble amplification primer,
(vi) two or more copies of the universal binding sequence for the soluble compressed oligonucleotide,
(vii) two or more copies of the sample barcode sequence and/or
(viii) two or more copies of a unique molecular indicator sequence.
A method further comprising any one or any combination of two or more. 282. The method of claim 281, wherein replacing the plurality of extended forward sequencing primer strands in step (d) comprises:
(i) removing the plurality of extended forward sequencing primer strands while maintaining the immobilized concatimer template molecule; and
(ii) a plurality of soluble amplification sequencing primers suitable for hybridizing a plurality of soluble amplification sequencing primers to a plurality of retained immobilized concatimer template molecules and hybridizing to the immobilized concatimer template molecules by performing a polymerase-catalyzed strand transfer reaction. Under conditions suitable for generating a forward extension strand and a plurality of partially transposed extended forward sequencing strands to form a plurality of fixed amplicons, the plurality of retained fixed concatimer molecules are combined with a plurality of soluble amplification primers. , contacting with a plurality of nucleotides and a plurality of strand transposition polymerases to generate a plurality of forward extension strands hybridized to the immobilized concatimer template molecule and a plurality of partially transposed extended forward sequencing strands to form a plurality of immobilized Steps to form an amplicon
Method, including. The method of claim 284, wherein the strand transfer polymerase is phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), E. coli (E. coli) DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. 282. The method of claim 281, wherein said forward sequencing of step (c)
a) contacting a plurality of sequencing polymerases and a plurality of soluble forward sequencing primers with a plurality of immobilized concatimer template molecules, wherein the contacting step each comprises a sequencing polymerase bound to a nucleic acid duplex. performing under conditions suitable for forming a plurality of complexed polymerases, wherein the nucleic acid duplex comprises an immobilized concatimer template molecule hybridized to a soluble forward sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides are labeled with a fluorophore. and comprising at least one nucleotide analog having a removable chain termination moiety at the 3' position of the sugar;
c) incorporating at least one nucleotide into the 3' end of the hybridized forward sequencing primer to generate a plurality of initial extended forward sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.
Method, including. 287. The method of claim 286, wherein in the plurality of nucleotides of step (b) at least one of the nucleotides comprises a removable chain termination moiety attached to the 3′ carbon position of the sugar group, wherein the removable chain termination moiety T is an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, and phosphate group. , a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, wherein the removable chain terminating moiety is cleavable by a chemical compound to produce an extendable 3'OH moiety on the sugar group, method. 282. The method of claim 281, wherein said reverse sequencing of step (e) comprises:
a) contacting the plurality of sequencing polymerases and the plurality of soluble reverse sequencing primers with the plurality of fixed partially translocated forward extension strands, wherein the contacting step involves sequencing polymerization linked to a nucleic acid duplex. performing under conditions suitable for forming a plurality of complexed polymerases, each comprising an enzyme, wherein the nucleic acid duplex comprises a soluble reverse sequencing primer hybridized to an immobilized partially translocated forward extension strand;
b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides under conditions suitable to bind at least one nucleotide to the complexed sequencing polymerase, wherein the plurality of nucleotides are labeled with a fluorophore. and comprising at least one nucleotide analog having a removable chain termination moiety at the 3' position of the sugar;
c) incorporating at least one nucleotide into the 3' end of the hybridized reverse sequencing primer to generate a plurality of initial extended reverse sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.
Method, including. 289. The method of claim 288, wherein in the plurality of nucleotides of step (b) at least one of the nucleotides comprises a removable chain termination moiety attached to the 3′ carbon position of the sugar group, wherein the removable chain termination moiety T is an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, and phosphate group. , a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group, wherein the removable chain terminating moiety is cleavable by a chemical compound to produce an extendable 3'OH moiety on the sugar group, method. 282. The method of claim 281, wherein said forward sequencing of step (b) and said reverse sequencing of step (e)
a) contacting a plurality of first sequencing polymerases and a plurality of soluble sequencing primers with a plurality of nucleic acid template molecules, wherein the contacting step includes nucleic acid comprising the nucleic acid template molecules hybridized to the soluble sequencing primers. carried out under conditions suitable to form a plurality of first complexed polymerases, each comprising a first sequencing polymerase linked to a duplex, wherein (1) the plurality of nucleic acid template molecules are (2) the plurality of nucleic acid template molecules comprise a plurality of fixed, partially translocated forward extension strands, and , wherein the plurality of sequencing primers comprises a plurality of the soluble reverse sequencing primers;
b) contacting the plurality of first complexed polymerases with a plurality of detectably labeled multivalent molecules to form a plurality of multivalent-complexed polymerases, wherein each multivalent molecule of the plurality of multivalent molecules comprises a core attached to multiple nucleotide arms, each nucleotide arm attached to a nucleotide unit, wherein the contacts bind complementary nucleotide units of the multivalent molecule to at least two of the plurality of first complexed polymerases. is carried out under conditions suitable for forming the plurality of multivalent-complexed polymerases, and the conditions inhibit the incorporation of the complementary nucleotide units of the plurality of multivalent-complexed polymerases into the sequencing primer. to do, step;
c) detecting the plurality of multivalent-complexed polymerases; and
d) determining the sequence of the nucleic acid template by identifying the nucleobase of the complementary nucleotide unit bound to the plurality of first complexed polymerases in the plurality of multivalent-complexed polymerases.
Method, including. 291. The method of claim 290, further comprising:
e) dissociating the plurality of multivalent-complexed polymerases and removing the plurality of first sequencing polymerases and their associated multivalent molecules, and maintaining the plurality of nucleic acid duplexes;
f) contacting the plurality of maintained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein contacting causes the plurality of second sequencing polymerases to bind to the plurality of maintained nucleic acid duplexes. , the contacting step being carried out under conditions suitable to form a plurality of second complexed polymerases;
g) linking complementary nucleotides from the plurality of nucleotides to at least two of the second complexed polymerases of step (f) to form a plurality of nucleotide-complexed polymerases under conditions suitable for forming a plurality of second complexed polymerases. contacting the complexed polymerase with a plurality of nucleotides, wherein conditions are suitable to promote incorporation of the linked complementary nucleotide into the sequencing primer of the nucleotide-complexed polymerase. . In clause 291,
h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.
A method further comprising: In clause 291,
h) detecting the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase; and
i) identifying the nucleobase of the complementary nucleotide incorporated into the sequencing primer of the nucleotide-complexed polymerase.
A method further comprising: 291. The method of claim 290, further comprising forming at least one avidity complex of step (b),
a) linking a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a nucleic acid template molecule to form a first binding complex, wherein the first nucleotide unit of the first multivalent molecule is Binding to the first sequencing polymerase; and
b) linking a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same nucleic acid template molecule to form a second binding complex, wherein the first multivalent molecule of the second multivalent molecule 2 nucleotide units bind to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex.
Method, including. 294. The method of claim 294, wherein (i) the first sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an immobilized concatimer template molecule, and (ii) the second sequencing primer comprises a soluble forward sequencing primer, the nucleic acid template molecule comprises an identical immobilized concatimer template molecule, and (iii) the first sequencing primer and the second sequencing primer have identical sequences. . 294. The method of claim 294, wherein (i) the first sequencing primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises a fixed, partially translocated forward extension strand, and (ii) the second sequence The assay primer comprises a soluble reverse sequencing primer, the nucleic acid template molecule comprises the identical fixed partially translocated forward extension strand, and (iii) the first sequencing primer and the second sequencing primer are identical. Method having a sequence. The method of claim 290, wherein each multivalent molecule among the plurality of multivalent molecules includes (a) a core; and (b) a plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core comprises a core attachment moiety. Attached to a plurality of nucleotide arms via a tee, wherein the spacer is attached to the linker, and the linker is attached to a nucleotide unit. In clause 297,
(i) the core comprises streptavidin and the core attachment moiety comprises biotin;
(ii) the linker comprises an aliphatic chain having 2 to 6 subunits or an oligo ethylene glycol chain having 2 to 6 subunits; and
(iii) the nucleotide unit comprises an aromatic base, a pentose sugar and 1 to 10 phosphate groups. 298. The method of claim 297, wherein the plurality of nucleotide arms attached to the core have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. 297. The method of claim 297, wherein the plurality of multivalent molecules comprises one type of multivalent molecule, wherein each multivalent molecule of the plurality contains the same type of nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. Having, way. 298. The method of claim 297, wherein the plurality of multivalent molecules comprises a mixture of any combination of two or more types of multivalent molecules, each type having nucleotide units selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. , method. 291. The method of claim 290, wherein the plurality of detectably labeled multivalent molecules comprise fluorescently labeled multivalent molecules. 282. The method of claim 281, wherein the support includes at least one hydrophilic polymer coating layer and a plurality of surface primers secured to the at least one hydrophilic polymer coating layer, wherein the at least one hydrophilic polymer coating layer has a water contact angle of 45 degrees or less. . 303. The method of claim 303, wherein the at least one hydrophilic polymer coating layer is polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid). (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo A method comprising a molecule selected from the group consisting of (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran. 303. The method of claim 303, wherein the at least one hydrophilic polymer coating layer comprises polymer molecules having a molecular weight of at least 1000 daltons. 304. The method of claim 303, wherein the at least one hydrophilic polymer coating layer comprises branched hydrophilic polymer molecules having 4 to 8 branches. 304. The method of claim 303, wherein the at least one hydrophilic polymer coating layer comprises a plurality of surface primers with a surface density of at least 1000/μm ² . 303. The method of claim 303, wherein the support is
a) a first coating layer comprising a first monolayer of hydrophilic polymer molecules tethered to the support;
b) a second coating layer comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and
c) a third coating layer comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first, second or third layer form a branched polymer layer. Comprising, the third coating layer
Including,
The surface primer is fixed to the hydrophilic polymer molecules of the second or third monolayer, and the surface primer is distributed to a plurality of depths throughout the second or third layer. 303. The method of claim 303, wherein the hydrophilic coating layer on the support is formed by contacting the support with a fluorescently labeled nucleotide (Cy3-labeled nucleotide) and acquiring a fluorescence image using an inverted fluorescence microscope and camera under non-signal saturating conditions. exhibiting a contrast-to-noise (CNR) ratio of at least 20 when a fluorescence image is obtained, while the support is immersed in a buffer.