WO2024031061A1 - Methods for improving strand invasion efficiency - Google Patents

Methods for improving strand invasion efficiency Download PDF

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Publication number
WO2024031061A1
WO2024031061A1 PCT/US2023/071692 US2023071692W WO2024031061A1 WO 2024031061 A1 WO2024031061 A1 WO 2024031061A1 US 2023071692 W US2023071692 W US 2023071692W WO 2024031061 A1 WO2024031061 A1 WO 2024031061A1
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Prior art keywords
strand
invasion
primer
sequence
polynucleotide
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PCT/US2023/071692
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French (fr)
Inventor
Andrew King
Daan WITTERS
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Singular Genomics Systems, Inc.
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Publication of WO2024031061A1 publication Critical patent/WO2024031061A1/en

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • compositions and methods provided herein reduce the amount of nucleic acid manipulation and duplication required by traditional NGS Attorney Docket No.: 051385-585001WO techniques.
  • cluster-based sequencing processes would include cleaving and removing one strand from double-stranded molecules in a cluster before generating a first read, without which the second strand would effectively compete with hybridization of the sequencing primer. Generating a sequencing read for the second (cleaved) strand would then require creating a new complementary strand from the sequenced first strand (i.e., a new second strand).
  • the methods disclosed herein permit obtaining sequence information (i.e., reading) from the original first and second strands (e.g., original strands from the initial cluster amplification, or amplicons), thereby reducing the time, reagents, expense, and risk of polymerase errors inherent in previous methods.
  • a method of sequencing including: hybridizing an invasion primer including a first sequence (e.g., a binding sequence complementary to a portion of the polynucleotide) and a second sequence (e.g., a tail sequence) to a second strand of a double-stranded polynucleotide and extending the binding sequence (e.g., extending the 3’ end of the binding sequence) with a polymerase, thereby generating an invasion strand and displacing the first strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; extending the second strand along the tail sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension
  • a method of forming a single-stranded polynucleotide attached to a solid support including: contacting a plurality of double-stranded polynucleotides including a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers include a binding sequence and a tail sequence; hybridizing the binding sequence of one of the invasion primers to one of the second strands; and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the Attorney Docket No.: 051385-585001WO invasion primer hybridized to the second strand to generate an extended second strand including a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support.
  • a method of incorporating a sequence including: hybridizing an invasion primer including a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double- stranded polynucleotide.
  • a method of sequencing including: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the extension strand, thereby sequencing the first strand of the double-strand
  • FIGS.1A-1B illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase.
  • the hashed boxes on each end represent a polymer scaffold that is anchored to a solid support, such as glass or silicon support.
  • FIG.1A illustrates two dsDNA duplex strands, each duplex having a first strand hybridized to a second strand, wherein each strand is attached to the solid support.
  • duplexes double-stranded amplification products
  • An invasion oligonucleotide also referred to herein as an invasion primer or invasion oligo
  • FIG.1B After extension of the invasion oligonucleotide has been completed, one strand of the initial dsDNA molecule is now single- stranded and available for a first sequencing read, as shown in FIG.1B.
  • the sequenced strand may optionally be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing.
  • a single X is depicted, however it is understood that the cleavable site may include multiple chemical, enzymatic, or photochemical entities capable of being cleaved.
  • FIGS.2A-2B illustrate an embodiment of strand invasion including an invasion primer with a 5’ tail sequence.
  • the hashed box represents a polymer scaffold that is anchored to a solid support, such as glass or silicon support.
  • FIG.2A illustrates two dsDNA duplex strands, each duplex having a first strand hybridized to a second strand, and each strand is attached to the solid support.
  • An invasion primer including a binding sequence e.g., a sequence complementary to a region within one of the strands of the dsDNA
  • a 5’ tail sequence wherein the tail sequence is not complementary to the dsDNA
  • An invasion mixture as described above may be used to introduce and hybridize the elongated invasion primer to the dsDNA.
  • a blocking strand hybridized to one of the strands of the dsDNA (e.g., a blocking strand, also referred to herein as an invasion strand, is now hybridized to the second strand, for example, resulting in a single-stranded first strand that is accessible to a primer).
  • the second strand is extended along the elongated invasion primer to incorporate the complement of the 5’ tail sequence, thereby modifying the 3’ end of the second strand such that it is no longer complementary to the 5’ end of the first strand.
  • FIG.2B illustrates an additional embodiment of strand invasion, wherein after generating an extended second strand as shown in FIG.2A the blocking strand is removed (e.g., removed via exonuclease digestion). This also enables the use of distinct invasion primer hybridization and extension conditions to used.
  • the first strand may reanneal to the second strand, but the incorporated tail sequence at the 3’ end of the second strand is free, allowing for annealing of a primer (e.g., a second invasion primer complementary to the tail sequence), followed by extension with a polymerase (e.g., a strand-displacing polymerase) thereby generating a second blocking strand hybridized to the second strand.
  • a primer e.g., a second invasion primer complementary to the tail sequence
  • a polymerase e.g., a strand-displacing polymerase
  • a bridged dsDNA complex is present on a solid support, wherein the first strand and the second strand of the dsDNA are immobilized on the solid support.
  • a second oligonucleotide primer e.g., a second competitive oligonucleotide
  • a competitor strand at the 5’ end of the immobilized first strand that may reduce reannealing of a portion of the second strand (i.e., prevent the 3’ end of the second strand from reannealing to the 5’ end of the first strand).
  • FIGS.4A-4B illustrate an embodiment of strand invasion using an invasion primer that contains peptide nucleic acids (PNAs) into dsDNA clusters.
  • PNA oligos can invade into dsDNA at low ionic strength ( ⁇ 25 mM NaCl) conditions.
  • the PNA-containing invasion primer is designed to invade at the common adapter sequence of the 5′ end of one of the solid phase-bound amplicons. This, in turn, makes the displaced complementary 3′ DNA end on the complementary strand accessible for binding with another invasion oligonucleotide, referred to as a runoff primer in FIG.4B, that can be extended by a strand-displacing DNA polymerase.
  • FIGS 5A-5B illustrate an embodiment of strand invasion into dsDNA monoclonal clusters by using a recombinase and an invasion oligonucleotide.
  • the pre-synaptic filament (alternatively referred to as a pre-synaptic complex), consisting of an invasion oligonucleotide complexed with recombinase enzymes searches dsDNA fragments for homology.
  • the invasion oligonucleotide can be inserted to its complementary sequence in the dsDNA amplicons, after Attorney Docket No.: 051385-585001WO which the invasion oligonucleotide can be extended by a strand-displacing polymerase. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process.
  • FIGS.6A-6D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase.
  • FIG.6A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion.
  • the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage).
  • a cleavable site also referred to herein as a scissile linkage.
  • the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo.
  • the sequenced strand may further optionally be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.6B.
  • the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicking the dU using suitable conditions), leaving behind a 5’-phosphate in the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.6C-6D.
  • FIGS.7A-7D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase.
  • FIG.7A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion.
  • the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage).
  • the cleavable site may Attorney Docket No.: 051385-585001WO be a deoxyuracil (dU) towards the 3’ end of the invasion oligo.
  • runoff extension i.e., extension to a sufficient length
  • one strand of the initial dsDNA molecule is now single-stranded and available for a first sequencing read, as shown in FIG.7B. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process.
  • sequenced strand may further be extended with native dNTPs to complete the extension of the sequenced strand, as illustrated in FIG.7B as the solid line beyond the star. Further extending the sequencing primer with unmodified nucleotides eliminates any remaining single-stranded region.
  • the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., cleaving the dU using a uracil DNA glycosylase or formamidopyrimidine DNA glycosylase (Fpg) as described herein), leaving behind a 5’-phosphate in the extended part of the invasion primer that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.7C-7D.
  • a cleavable site e.g., cleaving the dU using a uracil DNA glycosylase or formamidopyrimidine DNA glycosylase (Fpg) as described herein
  • FIGS.8A-8D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase.
  • FIG.8A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion.
  • the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage).
  • the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo.
  • dU deoxyuracil
  • one strand of the initial dsDNA molecule is now single-stranded and available for a first sequencing read, as shown in FIG.8B.
  • the sequenced strand may further be extended with one or more dideoxynucleotide triphosphates (ddNTPs) to prevent further extension, as illustrated in FIG.8B as the hexagon.
  • ddNTPs dideoxynucleotide triphosphates
  • the cleavable site at the 3’ end of the invasion primer may be cleaved (e.g., the dU), leaving behind a 5’-phosphate in the extended part of the invasion primer that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.8C-8D.
  • FIGS.9A-9D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase.
  • FIG.9A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion.
  • the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage).
  • the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo.
  • Runoff extension of the invasion oligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.9B.
  • the sequenced strand may optionally further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.9B.
  • the invasion strand may be nicked at internal scissile sites (e.g., resulting from amplification with the dUTP), leaving behind small, low Tm fragments that may be denatured and removed under suitable conditions, as shown in FIG.9C. Additionally, this cleavage and denaturation step exposes the 3’ end of the invasion oligo, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.9C- 9D.
  • FIGS.10A-10E illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase.
  • FIG.10A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion.
  • the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage).
  • the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo.
  • Runoff extension of the invasion oligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.10B.
  • the 3’ end of the first sequencing read is capped by ddNTP incorporation.
  • a second sequencing read is then obtained by annealing and extending a second sequencing primer 3’ of the terminated first Attorney Docket No.: 051385-585001WO sequencing read.
  • a ddNTP is incorporated into the 3’ end of the second sequencing read, and thereafter the invasion strand may be nicked at internal scissile sites (e.g., resulting from amplification with the dUTP), leaving behind small fragments with exposed 5’ ends that may be removed under suitable conditions, for example, by lambda exonuclease digestion, as shown in FIGS.10C-10D.
  • This cleavage and removal step exposes the 3’ end of the second strand, making it available for a third sequencing read, as shown in FIG.10E.
  • the aspects and embodiments described herein relate to sequencing a polynucleotide.
  • the methods relate to sequencing a first strand of a double- stranded polynucleotide, and optionally sequencing the complement of first strand (i.e., the second strand) of the same double-stranded polynucleotide.
  • cluster and “colony” are used interchangeably throughout this application and refer to a discrete site on a solid support comprised of a plurality of immobilized nucleic acid strands.
  • the term “clustered array” refers to an array formed from such clusters or colonies.
  • the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about means the specified value.
  • control or “control experiment” is used in accordance with its plain and ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects.
  • association or “associated with” can mean that two or more species are identifiable as being co-located at a point in time. An association can mean that two or more species are or were within a similar container.
  • An association can be an informatics association, where for example digital information regarding two or more species is stored and can be used to determine that one or more of the species were co-located at a point in time.
  • An association can also be a physical association.
  • two or more associated species are "tethered”, “coated”, “attached”, or “immobilized” to one another or to a common solid or semisolid support (e.g. a receiving substrate).
  • An association may refer to a relationship, or connection, between two entities. Associated may refer to the relationship between a sample and the DNA molecules, RNA molecules, or polynucleotides originating from or derived from that sample. These relationships may be encoded in oligonucleotide barcodes, as described herein.
  • a polynucleotide is associated with a sample if it is an endogenous polynucleotide, i.e., it occurs in the sample at the time the sample is obtained, or is derived from an endogenous polynucleotide.
  • the RNAs endogenous to a cell are associated with that cell.
  • cDNAs resulting from reverse transcription of these RNAs, and DNA amplicons resulting from PCR amplification of the cDNAs contain the sequences of the RNAs and are also associated with the cell.
  • the polynucleotides associated with a sample need not be located or synthesized in the sample, and are considered associated with the sample even after the sample has been destroyed (for example, after a cell has been lysed).
  • Barcoding can be used to determine which polynucleotides in a mixture are associated with a particular sample.
  • the term “complementary” or “substantially complementary” refers to the hybridization, base pairing, or the formation of a duplex between nucleotides or nucleic acids.
  • the term “complement,” as used herein refers to a nucleotide (e.g., RNA Attorney Docket No.: 051385-585001WO or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides.
  • complementarity exists between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides is capable of base pairing with a respective cognate nucleotide or cognate sequence of nucleotides.
  • a nucleotide e.g., RNA or DNA
  • sequence of nucleotides is capable of base pairing with a respective cognate nucleotide or cognate sequence of nucleotides.
  • a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence.
  • the nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence.
  • nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence.
  • complementary sequences include coding and non- coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence.
  • complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.
  • Duplex means at least two oligonucleotides and/or polynucleotides that are fully or partially complementary undergo Watson-Crick type base pairing among all or most of their nucleotides so that a stable complex is formed.
  • Complementary single stranded nucleic acids and/or substantially complementary single stranded nucleic acids can hybridize to each other under hybridization conditions, thereby forming a nucleic acid that is partially or fully double stranded.
  • a double-stranded polynucleotide including a first strand hybridized to a second strand it is understood that each of the first strand and the second strand are independently single-stranded polynucleotides.
  • nucleic acid sequence may be substantially complementary to another nucleic acid sequence, in some embodiments.
  • Attorney Docket No.: 051385-585001WO As referred to herein, “substantially complementary” refers to nucleotide sequences that can hybridize with each other under suitable hybridization conditions. Hybridization conditions can be altered to tolerate varying amounts of sequence mismatch within complementary nucleic acids that are substantially complementary.
  • Substantially complementary portions of nucleic acids that can hybridize to each other can be 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more complementary to each other.
  • substantially complementary portions of nucleic acids that can hybridize to each other are 100% complementary.
  • Nucleic acids, or portions thereof, that are configured to hybridize to each other often comprise nucleic acid sequences that are substantially complementary to each other.
  • the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.
  • two sequences that are complementary to each other may have a specified percentage of nucleotides that complement one another (e.g., about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher complementarity over a specified region).
  • sequences in a pair of complementary sequences form portions of a single polynucleotide with non-base-pairing nucleotides (e.g., as in a hairpin or loop structure, with or without an overhang) or portions of separate polynucleotides.
  • one or both sequences in a pair of complementary sequences form portions of longer polynucleotides, which may or may not include additional regions of complementarity.
  • the term “contacting” may include Attorney Docket No.: 051385-585001WO allowing two species to react, interact, or physically touch, wherein the two species may be a compound, a protein (e.g., an antibody), or enzyme.
  • nucleic acid As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “strand,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown.
  • Non- limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer.
  • Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.
  • nucleic acid oligomer and “oligonucleotide” are used interchangeably and are intended to include, but are not limited to, nucleic acids having a length of 200 nucleotides or less.
  • an oligonucleotide is a nucleic acid having a length of 2 to 200 nucleotides, 2 to 150 nucleotides, 5 to 150 nucleotides or 5 to 100 nucleotides.
  • polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides.
  • Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length.
  • an oligonucleotide is a primer configured for extension by a polymerase when the primer is annealed completely or partially to a complementary nucleic acid template.
  • a primer is often a single stranded nucleic acid.
  • a primer, or portion thereof is substantially complementary to a portion of an adapter.
  • a primer has a length of 200 nucleotides or less.
  • a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides.
  • an oligonucleotide may be immobilized to a solid support.
  • An association may refer to a relationship, or connection, between two entities.
  • an immobilized polynucleotide or an immobilized primer refers to a polynucleotide or a primer that is attached to a solid surface, such as a solid support.
  • the immobilized polynucleotide and/or immobilized primer may be attached covalently (e.g. through a linker) or non-covalently to a solid support.
  • immobilized polynucleotide and/or immobilized primer is covalently attached to a solid support.
  • polynucleotide primer and “primer” refers to any polynucleotide molecule that may hybridize to a polynucleotide template, be bound by a polymerase, and be extended in a template-directed process for nucleic acid synthesis.
  • the primer may be a separate polynucleotide from the polynucleotide template, or both may be portions of the same polynucleotide (e.g., as in a hairpin structure having a 3’ end that is extended along another portion of the polynucleotide to extend a double-stranded portion of the hairpin).
  • Primers may be attached to a solid support (e.g., a polymer coated solid support).
  • a solid support e.g., a polymer coated solid support.
  • forward primers anneal to the antisense strand of the double-stranded DNA, which runs from the 3’ to 5’ direction.
  • Forward primers for example, initiate the synthesis of a gene in the 5’ to 3’ direction.
  • reverse primers anneal to the sense strand of the double-stranded DNA, which runs from the 5’ to 3’ direction.
  • Reverse primers for example, initiate the synthesis of a gene in the 3’ to 5’ direction.
  • a primer can be of any length depending on the particular technique it will be used for.
  • PCR primers are generally between 10 and 40 nucleotides in length.
  • the length and complexity of the nucleic acid fixed onto the nucleic acid template may vary.
  • a primer has a length of 200 nucleotides or less.
  • a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides.
  • a primer typically has a length of 10 to 50 nucleotides.
  • a primer may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides.
  • a primer has a length of 18 to 24 nucleotides.
  • the primer permits the addition of a nucleotide residue thereto, or oligonucleotide or polynucleotide synthesis therefrom, under suitable conditions.
  • the primer is a DNA primer, i.e., a primer consisting of, or largely consisting of, deoxyribonucleotide residues.
  • the primers are designed to have a sequence that is the complement of a region of template/target DNA to which the primer hybridizes.
  • the primer is an RNA primer.
  • a primer is hybridized to a target polynucleotide.
  • a “primer” is complementary to a polynucleotide template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded bases linked at its 3′ end complementary to the template in the process of DNA synthesis.
  • the term “primer binding sequence” or simply “binding sequence” refers to a polynucleotide sequence that is complementary to at least a portion of a primer (e.g., an invasion primer, a sequencing primer, or an amplification primer). Primer binding sequences can be of any suitable length.
  • a primer binding sequence is about or at least about 10, 15, 20, 25, 30, or more nucleotides in length. In embodiments, a primer binding sequence is 10-50, 15-30, or 20-25 nucleotides in length.
  • the primer binding sequence may be selected such that the primer (e.g., sequencing primer) has the preferred characteristics to minimize secondary structure formation or minimize non-specific amplification, for example having a length of about 20-30 nucleotides; approximately 50% GC content, and a Tm of about 55°C to about 65°C.
  • a platform primer is a primer oligonucleotide immobilized or otherwise bound to a solid support (i.e., an immobilized oligonucleotide).
  • platform primers include P7 and P5 primers, or S1 and S2 sequences, or the reverse complements thereof.
  • a “platform primer binding sequence” refers to a sequence or portion of an oligonucleotide that is capable of binding to a platform primer (e.g., the platform primer binding sequence is complementary to the platform primer).
  • a platform primer binding sequence may form part of an adapter.
  • a platform primer binding sequence is Attorney Docket No.: 051385-585001WO complementary to a platform primer sequence.
  • a platform primer binding sequence is complementary to a primer.
  • the term “capture domain” refers to an oligonucleotide sequence (e.g., an oligonucleotide sequence included in a primer, for example a surface-immobilized capture primer).
  • the capture domain may be any suitable domain capable of hybridizing to RNA or a transcript thereof, such as mRNA.
  • the capture domain includes a poly- T oligonucleotide.
  • a poly-T oligonucleotide includes a series of consecutive deoxythymidine residues linked by phosphodiester bonds.
  • a poly-T oligonucleotide is capable of hybridizing to the poly-A tail of mRNA.
  • the capture domain may further include additional sequences to facilitate the capture of a particular RNA (e.g., mRNA) corresponding to select genes or groups of genes.
  • a capture primer may be selected or designed based on sequence of the RNA it is desired to capture. Accordingly, the capture primer may be a sequence-specific capture primer as described herein.
  • the capture domain may target DNA, instead of RNA.
  • the capture domain may target non-specific or specific DNA sequences.
  • the capture domain may include a nucleic acid sequence to facilitate the capture of a target DNA sequence. The type of target may depend on the specific capture domain used and/or the presence of additional capture moieties on the substrate.
  • capture domains including a poly-dT tail are suited for spatial detection of RNA with poly-A tail.
  • RNA that does not have poly-A tail may be labeled with poly-A before being captured by the substrate.
  • Capture domains including a nucleic acid sequence against a target DNA sequence are useful for spatial detection of DNA.
  • Substrates include a capture primer and an additional capture moiety (e.g. an antibody targeting protein or DNA/RNA probes targeting specific nucleic acid sequence) are useful for multiplex detection of nucleic acid and non-nucleic acid targets.
  • the term “spatial barcode” refers to a known nucleic acid sequence that allows the location of a biological molecule with which the barcode is associated to be resolved.
  • a barcode can be a spatial barcode.
  • the barcode or spatial barcode may be associated with an oligonucleotide as described herein (e.g., a capture oligonucleotide or capture probe).
  • the barcodes can be designed for precision sequence performance, e.g., GC content between 40% and 60%, no homo-polymer runs longer than two, no self-complementary stretches longer than 3, Attorney Docket No.: 051385-585001WO and be comprised of sequences not present in a human genome reference.
  • a barcode sequence can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.
  • a barcode sequence can be at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.
  • a barcode sequence can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases.
  • An oligonucleotide e.g., primer or adapter
  • Barcodes can be of sufficient length and include sequences that can be sufficiently different to allow the identification of the spatial position of each biological molecule based on barcode(s) with which each biological molecule is associated.
  • each barcode is, for example, four deletions or insertions or substitutions away from any other barcode in an array.
  • the oligonucleotides in each array spot on the barcoded oligonucleotide array can include the same barcode sequence and oligonucleotides in different array spots can include different barcode sequences.
  • the barcode sequence used in one array spot can be different from the barcode sequence in any other array spot.
  • the barcode sequence used in one array spot can be the same as the barcode sequence used in another array spot, as long as the two array spots are not adjacent. Barcode sequences corresponding to particular array spots can be known from the controlled synthesis of the array.
  • barcode sequences corresponding to particular array spots can be known by retrieving and sequencing material from particular array spots.
  • the spatial barcode sequence is indicative of the location of the immobilized capture probe on the solid support to within about 2 ⁇ m, about 1 ⁇ m, about 0.5 ⁇ m, about 0.2 ⁇ m, or about 0.1 ⁇ m.
  • invasion primer refers to a polynucleotide molecule that may hybridize to a single-stranded nucleic acid sequence of a double-stranded polynucleotide and be extended in a template- directed process (e.g., extended with a polymerase) for nucleic acid synthesis.
  • the invasion primer includes a binding sequence (e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof) and a 5’ tail sequence (i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof).
  • a binding sequence e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof
  • a 5’ tail sequence i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof.
  • an invasion primer hybridizes at or near the end of the single-stranded nucleic acid sequence (e.g., the 5’ end or the 3’ end), or the invasion primer hybridizes at an internal sequence.
  • extension of an invasion primer results in the formation of an “invasion strand” complementary to either the first strand or the second strand of the double-stranded polynucleotide. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the sequencing process.
  • the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’- O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof.
  • the invasion primer includes phosphorothioate nucleic acids.
  • the invasion primer includes one or more locked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene- functionalized oligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids.
  • the invasion primer includes 10 to 15 locked nucleic acids (LNAs).
  • the invasion primer includes a sequence described herein, for example within Table 1.
  • the invasion primer includes one or more phosphorothioates at the 5′ end.
  • the invasion primer includes one or more LNAs at the 5′ end. In embodiments, the invasion primer includes two or more consecutive LNAs at the 3′ end. In embodiments, the invasion primer includes two or more consecutive LNAs at the 5′ end. In embodiments, the invasion primer includes a plurality (e.g., 2 to 10) of synthetic nucleotides (e.g., LNAs) and a plurality (e.g., 2 to 10) canonical or native nucleotides (e.g., dNTPs). In embodiments, the invasion primer includes one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU).
  • dU deoxyuracil nucleobases
  • the one or more dU nucleobases are at or near the 3’ end of the invasion primer (e.g., within 5 nucleotides of the 3’ end). In embodiments, the one or more dU nucleobases are distributed through the invasion primer.
  • the invasion primer includes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followed by a plurality of synthetic nucleotides (e.g., LNAs), Attorney Docket No.: 051385-585001WO and a plurality (e.g., 2 to 10) of canonical bases.
  • the invasion primer includes a plurality of canonical bases, wherein the canonical bases terminate (i.e., at the 3′ end) with a deoxyuracil nucleobase (dU).
  • the invasion primer is about 10 to 100 nucleotides in length.
  • the invasion primer is about 15 to about 40 nucleotides in length.
  • the calculated or predicted melting temperature (Tm) of the invasion primer is about 70°C to about 95°C.
  • the calculated or predicted melting temperature (Tm) of the invasion primer is about 75°C to about 85°C.
  • the calculated or predicted melting temperature (Tm) of the invasion primer is 75°C to 85°C.
  • the invasion primer is capable of hybridizing (i.e., capable of hybridizing under suitable hybridization conditions) to one strand of a double-stranded polynucleotide molecule in a process of strand invasion.
  • the invasion primer may include nucleic acids having a binding affinity greater than the binding affinity of standard or canonical DNA oligonucleotides, such as locked nucleic acids (LNA), peptide nucleic acids (PNAs), 2’-O-methyl RNA:DNA chimeras, minor groove binder probes (MGB), or morpholino probes.
  • LNA locked nucleic acids
  • PNAs peptide nucleic acids
  • MGB minor groove binder probes
  • invasion primers can undergo spontaneous strand invasion into dsDNA (e.g., hybridizing to a sequence near the end or terminus of the dsDNA), as is the case for example for PNA invasion primers under low ionic strength conditions, while other invasion primers may need assistance of additives such as DMSO, ethylene glycol, formamide, betaine, or other denaturants that assist strand invasion by inducing more breathability within dsDNA amplicons.
  • the invasion primer may be introduced without a polymerase and allowed to invade and anneal to the complementary region of one strand of a dsDNA molecule, or it may be introduced together with a polymerase for runoff extension.
  • the invasion primer includes a primer binding sequence and a tail sequence.
  • a “tail sequence” refers to a nucleic acid sequence that is upstream of the primer binding sequence.
  • an invasion primer may include, from 5’ to 3’, the tail sequence and the binding sequence.
  • the tail sequence does not hybridize to the double-stranded polynucleotide (e.g., upon initial contact, the invasion primer hybridizes to the second strand, though it is understood that following extension of the second strand, the tail sequence is complementary to the extended second strand).
  • strand invasion refers to the displacement of one strand of a double-stranded nucleic acid molecule by a nucleic acid molecule (e.g., single stranded nucleic acid molecule, such as an invasion primer).
  • the nucleic acid molecule includes a nucleotide sequence that is substantially identical to a portion of the displaced strand and can selectively hybridize to the strand complementary to the displaced strand.
  • Strand displacement can occur without degradation of the displaced strands, thus being distinct from exonuclease activity.
  • the displacement occurs as a result of an extension reaction of a primer hybridized to a polynucleotide and a strand displacing enzyme.
  • invasion strand refers to an extended invasion primer (e.g., an invasion primer that has been hybridized to a first strand of a dsDNA molecule and extended by, for example, a strand-displacing polymerase in runoff extension to generate an invasion strand hybridized to the first strand of the dsDNA molecule).
  • the invasion strand for example, when hybridized to the first strand of a dsDNA molecule, prevents or blocks hybridization of the second strand of the dsDNA molecule to the first strand.
  • the invasion strand may be removed (e.g., the invasion strand may be digested with an exonuclease enzyme or denatured and washed away), allowing re-hybridization of the second strand of the dsDNA molecule to the first strand.
  • substrate refers to a solid support material.
  • the substrate can be non-porous or porous.
  • the substrate can be rigid or flexible.
  • solid support and “solid surface” refers to discrete solid or semi-solid surface.
  • a solid support may encompass any type of solid, porous, or hollow sphere, ball, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently).
  • a solid support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non- spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. Solid supports may be in the form of discrete particles, which alone does not imply or require any particular shape.
  • particle means a small body made of a rigid or semi-rigid material.
  • the body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions.
  • discrete particles refers to physically distinct particles Attorney Docket No.: 051385-585001WO having discernible boundaries.
  • the term “particle” does not indicate any particular shape.
  • the shapes and sizes of a collection of particles may be different or about the same (e.g., within a desired range of dimensions, or having a desired average or minimum dimension).
  • a particle may be substantially spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like.
  • the particle has the shape of a sphere, cylinder, spherocylinder, or ellipsoid.
  • Discrete particles collected in a container and contacting one another will define a bulk volume containing the particles, and will typically leave some internal fraction of that bulk volume unoccupied by the particles, even when packed closely together.
  • cores and/or core-shell particles are approximately spherical.
  • spherical refers to structures which appear substantially or generally of spherical shape to the human eye, and does not require a sphere to a mathematical standard.
  • spherical cores or particles are generally spheroidal in the sense of resembling or approximating to a sphere.
  • the diameter of a spherical core or particle is substantially uniform, e.g., about the same at any point, but may contain imperfections, such as deviations of up to 1, 2, 3, 4, 5 or up to 10%. Because cores or particles may deviate from a perfect sphere, the term “diameter” refers to the longest dimension of a given core or particle.
  • a solid support may further comprise a polymer or hydrogel on the surface to which the primers are attached (e.g., the primers are covalently attached to the polymer, wherein the polymer is in direct contact with the solid support).
  • Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, cyclic olefin copolymers, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, photopatternable dry film resists, UV-cured adhesives and polymers.
  • the solid supports for some embodiments have at least one surface located within a flow cell.
  • the solid support, or regions thereof, can be substantially flat.
  • the solid support can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts or the like.
  • Attorney Docket No.: 051385-585001WO The term solid support is encompassing of a substrate (e.g., a flow cell) having a surface comprising a polymer coating covalently attached thereto.
  • the solid support is a flow cell.
  • flow cell refers to a chamber including a solid surface across which one or more fluid reagents can be flowed. Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008).
  • a substrate comprises a surface (e.g., a surface of a flow cell, a surface of a tube, a surface of a chip, surface of a particle), for example a metal surface (e.g., steel, gold, silver, aluminum, silicon and copper).
  • a substrate e.g., a substrate surface
  • a substrate is coated and/or comprises functional groups and/or inert materials.
  • a substrate comprises a bead, a chip, a capillary, a plate, a membrane, a wafer (e.g., silicon wafers), a comb, or a pin for example.
  • a substrate comprises a bead and/or a nanoparticle.
  • a substrate can be made of a suitable material, non-limiting examples of which include a plastic or a suitable polymer (e.g., polycarbonate, poly(vinyl alcohol), poly(divinylbenzene), polystyrene, polyamide, polyester, polyvinylidene difluoride (PVDF), polyethylene, polyurethane, polypropylene, and the like), borosilicate, silica, nylon, Wang resin, Merrifield resin, metal (e.g., iron, a metal alloy, sepharose, agarose, polyacrylamide, dextran, cellulose and the like or combinations thereof.
  • a substrate comprises a magnetic material (e.g., iron, nickel, cobalt, platinum, aluminum, and the like).
  • a substrate comprises a magnetic bead (e.g., DYNABEADS®, hematite, AMPure XP). Magnets can be used to purify and/or capture nucleic acids bound to certain substrates (e.g., substrates comprising a metal or magnetic material).
  • a polymer refers to macromolecules having one or more structurally unique repeating units. The repeating units are referred to as “monomers,” which are polymerized for the polymer. Typically, a polymer is formed by monomers linked in a chain- like structure.
  • a polymer formed entirely from a single type of monomer is referred to as a “homopolymer.”
  • a polymer formed from two or more unique repeating structural units may be referred to as a “copolymer.”
  • a polymer may be linear or branched, and may be random, block, polymer brush, hyperbranched polymer, bottlebrush polymer, dendritic polymer, or polymer micelles.
  • the term “polymer” includes homopolymers, copolymers, tripolymers, tetra polymers and other polymeric molecules made from monomeric subunits.
  • Copolymers include alternating Attorney Docket No.: 051385-585001WO copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, linear copolymers and branched copolymers.
  • the term “polymerizable monomer” is used in accordance with its meaning in the art of polymer chemistry and refers to a compound that may covalently bind chemically to other monomer molecules (such as other polymerizable monomers that are the same or different) to form a polymer.
  • Polymers can be hydrophilic, hydrophobic, or amphiphilic, as known in the art.
  • hydrophilic polymers are substantially miscible with water and include, but are not limited to, polyethylene glycol and the like.
  • Hydrophilic polymers are substantially immiscible with water and include, but are not limited to, polyethylene, polypropylene, polybutadiene, polystyrene, polymers disclosed herein, and the like.
  • Amphiphilic polymers have both hydrophilic and hydrophobic properties and are typically copolymers having hydrophilic segment(s) and hydrophobic segment(s). Polymers include homopolymers, random copolymers, and block copolymers, as known in the art.
  • the term “homopolymer” refers, in the usual and customary sense, to a polymer having a single monomeric unit.
  • copolymer refers to a polymer derived from two or more monomeric species.
  • random copolymer refers to a polymer derived from two or more monomeric species with no preferred ordering of the monomeric species.
  • block copolymer refers to polymers having two or homopolymer subunits linked by covalent bond.
  • hydrophobic homopolymer refers to a homopolymer which is hydrophobic.
  • hydrophobic block copolymer refers to two or more homopolymer subunits linked by covalent bonds and which is hydrophobic.
  • hydrogel refers to a three-dimensional polymeric structure that is substantially insoluble in water, but which is capable of absorbing and retaining large quantities of water to form a substantially stable, often soft and pliable, structure.
  • water can penetrate in between polymer chains of a polymer network, subsequently causing swelling and the formation of a hydrogel.
  • hydrogels are super-absorbent (e.g., containing more than about 90% water) and can be comprised of natural or synthetic polymers.
  • Hydrogels can contain over 99% water and may include natural or synthetic polymers, or a combination thereof. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content.
  • hydrogel subunits or “hydrogel precursors” is meant hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or “polymerized”, to form a three-dimensional (3D) hydrogel network.
  • the alternating layers of polymeric gels described herein are hydrogels.
  • Hydrogels may be prepared by cross-linking hydrophilic biopolymers or synthetic polymers.
  • the hydrogel may include a crosslinker.
  • crosslinker refers to a molecule that can form a three-dimensional network when reacted with the appropriate base monomers.
  • hydrogel polymers which may include one or more crosslinkers, include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO—PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine),
  • a combination may include a polymer and a crosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO).
  • the hydrogel includes chemical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a covalent bond) and may be referred to as a chemical hydrogel.
  • the hydrogel includes physical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a non- covalent bond) and may be referred to as a physical hydrogel.
  • the physical hydrogel include one or more crosslinks including hydrogen bonds, hydrophobic interactions, and/or polymer chain entanglements.
  • Hydrogels may be prepared by cross-linking hydrophilic biopolymers or synthetic polymers.
  • the hydrogel may include a crosslinker.
  • crosslinker refers to a molecule that can form a three-dimensional network when reacted with the appropriate base monomers.
  • hydrogel polymers which may include one or more crosslinkers, include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO—PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO- PL(G)A copolymers, poly(ethylene imine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cys
  • a combination may include a polymer and a crosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO).
  • the hydrogel includes chemical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a covalent bond) and may be referred to as a chemical hydrogel.
  • the hydrogel includes physical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a non- covalent bond) and may be referred to as a physical hydrogel.
  • the physical hydrogel include one or more crosslinks including hydrogen bonds, hydrophobic interactions, and/or polymer chain entanglements.
  • array refers to a container (e.g., a microplate, tube, or flow cell) including a plurality of features (e.g., wells).
  • a container e.g., a microplate, tube, or flow cell
  • a plurality of features e.g., wells.
  • an array may include a container with a plurality of wells.
  • the array is a microplate.
  • the array is a flow cell.
  • the array is a multiwell container.
  • microplate refers to a substrate including a surface, the surface including a plurality of chambers or wells separated from each other by interstitial regions on the surface.
  • the microplate has dimensions as provided and described by American National Standards Institute (ANSI) and Society for Laboratory Automation And Screening (SLAS); for example the tolerances and dimensions set forth in ANSI SLAS 1-2004 (R2012); ANSI SLAS 2- 2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSI SLAS 4-2004 (R2012); and ANSI SLAS 6- 2012, which are incorporated herein by reference.
  • High-throughput screening refers to a process that uses a combination of modern robotics, data processing and control software, liquid handling devices, and/or sensitive detectors, to efficiently process a large amount of (e.g., thousands, hundreds of thousands, or millions) samples in biochemical, genetic, or pharmacological experiments, either in parallel or in sequence, within a reasonably short period of time (e.g., days).
  • the process is amenable to automation, such as robotic simultaneous handling of 96 samples, 384 samples, 1536 samples or more.
  • a typical HTS robot tests up to 100,000 to a few hundred thousand compounds per day.
  • the samples are often in small volumes, such as no more than 1 mL, 500 ⁇ l, 200 ⁇ l, 100 ⁇ l, 50 ⁇ l or less. Through this process, one can rapidly identify active compounds, small molecules, antibodies, proteins, or polynucleotides in a cell.
  • the reaction chambers may be provided as wells, for example an array or microplate may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells.
  • the 96 and 384 wells are arranged in a 2:3 rectangular matrix.
  • the 24 wells are arranged in a 3:8 rectangular matrix.
  • the 48 wells are arranged in a 3:4 rectangular matrix.
  • the reaction chamber is a microscope slide (e.g., a glass slide about 75 mm by about 25 mm).
  • the slide is a concavity slide (e.g., the slide includes a depression).
  • the slide includes a coating for enhanced cell adhesion (e.g., poly- L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, or gold).
  • the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 5 mm diameter wells.
  • the microplate is about 5 inches by about 3.33 inches, and includes a Attorney Docket No.: 051385-585001WO plurality of 6 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 7 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the microplate is 5 inches by 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 8 mm diameter wells.
  • the microplate is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples.
  • the term “surface” is intended to mean an external part or external layer of a substrate.
  • the surface can be in contact with another material such as a gas, liquid, gel, polymer, organic polymer, second surface of a similar or different material, metal, or coating.
  • the surface, or regions thereof, can be substantially flat.
  • the substrate and/or the surface can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts or the like.
  • well refers to a discrete concave feature in a substrate having a surface opening that is completely surrounded by interstitial region(s) of the surface.
  • Wells can have any of a variety of shapes at their opening in a surface including but not limited to round, elliptical, square, polygonal, or star shaped (i.e., star shaped with any number of vertices).
  • the cross section of a well taken orthogonally with the surface may be curved, square, polygonal, hyperbolic, conical, or angular.
  • the wells of a microplate are available in different shapes, for example F-Bottom: flat bottom; C-Bottom: bottom with minimal rounded edges; V-Bottom: V- shaped bottom; or U-Bottom: U-shaped bottom.
  • the well is substantially square.
  • the well is square.
  • the well is F-bottom.
  • the microplate includes 24 substantially round flat bottom wells.
  • the microplate includes 48 substantially round flat bottom wells.
  • the microplate includes 96 substantially round flat bottom wells.
  • the microplate includes 384 substantially square flat bottom wells.
  • the wells (alternatively referred to as reaction chambers) of a solid support and/or support insert may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells.
  • the 96 and 384 wells are arranged in a 2:3 rectangular matrix.
  • the 24 wells are arranged in a 3:8 rectangular matrix.
  • the 48 wells are arranged in a 3:4 rectangular matrix.
  • the solid support is a microscope slide (e.g., a glass slide Attorney Docket No.: 051385-585001WO about 75 mm by about 25 mm).
  • the slide is a concavity slide (e.g., the slide includes a depression).
  • the slide includes a coating for enhanced cell adhesion (e.g., poly-L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, or gold).
  • the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 5 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 6 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 7 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 7.5 mm diameter wells.
  • the solid support is 5 inches by 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 8 mm diameter wells.
  • the solid support insert is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples. In embodiments, the solid support is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples.
  • the solid support has a rectangular shape that measures 127.7 mm ⁇ 0.5 mm in length by 85.4 mm ⁇ 0.5 mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each well has an average diameter of about 5-7 mm. In embodiments, the solid support has a rectangular shape that measures 127.7 mm ⁇ 0.5 mm in length by 85.4 mm ⁇ 0.5 mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each well has an average diameter of about 6 mm. In embodiments, the solid support includes an array of femtoliter wells, array of nanoliter wells, or array of microliter wells. In embodiments, the wells in an array may all have substantially the same volume.
  • the array of wells may have a volume up to 100 e.g., about 0.1 femtoliter, 1 femtoliter, 10 femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50 microliter, or 100 microliter.
  • nanowell refers to a discrete concave feature or depression in a substrate having a surface opening that is completely surrounded by interstitial region(s) of the surface, wherein the diameter of the feature is less than or equal to 1000 nanometers.
  • the discrete regions (i.e., features, wells) of the microplate may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like.
  • the pattern of wells includes concentric circles of regions, spiral patterns, rectilinear patterns, hexagonal patterns, and the like.
  • the pattern of wells is arranged in a rectilinear or hexagonal pattern
  • a regular array of such regions is advantageous for detection and data analysis of signals collected from the arrays during an analysis.
  • These discrete regions are separated by interstitial regions.
  • the term “interstitial region” refers to an area in a substrate or on a surface that separates other areas of the substrate or surface.
  • an interstitial region can separate one concave feature of an array from another concave feature of the array.
  • the two regions that are separated from each other can be discrete, lacking contact with each other.
  • an interstitial region can separate a first portion of a feature from a second portion of a feature.
  • interstitial region is continuous whereas the features are discrete, for example, as is the case for an array of wells in an otherwise continuous surface.
  • the separation provided by an interstitial region can be partial or full separation.
  • interstitial regions have a surface material that differs from the surface material of the wells (e.g., the interstitial region contains a photoresist and the surface of the well is glass).
  • interstitial regions have a surface material that is the same as the surface material of the wells (e.g., both the surface of the interstitial region and the surface of well contain a polymer or copolymer).
  • cluster and “colony” are used interchangeably to refer to a discrete site on a solid support that includes a plurality of immobilized polynucleotides and a plurality of immobilized complementary polynucleotides.
  • the term “clustered array” refers to an array formed from such clusters or colonies. In this context the term “array” is not to be understood as requiring an ordered arrangement of clusters.
  • array is used in accordance with its ordinary meaning in the art, and refers to a population of different molecules that are attached to one or more solid-phase substrates such that the different molecules can be differentiated from each other according to their relative location.
  • An array can include different molecules that are each located at different addressable features on a solid-phase substrate.
  • the molecules of the array can be nucleic acid primers, nucleic acid probes, nucleic acid templates or nucleic acid enzymes such as polymerases or ligases.
  • Arrays useful in the invention can have densities that ranges from about 2 different features to many millions, billions or higher.
  • the Attorney Docket No.: 051385-585001WO density of an array can be from 2 to as many as a billion or more different features per square cm.
  • an array can have at least about 100 features/cm 2 , at least about 1,000 features/cm 2 , at least about 10,000 features /cm 2 , at least about 100,000 features /cm 2 , at least about 10,000,000 features /cm 2 , at least about 100,000,000 features /cm 2 , at least about 1,000,000,000 features /cm 2 , at least about 2,000,000,000 features /cm 2 or higher.
  • the arrays have features at any of a variety of densities including, for example, at least about 10 features/cm 2 , 100 features/cm 2 , 500 features/cm 2 , 1,000 features/cm 2 , 5,000 features/cm 2 , 10,000 features/cm 2 , 50,000 features/cm 2 , 100,000 features/cm 2 , 1,000,000 features/cm 2 , 5,000,000 features/cm 2 , or higher.
  • Nucleic acids including e.g., nucleic acids with a phosphorothioate backbone, can include one or more reactive moieties.
  • the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions.
  • the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction.
  • template polynucleotide or “template nucleic acid” refers to any polynucleotide molecule that may be bound by a polymerase and utilized as a template for nucleic acid synthesis.
  • a template polynucleotide may be a target polynucleotide.
  • target polynucleotide refers to a nucleic acid molecule or polynucleotide in a starting population of nucleic acid molecules having a target sequence whose presence, amount, and/or nucleotide sequence, or changes in one or more of these, are desired to be determined.
  • target sequence refers to a nucleic acid sequence on a single strand of nucleic acid.
  • single strand and ssDNA are used in accordance with its plain and ordinary meaning and refer to a single-stranded polynucleotide.
  • the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others.
  • the target sequence may be a target sequence from a sample or a secondary target such as a product of an amplification reaction.
  • a target polynucleotide is not necessarily any single molecule or sequence.
  • a target polynucleotide may be any one of a plurality of target polynucleotides in a reaction, or all polynucleotides in a given reaction, depending on the reaction conditions.
  • all polynucleotides in a reaction may be amplified.
  • a collection of targets may be simultaneously assayed using polynucleotide primers directed to a plurality of targets in a single reaction.
  • all or a subset of polynucleotides in a sample may be modified by the addition of a primer-binding sequence (such as by the ligation of adapters containing the primer binding sequence), rendering each modified polynucleotide a target polynucleotide in a reaction with the corresponding primer polynucleotide(s).
  • target polynucleotide(s) refers to the subset of polynucleotide(s) to be sequenced from within a starting population of polynucleotides.
  • a target polynucleotide is a cell-free polynucleotide.
  • the terms “cell-free,” “circulating,” and “extracellular” as applied to polynucleotides e.g.
  • cell-free DNA cfDNA
  • cfRNA cell-free RNA
  • cfDNA cfDNA
  • cfRNA cell-free RNA
  • Cell-free polynucleotides are thus unencapsulated or “free” from the cells or viruses from which they originate, even before a sample of the subject is collected.
  • Cell-free polynucleotides may be produced as a byproduct of cell death (e.g. apoptosis or necrosis) or cell shedding, releasing polynucleotides into surrounding body fluids or into circulation.
  • cell-free polynucleotides may be isolated from a non-cellular fraction of blood (e.g. serum or plasma), from other bodily fluids (e.g. urine), or from non-cellular fractions of other types of samples.
  • a polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself.
  • This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides.
  • analogue in reference to a chemical compound, refers to compound having a structure similar to that of another one, but differing Attorney Docket No.: 051385-585001WO from it in respect of one or more different atoms, functional groups, or substructures that are replaced with one or more other atoms, functional groups, or substructures.
  • a nucleotide analog refers to a compound that, like the nucleotide of which it is an analog, can be incorporated into a nucleic acid molecule (e.g., an extension product) by a suitable polymerase, for example, a DNA polymerase in the context of a nucleotide analogue.
  • a suitable polymerase for example, a DNA polymerase in the context of a nucleotide analogue.
  • the terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphorothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see, e.g., see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine; and peptide nucleic acid backbones and linkages.
  • phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphoroth
  • nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA)), including those described in U.S. Patent Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids.
  • LNA locked nucleic acids
  • Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip.
  • Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.
  • Other analog nucleic acids include bis-locked nucleic acids (bisLNAs; e.g., including those described in Moreno PMD et al.
  • TAAs intercalating nucleic acids
  • BNAs bridged nucleic acids
  • 2’-O-methyl RNA:DNA chimeric nucleic acids e.g., including those described in Wang S and Kool ET.
  • Nucleic Acids Res.1995; 23(7):1157-1164 minor groove binder (MGB) nucleic acids (e.g., including those described in Kutyavin IV et al. Nucleic Acids Res.2000; 28(2):655-61), morpholino nucleic acids (e.g., including those described in Summerton J and Weller D. Antisense Nucleic Acid Drug Dev.1997; 7(3):187-95), C5-modified pyrimidine nucleic acids (e.g., including those described in Kumar P et al. J. Org.
  • MGB minor groove binder
  • PNAs peptide nucleic acids
  • phosphorothioate nucleotides e.g., including those described in Eckstein F. Nucleic Acid Ther.2014; 24(6):374-87.
  • nucleotide As used herein, a "native" nucleotide is used in accordance with its plain and ordinary meaning and refers to a naturally occurring nucleotide that does not include an exogenous label (e.g., a fluorescent dye, or other label) or chemical modification such as may characterize a nucleotide analog (e.g., a reversible terminating moiety).
  • exogenous label e.g., a fluorescent dye, or other label
  • chemical modification such as may characterize a nucleotide analog (e.g., a reversible terminating moiety).
  • native nucleotides useful for carrying out procedures described herein include: dATP (2'-deoxyadenosine-5'-triphosphate); dGTP (2'-deoxyguanosine-5'-triphosphate); dCTP (2'-deoxycytidine-5'-triphosphate); dTTP (2'- deoxythymidine-5'-triphosphate); and dUTP (2'-deoxyuridine-5'-triphosphate).
  • a “canonical” nucleotide is an unmodified nucleotide.
  • the term “modified nucleotide” refers to nucleotide modified in some manner.
  • a nucleotide typically contains a single 5-carbon sugar moiety, a single nitrogenous base moiety and 1 to three phosphate moieties.
  • a nucleotide can include a blocking moiety (alternatively referred to herein as a reversible terminator moiety) and/or a label moiety.
  • a blocking moiety on a nucleotide prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide.
  • a blocking moiety on a nucleotide can be reversible, whereby the blocking moiety can be removed or modified to allow the 3' hydroxyl to form a covalent bond with the 5' phosphate of another nucleotide.
  • a blocking moiety can be effectively irreversible under particular conditions used in a method set forth herein.
  • the blocking moiety is attached to the 3’ oxygen of .
  • a label moiety of a nucleotide can be using a spectroscopic method.
  • Exemplary label moieties are fluorescent labels, mass labels, chemiluminescent labels, electrochemical labels, detectable labels and the like.
  • One or more of the above moieties can be absent from a nucleotide used in the methods and compositions set forth herein.
  • a nucleotide can lack a label moiety or a blocking moiety or both.
  • nucleotide analogues include, without limitation, 7-deaza-adenine, 7-deaza-guanine, the analogues of deoxynucleotides shown herein, analogues in which a label is attached through a cleavable linker to the 5-position of cytosine or thymine or to the 7-position of deaza-adenine or deaza-guanine, and analogues in which a small chemical moiety is used to cap the OH group at the 3'-position of deoxyribose. Nucleotide analogues and DNA polymerase-based DNA sequencing are also described in U.S.
  • cleavable complement refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides, wherein the complementary nucleotide or sequence of nucleotides includes a cleavable site, and the cleavable complement also includes a complement to the cleavable site.
  • the cleavable complement of the cleavable site and the cleavable site are cleaved by the same mechanism (e.g., restriction enzyme digestion of the duplexed cleavable site and cleavable complement of the cleavable site).
  • cleavable linker or “cleavable moiety” as used herein refers to a divalent or monovalent, respectively, moiety which is capable of being separated (e.g., detached, split, disconnected, hydrolyzed, a stable bond within the moiety is broken) into distinct entities.
  • a cleavable linker is cleavable (e.g., specifically cleavable) in response to external stimuli (e.g., enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents).
  • external stimuli e.g., enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents.
  • a chemically cleavable linker refers to a linker which is capable of being split in response to the presence of a chemical (e.g., acid, base, oxidizing agent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite (Na2S2O4), or hydrazine (N 2 H 4 )).
  • a chemically cleavable linker is non-enzymatically cleavable.
  • the cleavable linker is cleaved by contacting the cleavable linker with a cleaving agent.
  • the cleaving agent is a phosphine containing reagent (e.g., TCEP or THPP), sodium dithionite (Na 2 S 2 O 4 ), weak acid, hydrazine (N 2 H 4 ), Pd(0), or light-irradiation (e.g., ultraviolet radiation).
  • cleaving includes removing.
  • cleavable linker and “cleavable site” are different terms with different meanings as used herein.
  • a cleavable linker may include a covalent linker that includes one or more cleavable sites.
  • a “cleavable site” or “scissile linkage” in the context of a polynucleotide is a site which allows controlled cleavage of the polynucleotide strand (e.g., the linker, the primer, or the polynucleotide) by chemical, enzymatic, or photochemical means known in the art and described herein.
  • a scissile site may refer to the linkage of a nucleotide between two other nucleotides in a nucleotide strand (i.e., an internucleosidic linkage).
  • the scissile linkage can be located at any position within the one or more nucleic acid molecules, including at or near a terminal end (e.g., the 3′ end of an oligonucleotide) or in an interior portion of the one or more nucleic acid molecules.
  • conditions suitable for separating a scissile linkage include a modulating the pH and/or the temperature.
  • a scissile site can include at least one acid-labile linkage.
  • an acid-labile linkage may include a phosphoramidate linkage.
  • a phosphoramidate linkage can be hydrolysable under acidic conditions, including mild acidic conditions such as trifluoroacetic acid and a suitable temperature (e.g., 30°C), or other conditions known in the art, for example Matthias Mag, et al Tetrahedron Letters, Volume 33, Issue 48, 1992, 7319-7322.
  • the Attorney Docket No.: 051385-585001WO scissile site can include at least one photolabile internucleosidic linkage (e.g., o-nitrobenzyl linkages, as described in Walker et al, J. Am. Chem.
  • the scissile site includes at least one uracil nucleobase.
  • a uracil nucleobase can be cleaved with a uracil DNA glycosylase (UDG) or Formamidopyrimidine DNA Glycosylase Fpg.
  • the scissile linkage site includes a sequence-specific nicking site having a nucleotide sequence that is recognized and nicked by a nicking endonuclease enzyme or a uracil DNA glycosylase.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site blast.ncbi.nlm.nih.gov/Blast.cgi or the like).
  • sequences are then said to be "substantially identical.”
  • This definition also refers to, or may be applied to, the complement of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • the term “determine” can be used to refer to the act of ascertaining, establishing or estimating. A determination can be probabilistic.
  • a determination can have an apparent likelihood of at least 50%, 75%, 90%, 95%, 98%, 99%, 99.9% or higher. In some cases, a determination can have an apparent likelihood of 100%.
  • An exemplary determination is a maximum likelihood analysis or report.
  • the term “identify,” when used in reference to a thing, can be used to refer to recognition of the thing, distinction of the thing from at least one other thing or categorization of the thing with at least one other thing. Attorney Docket No.: 051385-585001WO
  • the recognition, distinction or categorization can be probabilistic. For example, a thing can be identified with an apparent likelihood of at least 50%, 75%, 90%, 95%, 98%, 99%, 99.9% or higher.
  • a thing can be identified based on a result of a maximum likelihood analysis. In some cases, a thing can be identified with an apparent likelihood of 100%.
  • the term “removable” group e.g., a label or a blocking group or protecting group, is used in accordance with its plain and ordinary meaning and refers to a chemical group that can be removed from a nucleotide analogue such that a DNA polymerase can extend the nucleic acid (e.g., a primer or extension product) by the incorporation of at least one additional nucleotide. Removal may be by any suitable method, including enzymatic, chemical, or photolytic cleavage.
  • Removal of a removable group does not require that the entire removable group be removed, only that a sufficient portion of it be removed such that a DNA polymerase can extend a nucleic acid by incorporation of at least one additional nucleotide using a nucleotide or nucleotide analogue.
  • the conditions under which a removable group is removed are compatible with a process employing the removable group (e.g., an amplification process or sequencing process).
  • reversible blocking groups and “reversible terminators” are used in accordance with their plain and ordinary meanings and refer to a blocking moiety located, for example, at the 3' position of the nucleotide and may be a chemically cleavable moiety such as an allyl group, an azidomethyl group or a methoxymethyl group, or may be an enzymatically cleavable group such as a phosphate ester.
  • nucleotide blocking moieties are described in applications WO 2004/018497, U.S. Pat. Nos.7,057,026, 7,541,444, WO 96/07669, U.S. Pat.
  • nucleotides may be labelled or unlabeled. They may be modified with reversible terminators useful in methods provided herein and may be 3'-O-blocked reversible or 3'-unblocked reversible terminators.
  • the blocking group –OR reversible terminating (capping) group
  • the label is linked to the base, which acts as a reporter and can be cleaved.
  • the 3'-O-blocked reversible terminators are known in the art, and may be, for instance, a 3'-ONH 2 reversible terminator, a 3'-O-allyl reversible terminator, or a 3'-O-azidomethyl reversible terminator.
  • the reversible terminator moiety , .
  • the reversible terminator moiety as described in U.S.
  • a nucleotide including a reversible terminator moiety may be represented by the formula: , where the nucleobase is adenine or adenine analogue, thymine or thymine analogue, guanine or guanine analogue, or cytosine or cytosine analogue.
  • a nucleic acid comprises a molecular identifier or a molecular barcode.
  • molecular barcode refers to any material (e.g., a nucleotide sequence, a nucleic acid molecule feature) that is capable of distinguishing an individual molecule in a large heterogeneous population of molecules.
  • a barcode is unique in a pool of barcodes that differ from one another in sequence, or is uniquely associated with a particular sample polynucleotide in a pool of sample polynucleotides.
  • every barcode in a pool of adapters is unique, such that sequencing reads comprising the barcode can be identified as originating from a single sample polynucleotide molecule on the basis of the barcode alone.
  • individual barcode sequences may be used more than once, but adapters comprising the duplicate Attorney Docket No.: 051385-585001WO barcodes are associated with different sequences and/or in different combinations of barcoded adapters, such that sequence reads may still be uniquely distinguished as originating from a single sample polynucleotide molecule on the basis of a barcode and adjacent sequence information (e.g., sample polynucleotide sequence, and/or one or more adjacent barcodes).
  • barcodes are about or at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75 or more nucleotides in length. In embodiments, barcodes are shorter than 20, 15, 10, 9, 8, 7, 6, or 5 nucleotides in length. In embodiments, barcodes are about 10 to about 50 nucleotides in length, such as about 15 to about 40 or about 20 to about 30 nucleotides in length. In a pool of different barcodes, barcodes may have the same or different lengths. In general, barcodes are of sufficient length and include sequences that are sufficiently different to allow the identification of sequencing reads that originate from the same sample polynucleotide molecule.
  • each barcode in a plurality of barcodes differs from every other barcode in the plurality by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions.
  • substantially degenerate barcodes may be known as random.
  • a barcode may include a nucleic acid sequence from within a pool of known sequences.
  • the barcodes may be pre-defined.
  • a nucleic acid e.g., an adapter or primer
  • sample barcode is a nucleotide sequence that is sufficiently different from other sample barcode to allow the identification of the sample source based on sample barcode sequence(s) with which they are associated.
  • a plurality of nucleotides e.g., all nucleotides from a particular sample source, or sub-sample thereof
  • a different plurality of nucleotides e.g., all nucleotides from a different sample source, or different subsample
  • each sample barcode in a plurality of sample barcodes differs from every other sample barcode in the plurality by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions.
  • substantially degenerate sample barcodes may be known as random.
  • a sample barcode may include a nucleic acid sequence from within a pool of known sequences.
  • the sample barcodes may be pre-defined.
  • the sample barcode includes about 1 to about 10 nucleotides.
  • the sample barcode includes about 3, 4, 5, 6, 7, 8, 9, or about 10 Attorney Docket No.: 051385-585001WO nucleotides.
  • the sample barcode includes about 3 nucleotides. In embodiments, the sample barcode includes about 5 nucleotides. In embodiments, the sample barcode includes about 7 nucleotides. In embodiments, the sample barcode includes about 10 nucleotides. In embodiments, the sample barcode includes about 6 to about 10 nucleotides. [0072] In some embodiments, the reaction conditions for a plurality of invasion-primer extension cycles includes incubation in a denaturant.
  • the terms “denaturant” or plural “denaturants” are used in accordance with their plain and ordinary meanings and refer to an additive or condition that disrupts the base pairing between nucleotides within opposing strands of a double-stranded polynucleotide molecule.
  • denaturation includes rendering at least some portion or region of two strands of the double-stranded polynucleotide molecule or sequence single-stranded or partially single-stranded.
  • denaturation includes separation of at least some portion or region of two strands of the double-stranded polynucleotide molecule or sequence from each other.
  • the denatured region or portion is then capable of hybridizing to another polynucleotide molecule or sequence.
  • Complete denaturation conditions are, for example, conditions that would result in complete separation of a significant fraction (e.g., more than 10%, 20%, 30%, 40% or 50%) of a large plurality of strands from their extended and/or full-length complements.
  • complete or total denaturation disrupts all of the base pairing between the nucleotides of the two strands with each other.
  • a nucleic acid sample is optionally considered fully denatured when more than 80% or 90% of individual molecules of the sample lack any double-strandedness (or lack any hybridization to a complementary strand).
  • the double-stranded polynucleotide molecule or sequence can be partially or incompletely denatured.
  • a given nucleic acid molecule can be considered partially denatured when a portion of at least one strand of the nucleic acid remains hybridized to a complementary strand, while another portion is in an unhybridized state (even if it is in the presence of a Attorney Docket No.: 051385-585001WO complementary sequence).
  • the unhybridized portion is optionally at least 5, 10, 15, 20, 50, or more nucleotides in length.
  • the hybridized portion is optionally at least 5, 10, 15, 20, 50, or more nucleotides in length.
  • Partial denaturation includes situations where some, but not all, of the nucleotides of one strand or sequence, are based paired with some nucleotides of the other strand or sequence within a double-stranded polynucleotide. In some embodiments, at least 20% but less than 100% of the nucleotide residues of one strand of the partially denatured polynucleotide (or sequence) are not base paired to nucleotide residues within the opposing strand.
  • nucleic acid sample can be considered to be partially denatured when a substantial fraction of individual nucleic acid molecules of the sample (e.g., above 20%, 30%, 50%, or 70%) are in a partially denatured state.
  • nucleic acid sample can be non-denatured when a minority of individual nucleic acid molecules in the sample are partially or completely denatured.
  • partially denaturing conditions are achieved by maintaining the duplexes as a suitable temperature range.
  • the nucleic acid is maintained at temperature sufficiently elevated to achieve some heat-denaturation (e.g., above 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C) but not high enough to achieve complete heat-denaturation (e.g., below 95°C or 90°C or 85°C or 80°C or 75°C).
  • the nucleic acid is partially denatured using substantially isothermal conditions.
  • chemical denaturation can be accomplished by contacting the double-stranded polynucleotide to be denatured with appropriate chemical denaturants, such as strong alkalis, strong acids, chaotropic agents, and the like and can include, for example, NaOH, urea, or guanidine-containing compounds.
  • appropriate chemical denaturants such as strong alkalis, strong acids, chaotropic agents, and the like and can include, for example, NaOH, urea, or guanidine-containing compounds.
  • partial or complete denaturation is achieved by exposure to chemical denaturants such as urea or Attorney Docket No.: 051385-585001WO formamide, with concentrations suitably adjusted, or using high or low pH (e.g., pH between 4-6 or 8-9).
  • the denaturant is a buffered solution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4- methylmorpholine 4-oxide (NMO), or a mixture thereof.
  • the first denaturant is a buffered solution including about 0% to about 50% dimethyl sulfoxide (DMSO); about 0% to about 50% ethylene glycol; about 0% to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof.
  • partial denaturation and/or amplification can be achieved using a recombinase and/or single-stranded binding protein.
  • complete or partial denaturation of a double-stranded polynucleotide sequence is accomplished by contacting the double-stranded polynucleotide sequence using appropriate denaturing agents.
  • the double-stranded polynucleotide can be subjected to heat-denaturation (also referred to interchangeably as thermal denaturation) by raising the temperature to a point where the desired level of denaturation is accomplished.
  • thermal denaturation of a double-stranded polynucleotide includes adjusting the temperature to achieve complete separation of the two strands of the polynucleotide, such that 90% or greater of the strands are in single-stranded form across their entire length.
  • a completely denatured double-stranded polynucleotide results in a separated first strand and a second strand, each of which is a single-stranded polynucleotide.
  • complete thermal denaturation of a polynucleotide molecule is accomplished by exposing the polynucleotide molecule (or sequence) to a temperature that is at least 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 50°C, or 100°C, above the calculated or predict melting temperature (Tm) of the polynucleotide molecule or sequence.
  • complete or partial denaturation is accomplished by treating the double-stranded polynucleotide sequence to be denatured using a denaturant mixture including an SSB protein (e.g., T4 gp32 protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNA Binding Protein (ET- SSB)), a strand-displacing polymerase (e.g., Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst 2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 Attorney Docket No.: 051385-585001WO poly
  • SSB protein e
  • the crowding agent is poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000), dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, ⁇ -lactoglobulin, hemoglobin, bovine serum albumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS).
  • poly(ethylene glycol) e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000
  • BPTI bovine pancreatic trypsin inhibitor
  • ribonuclease A e.g., ribonuclease A,
  • the denaturant mixture including an SSB, a strand-displacing polymerase, and one or more crowding agents does not include a chemical denaturant (e.g., betaine, DMSO, ethylene glycol, formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof).
  • a chemical denaturant e.g., betaine, DMSO, ethylene glycol, formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof.
  • a nucleic acid comprises a label.
  • label or “labels” are used in accordance with their plain and ordinary meanings and refer to molecules that can directly or indirectly produce or result in a detectable signal either by themselves or upon interaction with another molecule.
  • Non-limiting examples of detectable labels include fluorescent dyes, biotin, digoxin, haptens, and epitopes.
  • a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal.
  • the label is a dye.
  • the dye is a fluorescent dye.
  • Non- limiting examples of dyes include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.).
  • CF dyes Biotium, Inc.
  • Alexa Fluor dyes Thermo Fisher
  • DyLight dyes Thermo Fisher
  • Cy dyes GE Healthscience
  • IRDyes Li-Cor Biosciences, Inc.
  • HiLyte dyes HiLyte dyes
  • the label is luciferin that reacts with luciferase to produce a detectable signal in response to one or more bases being incorporated into an elongated complementary strand, such as in pyrosequencing.
  • a nucleotide comprises a label (such as a dye).
  • the label is not associated with any particular nucleotide, but detection of the label identifies whether one or more nucleotides having a known identity were added during an extension step (such as in the case of pyrosequencing).
  • the detectable label is a fluorescent dye.
  • the detectable label is a fluorescent dye capable of exchanging energy with another fluorescent dye (e.g., fluorescence resonance energy transfer (FRET) chromophores).
  • fluorescent dye e.g., fluorescence resonance energy transfer (FRET) chromophores
  • detectable agents include imaging agents, including fluorescent and luminescent substances, including, but not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes.
  • the detectable moiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye).
  • the detectable moiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye).
  • the detectable moiety is a moiety of a derivative of one of the detectable moieties described immediately above, wherein the derivative differs from one of the detectable moieties immediately above by a modification resulting from the conjugation of the detectable moiety to a compound described herein.
  • the term “cyanine” or “cyanine moiety” as described herein refers to a detectable moiety containing two nitrogen groups separated by a polymethine chain. In embodiments, the cyanine moiety has 3 methine structures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moiety has 5 methine structures (i.e., cyanine 5 or Cy5).
  • the cyanine moiety has 7 methine structures (i.e., cyanine 7 or Cy7).
  • DNA polymerase and “nucleic acid polymerase” are used in accordance with their plain ordinary meanings and refer to enzymes capable of synthesizing nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides to the 3’- end of a DNA strand, one nucleotide at a time.
  • the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase
  • the DNA polymerase is a modified archaeal DNA polymerase.
  • the polymerase is a reverse transcriptase.
  • the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044).
  • the polymerase is an enzyme described in US 2021/0139884.
  • a polymerase catalyzes the addition of a next correct nucleotide to the 3′-OH group of the primer via a phosphodiester bond, thereby chemically incorporating the nucleotide into the primer.
  • the polymerase used in the provided methods is a processive polymerase.
  • the polymerase used in the provided methods is a distributive polymerase.
  • thermophilic nucleic acid polymerase refers to a family of DNA polymerases (e.g.,9°N TM ) and mutants thereof derived from the DNA polymerase originally isolated from the hyperthermophilic archaea, Thermococcus sp.9 degrees N-7, found in hydrothermal vents at that latitude (East Pacific Rise) (Southworth MW, et al. PNAS. 1996;93(11):5281-5285).
  • a thermophilic nucleic acid polymerase is a member of the family B DNA polymerases.
  • thermophilic nucleic acid polymerases may be found in (Southworth MW, et al. PNAS. 1996;93(11):5281-5285; Bergen K, et al. ChemBioChem.2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012;2:684; Fuller CW, et al.2016;113(19):5233-5238; Guo J, et al. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(27):9145-9150), which are incorporated herein in their entirety for all purposes.
  • exonuclease activity is used in accordance with its ordinary meaning in the art, and refers to the removal of a nucleotide from a nucleic acid by an enzyme (e.g. DNA polymerase, a lambda exonuclease, Exo I, Exo III, T5, Exo V, Exo VII or the like). For example, during polymerization, nucleotides are added to the 3’ end of the primer strand.
  • an enzyme e.g. DNA polymerase, a lambda exonuclease, Exo I, Exo III, T5, Exo V, Exo VII or the like.
  • a DNA polymerase incorporates an incorrect nucleotide to the 3′-OH terminus of the primer strand, wherein the incorrect nucleotide cannot form a hydrogen bond to the corresponding base in the template strand.
  • exonuclease activity may be referred to as “proofreading.”
  • the DNA polymerase facilitates a hydrolyzing reaction that breaks phosphodiester bonds at the 3’ end of a polynucleotide chain to excise the nucleotide.
  • 3’-5’ exonuclease activity refers to the successive removal of nucleotides in single-stranded DNA in a 3’ ⁇ 5’ direction, releasing deoxyribonucleoside 5’-monophosphates one after another. Methods for quantifying exonuclease activity are known in the art, see for example Southworth et al, PNAS Vol 93, 8281-8285 (1996).
  • 5’-3’ exonuclease activity refers to the successive removal of nucleotides in double-stranded DNA in a 5’ ⁇ 3’ direction.
  • the 5’-3’ exonuclease is lambda exonuclease.
  • lambda exonuclease catalyzes the removal of 5’ mononucleotides from duplex DNA, with a preference for 5’ phosphorylated double-stranded DNA.
  • the 5’-3’ exonuclease is E. coli DNA Polymerase I.
  • the term “selective” or “selectivity” or the like of a compound refers to the compound’s ability to discriminate between molecular targets.
  • this term refers to sequencing one or more target polynucleotides from an original starting population of polynucleotides, and not sequencing non-target polynucleotides from the starting population.
  • selectively sequencing one or more target polynucleotides involves differentially manipulating the target polynucleotides based on known sequence.
  • target polynucleotides may be Attorney Docket No.: 051385-585001WO hybridized to a probe oligonucleotide that may be labeled (such as with a member of a binding pair) or bound to a surface.
  • hybridizing a target polynucleotide to a probe oligonucleotide includes the step of displacing one strand of a double-stranded nucleic acid.
  • Probe-hybridized target polynucleotides may then be separated from non-hybridized polynucleotides, such as by removing probe-bound polynucleotides from the starting population or by washing away polynucleotides that are not bound to a probe. The result is a selected subset of the starting population of polynucleotides, which is then subjected to sequencing, thereby selectively sequencing the one or more target polynucleotides.
  • the terms “specific”, “specifically”, “specificity”, or the like of a compound refers to the agent’s ability to cause a particular action, such as binding, to a particular molecular target with minimal or no action to other proteins in the cell.
  • bound and bound are used in accordance with their plain and ordinary meanings and refer to an association between atoms or molecules.
  • the association can be direct or indirect.
  • bound atoms or molecules may be directly bound to one another, e.g., by a covalent bond or non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole- induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).
  • electrostatic interactions e.g., ionic bond, hydrogen bond, halogen bond
  • van der Waals interactions e.g., dipole-dipole, dipole- induced dipole, London dispersion
  • ring stacking pi effects
  • hydrophobic interactions and the like.
  • two molecules may be bound indirectly to one another by way of direct binding to one or more intermediate molecules, thereby forming a complex.
  • “Specific binding” is where the binding is selective between two molecules.
  • a particular example of specific binding is that which occurs between an antibody and an antigen.
  • specific binding can be distinguished from non-specific when the dissociation constant (KD) is less than about 1 ⁇ 10 ⁇ 5 M or less than about 1 ⁇ 10 ⁇ 6 M or 1 ⁇ 10 ⁇ 7 M.
  • KD dissociation constant
  • Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslinking, two-hybrid assays and the like.
  • the K D (equilibrium dissociation constant) between two specific binding molecules is less than 10 ⁇ 6 M, less than 10 ⁇ 7 M, less than 10 ⁇ 8 M, less than 10 ⁇ 9 M, less than 10 ⁇ 9 M, less than 10 ⁇ 11 M, or less than about 10 ⁇ 12 M or less.
  • rolling circle amplification refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., single-stranded DNA Attorney Docket No.: 051385-585001WO circles) via a rolling circle mechanism.
  • Rolling circle amplification reaction is initiated by the hybridization of a primer to a circular, often single-stranded, nucleic acid template.
  • the nucleic acid polymerase then extends the primer that is hybridized to the circular nucleic acid template by continuously progressing around the circular nucleic acid template to replicate the sequence of the nucleic acid template over and over again (rolling circle mechanism).
  • the rolling circle amplification typically produces concatemers comprising tandem repeat units of the circular nucleic acid template sequence.
  • the rolling circle amplification may be a linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCA using a single specific primer), or may be an exponential RCA (ERCA) exhibiting exponential amplification kinetics.
  • LRCA linear RCA
  • ERCA exponential RCA
  • Rolling circle amplification may also be performed using multiple primers (multiply primed rolling circle amplification or MPRCA) leading to hyper-branched concatemers.
  • MPRCA multiply primed rolling circle amplification
  • one primer may be complementary, as in the linear RCA, to the circular nucleic acid template, whereas the other may be complementary to the tandem repeat unit nucleic acid sequences of the RCA product. Consequently, the double-primed RCA may proceed as a chain reaction with exponential (geometric) amplification kinetics featuring a ramifying cascade of multiple-hybridization, primer-extension, and strand-displacement events involving both the primers.
  • the rolling circle amplification may be performed in-vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.
  • RCA may be performed by using any of the DNA polymerases that are known in the art (e.g., a Phi29 DNA polymerase, a Bst DNA polymerase, or SD polymerase).
  • sequence determination As used herein, the terms “sequencing”, “sequence determination”, “determining a nucleotide sequence”, and the like include determination of a partial or complete sequence information, including the identification, ordering, or locations of the nucleotides that comprise the polynucleotide being sequenced, and inclusive of the physical processes for generating such sequence information. That is, the term includes sequence comparisons, consensus sequence determination, contig assembly, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of nucleotides in a target polynucleotide.
  • a sequencing process described herein comprises contacting a template and Attorney Docket No.: 051385-585001WO an annealed primer with a suitable polymerase under conditions suitable for polymerase extension and/or sequencing.
  • the sequencing methods are preferably carried out with the target polynucleotide arrayed on a solid substrate.
  • Multiple target polynucleotides can be immobilized on the solid support through linker molecules, or can be attached to particles, e.g., microspheres, which can also be attached to a solid substrate.
  • the solid substrate is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, or a column.
  • the solid substrate is gold, quartz, silica, plastic, glass, diamond, silver, metal, or polypropylene. In embodiments, the solid substrate is porous.
  • sequencing cycle is used in accordance with its plain and ordinary meaning and refers to incorporating one or more nucleotides (e.g., nucleotide analogues) to the 3’ end of a polynucleotide with a polymerase, and detecting the one or more nucleotides incorporated.
  • one nucleotide e.g., a modified nucleotide
  • the sequencing may be accomplished by, for example, sequencing by synthesis, pyrosequencing, and the like.
  • a sequencing cycle includes extending a complementary polynucleotide by incorporating a first nucleotide using a polymerase, wherein the polynucleotide is hybridized to a template nucleic acid, detecting the first nucleotide, and identifying the first nucleotide.
  • An “extension strand” is formed as the one or more nucleotides are incorporated into a complementary polynucleotide hybridized to a template nucleic acid. The extension strand is complementary to the template nucleic acid.
  • one or more differently labeled nucleotides and a DNA polymerase can be introduced.
  • signals produced can be detected to determine the identity of the incorporated nucleotide (based on the labels on the nucleotides).
  • Reagents can then be added to remove the 3’ reversible terminator and to remove labels from each incorporated base. Reagents, enzymes, and other substances can be removed between steps by washing. Cycles may include repeating these steps, and the sequence of each cluster is read over the multiple repetitions.
  • sequencing reaction mixture is used in accordance with its plain and ordinary meaning and refers to an aqueous mixture that contains the reagents necessary to allow a nucleotide or nucleotide analogue to be added (i.e., incorporated) to a DNA strand by a DNA polymerase.
  • the term “invasion-reaction mixture” is used in accordance Attorney Docket No.: 051385-585001WO with its plain and ordinary meaning and refers to an aqueous mixture that contains the reagents necessary to allow a nucleotide or nucleotide analogue to be added to a DNA strand by a DNA polymerase that extends the invasion primer.
  • extension As used herein, the term “extending”, “extension” or “elongation” is used in accordance with their plain and ordinary meanings and refer to synthesis by a polymerase of a new polynucleotide strand (i.e., an “extension strand”) complementary to a template strand by adding free nucleotides (e.g., dNTPs) from a reaction mixture that are complementary to the template in a 5’-to-3’ direction, including condensing a 5’-phosphate group of a dNTPs with a 3’-hydroxy group at the end of the nascent (elongating) DNA strand.
  • free nucleotides e.g., dNTPs
  • sequencing read is used in accordance with its plain and ordinary meaning and refers to an inferred sequence of nucleotide bases (or nucleotide base probabilities) corresponding to all or part of a single polynucleotide fragment.
  • a sequencing read may include 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more nucleotide bases.
  • Reads of length 20-40 base pairs (bp) are referred to as ultra-short. Typical sequencers produce read lengths in the range of 100-500 bp. Read length is a factor which can affect the results of biological studies. For example, longer read lengths improve the resolution of de novo genome assembly and detection of structural variants.
  • a sequencing read includes reading a barcode and a template nucleotide sequence. In embodiments, a sequencing read includes reading a template nucleotide sequence. In embodiments, a sequencing read includes reading a barcode and not a template nucleotide sequence. In embodiments, a sequencing read includes a computationally derived string corresponding to the detected label. In some embodiments, a sequencing read may include 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or more nucleotide bases. In embodiments, a sequencing read is a string of characters representing the sequence of nucleotides. In embodiments, the length of a sequencing read corresponds to the length of the target sequence.
  • the length of a sequencing read corresponds to the number of sequencing cycles.
  • a sequencing read may be subjected to initial processing (often termed “pre-processing”) prior to annotation. Pre- processing includes filtering out low-quality sequences, sequence trimming to remove continuous low-quality nucleotides, merging paired-end sequences, or identifying and filtering out PCR repeats using known techniques in the art.
  • the sequenced reads may then be assembled Attorney Docket No.: 051385-585001WO and aligned using bioinformatic algorithms known in the art.
  • a sequencing read may be aligned to a reference sequence.
  • a sequencing read includes a computationally derived string corresponding to the detected complementary nucleotide (e.g., a labeled nucleotide).
  • the sequence reads are optionally stored in an appropriate data structure for further evaluation.
  • a first sequencing reaction can generate a first sequencing read.
  • the first sequencing read can provide the sequence of a first region of the polynucleotide fragment.
  • a second sequencing primer can initiate sequencing at a second location on the nucleic acid template. The second location can be distinct from the first location.
  • a 3′ terminal nucleotide of the second primer can hybridize to a location that is more than 5 nucleotides away from a binding site of a 3′ terminal nucleotide of the first primer.
  • the second sequencing reaction can generate a second sequencing read.
  • the second sequencing read can provide the sequence of a second region of the nucleic acid template which is distinct from the first region of the nucleic acid template.
  • the nucleic acid template is optionally subjected to one or more additional rounds of sequencing using additional sequencing primers, thereby generating additional sequencing reads.
  • a sequencing read is about 25 nucleotide bases. In embodiments, a sequencing read is about 35 nucleotide bases.
  • a sequencing read is about 45 nucleotide bases. In embodiments, a sequencing read is about 55 nucleotide bases. In embodiments, a sequencing read is about 65 nucleotide bases. In embodiments, a sequencing read is about 75 nucleotide bases. In embodiments, a sequencing read is about 85 nucleotide bases.
  • multiplexing refers to an analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid target sequences, can be assayed simultaneously by using the methods and devices as described herein, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic.
  • fluorescence characteristic for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime
  • Hybridize shall mean the annealing of a nucleic acid sequence to another nucleic acid sequence (e.g., one single-stranded nucleic acid (such as a primer) to another nucleic acid) based on the well-understood principle of sequence complementarity.
  • another nucleic acid sequence e.g., one single-stranded nucleic acid (such as a primer) to another nucleic acid
  • the other Attorney Docket No.: 051385-585001WO nucleic acid is a single-stranded nucleic acid.
  • one portion of a nucleic acid hybridizes to itself, such as in the formation of a hairpin structure.
  • the propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity.
  • hybridization of a primer, or of a DNA extension product, respectively is extendable by creation of a phosphodiester bond with an available nucleotide or nucleotide analogue capable of forming a phosphodiester bond, therewith.
  • hybridization can be performed at a temperature ranging from 15° C to 95° C.
  • the hybridization is performed at a temperature of about 20° C, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, about 65° C, about 70° C, about 75° C, about 80° C, about 85° C, about 90° C, or about 95° C.
  • the stringency of the hybridization can be further altered by the addition or removal of components of the buffered solution.
  • specific hybridizes refers to preferential hybridization under hybridization conditions where two nucleic acids, or portions thereof, that are substantially complementary, hybridize to each other and not to other nucleic acids that are not substantially complementary to either of the two nucleic acids.
  • specific hybridization includes the hybridization of a primer or capture nucleic acid to a portion of a target nucleic acid (e.g., a template, or adapter portion of a template) that is substantially complementary to the primer or capture nucleic acid.
  • nucleic acids, or portions thereof, that are configured to specifically hybridize are often about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100% complementary to each other over a contiguous portion of nucleic acid sequence.
  • a specific hybridization discriminates over non-specific hybridization interactions (e.g., two nucleic acids that a not configured to specifically hybridize, e.g., two nucleic acids that are 80% or less, 70% or less, 60% or less or 50% or less complementary) by about 2-fold or more, often about 10-fold or more, and sometimes about 100- fold or more, 1000-fold or more, 10,000-fold or more, 100,000-fold or more, or 1,000,000-fold Attorney Docket No.: 051385-585001WO or more.
  • Two nucleic acid strands that are hybridized to each other can form a duplex which includes a double stranded portion of nucleic acid.
  • a nucleic acid can be amplified by a suitable method.
  • amplification refers to subjecting a target nucleic acid in a sample to a process that linearly or exponentially generates amplicon nucleic acids having the same or substantially the same (e.g., substantially identical) nucleotide sequence as the target nucleic acid, or segment thereof, and/or a complement thereof (which may be referred to herein as an “amplification product” or “amplification products”).
  • an amplification reaction comprises a suitable thermal stable polymerase. Thermal stable polymerases are known and are stable for prolonged periods of time, at temperature greater than 80° C when compared to common polymerases found in most mammals.
  • amplification refers to a method that comprises a polymerase chain reaction (PCR).
  • Conditions conducive to amplification i.e., amplification conditions
  • amplification conditions are known and often comprise at least a suitable polymerase, a suitable template, a suitable primer or set of primers, suitable nucleotides (e.g., dNTPs), a suitable buffer, and application of suitable annealing, hybridization and/or extension times and temperatures.
  • an amplified product e.g., an amplicon
  • bPCR bridge-PCR
  • Bridge-PCR involves repeated polymerase chain reaction cycles, cycling between denaturation, annealing, and extension conditions and enables controlled, spatially-localized, amplification, to generate amplification products (e.g., amplicons) immobilized on a solid support in order to form arrays comprised of colonies (or “clusters”) of immobilized nucleic acid molecule.
  • amplification products e.g., amplicons
  • a nucleic acid can be amplified by a thermocycling method or by an isothermal amplification method. In some embodiments, a rolling circle amplification method is used.
  • amplification takes place on a solid support (e.g., within a flow cell) where a nucleic acid, nucleic acid library or portion thereof is immobilized.
  • a nucleic acid library is added to a flow cell and immobilized by hybridization to anchors under suitable conditions. This type of nucleic acid amplification is often referred to as solid phase amplification.
  • all or a portion of the amplified products are synthesized by an extension initiating from an immobilized primer.
  • Solid phase amplification reactions are analogous to standard solution phase amplifications except that at least one of the amplification oligonucleotides (e.g., primers) is immobilized on a solid support.
  • solid phase amplification comprises a nucleic acid amplification reaction comprising only one species of oligonucleotide primer immobilized to a surface or substrate.
  • solid phase amplification comprises a plurality of different immobilized oligonucleotide primer species.
  • solid phase amplification may comprise a nucleic acid amplification reaction comprising one species of oligonucleotide primer immobilized on a solid surface and a second different oligonucleotide primer species in solution. Multiple different species of immobilized or solution-based primers can be used.
  • solid phase nucleic acid amplification reactions include interfacial amplification, bridge amplification, emulsion PCR, WildFire amplification (e.g., US patent publication US20130012399), the like or combinations thereof.
  • biomolecule refers to an agent (e.g., a compound, macromolecule, or small molecule), and the like derived from a biological system (e.g., an organism).
  • the biomolecule may contain multiple individual components that collectively construct the biomolecule, for example, in embodiments, the biomolecule is a polynucleotide wherein the polynucleotide is composed of nucleotide monomers.
  • the biomolecule may be or may include DNA, RNA, organelles, carbohydrates, lipids, proteins, or any combination thereof. These components may be extracellular. In some examples, the biomolecule may be referred to as a clump or aggregate of combinations of components.
  • the biomolecule may include one or more constituents of a cell but may not include other constituents of the cell.
  • a biomolecule is a molecule produced by a biological system (e.g., an organism).
  • a biomolecule may be referred to as an analyte.
  • Analytes can be broadly Attorney Docket No.: 051385-585001WO classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
  • non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments.
  • viral proteins e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.
  • the analytes within a cell can be localized to subcellular locations, including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • organelles e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • analyte(s) can be peptides or proteins, including antibodies and/or enzymes.
  • an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an analyte capture agent e.g., an oligonucleotide-conjugated antibody
  • Examples of a cell organelle include, without limitation, a nucleus, endoplasmic reticulum, a ribosome, a Golgi apparatus, an endoplasmic reticulum, a chloroplast, an endocytic vesicle, an exocytic vesicle, a vacuole, and a lysosome.
  • the biological system e.g., an organism
  • the biological system may contain multiple individual components, such as viruses, cells, cell derivatives, cell nuclei, cell organelles and cell constituents, including combinations of different of these and other components.
  • the biological system may include DNA, RNA, organelles, proteins, or any combination thereof. These components may be extracellular.
  • the biological system may be referred to as a clump or aggregate of combinations of components.
  • the biological system may include one or more constituents of a cell but may not include other constituents of the cell.
  • An example of such constituents include nucleus or an organelle.
  • a cell may be a live or viable cell.
  • the live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix or cultured when including a gel or polymer matrix.
  • a biological system may include a single cell and/or a single nuclei from a cell.
  • a sample e.g., a sample comprising nucleic acid
  • a sample can be obtained from a suitable subject.
  • a sample can be isolated or obtained directly from a subject or part thereof. In some embodiments, a sample is obtained indirectly from an individual or medical professional.
  • a sample can be any specimen that is isolated or obtained from a subject or part thereof.
  • a sample can be any specimen that is isolated or obtained from multiple subjects.
  • specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof.
  • a blood product e.g., serum, plasma, platelets, buffy coats, or the like
  • a fluid or tissue sample from which nucleic acid is extracted may be acellular (e.g., cell-free).
  • tissues include organ tissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder, reproductive organs, intestine, colon, spleen, brain, the like or parts thereof), epithelial tissue, hair, hair follicles, ducts, canals, bone, eye, nose, mouth, throat, ear, nails, the like, parts thereof or combinations thereof.
  • a sample may comprise cells or tissues that are normal, healthy, diseased (e.g., infected), and/or cancerous (e.g., cancer cells).
  • a sample obtained from a subject may comprise cells or cellular material (e.g., nucleic acids) of multiple organisms (e.g., virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasite nucleic acid).
  • cells or cellular material e.g., nucleic acids
  • organisms e.g., virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasite nucleic acid.
  • an extracted human cell obtained is considered in situ when the cell is retained in its local microenvironment so as to avoid extracting the target (e.g., nucleic acid molecules or proteins) away from their native environment.
  • An in situ sample e.g., a cell
  • An in situ cell sample may refer to a cell and its surrounding milieu, or a tissue.
  • a sample can be isolated or obtained directly from a subject or part thereof.
  • the Attorney Docket No.: 051385-585001WO methods described herein are applied to an isolated cell (i.e., a cell not surrounded by least a portion of its native environment).
  • a sample is obtained indirectly from an individual or medical professional.
  • a sample can be any specimen that is isolated or obtained from a subject or part thereof.
  • a sample can be any specimen that is isolated or obtained from multiple subjects.
  • specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof.
  • a blood product e.g., serum, plasma, platelets, buffy coats, or the like
  • Non-limiting examples of tissues include organ tissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder, reproductive organs, intestine, colon, spleen, brain, the like or parts thereof), epithelial tissue, hair, hair follicles, ducts, canals, bone, eye, nose, mouth, throat, ear, nails, the like, parts thereof or combinations thereof.
  • a sample may include cells or tissues that are normal, healthy, diseased (e.g., infected), and/or cancerous (e.g., cancer cells).
  • a sample obtained from a subject may include cells or cellular material (e.g., nucleic acids) of multiple organisms (e.g., virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasite nucleic acid).
  • a sample may include a cell and RNA transcripts.
  • a sample can include nucleic acids obtained from one or more subjects.
  • a sample includes nucleic acid obtained from a single subject.
  • a subject can be any living or non-living organism, including but not limited to a human, non-human animal, plant, bacterium, fungus, virus, or protist.
  • a subject may be any age (e.g., an embryo, a fetus, infant, child, adult).
  • a subject can be of any sex (e.g., male, female, or combination thereof).
  • a subject may be pregnant.
  • a subject is a mammal.
  • a subject is a plant.
  • a subject is a human subject.
  • a subject can be a patient (e.g., a human patient).
  • a subject is suspected of having a genetic variation or a disease or condition associated with a genetic variation.
  • a “tissue section” as used herein refers to a portion of a biological tissue Attorney Docket No.: 051385-585001WO derived from a biological sample, typically from an organism (e.g., a human or animal subject or patient).
  • fresh tissue generally in the context of a fresh tissue means that the tissue has recently been obtained from an organism, generally before any subsequent fixation steps, for example, flash freezing or chemical fixation.
  • a fresh tissue is obtained from an organism about 1 second up to about 20 minutes before any fixation steps are performed.
  • a fresh tissue is obtained from an organism about 1 second up to about 60 seconds before any fixation steps are performed.
  • a fresh tissue is obtained from an organism about 30 seconds up to about 60 seconds before any fixation steps are performed.
  • a fresh tissue is obtained from an organism about 1 minutes up to about 20 minutes before any fixation steps are performed.
  • a fresh tissue is obtained from an organism about 1 minutes up to about 10 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 5 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before any fixation steps are performed. [0107] As used herein, the term “fix,” refers to formation of covalent bonds, such as crosslinks, between biomolecules or within molecules.
  • fixation The process of fixing tissue samples or biological samples (e.g., cells and nuclei) for example, is called “fixation.”
  • fixative The agent that causes fixation is generally referred to as a “fixative” or “fixing agent.”
  • “Fixed biological samples” e.g., fixed cells or nuclei
  • fixed tissues refers to biological samples (e.g., cells or nuclei) or tissues that have been in contact with a fixative under conditions sufficient to allow or result in formation of intra- and inter-molecular crosslinks between biomolecules in the biological sample.
  • Unfixing or decrosslinking refers to breaking or reversing the formation of covalent bonds in biomolecules formed by fixatives.
  • the tissue fixed is fresh tissue.
  • the tissue fixed may be frozen tissue.
  • the tissue fixed may not be dissociated.
  • the tissue fixed may be dissociated or partially dissociated (e.g., chopped, cut).
  • tissue that has been rapidly frozen Attorney Docket No.: 051385-585001WO and, perhaps, cut or chopped into pieces (e.g., small enough to fit into a tube or container used for fixation) may be used.
  • tissue may be dissociated or partially dissociated (e.g., cut, chopped) before or during fixation.
  • tissue that is fixed may not be dissociated.
  • the frozen biological tissue can be fixed using a fixing agent, which is suitably an organic fixing agent.
  • Suitable organic fixing agents include without limitation alcohols, ketones, aldehydes (e.g., glutaraldehyde), cross-linking agents, disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), ethylene glycol bis (succinimidyl succinate) (EGS), bis(sulfosuccinimidyl)suberate (BS3) and combinations thereof.
  • a particularly suitable fixing agent is a formaldehyde-based fixing agent such as formalin, which is a mixture of formaldehyde and water.
  • the formalin may include about 1% to about 15% by weight formaldehyde and about 85% to about 99% by weight water, suitable about 2% to about 8% by weight formaldehyde and about 92% to about 98% by weight water, or about 4% by weight formaldehyde and about 96% by weight water.
  • tissues may be fixed in 4% paraformaldehyde.
  • Other suitable fixing agents will be appreciated by those of ordinary skill in the art (e.g., International PCT App. No. PCT/US2020/066705, which is incorporated herein by reference in its entirety).
  • the term “permeable” refers to a property of a substance that allows certain materials to pass through the substance.
  • Permeable may be used to describe a biological sample, such as a cell or nucleus, in which analytes in the biological sample can leave the biological sample. “Permeabilize” is an action taken to cause, for example, a biological sample (e.g., a cell) to release its analytes. In some examples, permeabilization of a biological sample is accomplished by affecting the integrity (e.g., compromising) of a biological sample membrane (e.g., a cellular or nuclear membrane) such as by application of a protease or other enzyme capable of disturbing a membrane allowing analytes to diffuse out of the biological sample.
  • a biological sample membrane e.g., a cellular or nuclear membrane
  • single biological sample such as a single cell or a single nucleus generally refers to a biological sample that is not present in an aggregated form or clump.
  • Single biological samples such as cells and/or nuclei may be the result of dissociating a tissue sample.
  • disease state is used in accordance with its plain and ordinary meaning and refers to any abnormal biological or aberrant state of a cell or organism. The presence of a disease state may be identified by the same collection of biological constituents used to determine the cell’s biological state. In general, a disease state will be detrimental to a biological system.
  • a disease state may be a consequence of, inter alia, an environmental pathogen, for example a viral infection (e.g., HIV/AIDS, hepatitis B, hepatitis C, influenza, measles, etc.), a bacterial infection, a parasitic infection, a fungal infection, or infection by some other organism.
  • a disease state may also be the consequence of some other environmental agent, such as a chemical toxin or a chemical carcinogen.
  • a disease state further includes genetic disorders wherein one or more copies of a gene is altered or disrupted, thereby affecting its biological function.
  • Exemplary genetic diseases include, but are not limited to polycystic kidney disease, familial multiple endocrine neoplasia type I, neurofibromatoses, Tay-Sachs disease, Huntington's disease, sickle cell anemia, thalassemia, and Down's syndrome, as well as others (see, e.g., The Metabolic and Molecular Bases of Inherited Diseases, 7th ed., McGraw-Hill Inc., New York).
  • Other exemplary diseases include, but are not limited to, cancer, hypertension, Alzheimer's disease, neurodegenerative diseases, and neuropsychiatric disorders such as bipolar affective disorders or paranoid schizophrenic disorders.
  • Disease states are monitored to determine the level or severity (e.g., the stage or progression) of one or more disease states of a subject and, more specifically, detect changes in the biological state of a subject which are correlated to one or more disease states (see, e.g., U.S. Pat. No.6,218,122, which is incorporated by reference herein in its entirety).
  • methods provided herein are also applicable to monitoring the disease state or states of a subject undergoing one or more therapies.
  • the present disclosure also provides, in some embodiments, methods for determining or monitoring efficacy of a therapy or therapies (i.e., determining a level of therapeutic effect) upon a subject.
  • methods of the present disclosure can be used to assess therapeutic efficacy in a clinical trial, e.g., as an early surrogate marker for success or failure in such a clinical trial.
  • a clinical trial e.g., as an early surrogate marker for success or failure in such a clinical trial.
  • perturbations in the function of proteins within a cell have numerous effects on other proteins and the transcription of other genes that are connected by primary, secondary, and sometimes tertiary pathways.
  • This extensive interconnection between the function of various proteins means that the alteration of Attorney Docket No.: 051385-585001WO any one protein is likely to result in compensatory changes in a wide number of other proteins.
  • the partial disruption of even a single protein within a cell results in characteristic compensatory changes in the transcription of enough other genes that these changes in transcripts can be used to define a “signature” of particular transcript alterations which are related to the disruption of function, e.g., a particular disease state or therapy, even at a stage where changes in protein activity are undetectable.
  • the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may optionally be conjugated to a moiety that does not consist of amino acids.
  • a protein may refer to a protein expressed in a cell.
  • a polypeptide, or a cell is “recombinant” when it is artificial or engineered, or derived from or contains an artificial or engineered protein or nucleic acid (e.g., non-natural or not wild type).
  • a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide.
  • a protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide.
  • a polynucleotide sequence that does not appear in nature for example a variant of a naturally occurring gene, is recombinant.
  • a “single cell” refers to one cell.
  • Single cells useful in the methods described herein can be obtained from a tissue of interest, or from a biopsy, blood sample, or cell culture. Additionally, cells from specific organs, tissues, tumors, neoplasms, or the like can be obtained and used in the methods described herein. In general, cells from any population can be used in the methods, such as a population of prokaryotic or eukaryotic organisms, including bacteria or yeast.
  • tissue is used in accordance with its plain and ordinary meaning and refers to an organization of cells in a structure, where the structure generally Attorney Docket No.: 051385-585001WO functions as a unit in an organism (e.g., mammals) and may carry out specific functions.
  • cells in a tissue are configured in a mass and may not be free from one another.
  • This disclosure describes methods of obtaining single biological samples (e.g., cells or nuclei) from tissues that can be used in various single biological samples (e.g., single-cell/nucleus) workflows.
  • blood cells e.g., lymphocytes
  • tissue like lymphocytes, generally are free from one another in the blood.
  • the methods disclosed herein can be used to process those cells to obtain cells and/or nuclei, although dissociation steps may not be necessary when using those types of tissues.
  • any type of tissue can be used in the methods described herein. Examples of tissues that may be used in the disclosed methods include, but are not limited to connective, epithelial, muscle and nervous tissue. In some examples, the tissues are from mammals. Tissues that contain any type of cells may be used.
  • the tissue may be normal or tumor tissue (e.g., malignant). This example is not meant to be limiting.
  • tumor tissue e.g., malignant
  • the methods may be applied to any tissue.
  • the tissues used in the disclosed methods may be in various states. In some examples, the tissues used in the disclosed methods may be fresh, frozen, or fixed.
  • cellular component is used in accordance with its ordinary meaning in the art and refers to any organelle, nucleic acid, protein, or analyte that is found in a prokaryotic, eukaryotic, archaeal, or other organismic cell type.
  • cellular components e.g., a component of a cell
  • examples of cellular components include RNA transcripts, proteins, membranes, lipids, and other analytes.
  • a cellular component is a biomolecule.
  • a sample comprises nucleic acid, or fragments thereof.
  • a sample can comprise nucleic acids obtained from one or more subjects.
  • a sample comprises nucleic acid obtained from a single subject.
  • a sample comprises a mixture of nucleic acids.
  • a mixture of nucleic acids can comprise two or more nucleic acid species having different nucleotide sequences, different fragment lengths, different origins (e.g., genomic origins, cell or tissue origins, subject origins, the like or combinations thereof), or combinations thereof.
  • a sample may comprise synthetic nucleic acid.
  • a subject can be any living or non-living organism, including but not limited to a human, non-human animal, plant, bacterium, fungus, virus or protist.
  • a subject may be any age (e.g., an embryo, a fetus, infant, child, adult).
  • a subject can be of any sex (e.g., male, female, or combination thereof).
  • a subject may be pregnant.
  • a subject is a mammal.
  • a subject is a human subject.
  • a subject can be a patient (e.g., a human patient).
  • a subject is suspected of having a genetic variation or a disease or condition associated with a genetic variation.
  • the term “protein-specific binding agent” refers to an agent to a protein or polypeptide molecule, or portion thereof, capable of selectively binding or interacting with a protein.
  • a protein-specific binding agent specifically binds a particular protein (e.g., a protein antigen or epitope thereof).
  • a protein-specific binding agent is an immunoglobulin (IgA, IgD, IgE, IgG, or IgM).
  • Intact immunoglobulins also known as antibodies, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each, and two heavy (H) chains of approximately 50 kDa each.
  • the protein binding moiety is an antigen-specific antibody.
  • Non-limiting examples of protein-specific binding agent encompassed within the term “antigen-specific antibody” used herein include: (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated CDR.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they may be recombinantly joined by a synthetic linker, creating a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv)).
  • the most commonly used linker is a 15-residue (Gly4Ser)3 peptide, but other linkers are also known in the art.
  • Single chain antibodies are also intended to be encompassed within the terms “protein-specific binding agent,” of an antibody.
  • the antibody can also be a polyclonal antibody, monoclonal antibody, chimeric antibody, antigen-binding fragment, Fc fragment, single chain antibodies, or any derivatives thereof.
  • the protein-specific binding agent is the antigen-binding site (e.g., fragment antigen-binding (Fab) variable region) of an antibody.
  • antigen-binding site e.g., fragment antigen-binding (Fab) variable region
  • antibody e.g., fragment antigen-binding (Fab) variable region
  • antigen-binding site refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • An “antibody” (Ab) is a protein that binds specifically to a particular substance, known as an “antigen” (Ag).
  • an “antibody” or “antigen-binding fragment” is an immunoglobulin that binds a specific “epitope.”
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies. In nature, antibodies are generally produced by lymphocytes in response to immune challenge, such as by infection or immunization.
  • An “antigen” (Ag) is any substance that reacts specifically with antibodies or T lymphocytes (T cells).
  • An antibody may include the entire antibody as well as any antibody fragments capable of binding the antigen or antigenic fragment of interest. Examples include complete antibody molecules, antibody fragments, such as Fab, F(ab')2, CDRs, VL, VH, and any other portion of an antibody which is capable of specifically binding to an antigen.
  • Antibodies used herein are immunospecific for, and therefore specifically and selectively bind to, for example, proteins either detected (e.g., biological targets of interest) or used for detection (e.g., probes containing oligonucleotide barcodes) in the methods and devices as described herein.
  • the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., packaging, buffers, written instructions for performing a method, etc.) from one location to another.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
  • the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides.
  • a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components).
  • kit includes both fragmented and combined kits.
  • bioconjugate group refers to a chemical moiety which participates in a reaction to form a bioconjugate linker (e.g., covalent linker).
  • bioconjugate groups include —NH2, –COOH, –COOCH 3 , –N-hydroxysuccinimide,
  • the bioconjugate reactive group may be .
  • the bioconjugate reactive moiety , , , or -NH2.
  • Bioconjugate reactive group 1 Bioconjugate reactive group 2 (e.g., electrophilic bioconjugate (e.g., nucleophilic bioconjugate Resulting Bioconjugate reactive moiety) reactive moiety) reactive linker activated esters amines/anilines carboxamides acrylamides thiols thioethers acyl azides amines/anilines carboxamides acyl halides amines/anilines carboxamides acyl halides alcohols/phenols esters acyl nitriles alcohols/phenols esters acyl nitriles amines/anilines carboxamides aldehydes amines/anilines imines aldehydes or ketones hydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkyl halides amines/anilines alkyl amines
  • the association can be direct or indirect.
  • a conjugate between a first bioconjugate reactive group e.g., –NH 2 , –COOH, –N-hydroxysuccinimide, or –maleimide
  • a second Attorney Docket No.: 051385-585001WO bioconjugate reactive group e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate
  • a conjugate between a first bioconjugate reactive group e.g., –NH 2 , –COOH, –N-hydroxysuccinimide, or –maleimide
  • a second Attorney Docket No.: 051385-585001WO bioconjugate reactive group e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate
  • covalent bond or linker e.g., a first linker of second link
  • bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e., the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
  • bioconjugate chemistry i.e., the association of two bioconjugate reactive groups
  • nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
  • electrophilic substitutions e.g., enamine reactions
  • additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
  • the first bioconjugate reactive group e.g., maleimide moiety
  • the second bioconjugate reactive group e.g., a sulfhydryl
  • the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl).
  • the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl).
  • the first bioconjugate reactive group e.g., –N-hydroxysuccinimide moiety
  • is covalently attached to the second bioconjugate reactive group (e.g., an amine).
  • the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl).
  • the first bioconjugate reactive group (e.g., – sulfo–N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., an amine).
  • bioconjugate reactive groups used for bioconjugate chemistries herein include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.; (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an Attorney Docket No.: 051385-585001WO amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophil
  • covalent linker is used in accordance with its ordinary meaning and refers to a divalent moiety which connects at least two moieties to form a molecule.
  • non- covalent linker is used in accordance with its ordinary meaning and refers to a divalent moiety which includes at least two molecules that are not covalently linked to each other but are capable of interacting with each other via a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond) or van der Waals interactions (e.g., dipole-dipole, dipole- induced dipole, London dispersion).
  • the non-covalent linker is the result of two molecules that are not covalently linked to each other that interact with each other via a non- covalent bond.
  • the term “adapter” as used herein refers to any oligonucleotide that can be ligated to a nucleic acid molecule, thereby generating nucleic acid products that can be sequenced on a sequencing platform (e.g., an Illumina or Singular Genomics G4TM sequencing platform).
  • a sequencing platform e.g., an Illumina or Singular Genomics G4TM sequencing platform.
  • adapters include two reverse complementary oligonucleotides forming a double- Attorney Docket No.: 051385-585001WO stranded structure.
  • an adapter includes two oligonucleotides that are complementary at one portion and mismatched at another portion, forming a Y-shaped or fork- shaped adapter that is double stranded at the complementary portion and has two overhangs at the mismatched portion. Since Y-shaped adapters have a complementary, double-stranded region, they can be considered a special form of double-stranded adapters. When this disclosure contrasts Y-shaped adapters and double stranded adapters, the term “double-stranded adapter” or “blunt-ended” is used to refer to an adapter having two strands that are fully complementary, substantially (e.g., more than 90% or 95%) complementary, or partially complementary.
  • adapters include sequences that bind to sequencing primers.
  • adapters include sequences that bind to immobilized oligonucleotides (e.g., P7 and P5 sequences) or reverse complements thereof.
  • the adapter is substantially non- complementary to the 3' end or the 5' end of any target polynucleotide present in the sample.
  • the adapter can include a sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a universal primer.
  • the adapter can include an index sequence (also referred to as barcode or tag) to assist with downstream error correction, identification or sequencing.
  • hairpin adapter refers to a polynucleotide including a double-stranded stem portion and a single-stranded hairpin loop portion.
  • an adapter is hairpin adapter.
  • a hairpin adapter comprises a single nucleic acid strand comprising a stem-loop structure.
  • a hairpin adapter comprises a nucleic acid having a 5’-end, a 5’-portion, a loop, a 3’-portion and a 3’-end (e.g., arranged in a 5’ to 3’ orientation).
  • the 5’ portion of a hairpin adapter is annealed and/or hybridized to the 3’ portion of the hairpin adapter, thereby forming a stem portion of the hairpin adapter.
  • the 5’ portion of a hairpin adapter is substantially complementary to the 3’ portion of the hairpin adapter.
  • a hairpin adapter comprises a stem portion (i.e., stem) and a loop, wherein the stem portion is substantially double stranded thereby forming a duplex.
  • the loop of a hairpin adapter comprises a nucleic acid strand that is not complementary (e.g., not substantially complementary) to itself or to any other portion of the hairpin adapter.
  • a method herein comprises ligating a first adapter to a first end of a double stranded nucleic acid, and ligating a second adapter to a second end of a double stranded nucleic acid.
  • the first adapter and the second adapter are different.
  • the first adapter and the second adapter may comprise different nucleic acid sequences or different structures.
  • the first adapter is a Y-adapter and the second adapter is a hairpin adapter.
  • the first adapter is a hairpin adapter and a second adapter is a hairpin adapter.
  • the first adapter and the second adapter may comprise different primer binding sites, different structures, and/or different capture sequences (e.g., a sequence complementary to a capture nucleic acid).
  • some, all or substantially all of the nucleic acid sequence of a first adapter and a second adapter are the same.
  • some, all or substantially all of the nucleic acid sequence of a first adapter and a second adapter are substantially different.
  • a nucleic acid comprises a capture nucleic acid.
  • a capture nucleic acid refers to a nucleic acid that is attached to a substrate.
  • a capture nucleic acid comprises a primer.
  • a capture nucleic acid is a nucleic acid configured to specifically hybridize to a portion of one or more nucleic acid templates (e.g., a template of a library). In some embodiments a capture nucleic acid configured to specifically hybridize to a portion of one or more nucleic acid templates is substantially complementary to a suitable portion of a nucleic acid template, or an amplicon thereof. In some embodiments a capture nucleic acid is configured to specifically hybridize to a portion of an adapter, or a complement thereof. In some embodiments a capture nucleic acid, or portion thereof, is substantially complementary to a portion of an adapter, or a complement thereof.
  • a capture nucleic acid is a probe oligonucleotide.
  • a probe oligonucleotide is complementary to a target polynucleotide or portion thereof, and further comprises a label (such as a binding moiety) or is attached to a surface, such that hybridization to the probe oligonucleotide permits the selective isolation of probe-bound polynucleotides from unbound polynucleotides in a population.
  • a probe oligonucleotide may or may not also be used as a primer.
  • nucleic acid sequencing device and the like means an integrated system of one or more chambers, ports, and channels that are interconnected and in fluid communication and designed for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions, such as sample introduction, fluid and/or reagent driving means, temperature control, detection systems, data collection and/or integration systems, for the purpose of determining the nucleic acid sequence of a template polynucleotide.
  • Nucleic acid sequencing devices may further include valves, pumps, and specialized functional coatings on interior walls.
  • Nucleic acid sequencing devices may include a receiving unit, or platen, that orients the flow cell such that a maximal surface area of the flow cell is available to be exposed to an optical lens.
  • nucleic acid sequencing devices include those provided by Singular GenomicsTM (e.g., the G4TM system), IlluminaTM (e.g., HiSeqTM, MiSeqTM, NextSeqTM, or NovaSeqTM systems), Life TechnologiesTM (e.g., ABI PRISMTM, or SOLiDTM systems), Pacific Biosciences (e.g., systems using SMRTTM Technology such as the SequelTM or RS IITM systems), or Qiagen (e.g., GenereaderTM system). Nucleic acid sequencing devices may further include fluidic reservoirs (e.g., bottles), valves, pressure sources, pumps, sensors, control systems, valves, pumps, and specialized functional coatings on interior walls.
  • fluidic reservoirs e.g., bottles
  • valves e.g., pressure sources, pumps, sensors, control systems, valves, pumps, and specialized functional coatings on interior walls.
  • the device includes a plurality of a sequencing reagent reservoirs and a plurality of clustering reagent reservoirs.
  • the clustering reagent reservoir includes amplification reagents (e.g., an aqueous buffer containing enzymes, salts, and nucleotides, denaturants, crowding agents, etc.)
  • the reservoirs include sequencing reagents (such as an aqueous buffer containing enzymes, salts, and nucleotides); a wash solution (an aqueous buffer); a cleave solution (an aqueous buffer containing a cleaving agent, such as a reducing agent); or a cleaning solution (a dilute bleach solution, dilute NaOH solution, dilute HCl solution, dilute antibacterial solution, or water).
  • the fluid of each of the reservoirs can vary.
  • the fluid can be, for example, an aqueous solution which may contain buffers (e.g., saline- sodium citrate (SSC), ascorbic acid, tris(hydroxymethyl)aminomethane or “Tris”), aqueous salts Attorney Docket No.: 051385-585001WO (e.g., KCl or (NH4)2SO4)), nucleotides, polymerases, cleaving agent (e.g., tri-n-butyl-phosphine, triphenyl phosphine and its sulfonated versions (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), and tri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agent scavenger compounds (e.g., 2′- Dithiobisethanamine or 11-Azido-3,6,9
  • Non- limited examples of reservoirs include cartridges, pouches, vials, containers, and eppendorf tubes.
  • the device is configured to perform fluorescent imaging.
  • the device includes one or more light sources (e.g., one or more lasers).
  • the illuminator or light source is a radiation source (i.e., an origin or generator of propagated electromagnetic energy) providing incident light to the sample.
  • a radiation source can include an illumination source producing electromagnetic radiation in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 390 to 770 nm), or infrared (IR) range (about 0.77 to 25 microns), or other range of the electromagnetic spectrum.
  • the illuminator or light source is a lamp such as an arc lamp or quartz halogen lamp. In embodiments, the illuminator or light source is a coherent light source. In embodiments, the light source is a laser, LED (light emitting diode), a mercury or tungsten lamp, or a super-continuous diode. In embodiments, the light source provides excitation beams having a wavelength between 200 nm to 1500 nm.
  • the laser provides excitation beams having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm.
  • the illuminator or light source is a light-emitting diode (LED).
  • the LED can be, for example, an Organic Light Emitting Diode (OLED), a Thin Film Electroluminescent Device (TFELD), or a Quantum dot based inorganic organic LED.
  • the nucleic acid sequencing device includes an imaging system (e.g., an imaging system as described herein).
  • the imaging system capable of exciting one or more of the identifiable labels (e.g., a fluorescent label) linked to a nucleotide and thereafter obtain image data for the identifiable labels.
  • the image data (e.g., detection data) may be analyzed by another component within the device.
  • the imaging system may include a system described herein and may include a fluorescence spectrophotometer including an objective lens and/or a solid-state imaging device.
  • the solid-state imaging device may include a charge coupled device (CCD) and/or a complementary metal oxide semiconductor (CMOS).
  • CCD charge coupled device
  • CMOS complementary metal oxide semiconductor
  • the system may also include circuitry and Attorney Docket No.: 051385-585001WO processors, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate array (FPGAs), logic circuits, and any other circuit or processor capable of executing functions described herein.
  • the set of instructions may be in the form of a software program.
  • the terms “software” and “firmware” are interchangeable, and include any computer program stored in memory for execution by a computer, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory.
  • the device includes a thermal control assembly useful to control the temperature of the reagents.
  • Synthetic agents refer to non-naturally occurring agents, such as enzymes or nucleotides derived or constructed using man-made techniques.
  • Synthetic DNA polymerases refer to non-naturally occurring DNA polymerases such as those constructed by synthetic methods, mutated parent DNA polymerases such as truncated DNA polymerases and fusion DNA polymerases (e.g. U.S. Pat. No.7,541,170).
  • Variants of the parent DNA polymerase have been engineered by mutating residues using site-directed or random mutagenesis methods known in the art.
  • the mutations are in any of Motifs I-VI.
  • the variant is expressed in an expression system such as E. coli by methods known in the art.
  • GC bias describes the relationship between GC content and read coverage across a genome. For example, a genomic region of a higher GC content tends to have more (or less) sequencing reads covering that region. As described herein, GC bias can be introduced during amplification of library, cluster amplification, and/or the sequencing reactions.
  • biological activity may include the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture.
  • Biological activity thus, may encompass therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed in vitro systems designed to test or use such activities.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a polypeptide naturally present in a living animal is not isolated, but the same Attorney Docket No.: 051385-585001WO nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated refers to a nucleic acid, polynucleotide, polypeptide, protein, or other component that is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, etc.).
  • a “plurality” refers to two or more.
  • cellular component is used in accordance with its ordinary meaning in the art and refers to any organelle, nucleic acid, protein, or analyte that is found in a prokaryotic, eukaryotic, archaeal, or other organismic cell type.
  • cellular components e.g., a component of a cell
  • examples of cellular components include RNA transcripts, proteins, membranes, lipids, and other analytes.
  • a “gene” refers to a polynucleotide that is capable of conferring biological function after being transcribed and/or translated. Functionally, a genome is subdivided into genes. Each gene is a nucleic acid sequence that encodes an RNA or polypeptide.
  • RNA is transcribed from DNA into RNA, which can either be non-coding (ncRNA) with a direct function, or an intermediate messenger (mRNA) that is then translated into protein.
  • a gene includes multiple sequence elements, such as for example, a coding element (i.e., a sequence that encodes a functional protein), non-coding element, and regulatory element. Each element may be as short as a few bp to 5kb.
  • the gene is the protein coding sequence of RNA.
  • Non- limiting examples of genes include developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF
  • a gene includes at least one mutation associated with a disease or condition mediated by a mutant form of the gene.
  • the term “genetic locus,” or “locus” as used herein refers to a genome or target polynucleotide, specifically a contiguous subregion or segment of the genome or target polynucleotide.
  • genetic locus, or locus may refer to the position of a nucleotide, a gene, or a portion of a gene in a genome, including mitochondrial DNA, or it may refer to any contiguous portion of genomic sequence whether or not it is within, or associated with, a gene.
  • a genetic locus refers to any portion of genomic sequence, including mitochondrial DNA, from a single nucleotide to a segment of few hundred nucleotides, e.g.100-300, in length. Usually, a particular genetic locus may be identified by its nucleotide sequence, or the nucleotide sequence, or sequences, of one or both adjacent or flanking regions. In another aspect, a genetic locus refers to the expressed nucleic acid product of a gene, such as an RNA molecule or a cDNA copy thereof.
  • multiplexing refers to an analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid target sequences, can be assayed simultaneously by using the methods and devices as described herein, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic.
  • fluorescence characteristic for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime
  • multiplex is used to refer to an assay in which multiple (i.e.
  • At least two) different biomolecules are assayed at the same time, and more particularly in the same aliquot of the sample, or in the same reaction mixture.
  • more than two Attorney Docket No.: 051385-585001WO different biomolecules are assayed at the same time.
  • at least 2, 4, 6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more biomolecules are detected according to the present method.
  • adjacent refers to two nucleotide sequences in a nucleic acid, can refer to nucleotide sequences separated by 0 to about 20 nucleotides, more specifically, in a range of about 1 to about 10 nucleotides, or to sequences that directly abut one another. As those of skill in the art appreciate, two nucleotide sequences that that are to ligated together will generally directly abut one another.
  • incubate and “incubation refer collectively to altering the temperature of an object in a controlled manner such that conditions are sufficient for conducting the desired reaction.
  • the terms encompass heating a receptacle (e.g., a microplate) to a desired temperature and maintaining such temperature for a fixed time interval. Also included in the terms is the act of subjecting a receptacle to one or more heating and cooling cycles (i.e., “temperature cycling” or “thermal cycling”). While temperature cycling typically occurs at relatively high rates of change in temperature, the term is not limited thereto, and may encompass any rate of change in temperature.
  • image is used according to its ordinary meaning and refers to a representation of all or part of an object. The representation may be an optically detected reproduction. For example, an image can be obtained from fluorescent, luminescent, scatter, or absorption signals.
  • the part of the object that is present in an image can be the surface or other xy plane of the object.
  • an image is a 2 dimensional representation of a 3 dimensional object.
  • An image may include signals at differing intensities (i.e., signal levels).
  • An image can be provided in a computer readable format or medium.
  • An image is derived from the collection of focus points of light rays coming from an object (e.g., the sample), which may be detected by any image sensor.
  • the term “signal” is intended to include, for example, fluorescent, luminescent, scatter, or absorption impulse or electromagnetic wave transmitted or received.
  • Signals can be detected in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 391 to 770 nm), infrared (IR) range (about 0.771 to 25 microns), or other range of the electromagnetic spectrum.
  • UV ultraviolet
  • VIS visible
  • IR infrared
  • signal level refers to an amount or quantity of detected Attorney Docket No.: 051385-585001WO energy or coded information.
  • a signal may be quantified by its intensity, wavelength, energy, frequency, power, luminance, or a combination thereof.
  • Other signals can be quantified according to characteristics such as voltage, current, electric field strength, magnetic field strength, frequency, power, temperature, etc. Absence of signal is understood to be a signal level of zero or a signal level that is not meaningfully distinguished from noise.
  • xy coordinates refers to information that specifies location, size, shape, and/or orientation in an xy plane.
  • the information can be, for example, numerical coordinates in a Cartesian system.
  • the coordinates can be provided relative to one or both of the x and y axes or can be provided relative to another location in the xy plane (e.g., a fiducial).
  • xy plane refers to a 2 dimensional area defined by straight line axes x and y. When used in reference to a detecting apparatus and an object observed by the detector, the xy plane may be specified as being orthogonal to the direction of observation between the detector and object being detected.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
  • the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)).
  • “capable of hybridizing” is used in accordance with its ordinary meaning in the art and refers to two oligonucleotides that, under suitable conditions, can form a duplex (e.g., Watson-Crick pairing) which includes a double-stranded portion of nucleic acid.
  • a duplex e.g., Watson-Crick pairing
  • Such conditions depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions.
  • the stringency of hybridization can be influenced by various parameters, including degree of identity and/or complementarity between the polynucleotides (or any target sequences within the polynucleotides) to be hybridized; melting point of the polynucleotides and/or target sequences to be hybridized, referred to as “Tm”; parameters such as salts, buffers, pH, temperature, GC % content of the Attorney Docket No.: 051385-585001WO polynucleotide and primers, and/or time.
  • Tm melting point of the polynucleotides and/or target sequences to be hybridized
  • parameters such as salts, buffers, pH, temperature, GC % content of the Attorney Docket No.: 051385-585001WO polynucleotide and primers, and/or time.
  • hybridization is favored in lower temperatures and/or increased salt concentrations, as well as reduced concentrations of organic solvents.
  • hybridization or wash solutions can include about 10-75% formamide and/or about 0.01- 0.7% sodium dodecyl sulfate (SDS).
  • a hybridization solution can be a stringent hybridization solution which can include any combination of 50% formamide, 5 ⁇ SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 ⁇ Denhardt’s solution, 0.1% SDS, and/or 10% dextran sulfate.
  • the hybridization or washing solution can include BSA (bovine serum albumin).
  • hybridization or washing can be conducted at a temperature range of about 20-25 °C, or about 25-30 °C, or about 30-35 °C, or about 35-40 °C, or about 40-45 °C, or about 45-50 °C, or about 50-55 °C, or higher.
  • hybridization or washing can be conducted for a time range of about 1-10 minutes, or about 10-20 minutes, or about 20-30 minutes, or about 30-40 minutes, or about 40-50 minutes, or about 50-60 minutes, or longer.
  • hybridization or wash conditions can be conducted at a pH range of about 5-10, or about pH 6-9, or about pH 6.5-8, or about pH 6.5-7.
  • upstream refers to a region in the nucleic acid sequence that is towards the 5’ end of a particular reference point
  • downstream refers to a region in the nucleic acid sequence that is toward the 3’ end of the reference point.
  • feature refers a site (i.e., a physical location) on a solid support for one or more molecule(s).
  • a feature can contain only a single molecule or it can contain a population of several molecules of the same species (i.e., a cluster).
  • Features of an array are typically discrete.
  • the discrete features can be contiguous, or they can have spaces between each other.
  • An “optically resolvable feature” refers to a feature capable of being distinguished from other features.
  • Optics and sensor resolution has a finite limit as to a resolvable area. The Rayleigh criterion for the diffraction limit to resolution states that two images are just resolvable when the center of the diffraction pattern of one object is directly over the first minimum of the diffraction pattern of the other object.
  • NA numerical aperture
  • a substrate e.g., a solid support
  • a substrate including a first polynucleotide attached to the substrate; a second polynucleotide attached to the substrate, wherein the second polynucleotide includes a complementary sequence to the first polynucleotide; and a third polynucleotide (alternatively referred to herein as an invasion primer or extended invasion primer) hybridized to the second polynucleotide.
  • the invasion primer includes a binding sequence (e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof) and a tail sequence (i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof).
  • the binding sequence is also referred to herein as the first region of the third polynucleotide
  • the tail sequence is also referred to herein as the second region of the third polynucleotide.
  • the third polynucleotide includes the Attorney Docket No.: 051385-585001WO first region.
  • the third polynucleotide includes the first region and the second region. In embodiments, the third polynucleotide includes, from 5’ to 3’, the tail sequence and the binding sequence (e.g., wherein the binding sequence is downstream from the tail sequence). In embodiments, the third polynucleotide includes three or more regions, wherein the additional regions may or may not be complementary to the first or the second polynucleotide, or a complement thereof.
  • the third polynucleotide may include a third region (e.g., a third region at the 5’ end of the third polynucleotide) that is complementary to a capture oligonucleotide (e.g., an oligonucleotide including a functional group that allows for affinity capture of the third polynucleotide).
  • a capture oligonucleotide e.g., an oligonucleotide including a functional group that allows for affinity capture of the third polynucleotide.
  • a composition including a first invasion strand hybridized to a second strand (e.g., a second polynucleotide) of a double-stranded polynucleotide, wherein the double-stranded polynucleotide includes a first strand (e.g., a first polynucleotide) and the second strand, wherein the first strand and the second strand are both attached to a solid support; and wherein a 5’ end of the first strand is hybridized to a second invasion strand.
  • the first invasion strand includes an invasion primer (e.g., an extended third polynucleotide).
  • the invasion primer includes a binding sequence. In embodiments, the invasion primer includes a binding sequence and a tail sequence (e.g., a 5’ tail sequence). In embodiments, the second invasion strand includes a blocking primer (e.g., an extended third polynucleotide).
  • a cell including a first polynucleotide attached to the cell (e.g., attached to a cellular biomolecule in the cell, such as a cellular compartment, or attached to an exogenous matrix or polymer in the cell); a second polynucleotide attached to the cell, wherein the second polynucleotide includes a complementary sequence to the first polynucleotide; and a third polynucleotide (alternatively referred to herein as an invasion primer or extended invasion primer) hybridized to the second polynucleotide.
  • a first polynucleotide attached to the cell (e.g., attached to a cellular biomolecule in the cell, such as a cellular compartment, or attached to an exogenous matrix or polymer in the cell); a second polynucleotide attached to the cell, wherein the second polynucleotide includes a complementary sequence to the first polynucleotide; and a third polynucleotide
  • the invasion primer includes a binding sequence (e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof) and a 5’ tail sequence (i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof).
  • the binding sequence is also referred to herein as the first region of the third polynucleotide
  • the 5’ tail sequence is also referred to herein as the second region of the third polynucleotide.
  • the third polynucleotide includes the first region.
  • the third polynucleotide includes the first region and the second region. In embodiments, the third polynucleotide includes three or more regions, wherein the additional regions may or may not be complementary to the first or the second polynucleotide, or a complement thereof.
  • the third polynucleotide may include a third region (e.g., a third region at the 5’ end of the third polynucleotide) that is complementary to a capture oligonucleotide (e.g., an oligonucleotide including a functional group that allows for affinity capture of the third polynucleotide).
  • the cell is attached to a substrate.
  • the cell is attached to the substrate via a bioconjugate reactive moiety.
  • the composition is within a cell or tissue sample.
  • the cell or tissue sample is cleared (e.g., digested) of proteins, lipids, or proteins and lipids.
  • the cell or tissue sample is processed according to a known technique in the art, for example CLARITY (Chung K., et al. Nature 497, 332–337 (2013)), PACT-PARS (Yang Bet al. Cell 158, 945–958 (2014).), CUBIC (Susaki E. A. et al. Cell 157, 726–739 (2014)., 18), ScaleS (Hama H., et al. Nat.
  • the substrate further includes a plurality of immobilized oligonucleotides (e.g., immobilized primers, such as immobilized forward and immobilized reverse primers) attached to the substrate via a linker.
  • immobilized oligonucleotides e.g., immobilized primers, such as immobilized forward and immobilized reverse primers
  • the first and second polynucleotides are covalently attached to the substrate.
  • the 5 ⁇ end of the first and second polynucleotides contains a functional group that serves to tether the first and second polynucleotides to the substrate (e.g., a bioconjugate linker).
  • covalent attachment include amine-modified polynucleotides reacting with epoxy or isothiocyanate groups on the substrate, succinylated polynucleotides reacting with aminophenyl or aminopropyl functional groups on the substrate, dibenzocycloctyne-modified polynucleotides reacting with azide functional groups on the substrate (or vice versa), trans-cyclooctyne-modified Attorney Docket No.: 051385-585001WO polynucleotides reacting with tetrazine or methyl tetrazine groups on the substrate (or vice versa), disulfide modified polynucleotides reacting with mercapto-functional groups on the substrate, amine-
  • the primer is attached to the substrate polymer through electrostatic binding.
  • the negatively charged phosphate backbone of the primer may be bound electrostatically to positively charged monomers in the substrate.
  • the third polynucleotide is not covalently attached to the substrate.
  • the substrate includes a plurality of first polynucleotides attached to a solid support; a plurality of second polynucleotides attached to a solid support; and a plurality of third polynucleotides hybridized to each of the second polynucleotides. It is understood that when referring to first, second, and third polynucleotides it is in reference to a class of polynucleotide types.
  • the polynucleotides of the first polynucleotides are substantially similar to each other insomuch as they contain substantially identical sequences.
  • the third polynucleotide which may also be referred to as the invasion primer and is interchangeable with the third polynucleotide, includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), or combinations thereof.
  • LNAs locked nucleic acids
  • bisLNAs Bis-locked nucleic acids
  • TAAs twisted intercalating nucleic acids
  • BNAs bridged nucleic acids
  • MGB minor groove binder
  • PNAs morph
  • the third polynucleotide includes Bis-locked nucleic acids (bisLNAs). In embodiments, the third polynucleotide includes twisted intercalating nucleic acids (TINAs). In embodiments, the third polynucleotide includes bridged nucleic acids (BNAs). In embodiments, the third polynucleotide includes 2’-O-methyl RNA:DNA chimeric nucleic acids. In embodiments, the third polynucleotide includes minor groove binder (MGB) nucleic acids. In embodiments, the third polynucleotide includes morpholino nucleic acids.
  • bisLNAs Bis-locked nucleic acids
  • TAAs twisted intercalating nucleic acids
  • BNAs bridged nucleic acids
  • MGB minor groove binder
  • the third polynucleotide includes morpholino nucleic acids.
  • Morpholino nucleic acids are synthetic nucleotides that have standard Attorney Docket No.: 051385-585001WO nucleic acid bases (e.g., adenine, guanine, cytosine, and thymine) wherein those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates. Morpholino nucleic acids may be referred to as phosphorodiamidate morpholino oligomers (PMOs).
  • the third polynucleotide includes C5-modified pyrimidine nucleic acids.
  • the third polynucleotide includes peptide nucleic acids (PNAs).
  • the third polynucleotide includes from 5′ to 3′ a plurality of synthetic nucleotides (e.g., LNAs) followed by a plurality (e.g., 2 to 5) canonical or native nucleotides (e.g., dNTPs).
  • the third polynucleotide comprises one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU).
  • the one or more dU nucleobases are at or near the 3’ end of the third polynucleotide (e.g., within 5 nucleotides of the 3’ end).
  • the third polynucleotide includes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followed by a plurality of synthetic nucleotides (e.g., LNAs), and subsequently followed by a plurality (e.g., 2 to 5) of canonical nucleobases.
  • the third polynucleotide includes a plurality of canonical nucleobases, wherein the canonical nucleobases terminate (i.e., at the 3′ end) with a deoxyuracil nucleobase (dU).
  • the third polynucleotide includes a plurality of LNAs interspersed throughout the polynucleotide.
  • the third polynucleotide includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the polynucleotide.
  • the entire composition of the third polynucleotide includes less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of LNAs.
  • the entire composition of the third polynucleotide includes up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, or up to about 5% of LNAs. In embodiments, the entire composition of the third polynucleotide includes more than 60%, more than 50%, more than 40%, more than 30%, more than 20%, more than 10%, or more than 5% of LNAs. In embodiments, the entire composition of the third polynucleotide includes about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 60% to about 70% of LNAs.
  • the entire composition of the third polynucleotide includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs.
  • the entire Attorney Docket No.: 051385-585001WO composition of the third polynucleotide includes about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of canonical dNTPs.
  • the entire composition of the third polynucleotide includes less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, or up to about 30% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of canonical dNTPs.
  • the third polynucleotide includes a first region and a second region (e.g., a first region of the polynucleotide that is complementary to a polynucleotide, or complement thereof, and a second region that is not complementary to one or more polynucleotides or complements thereof).
  • the second region of the third polynucleotide is located at a 5’ end of the polynucleotide.
  • the third polynucleotide includes a plurality of LNAs interspersed throughout the first region of the polynucleotide (e.g., the plurality of LNAs are interspersed throughout the region of the third polynucleotide that is complementary to a polynucleotide, or complement thereof, for example, a first or a second polynucleotide or complements thereof, wherein the first and second polynucleotides form a dsDNA complex).
  • the second region of the third polynucleotide does not include LNAs.
  • the third polynucleotide includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the the first region of polynucleotide.
  • the first region of the third polynucleotide includes less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of LNAs.
  • the first region of the third polynucleotide includes up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, or up to about 5% of LNAs.
  • the first region of the third polynucleotide includes more than 60%, more than 50%, more than 40%, more than 30%, more than 20%, more than 10%, or more than 5% of LNAs. In embodiments, the first region of the third polynucleotide includes about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to Attorney Docket No.: 051385-585001WO about 50%, about 50% to about 60%, or about 60% to about 70% of LNAs.
  • the first region of the third polynucleotide includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs. In embodiments, the first region of the third polynucleotide includes about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of canonical dNTPs.
  • the first region of the third polynucleotide includes less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, or up to about 30% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of canonical dNTPs.
  • the entire composition of the third polynucleotide includes about 70% of LNAs and about 30% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 65% of LNAs and about 35% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 60% of LNAs and about 40% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 55% of LNAs and about 45% of canonical dNTPs.
  • the entire composition of the third polynucleotide includes about 50% of LNAs and about 50% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 45% of LNAs and about 55% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 40% of LNAs and about 60% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 35% of LNAs and about 65% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 30% of LNAs and about 70% of canonical dNTPs.
  • the entire composition of the third polynucleotide includes about 25% of LNAs and about 75% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 20% of LNAs and about 80% of canonical dNTPs. In embodiments, the entire composition of the third Attorney Docket No.: 051385-585001WO polynucleotide includes about 15% of LNAs and about 85% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 10% of LNAs and about 90% of canonical dNTPs.
  • the entire composition of the third polynucleotide includes about 5% of LNAs and about 95% of canonical dNTPs.
  • the first region of the third polynucleotide includes about 70% of LNAs and about 30% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 65% of LNAs and about 35% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 60% of LNAs and about 40% of canonical dNTPs.
  • the first region of the third polynucleotide includes about 55% of LNAs and about 45% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 50% of LNAs and about 50% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 45% of LNAs and about 55% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 40% of LNAs and about 60% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 35% of LNAs and about 65% of canonical dNTPs.
  • the first region of the third polynucleotide includes about 30% of LNAs and about 70% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 25% of LNAs and about 75% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 20% of LNAs and about 80% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 15% of LNAs and about 85% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 10% of LNAs and about 90% of canonical dNTPs.
  • the first region of the third polynucleotide includes about 5% of LNAs and about 95% of canonical dNTPs.
  • the third polynucleotide (e.g., the first, second, and/or other region of the third polynucleotide) includes one or more dT nucleobases that are replaced with dU nucleobases.
  • one or both of the first region and the second region of the third polynucleotide includes one or more dT nucleobases that are replaced with dU nucleobases.
  • the third polynucleotide includes a plurality of dT nucleobases that are replaced with dU nucleobases. In embodiments, the third polynucleotide includes all dT nucleobases replaced with dU nucleobases. In embodiments, the third polynucleotide includes dU Attorney Docket No.: 051385-585001WO nucleobases and LNA nucleotides. In embodiments, the third polynucleotide includes dU nucleobases and LNA nucleotides, wherein the LNA nucleotides are not adjacent to the dU nucleobases.
  • the third polynucleotide (e.g., the first, second, and/or other region of the third polynucleotide) includes a homologous recombination complex including a recombinase bound thereto.
  • the homologous recombination complex further includes a loading factor, a single-stranded binding (SSB) protein, or both.
  • the substrate includes a silica surface including a polymer coating.
  • the substrate is silica or quartz, such as a microscope slide, having a surface that is uniformly silanized.
  • the substrate surface further includes a polymer coating, which contains functional groups capable of immobilizing primers.
  • the substrate includes a patterned surface suitable for immobilization of primers in an ordered pattern.
  • a patterned surface refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more of the regions can be features where one or more primers are present. The features can be separated by interstitial regions where capture primers are not present.
  • the pattern can be an x-y format of features that are in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and/or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and/or interstitial regions. In some embodiments, the primers are randomly distributed upon the substrate. In some embodiments, the primers are distributed on a patterned surface. [0164] In embodiments, the first polynucleotide is immobilized on the substrate via a first linker and the second polynucleotide is immobilized to the substrate via a second linker. The linkers may also include spacer nucleotides.
  • Including spacer nucleotides in the linker puts the polynucleotide in an environment having a greater resemblance to free solution. This can be beneficial, for example, in enzyme-mediated reactions such as sequencing-by-synthesis. It is believed that such reactions suffer less steric hindrance issues that can occur when the polynucleotide is directly attached to the solid support or is attached through a very short linker Attorney Docket No.: 051385-585001WO (e.g., a linker comprising about 1 to 3 carbon atoms).
  • Spacer nucleotides form part of the polynucleotide but do not participate in any reaction carried out on or with the polynucleotide (e.g. a hybridization or amplification reaction).
  • the spacer nucleotides include 1 to 20 nucleotides.
  • the linker includes 10 spacer nucleotides.
  • the linker includes 12 spacer nucleotides.
  • the linker includes 15 spacer nucleotides. It is preferred to use polyT spacers, although other nucleotides and combinations thereof can be used.
  • the linker includes 10, 11, 12, 13, 14, or 15 T spacer nucleotides.
  • the linker includes 12 T spacer nucleotides. Spacer nucleotides are typically included at the 5′ ends of polynucleotides which are attached to a suitable support.
  • Attachment can be achieved via a phosphorothioate present at the 5′ end of the polynucleotide, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, or any other bioconjugate reactive moiety.
  • the linker may be a carbon-containing chain such as those of formula -(CH2)n- wherein “n” is from 1 to about 1000. However, a variety of other linkers may be used so long as the linkers are stable under conditions used in DNA sequencing.
  • the linker includes polyethylene glycol (PEG) having a general formula of -(CH2—CH2—O)m-, wherein m is from about 1 to 500, 1 to 100, or 1 to 12.
  • the linker, or the immobilized oligonucleotides include a cleavable site.
  • the invasion primer e.g., the first, second, and/or other region of the invasion primer
  • a cleavable site is a location which allows controlled cleavage of the immobilized polynucleotide strand (e.g., the linker, the primer, or the polynucleotide) by chemical, enzymatic or photochemical means.
  • the cleavable site includes one or more deoxyuracil nucleobases (dUs).
  • Any suitable enzymatic, chemical, or photochemical cleavage reaction may be used to cleave the cleavable site.
  • the cleavage reaction may result in removal of a part or the whole of the strand being cleaved.
  • Suitable cleavage means include, for example, restriction enzyme digestion, in which case the cleavable site is an appropriate restriction site for the enzyme which directs cleavage of one or both strands of a duplex template; RNase digestion or chemical cleavage of a bond between a deoxyribonucleotide and a ribonucleotide, in which case the cleavable site may include one or more ribonucleotides; chemical reduction of a disulfide linkage with a reducing agent (e.g., THPP or TCEP), in which case the cleavable site should include an Attorney Docket No.: 051385-585001WO appropriate disulfide linkage; chemical cleavage of a diol linkage with periodate, in which case the cleavable site should include a diol linkage; generation of an abasic site and subsequent hydrolysis, etc.
  • restriction enzyme digestion in which case the cleavable site is an appropriate restriction
  • the cleavable site is included in the surface immobilized primer (e.g., within the polynucleotide sequence of the primer).
  • the linker, the primer, or the first or second polynucleotide includes a diol linkage which permits cleavage by treatment with periodate (e.g., sodium periodate). It will be appreciated that more than one diol can be included at the cleavable site.
  • One or more diol units may be incorporated into a polynucleotide using standard methods for automated chemical DNA synthesis. Polynucleotide primers including one or more diol linkers can be conveniently prepared by chemical synthesis.
  • the diol linker is cleaved by treatment with any substance which promotes cleavage of the diol (e.g., a diol-cleaving agent).
  • the diol-cleaving agent is periodate, e.g., aqueous sodium periodate (NaIO4).
  • the cleaved product may be treated with a “capping agent” in order to neutralize reactive species generated in the cleavage reaction.
  • Suitable capping agents for this purpose include amines, e.g., ethanolamine or propanolamine.
  • cleavage may be accomplished by using a modified nucleotide as the cleavable site (e.g., uracil, 8oxoG, 5-mC, 5-hmC) that is removed or nicked via a corresponding DNA glycosylase, endonuclease, or combination thereof.
  • a modified nucleotide e.g., uracil, 8oxoG, 5-mC, 5-hmC
  • each of the plurality of immobilized oligonucleotides is about 5 to about 25 nucleotides in length.
  • each of the plurality of immobilized oligonucleotides is about 10 to about 40 nucleotides in length.
  • each of the plurality of immobilized oligonucleotides is about 5 to about 100 nucleotides in length. In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) is about 20 to 200 nucleotides in length. In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) about or at least about 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50 or more nucleotides in length. In embodiments, one or more immobilized oligonucleotides include blocking groups at their 3’ ends that prevent polymerase extension.
  • a blocking moiety prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide.
  • the 3’ modification is a 3’-phosphate modification, including a 3’ phosphate moiety, which is removed by a PNK enzyme or a Attorney Docket No.: 051385-585001WO phosphatase enzyme.
  • abasic site cleavage with certain endonucleases results in a 3’-OH at the cleavable site from the 3’-diesterase activity.
  • the immobilized oligonucleotides includes one or more phosphorothioate nucleic acids. In embodiments, the immobilized oligonucleotides includes a plurality of phosphorothioate nucleic acids. In embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100% of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, most of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids.
  • all of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, none of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, the 5’ end of the immobilized oligonucleotide includes one or more phosphorothioate nucleic acids. In embodiments, the 5’ end of the immobilized oligonucleotide includes between one and five phosphorothioate nucleic acids.
  • the first and second polynucleotides are each attached to the solid support (i.e., immobilized on the surface of a solid support).
  • the polynucleotide molecules can be fixed to surface by a variety of techniques, including covalent attachment and non-covalent attachment.
  • the polynucleotides are confined to an area of a discrete region (referred to as a cluster).
  • the discrete regions may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. A regular array of such regions is advantageous for detection and data analysis of signals collected from the arrays during an analysis. These discrete regions are separated by interstitial regions.
  • an interstitial region refers to an area in a substrate or on a surface that separates other areas of the substrate or surface.
  • an interstitial region can separate one concave feature of an array from another concave feature of the array.
  • the two regions that are separated from each other can be discrete, lacking contact with each other.
  • an interstitial region can separate a first portion of a feature from a second portion of a feature.
  • the interstitial region is continuous whereas the features are discrete, for example, as is the case for an array of wells in an otherwise continuous surface.
  • the separation provided by an interstitial region can be partial or full separation.
  • Interstitial regions will typically have a surface material that differs from the surface material of the features on the Attorney Docket No.: 051385-585001WO surface.
  • features of an array can have polynucleotides that exceeds the amount or concentration present at the interstitial regions.
  • the polynucleotides and/or primers may not be present at the interstitial regions.
  • at least two different primers are attached to the solid support (e.g., a forward and a reverse primer), which facilitates generating multiple amplification products from the first extension product or a complement thereof.
  • the clusters have a mean or median separation from one another of about 0.5-5 ⁇ m.
  • the mean or median separation is about 0.1-10 microns, 0.25-5 microns, 0.5-2 microns, 1 micron, or a number or a range between any two of these values. In embodiments, the mean or median separation is about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 ⁇ m or a number or a range between any two of these values.
  • the mean or median separation is about 0.1-10 microns. In embodiments, the mean or median separation is about 0.25-5 microns. In embodiments, the mean or median separation is about 0.5-2 microns. In embodiments, the mean or median separation is about or at least about 0.1 ⁇ m. In embodiments, the mean or median separation is about or at least about 0.25 ⁇ m. In embodiments, the mean or median separation is about or at least about 0.5 ⁇ m. In embodiments, the mean or median separation is about or at least about 1.0 ⁇ m. In embodiments, the mean or median separation is about or at least about 2.0 ⁇ m. In embodiments, the mean or median separation is about or at least about 5.0 ⁇ m.
  • the mean or median separation is about or at least about 10 ⁇ m.
  • the mean or median separation may be measured center-to-center (i.e., the center of one cluster to the center of a second cluster).
  • the amplicon clusters have a mean or median separation (measured center-to-center) from one another of about 0.5-5 ⁇ m.
  • the mean or median separation may be measured edge-to-edge (i.e., the edge of one amplicon cluster to the edge of a second amplicon cluster).
  • the amplicon clusters have a mean or median separation (measured edge-to-edge) from one another of about 0.2-5 ⁇ m.
  • the amplicon clusters have a mean or median diameter of about 100-2000 nm, or about 200-1000 nm.
  • the mean or median diameter is about 100-3000 nanometers, about 500-2500 nanometers, about 1000-2000 nanometers, or a number or a range between any two of these values.
  • the mean or median diameter is about or at most about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2000 nanometers or a number or a range between any two of these values.
  • the mean or median diameter is about 100-3,000 nanometers. In embodiments, the mean or median diameter is about 100-2,000 nanometers. In embodiments, the mean or median diameter is about 500-2500 nanometers. In embodiments, the mean or median diameter is about 200-1000 nanometers. In embodiments, the mean or median diameter is about 1,000-2,000 nanometers. In embodiments, the mean or median diameter is about or at most about 100 nanometers. In embodiments, the mean or median diameter is about or at most about 200 nanometers. In embodiments, the mean or median diameter is about or at most about 500 nanometers. In embodiments, the mean or median diameter is about or at most about 400 nanometers. In embodiments, the mean or median diameter is about or at most about 500 nanometers.
  • the mean or median diameter is about or at most about 600 nanometers. In embodiments, the mean or median diameter is about or at most about 700 nanometers. In embodiments, the mean or median diameter is about or at most about 1,000 nanometers. In embodiments, the mean or median diameter is about or at most about 2,000 nanometers. In embodiments, the mean or median diameter is about or at most about 2,500 nanometers. In embodiments, the mean or median diameter is about or at most about 3,000 nanometers.
  • each amplicon cluster (e.g., an amplicon cluster having a mean or median diameter of about 100-2000 nm, or about 200-1000 nm) includes about or at least about 100, 500, 1,000, 2,500, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 dsDNA molecules.
  • each amplicon cluster includes about 100 dsDNA molecules.
  • each amplicon cluster includes about 500 dsDNA molecules.
  • each amplicon cluster includes about 1000 dsDNA molecules.
  • each amplicon cluster includes about 500 dsDNA molecules.
  • each amplicon cluster includes about 1,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 2,500 dsDNA molecules. In embodiments, Attorney Docket No.: 051385-585001WO each amplicon cluster includes about 5,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 10,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 20,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 30,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 40,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 50,000 dsDNA molecules. In embodiments, each amplicon cluster includes more than about 50,000 dsDNA molecules.
  • the substrate is a particle.
  • the substrate is a multiwell container.
  • the substrate is a polymer coated particle or polymer coated planar support.
  • the substrate includes a polymer.
  • the particle includes polymerized units of polyacrylamide (AAm), poly-N-isopropylacrylamide, poly N- isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate, methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N′- bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide (PPO), polyacrylamide (AAm), polyacrylamide (AAm), poly-
  • the particle shell includes polymerized units of polyacrylamide (AAm), glicydyl methacrylate (GMA), polyethylene glycol methacrylate (PEGMA), polyethylene glycol methacrylate (PEGMA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof.
  • the particle includes polymerized units of polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate (GMA).
  • the particle includes polymerized units of polyethylene glycol methacrylate (PEGMA) and isocyanatoethyl methacrylate (IEM).
  • the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate, 2-azido-3-hydroxypropyl methacrylate, 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate, Attorney Docket No.: 051385-585001WO 3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or 2-(((2- azidoethoxy)carbonyl)amino)ethyl acrylate.
  • the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate, 2-azido-3-hydroxypropyl methacrylate, or 2- (((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.
  • the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate.
  • the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate 2-azido-3-hydroxypropyl methacrylate.
  • the particle includes polymerized units of 3-azido-2- hydroxypropyl methacrylate 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.
  • the kit includes the substrate as described herein.
  • the kit includes one or more containers providing a composition and one or more additional reagents (e.g., a buffer suitable for polynucleotide extension).
  • the kit may also include a template nucleic acid (DNA and/or RNA), one or more primer polynucleotides, nucleoside triphosphates (including, e.g., deoxyribonucleotides, ribonucleotides, labeled nucleotides, and/or modified nucleotides), buffers, salts, and/or labels (e.g., fluorophores).
  • the kit includes a sequencing polymerase, and one or more amplification polymerases.
  • the sequencing polymerase is capable of incorporating modified nucleotides.
  • the polymerase is a DNA polymerase.
  • the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase, Pol ⁇ DNA polymerase
  • the DNA polymerase is a thermophilic nucleic acid polymerase. In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044, each of which are incorporated herein by reference for all purposes). In embodiments, the kit includes a strand-displacing polymerase.
  • the kit includes a strand-displacing polymerase, Attorney Docket No.: 051385-585001WO such as a phi29 polymerase, phi29 mutant polymerase or a thermostable phi29 mutant polymerase.
  • the kit includes a buffered solution.
  • the buffered solutions contemplated herein are made from a weak acid and its conjugate base or a weak base and its conjugate acid.
  • sodium acetate and acetic acid are buffer agents that can be used to form an acetate buffer.
  • buffer agents that can be used to make buffered solutions include, but are not limited to, Tris, bicine, tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, other buffer agents that can be used in enzyme reactions, hybridization reactions, and detection reactions are known in the art.
  • the buffered solution can include Tris.
  • the pH of the buffered solution can be modulated to permit any of the described reactions.
  • the buffered solution can have a pH greater than pH 7.0, greater than pH 7.5, greater than pH 8.0, greater than pH 8.5, greater than pH 9.0, greater than pH 9.5, greater than pH 10, greater than pH 10.5, greater than pH 11.0, or greater than pH 11.5.
  • the buffered solution can have a pH ranging, for example, from about pH 6 to about pH 9, from about pH 8 to about pH 10, or from about pH 7 to about pH 9.
  • the buffered solution can comprise one or more divalent cations. Examples of divalent cations can include, but are not limited to, Mg 2+ , Mn 2+ , Zn 2+ , and Ca 2+ .
  • the buffered solution can contain one or more divalent cations at a concentration sufficient to permit hybridization of a nucleic acid.
  • a polynucleotide e.g., an invasion primer.
  • the polynucleotide includes a plurality of LNA nucleotides; one or more cleavable sites, wherein the one or more cleavable sites partition the invasion primer into two or more regions; and a plurality of native nucleotides.
  • a polynucleotide e.g., an invasion primer.
  • the polynucleotide includes a plurality of LNA nucleotides; one or more dU nucleobases, wherein the one or more dU nucleobases partition the invasion primer into two or more regions; and a plurality of native nucleotides.
  • the polynucleotide is 20 to 40 nucleotides in length. In embodiments, the polynucleotide is about 10 to 100 nucleotides in length. In embodiments, the polynucleotide is about 15 to about 75 nucleotides in length.
  • the polynucleotide is about 15 to Attorney Docket No.: 051385-585001WO about 90 nucleotides in length. In embodiments, the polynucleotide is about 30 to about 95 nucleotides in length. In embodiments, the polynucleotide is about 20 to about 80 nucleotides in length. In embodiments, the polynucleotide is about 25 to about 75 nucleotides in length. In embodiments, the polynucleotide is about 15 to about 50 nucleotides in length. In embodiments, the polynucleotide is about 10 to about 20 nucleotides in length.
  • the polynucleotide is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length. In embodiments, the polynucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 nucleotides in length. In embodiments, the polynucleotide is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 nucleotides in length. In embodiments, the polynucleotide is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length.
  • the polynucleotide is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 nucleotides in length. In embodiments, the polynucleotide is about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 nucleotides in length. In embodiments, the polynucleotide is about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80 nucleotides in length.
  • the polynucleotides is about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 nucleotides in length. In embodiments, the polynucleotide is greater than 30 nucleotides in length. In embodiments, the polynucleotide is greater than 40 nucleotides in length. In embodiments, the polynucleotide is greater than 50 nucleotides in length. In embodiments, the polynucleotide is greater than 60 nucleotides in length. In embodiments, the polynucleotide is greater than 70 nucleotides in length.
  • the polynucleotide is greater than 80 nucleotides in length. In embodiments, the polynucleotide is greater than 90 nucleotides in length. In embodiments, the polynucleotide is no less than 15 nucleotides. In embodiments, the polynucleotide is no less than 20 nucleotides. In embodiments, the polynucleotide is about 15 to about 35 nucleotides in length. In embodiments, the polynucleotide is about 15 to about 90 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides.
  • the polynucleotide is about 25 to about 90 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 35 to about 90 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 25 to about 35 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 25 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides.
  • the polynucleotide is about 30 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides.
  • the Attorney Docket No.: 051385-585001WO polynucleotide is 30, 31, 32, or 33 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides.
  • the calculated or predicted melting temperature (Tm) of the polynucleotide is about 70°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the polynucleotide is about 80°C to about 95°C.
  • the calculated or predicted melting temperature (Tm) of the polynucleotide is about 85°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the polynucleotide is about 85°C to about 90°C. In embodiments, the plurality of LNA nucleotides are interspersed throughout the polynucleotide (e.g., throughout the invasion primer).
  • the one or more dU nucleobases partition the polynucleotide into two or more regions or sequences of nucleotides (e.g., a first plurality of consecutive nucleotides and a second plurality of consecutive nucleotides are separated by the one or more dU nucleobases).
  • the one or more dU nucleobases partition the polynucleotide into a first binding region and a 5’ tail sequence (e.g., a first binding region or first region complementary to a first polynucleotide, a second polynucleotide, or a complement thereof, and a 5’ tail sequence or second region that is not complementary to the first polynucleotide, second polynucleotide, or a complement thereof).
  • each of the two or more regions of consecutive nucleotides are each about 3 to about 30 nucleotides in length, or about 15 to about 45 nucleotides in length.
  • each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length, or about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each at least about 3, 5, 7, 10, 13, or 15 nucleotides in length. In embodiments, each of the two or more regions of consecutive nucleotides is greater than about 15 nucleotides in length.
  • the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 50°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 60°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the Attorney Docket No.: 051385-585001WO two or more regions of consecutive nucleotides is about 50°C to about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 75°C.
  • the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 60°C. III.
  • a method of sequencing including: hybridizing an invasion primer including a binding sequence and a tail sequence (e.g., annealing an invasion oligonucleotide to the 3’ end of one strand of a double-stranded polynucleotide, wherein a portion of the invasion oligonucleotide, for example the sequence at the 5’ end of the invasion oligonucleotide, does not anneal to the double-stranded polynucleotide) to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand; extending the second strand along the tail sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating
  • the method further includes removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand. In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. In embodiments, both the first strand and the second strand are both attached to a solid support (e.g., each strand is attached via their 5’ end).
  • the method includes nicking and/or cleaving the invasion strand to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the invasion primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand.
  • a method of forming a single-stranded polynucleotide attached to a solid support including: contacting a plurality of double-stranded polynucleotides including a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers include a binding sequence and a tail sequence; hybridizing the binding sequence of one of the invasion primers to one of the second strands; and extending the invasion primer hybridized to the second strand with a polymerase (e.g., Bst large fragment (Bst LF) polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, T4 DNA polymerase, T7 DNA polymerase, or a mutant thereof) to generate a polymerase (e.g., Bst large fragment (Bst LF) polymerase
  • the method further includes sequencing the single-stranded polynucleotide. In embodiments, the method further includes removing the invasion strand and sequencing the second strand. [0185] In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on the second strand, and extending the second invasion primer with a polymerase, thereby generating a second invasion strand hybridized to the second strand. In embodiments, the second invasion primer includes a different tail sequence than the first invasion primer. In embodiments, the second invasion primer includes the same tail sequence as the first invasion primer.
  • the invasion primer includes a first primer binding sequence and a second primer binding sequence, wherein the second primer binding sequence may be referred to as the tail sequence. In embodiments, the invasion primer includes multiple copies of the primer binding sequence.
  • the solid support includes about 100, 500, 1000, 5000, 10000, or more dsDNA molecules in a 2 ⁇ m 2 area. In embodiments, the solid support includes about 1,000 to about 10,000 dsDNA molecules in a 2 ⁇ m 2 area. In embodiments, the solid support includes about 1,000 to about 10,000 dsDNA molecules in a 0.5 ⁇ m diameter feature.
  • the solid support includes about 1,000 to about 50,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. In embodiments, the solid support includes about Attorney Docket No.: 051385-585001WO 10,000 to about 50,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. In embodiments, the solid support includes about 20,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. In embodiments, the solid support includes about 30,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature.
  • a feature may be a wells, pits, channels, ridges, raised regions, pegs, or posts on a solid support.
  • Each feature includes a colony and refers to a discrete site on a solid support that includes a plurality of immobilized polynucleotides.
  • removing the invasion strand includes digesting (i.e., cleaving internal phosphodiester bonds of a polynucleotide) all or portions thereof of the invasion strand using an exonuclease enzyme.
  • Exonucleases can be active on ssDNA and/or dsDNA, initiate from the 5′ end and/or the 3′ end of polynucleotides, and can also act on RNA polynucleotides.
  • the exonuclease enzyme is a DNA specific exonuclease.
  • the exonuclease catalyzes the removal of nucleotides from linear, and/or nicked double-stranded DNA in the 5' to 3' direction.
  • removing the invasion strand includes contacting the double-stranded polynucleotide with a denaturant (e.g., NaOH or formamide) or increasing the temperature to denature the double-stranded polynucleotide (e.g., increasing the temperature is increased to greater than 90°C) and washing away the invasion strand.
  • removing include washing (e.g., contacting the solid support with a wash buffer) and flowing the un-attached strands away from the solid support.
  • the first invasion strand blocks and/or prevents, and/or reduces rehybridization of the complementary first strand.
  • the invasion primer is not covalently attached to the solid support.
  • the invasion strand alternatively referred to herein as the third polynucleotide, is not covalently attached to the solid support.
  • the invasion primer is in solution.
  • the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing an invasion primer to the second strand, and extending the invasion primer, wherein the invasion primer is not covalently attached to the Attorney Docket No.: 051385-585001WO solid support; and generating a first sequencing read by hybridizing one or more sequencing primers to the first strand, and extending the one or more first sequencing primers.
  • the invasion primer does not hybridize at the end of the strand, rather the invasion primer hybridizes about 5 to about 50 nucleotides from the end of the strand. In embodiments, the invasion primer hybridizes about 10 to about 30 nucleotides, about 12 to about 24, or about 15 to about 30 from the end of the strand. In embodiments, the invasion primer hybridizes towards the 5′ end of the strand. In embodiments, the invasion primer hybridizes towards the 3′ end of the strand. In embodiments, the invasion primer hybridizes at the end of the strand (e.g., the invasion primer hybridizes to the last few nucleotides on the strand).
  • a 5’ region (e.g., a tail sequence) of the invasion primer does not hybridize to the end of the strand (e.g., the invasion primer includes a 5’ tail sequence that does not hybridize).
  • the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing an invasion primer (e.g., a first invasion primer) to the second strand, and extending the first invasion primer (e.g., extending the first invasion primers with a polymerase under strand-displacing conditions); and generating a first sequencing read by hybridizing one or more first sequencing primers to the first strand, and extending the one or more first sequencing
  • the first invasion strand when hybridized to the second strand, blocks and/or prevents rehybridization of the complementary first strand.
  • the first invasion primer is not covalently attached to the solid support.
  • the invasion strand is not covalently attached to the solid support.
  • the invasion strand includes substantially the same sequence as the first strand. [0191]
  • each invasion primer of the one or more invasions primers is complementary to the same sequence (e.g., the same sequence in the first strand or the same sequence in the second strand).
  • each invasion primer of the one or more invasion primers is not complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand).
  • each invasion primer Attorney Docket No.: 051385-585001WO of the one or more invasion primers is complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand).
  • one or more invasions primers is complementary to the same sequence (e.g., the same sequence in the first strand or the same sequence in the second strand).
  • one or more invasion primers is not complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand). In embodiments, one or more invasion primers is complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand).
  • the first strand is covalently attached to the solid support via a first linker and the second strand is covalently attached to the solid support via a second linker.
  • the linker tethering the polynucleotide strands may be any linker capable of localizing nucleic acids to arrays. The linkers may be the same, or the linkers may be different.
  • Solid-supported molecular arrays have been generated previously in a variety of ways, for example, the attachment of biomolecules (e.g., proteins and nucleic acids) to a variety of substrates (e.g., glass, plastics, or metals) underpins modern microarray and biosensor technologies employed for genotyping, gene expression analysis and biological detection.
  • substrates e.g., glass, plastics, or metals
  • Silica-based substrates are often employed as supports on which molecular arrays are constructed, and functionalized silanes are commonly used to modify glass to permit a click-chemistry enabled linker to tether the biomolecule (e.g., polynucleotide strand).
  • the method further includes generating a second invasion strand hybridized to the first strand by hybridizing one or more second invasion primers to the first strand, and extending the one or more second invasion primers; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers.
  • the second invasion strand is not covalently attached to the solid support.
  • the method further includes removing the first invasion strand; generating a second invasion strand hybridized to the first strand by hybridizing one or more invasion primers to the first strand, and extending the one or more second invasion primers; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers.
  • the method further includes generating a second invasion Attorney Docket No.: 051385-585001WO strand hybridized to the first strand by hybridizing a second invasion primer to the first strand, and extending the second invasion primer; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers.
  • the second invasion strand is not covalently attached to the solid support.
  • the method further includes removing the first invasion strand; generating a second invasion strand hybridized to the first strand by hybridizing a second invasion primer to the first strand, and extending the second invasion primers; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers.
  • the method includes sequencing both strands (i.e., the first and the second strand) of the sample double-stranded amplification product.
  • the method includes sequencing both strands (i.e., the first and the second strand) of the template polynucleotide.
  • the double-stranded amplification product includes common sequences at their 5′ and 3′ ends.
  • the term “common” is interpreted as meaning common to all templates in the library, such as a synthetic amplification primer binding sequence.
  • the double-stranded amplification product may include a first adapter sequence at the 5′ end and a second adapter sequence at the 3′ end.
  • the first adapter sequence and the second adapter sequence will consist of no more than 100, or no more than 50, or no more than 40 consecutive nucleotides at the 5′ and 3′ ends, respectively, of each strand of each template polynucleotide.
  • the precise length of the two sequences may or may not be identical.
  • the precise sequences of the common regions are generally not material to the invention and may be selected by the user.
  • the common sequences must at least include primer- binding sequences (i.e., regions of complementarity for a primer) which enable specific annealing of primers when the template polynucleotides are in used in a solid-phase amplification reaction.
  • the primer-binding sequences are thus determined by the sequence of the primers to be ultimately used for solid-phase amplification.
  • generating the invasion strand i.e., generating the first invasion strand or the second invasion strand
  • generating the invasion strand includes hybridizing one Attorney Docket No.: 051385-585001WO primer to a common sequence in the double-stranded amplification product.
  • generating the invasion strand includes hybridizing a primer to at or near the 3′ end of the double-stranded amplification product.
  • generating the invasion strand includes hybridizing a primer to at or near the 3′ end of the double-stranded amplification product, wherein the primer is not covalently attached to the solid support (e.g., the primer is in solution prior to hybridization).
  • the invasion primer does not hybridize at the terminus of the strand, rather the invasion primer hybridizes about 10, about 20, about 30, or about 50 nucleotides from the terminus of the strand. In embodiments, the invasion primer hybridizes about 10 to about 30 nucleotides from the terminus of the strand.
  • the invasion primer hybridizes to a common sequence (e.g., a sequence or the complement thereof as described in U.S. Patent Publication 2016/0256846, which is incorporated herein by reference, for example SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 11, or complements thereof, of U.S. Patent Publication 2016/0256846).
  • the invasion primer includes a tail sequence (e.g., a 5’ tail sequence) that does not hybridize to a common sequence.
  • the tail sequence is complementary to a second primer (e.g., a second invasion primer).
  • the method further includes removing the first strand by cleaving the first strand at a cleavable site, washing away the cleaved strand, and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand; and extending the one or more second sequencing primers.
  • removing the first strand is optional.
  • the one or more cleavable sites may include a modified nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleavage agent.
  • the cleavable site(s) may be deoxyuracil triphosphate (dUTP), deoxy-8-Oxo- guanine triphosphate (d-8-oxoG), or other modified nucleotide(s), such as those described, for example, in US 2012/0238738, which is incorporated herein by reference for all purposes.
  • the cleavable site includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent.
  • the cleavable site includes one or more ribonucleotides.
  • the cleavable site includes 2 to 5 Attorney Docket No.: 051385-585001WO ribonucleotides.
  • the cleavable site includes one ribonucleotide.
  • the cleavable sites can be cleaved at or near a modified nucleotide or bond by enzymes or chemical reagents, collectively referred to here and in the claims as “cleaving agents.”
  • cleaving agents include DNA repair enzymes, glycosylases, DNA cleaving endonucleases, or ribonucleases.
  • cleavage at dUTP may be achieved using uracil DNA glycosylase and endonuclease VIII (USERTM, NEB, Ipswich, Mass.), as described in U.S. Pat. No.7,435,572.
  • cleavable site when the modified nucleotide is a ribonucleotide, the cleavable site can be cleaved with an endoribonuclease.
  • cleaving an extension product includes contacting the cleavable site with a cleaving agent, wherein the cleaving agent includes a reducing agent, sodium periodate, RNase, formamidopyrimidine DNA glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase (UDG).
  • the cleaving agent includes a reducing agent, sodium periodate, RNase, formamidopyrimidine DNA glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase (UDG).
  • the cleaving agent is an endonuclease enzyme such as nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Micrococcal nuclease, Endonuclease II (endo VI, exo III), nuclease BAL-31 or mung bean nuclease.
  • the cleaving agent includes a restriction endonuclease, including, for example a type IIS restriction endonuclease.
  • the cleaving agent is an exonuclease (e.g., RecBCD), restriction nuclease, endoribonuclease, exoribonuclease, or RNase (e.g., RNAse I, II, or III).
  • the cleaving agent is a restriction enzyme.
  • the cleaving agent includes a glycosylase and one or more suitable endonucleases.
  • cleavage is performed under alkaline (e.g., p/’H greater than 8) buffer conditions at between 40°C to 80°C (e.g., 65°C).
  • cleaving includes contacting the cleavable site with a Uracil DNA glycosylase (UDG) enzyme to catalyze the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact, and contacting the abasic site with an endonuclease VIII enzyme to cleave the phosphodiester bond.
  • UDG Uracil DNA glycosylase
  • the method further includes removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand.
  • the method further includes removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on the second strand, and extending the second invasion primer with a polymerase, thereby generating a second invasion strand.
  • the second invasion primer does not include a tail sequence.
  • the method prior to generating a first invasion strand, includes removing immobilized primers that do not contain a first or second strand (i.e., unused primers). Methods of removing immobilized primers can include digestion using an enzyme with exonuclease activity. Removing unused primers may serve to increase the free volume and allow for greater accessibility of the invasion primer.
  • Removal of unused primers may also prevent opportunities for the newly released first strand to rehybridize to an available surface primer, producing a priming site off the available surface primer, thereby facilitating the “reblocking” of the released first strand.
  • the method prior to generating a first invasion strand, includes contacting the immobilized primers with an exonuclease enzyme. [0199]
  • the method prior to generating a first invasion strand, includes blocking the immobilized primers that do not include a first or second strand.
  • the immobilized oligonucleotides include blocking groups at their 3’ ends that prevent polymerase extension.
  • a blocking moiety prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide.
  • the method prior to generating a first invasion strand the method includes incubating the amplification products with dideoxynucleotide triphosphates (ddNTPs) to block the 3′-OH of the immobilized oligonucleotides from future extension.
  • the method prior to generating a first invasion strand, includes incorporating a dideoxynucleotide triphosphate (ddNTP) into an immobilized primer.
  • the method prior to generating a first invasion strand, includes contacting the immobilized primer with a polymerase. In embodiments, during generation of a first invasion strand, the method includes contacting the immobilized primer with a polymerase buffer (e.g., incubating the solid support with a buffered solution including a polymerase).
  • a polymerase buffer e.g., incubating the solid support with a buffered solution including a polymerase.
  • the first strand is cleaved after generating the first sequencing read but before generating the second sequencing read. In embodiments, the first strand is not cleaved after generating the first sequencing read. Cleaving one strand of the double-stranded amplification product may be referred to as linearization.
  • the first strand may be cleaved by exposing the first strand to a mixture containing a glycosylase and one Attorney Docket No.: 051385-585001WO or more suitable endonucleases.
  • the first strand is attached to the surface in a way that allows for selective removal. If the first template strand is removed from the surface, and the partially double-stranded amplification product is denatured, for example by treatment with hydroxide or formamide, then the second strand remains immobilized as a linearized single strand.
  • one of the surface immobilized primers includes a cleavable site such that it can be cleaved from the surface, (e.g., diol linkage) the resulting partially double-stranded amplification product can be made single-stranded using heat, or chemical denaturing agents, or a combination thereof providing conditions to give a single strand containing a primer hybridization site.
  • Any suitable enzymatic, chemical, or photochemical cleavage reaction may be used to cleave the cleavable site. The cleavage reaction may result in removal of a part or the whole of the strand being cleaved.
  • Suitable cleavage means include, for example, restriction enzyme digestion, in which case the cleavable site is an appropriate restriction site for the enzyme which directs cleavage of one or both strands of a duplex template; RNase digestion or chemical cleavage of a bond between a deoxyribonucleotide and a ribonucleotide, in which case the cleavable site may include one or more ribonucleotides; chemical reduction of a disulfide linkage with a reducing agent (e.g., THPP or TCEP), in which case the cleavable site should include an appropriate disulfide linkage; chemical cleavage of a diol linkage with periodate, in which case the cleavable site should include a diol linkage; generation of an abasic site and subsequent hydrolysis, etc.
  • restriction enzyme digestion in which case the cleavable site is an appropriate restriction site for the enzyme which directs cleavage of one or both
  • the cleavable site is included in the surface immobilized primer (e.g., within the polynucleotide sequence of the primer).
  • one strand of the double-stranded amplification product (or the surface immobilized primer) may include a diol linkage which permits cleavage by treatment with periodate (e.g., sodium periodate). It will be appreciated that more than one diol can be included at the cleavable site.
  • One or more diol units may be incorporated into a polynucleotide using standard methods for automated chemical DNA synthesis. Polynucleotide primers including one or more diol linkers can be conveniently prepared by chemical synthesis.
  • the diol linker is cleaved by treatment with any substance which promotes cleavage of the diol (e.g., a diol-cleaving agent).
  • the diol- cleaving agent is periodate, e.g., aqueous sodium periodate (NaIO4).
  • the cleaved product may be treated with a “capping agent” in order to neutralize reactive species generated in the cleavage reaction.
  • Suitable capping agents for this purpose include amines, e.g., ethanolamine or propanolamine.
  • the cleavable site is not in the immobilized primer sequence (e.g., within the polynucleotide sequence of the primer). In embodiments, the cleavable site is included in the linking moiety responsible for tethering the primer to the substrate. In embodiments, the cleavable site is a cleavable linker (e.g., a disulfide containing linker that cleaves when exposed to a reducing agent). In embodiments, the cleavable site is a diol linker. [0203] In embodiments, the first strand includes at least one cleavable site.
  • the first linker includes at least one cleavable site.
  • the cleavable site includes deoxyuracil triphosphate (dUTP).
  • the enzyme uracil DNA glycosylase (UDG) may then be used to remove dUTP, generating an abasic site on one strand.
  • the polynucleotide strand including the abasic site may then be cleaved at the abasic site by treatment with endonuclease (e.g EndoIV endonuclease, AP lyase, FPG glycosylase/AP lyase, EndoVIII glycosylase/AP lyase), heat or alkali.
  • endonuclease e.g EndoIV endonuclease, AP lyase, FPG glycosylase/AP lyase, EndoVIII glycosylase/AP lyase
  • the USER TM reagent available from New England Biolabs (NEB catalog #M5508) is used for the creation of a single nucleotide gap at a uracil base in a duplex strand and subsequent cleavage.
  • the cleavable site includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent.
  • the cleavable site includes one or more ribonucleotides. In embodiments, the cleavable site includes 2 to 5 ribonucleotides.
  • the cleavable site includes one ribonucleotide. In embodiments, the cleavable site includes more than one ribonucleotide. In embodiments, the cleavable site includes deoxyuracil triphosphate (dUTP) or deoxy-8-oxo-guanine triphosphate (d-8-oxoG). In embodiments, the cleavable site includes two or more deoxyuracil triphosphate (dUTP). In embodiments, the cleavable site includes 2 to 15 dUTPs. In embodiments, the cleavable site includes 2 to 4 dUTPs.
  • dUTP deoxyuracil triphosphate
  • d-8-oxoG deoxy-8-oxo-guanine triphosphate
  • the cleavable site includes two or more deoxyuracil triphosphate (dUTP). In embodiments, the cleavable site includes 2 to 15 dUTPs. In embodiments, the clea
  • cleaving includes enzymatically cleaving the first strand at the at least one cleavable site (e.g., enzymatically cleaving with an endonuclease).
  • the first strand includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, Attorney Docket No.: 051385-585001WO ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent.
  • cleaving the first strand includes contacting the cleavable site with a cleaving agent, wherein the cleaving agent includes a reducing agent, sodium periodate, RNase, Formamidopyrimidine DNA Glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase (UDG).
  • a cleaving agent includes a reducing agent, sodium periodate, RNase, Formamidopyrimidine DNA Glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase (UDG).
  • the cleaving agent is an endonuclease enzyme such as nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Micrococcal nuclease, Endonuclease II (endo VI, exo III), nuclease BAL-31 or mung bean nuclease.
  • the cleaving agent includes a restriction endonuclease, including, for example a type IIS restriction endonuclease.
  • the cleaving agent is an exonuclease (e.g., RecBCD), restriction nuclease, endoribonuclease, exoribonuclease, or RNase (e.g., RNAse I, II, or III).
  • the cleaving agent is a restriction enzyme.
  • the cleaving agent includes a glycosylase and one or more suitable endonucleases.
  • cleavage is performed under alkaline (e.g., pH greater than 8) buffer conditions at between 40°C to 80°C (e.g., 65°C).
  • cleaving includes chemically cleaving the first strand at the at least one cleavable site.
  • the first linker includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent.
  • the invasion primer is not covalently attached to the solid support.
  • the invasion primer includes synthetic nucleotides.
  • the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5- modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), or combinations thereof.
  • LNAs locked nucleic acids
  • bisLNAs Bis-locked nucleic acids
  • TAAs twisted intercalating nucleic acids
  • BNAs bridged nucleic acids
  • MGB minor groove binder
  • morpholino nucleic acids C5- modified pyrimidine nucleic acids
  • PNAs peptide nucleic acids
  • the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs), or combinations thereof.
  • the invasion primer includes locked nucleic acids (LNAs).
  • the invasion primer includes Bis-locked Attorney Docket No.: 051385-585001WO nucleic acids (bisLNAs).
  • the invasion primer includes twisted intercalating nucleic acids (TINAs).
  • the invasion primer includes bridged nucleic acids (BNAs).
  • the invasion primer includes 2’-O-methyl RNA:DNA chimeric nucleic acids.
  • the invasion primer includes minor groove binder (MGB) nucleic acids.
  • the invasion primer includes morpholino nucleic acids.
  • the invasion primer includes C5-modified pyrimidine nucleic acids.
  • the invasion primer includes peptide nucleic acids (PNAs).
  • the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs), or combinations thereof.
  • the binding sequence of the invasion primer includes synthetic nucleotides (e.g., one or more LNAs), and the tail sequence includes native nucleotides.
  • the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof.
  • the invasion primer includes phosphorothioate nucleic acids.
  • the invasion primer includes one or more locked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalized oligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids.
  • the invasion primer includes one or more locked nucleic acids (LNAs).
  • the invasion primer includes one or more 2-amino-deoxyadenosine (2- amino-dA). In embodiments, the invasion primer includes one or more trimethoxystilbene- functionalized oligonucleotides (TFOs). In embodiments, the invasion primer includes one or more Pyrene-functionalized oligonucleotides (PFOs). In embodiments, the invasion primer includes one or more peptide nucleic acids (PNAs). In embodiments, the invasion primer includes one or more aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In embodiments, the invasion primer includes 10 to 15 locked nucleic acids (LNAs).
  • the invasion primer includes a sequence described herein, for example within Table 1.
  • the invasion primer includes one or more phosphorothioates at the 5′ end.
  • the invasion primer includes one or more LNAs at the 5′ end.
  • the invasion primer Attorney Docket No.: 051385-585001WO includes two or more consecutive LNAs at the 3′ end.
  • the invasion primer includes two to four consecutive LNAs at the 3′ end.
  • the invasion primer includes two or more consecutive LNAs at the 5′ end.
  • the invasion primer includes two to four consecutive LNAs at the 5′ end.
  • the invasion primer includes one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence.
  • the invasion primer includes 2, 3, 4, 5, or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence.
  • the invasion primer includes a plurality of locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence.
  • the invasion primer includes one locked nucleic acid (LNA) at the 3′ end of the invasion primer sequence.
  • the invasion primer includes 2 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence.
  • the invasion primer includes 3 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 4 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 5 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. [0212] In embodiments, the invasion primer includes from 5′ to 3′ a plurality of synthetic nucleotides (e.g., LNAs) followed by a plurality (e.g., 2 to 5) canonical or native nucleotides (e.g., dNTPs).
  • LNAs synthetic nucleotides
  • dNTPs native nucleotides
  • the invasion primer comprises one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU).
  • the one or more dU nucleobases are at or near the 3’ end of the invasion primer (e.g., within 5 nucleotides of the 3’ end).
  • the invasion primer includes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followed by a plurality of synthetic nucleotides (e.g., LNAs), and subsequently followed by a plurality (e.g., 2 to 5) of canonical bases.
  • the invasion primer includes a plurality of canonical bases, wherein the canonical bases terminate (i.e., at the 3′ end) with a deoxyuracil nucleobase (dU).
  • the invasion primer includes the sequence provided in Table 1.
  • the first region of the invasion primer e.g., a first 3’ region of an invasion primer, wherein the invasion primer comprises two or more regions
  • the 5′ end of the sequences provided in Table 1 include one or more Attorney Docket No.: 051385-585001WO phosphorothioate nucleic acids.
  • the binding sequence of the invasion primer includes a sequence provided in Table 1. [0214] Table 1.
  • Morpholino nucleic acids are synthetic nucleotides that have standard nucleic acid bases (e.g., adenine, guanine, cytosine, and thymine) wherein those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates. Morpholino nucleic acids may be referred to as phosphorodiamidate morpholino oligomers (PMOs).
  • the invasion primer includes locked nucleic acids (LNAs).
  • the invasion primer includes LNAs dispersed throughout the primer, wherein about 2 to 5 nucleotides on the 3′ end are canonical dNTPs.
  • the entire composition of the invasion primer includes less than 50%, less than 40%, or less than 30% of LNAs.
  • the invasion primer includes peptide nucleic acids (PNAs).
  • PNA peptide nucleic acids
  • a PNA is a synthetic nucleic acid analogue wherein the nucleobases are arrayed along a neutral N-(2- aminoethyl)-glycine backbone in place of the negatively charged phosphate backbone of canonical DNA.
  • the unique pseudopeptide backbone is considered to be responsible for dramatically altering the interactions of nucleic acids and proteins with PNA. For example resulting in increased thermostability of PNA hybridization with DNA.
  • the invasion primer includes one or more PNAs and anneals to the dsDNA (e.g., the second strand) in a buffer containing less than 200nM NaCl, less than about 100 nM NaCl, or less than about 50 nM NaCl.
  • the invasion primer includes a plurality of LNAs interspersed throughout the polynucleotide.
  • the invasion primer includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the Attorney Docket No.: 051385-585001WO polynucleotide.
  • the entire composition of the invasion primer includes less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of LNAs.
  • the entire composition of the invasion primer includes up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, or up to about 5% of LNAs.
  • the entire composition of the invasion primer includes more than 60%, more than 50%, more than 40%, more than 30%, more than 20%, more than 10%, or more than 5% of LNAs. In embodiments, the entire composition of the invasion primer includes about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 60% to about 70% of LNAs. In embodiments, the entire composition of the invasion primer includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs.
  • the entire composition of the invasion primer includes about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of canonical dNTPs. In embodiments, the entire composition of the invasion primer includes less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of canonical dNTPs. In embodiments, the entire composition of the invasion primer includes up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, or up to about 30% of canonical dNTPs.
  • the entire composition of the invasion primer includes more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of canonical dNTPs.
  • the invasion primer includes about 70% of LNAs and about 30% of canonical dNTPs. In embodiments, the invasion primer includes about 65% of LNAs and about 35% of canonical dNTPs. In embodiments, the invasion primer includes about 60% of LNAs and about 40% of canonical dNTPs. In embodiments, the invasion primer includes about 55% of LNAs and about 45% of canonical dNTPs.
  • the invasion primer includes about 50% of LNAs and about 50% of canonical dNTPs. In embodiments, the invasion primer includes about 45% of LNAs and about 55% of canonical dNTPs. In embodiments, the invasion primer includes about 40% of LNAs and about 60% of canonical dNTPs. In embodiments, the invasion primer includes about 35% of LNAs and about 65% of canonical dNTPs. In embodiments, the Attorney Docket No.: 051385-585001WO invasion primer includes about 30% of LNAs and about 70% of canonical dNTPs. In embodiments, the invasion primer includes about 25% of LNAs and about 75% of canonical dNTPs.
  • the invasion primer includes about 20% of LNAs and about 80% of canonical dNTPs. In embodiments, the invasion primer includes about 15% of LNAs and about 85% of canonical dNTPs. In embodiments, the invasion primer includes about 10% of LNAs and about 90% of canonical dNTPs. In embodiments, the invasion primer includes about 5% of LNAs and about 95% of canonical dNTPs. [0220] In embodiments, the invasion primer includes one or more dT nucleobases that are replaced with dU nucleobases. In embodiments, the invasion primer includes a plurality of dT nucleobases that are replaced with dU nucleobases.
  • the invasion primer includes all dT nucleobases replaced with dU nucleobases.
  • the one or more dU nucleobases partition the invasion primer into two or more regions of consecutive nucleotides (e.g., a first plurality of consecutive nucleotides and a second plurality of consecutive nucleotides are separated by the one or more dU nucleobases).
  • each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length, or about 3 to about 15 nucleotides in length.
  • each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length.
  • each of the two or more regions of consecutive nucleotides are each about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each at least about 3, 5, 7, 10, 13, or 15 nucleotides in length. In embodiments, each of the two or more regions of consecutive nucleotides is greater than about 15 nucleotides in length. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 50°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 60°C to about 75°C.
  • the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 50°C to about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less Attorney Docket No.: 051385-585001WO than about 60°C. In embodiments, the dU and the LNA nucleotides are not adjacent to each other.
  • the dU and the LNA nucleotides are separated by one or more native nucleotides.
  • the invasion primer is about 10 to 100 nucleotides in length. In embodiments, the invasion primer is about 15 to about 90 nucleotides in length. In embodiments, the invasion primer is about 15 to about 75 nucleotides in length. In embodiments, the invasion primer is about 25 to about 75 nucleotides in length. In embodiments, the invasion primer is about 15 to about 50 nucleotides in length. In embodiments, the invasion primer is about 10 to about 20 nucleotides in length. In embodiments, the invasion primer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length.
  • the invasion primer is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 nucleotides in length. In embodiments, the invasion primer is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 nucleotides in length. In embodiments, the invasion primer is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length. In embodiments, the invasion primer is greater than 30 nucleotides in length. In embodiments, the invasion primer is greater than 40 nucleotides in length. In embodiments, the invasion primer is greater than 50 nucleotides in length. In embodiments, the invasion primer is no less than 20 nucleotides.
  • the invasion primer is about 15 to about 35 nucleotides in length. In embodiments, the invasion primer is about 25 to about 35 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the invasion primer is about 25 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, the invasion primer is about 30 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, the invasion primer is 30, 31, 32, or 33 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides.
  • the calculated or predicted melting temperature (Tm) of the invasion primer is about 70°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 80°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 85°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 85°C to about 90°C.
  • a method of incorporating a sequence including: hybridizing an invasion primer including a first sequence (e.g., a primer binding sequence) and a second sequence (e.g., a tail sequence) to a second strand of a double-stranded polynucleotide and extending the first sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand (e.g., partially hybridized), wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the second sequence of the invasion primer to generate an extended second strand including a complement of the second sequence, thereby incorporating a sequence (i.e., the complement of the second sequence) into the second strand of the double-stranded polynucleotide.
  • an invasion primer including a first sequence (e.g., a primer binding sequence) and a second sequence (e.g.,
  • the invasion primer hybridizes to a 3’ end of the second strand.
  • the polymerase extends the first sequence and the second strand simultaneously.
  • the same polymerase e.g., Bst polymerase
  • the method includes contacting the double stranded polynucleotide with amplification reagents (e.g., nucleotides and an enzyme).
  • the tail sequence includes a barcode sequence.
  • a barcode includes both a UMI and a spatial barcode.
  • a barcode includes two or more sub-barcodes that together function as a single barcode.
  • a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode (i.e., non-identifying) sequences.
  • the binding sequence includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and the tail sequence includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  • the binding sequence includes 10 to 25 nucleotides and the tail sequence comprises 5 to 25 nucleotides.
  • the binding sequence is greater than the tail sequence (e.g., the binding sequence include more nucleotides than the tail sequence).
  • the tail sequence includes a capture sequence, wherein the capture sequence is capable of hybridizing to a target polynucleotide.
  • the capture sequence includes a sequence capable of hybridizing to an endogenous region of a target polynucleotide.
  • the tail sequence is 10 to 25 nucleotides.
  • the Attorney Docket No.: 051385-585001WO tail sequence is downstream of the primer binding sequence.
  • the tail sequence is at the 5’ end of the invasion primer.
  • the tail sequence includes 10 to 30 nucleotides.
  • the tail sequence includes 2 to 20 nucleotides. In embodiments, the tail sequence includes 5 to 10 nucleotides. In embodiments, the tail sequence includes an index sequence and a barcode sequence.
  • endogenous is used in accordance with its ordinary meaning in the art and refers to an internal origin.
  • an endogenous gene sequence (also referred to herein as an endogenous region) is a polynucleotide sequence found within the original polynucleotide sequence.
  • an endogenous gene sequence is a polynucleotide sequence found within the original polynucleotide sequence in a biological sample.
  • the endogenous region includes a gene or a fragment thereof.
  • the first endogenous region includes a gene or a fragment thereof.
  • the second endogenous region includes a gene or a fragment thereof.
  • both the first endogenous region and the second endogenous region include a gene or a fragment thereof.
  • the endogenous region includes mRNA.
  • the endogenous region includes genomic DNA (e.g., exons, single nucleotide polymorphisms, mutable regions and/or highly conserved regions).
  • the endogenous region includes a genetic locus.
  • the endogenous region includes autosomal DNA and/or mitochondrial DNA.
  • the endogenous region includes a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, or rRNA.
  • an endogenous region may refer to the post hoc ligation (i.e., ligation after the fragmentation) of an exogenous sequence to one of the ends of the target polynucleotide.
  • an endogenous region of a target polynucleotide is a sequence of the target polynucleotide present prior to fragmentation.
  • the endogenous region of a target polynucleotide is not an adapter sequence.
  • the endogenous region of a target polynucleotide does not include a universal priming binding sequence.
  • the endogenous region does not include the P5, P7, or complementary sequences thereof (i.e., P5’ or P7’).
  • the P5 and P7 primers are used on the surface of commercial flow cells for sequencing on various Illumina platforms.
  • the P5 and P7 adapter sequences are described in U.S. Patent Publication No.2011/0059865 A1, which is incorporated herein by reference in its entirety.
  • the terms P5 and P7 may be used when referring to amplification primers, e.g., universal primers.
  • P5 P5 prime
  • P7 P7 Attorney Docket No.: 051385-585001WO prime
  • an endogenous sequence is not a synthetic sequence.
  • an endogenous sequence is a sequence found in nature.
  • an endogenous sequence is not a synthetic or engineered sequence.
  • the tail sequence includes a homopolymer sequence.
  • the homopolymer sequence includes consecutive identical nucleotides (e.g., a 5-mer of C nucleotides). In embodiments, the homopolymer sequence includes 10 to 30 consecutive identical nucleotides.
  • the homopolymer sequence includes 2 to 20 consecutive identical nucleotides. In embodiments, the homopolymer sequence includes 5 to 10 consecutive identical nucleotides. In embodiments, the homopolymer sequence includes poly (dA), poly (dT), poly (dC), poly (dG), or poly (dU) nucleotides. In embodiments, the tail sequence includes a poly(dT) sequence. In embodiments, the poly(dT) sequence includes 10 to 30 dT nucleotides. In embodiments, the poly(dT) sequence includes 2 to 20 dT nucleotides. In embodiments, the poly(dT) sequence includes 5 to 10 dT nucleotides.
  • the capture sequence includes nucleotides which are functionally or structurally analogous to poly-T and retain the functional property of binding to poly-A.
  • the capture sequence may include a poly-U oligonucleotide.
  • the capture sequence is nonspecific (e.g., intended to capture all RNAs containing a poly-A tail).
  • the capture sequence may further include additional sequences, such as random sequences, to facilitate the capture of specific subtypes of RNA.
  • the capture sequence may further include additional sequences to capture a desired subtype of RNA, such as mRNA or rRNA.
  • the capture sequence for each primer is the same.
  • the capture sequence for one or more probes is different from the capture sequence from at least one other probe. Additional embodiments of capture sequence may be found, for example, in PCT Publication No. WO2022/015913 and U.S. Patent Pub. No.2021/0317524, each of which is incorporated herein by reference in its entirety. [0231]
  • the capture sequence can be based on a particular gene sequence or particular motif sequence or common/conserved sequence, that it is designed to capture (i.e., a sequence-specific capture sequence).
  • the capture sequence is capable of binding selectively to a desired sub-type or subset of nucleic acid, for example a particular type of RNA, Attorney Docket No.: 051385-585001WO such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of RNA.
  • a desired sub-type or subset of nucleic acid for example a particular type of RNA, Attorney Docket No.: 051385-585001WO such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA,
  • the capture sequence can be capable of binding selectively to a desired subset of ribonucleic acids, for example, microbiome RNA, such as 16S rRNA.
  • a capture sequence includes an “anchor” or “anchoring sequence”, which is a sequence of nucleotides that is designed to ensure that the capture sequence hybridizes to the intended biological analyte.
  • an anchor sequence includes a sequence of nucleotides, including a 1-mer, 2-mer, 3-mer or longer sequence.
  • the short sequence is random.
  • a capture sequence including a poly(T) sequence can be designed to capture an mRNA.
  • an anchoring sequence can include a random 3-mer (e.g., GGG) that helps ensure that the poly(T) capture sequence hybridizes to an mRNA.
  • an anchoring sequence can be VN, N, or NN.
  • the sequence can be designed using a specific sequence of nucleotides.
  • the anchor sequence is at the 3′ end of the capture sequence. In some embodiments, the anchor sequence is at the 5′ end of the capture sequence. [0233] In some embodiments, capture sequences of capture probes are blocked prior to contacting the biological sample with the array, and blocking probes are used when the nucleic acid in the biological sample is modified prior to its capture on the array.
  • the blocking probe is used to block or modify the free 3′ end of the capture sequence.
  • blocking probes can be hybridized to the capture probes to mask the free 3′ end of the capture sequence, e.g., hairpin probes or partially double stranded probes.
  • the free 3′ end of the capture sequence can be blocked by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture probes do not include a free 3′ end.
  • 3′ modifications include dideoxy C-3′ (3′-ddC), 3′ inverted dT, 3′ C3 spacer, 3′Amino, and 3′ phosphorylation.
  • the nucleic acid in the Attorney Docket No.: 051385-585001WO biological sample can be modified such that it can be captured by the capture sequence.
  • an adaptor sequence (including a binding domain capable of binding to the capture domain of the capture probe) can be added to the end of the nucleic acid, e.g., fragmented genomic DNA. In some embodiments, this is achieved by ligation of the adaptor sequence or extension of the nucleic acid. In some embodiments, an enzyme is used to incorporate additional nucleotides at the end of the nucleic acid sequence, e.g., a poly(A) tail. In some embodiments, the capture probes can be reversibly masked or modified such that the capture sequence of the capture probe does not include a free 3′ end.
  • the 3′ end is removed, modified, or made inaccessible so that the capture domain is not susceptible to the process used to modify the nucleic acid of the biological sample, e.g., ligation or extension.
  • the capture sequence of the capture probe is modified to allow the removal of any modifications of the capture probe that occur during modification of the nucleic acid molecules of the biological sample.
  • the capture probes can include an additional sequence downstream of the capture sequence, i.e., 3′ to the capture domain, namely a blocking domain.
  • the capture sequence of the capture probe can be a non-nucleic acid domain.
  • the invasion primer includes, from 5’ to 3’, the capture sequence, optionally the barcode sequence, and the binding sequence.
  • the double-stranded polynucleotide includes a cleavage domain (i.e., a polynucleotide sequence including a cleavable site). In embodiments, the double-stranded polynucleotide includes one or more cleavable sites.
  • the first strand of the double-stranded polynucleotide includes a cleavable site.
  • the double-stranded polynucleotide includes a spatial barcode.
  • a “spatial barcode” is a nucleic acid sequence capable of conveying spatial information (e.g., xy coordinates) upon detection.
  • the barcode is associated with a particular location within an array or a particular location on a substrate. Attorney Docket No.: 051385-585001WO [0240]
  • the double-stranded polynucleotide does not include genomic DNA.
  • the plurality of target polynucleotides do not include a common sequence (e.g., a sequence universal to a substantial majority of the plurality, such as for example a sequence of an adapter).
  • the target polynucleotides do not include a common sequence (e.g., the same sequence within the plurality).
  • the target polynucleotides do not include a synthetic sequence (e.g., a primer binding sequence).
  • the target polynucleotide does not include a universal primer binding sequence (e.g., a polynucleotide sequence that is common to a majority of the target polynucleotides).
  • the target polynucleotide is a fragmented polynucleotide.
  • the target polynucleotide is genomic DNA (gDNA).
  • the target polynucleotide is genomic DNA (gDNA) including a sequence that encodes for a protein.
  • the method includes generating a first invasion strand by hybridizing a first invasion primer (e.g., an invasion primer that includes one or more PNAs or LNAs) to the first strand.
  • a first invasion primer e.g., an invasion primer that includes one or more PNAs or LNAs
  • the invasion primer does not hybridize at the end of the strand, rather the invasion primer hybridizes about 5 to about 50 nucleotides from the end of the strand.
  • the invasion primer hybridizes about 10 to about 30 nucleotides, about 12 to about 24, or about 15 to about 30 from the end of the strand.
  • the first invasion primer creates a “bubble” in the duplex (e.g., as depicted in FIGS.4A-4B).
  • a second invasion primer anneals to the second strand (e.g., within the bubble formed by annealing the first invasion primer) and is extended thereby generating a first invasion strand hybridized to the second strand.
  • the first invasion primer may remain during the first sequencing read, or may be removed prior to starting the first sequencing read.
  • the first invasion primer and the second invasion primer are not covalently attached to the solid support.
  • generating the invasion strand includes a plurality of invasion primer extension cycles, wherein each invasion primer extension cycle includes incorporating one or more nucleotides into the invasion primer.
  • generating the invasion strand includes extending the invasion primer by incorporating one or more nucleotides (e.g., dNTPs) using Bst large fragment (Bst LF) polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, or a mutant thereof.
  • the polymerase extends by incorporating a nucleotide to the 3’ end of the invasion primer.
  • the polymerase extends by incorporating a nucleotide to the 3’ end of an LNA nucleotide of the invasion primer.
  • generating the invasion strand includes a plurality of invasion-primer extension cycles by incorporating universal nucleobases (e.g., 5-nitroindole and/or inosine nucleobases) into the invasion primer.
  • the blocking strand does not need to be a faithful representation (i.e., an exact copy) of the strand to which the invasion primer is hybridized.
  • one or more inosine nucleotides or “universal” nucleotides may be incorporated into the primer to generate a blocking strand.
  • the term “universal nucleotide,” as used herein, refers to a nucleotide analog that is capable of forming a base pair to two or more (e.g., any of the four) natural nucleotide bases (e.g., cytosine (C), guanine (G), adenine (A), or thymine (T)).
  • any other base may be paired with a universal base analog in a double-stranded polynucleotide.
  • Universal nucleotides may be divided into hydrogen bonding bases and pi-stacking bases.
  • Hydrogen bonding bases form hydrogen bonds with any of the natural nucleobases.
  • the hydrogen bonds formed by hydrogen bonding bases are weaker than the hydrogen bonds between natural nucleobases.
  • Pi-stacking nucleobases are non-hydrogen bonding, hydrophobic, aromatic bases that stabilize duplex polynucleotides by stacking interactions.
  • Examples of hydrogen bonding bases include, but are not limited to, hypoxanthine (inosine), 7-deazahypoxanthine, 2-azahypoxanthine, 2-hydroxypurine, purine, and 4-Amino-1H- pyrazolo [3,4-d]pyrimidine.
  • pi-stacking bases include, but are not limited to, nitroimidazole, indole, benzimidazole, 5-fluoroindole, 5-nitroindole, N-indol-5-yl-formamide, isoquinoline, and methylisoquinoline.
  • the invasion strand includes at least a subset of nucleotides that are not universal nucleotides. In embodiments, at least 1% to 10% of the nucleotides in the invasion strand are universal nucleotides.
  • the blocking strand includes universal nucleobases.
  • the invasion strand is generated using an error-prone polymerase, for example Taq, a Y-family member Dpo4, or others known in the art (e.g., Rattray AJ and Strathern JN. Annu Rev Genet. 2003;37:31-66).
  • the blocking strand is not a copy of the strand the invasion primer is hybridized to. In embodiments, the blocking strand does not replicate the exact sequence of the strand to which the invasion primer is hybridized.
  • generating the invasion strand includes a first plurality of invasion- primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles.
  • generating the invasion strand includes alternating between a first plurality of invasion-primer extension cycles and a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles.
  • the reaction conditions for the first plurality of invasion-primer extension cycles include higher stringency hybridization conditions relative to the second plurality of invasion-primer extension cycles.
  • the reaction conditions for the first plurality of invasion-primer extension cycles include incubation in a first denaturant.
  • the first denaturant includes additives such as ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine, or tetramethylammonium chloride (TMAC).
  • the first denaturant is a buffered solution including about 0% to about 50% dimethyl sulfoxide (DMSO); about 0% to about 50% ethylene glycol; about 0% to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof.
  • the reaction conditions for the first plurality of invasion-primer extension cycles include incubation in a first denaturant, wherein the first denaturant is a buffered solution including about 15% to about 50% dimethyl sulfoxide (DMSO); about 15% to about 50% ethylene glycol; about 10% to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof.
  • the temperature is between 50°C and about 75°C, inclusive of the endpoints (i.e., the temperature may be 50°C, 52°C, or 75°C, etc.). In embodiments, the temperature is about 50°C to about 75°C.
  • the temperature is Attorney Docket No.: 051385-585001WO about 55°C to about 70°C. In embodiments, the temperature is about 60°C to about 70°C. In embodiments, the temperature is about 55°C to about 68°C. In embodiments, the buffered solution includes 5 ⁇ SSC. [0248] In embodiments, the reaction conditions for the second plurality of invasion-primer extension cycles include incubation in a second denaturant.
  • the second denaturant includes additives such as ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine, or tetramethylammonium chloride (TMAC), wherein the concentrations of the additives in the second denaturant differ than the concentrations of the additives in the first denaturant.
  • the second denaturant is a buffered solution including about 0 to about 50% dimethyl sulfoxide (DMSO); about 0 to about 50% ethylene glycol; about 0 to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof.
  • the reaction conditions for the second plurality of invasion-primer extension cycles include incubation in a second denaturant, wherein the second denaturant is a buffered solution including about 0% to about 15% dimethyl sulfoxide (DMSO); about 0 to about 15% ethylene glycol; about 0 to about 10% formamide; or about 0 to about 3M betaine, or a mixture thereof.
  • the temperature is between 50°C and about 75°C, inclusive of the endpoints (i.e., the temperature may be 50°C, 52°C, or 75°C, etc.). In embodiments, the temperature is about 50°C to about 75°C. In embodiments, the temperature is about 55°C to about 70°C.
  • the temperature is about 60°C to about 70°C. In embodiments, the temperature is about 55°C to about 68°C. In embodiments, the buffered solution includes 5 ⁇ SSC.
  • the first denaturant is a buffered solution including dimethyl sulfoxide (DMSO); and the second denaturant is a buffered solution including dimethyl sulfoxide (DMSO) and betaine. In embodiments, the first denaturant is a buffered solution including about 25 to about 35% DMSO; and the second denaturant is a buffered solution including about 0 to about 10% DMSO and about 1M to about 4M betaine.
  • the first denaturant is a buffered solution including about 30% DMSO; and the second denaturant is a buffered solution including about 5% DMSO, about 2.5M betaine.
  • the reaction conditions for the second plurality of invasion-primer extension cycles further includes incubation with a SSB protein. Attorney Docket No.: 051385-585001WO [0251]
  • generating the invasion strand comprises contacting the polynucleotide with one or more invasion- reaction mixtures.
  • generating the invasion strand includes contacting the double-stranded amplification product with one or more invasion-reaction mixtures; each of the invasion-reaction mixture including a plurality of invasion primers, a plurality of deoxyribonucleotide triphosphate (dNTPs), and a polymerase.
  • each of the invasion-reaction mixture including a plurality of invasion primers, a plurality of deoxyribonucleotide triphosphate (dNTPs), and a polymerase.
  • dNTPs deoxyribonucleotide triphosphate
  • generating the invasion strand includes contacting the double-stranded amplification product with a first invasion-reaction mixture followed by contacting the double-stranded amplification product with a second invasion-reaction mixture; the first invasion-reaction mixture including a plurality of invasion primers and no polymerase; and the second invasion-reaction mixture includes a plurality of deoxyribonucleotide triphosphate (dNTPs) and a polymerase.
  • the polymerase is a strand-displacing polymerase.
  • the strand-displacing polymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, or a mutant thereof.
  • the polymerase is template dependent.
  • the polymerase is not a TdT polymerase.
  • the polymerase is a strand-displacing or non-strand displacing polymerase.
  • the polymerase is a strand-displacing polymerase.
  • the strand-displacing polymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, or a mutant thereof.
  • the polymerase is Bst DNA Polymerase, Vent (exo-) DNA Polymerase, Pfu DNA polymerase, Taq polymerase, Phusion High-Fidelity DNA Polymerase, Q5 High-Fidelity DNA Polymerase, or mutant of any one of the foregoing.
  • the polymerase is Bst DNA Polymerase, Vent (exo-) DNA Polymerase, Phusion High-Fidelity DNA Polymerase, or Q5 High-Fidelity DNA Polymerase.
  • the polymerase is a Pyrococcus polymerase (e.g., a polymerase described in WO 2018/148723 or WO 2020/056044, each of which are incorporated herein by reference for all purposes).
  • the polymerase is a Bst DNA polymerase (e.g., exonuclease minus Bst), phi29 DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-) DNA polymerase, Klenow fragment of E.
  • the polymerase is a phi29 DNA Attorney Docket No.: 051385-585001WO polymerase 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).
  • the polymerase is a non-strand displacing polymerase.
  • the non-strand displacing polymerase is T4 DNA polymerase.
  • each of the plurality of invasion-reaction mixtures include a plurality of invasion primers, a plurality of deoxyribonucleotide triphosphate (dNTPs), a polymerase, or a combination thereof.
  • each of the plurality of invasion-reaction mixtures include a denaturant, single-stranded DNA binding protein (SSB), or both a denaturant and single- stranded DNA binding protein (SSB).
  • each invasion-reaction mixture further includes a denaturant, single-stranded DNA binding protein (SSB), or a combination thereof.
  • each invasion-reaction mixture includes a different amount of a denaturant, single- stranded DNA binding protein (SSB), or a combination thereof.
  • the denaturant is a buffered solution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4- methylmorpholine 4-oxide (NMO), TMAC, or a mixture thereof.
  • the denaturant is a buffered solution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, or a mixture thereof.
  • each invasion-reaction mixture includes a denaturant including an SSB, a strand-displacing polymerase, and one or more crowding agents.
  • the denaturant does not include a chemical denaturant (e.g., betaine, DMSO, ethylene glycol, formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof).
  • the SSB in the denaturant is T4 gp32 protein, SSB protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNA Binding Protein (ET- SSB).
  • KOD Thermococcus kodakarensis
  • TTH Thermus thermophilus
  • SSO Sulfolobus solfataricus
  • ET- SSB Extreme Thermostable Single-Stranded DNA Binding Protein
  • the strand-displacing polymerase in the denaturant is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Bsm DNA Polymerase, Phi29 polymerase, or a mutant thereof.
  • the crowding agent in the denaturant is poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or Attorney Docket No.: 051385-585001WO PEG 35,000).
  • PEG is present in the denaturant at a concentration of 1% to 25%. In embodiments, PEG is present in the denaturant at a concentration of about 1%, about 5%, about 10%, about 15%, about 20%, or about 25%. In embodiments, the denaturant is a buffered solution including T4 gp32 protein, Bsu polymerase, and 5 to 10% PEG 20,000. In embodiments, the denaturant is a buffered solution including T4 gp32 protein, Bsu polymerase, and 5% PEG 20,000. In embodiments, the denaturant is a buffered solution including T4 gp32 protein, Bsu polymerase, and 10% PEG 20,000.
  • the SSB is T4 gp32 protein, SSB protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB).
  • the SSB is active (i.e., has measurable activity) at temperatures less than about 72°C. In embodiments, the SSB is active (i.e., has measurable activity) at temperatures about 72°C.
  • the SSB is active (i.e., has measurable activity) at temperatures greater than about 72°C.
  • the method further includes contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof.
  • generating the invasion strand includes (i) forming a complex including a portion of the double-stranded amplification product, an invasion primer, and a homologous recombination complex including a recombinase, (ii) releasing the recombinase, and (iii) in a primer extension reaction, extending the invasion primer with a strand-displacing polymerase.
  • the strand-displacing polymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Bsm DNA Polymerase, Phi29 polymerase, or a mutant thereof.
  • the recombinase is a T4 UvsX, RecA, RecT, RecO, or Rad51 protein.
  • the homologous recombination complex further includes a crowding agent.
  • the crowding agent includes poly(ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), dextran, Ficoll (e.g., Ficoll 70 or Ficoll 400), glycerol, or a combination thereof.
  • PEG poly(ethylene glycol)
  • PVP polyvinylpyrrolidone
  • BSA bovine serum albumin
  • dextran dextran
  • Ficoll e.g., Ficoll 70 or Ficoll 400
  • glycerol e.g., Ficoll 70 or Ficoll 400
  • the crowding agent is poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, Attorney Docket No.: 051385-585001WO PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000), dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, ⁇ -lactoglobulin, hemoglobin, bovine serum albumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS).
  • PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000 Attorney Docket No.: 051385-585001WO PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000
  • dextran sulfate e.g., PEG 200, PEG 600, PEG
  • the crowding agent is PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000. In embodiments, the crowding agent is PEG 10,000, PEG 20,000, or PEG 35,000.
  • the homologous recombination complex further includes a loading factor, a single-stranded binding (SSB) protein, or both. In embodiments, the homologous recombination complex includes a single-stranded binding (SSB) protein.
  • the SSB protein is T4 gp32 protein, SSB protein, Extreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB), T7 gene 2.5 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or phi29 SSB protein.
  • the homologous recombination complex further includes a loading factor.
  • the loading factor includes a T4 UvsY protein.
  • generating the invasion strand includes thermally cycling between (i) about 72-80°C for about 5 seconds to about 30 seconds (referred to as cycle 1); and (ii) about 60- 70°C for about 30 to 90 seconds (referred to as cycle 2).
  • the method includes a plurality of thermal cycles in a periodic order (e.g., cycle type 1, cycle 2, cycle 1, etc.).
  • generating the invasion strand includes thermally cycling between (i) about 67- 80°C for about 5 seconds to about 30 seconds (referred to as cycle 1); and (ii) about 60-70°C for about 30 to 90 seconds (referred to as cycle 2).
  • the method includes a plurality of thermal cycles in a periodic order (e.g., cycle type 1, cycle 2, cycle 1, etc.).
  • one or more invasion primers transiently hybridize to the first or second strand.
  • the denaturing conditions in the invasion-reaction mix may be too stringent for the invasion primer to fully and stably hybridize for a significant time, however if a polymerase is present in the invasion-reaction mixture, the polymerase could still extend the invasion primer.
  • generating the first invasion strand includes transient hybridization of one or more invasion primers to the second strand, and extending the one or more invasion strand during their transient hybridization by a polymerase.
  • the invasion primer partially hybridizes (e.g., less than 100% of the invasion primer hybridizes) to Attorney Docket No.: 051385-585001WO the second strand.
  • the invasion primer hybridizes to the second strand and is extended with a polymerase.
  • the invasion primer does not remain fully annealed to the second strand while the polymerase extends the invasion primer.
  • at least three nucleotides of the invasion primer e.g., the three nucleotides at the 3′ end of the invasion primer
  • the strand-displacing enzyme is an SD polymerase, Bst large fragment polymerase, or a phi29 polymerase or mutant thereof.
  • the strand-displacing polymerase is phi29 polymerase, phi29 mutant polymerase or a thermostable phi29 mutant polymerase.
  • a “phi polymerase” is a DNA polymerase from the ⁇ 29 phage or from one of the related phages that, like ⁇ 29, contain a terminal protein used in the initiation of DNA replication.
  • phi29 polymerases include the B103, GA-1, PZA, ⁇ 15, BS32, M2Y (also known as M2), Nf, G1, Cp-1, PRD1, PZE, SFS, Cp-5, Cp-7, PR4, PR5, PR722, L17, ⁇ 21, and AV-1 DNA polymerases, as well as chimeras thereof.
  • a phi29 mutant DNA polymerase includes one or more mutations relative to naturally-occurring wild-type phi29 DNA polymerases, for example, one or more mutations that alter interaction with and/or incorporation of nucleotide analogs, increase stability, increase read length, enhance accuracy, increase phototolerance, and/or alter another polymerase property, and can include additional alterations or modifications over the wild-type phi29 DNA polymerase, such as one or more deletions, insertions, and/or fusions of additional peptide or protein sequences.
  • Thermostable phi29 mutant polymerases are known in the art, see for example US 2014/0322759, which is incorporated herein by reference for all purposes.
  • thermostable phi29 mutant polymerase refers to an isolated bacteriophage phi29 DNA polymerase including at least one mutation selected from the group consisting of M8R, V51A, M97T, L123S, G197D, K209E, E221K, E239G, Q497P, K512E, E515A, and F526 (relative to wild type phi29 polymerase).
  • the template polynucleotide includes genomic DNA, complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), or noncoding RNA (ncRNA).
  • cDNA complementary DNA
  • cfDNA cell-free DNA
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • cfRNA cell-free RNA
  • ncRNA noncoding RNA
  • the template polynucleotide is about 100 to 1000 nucleotides in length. In embodiments, the template polynucleotide is about 500 to 2000 nucleotides in length. In embodiments, the template polynucleotide is about 1000 to 1000 nucleotides in length.
  • the template polynucleotide is about 50 to 500 nucleotides in length. In embodiments, the template polynucleotide is about 500 to 1000 nucleotides in length. In embodiments, the template polynucleotide is about 350 nucleotides in length. In embodiments, the template polynucleotide is about 10, 20, 50, 100, 150, 200, 300, or 500 nucleotides in length.
  • the template polynucleotide molecules can vary length, such as about 100-300 nucleotides long, about 300-500 nucleotides long, or about 500-1000 nucleotides long.
  • the template polynucleotide molecular is about 100-1000 nucleotides, about 150-950 nucleotides, about 200-900 nucleotides, about 250-850 nucleotides, about 300-800 nucleotides, about 350- 750 nucleotides, about 400-700 nucleotides, or about 450-650 nucleotides.
  • the template polynucleotide molecule is about 150 nucleotides.
  • the template polynucleotide is about 100-1000 nucleotides long. In embodiments, the template polynucleotide is about 100-300 nucleotides long.
  • the template polynucleotide is about 300- 500 nucleotides long. In embodiments, the template polynucleotide is about 500-1000 nucleotides long. In embodiments, the template polynucleotide molecule is about 100 nucleotides. In embodiments, the template polynucleotide molecule is about 300 nucleotides. In embodiments, the template polynucleotide molecule is about 500 nucleotides. In embodiments, the template polynucleotide molecule is about 1000 nucleotides.
  • the template polynucleotide e.g., genomic template DNA
  • single-stranded linear fragments e.g., ranging in length from about 50 to about 600 nucleotides.
  • Treatment typically entails fragmentation, such as by chemical fragmentation, enzymatic fragmentation, or mechanical fragmentation, followed by denaturation to produce single-stranded DNA fragments.
  • the template polynucleotide includes an adapter.
  • the adapter may have other functional elements including tagging sequences (i.e., a barcode), attachment sequences, palindromic sequences, restriction sites, sequencing primer Attorney Docket No.: 051385-585001WO binding sites, functionalization sequences, and the like.
  • Barcodes can be of any of a variety of lengths.
  • the primer includes a barcode that is 10-50, 20-30, or 4-12 nucleotides in length.
  • the adapter includes a primer binding sequence that is complementary to at least a portion of a primer (e.g., a sequencing primer).
  • Primer binding sites can be of any suitable length.
  • a primer binding site is about or at least about 10, 15, 20, 25, 30, or more nucleotides in length.
  • a primer binding site is 10-50, 15-30, or 20- 25 nucleotides in length.
  • the template polynucleotide and the double-stranded amplification products include known adapter sequences on the 5′ and 3′ ends.
  • the template polynucleotide includes known adapter sequences on the 5′ and 3′ ends.
  • the double-stranded amplification products include known adapter sequences on the 5′ and 3′ ends.
  • the method prior to hybridizing the invasion primer the method includes amplifying the double-stranded polynucleotides with bridge polymerase chain reaction (bPCR) amplification, solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR, or combinations of said methods.
  • bPCR bridge polymerase chain reaction
  • RCA solid-phase rolling circle amplification
  • eRCA solid-phase exponential rolling circle amplification
  • RPA solid-phase recombinase polymerase amplification
  • HDA solid- phase helicase dependent amplification
  • template walking amplification or emulsion PCR, or combinations of said methods.
  • generating a double-stranded amplification product includes bridge polymerase chain reaction (bPCR) amplification, solid- phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR on particles, or combinations of the methods.
  • generating a double-stranded amplification product includes a bridge polymerase chain reaction amplification.
  • generating a double-stranded amplification product includes a thermal bridge polymerase chain reaction (t- bPCR) amplification.
  • generating a double-stranded amplification product includes a chemical bridge polymerase chain reaction (c-bPCR) amplification.
  • Chemical bridge polymerase chain reactions include fluidically cycling a denaturant (e.g., formamide) and maintaining the temperature within a narrow temperature range (e.g., +/-5°C).
  • thermal bridge polymerase chain reactions include thermally cycling between high temperatures (e.g., 85°C-95°C) and low temperatures (e.g., 60°C-70°C).
  • Thermal bridge polymerase chain Attorney Docket No.: 051385-585001WO reactions may also include a denaturant, typically at a much lower concentration than traditional chemical bridge polymerase chain reactions.
  • the solid support includes a plurality of polynucleotides, wherein each polynucleotide is attached to the solid support at a 5′ end of the polynucleotide.
  • generating a double-stranded amplification product includes amplifying the template polynucleotide or complement thereof on a solid support including a plurality of primers attached to the solid support, wherein the plurality of primers include a plurality of forward primers with complementarity to the template polynucleotide and a plurality of reverse primers with complementarity to a complement of the template polynucleotide, and the amplifying includes a plurality of cycles of strand denaturation, primer hybridization, and primer extension.
  • the plurality of strand denaturation cycles are different for one or more cycles, wherein the initial denaturation cycle is maintained at different conditions from the remaining denaturation cycles.
  • the initial denaturation cycle is at about 85°C-95°C for about 1 minute to about 10 minutes, whereas denaturation in the remaining cycles is different (e.g., about 85°C for about 15-30 sec).
  • the initial denaturation is maintained at about 85°C-95°C for about 5 minutes to about 10 minutes.
  • the initial denaturation is maintained at 90°C-95°C for about 1 to 10 minutes.
  • the initial denaturation is maintained at 80°C-85°C for about 1 to 10 minutes.
  • the initial denaturation is maintained at 85°C-90°C for about 1 to 10 minutes. In embodiments, the initial denaturation is maintained at about 85°C-95°C for about 1 minutes to about 10 minutes. In embodiments, the initial denaturation is maintained at about 95°C for about 5 minutes to about 10 minutes. In embodiments, the initial denaturation is maintained at about 85°C-95°C for about 5 minutes to about 10 minutes.
  • generating a double-stranded amplification product includes a thermal bridge polymerase chain reaction (t-bPCR) amplification.
  • the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 30 seconds for annealing/extension of the primer. Attorney Docket No.: 051385-585001WO [0274] In embodiments, the plurality of cycles includes thermally cycling between (i) about 80°C to 90°C for denaturation, and (ii) about 55°C to about 65°C for annealing/extension of the primer.
  • the plurality of cycles includes thermally cycling between (i) about 85°C for denaturation, and (ii) about 55°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for denaturation, and (ii) about 65°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) less than 80°C (e.g., 70 to 80°C) for denaturation, and (ii) about 55°C to about 65°C for annealing/extension of the primer.
  • 80°C e.g., 70 to 80°C
  • the plurality of cycles includes thermally cycling between (i) about 70°C for denaturation, and (ii) about 65°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 75°C for denaturation, and (ii) about 55°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for denaturation, and (ii) about 65°C for annealing/extension of the primer.
  • the plurality of cycles includes thermally cycling between (i) about 85°C for less than 1 minute for denaturation, and (ii) about 65°C for about 1 to 2 minutes for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for less than 1 minute for denaturation, and (ii) about 60°C to about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation and (ii) about 65°C for about 1 minute for annealing/extension of the primer.
  • the plurality of cycles includes thermally cycling between (i) about 85°C for about 30 sec for denaturation and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 30 seconds for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the temperature and duration for the annealing of the primer and the extension of the primer are different.
  • the plurality of cycles includes thermally cycling between (i) about 90°C to 95°C for about 15 to 30 sec for denaturation and (ii) about 55°C to about 65°C for about 30 to 60 Attorney Docket No.: 051385-585001WO seconds for annealing and about 65°C to 70°C for about 30 to 60 seconds for extension of the primer.
  • the plurality of denaturation steps is at a temperature of about 80°C- 95°C.
  • the plurality of denaturation steps is at a temperature of about 80°C- 90°C.
  • the plurality of denaturation steps is at a temperature of about 85°C- 90°C.
  • the plurality of denaturation steps is at a temperature of about 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, or about 90°C. In embodiments, the plurality of denaturation steps is at a temperature of about 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or about 99°C.
  • the plurality of denaturation steps is at a temperature of about 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or about 95°C. In embodiments, the plurality of denaturation steps is at a temperature of about 90°C, 91°C, 92°C, 93°C, 94°C, or about 95°C. In embodiments, the plurality of denaturation steps is at a temperature of about 70°C-85°C. In embodiments, the plurality of denaturation steps is at a temperature of about 70°C-80°C.
  • the plurality of denaturation steps is at a temperature of about 75°C-80°C. In embodiments, the plurality of denaturation steps is at a temperature of about 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or about 80°C. In embodiments, the annealing/extension of the primer cycle is at a temperature of about 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or about 65°C.
  • amplifying includes incubation in a denaturant.
  • the denaturant is acetic acid, ethylene glycol, hydrochloric acid, nitric acid, formamide, guanidine, sodium salicylate, sodium hydroxide, dimethyl sulfoxide (DMSO), propylene glycol, urea, or a mixture thereof.
  • the denaturant is an additive that lowers a DNA denaturation temperature.
  • the denaturant is betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4-methylmorpholine 4-oxide (NMO), or a mixture thereof.
  • the denaturant is betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, or 4-methylmorpholine 4-oxide (NMO).
  • amplifying includes a plurality of cycles of strand denaturation, primer hybridization, and primer extension. Although each cycle will include each of these three events (denaturation, hybridization, and extension), events within a cycle may or may not be discrete. For example, each step may have different reagents and/or reaction conditions (e.g., Attorney Docket No.: 051385-585001WO temperatures). Alternatively, some steps may proceed without a change in reaction conditions.
  • extension may proceed under the same conditions (e.g., same temperature) as hybridization. After extension, the conditions are changed to start a new cycle with a new denaturation step, thereby amplifying the amplicons.
  • Primer extension products from an earlier cycle may serve as templates for a later amplification cycle.
  • the plurality of cycles is about 5 to about 50 cycles. In embodiments, the plurality of cycles is about 10 to about 45 cycles. In embodiments, the plurality of cycles is about 10 to about 20 cycles. In embodiments, the plurality of cycles is about 20 to about 30 cycles. In embodiments, the plurality of cycles is 10 to 45 cycles. In embodiments, the plurality of cycles is 10 to 20 cycles. In embodiments, the plurality of cycles is 20 to 30 cycles.
  • the plurality of cycles is about 10 to about 45 cycles. In embodiments, the plurality of cycles is about 20 to about 30 cycles.
  • the double-stranded amplification product is provided in a clustered array. In embodiments, the clustered array includes a plurality of double-stranded amplification products localized to discrete sites on a solid support. In embodiments, the solid support is a bead. In embodiments, the solid support is substantially planar. In embodiments, the solid support is contained within a flow cell.
  • the method further includes removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on the second strand, and extending the second invasion primer with a polymerase, thereby generating a second invasion strand.
  • a method of sequencing including: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides Attorney Docket No.: 051385-585001WO so as to identify each incorporated nucleotide in the extension
  • the sequencing includes sequencing-by-synthesis, sequencing-by- binding, sequencing by ligation, or pyrosequencing.
  • generating a first sequencing read or a second sequencing read includes a sequencing by synthesis process.
  • generating a first sequencing read or a second sequencing read includes a sequencing-by-binding.
  • sequencing-by-binding refers to a sequencing technique wherein specific binding of a polymerase and cognate nucleotide to a primed template nucleic acid molecule (e.g., blocked primed template nucleic acid molecule) is used for identifying the next correct nucleotide to be incorporated into the primer strand of the primed template nucleic acid molecule.
  • the specific binding interaction need not result in chemical incorporation of the nucleotide into the primer.
  • the specific binding interaction can precede chemical incorporation of the nucleotide into the primer strand or can precede chemical incorporation of an analogous, next correct nucleotide into the primer.
  • the “next correct nucleotide” (sometimes referred to as the “cognate” nucleotide) is the nucleotide having a base complementary to the base of the next template nucleotide.
  • the next correct nucleotide will hybridize at the 3′-end of a primer to complement the next template nucleotide.
  • the next correct nucleotide can be, but need not necessarily be, capable of being incorporated at the 3′ end of the primer.
  • next correct nucleotide can be a member of a ternary complex that will complete an incorporation reaction or, alternatively, the next correct nucleotide can be a member of a stabilized ternary complex that does not catalyze an incorporation reaction.
  • a nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect” (or “non-cognate”) nucleotide.
  • sequencing includes a plurality of sequencing cycles. In embodiments, sequencing includes 10 to 100 sequencing cycles. In embodiments, sequencing includes 50 to 100 sequencing cycles. In embodiments, sequencing includes 50 to 300 sequencing cycles. In embodiments, sequencing includes 50 to 150 sequencing cycles.
  • sequencing includes at least 10, 20, 3040, or 50 sequencing cycles. In embodiments, sequencing includes at Attorney Docket No.: 051385-585001WO least 10 sequencing cycles. In embodiments, sequencing includes 10 to 20 sequencing cycles. In embodiments, sequencing includes 10, 11, 12, 13, 14, or 15 sequencing cycles. In embodiments, sequencing includes (a) extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue and (b) detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue. In embodiments, detecting includes two-dimensional (2D) or three-dimensional (3D) fluorescent microscopy.
  • the imaging is accomplished by confocal microscopy.
  • Confocal fluorescence microscopy involves scanning a focused laser beam across the sample, and imaging the emission from the focal point through an appropriately-sized pinhole. This suppresses the unwanted fluorescence from sections at other depths in the sample.
  • the imaging is accomplished by multi-photon microscopy (e.g., two-photon excited fluorescence or two-photon-pumped microscopy).
  • multi-photon microscopy can utilize much longer excitation wavelength up to the red or near-infrared spectral region. This lower energy excitation requirement enables the implementation of semiconductor diode lasers as pump sources to significantly enhance the photostability of materials. Scanning a single focal point across the field of view is likely to be too slow for many sequencing applications. To speed up the image acquisition, an array of multiple focal points can be used. The emission from each of these focal points can be imaged onto a detector, and the time information from the scanning mirrors can be translated into image coordinates.
  • the multiple focal points can be used just for the purpose of confining the fluorescence to a narrow axial section, and the emission can be imaged onto an imaging detector, such as a CCD, EMCCD, or s-CMOS detector.
  • an imaging detector such as a CCD, EMCCD, or s-CMOS detector.
  • a scientific grade CMOS detector offers an optimal combination of sensitivity, readout speed, and low cost.
  • One configuration used for confocal microscopy is spinning disk confocal microscopy.
  • 2-photon microscopy the technique of using multiple focal points simultaneously to parallelize the readout has been called Multifocal Two-Photon Microscopy (MTPM).
  • MTPM Multifocal Two-Photon Microscopy
  • the imaging is accomplished by light sheet fluorescence microscopy (LSFM).
  • detecting includes 3D structured illumination (3DSIM).
  • 3DSIM patterned light Attorney Docket No.: 051385-585001WO is used for excitation, and fringes in the Moiré pattern generated by interference of the illumination pattern and the sample, are used to reconstruct the source of light in three dimensions.
  • multiple spatial patterns are used to excite the same physical area, which are then digitally processed to reconstruct the final image. See York, Andrew G., et al. “Instant super-resolution imaging in live cells and embryos via analog image processing.” Nature methods 10.11 (2013): 1122-1126 which is incorporated herein by reference.
  • detecting includes selective planar illumination microscopy, light sheet microscopy, emission manipulation, pinhole confocal microscopy, aperture correlation confocal microscopy, volumetric reconstruction from slices, deconvolution microscopy, or aberration- corrected multifocus microscopy.
  • detecting includes digital holographic microscopy (see for example Manoharan, V. N. Frontiers of Engineering: Reports on Leading- edge Engineering from the 2009 Symposium, 2010, 5-12, which is incorporated herein by reference).
  • detecting includes confocal microscopy, light sheet microscopy, or multi-photon microscopy.
  • the method includes sequencing the first and/or the second strand of a double-stranded amplification product by extending a sequencing primer hybridized thereto.
  • a variety of sequencing methodologies can be used such as sequencing-by-synthesis (SBS), pyrosequencing, sequencing by ligation (SBL), or sequencing by hybridization (SBH).
  • Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res.11(1), 3-11 (2001); Ronaghi et al.
  • SBL methods include those described in Shendure et al. Science 309:1728-1732 (2005); U.S. Pat. Nos.5,599,675; and 5,750,341, each of which is incorporated herein by reference in its entirety; and the SBH methodologies are as described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Attorney Docket No.: 051385-585001WO Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which is incorporated herein by reference in its entirety.
  • sequencing is performed according to a “sequencing-by-binding” method (see, e.g., U.S. Pat. Pubs. US2017/0022553 and US2019/0048404, each of which is incorporated herein by reference in its entirety), which refers to a sequencing technique wherein specific binding of a polymerase and cognate nucleotide to a primed template nucleic acid molecule (e.g., blocked primed template nucleic acid molecule) is used for identifying the next correct nucleotide to be incorporated into the primer strand of the primed template nucleic acid molecule.
  • the specific binding interaction need not result in chemical incorporation of the nucleotide into the primer.
  • the specific binding interaction can precede chemical incorporation of the nucleotide into the primer strand or can precede chemical incorporation of an analogous, next correct nucleotide into the primer.
  • detection of the next correct nucleotide can take place without incorporation of the next correct nucleotide.
  • the “next correct nucleotide” (sometimes referred to as the “cognate” nucleotide) is the nucleotide having a base complementary to the base of the next template nucleotide. The next correct nucleotide will hybridize at the 3′-end of a primer to complement the next template nucleotide.
  • the next correct nucleotide can be, but need not necessarily be, capable of being incorporated at the 3′ end of the primer.
  • the next correct nucleotide can be a member of a ternary complex that will complete an incorporation reaction or, alternatively, the next correct nucleotide can be a member of a stabilized ternary complex that does not catalyze an incorporation reaction.
  • a nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect” (or “non-cognate”) nucleotide.
  • sequencing includes extending a sequencing primer to incorporate a nucleotide containing a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting of steps.
  • the methods include sequencing one or more bases of a target nucleic acid by extending a sequencing primer hybridized to a target nucleic acid (e.g., an amplification product of a target nucleic acid).
  • the sequencing includes sequencing-by-synthesis, sequencing-by-binding, sequencing by ligation, sequencing-by-hybridization, or pyrosequencing, and generates a sequencing read.
  • generating a sequencing read includes Attorney Docket No.: 051385-585001WO executing a plurality of sequencing cycles, each cycle including extending the sequencing primer by incorporating a nucleotide or nucleotide analogue using a polymerase and detecting a characteristic signature indicating that the nucleotide or nucleotide analogue has been incorporated.
  • SBS extension of a nucleic acid primer along a nucleic acid template is monitored to determine the sequence of nucleotides in the template.
  • the underlying chemical process can be catalyzed by a polymerase, wherein fluorescently labeled nucleotides are added to a primer (thereby extending the primer) in a template dependent fashion such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template.
  • a plurality of different nucleic acid fragments that have been attached at different locations of an array can be subjected to an SBS technique under conditions where events occurring for different templates can be distinguished due to their location in the array.
  • the sequencing step includes annealing and extending a sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting steps.
  • the methods include sequencing one or more bases of a target nucleic acid by extending a sequencing primer hybridized to a target nucleic acid (e.g., an amplification product produced by the amplification methods described herein).
  • the sequencing step may be accomplished by a sequencing-by-synthesis (SBS) process.
  • SBS sequencing-by-synthesis
  • sequencing comprises a sequencing by synthesis process, where individual nucleotides are identified iteratively, as they are polymerized to form a growing complementary strand.
  • nucleotides added to a growing complementary strand include both a label and a reversible chain terminator that prevents further extension, such that the nucleotide may be identified by the label before removing the terminator to add and identify a further nucleotide.
  • reversible chain terminators include removable 3’ blocking groups, for example as described in U.S. Pat. Nos. 10,738,072, 7,541,444 and 7,057,026.
  • Sequencing includes, for example, detecting a sequence of signals.
  • sequencing examples include, but are not limited to, sequencing by synthesis (SBS) processes in which reversibly terminated nucleotides carrying fluorescent dyes are incorporated into a growing strand, complementary to the target strand being sequenced.
  • SBS sequencing by synthesis
  • the nucleotides are labeled with up to four unique fluorescent dyes.
  • the nucleotides are labeled with at least two unique fluorescent dyes.
  • the readout is accomplished by epifluorescence imaging.
  • a variety of sequencing chemistries are available, non-limiting examples of which are described herein.
  • the method includes determining the nucleic acid sequence of the target polynucleotide.
  • the molecule further includes quantifying the target nucleic acid molecule or amplicons.
  • Methods for quantifying a target polynucleotide or amplicon are well known to one of skilled in the art. For example, during amplification of the target nucleic acid, quantitative techniques such as real-time polymerase chain reaction (RT-PCR) can be used to quantify the copy number of target nucleic acid molecules present in the clonal object as discussed in Logan et al. Real-Time PCR: Current Technology and Applications, Caister Academic Press. (2009).
  • RT-PCR real-time polymerase chain reaction
  • RT-PCR follows the general principle of polymerase chain reaction, however inclusion of detection molecules, such as non-specific fluorescent dyes that intercalate with any double-stranded DNA, or sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary DNA target, allows for the detection of nucleic acid formed during amplification.
  • detection molecules such as non-specific fluorescent dyes that intercalate with any double-stranded DNA, or sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary DNA target, allows for the detection of nucleic acid formed during amplification.
  • the rate of detectable molecules is proportional to the copy number of target nucleic acid molecules present in the clonal object.
  • quantifying the target nucleic acid molecule or amplicons can be done following amplification using standard gel electrophoresis and/or Southern
  • the method further includes sequencing the amplification product(s).
  • Sequencing includes, for example, detecting a sequence of signals within the particle.
  • Examples of sequencing include, but are not limited to, sequencing by synthesis (SBS) processes in which reversibly terminated nucleotides carrying fluorescent dyes are incorporated into a growing strand, complementary to the target strand being sequenced.
  • the nucleotides are labeled with up to four unique fluorescent dyes.
  • the readout is accomplished by epifluorescence imaging.
  • a variety of sequencing chemistries are available, non-limiting examples of which are described herein.
  • each cluster of capture probes e.g., each cluster of a plurality of immobilized double-stranded polynucleotides including the tail sequence, wherein the tail sequence includes a capture sequence
  • the substrate may be manufactured by immobilizing one or more polynucleotides on the surface of the substrate (e.g., by binding to a surface probe on the substrate) and generating clusters of double-stranded polynucleotides (e.g., by bridge amplification), as described herein.
  • the double-stranded polynucleotides may include a spatial barcode, as described above.
  • the determination of the location of each cluster of double-stranded polynucleotides on the surface may be determined by sequencing the double-stranded polynucleotides on the substrate.
  • sequencing primers targeting the spatial barcode may be utilized, and the sequence of the spatial barcode may be determined.
  • the sequence of the spatial barcode for each cluster may be assigned to a specific location on the substrate (e.g., an XY coordinate on the substrate).
  • a high- resolution map of the substrate may be generated based upon the signal detected during sequencing (e.g., the fluorescent signal) and used to assign an XY coordinate to each cluster on the substrate.
  • the method further includes incorporating a tail sequence into a strand of the double-stranded polynucleotide (e.g., the capture probe).
  • the method further includes hybridizing an invasion primer including a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail Attorney Docket No.: 051385-585001WO sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence, thereby incorporating a sequence (e.g., a capture sequence) into the second strand of the double-stranded polynucle
  • the methods for spatial detection of nucleic acid (e.g. RNA) in a tissue sample further include correlating the sequence of the spatial barcode for each sequenced cDNA molecule with the location of the cluster of capture probes on the substrate having the corresponding spatial barcode.
  • the first strand cDNA will contain the same spatial barcode as the capture probe, whereas the second strand cDNA will contain the complement to the spatial barcode of the capture probe.
  • “Corresponding” as used herein covers each of these possibilities, depending on which cDNA strand is sequenced. For instance, if the second strand cDNA is sequenced, the sequence of the second strand cDNA is correlated with the location of the cluster of capture probes on the substrate having the complementary spatial barcode.
  • the sequence of the first strand cDNA is correlated with the location of the cluster of capture probes on the substrate having the same spatial barcode.
  • the plurality of capture probes e.g., a plurality of immobilized double-stranded polynucleotides including the tail sequence, wherein the tail sequence includes a capture sequence
  • the plurality of capture probes is arranged in clusters on the surface of the substrate, each cluster including multiple capture probes.
  • each capture probe in a cluster includes the same spatial barcode.
  • the spatial barcode for each cluster is unique.
  • the methods further include generating cDNA molecules from the bound RNA molecules.
  • the cDNA generated is considered to be indicative of the RNA present in a cell at the time in which a tissue sample was taken. Therefore, cDNA represents all or some of the genes that were expressed in the cell at the time the tissue sample was taken.
  • the capture probe acts as a primer for reverse transcription, such that the sequence of the capture probe is incorporated into the sequence of the first strand cDNA molecule along with the sequence complementary to the captured RNA strand.
  • the spatial barcode of the Attorney Docket No.: 051385-585001WO capture probe is incorporated into the sequence of the first strand cDNA molecule.
  • Generating cDNA molecules from the bound RNA molecules may be performed by any suitable method. For example, generating cDNA molecules from the bound RNA molecules may be performed by addition of a reverse transcriptase to facilitate reverse transcription of the RNA (e.g., mRNA) to generate a complementary or copy DNA (i.e., cDNA).
  • the cDNA resulting from the reverse transcription of RNA is referred to herein as “first strand cDNA”.
  • First strand cDNA synthesis (e.g., reverse transcription) may be performed directly on the substrate.
  • the reverse transcription reaction includes a reverse transcriptase, dNTPs and a suitable buffer.
  • the reaction mixture may include other components, such as RNase inhibitor(s).
  • Each dNTP is typically present in an amount ranging from about 10 to 5000 mM, usually from about 20 to 1000 mM.
  • Any suitable reverse transcriptase enzyme may be used. Suitable enzymes include: M-MLV, MuLV, AMV, HIV, ArrayScriptTM, MultiScribeTM, ThermoScriptTM, and Superscript® I, II, and III enzymes.
  • the reverse transcriptase reaction may be carried out at any suitable temperature, which is dependent on the properties of the enzyme.
  • reverse transcriptase reactions are performed between 37-55°C, although temperatures outside of this range may also be appropriate.
  • the reaction time may be as little as 1, 2, 3, 4 or 5 minutes or as much as 48 hours.
  • the reaction is carried out for between 3-12 hours, although other suitable reaction times (e.g., overnight) may be used.
  • a strand complementary to the first strand cDNA may be synthesized.
  • the strand complementary to the first strand cDNA is referred to herein as “second strand cDNA”.
  • the term “cDNA” as used herein is used in the broadest sense and refers to any cDNA, including first strand cDNA and second strand cDNA.
  • “generating cDNA” includes performing second strand synthesis (e.g., following the reverse transcription reaction) to generate second strand cDNA.
  • second strand cDNA synthesis may occur without increasing the number of copies of the second strand cDNA (e.g., without amplifying the second strand).
  • second strand cDNA may be synthesized and amplified, resulting in multiple copies of the second strand.
  • Second strand cDNA synthesis if performed, may be performed on the substrate (e.g., while the cDNA is immobilized on the substrate). Alternatively, the first strand cDNA may be released from the substrate and second strand cDNA synthesis may be performed in solution.
  • the second strand Attorney Docket No.: 051385-585001WO cDNA includes a complement of the capture probe and therefore includes a complement of the spatial barcode sequence of the capture probe.
  • the second strand cDNA may be amplified using a suitable primer or combination of primers upstream of the complement to the spatial barcode sequence, such that the complement of the spatial barcode sequence is presence in each amplified second strand cDNA.
  • second strand cDNA synthesis is performed using random primers.
  • the first strand cDNA may be incubated with random primers, such as hexamer primers, and a DNA polymerase, under conditions sufficient for synthesis of the complementary DNA strand (e.g., second strand cDNA) to form.
  • the second strand cDNA may be isolated, purified and amplified following synthesis.
  • the second strand cDNA may be synthesized by a suitable method as described above (e.g., using random primers).
  • the secondary strand cDNA may be isolated through DNA denaturation in any solutions with high pH and/or organic solutions that can denature the DNA.
  • the secondary strand cDNA may be isolated through heat denaturation. The isolated second strand may be purified, and then amplified by PCR.
  • Primers for PCR amplification of the second strand cDNA may be any suitable primers, including primers targeting the additional features (e.g., primer binding sites, sequencing barcodes, unique molecular identifiers) added to the second strand cDNA. Any suitable number of isolation, amplification, and purification steps may be performed to generate the final library of cDNA prior to sequencing.
  • the capture probes used for the initial capture of RNA e.g., mRNA
  • the capture probes may contain one or more additional features (e.g., additional to the spatial barcode and capture domain) that facilitate sequencing library preparation.
  • the capture probes may contain a sequencing handle (e.g., sequencing barcode). Therefore, the complement of the sequencing barcode will be present in the cDNA.
  • cDNA generated by the methods described herein may include two distinct sequencing barcodes.
  • the cDNA may include sequencing barcode(s) compatible with a Singular Genomics TM or Illumina TM sequencing platform.
  • the cDNA includes sequencing barcode(s), a spatial barcode, and/or a unique molecular identifier. These additional features may facilitate library preparation, sequencing, and spatial detection of RNA by the methods described herein.
  • the generated cDNA may be sequenced with no intervening treatment steps prior to sequencing.
  • tissue samples that include large amounts of RNA may yield a sufficient amount of cDNA such that it may be sequenced directly.
  • it may be desirable to generate double stranded DNA without increasing the number of double stranded DNA molecules.
  • one or multiple amplification reactions may be conducted to generate multiple copies of single stranded or double stranded DNA.
  • generation of cDNA e.g., by reverse transcription of the RNA bound to the capture probes
  • the generated cDNA may be released from the substrate prior to subsequent treatment steps.
  • the cDNA may be generated on the substrate and the generated DNA may be released from the substrate and collected in a tube.
  • Subsequent steps e.g., second strand cDNA synthesis, amplification, sequencing, etc.
  • RNA may be removed prior to subsequent treatment of the cDNA strand.
  • RNA may be removed using an RNA digesting enzyme (e.g., RNase).
  • RNase RNA digesting enzyme
  • no specific RNA removal step is necessary, as RNA will degrade naturally and/or removal of the tissue from the substrate is sufficient for RNA removal.
  • the methods for spatial detection of nucleic acid (e.g. RNA) in a tissue sample further include sequencing the cDNA molecules.
  • the cDNA molecules may be sequenced on the substrate or may be released and collected into a suitable device (e.g., a tube) prior to sequencing. Sequencing may be performed by any suitable method.
  • the full length of the cDNA molecules may be sequenced.
  • RNA transcript specific sequence data may be obtained.
  • the sequencing may be performed to determine the sequence of the spatial barcode and at least 50, 60, 70, 80, 90, or 100 bases of RNA transcript specific data.
  • the methods for spatial detection of nucleic acid (e.g. RNA) in a tissue sample further include imaging the tissue after contacting the tissue with the substrate (e.g., after immobilizing the tissue on the substrate). Imaging the tissue may assist in the determination of the spatial location of RNA molecules within the tissue sample.
  • imaging the tissue is performed before generating cDNA.
  • imaging the tissue is performed after generating cDNA.
  • the method prior to imaging, the method further includes permeabilizing the immobilized tissue section.
  • the method does not include permeabilizing the immobilized tissue section.
  • the method further includes contacting the immobilized tissue section with one or more imaging reagents or stains.
  • the tissue section is contacted with one or more imaging reagents or stains.
  • the tissue section is contacted with one or more imaging reagents or stains without permeabilization.
  • the imaging reagents or stains include hematoxylin and eosin (H&E) staining reagents.
  • the imaging includes phase-contrast microscopy, bright-field microscopy, Nomarski differential-interference-contrast microscopy, dark field microscopy, electron microscopy, or cryo-electron microscopy.
  • the imaging reagents or stains include phase-contrast microscopy, bright-field microscopy, Nomarski differential- interference-contrast microscopy, or dark field microscopy imaging reagents.
  • the light transmittance of the sample is measured.
  • light transmittance may be measured with a visible near-infrared optical fiber spectrometer, wherein a circular spot of light (e.g., diameter, 5 mm) is irradiated on the central part a sample and the transmitted light is collected using an optical sensor.
  • a circular spot of light e.g., diameter, 5 mm
  • the transmitted light is collected using an optical sensor.
  • the imaging reagents or stains include electron microscopy (e.g., transmission electron microscopy or scanning electron microscopy) or cryo-electron microscopy imaging reagents.
  • Examples of electron microscopy contrast agents may include one or more heavy metals (e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium) and/or antibodies bound to one or more types of heavy metals (e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium).
  • heavy metals e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium
  • immunogold labels that may be used to contact the tissue section include may include different antibodies bound to gold particles of different sizes to image different molecules of interest.
  • the method may include contacting the tissue section with heavy metals.
  • the tissue section includes a tissue portion or a cell (e.g. plurality of cells such as blood cells). In embodiments, the tissue section includes one or more cells.
  • the tissue section is embedded in an embedding material including paraffin wax, polyepoxide polymer, polyacrylic polymer, agar, gelatin, celloidin, cryogel, optimal cutting temperature (OCT) compositions, glycols, or a combination thereof.
  • OCT optimal cutting temperature
  • the tissue section is embedded in an embedding material including paraffin wax.
  • the OCT composition includes about 10% polyvinyl alcohol and about 4% polyethylene glycol.
  • the OCT composition includes sucrose (e.g., 30% sucrose).
  • the OCT composition is Tissue Freezing Medium (TFM) available from Leica Microsystems, Catalog #14020108926.
  • the tissue is immobilized to the substrate (e.g., the solid support including one or more clusters of the first plurality of capture probes and the second plurality of capture probes) by covalently binding the tissue to one or more bioconjugate reactive moieties of the substrate.
  • the tissue is immobilized to the substrate by non-covalently binding the tissue to the substrate.
  • the tissue sections attach to the substrate surface due to surface interactions, such as Van der Waal forces, electrostatic forces, Attorney Docket No.: 051385-585001WO hydrophobic interactions and hydrogen bonds.
  • the physical adsorption efficiency can be enhanced by treating the material with air plasma to increase its hydrophilicity.
  • the substrate includes a functionalized glass surface or a functionalized plastic surface.
  • the functionalized glass surface includes (3- aminopropyl)triethoxysilane (APTES), (3-Aminopropyl)trimethoxysilane (APTMS), ⁇ - Aminopropylsilatrane (APS), N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6-epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof.
  • APTES (3- aminopropyl)triethoxysilane
  • APITMS (3-Aminopropyl)trimethoxysilane
  • APS ⁇ - Aminopropylsilatrane
  • AHAMTES N-(6-aminohexyl)aminomethyltriethoxysilane
  • PEI polyeth
  • Tissue sections include tissue or organ samples obtained from a subject, e.g., a mammal.
  • the subject is diagnosed with a disease or disorder, such as a cancerous tumor, or considered at risk of having or developing the disease or disorder.
  • Tissue sections may also be obtained from healthy donors, e.g., as normal control samples.
  • both a disease tissue (e.g., a tumor tissue) sample and a normal sample are obtained from the same subject.
  • the tissue section is obtained from a patient, e.g., a mammal such as a human.
  • a tissue section is obtained from an animal model of disease.
  • animal models of disease are known and available in the art.
  • tissue section includes a tissue or a cell.
  • Biological tissue samples suitable for use with the methods and systems described herein generally include any type of tissue samples collected from living or dead subjects, such as, for example, tumor tissue and autopsy samples.
  • Tissue samples may be collected and processed using the methods and systems described herein and subjected to microscopic analysis immediately following processing, or may be preserved and subjected to microscopic analysis at a future time, e.g., after storage for an extended period of time.
  • the methods described herein may be used to preserve tissue samples in a stable, accessible and fully intact form for future analysis.
  • tissue samples such as, e.g., human tumor tissue samples, may be processed as described herein and cleared to remove a plurality of cellular components, such as, e.g., lipids, and then stored for future analysis.
  • the methods and systems described herein may be used to analyze a fresh tissue section.
  • tissue sections are tumor tissue samples. Tumor samples may contain only tumor cells, or they may contain both tumor cells and non-tumor cells. In particular embodiments, a tissue section includes only non-tumor cells. In particular embodiments, the tumor is a solid tumor.
  • the tissue section is obtained from or includes an adrenal cortical cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, head or neck cancer, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, myelodysplasia syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal
  • the methods of the invention can be used to characterize a cancer or metastasis thereof, including without limitation, a carcinoma, a sarcoma, a lymphoma or leukemia, a germ cell tumor, a blastoma, or other cancers.
  • Carcinomas include without limitation epithelial neoplasms, squamous cell neoplasms squamous cell carcinoma, basal cell neoplasms basal cell carcinoma, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (glands), adenoma, adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma, grawitz tumor, multiple endocrine adenomas, endometrioid adenoma, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous
  • Sarcoma includes without limitation Askin's tumor, botryodies, chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcomas including: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and
  • Lymphoma and leukemia include without limitation chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, Attorney Docket No.: 051385-585001WO T cell prolymphocytic leukemia, T cell large gran
  • Germ cell tumors include without limitation germinoma, dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonal carcinoma, endodermal sinus turmor, choriocarcinoma, teratoma, polyembryoma, and gonadoblastoma.
  • Blastoma includes without limitation nephroblastoma, medulloblastoma, and retinoblastoma.
  • cancers include without limitation labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma
  • the cancer under analysis may be a lung cancer including non- small cell lung cancer and small cell lung cancer (including small cell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma, and combined small cell carcinoma), colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, or a solid tumor.
  • non- small cell lung cancer and small cell lung cancer including small cell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma, and combined small cell carcinoma
  • colon cancer breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblast
  • Tissue sections may be obtained from a subject by any means known and available in the art.
  • a tissue section e.g., a tumor tissue sample
  • the surgical biopsy is an incisional biopsy, which removes only part of the suspicious area.
  • the surgical biopsy is an excisional biopsy, which removes the entire diseased tissue (e.g., tumor) or abnormal area.
  • an excisional tumor tissue sample is obtained from a tumor that has been excised with the intent to “cure” a patient in the case of early stage disease, wherein in other embodiments, the excisional tumor tissue sample is obtained from an excised bulk of primary tumor in later stage disease.
  • Tumor tissue samples may include primary tumor tissue, metastastic tumor tissue and/or secondary tumor tissue.
  • Tumor tissue samples may be cell cultures, e.g., cultures of tumor-derived cell lines.
  • a tissue section is a cell line, e.g., a cell pellet of a cultured cell line, such as a tumor cell line.
  • the cell line or cell pellet is frozen or was previously frozen.
  • Tumor tissue samples may also be xenograft tumors, e.g., tumors obtained from animals administered with tumor cells, e.g., a human tumor cell line.
  • a first tumor tissue sample from a subject is a primary tumor tissue sample obtained during an initial surgery intended to remove the entire tumor
  • a second tumor tissue sample is obtained from the same subject is a metastatic tumor tissue sample or a secondary tumor tissue sample obtained during a later surgery.
  • Tissue sections e.g., tumor tissue samples, may be obtained surgically or using a laparoscope.
  • a tissue section may be a tissue sample obtained from any part of the body to examine it for disease or injury, e.g., presence of cancer tissue or cells, or the extent or characteristics thereof.
  • the tissue section includes abdominal tissue, bone, bone marrow, breast tissue, endometrial tissue, kidney tissue, liver tissue, lung or chest tissue, lymph node, nerve tissue, skin, testicular tissue, head or neck tissue, or thyroid tissue.
  • the tissue is obtained from brain, breast, skin, bone, joint, skeletal muscle, smooth muscle, red bone marrow, thymus, lymphatic vessel, thoracic duct, spleen, lymph node, nasal cavity, pharynx, larynx, trachea, bronchus, lung, oral cavity, esophagus, liver, stomach, small intestine, large intestine, rectum, anus, spinal cord, nerve, pineal gland, pituitary gland, Attorney Docket No.: 051385-585001WO thyroid gland, thymus, adrenal gland, pancreas, ovary, testis, heart, blood vessel, kidney, uterus, urinary bladder, urethra, prostate gland, penis, prostate, testis, scrotum, ductus deferens, mammary glands, ovary, uterus, vagina, or uterine tube.
  • a tissue section has a size greater than sections typically examined by traditional pathology thin section or immunohistochemical analysis, which are typically in the range of 4-10 microns thick.
  • a tissue section is greater than 20 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, greater than 500 microns, greater than 1 mm, greater than 2 mm, greater than 5 mm, greater than 10 mm or greater than 20 mm in thickness and/or length.
  • the tissue section has a length and/or a thickness between 20 microns and 20 mm, between 20 microns and 10 mm, or between 50 microns and 1 mm.
  • a tissue section is a cubic sample with each side greater than 10 microns, greater than 20 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, greater than 500 microns, greater than 1 mm, greater than 2 mm, greater than 5 mm, greater than 10 mm, or greater than 2 mm in thickness and/or length.
  • a tissue section is thinner, e.g., from about 4-10 or 4-20 microns in thickness.
  • RNA including mRNA, is highly susceptible to degradation upon exposure to one or more RNAses. RNAses are present in a wide range of locations, including water, many reagents, laboratory equipment and surfaces, skin, and mucous membranes.
  • RNAse cleaning product e.g., RNASEZAPTM and other commercially available products or 0.5% sodium dodecyl sulfate [SDS] followed by 3% H 2 O 2
  • SDS sodium dodecyl sulfate
  • barrier tips e.g., baking designated glassware (e.g., 300° C.
  • the tissue section forms part of a tissue in situ.
  • the tissue section includes one or more prokaryotic cells.
  • the tissue section includes one or more eukaryotic cells.
  • the tissue section includes a bacterial cell (e.g., a bacterial cell or bacterial spore), a fungal cell (e.g., a fungal spore), a plant cell, or a mammalian cell.
  • the tissue section includes a stem cell.
  • the stem cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, or an induced pluripotent stem cell.
  • the tissue section includes an endothelial cell, muscle cell, myocardial, smooth muscle cell, skeletal muscle cell, mesenchymal cell, epithelial cell; hematopoietic cell, such as lymphocytes, including T cell, e.g., (Th1 T cell, Th2 T cell, ThO T cell, cytotoxic T cell); B cell, pre-B cell; monocytes; dendritic cell; neutrophils; or a macrophage.
  • the tissue section includes a stem cell, an immune cell, a cancer cell (e.g., a circulating tumor cell or cancer stem cell), a viral-host cell, or a cell that selectively binds to a desired target.
  • the cell includes a T cell receptor gene sequence, a B cell receptor gene sequence, or an immunoglobulin gene sequence.
  • the cell includes a Toll-like receptor (TLR) gene sequence.
  • TLR Toll-like receptor
  • the cell includes a gene sequence corresponding to an immunoglobulin light chain polypeptide and a gene sequence corresponding to an immunoglobulin heavy chain polypeptide.
  • the tissue section includes a genetically modified cell.
  • the tissue section includes a circulating tumor cell or cancer stem cell.
  • the tissue section includes a prokaryotic cell.
  • the tissue section includes a bacterial cell.
  • the bacterial cell is a Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, or Bifidobacterium cell.
  • the bacterial cell is a Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Enterococcus faecalis, Escherichia coli, Enterobacter sp., Klebsiella sp., Bifidobacterium bifidum, Staphylococcus aureus, Lactobacillus, Clostridium perfringens, Proteus mirabilis, Clostridium tetani, Clostridium septicum, Pseudomonas aeruginosa, Salmonella enterica, Faecalibacterium prausnitzii, Peptostreptococcus sp., or Peptococcus sp.
  • tissue section includes a fungal cell.
  • the fungal cell is a Candida, Saccharomyces, Aspergillus, Penicillium, Rhodotorula, Trametes, Pleospora, Sclerotinia, Bullera, or a Galactomyces cell. Attorney Docket No.: 051385-585001WO [0318]
  • the tissue section includes a viral-host cell.
  • a “viral-host cell” is used in accordance with its ordinary meaning in virology and refers to a cell that is infected with a viral genome (e.g., viral DNA or viral RNA). The cell, prior to infection with a viral genome, can be any cell that is susceptible to viral entry.
  • the viral-host cell is a lytic viral-host cell. In embodiments, the viral-host cell is capable of producing viral protein. In embodiments, the viral-host cell is a lysogenic viral-host cell. In embodiments, the cell is a viral- host cell including a viral nucleic acid sequence, wherein the viral nucleic acid sequence is from a Hepadnaviridae, Adenoviridae, Herpesviridae, Poxviridae, Parvoviridae, Reoviridae, Coronaviridae, Retroviridae virus. [0319] In embodiments, the tissue section includes an adherent cell (e.g., epithelial cell, endothelial cell, or neural cell).
  • adherent cell e.g., epithelial cell, endothelial cell, or neural cell.
  • Adherent cells are usually derived from tissues of organs and attach to a substrate (e.g., epithelial cells adhere to an extracellular matrix coated substrate via transmembrane adhesion protein complexes).
  • Adherent cells typically require a substrate, e.g., tissue culture plastic, which may be coated with extracellular matrix (e.g., collagen and laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation.
  • the tissue section includes a neuronal cell, an endothelial cell, epithelial cell, germ cell, plasma cell, a muscle cell, peripheral blood mononuclear cell (PBMC), a myocardial cell, or a retina cell.
  • PBMC peripheral blood mononuclear cell
  • the tissue section includes a suspension cell (e.g., a cell free-floating in the culture medium, such a lymphoblast or hepatocyte).
  • the tissue section includes a glial cell (e.g., astrocyte, radial glia), pericyte, or stem cell (e.g., a neural stem cell).
  • the tissue section includes a neuronal cell.
  • the tissue section includes an endothelial cell.
  • the tissue section includes an epithelial cell.
  • the tissue section includes a germ cell.
  • the tissue section includes a plasma cell.
  • the tissue section includes a muscle cell.
  • the tissue section includes a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • the tissue section includes a myocardial cell.
  • the tissue section includes a retina cell.
  • the tissue section includes a lymphoblast.
  • the tissue section includes a hepatocyte.
  • the tissue section includes a glial cell.
  • the tissue section includes an astrocyte.
  • the tissue section includes a radial glia.
  • the tissue Attorney Docket No.: 051385-585001WO section includes a pericyte.
  • the tissue section includes a stem cell.
  • the tissue section includes a neural stem cell.
  • the tissue section includes a cell bound to a known antigen.
  • the cell is a cell that selectively binds to a desired target, wherein the target is an antibody, or antigen binding fragment, an aptamer, affimer, non-immunoglobulin scaffold, small molecule, or genetic modifying agent.
  • the cell is a leukocyte (i.e., a white-blood cell).
  • leukocyte is a granulocyte (neutrophil, eosinophil, or basophil), monocyte, or lymphocyte (T cells and B cells).
  • the cell is a lymphocyte.
  • the cell is a T cell, an NK cell, or a B cell.
  • the tissue section includes an immune cell.
  • the immune cell is a granulocyte, a mast cell, a monocyte, a neutrophil, a dendritic cell, or a natural killer (NK) cell.
  • the immune cell is an adaptive cell, such as a T cell, NK cell, or a B cell.
  • the cell includes a T cell receptor gene sequence, a B cell receptor gene sequence, or an immunoglobulin gene sequence.
  • the immune cell is a granulocyte.
  • the immune cell is a mast cell.
  • the immune cell is a monocyte.
  • the immune cell is a neutrophil. In embodiments, the immune cell is a dendritic cell. In embodiments, the immune cell is a natural killer (NK) cell. In embodiments, the immune cell is a T cell. In embodiments, the immune cell is a B cell. In embodiments, the cell includes a T cell receptor gene sequence. In embodiments, the cell includes a B cell receptor gene sequence. In embodiments, the cell includes an immunoglobulin gene sequence. In embodiments, the plurality of target nucleic acids includes non-contiguous regions of a nucleic acid molecule. In embodiments, the non-contiguous regions include regions of a VDJ recombination of a B cell or T cell.
  • the tissue section includes a cancer cell.
  • the cancer is lung cancer, colorectal cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, lymphoma, leukemia, or a cancer associated with aberrant K-Ras, aberrant APC, aberrant Smad4, aberrant p53, or aberrant TGF ⁇ .
  • the cancer cell includes a ERBB2, KRAS, TP53, PIK3CA, or FGFR2 gene.
  • the cancer cell includes a cancer-associated gene (e.g., an oncogene associated with kinases and genes involved in DNA repair) or a cancer-associated biomarker.
  • a “biomarker” is a substance that is associated with a Attorney Docket No.: 051385-585001WO particular characteristic, such as a disease or condition.
  • a change in the levels of a biomarker may correlate with the risk or progression of a disease or with the susceptibility of the disease to a given treatment.
  • the cancer is Acute Myeloid Leukemia, Adrenocortical Carcinoma, Bladder Urothelial Carcinoma, Breast Ductal Carcinoma, Breast Lobular Carcinoma, Cervical Carcinoma, Cholangiocarcinoma, Colorectal Adenocarcinoma, Esophageal Carcinoma, Gastric Adenocarcinoma, Glioblastoma Multiforme, Head and Neck Squamous Cell Carcinoma, Hepatocellular Carcinoma, Kidney Chromophobe Carcinoma, Kidney Clear Cell Carcinoma, Kidney Papillary Cell Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma, Lung Squamous Cell Carcinoma, Mesothelioma, Ovarian Serous Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Paraganglioma & Pheochromocytoma, Prostate A
  • the cancer-associated gene is a nucleic acid sequence identified within The Cancer Genome Atlas Program, accessible at www.cancer.gov/tcga.
  • the cancer-associated biomarker is MDC, NME-2, KGF, PlGF, Flt- 3L, HGF, MCP1, SAT-1, MIP-1-b, GCLM, OPG, TNF RII, VEGF-D, ITAC, MMP-10, GPI, PPP2R4, AKR1B1, Amy1A, MIP-1b, P-Cadherin, or EPO.
  • the cancer- associated gene is a AKT1, AKT2, AKT3, ALK, AR, ARAF, ARID1A, ATM, ATR, ATRX, AXL, BAP1, BRAF, BRCA1, BRCA2, BTK, CBL, CCND1, CCND2, CCND3, CCNE1, CDK12, CDK2, CDK4, CDK6, CDKN1B, CDKN2A, CDKN2B, CHEK1, CHEK2, CREBBP, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ERCC2, ERG, ESR1, ETV1, ETV4, ETV5, EZH2, FANCA, FANCD2, FANCI, FBXW7, FGF19, FGF3, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FOXL2, GATA2, GNA11, GNAQ, GNAS, H3F3A, HIST1H3B, H
  • the cancer-associated gene is a ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, or VHL gene.
  • the tissue section includes a cell (e.g., a T cell) within a tumor.
  • the tissue section includes a non-allogenic cell (i.e., native cell to the subject) within a tumor.
  • the tissue section includes a tumor infiltrating lymphocyte (TIL).
  • TIL tumor infiltrating lymphocyte
  • the tissue section includes an allogenic cell.
  • the tissue section includes a circulating tumor cell.
  • the tissue section is obtained from a subject (e.g., human or animal tissue). Once obtained, the tissue section is placed in an artificial environment in plastic or glass containers supported with specialized medium containing essential nutrients and growth factors to support proliferation.
  • the tissue section is permeabilized and immobilized to a solid support surface.
  • the tissue section is permeabilized and immobilized to an array (i.e., to discrete locations arranged in an array).
  • the tissue section is immobilized to a solid support surface.
  • the surface includes a patterned surface (e.g., suitable for immobilization of a plurality of cells in an ordered pattern.
  • the discrete regions of the ordered pattern may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. These discrete regions are separated by interstitial regions.
  • interstitial region refers to an area in a substrate or on a surface that separates other areas of the substrate or surface.
  • a plurality of cells are immobilized on a patterned surface that have a mean or median separation from one another of about 10-20 ⁇ m. In embodiments, a plurality of cells are immobilized on a patterned surface that have a mean or median separation from one another of about 10-20; 10-50; or 100 ⁇ m. In embodiments, a plurality of cells are arrayed on a substrate. In embodiments, a plurality of cells are immobilized in a 96-well microplate having a mean or median well-to-well spacing of about 8 mm to about 12 mm (e.g., about 9 mm).
  • a plurality of cells are immobilized in a 384-well microplate having a mean or median well-to-well spacing of about 3 mm to about 6 mm (e.g., about 4.5 mm).
  • Attorney Docket No.: 051385-585001WO [0325]
  • the tissue section is attached to the receiving substrate via a bioconjugate reactive linker.
  • the tissue section is attached to the substrate via a specific binding reagent.
  • the specific binding reagent includes an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer.
  • the specific binding reagent includes an antibody, or antigen binding fragment, an aptamer, affimer, or non-immunoglobulin scaffold.
  • the specific binding reagent is a peptide, a cell penetrating peptide, an aptamer, a DNA aptamer, an RNA aptamer, an antibody, an antibody fragment, a light chain antibody fragment, a single-chain variable fragment (scFv), a lipid, a lipid derivative, a phospholipid, a fatty acid, a triglyceride, a glycerolipid, a glycerophospholipid, a sphingolipid, a saccharolipid, a polyketide, a polylysine, polyethyleneimine, diethylaminoethyl (DEAE)-dextran, cholesterol, or a sterol moiety.
  • Substrates may be prepared for selective capture of particular cells of the tissue section.
  • a substrate containing a plurality of bioconjugate reactive moieties or a plurality of specific binding reagents optionally in an ordered pattern, contacts a plurality of cells of the tissue section. Only cells of the tissue section containing complementary bioconjugate reactive moieties or complementary specific binding reagents are capable of reacting, and thus adhering, to the substrate.
  • the methods are performed in situ in tissue sections that have been prepared according to methodologies known in the art.
  • the tissue section is cleared (e.g., digested) of proteins, lipids, or proteins and lipids.
  • the tissue section is exposed to paraformaldehyde (i.e., by contacting the cell with paraformaldehyde).
  • the method includes affixing single cells or tissues to a transparent substrate.
  • tissue include those from skin tissue, muscle tissue, bone tissue, organ tissue and the like.
  • the method includes immobilizing the tissue section in situ to a substrate and Attorney Docket No.: 051385-585001WO permeabilized for delivering probes, enzymes, nucleotides and other components required in the reactions.
  • the tissue section includes many cells from a tissue section in which the original spatial relationships of the cells are retained.
  • the tissue section in situ is within a Formalin-Fixed Paraffin-Embedded (FFPE) sample.
  • FFPE Formalin-Fixed Paraffin-Embedded
  • the tissue section is subjected to paraffin removal methods, such as methods involving incubation with a hydrocarbon solvent, such as xylene or hexane, followed by two or more washes with decreasing concentrations of an alcohol, such as ethanol.
  • the tissue section may be rehydrated in a buffer, such as PBS, TBS or MOPs.
  • the FFPE sample is incubated with xylene and washed using ethanol to remove the embedding wax, followed by treatment with Proteinase K to permeabilized the tissue.
  • the tissue section is fixed with a chemical fixing agent.
  • the chemical fixing agent is formaldehyde or glutaraldehyde.
  • the chemical fixing agent is glyoxal or dioxolane.
  • the chemical fixing agent includes one or more of ethanol, methanol, 2-propanol, acetone, and glyoxal.
  • the chemical fixing agent includes formalin, Greenfix ® , Greenfix ® Plus, UPM, CyMol ® , HOPE ® , CytoSkelFix TM , F-Solv ® , FineFIX ® , RCL2/KINFix, UMFIX, Glyo-Fixx ® , Histochoice ® , or PAXgene ® .
  • the tissue section is fixed within a synthetic three- dimensional matrix (e.g., polymeric material).
  • the synthetic matrix includes polymeric-crosslinking material.
  • the material includes polyacrylamide, poly- ethylene glycol (PEG), poly(acrylate-co-acrylic acid) (PAA), or Poly(N-isopropylacrylamide) (NIPAM).
  • the fixed tissue may be frozen tissue.
  • the frozen biological tissue can be fixed using a fixing agent, which is suitably an organic fixing agent.
  • the fixing agent can be chilled and can be at a temperature of about 0° C to about 100° C, suitably about zero to about 50° C, or about 1° C to about 50° C.
  • the fixing agent can be chilled by placing it over a bed of ice to maintain its temperature as close to 0° C as possible.
  • the frozen biological tissue can be treated with the fixing agent using any suitable technique, suitably by immersing it in the fixing agent for a period of time.
  • the treatment time can range from about 5 minutes to about 60 minutes, suitably about 10 minutes to about 30 minutes, or about 15 minutes to about 25 minutes, or about 20 minutes. In some embodiments, treatment time may be overnight.
  • the snap- Attorney Docket No.: 051385-585001WO frozen tissue will thaw but will suitably remain at a low temperature due to the low temperature environment of the fixing agent.
  • the type/identity of a fixation agent, the amount/concentration of a fixation agent, the temperature at which it is used, the duration for which it is used, and the like may be empirically determined or titrated. These parameters, and others, may need to be varied to obtain optimal results for different tissues, for different organisms, or for different days on which an experiment is performed. Insufficient fixation (e.g., too little fixing agent, too low temperature, too short duration) may not, for example, stabilize/preserve the cells/organelles/analytes of tissues.
  • Excess fixation may result in the single biological samples (e.g., cells/nuclei) obtained from the methods not yielding good results in single biological sample (e.g., single-cell or single nucleus) workflows or assays in which the biological samples (e.g., cells or nuclei) are used.
  • the quality of data obtained in these workflows/assays may be a good measure of the extent of the fixation process.
  • the fixative can be diluted in a buffer, e.g., saline, phosphate buffer (PB), phosphate buffered saline (PBS), citric acid buffer, potassium phosphate buffer, etc., usually at a concentration of about 1-10%, e.g.1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%, for example, 4% paraformaldehyde/0.1 M phosphate buffer; 2% paraformaldehyde/0.2% picric acid/0.1 M phosphate buffer; 4% paraformaldehyde/0.2% periodate/1.2% lysine in 0.1 M phosphate buffer; 4% paraformaldehyde/0.05% glutaraldehyde in phosphate buffer; etc.
  • a buffer e.g., saline, phosphate buffer (PB), phosphate buffered saline (PBS), citric acid buffer, potassium phosphate buffer, etc.
  • the type of fixative used and the duration of exposure to the fixative can depend on the sensitivity of the molecules of interest in the tissue section to denaturation by the fixative, and can be readily determined using conventional histochemical or immunohistochemical techniques, for example as described in Buchwalow and Bocker. Immunohistochemistry: Basics and Methods. Springer- Verlag Berlin Heidelberg 2010. [0331]
  • the biological tissue sample can be periodically cut into successively smaller segments while it is submerged in the fixation solution, to facilitate perfusion and fixation of the biological tissue sample by the organic fixing agent.
  • the tissue sample may have an initial length, width and/or diameter of about 0.25 cm to about 1.5 cm or may be initially cut into segments having such suitable dimensions.
  • the tissue sample or segments can be cut into smaller segments, and the smaller segments can remain immersed in the fixing agent.
  • This process can be repeated after a second periodic interval, after a third periodic interval, after a fourth periodic interval, and so on.
  • the periodic intervals can range from about 1 to about 10 minutes, or about 2 to about 8 minutes, or about 4 to about 6 minutes.
  • the sum of the periodic intervals can equal the entire fixing time and can range from about 5 to about 60 minutes, or about 10 to about 30 minutes, or about 15 to about 25 minutes, for example.
  • the resulting fixed tissue segments can have a length, width and/or diameter in a range of less than 1 mm to about 10 mm, by way of example.
  • the tissue is not cut into smaller segments during fixation. In some embodiments, this may be performed prior to fixation. In some embodiments, this may be performed after fixation. [0332] Once the biological tissue segments have been sufficiently fixed, the fixation process may be stopped and/or the tissue may be removed from the fixation and the tissue may be washed. Generally, fixation is stopped to cease additional activity of the fixative on the tissue. Fixation may also be stopped so that any subsequent biochemical reactions performed on the tissue (e.g., enzymatic cell dissociation) can function. In some embodiments, the tissue segments may be treated or contacted with a quenching medium to quench the fixation.
  • quenching means to stop the fixation reaction, i.e., the chemical interactions that cause the fixation. Quenching the fixation can be accomplished by immersing the fixed tissue segments in a suitable quenching medium.
  • the fixation quenching medium can be chilled and can have a temperature of about 0° C to about 100° C, or about 1° C to about 50° C.
  • the quenching medium is a phosphate buffer solution (PBS).
  • phosphate buffer solution is 1 ⁇ PBS, available from Sigma Aldrich Corp.1 ⁇ PBS has a pH of about 7.4 and the following composition in water: NaCl—137 mM, KCl—2.7 mM, Na2HPO4—10 mM, KH2PO4—1.8 mM.
  • the phosphate buffer solution can be combined with fetal bovine serum (FBS) to aid in quenching the fixation reaction.
  • FBS is the liquid fraction of clotted blood from fetal calves, depleted of cells, fibrin and clotting factors, but containing many nutritional and macromolecular factors essential for cell growth.
  • Bovine serum albumin (BSA) is the major component of FBS.
  • the fetal bovine serum can be combined with the phosphate buffer solution at a concentration of about 1% to about 25% by weight FBS and about 75% to about 99% by weight PBS, suitably about 5% to about 15% by weight FBS and about 85% to about 95% by Attorney Docket No.: 051385-585001WO weight PBS, or about 10% by weight FBS and about 90% by weight PBS.
  • a solution of concentrated ethanol in water can be used instead of the PBS in the quenching medium.
  • the ethanol solution can contain about 50% to about 90% by weight ethanol, or about 55% to about 85% by weight ethanol, or about 60% to about 80% by weight ethanol, or about 70% by weight ethanol.
  • fixation may be quenched using a quenching solution that does not contain serum.
  • Tris-based buffers may be used.
  • PBS + 50 mM Tris pH 8.0 + 0.02% BSA (RNAse free) + 0.1 U/ul of RNAse Inhibitor may be used.
  • the tissue may be removed from the fixative and washed using a quenching solution or biological buffer. [0333] In embodiments, the tissue section is lysed to release nucleic acid or other materials from the cells.
  • the tissue section may be lysed using reagents (e.g., a surfactant such as Triton-X or SDS, an enzyme such as lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase, proteinase K, etc.) or a physical lysing mechanism a physical condition (e.g., ultrasound, ultraviolet light, mechanical agitation, etc.).
  • reagents e.g., a surfactant such as Triton-X or SDS, an enzyme such as lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase, proteinase K, etc.
  • the cells may release, for instance, DNA, RNA, mRNA, proteins, or enzymes.
  • the cells may
  • the cells may be any cells for which nucleic acid from the cells is desired to be studied or sequenced, etc., and may include one, or more than one, cell type.
  • the cells may be for example, from a specific population of cells, such as from a certain organ or tissue (e.g., cardiac cells, immune cells, muscle cells, cancer cells, etc.), cells from a specific individual or species (e.g., human cells, mouse cells, bacteria, etc.), cells from different organisms, cells from a naturally-occurring sample (e.g., pond water, soil, etc.), or the like.
  • the cells may be dissociated from tissue.
  • the method does not include dissociating the cell from the tissue or the cellular microenvironment.
  • a permeabilization solution can contain additional reagents or a biological sample may be treated with additional reagents in order to optimize biological sample permeabilization.
  • an additional reagent is an RNA protectant.
  • RNA protectant typically refers to a reagent that protects RNA from RNA nucleases (e.g., RNases). Any appropriate RNA protectant that protects RNA from degradation can be used.
  • RNA protectant includes organic solvents (e.g., at Attorney Docket No.: 051385-585001WO least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% v/v organic solvent), which includes ethanol, methanol, propan-2-ol, acetone, trichloroacetic acid, propanol, polyethylene glycol, acetic acid, or a combination thereof.
  • the RNA protectant includes ethanol, methanol and/or propan-2-ol, or a combination thereof.
  • the RNA protectant includes RNAlater ICE (ThermoFisher Scientific).
  • the RNA protectant includes a salt.
  • the salt may include ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, potassium chloride, potassium sulfate, sodium chloride, sodium acetate, sodium sulfate, zinc chloride, zinc acetate and zinc sulfate.
  • the biological sample is treated with one or more RNA protectants before, contemporaneously with, or after permeabilization.
  • the method includes imaging the immobilized tissue section.
  • the method further includes an imaging modality, immunofluorescence (IF), or immunohistochemistry modality (e.g., immunostaining).
  • the method includes ER staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the endoplasmic reticula), Golgi staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the Golgi), F-actin staining (e.g., contacting the tissue section with a phalloidin-conjugated dye that binds to actin filaments), lysosomal staining (e.g., contacting the tissue section with a cell-permeable dye that accumulates in the lysosome via the lysosome pH gradient), mitochondrial staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the mitochondria), nucleolar staining, or plasma membrane staining.
  • ER staining e.g., contacting the tissue section with a cell-permeable dye which localizes to the endoplasm
  • the method includes live cell imaging (e.g., obtaining images of the tissue section) prior to or during fixing, immobilizing, and permeabilizing the tissue section.
  • Immunohistochemistry is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, commonly detected and examined with the light microscope.
  • Known IHC modalities may be used, such as the protocols described in Magaki, S., Hojat, S. A., Wei, B., So, A., & Yong, W. H. (2019). Methods in molecular biology (Clifton, N.J.), 1897, 289–298, which is incorporated herein by reference.
  • the additional imaging modality includes bright field microscopy, phase contrast microscopy, Nomarski differential-interference-contrast microscopy, or dark field microscopy.
  • the method further includes determining the cell morphology of the tissue section (e.g., the cell boundary or cell shape) using known methods in the art. For example, to determining the cell boundary includes comparing the pixel values of an image to a single intensity threshold, which may be determined quickly using histogram-based approaches as described in Carpenter, A. et al Genome Biology 7, R100 (2006) and Arce, S., Sci Rep 3, 2266 (2013)).
  • microscopic analysis is meant the analysis of a specimen using techniques that provide for the visualization of aspects of a specimen that cannot be seen with the unaided eye, i.e., that are not within the resolution range of the normal human eye.
  • Such techniques may include, without limitation, optical microscopy, e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal microscopy, CLARITY-optimized light sheet microscopy (COLM), light field microscopy, tissue expansion microscopy, etc., laser microscopy, such as, two photon microscopy, electron microscopy, and scanning probe microscopy.
  • the immobilized tissue section is imaged using “optical sectioning” techniques, such as laser scanning confocal microscopes, laser scanning 2-Photon microscopy, parallelized confocal (i.e. spinning disk), computational image deconvolution methods, and light sheet approaches.
  • optical sectioning microscopy methods provide information about single planes of a volume by minimizing contributions from other parts of the volume and do so without physical sectioning. The resulting “stack” of such optically sectioned images, represents a full reconstruction of the 3-dimensional features of a tissue volume.
  • a typical confocal microscope includes a 10 ⁇ /0.5 objective (dry; working distance, 2.0 mm) and/or a 20 ⁇ /0.8 objective (dry; working distance, 0.55 mm), with a s z-step interval of 1 to 5 ⁇ m.
  • a typical light sheet fluorescence microscope includes an sCMOS camera, a 2 ⁇ /0.5 objective lens, and zoom microscope body (magnification range of ⁇ 0.63 to ⁇ 6.3).
  • the z-step interval is 5 or 10 ⁇ m, and for image acquisition in the regions of interest, an interval in the range of 2 to 5 ⁇ m may be used.
  • Mounting medium is typically selected based on its suitability for the reagents used to visualize the cellular Attorney Docket No.: 051385-585001WO biomolecules, the refractive index of the tissue section, and the microscopic analysis to be performed.
  • the refractive index of the mounting medium should be different from the refractive index of the specimen, whereas for bright-field work the refractive indexes should be similar.
  • a mounting medium should be selected that reduces fading, photobleaching or quenching during microscopy or storage.
  • a mounting medium or mounting solution may be selected to enhance or increase the optical clarity of the cleared tissue specimen.
  • Nonlimiting examples of suitable mounting media include glycerol, CC/MountTM, FluoromountTM FluoroshieldTM, ImmunHistoMountTM, VectashieldTM, PermountTM, AcrytolTM, CureMountTM, FocusClearTM, or equivalents thereof.
  • the biological targets or molecules to be detected can be any biological molecules including but not limited to proteins, nucleic acids, lipids, carbohydrates, ions, or multicomponent complexes containing any of the above.
  • subcellular targets include organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • exemplary nucleic acid targets can include genomic DNA of various conformations (e.g., A-DNA, B-DNA, Z-DNA), mitochondria DNA (mtDNA), mRNA, tRNA, rRNA, hRNA, miRNA, and piRNA.
  • the sections may be fixed with methanol, permeabilized with 0.025% Triton in PBS solution, and stained with primary antibodies directed against vimentin (fibroblasts) and macrophages, followed by secondary antibody labeling (e.g., Alexa-594 conjugated secondary antibodies). Additional counterstaining may be performed, for example using 4,6-diamidino-2-phenylindole (DAPI) mounting media to counterstain nuclei.
  • DAPI 4,6-diamidino-2-phenylindole
  • signature may encompass any gene or genes, protein or proteins, or epigenetic element(s) whose expression profile or whose occurrence is associated with a specific cell type, subtype, or cell state of a specific cell type or subtype within a population of cells. It is to be understood that also when referring to proteins (e.g., differentially expressed proteins), such may fall within the definition of “gene” signature. Levels of expression or activity or prevalence may be compared between different cells in order to characterize or identify for instance signatures specific for cell (sub)populations. Increased or Attorney Docket No.: 051385-585001WO decreased expression or activity of signature genes may be compared between different cells in order to characterize or identify for instance specific cell (sub)populations.
  • the methods described herein may further include constructing a 3- dimensional pattern of abundance, expression, and/or activity of each target from spatial patterns of abundance, expression, and/or activity of each target of multiple samples.
  • the multiple samples can be consecutive tissue sections of a 3-dimensional tissue sample.
  • the method further includes digesting the tissue section by contacting the sample-carrier construct with an endopeptidase.
  • the endopeptidase is pepsin.
  • the method further includes removing the embedding material from the sample.
  • the embedding material is paraffin wax
  • the embedding material is removed by contacting the sample-carrier construct with a hydrocarbon solvent, such as xylene or hexane, followed by two or more washes with decreasing concentrations of an alcohol, such as ethanol.
  • the method includes measuring changes in the amount of biomaterial present in a well relative to a control (e.g., a sample obtained at a different time point or exposed to alternate conditions).
  • a control e.g., a sample obtained at a different time point or exposed to alternate conditions.
  • biomaterial refers to any biological material produced by an organism.
  • biomaterial includes secretions, extracellular matrix, proteins, lipids, organelles, membranes, cells, portions thereof, and combinations thereof.
  • cellular material includes secretions, extracellular matrix, proteins, lipids, organelles, membranes, cells, portions thereof, and combinations thereof.
  • biomaterial includes viruses.
  • the biomaterial is a replicating virus and thus includes virus infected cells.
  • additional methods may be performed to further characterize the sample. For example, in addition to sequencing, the method includes protein analysis, lipid analysis, metabolite analysis (e.g., glucose analysis), or measuring the transcriptomic profile, gene expression activity, genomic profile, protein expression activity, proteomic profile, protein interaction activity, cellular receptor expression activity, lipid profile, lipid activity, carbohydrate profile, microvesicle activity, glucose activity, and combinations thereof.
  • determining the spatial location of RNA molecules within a tissue sample includes correlating the location of the cluster of capture probes (e.g., the cluster including the first plurality of immobilized capture probes and second plurality of immobilized capture probes) on the substrate with a corresponding location within the tissue sample.
  • the spatial location of the RNA molecules in the tissue sample may allow identification of a single cell expressing the RNA molecules.
  • the methods described herein may include each of the following steps (in no particular order): a.
  • a solid support as described herein (e.g., a solid support including clusters including a plurality of immobilized double-stranded polynucleotides; b. determining the sequence of the spatial barcode for at least one polynucleotide in each cluster on the solid support; c. assigning each cluster a location (e.g., XY coordinate) on the solid support based upon the sequence of the spatial barcode; d. hybridizing an invasion primer including a capture sequence to a strand of the the double-stranded polynucleotide and incorporating the capture sequence into the strand; e. contacting the solid support with a tissue sample and allowing RNA molecules in the tissue sample to bind to the capture probes; f.
  • RNA e.g., gene
  • the methods described herein have a high enough resolution to enable determination of gene expression in a single cell.
  • the methods may further include analyzing the tissue sample for the presence of one or more additional targets, such as targets bound to the additional capture Attorney Docket No.: 051385-585001WO moieties on the substrate.
  • the methods may further include determining whether the tissue sample additionally contains one or more proteins of interest, which may be detected by an antibody conjugated capture moiety on the substrate.
  • the location of the additional capture moieties on the substrate may be known and thus used to determine the corresponding location of the additional target in the tissue sample.
  • the location of the additional capture moieties on the substrate may be known based upon the location of the cluster of capture probes in which the additional capture moieties are integrated.
  • Flow cells provide a convenient format for housing an array of clusters produced by the methods described herein, in particular when subjected to an SBS or other detection technique that involves repeated delivery of reagents in cycles. For example, to initiate a first SBS cycle, one or more labeled nucleotides and a DNA polymerase in a buffer, can be flowed into/through a flow cell that houses an array of clusters.
  • the nucleotides can further include a reversible termination moiety that temporarily halts further primer extension once a nucleotide has been added to a primer.
  • a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent (e.g., a reducing agent) is delivered to remove the moiety.
  • a deblocking reagent e.g., a reducing agent
  • a deblocking reagent can be delivered to the flow cell (before, during, or after detection occurs).
  • Washes can be carried out between the various delivery steps as needed.
  • the cycle can then be repeated N times to extend the primer by N nucleotides, thereby detecting a sequence of length N.
  • Example SBS procedures, fluidic systems and detection platforms that can be readily adapted for use with an array produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), US Patent Publication 2018/0274024, WO 2017/205336, US Patent Publication 2018/0258472, each of which are incorporated herein in their entirety for all purposes.
  • generating a sequencing read includes determining the identity of the nucleotides in the template polynucleotide (or complement thereof).
  • a sequencing read e.g., a first sequencing read or a second sequencing read, includes determining the identity of a portion (e.g., 1, 2, 5, 10, 20, 50 nucleotides) of the total template polynucleotide.
  • the first sequencing read determines the identity of 5-10 nucleotides and the second sequencing read determines the identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides).
  • the first sequencing read determines the identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides) and the second sequencing read determines the identity of 5-10 nucleotides.
  • subsequent extension is performed using a plurality of standard (e.g., non-modified) dNTPs until the complementary strand is copied.
  • subsequent extension is performed using a plurality of dideoxy nucleotide triphosphates (ddNTPs) to prevent further extension of the first sequencing read product during a second sequencing read.
  • ddNTPs dideoxy nucleotide triphosphates
  • subsequent extension is performed using a plurality of standard (e.g., non-modified) dNTPs until the complementary strand is copied.
  • ddNTPs dideoxy nucleotide triphosphates
  • the cleavable site located within the invasion primer is cleaved with a cleaving agent, thereby exposing a free 5’ phosphate in the invasion strand, and the invasion strand is removed (e.g., enzymatically digested using an exonuclease enzyme).
  • the invasion strand is cleaved at one or more cleavable sites.
  • the extension product of the invasion primer i.e., the invasion strand
  • suitable non-aggressive conditions Attorney Docket No.: 051385-585001WO (e.g., degraded or denatured under conditions that leave the complementary strand intact, and optionally still hybridized to at least a portion of the invasion primer).
  • the cleavable site is a dU.
  • the cleaving agent includes a glycosylase and one or more suitable endonucleases.
  • cleavage is performed under alkaline (e.g., pH greater than 8) buffer conditions at between 40°C to 80°C.
  • degradation of the invasion strand is enzymatic degradation. In embodiments, degradation of the invasion strand is accomplished with a 5’ to 3’ exonuclease. In embodiments, the 5’ to 3’ exonuclease is lambda exonuclease, or a mutant thereof.
  • the cleaved invasion primer following the degradation of the invasion strand, the cleaved invasion primer subsequently initiates a second sequencing read. In embodiments, the second sequencing read is generated without removal of the first sequencing read. In embodiments, the invasion primer (or a portion thereof) is the sequencing primer. [0352] In embodiments, the sequencing method relies on the use of modified nucleotides that can act as reversible reaction terminators.
  • the modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3’-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides.
  • the 3’ reversible terminator may be removed to allow addition of the next successive nucleotide.
  • the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3′ end of the extension strand and hybridizing a second sequencing primer to the second strand and incorporating one or more nucleotides into the second sequencing primer with a polymerase to create a second extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said second extension strand.
  • the method further includes terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the second extension strand; removing the invasion strand; hybridizing Attorney Docket No.: 051385-585001WO a third sequencing primer to the first strand and incorporating one or more nucleotides into the third sequencing primer with a polymerase to create a third extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said third extension strand.
  • the method includes terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the third extension strand; and hybridizing a fourth sequencing primer to the first strand and incorporating one or more nucleotides into the fourth sequencing primer with a polymerase to create a fourth extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said fourth extension strand.
  • the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3′ end of the extension strand; hybridizing a second sequencing primer to the second strand and incorporating one or more nucleotides into the second sequencing primer with a polymerase to create a second extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said second extension strand; terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the second extension strand; removing the invasion strand; hybridizing a third sequencing primer to the first strand and incorporating one or more nucleotides into the third sequencing primer with a polymerase to create a third extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said third extension strand; terminating extension by incorporating one or more
  • the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3′ end of the extension strand. In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs. In embodiments, the method further includes terminating extension by incorporating one or more ddNTPs into the 3′ end of the extension strand.
  • the method further includes hybridizing a second sequencing primer to the second strand and incorporating one or more nucleotides (e.g., labeled nucleotides) with a polymerase into the second sequencing primer to create a second extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said second extension strand.
  • the nucleotides are modified nucleotides including a label and a reversible terminator, as described herein.
  • the modified nucleotides may carry a label (e.g., a fluorescent label) to facilitate their detection.
  • Each nucleotide type may carry a different fluorescent label.
  • the detectable label need not be a fluorescent label. Any label can be used which allows the detection of an incorporated nucleotide.
  • One method for detecting fluorescently labeled nucleotides includes using laser light of a wavelength specific for the labeled nucleotides, or the use of other suitable sources of illumination. The fluorescence from the label on the nucleotide may be detected (e.g., by a CCD camera, CMOS camera, or other suitable detection means).
  • the methods of sequencing a nucleic acid include extending a complementary polynucleotide (e.g., a primer) that is hybridized to the nucleic acid by incorporating a first nucleotide.
  • the method includes a buffer exchange or wash step (e.g., between each sequencing cycle).
  • the methods of sequencing a nucleic acid include a sequencing solution.
  • the sequencing solution includes (a) an adenine nucleotide, or analog thereof; (b) (i) a thymine nucleotide, or analog thereof, or (ii) a uracil nucleotide, or analog thereof; (c) a cytosine nucleotide, or analog thereof; and (d) a guanine nucleotide, or analog thereof.
  • the sequencing methods provided herein comprises sequencing both strands of a double-stranded nucleic acid with an error rate of 5 x 10 -5 or less, 1 x 10 -5 or less, 5 x 10 -6 or less, 1 x 10 -6 or less, 5 x 10 -7 or less, 1 x 10 -7 or less, 5 x 10 -8 or less, or 1 x 10 -8 or less.
  • the sequencing methods provided herein comprises sequencing both strands of a double-stranded nucleic acid with an error rate of 5 x 10 -5 to 1 x 10 -8 , 1 x 10 -5 to 1 x 10 -8 , 5 x 10 -5 to 1 x 10 -7 , 1 x 10 -5 to 1 x 10 -7 , 5 x 10 -6 to 1 x 10 -8 , or 1 x 10 -6 to 1 x 10 -8 .
  • the sequencing methods provided herein comprises sequencing both strands of a double-stranded nucleic acid with an error rate of 1 x 10 -7 to 1 x 10 -8 .
  • a method of reducing GC bias in a plurality of sequencing reads including sequencing a template polynucleotide to generate a plurality of sequencing reads as described herein.
  • the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, as described herein, for example wherein generating the first invasion strand includes a first plurality of invasion-primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles and extending the one or more invasion primers; generating a first sequencing read by hybridizing one or more sequencing primers to the first strand, and extending the one or more first sequencing primers.
  • the invasion primer is not covalently attached to the solid support.
  • the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, wherein generating the invasion strand comprises alternating between a first plurality of invasion-primer extension cycles and a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles and extending the one or more invasion primers; generating a first sequencing read by hybridizing one or more sequencing primers to the first strand, and extending the one or more first sequencing primers.
  • generating the invasion strand includes a first plurality of invasion- primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles.
  • the Attorney Docket No.: 051385-585001WO method further includes a third plurality of invasion-primer extension cycles, wherein the reaction conditions for the third plurality of invasion-primer extension cycles are optionally different than the first or second plurality of invasion-primer extension cycles.
  • the method further includes a third plurality of invasion-primer extension cycles, wherein the reaction conditions for the third plurality of invasion-primer extension cycles are the same as the first plurality of invasion-primer extension cycles.
  • the method includes providing a solid support including a plurality of immobilized oligonucleotide primers attached to the solid support via a linker, wherein the plurality of oligonucleotide primers include a plurality of forward primers and a plurality of reverse primers, amplifying a template nucleic acid by using the oligonucleotide primers attached to the solid support to generate a plurality of double-stranded amplification products, each double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) each double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to the solid support; and generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, and extending the one or more invasion primers; thereby generating a template nucleic acid for a nucle
  • the method further includes hybridizing one or more sequencing primers to the first strand. In embodiments, the method includes generating a cluster of ssDNA templates. In embodiments, the invasion primer is not covalently attached to the solid support. In embodiments, the invasion strand is not covalently attached to the solid support.
  • a method including: amplifying a template nucleic acid by contacting the template nucleic acid with a plurality of oligonucleotide primers attached to a solid support to generate a plurality of double-stranded amplification products, each double- stranded amplification product including a first strand hybridized to a second strand, wherein the first strand and second strand are both attached to the solid support; and generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, and extending the one or more invasion primers to produce a single-stranded first strand.
  • the invasion primer is not covalently attached to the solid support.
  • a method of removing a polynucleotide hybridized to a first strand wherein the polynucleotide includes one or more of cleavable sites.
  • the method includes fragmenting a polynucleotide in the presence of a plurality of dsDNA polynucleotides.
  • the method includes contacting the polynucleotide with a cleaving agent thereby fragmenting the polynucleotide and generating two or more fragments.
  • the method includes denaturing the fragments (e.g., contacting the fragments with a chemical denaturant, increasing the temperature, or a combination thereof).
  • the method includes digesting the fragments (e.g., contacting the fragments with one or more exonuclease enzymes).
  • the method includes modulating the temperature to be at or below the calculated or predicted melting temperature (Tm) of the fragments (e.g., about 0°C to about 65°C).
  • Tm melting temperature
  • the method includes modulating the temperature to be at about 50°C to about 65°C.
  • the first strand is covalently attached to a solid support.
  • the polynucleotide is not attached to a solid support.
  • the first strand is attached to a solid support, wherein the solid support includes a plurality of double-stranded polynucleotides.
  • the first strand is in a colony of double-stranded polynucleotides.
  • the solid support includes a second strand hybridized to a sequenced strand, wherein the sequenced strand includes one or more sequenced nucleotides.
  • the sequenced nucleotides include a scar remnant (e.g., an alkynyl moiety attached to the nucleobase).
  • the nucleotides have the , wherein B is a nucleobase, R 1 is the scar remnant, and ” is the attachment point to the remainder of the sequenced strand polynucleotide.
  • B is a divalent nucleobase.
  • R 1 is hydrogen.
  • R 1 is -OH.
  • R 1 is -NH.
  • R 1 is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. In embodiments, R 1 is a substituted or unsubstituted alkenyl. In embodiments, R 1 is a substituted or unsubstituted alkynyl. In embodiments, R 1 is a substituted or unsubstituted heteroalkenyl. In embodiments, R 1 is a substituted or unsubstituted heteroalkynyl.
  • R 1 is a substituted (e.g., substituted with a substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl or substituted Attorney Docket No.: 051385-585001WO (e.g., substituted with a substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl.
  • R 1 is substituted with an oxo or -OH.
  • R 1 is substituted with an oxo and -OH.
  • R 1 is an oxo-substituted heteroalkyl (e.g., 2 to 10 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 4 to 8 membered heteroalkyl).
  • R 1 is an oxo-substituted heteroalkenyl (e.g., 2 to 10 membered heteroalkenyl, 2 to 8 membered heteroalkenyl, or 4 to 8 membered heteroalkenyl).
  • R 1 is an oxo-substituted heteroalkynyl (e.g., 2 to 10 membered heteroalkynyl, 2 to 8 membered heteroalkynyl, or 4 to 8 membered heteroalkynyl). In embodiments, R 1 is an oxo-substituted 10 membered heteroalkynyl. In embodiments, R 1 is an oxo-substituted 9 membered heteroalkynyl. In embodiments, R 1 is an oxo-substituted 8 membered heteroalkynyl. In embodiments, R 1 is an oxo-substituted 7 membered heteroalkynyl.
  • oxo-substituted heteroalkynyl e.g., 2 to 10 membered heteroalkynyl, 2 to 8 membered heteroalkynyl, or 4 to 8 membered heteroalkynyl.
  • R 1 is an o
  • R 1 is an oxo-substituted 6 membered heteroalkynyl.
  • the one or more nucleotides including a scar remnant include a , , Attorney Docket No.: 051385-585001WO , the fragments is about 50°C to about 75°C.
  • the calculated or predicted melting temperature (Tm) of the fragments is about 60°C to about 75°C.
  • the calculated or predicted melting temperature (Tm) of the fragments is about 50°C to about 65°C.
  • the calculated or predicted melting temperature (Tm) of the fragments is less than about 75°C.
  • the calculated or predicted melting temperature (Tm) of the fragments is less than about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of the fragments is less than about 60°C. In embodiments, two or more fragments are generated. In embodiments, three or more fragments are generated. In embodiments, four or more fragments are generated. In embodiments, at least three fragments are generated. In embodiments, four fragments are generated. [0371] In embodiments, the fragments are 3-10 nucleotides in length. In embodiments, the fragments are 3-15 nucleotides in length. In embodiments, the fragments are 5 to 20 nucleotides in length. In embodiments, the fragments are 4 to 6 nucleotides in length.
  • a functional library molecule typically includes the target molecule with specific adapter sequences added to the 3′ and 5′ ends, e.g., Illumina’s P5 and P7 adapters/primers, to ensure compatibility with the underlying flow cell, so it may be amplified appropriately.
  • specific adapter sequences e.g., Illumina’s P5 and P7 adapters/primers, to ensure compatibility with the underlying flow cell, so it may be amplified appropriately.
  • common platform primers include 5’-AATGATACGGCGACCACCG (P5) (SEQ ID NO:60), or the complement thereof, and 5’-CAAGCAGAAGACGGCATACGA (P7) (SEQ ID NO:61), or the complement thereof.
  • An example of an adapter ligation protocol includes phosphorylated template oligos at the 5′ end using a T4 polynucleotide kinase in 1x T4 ligase buffer for 30 minutes at 37 °C in a thermocycler. The kinase is then denatured (e.g., by heating) and the oligo reaction mixture is slowly cooled to 20 °C (e.g., by slowly changing the temperature by 0.1 °C every 2 seconds).
  • bridge PCR bridge amplification methodologies
  • amplification products e.g., amplicons
  • arrays comprised of colonies (or “clusters”) of immobilized nucleic acid molecules.
  • Each Attorney Docket No.: 051385-585001WO cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands.
  • the products of solid-phase amplification reactions are referred to as “bridged” structures when formed by annealed pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at the 5′ end, preferably via a covalent attachment.
  • RNA strands are denatured by flowing a denaturant such as formamide or NaOH over the DNA, which chemically denatures complementary strands. This is followed by washing out the denaturant and reintroducing a polymerase in buffer conditions that allow primer annealing and extension.
  • a denaturant such as formamide or NaOH
  • Sequencing two strands of the sample dsDNA template is a powerful technique to improve sequencing accuracy and is commonly performed in next-generation sequencing (NGS) workflows.
  • NGS next-generation sequencing
  • Sequencing by synthesis is a common implementation of NGS and paired-end sequencing is typically performed on monoclonal clusters generated by a clonal amplification process.
  • SBS Sequencing by synthesis
  • nucleic acid libraries that have common nucleic acid sequences referred to as adapter sequences
  • primers nucleic acid sequences within the flow cell.
  • the primers may be immobilized to a solid support (e.g., a flow cell or a bead); a solid support encompasses any type of solid, porous, or hollow sphere, ball, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently).
  • a polymerase will make an initial copy of the library nucleic acid molecule by extending the primer.
  • the complement of the initial library molecule is now attached to a solid support, and the initial library nucleic acid molecules can either be removed from the flow cell, or can stay present during subsequent steps, depending on which clonal amplification method is used.
  • spatially localized amplification of the initial single seed molecule will occur by means of a solid-phase clonal amplification process.
  • clonal amplification techniques include, but are not limited to, bridge PCR, solid-phase rolling circle amplification (RCA), solid- Attorney Docket No.: 051385-585001WO phase exponential rolling circle amplification, solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, emulsion PCR on particles (beads), or combinations of the aforementioned methods.
  • RPA solid-phase recombinase polymerase amplification
  • HDA solid-phase helicase dependent amplification
  • template walking amplification emulsion PCR on particles (beads)
  • beads template walking amplification
  • dsDNA double-stranded DNA
  • SBS workflows clusters of dsDNA are difficult to sequence effectively with high accuracy and read length, especially as miniaturization pushes the clusters to become more densely arranged on a solid support.
  • a sequencing primer needs to hybridize to a single-stranded region in the dsDNA and be extended by a polymerase. Individual strands in dsDNA clusters are difficult to access for hybridization of sequencing primers.
  • dNTPs reversibly terminated nucleotides
  • native dNTPs for example in pyrosequencing
  • dsDNA single-stranded DNA
  • the dsDNA structures may be linearized by cleavage of one or both strands with a restriction endonuclease or by cleavage of one strand with a nicking endonuclease.
  • Other methods of cleavage can be used as an alternative to restriction enzymes or nicking enzymes, including chemical cleavage (e.g., cleavage of a diol linkage with periodate), cleavage of abasic sites by cleavage with endonuclease, by exposure to heat or alkali, cleavage of ribonucleotides incorporated into amplification products otherwise comprised of deoxyribonucleotides, photochemical cleavage, or cleavage of a peptide linker.
  • chemical cleavage e.g., cleavage of a diol linkage with periodate
  • cleavage of abasic sites by cleavage with endonuclease by exposure
  • the primers may be attached to the solid support with a cleavable linker, such that upon exposure to a cleaving agent, all or a portion of the primer is removed from the surface.
  • a cleavable linker such that upon exposure to a cleaving agent, all or a portion of the primer is removed from the surface.
  • one linearization method requires one or both of the immobilized primers to have a cleavable site, such as a uracil, diol, 8- oxoG, disulfide, photocleavable moieties, an RNA base or an endonuclease cleaving site.
  • one of the two species of solid phase primers can be cleaved (chemically, enzymatically or optically), followed by a denaturation step to remove the cleaved molecules.
  • the monoclonal clusters can proceed to any necessary post-processing steps such as blocking of free 3’ ends, removal of select amplicons, or hybridization of a sequencing primer.
  • a first sequencing read is performed by hybridizing a first sequencing primer to a complementary region (e.g., a region within the adapter portion) of the ssDNA molecule.
  • a complementary region e.g., a region within the adapter portion
  • an enzyme e.g., a DNA polymerase
  • nucleotides e.g., labeled nucleotides
  • the second strand that was initially cleaved during linearization must be regenerated prior to starting the second read.
  • the methods disclosed herein permit reading of the original first and second strands (e.g., the first and second strand of the amplicons), reducing the time, reagents, expense, and risk of polymerase error inherent in previous methods.
  • methods described herein prevent the need for additional solid phase amplification between the two sequencing reads.
  • methods disclosed herein utilize strand invasion using invasion primers into dsDNA amplicons bound to a solid phase, followed by polymerase extension of the invasion primers.
  • Strand invasion into dsDNA can be challenging in general, but can be particularly challenging in dense monoclonal clusters of dsDNA where DNA Attorney Docket No.: 051385-585001WO molecules are packed tightly together in a spatially localized fashion on a solid phase. Because the local concentration of full-length complementary strands is very high, insertion of a traditional primer oligonucleotide is thermodynamically unfavorable.
  • the invasion primers are oligonucleotide sequences that binds to one strand of the dsDNA molecule in the cluster.
  • the invasion primer may bind to a portion of the common adapter sequence of only the forward, or only the reverse amplicons in clusters.
  • These invasion oligonucleotides may include nucleic acids having a binding affinity higher than the binding affinity of standard or canonical DNA oligonucleotides, such as locked nucleic acids (LNA), peptide nucleic acids (PNAs), 2’-O-methyl RNA:DNA chimeras, minor groove binder probes (MGB), or morpholino probes.
  • LNA locked nucleic acids
  • PNAs peptide nucleic acids
  • MGB minor groove binder probes
  • the invasion primers are introduced into a flow cell that contains monoclonal dsDNA clusters generated using a known amplification method or an amplification method described herein. Some of these invasion primers can undergo spontaneous strand invasion into dsDNA, as is the case for example for PNA invasion primers under low ionic strength conditions, while other invasion primers may need assistance of additives such as DMSO, ethylene glycol, formamide, betaine, or other denaturants that assist strand invasion by inducing more breathability within dsDNA amplicons.
  • additives may include a buffered solution containing about 0 to about 50% DMSO, about 0 to about 50% ethylene glycol, about 0 to about 20% formamide, or about 0 to about 3M betaine.
  • the invasion oligonucleotide can be introduced without a polymerase and allowed to invade and anneal to the complementary region, or it may be introduced together with a polymerase for runoff extension.
  • polymerases examples include strand-displacing polymerases such as Bst large fragment, Bst2.0 (New England Biolabs), Bsm DNA polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase or Phi29 polymerase.
  • strand-displacing polymerases such as Bst large fragment, Bst2.0 (New England Biolabs), Bsm DNA polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase or Phi29 polymerase.
  • the invasion oligonucleotide e.g., a 15-75 bp invasion primer
  • the hybridization of the invasion oligo to one of the DNA strands is Attorney Docket No.: 051385-585001WO often transient, and can be outcompeted easily by the reannealing of the full-length forward and reverse strands of the dsDNA molecules.
  • the dsDNA genomic library was introduced into a proprietary flow cell at a 1 pM concentration in presence of 5x SSC buffer and 30% ethylene glycol.
  • the flow cell was heated to 95°C to denature the dsDNA library molecules, followed by cooling the flow cell to 45°C to allow the denatured library molecules to bind to the immobilized primers on the surface of the flow cell.
  • a strand displacing (SD) polymerase (with SD polymerase buffer, 3 mM MgCl 2 , and 0.2 mM of each dNTP) was subsequently introduced into the flow cell and heated to 60°C for 10 min to make an initial first copy of the library molecules that were hybridized to the flow cell primers.
  • the initial library molecules were subsequently removed from the flow cell by flushing 0.1M NaOH through the lanes. This was followed by 45 bridge PCR cycles with a Bst LF polymerase and formamide as a chemical denaturant, which were cyclically introduced into the flow cell.
  • a positive control lane was subsequently treated with USER enzyme mix which cleaved the forward amplicons which contained a uracil base and were formed by extending the forward flow cell primers from the flow cell surface.
  • a second lane of the flow cell did not go through cleavage protocols and instead went through a strand invasion and runoff protocol, according to the following steps.
  • dsDNA clusters immobilized in a lane of the flow cell are exposed to a reagent mix that contains a plurality of LNA invasion oligos (at 1 uM concentration) capable of invading and hybridizing to a portion of the common adapter sequence, Attorney Docket No.: 051385-585001WO 0.56 units/uL of Bst polymerase, 30% DMSO, and 0.2 mM of each dNTP.
  • This reaction mix is incubated at 65°C for 5 min, followed by flowing in fresh reagent mix.
  • FIGS.2A-2B illustrate an embodiment of strand invasion including an invasion primer with a 5’ tail sequence.
  • the hashed box represents a polymer scaffold that is anchored to a solid support, such as glass or silicon support.
  • FIG.2A illustrates two dsDNA duplex strands, each duplex having a first strand hybridized to a second strand, and each strand is attached to the solid support.
  • An invasion primer including a binding sequence (e.g., a sequence complementary to a region within one of the strands of the dsDNA) and a 5’ tail sequence (wherein the tail sequence is not complementary to the dsDNA) is introduced, wherein the binding sequence of the elongated invasion primer hybridizes to one of the strands.
  • An invasion mixture as described above may be used to introduce and hybridize the elongated invasion primer to the dsDNA.
  • a blocking strand hybridized to one of the strands of the dsDNA (e.g., a blocking strand, also referred to herein as an invasion strand, is now hybridized to the second strand, for example, resulting in a single-stranded first strand that is accessible to a primer).
  • the second strand is extended along the elongated invasion primer to incorporate the complement of the 5’ tail sequence, thereby modifying the 3’ end of the second strand such that it is no longer complementary to the 5’ end of the first strand.
  • FIG.2B illustrates an additional embodiment of strand invasion, wherein after generating an extended second strand as shown in FIG.2A (and after generating a first sequencing read), the blocking strand is removed (e.g., removed via Attorney Docket No.: 051385-585001WO exonuclease digestion). Following removal of the blocking strand, the first strand may reanneal to the second strand, but the incorporated tail sequence at the 3’ end of the second strand is free, allowing for annealing of a primer (e.g., a second invasion primer), followed by extension with a polymerase (e.g., a strand-displacing polymerase) thereby generating a second blocking strand hybridized to the extended second strand.
  • a primer e.g., a second invasion primer
  • a polymerase e.g., a strand-displacing polymerase
  • Such an approach may be useful, for example, if the reaction conditions (e.g., buffer composition, such as high denaturant concentration) for hybridization of the invasion primer are not equally optimal for extension of the invasion primer, or the invasion primer melts off during invasion or is outcompeted by residual surface primers.
  • the invasion/blocking strand/primer may be removed by exonuclease digestion, for example, and then a new invasion primer hybrized to the incorporated tail sequence at the 3’ end of the second strand. This then allows for optimal strand-displacing extension conditions to be used for generating the second blocking strand.
  • FIG.3 illustrates an alternate embodiment of strand invasion that may be used, for example, in combination with any of the strand invasion embodiments described herein.
  • a bridged dsDNA complex is present on a solid support, wherein the first strand and the second strand of the dsDNA are immobilized on the solid support.
  • a second oligonucleotide primer e.g., a second competitive oligonucleotide
  • a competitor strand at the 5’ end of the immobilized first strand that may reduce reannealing of a portion of the second strand (prevent the 3’ end of the second strand from reannealing to the 5’ end of the first strand).
  • the 5’ end of the immobilized first strand is double-stranded, and no longer able to anneal to the 3’ end of the second strand, for example.
  • the probability that one or more sequencing primers may be introduced and extended successfully may be increased, resulting in a higher efficiency of paired-end sequencing according to the methods herein.
  • the initiation point for the first sequencing reaction was provided by annealing a Attorney Docket No.: 051385-585001WO sequencing primer complementary to a region within one of the strands.
  • an enzyme e.g., a DNA polymerase
  • nucleotides e.g., labeled nucleotides
  • an enzyme e.g., a DNA polymerase
  • the first sequencing reaction may include hybridizing a sequencing primer to a region of an amplification product, sequentially incorporating one or more nucleotides into a polynucleotide strand complementary to the region of amplified template strand to be sequenced, identifying the base present in one or more of the incorporated nucleotide(s) and thereby determining the sequence of a region of the template strand.
  • the second sequenced strand is present while sequencing the first strand, albeit the second strand is hybridized to the invasion strand.
  • PNAs within invasion primers Peptide nucleic acids (PNAs) can be used as invasion oligonucleotides in another example of this invention; see the schematic illustrated in FIGS.4A-4B.
  • Peptide nucleic acids consist of a pseudopeptide backbone, which has been shown to be capable of invading dsDNA.
  • MiniPEG- ⁇ PNAs are particularly beneficial because they have better water solubility (Bahal et al. ChemBioChem, 13(1), 56–60).
  • PNAs typically do not have a 3′-OH that is extendible by a DNA polymerase, though one can consider PNA-DNA chimeras that have 3-7 bp of canonical DNA nucleotides at the 3′ end of the oligonucleotide to be extendable by a DNA polymerase.
  • MiniPEG- ⁇ PNAs can be designed to invade into dsDNA clusters by targeting a sequence region in the common adapter sequence of all clusters.
  • PNAs can be designed for strand invasion into any part of the common adapter sequences, but targeting near the 5′ end of one of the amplicons is beneficial because it renders the complementary strand available for hybridization of another oligonucleotide, as shown in FIG.4A.
  • a second invasion oligonucleotide that hybridizes on the “liberated” ssDNA fragment opposite of the PNA invasion site can then be extended by a strand- displacing DNA polymerase.
  • a strand- displacing DNA polymerase As a result, one of the two strands of every dsDNA duplex has now been rendered into a ssDNA fragment that can be sequenced by hybridizing a sequencing primer followed a plurality of sequencing reactions.
  • Example 3 Recombinase-assisted invasion [0387] Another possibility for enabling strand invasion into dsDNA molecules in monoclonal clusters is by using a recombinase enzyme that enables the insertion of a DNA oligonucleotide Attorney Docket No.: 051385-585001WO complementary to part of the common adapter sequence; see FIGS.5A-5B.
  • a reagent mixture consisting of an invasion oligonucleotide, a recombinase, and necessary cofactors for forming a pre-synaptic filament (i.e.
  • an oligonucleotide complexed with recombinase enzymes is flowed into the flow cell that contains dsDNA clusters.
  • the pre-synaptic filaments search the dsDNA molecules in monoclonal clusters until homology is found, after which the invasion oligonucleotide is inserted into the dsDNA to form a D-loop.
  • a strand- displacing polymerase can be introduced that extends the invasion oligonucleotide, thereby rendering the opposite strand of the original dsDNA duplex into a single-stranded form.
  • the ssDNA molecule that is generated is then available for hybridization of a sequencing primer and the subsequent start of a first sequencing read.
  • recombinases include, but are not limited to, T4 UvsX (and possibly its cofactor UvsY, and single-stranded binding protein gp32), Rad51, and RecA.
  • the recombinase can be present in the same reaction mix as the strand- displacing polymerase, or the strand-displacing polymerase can be introduced after strand invasion with the recombinase has been done first.
  • Example 4 Additional approaches to sequencing two strands of the same polynucleotide [0388] Sequencing two strands of the sample dsDNA template is a powerful technique to improve sequencing accuracy and is commonly performed in next-generation sequencing (NGS) workflows.
  • NGS next-generation sequencing
  • SBS Sequencing by synthesis
  • dsDNA double-stranded DNA
  • a sequencing primer needs to hybridize to a single-stranded region in the dsDNA and be extended by a polymerase. Individual strands in dsDNA clusters are difficult to access for hybridization of sequencing primers. Additionally, the polymerases used during SBS to incorporate 3’ reversibly terminated nucleotides (dNTPs) or native dNTPs (for example in pyrosequencing) typically do not have strand-displacement capabilities, and so even if one is Attorney Docket No.: 051385-585001WO successful in incorporating sequencing primers into dsDNA molecules, it is still challenging to extend said sequencing primers when the vast majority of DNA molecules are in dsDNA format.
  • dNTPs reversibly terminated nucleotides
  • native dNTPs for example in pyrosequencing
  • dsDNA amplicons in clusters are typically processed into single-stranded DNA (ssDNA), sometimes referred to as linearization, by a variety of methods.
  • the dsDNA structures may be linearized by cleavage of one or both strands with a restriction endonuclease or by cleavage of one strand with a nicking endonuclease.
  • cleavage can be used as an alternative to restriction enzymes or nicking enzymes, including chemical cleavage (e.g., cleavage of a diol linkage with periodate), cleavage of abasic sites by cleavage with endonuclease, by exposure to heat or alkali, cleavage of ribonucleotides incorporated into amplification products otherwise comprised of deoxyribonucleotides, photochemical cleavage, or cleavage of a peptide linker.
  • the primers may be attached to the solid support with a cleavable linker, such that upon exposure to a cleaving agent, all or a portion of the primer is removed from the surface.
  • one linearization method requires one or both of the immobilized primers to have a cleavable site, such as a uracil, diol, 8- oxoG, disulfide, photocleavable moieties, an RNA base or an endonuclease cleaving site.
  • a cleavable site such as a uracil, diol, 8- oxoG, disulfide, photocleavable moieties, an RNA base or an endonuclease cleaving site.
  • one of the two species of solid phase primers can be cleaved (chemically, enzymatically or optically), followed by a denaturation step to remove the cleaved molecules. This transforms the dsDNA molecules into ssDNA molecules within the cluster and provides a region available for hybridization of a sequencing primer to initiate a sequencing reaction.
  • the monoclonal clusters can proceed to any necessary post-processing steps such as blocking of free 3’-OH ends, removal of select amplicons, or hybridization of a sequencing primer.
  • a first sequencing read is performed by hybridizing a first sequencing primer to a complementary region (e.g., a region within the adapter portion) of the ssDNA molecule.
  • an enzyme e.g., a DNA polymerase
  • nucleotides e.g., labeled nucleotides
  • detected such that the identity of the incorporated nucleotides allows for the identification of the first strand.
  • the second strand that was initially cleaved during linearization must be regenerated prior to starting the second read. This can be done by additional amplification steps, such as Attorney Docket No.: 051385-585001WO additional rounds of bridge PCR or another amplification process. Following an additional amplification step after the first sequencing read, the second strand may then be sequenced. All of these steps add complexity and time to the DNA sequencing workflow and can also introduce additional errors made by the polymerase used during solid phase amplification. Highly accurate sequencing methods would greatly benefit from novel methods that bypass the need for additional amplification steps between the two sequencing reads of conventional paired-end sequencing workflows.
  • the methods disclosed herein permit reading of the original first and second strands (e.g., the first and second strand of the amplicons), reducing the time, reagents, expense, and risk of polymerase error inherent in previous methods. Importantly, methods described herein prevent the need for additional solid phase amplification between the two sequencing reads.
  • methods disclosed herein utilize strand invasion using invasion primers into dsDNA amplicons bound to a solid phase, followed by polymerase extension of the invasion primers. Strand invasion into dsDNA can be challenging in general, but can be particularly challenging in dense monoclonal clusters of dsDNA where DNA molecules are packed tightly together in a spatially localized fashion on a solid phase.
  • the invasion primers are oligonucleotide sequences that binds to one strand of the dsDNA molecule in the cluster.
  • the invasion primer may bind to a portion of the common adapter sequence of only the forward, or only the reverse amplicons in clusters.
  • invasion oligonucleotides may include nucleic acids having a binding affinity higher than the binding affinity of standard or canonical DNA oligonucleotides, such as locked nucleic acids (LNA), peptide nucleic acids (PNAs), 2’-O-methyl RNA:DNA chimeras, minor groove binder probes (MGB), or morpholino probes.
  • LNA locked nucleic acids
  • PNAs peptide nucleic acids
  • MGB minor groove binder probes
  • the invasion primers may include one or more deoxyuracils(dUs).
  • the invasion primers may include one or more phosphorothioate groups.
  • the invasion primers are introduced into a flow cell that contains monoclonal dsDNA clusters generated using a known amplification method or an amplification method described herein.
  • invasion primers can undergo spontaneous strand invasion into dsDNA, as is the case for example for PNA invasion primers under low ionic strength conditions, while other Attorney Docket No.: 051385-585001WO invasion primers may need assistance of additives such as DMSO, ethylene glycol, formamide, betaine, or other denaturants or additives that assist strand invasion by inducing more breathability within dsDNA amplicons.
  • additives may include a buffered solution containing about 0 to about 50% DMSO, about 0 to about 50% ethylene glycol, about 0 to about 20% formamide, or about 0 to about 3M betaine.
  • the invasion oligonucleotide can be introduced without a polymerase and allowed to invade and anneal to the complementary region, or it may be introduced together with a polymerase for runoff extension. In certain experiments, it is preferable to introduce the invasion oligonucleotide (e.g., a 15-75 bp invasion primer) together with a polymerase in the same reaction mixture.
  • the invasion oligonucleotide e.g., a 15-75 bp invasion primer
  • the initiation point for the first sequencing reaction is provided by annealing a sequencing primer complementary to a region within one of the strands.
  • the first sequencing reaction may include hybridizing a sequencing primer to a region of an amplification product, sequentially incorporating one or more nucleotides into a polynucleotide strand complementary to the region of amplified template strand to be sequenced, identifying the base present in one or more of the incorporated nucleotide(s) and thereby determining the sequence of a region of the template strand.
  • an enzyme e.g., a DNA polymerase
  • FIG.6A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more 5’ phosphorothioate groups (e.g., 3-5 phosphorothioate linking groups) to protect from exonuclease digestion.
  • the invasion primer further includes a cleavable site (e.g., a 3’ deoxyuracil triphosphate (dUTP)).
  • a cleavable site e.g., a 3’ deoxyuracil triphosphate (dUTP)
  • dUTP deoxyuracil triphosphate
  • the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicked at the dU), leaving behind a 5’-phosphate in the extended part of the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.6C- 6D.
  • the invasion primer is treated with a 3’ phosphatase (for example Endonuclease IV or PNK) to generate a 3’ hydroxyl group prior to sequencing.
  • a 3’ phosphatase for example Endonuclease IV or PNK
  • one strand of the initial dsDNA molecule is now single- stranded and available for a first sequencing read, as shown in FIG.7B.
  • the sequenced strand may further be extended with natural dNTPs after sequencing the first read to complete the extension of the sequenced strand, as illustrated in FIG.7B, thereby preventing any rehybridization of any non-sequenced amplicon to the complement.
  • the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicked at the dU and removed), leaving behind a 5’-phosphate in the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.7C-7D.
  • the sequenced strand may further be extended with a one or more ddNTPs to prevent further extension, as illustrated in FIG.8B.
  • the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicked at the dU and removed), leaving behind a 5’-phosphate in Attorney Docket No.: 051385-585001WO the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS. 8C-8D.
  • the invasion primer is treated with a 3’ phosphatase (for example Endonuclease IV or PNK) to generate a 3’ hydroxyl group prior to sequencing.
  • a 3’ phosphatase for example Endonuclease IV or PNK
  • FIG.9A illustrates an embodiment wherein the invasion primer is annealed to the 3′ end of one of the strands.
  • the invasion primer includes one or more phosphorothioate nucleic acids at the 5′ end to protect from exonuclease digestion, and a cleavable site at the 3′ end (e.g., one or more deoxyuracil nucleobases).
  • Runoff extension of the invasion oligonucleotide is then performed with an amplification mixture that provides cleavable sites (e.g., a mixture of dUTP, dATP, dGTP, and dCTP nucleotides) leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.9B.
  • the sequenced strand may optionally further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.9B.
  • the invasion strand may be cleaved at internal cleavable sites (e.g., cleaved at the dU sites), leaving behind small, low Tm fragments (e.g., melting temperatures in the range of 0°C to about 60°C) that may be thermally denatured away, as shown in FIG.9C.
  • this cleavage and denaturation step exposes the 3’ end of the invasion oligo, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.9C-9D.
  • the invasion primer is treated with a 3’ phosphatase (for example Endonuclease IV or PNK) to generate a 3’ hydroxyl group prior to sequencing.
  • a 3’ phosphatase for example Endonuclease IV or PNK
  • internal cleavable sites e.g., cleavable internucleosidic bonds
  • the small products annealed to the second strand may then be digested away.
  • FIG.10A illustrates an invasion primer annealed to the 3′ end of one of the strands.
  • the invasion Attorney Docket No.: 051385-585001WO primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion.
  • the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage).
  • the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo.
  • Runoff extension of the invasion oligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.10B.
  • the 3’ end of the first sequencing read is capped by ddNTP incorporation.
  • a second sequencing read is then obtained by annealing and extending a second sequencing primer 3’ of the terminated first sequencing read.
  • a ddNTP is incorporated into the 3’ end of the second sequencing read, and thereafter the invasion strand may be nicked at internal scissile sites (e.g., resulting from amplification with the dUTP), leaving behind small fragments with exposed 5’ ends that may be removed under suitable conditions, for example, by lambda exonuclease digestion, as shown in FIGS.10C-10D.
  • This cleavage and removal step exposes the 3’ end of the second strand, making it available for a third sequencing read, as shown in FIG.10E.
  • the 3’ end of the third sequencing read is capped by ddNTP incorporation.
  • a fourth sequencing read is then obtained by annealing and extending a fourth sequencing primer 3’ of the terminated third sequencing read, as illustrated in FIG.10E.
  • Example 5 Spatially barcoded arrays [0399] The position of any given cell, relative to its neighbors and non-cellular structures, can provide useful information for defining cellular phenotype, cell state, and ultimately cell and tissue function. Location can determine the signals to which cells are exposed. While endocrine signals act at macroscopic scales, many other types of signals act upon neighboring cells via cell- cell interactions or via soluble signals acting in the vicinity.
  • RNA sequencing Single-cell RNA sequencing (scRNA-seq) has greatly advanced our understanding of cellular heterogeneity by profiling individual cell transcriptomes.
  • ISH RNA in situ hybridization
  • spatial transcriptomics techniques aim to examine all genes expressed from the genome from a single histological slide (see, e.g., Asp M et al. Bioessays.2020; 42(10): e1900221).
  • spatial transcriptomics There are three major methodologies to experimentally implement spatial transcriptomics.
  • in situ sequencing can identify RNA sequences from the tissue through fluorescence- based direct sequencing.
  • spatial barcoding methods associate RNA sequences and their spatial locations by capturing tissue RNA using a spatially barcoded oligonucleotide array.
  • one iteration of the 10X Genomics Visium platform includes the use of glass slides containing marked 6.5 ⁇ 6.5 mm areas where thin tissue sections are placed and imaged. Each area contains 5000 printed regions of barcoded mRNA capture probes with the dimensions of 55 ⁇ m in diameter and a center-to-center distance of 100 ⁇ m. Tissue is permeabilized and mRNAs are hybridized to the barcoded capture probes directly underneath. cDNA synthesis connects the spatial barcode and the captured mRNA, and sequencing reads are later overlaid with the captured tissue image.
  • NanoString’s GeoMX Digital Spatial Profiler wherein a slide-fixed tissue section is first exposed to a pre-known panel of barcoded hybridization probes, after which regions of interest (ROIs) are manually selected. The ROIs are then subjected to UV light and target specific barcodes are cleaved off and digital counting performed with the nCounter system.
  • ROIs regions of interest
  • target specific barcodes are cleaved off and digital counting performed with the nCounter system.
  • the techniques for spatial transcriptomics with barcoded oligonucleotide capture arrays described have limitations in the spatial resolution of up to 55-100 ⁇ m due to the physical size of capturing spots. To resolve the issue of low spatial resolution in capture-based sequencing methodology, bead-based capturing sequencing was developed.
  • HDST high-definition spatial transcriptomics
  • Seq-Scope a high-resolution spatial barcoding technology, Seq-Scope, with a 0.5-0.8 ⁇ m center-to-center resolution (see, e.g., Cho CS et al. Cell.2021; 184(13): 3559-3572).
  • This technique was developed based on the NGS technology that utilizes randomly barcoded single-molecule oligonucleotides with a transcriptome capture capacity of approximately 4,700 UMIs/cell on average, comparable to conventional scRNA-seq methods. This technical improvement enabled visualizing the histological organization of the transcriptome architecture in liver tissues at subcellular level.
  • Step-seq SpaTial Enhanced Re-solution Omics-sequencing
  • This method applies microfluidic channels perpendicularly for unique pair-wise barcoding on each spot of the tissue section.
  • Stereo-seq can capture up to 133,775 UMIs per 100 ⁇ m diameter bin, and was capable of profiling a whole mouse embryo.
  • Stereo-seq is currently undergoing commercial development by BGI as its STOmics platform, currently in early access.
  • a common approach for in situ spatial transcriptomic analysis of mRNAs is to use a capture array including poly-d(T) capture probes which then hybridize to polyadenylated RNA species.
  • the Seq-Scope method relies on the targeting of poly-A tails of mRNA molecules for capture onto a physical array of spatially barcoded RNA-capture molecules, and a spatial map of barcodes where each barcoded sequence is associated with a spatial coordinate.
  • the RNA-capture molecules include a poly-d(T) capture domain for associating with the mRNA molecules.
  • the poly-d(T) approach introduces several limitations, including, for example, the inability to target RNA species which are not poly-A tailed (e.g., non- polyadenylated), for example, histone mRNAs, lncRNAs, rRNAs, and snRNAs (see, e.g., Yang L et al. Genome Biol.2011; 12(2): R16, which is incorporated herein by reference in its entirety). Additionally, biases may be introduced towards polyadenylated RNA molecules due to the varying degree of lengths of poly-A tails present.
  • target RNA species which are not poly-A tailed e.g., non- polyadenylated
  • histone mRNAs lncRNAs
  • rRNAs rRNAs
  • snRNAs see, e.g., Yang L et al. Genome Biol.2011; 12(2): R16, which is incorporated herein by reference in its entirety.
  • the methods described herein include hybridizing an invasion primer including a binding sequence and a tail sequence to a strand of a double-stranded nucleic acid molecule immobilized on a solid support (e.g., a spatial array), wherein the tail sequence of the invasion primer includes a capture sequence capable of hybridizing to an endogenous region of a target polynucleotide of a cell in situ, and extending the strand along the tail sequence to generate an extended strand including the capture sequence.
  • a solid support e.g., a spatial array
  • the Seq-Scope method described in Cho et al. used capture probes including an oligo- dT tail for targeting mRNA molecules in cells.
  • the capture sequence may instead include an oligo-dT sequence for capture of polyadenylated mRNA species.
  • a plurality of polynucleotides each including a spatial barcode and a cleavage domain are immobilized on a solid support.
  • the polynucleotides include a platform primer binding sequence and are bound to the solid support by interactions with a corresponding platform primer attached to the solid support.
  • the polynucleotides are then amplified, for example, by bridge amplification, resulting in the generation of multiple clusters including immobilized pluralities of polynucleotides on the surface of the solid support.
  • the resulting substrate includes millions of clusters, each cluster containing pluralities of immobilized polynucleotides including the same spatial barcode.
  • An invasion primer including a binding sequence and a tail sequence is then hybridized to one strand of each double-stranded polynucleotide immobilized on the solid support (e.g., the spatial array), wherein the tail sequence of the invasion primer includes, for example, a capture sequence capable of hybridizing to an endogenous region of a target polynucleotide of a cell in situ.
  • the strand of the double- Attorney Docket No.: 051385-585001WO stranded polynucleotide hybridized to the invasion primer is then extended along the tail sequence to generate an extended strand including the capture sequence.
  • the tail sequence may also include a barcode sequence, or a primer binding sequence.
  • the invasion primer may be blocked (e.g., blocked with a non-extendible 3’ blocking moiety) such that it is not extended.
  • the invasion primer is extended to generate an invasion strand hybridized to the strand of the double-stranded polynucleotide. Once the extended strand including the capture sequence is generated, the invasion strand may be removed (e.g., removed by denaturation or by exonuclease digestion). Sequencing is performed to determine the sequence of the spatial barcodes for each cluster. The sequence is then used to assign each cluster to a specific location on the solid support.
  • the solid support is then further processed (e.g., removing unused platform primer oligonucleotides and other non-specific ssDNA) to generate a spatial barcode array that can capture target nucleic acids (e.g., mRNAs) released from a tissue section or plurality of cells, as described in PCT Pub. No. WO2022/015913, which is incorporated by reference herein in its entirety.
  • the quality of the clustered array may be inspected by staining with a DNA dye, such as SYBR Gold.
  • Tissue for example OCT-mounted fresh frozen tissue, is sectioned in a cryostat using methods known in the art.
  • tissue sections are then placed on the spatial capture array and fixed, for example using 4% formaldehyde.
  • the tissues may then be hematoxylin and eosin stained using methods known in the art and imaged under a light microscope.
  • the tissues are treated, for example, with 0.2U/uL collagenase I at 37 °C 20 min, and then with l mg/mL pepsin in 0.1M HC1 at 37 °C 10 min, as previously described (see, Salmen F et al. Nat. Protoc.2018; 13(11): 2501-34, which is incorporated herein by reference in its entirety).
  • tissue is then washed with a reverse-transcription buffer including, for example, Maxima 5x RT Buffer (Cat. No. EP0751, Thermofisher) and RNase Inhibitor (Cat. No.30281, Lucigen).
  • a reverse-transcription buffer including, for example, Maxima 5x RT Buffer (Cat. No. EP0751, Thermofisher) and RNase Inhibitor (Cat. No.30281, Lucigen).
  • reverse transcription is performed by incubating the tissue-attached spatial barcode array in an RT buffered reaction solution containing, for example, Maxima 5x RT Buffer (Cat. No. EP0751, Thermofisher), 20% Ficoll PM-400 (Cat. No. F4375-10G, Sigma), dNTPs (Cat. No. N0477L, NEB), RNase Inhibitor (Cat.
  • tissue digestion buffer 100 mM Tris pH 8.0, 100 mM NaCl, 2% SDS, 5 mM EDTA, 16 U/mL Proteinase K (Cat. No. P8107S, NEB) and incubated at 37 °C for 40 min.
  • the array is washed with water 3 times, 0. IN NaOH 3 times (each with 5 min incubation at room temperature), 0.1M Tris (pH7.5) 3 times (each with a brief wash), and then water 3 times (each with brief wash). This will eliminate all mRNA from the spatially barcoded array.
  • second strand synthesis is performed by adding to the tissue a second strand synthesis mix including a sequencing primer-conjugated random primer, dNTP mix (Cat. No. N0477, NEB), and Klenow Fragment (exonuclease-deficient; Cat. No. M0212, NEB). Then the spatially barcoded array is incubated at 37 °C for 2 hr in a humidity-controlled chamber. After second strand synthesis, the array is washed with water to remove all DNAs that are taken off from the array, so that each spatial barcode molecule corresponds to each single copy of second strand. Then the array is treated with 0.1 N NaOH to elute the second strand.
  • the elution step is duplicated and combined with the first elution.
  • the second strand product elution is then neutralized by mixing with 3 M potassium acetate, pH 5.5.
  • the neutralized secondary strand product is then subjected to AMPure XP purification (Cat. No. A63881, Beckman Coulter) using 1.8X bead/sample ratio, according to the manufacturer’s protocol.
  • the final elution is performed using water. Sequencing libraries are then prepared according to manufacturer’s protocols.
  • the libraries are subjected to, for example, paired-end (e.g., 100- 150bp) sequencing processes on, for example, a Singular GenomicsTM sequencer (e.g., the G4TM system) or IlluminaTM sequencer (e.g., HiSeqTM, MiSeqTM, NextSeqTM, or NovaSeqTM systems).
  • a plurality of sequencing cycles then occur, wherein each cycle includes extension and detection of an incorporated nucleotide.
  • the entire array may be selectively sequenced by choosing the appropriate initiator, i.e., the appropriate sequencing primer.
  • Attorney Docket No.: 051385-585001WO P-EMBODIMENTS [0413] The present disclosure provides the following illustrative embodiments.
  • Embodiment P1 A method of sequencing, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; extending the second strand along the tail sequence of said invasion primer to generate an extended second strand comprising a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide.
  • Embodiment P2 The method of Embodiment P1, further comprising removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand.
  • Embodiment P3. The method of Embodiment P1, further comprising removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand.
  • Embodiment P4 The method of Embodiment P1, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand.
  • Embodiment P5. A method of forming a single-stranded polynucleotide attached to a solid support, said method comprising: contacting a plurality of double-stranded polynucleotides comprising a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers comprise a binding sequence and a tail sequence; hybridizing the binding sequence of one of said invasion primers to one of said second strands; Attorney Docket No.: 051385-585001WO and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the invasion primer hybridized to the second strand to generate an extended second strand comprising a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support
  • Embodiment P6 The method of Embodiment P5, further comprising sequencing the single-stranded polynucleotide.
  • Embodiment P7 The method of Embodiment P5 or Embodiment P6, further comprising removing the invasion strand and sequencing the second strand.
  • Embodiment P8 The method of any one of Embodiment P5 to Embodiment P7, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand.
  • Embodiment P1 comprising nicking the invasion strand to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the invasion primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand.
  • Embodiment P10 The method of any one of Embodiment P2 to Embodiment P9, wherein removing the invasion strand comprises digesting the invasion strand using an exonuclease enzyme.
  • Embodiment P12 The method of any one of Embodiment P1 to Embodiment P9, wherein the double-stranded polynucleotides comprise known adapter sequences on 5′ and 3′ ends.
  • Embodiment P13 The method of any one of Embodiment P1 to Embodiment P9, wherein the double-stranded polynucleotides comprise known adapter sequences on 5′ and 3′ ends.
  • the invasion primer comprises locked nucleic acids (LNAs), Bis-locked nucleic acids Attorney Docket No.: 051385-585001WO (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O- methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof.
  • LNAs locked nucleic acids
  • Bis-locked nucleic acids Attorney Docket No.: 051385-585001WO
  • bisLNAs twisted intercalating nucleic acids
  • BNAs bridged nucleic acids
  • MGB minor groove binder
  • morpholino nucleic acids C5-modified pyrimidine nucleic acids
  • PNAs peptide nucleic acids
  • Embodiment P11 The method of any one of Embodiment P1 to Embodiment P11, wherein the invasion primer is about 15 to about 90 nucleotides in length.
  • Embodiment P15 The method of any one of Embodiment P1 to Embodiment P12, wherein the invasion primer comprises one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence.
  • Embodiment P16 The method of any one of Embodiment P1 to Embodiment P13, further comprising contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof.
  • Embodiment P17 Embodiment P17.
  • Embodiment P18 The method of any one of Embodiment P1 to Embodiment P14, wherein generating the invasion strand comprises contacting the polynucleotide with a buffered solution comprising dimethyl sulfoxide (DMSO), betaine, or a combination of dimethyl sulfoxide (DMSO) and betaine.
  • DMSO dimethyl sulfoxide
  • betaine a buffered solution comprising dimethyl sulfoxide
  • DMSO dimethyl sulfoxide
  • betaine a combination of dimethyl sulfoxide (DMSO) and betaine.
  • Embodiment P19 The method of any one of Embodiment P1 to Embodiment P15, wherein prior to hybridizing the invasion primer the method comprises amplifying the double- stranded polynucleotides with bridge polymerase chain reaction (bPCR) amplification, solid- phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR, or combinations of said methods.
  • bPCR bridge polymerase chain reaction
  • RCA solid- phase rolling circle amplification
  • eRCA solid-phase exponential rolling circle amplification
  • RPA solid-phase recombinase polymerase amplification
  • HDA solid-phase helicase dependent amplification
  • template walking amplification or emulsion PCR, or combinations of said methods.
  • a method of incorporating a sequence comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail Attorney Docket No.: 051385-585001WO sequence of said invasion primer to generate an extended second strand comprising a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double-stranded polynucleotide.
  • Embodiment P20 The method of any one of Embodiment P1 to Embodiment P19, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence.
  • Embodiment P21 The method of any one of Embodiment P1 to Embodiment P19, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence.
  • a method of sequencing comprising: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide
  • Embodiment 1 A method of incorporating a sequence, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand and said second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail sequence of said invasion primer with a polymerase to generate an extended second strand comprising a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double-stranded polynucleotide.
  • Embodiment 2 The method of Embodiment 1, wherein the tail sequence comprises a barcode sequence. Attorney Docket No.: 051385-585001WO [0437] Embodiment 3. The method of Embodiment 1 or 2, wherein the tail sequence comprises a capture sequence, wherein the capture sequence is capable of hybridizing to a target polynucleotide. [0438] Embodiment 4. The method of Embodiment 3, wherein the capture sequence comprises a sequence capable of hybridizing to an endogenous sequence of a target polynucleotide. [0439] Embodiment 5. The method of Embodiment 1 or 2, wherein the tail sequence comprises a poly(dT) sequence. [0440] Embodiment 6.
  • Embodiment 7 The method of any one of Embodiments 1 to 6, wherein the double-stranded polynucleotide comprises a cleavable site.
  • Embodiment 8 The method of any one of Embodiments 1 to 7, wherein the binding sequence comprises 10 to 25 nucleotides and the tail sequence comprises 5 to 25 nucleotides.
  • Embodiment 9 The method of any one of Embodiments 1 to 8, wherein the double-stranded polynucleotide does not comprise genomic DNA.
  • Embodiment 10 The method of any one of Embodiments 1 to 9, further comprising sequencing the first strand.
  • Embodiment 11 The method of any one of Embodiments 1 to 9, further comprising hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide.
  • Embodiment 12 The method of Embodiments 10 or 11, further comprising removing the invasion strand and sequencing the second strand.
  • Embodiment 13 A method of sequencing, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; extending the second strand along the tail sequence of said invasion primer to generate an extended second strand comprising a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-
  • Embodiment 14 The method of Embodiment 13, further comprising removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand.
  • Embodiment 15 The method of Embodiment 13, further comprising removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand.
  • Embodiment 16 The method of Embodiment 13, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand.
  • Embodiment 17 A method of forming a single-stranded polynucleotide attached to a solid support, said method comprising: contacting a plurality of double-stranded polynucleotides comprising a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers comprise a binding sequence and a tail sequence; hybridizing the binding sequence of one of said invasion primers to one of said second strands; and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the invasion primer hybridized to the second strand to generate an extended second Attorney Docket No.: 051385-585001WO strand comprising a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support
  • Embodiment 18 The method of Embodiment 17, further comprising sequencing the single-stranded polynucleotide.
  • Embodiment 19 The method of Embodiment 17 or 18, further comprising removing the invasion strand and sequencing the second strand.
  • Embodiment 20 The method of any one of Embodiments 17 to 19, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand.
  • Embodiment 21 Embodiment 21.
  • Embodiment 13 comprising nicking the invasion strand to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the invasion primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand.
  • Embodiment 22 The method of any one of Embodiments 14 to 21, wherein removing the invasion strand comprises digesting the invasion strand using an exonuclease enzyme.
  • Embodiment 24 The method of any one of Embodiments 1 to 21, wherein the double-stranded polynucleotides comprise known adapter sequences on 5′ and 3′ ends.
  • Embodiment 25 The method of any one of Embodiments 1 to 19, wherein the first strand is covalently attached to the solid support via a first linker and the second strand is covalently attached to the solid support via a second linker.
  • the invasion primer comprises locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof.
  • LNAs locked nucleic acids
  • bisLNAs Bis-locked nucleic acids
  • TAAs twisted intercalating nucleic acids
  • BNAs bridged nucleic acids
  • MGB minor groove binder
  • PNAs peptide nucleic acids
  • PNAs phosphorothioate nucleic acids, or combinations thereof.
  • Embodiment 27 The method of any one of Embodiments 1 to 24, wherein the invasion primer comprises one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence.
  • Embodiment 28 The method of any one of Embodiments 1 to 25, further comprising contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof.
  • Embodiment 29 Embodiment 29.
  • Embodiment 30 The method of any one of Embodiments 1 to 26, wherein generating the invasion strand comprises contacting the polynucleotide with a buffered solution comprising dimethyl sulfoxide (DMSO), betaine, or a combination of dimethyl sulfoxide (DMSO) and betaine.
  • Embodiment 31 The method of any one of Embodiments 1 to 30, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence.
  • Embodiment 32 The method of any one of Embodiments 1 to 30, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence.
  • a method of sequencing comprising: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer Attorney Docket No.: 051385-585001WO with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first
  • Embodiment of any one of claims 1 to 30, prior to hybridizing an invasion primer said method comprises amplifying a nucleic acid molecule thereby forming a plurality of double- stranded polynucleotides immobilized to the solid support.

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Abstract

Disclosed herein, inter alia, are substrates, kits, and efficient methods of preparing and sequencing a double-stranded polynucleotide.

Description

Attorney Docket No.: 051385-585001WO METHODS FOR IMPROVING STRAND INVASION EFFICIENCY CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/483,812 filed February 8, 2023 and U.S. Provisional Application No.63/395,644, filed August 5, 2022; each of which is incorporated herein by reference in their entirety and for all purposes. SEQUENCE LISTING [0002] The Sequence Listing written in file 051385-585001WO_ST26.xml, created July 27, 2023, 121,528 bytes, machine format IBM-PC, MS Windows operating system, is hereby incorporated by reference. BACKGROUND [0003] Genetic analysis is taking on increasing importance in modern society as a diagnostic, prognostic, and as a forensic tool. DNA sequencing is a fundamental tool in biological and medical research; it is an essential technology for the paradigm of personalized precision medicine. Sanger sequencing, where the sequence of a nucleic acid is determined by selective incorporation and detection of dideoxynucleotides, enabled the mapping of the first human reference genome. While this methodology is still useful for validating newer sequencing technologies, efforts to sequence and assemble genomes using the Sanger method are an expensive and laborious undertaking, requiring specialized equipment and expertise. Next generation sequencing (NGS) methodologies make use of simultaneously sequencing millions of fragments of nucleic acids in a single run. However, traditional next generation sequencing still has shortcomings, such as challenges with detecting rare sequence variants in the context of polymerase errors. BRIEF SUMMARY [0004] In view of the foregoing, innovative approaches to address issues with existing sequencing technologies are needed. Disclosed herein are solutions to these and other problems in the art which, in embodiments, increase the fidelity and accuracy of high throughput sequencing methods. In certain embodiments, the compositions and methods provided herein reduce the amount of nucleic acid manipulation and duplication required by traditional NGS Attorney Docket No.: 051385-585001WO techniques. Prior to the present disclosure, cluster-based sequencing processes would include cleaving and removing one strand from double-stranded molecules in a cluster before generating a first read, without which the second strand would effectively compete with hybridization of the sequencing primer. Generating a sequencing read for the second (cleaved) strand would then require creating a new complementary strand from the sequenced first strand (i.e., a new second strand). In accordance with various embodiments, the methods disclosed herein permit obtaining sequence information (i.e., reading) from the original first and second strands (e.g., original strands from the initial cluster amplification, or amplicons), thereby reducing the time, reagents, expense, and risk of polymerase errors inherent in previous methods. [0005] In an aspect is provided a method of sequencing, the method including: hybridizing an invasion primer including a first sequence (e.g., a binding sequence complementary to a portion of the polynucleotide) and a second sequence (e.g., a tail sequence) to a second strand of a double-stranded polynucleotide and extending the binding sequence (e.g., extending the 3’ end of the binding sequence) with a polymerase, thereby generating an invasion strand and displacing the first strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; extending the second strand along the tail sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. [0006] In an aspect is provided a method of forming a single-stranded polynucleotide attached to a solid support, the method including: contacting a plurality of double-stranded polynucleotides including a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers include a binding sequence and a tail sequence; hybridizing the binding sequence of one of the invasion primers to one of the second strands; and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the Attorney Docket No.: 051385-585001WO invasion primer hybridized to the second strand to generate an extended second strand including a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support. [0007] In an aspect is provided a method of incorporating a sequence, the method including: hybridizing an invasion primer including a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double- stranded polynucleotide. [0008] In an aspect is provided a method of sequencing, the method including: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. BRIEF DESCRIPTION OF THE DRAWINGS [0009] FIGS.1A-1B illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase. The hashed boxes on each end represent a polymer scaffold that is anchored to a solid support, such as glass or silicon support. FIG.1A illustrates two dsDNA duplex strands, each duplex having a first strand hybridized to a second strand, wherein each strand is attached to the solid support. By way of simplification, only one Attorney Docket No.: 051385-585001WO duplex is shown, however it is understood that a plurality of duplexes (double-stranded amplification products) is present on the solid support, typically in a plurality of localized monoclonal clusters. An invasion oligonucleotide (also referred to herein as an invasion primer or invasion oligo) anneals at the 3′ end of one of the strands. As illustrated, the invasion oligonucleotide is not attached to the solid support. After extension of the invasion oligonucleotide has been completed, one strand of the initial dsDNA molecule is now single- stranded and available for a first sequencing read, as shown in FIG.1B. Also illustrated in FIG. 1B, the sequenced strand may optionally be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing. For clarity in the figure, a single X is depicted, however it is understood that the cleavable site may include multiple chemical, enzymatic, or photochemical entities capable of being cleaved. [0010] FIGS.2A-2B illustrate an embodiment of strand invasion including an invasion primer with a 5’ tail sequence. The hashed box represents a polymer scaffold that is anchored to a solid support, such as glass or silicon support. FIG.2A illustrates two dsDNA duplex strands, each duplex having a first strand hybridized to a second strand, and each strand is attached to the solid support. An invasion primer including a binding sequence (e.g., a sequence complementary to a region within one of the strands of the dsDNA) and a 5’ tail sequence (wherein the tail sequence is not complementary to the dsDNA) is introduced, wherein the binding sequence of the elongated invasion primer hybridizes to one of the strands. An invasion mixture as described above may be used to introduce and hybridize the elongated invasion primer to the dsDNA. Following invasion, extension of the elongated invasion primer using an extension reaction mixture as described herein generates a blocking strand hybridized to one of the strands of the dsDNA (e.g., a blocking strand, also referred to herein as an invasion strand, is now hybridized to the second strand, for example, resulting in a single-stranded first strand that is accessible to a primer). To reduce the probability of the second strand re-annealing to the first strand, the second strand is extended along the elongated invasion primer to incorporate the complement of the 5’ tail sequence, thereby modifying the 3’ end of the second strand such that it is no longer complementary to the 5’ end of the first strand. FIG.2B illustrates an additional embodiment of strand invasion, wherein after generating an extended second strand as shown in FIG.2A the blocking strand is removed (e.g., removed via exonuclease digestion). This also enables the use of distinct invasion primer hybridization and extension conditions to used. Following removal of Attorney Docket No.: 051385-585001WO the blocking strand, the first strand may reanneal to the second strand, but the incorporated tail sequence at the 3’ end of the second strand is free, allowing for annealing of a primer (e.g., a second invasion primer complementary to the tail sequence), followed by extension with a polymerase (e.g., a strand-displacing polymerase) thereby generating a second blocking strand hybridized to the second strand. [0011] FIG.3 illustrates an alternate embodiment of strand invasion. As illustrated, a bridged dsDNA complex is present on a solid support, wherein the first strand and the second strand of the dsDNA are immobilized on the solid support. Following invasion by an invasion oligonucleotide and strand synthesis along the second strand, for example as illustrated in FIG.1, a second oligonucleotide primer (e.g., a second competitive oligonucleotide) is annealed to the 5’ end of the immobilized first strand and extended towards the solid support, generating a competitor strand at the 5’ end of the immobilized first strand that may reduce reannealing of a portion of the second strand (i.e., prevent the 3’ end of the second strand from reannealing to the 5’ end of the first strand). [0012] FIGS.4A-4B illustrate an embodiment of strand invasion using an invasion primer that contains peptide nucleic acids (PNAs) into dsDNA clusters. PNA oligos can invade into dsDNA at low ionic strength (<25 mM NaCl) conditions. As described herein, the PNA-containing invasion primer is designed to invade at the common adapter sequence of the 5′ end of one of the solid phase-bound amplicons. This, in turn, makes the displaced complementary 3′ DNA end on the complementary strand accessible for binding with another invasion oligonucleotide, referred to as a runoff primer in FIG.4B, that can be extended by a strand-displacing DNA polymerase. At the end of that process, one of the two strands of the initially dsDNA cluster is now single- stranded and accessible for hybridization with a sequencing primer for a first sequencing read. The sequenced strand may further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing. [0013] FIGS 5A-5B illustrate an embodiment of strand invasion into dsDNA monoclonal clusters by using a recombinase and an invasion oligonucleotide. The pre-synaptic filament (alternatively referred to as a pre-synaptic complex), consisting of an invasion oligonucleotide complexed with recombinase enzymes searches dsDNA fragments for homology. The invasion oligonucleotide can be inserted to its complementary sequence in the dsDNA amplicons, after Attorney Docket No.: 051385-585001WO which the invasion oligonucleotide can be extended by a strand-displacing polymerase. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process. The sequenced strand may further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.5B. [0014] FIGS.6A-6D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase. FIG.6A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion. In embodiments, the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage). For example, as depicted as a ‘U’ in FIGS.6A-6C, the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo. After runoff extension of the invasion oligonucleotide has been completed, one strand of the initial dsDNA molecule is now single-stranded and available for a first sequencing read, as shown in FIG.6B. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process. The sequenced strand may further optionally be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.6B. Subsequently, the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicking the dU using suitable conditions), leaving behind a 5’-phosphate in the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.6C-6D. [0015] FIGS.7A-7D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase. FIG.7A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion. In embodiments, the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage). For example, as depicted as a ‘U’, the cleavable site may Attorney Docket No.: 051385-585001WO be a deoxyuracil (dU) towards the 3’ end of the invasion oligo. After runoff extension (i.e., extension to a sufficient length) of the invasion oligonucleotide has been completed, one strand of the initial dsDNA molecule is now single-stranded and available for a first sequencing read, as shown in FIG.7B. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process. The sequenced strand may further be extended with native dNTPs to complete the extension of the sequenced strand, as illustrated in FIG.7B as the solid line beyond the star. Further extending the sequencing primer with unmodified nucleotides eliminates any remaining single-stranded region. Subsequently, the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., cleaving the dU using a uracil DNA glycosylase or formamidopyrimidine DNA glycosylase (Fpg) as described herein), leaving behind a 5’-phosphate in the extended part of the invasion primer that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.7C-7D. [0016] FIGS.8A-8D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase. FIG.8A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion. In embodiments, the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage). For example, as depicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo. Following runoff extension of the invasion oligonucleotide, one strand of the initial dsDNA molecule is now single-stranded and available for a first sequencing read, as shown in FIG.8B. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process. The sequenced strand may further be extended with one or more dideoxynucleotide triphosphates (ddNTPs) to prevent further extension, as illustrated in FIG.8B as the hexagon. Further extending the sequencing primer with ddNTPs eliminates any prevents any further extension. Subsequently, the cleavable site at the 3’ end of the invasion primer may be cleaved (e.g., the dU), leaving behind a 5’-phosphate in the extended part of the invasion primer that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.8C-8D. Attorney Docket No.: 051385-585001WO [0017] FIGS.9A-9D illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase. FIG.9A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion. In embodiments, the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage). For example, as depicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo. Runoff extension of the invasion oligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.9B. The sequenced strand may optionally further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.9B. Subsequently, the invasion strand may be nicked at internal scissile sites (e.g., resulting from amplification with the dUTP), leaving behind small, low Tm fragments that may be denatured and removed under suitable conditions, as shown in FIG.9C. Additionally, this cleavage and denaturation step exposes the 3’ end of the invasion oligo, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.9C- 9D. [0018] FIGS.10A-10E illustrate an embodiment of paired-strand sequencing by strand invasion of an invasion primer at the 3′ end of a first strand of a duplex, followed by runoff extension of the invasion primer by a strand-displacing polymerase. FIG.10A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion. In embodiments, the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage). For example, as depicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo. Runoff extension of the invasion oligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.10B. Once the first sequencing read has been obtained, the 3’ end of the first sequencing read is capped by ddNTP incorporation. A second sequencing read is then obtained by annealing and extending a second sequencing primer 3’ of the terminated first Attorney Docket No.: 051385-585001WO sequencing read. Subsequently, a ddNTP is incorporated into the 3’ end of the second sequencing read, and thereafter the invasion strand may be nicked at internal scissile sites (e.g., resulting from amplification with the dUTP), leaving behind small fragments with exposed 5’ ends that may be removed under suitable conditions, for example, by lambda exonuclease digestion, as shown in FIGS.10C-10D. This cleavage and removal step exposes the 3’ end of the second strand, making it available for a third sequencing read, as shown in FIG.10E. Once the third sequencing read has been obtained, the 3’ end of the third sequencing read is capped by ddNTP incorporation. A fourth sequencing read is then obtained by annealing and extending a fourth sequencing primer 3’ of the terminated third sequencing read, as illustrated in FIG.10E. DETAILED DESCRIPTION [0019] The aspects and embodiments described herein relate to sequencing a polynucleotide. In embodiments, as described herein, the methods relate to sequencing a first strand of a double- stranded polynucleotide, and optionally sequencing the complement of first strand (i.e., the second strand) of the same double-stranded polynucleotide. The terms “cluster” and “colony” are used interchangeably throughout this application and refer to a discrete site on a solid support comprised of a plurality of immobilized nucleic acid strands. The term “clustered array” refers to an array formed from such clusters or colonies. I. Definitions [0020] All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby expressly incorporated herein by reference in their entireties. The practice of the technology described herein will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, bioinformatics, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Examples of such techniques are available in the literature. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); and Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012). Methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. Attorney Docket No.: 051385-585001WO [0021] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those of skill in the art. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. [0022] As used herein, the singular terms “a”, “an”, and “the” include the plural reference unless the context clearly indicates otherwise. Reference throughout this specification to, for example, "one embodiment", "an embodiment", "another embodiment", "a particular embodiment", "a related embodiment", "a certain embodiment", "an additional embodiment", or "a further embodiment" or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. [0023] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about means the specified value. [0024] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of" is meant including, and limited to, whatever Attorney Docket No.: 051385-585001WO follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. [0025] As used herein, the term “control” or “control experiment” is used in accordance with its plain and ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects. [0026] As used herein, the term "associated" or "associated with" can mean that two or more species are identifiable as being co-located at a point in time. An association can mean that two or more species are or were within a similar container. An association can be an informatics association, where for example digital information regarding two or more species is stored and can be used to determine that one or more of the species were co-located at a point in time. An association can also be a physical association. In some instances, two or more associated species are "tethered", "coated”, "attached", or "immobilized" to one another or to a common solid or semisolid support (e.g. a receiving substrate). An association may refer to a relationship, or connection, between two entities. Associated may refer to the relationship between a sample and the DNA molecules, RNA molecules, or polynucleotides originating from or derived from that sample. These relationships may be encoded in oligonucleotide barcodes, as described herein. A polynucleotide is associated with a sample if it is an endogenous polynucleotide, i.e., it occurs in the sample at the time the sample is obtained, or is derived from an endogenous polynucleotide. For example, the RNAs endogenous to a cell are associated with that cell. cDNAs resulting from reverse transcription of these RNAs, and DNA amplicons resulting from PCR amplification of the cDNAs, contain the sequences of the RNAs and are also associated with the cell. The polynucleotides associated with a sample need not be located or synthesized in the sample, and are considered associated with the sample even after the sample has been destroyed (for example, after a cell has been lysed). Barcoding can be used to determine which polynucleotides in a mixture are associated with a particular sample. [0027] As used herein, the term “complementary” or “substantially complementary” refers to the hybridization, base pairing, or the formation of a duplex between nucleotides or nucleic acids. As used herein, the term “complement,” as used herein, refers to a nucleotide (e.g., RNA Attorney Docket No.: 051385-585001WO or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. For example, complementarity exists between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides is capable of base pairing with a respective cognate nucleotide or cognate sequence of nucleotides. When referring to a double-stranded polynucleotide including a first strand hybridized to a second strand, it is to be understood that each of the terms “first strand” and “second strand” refer to single-stranded polynucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine (A) is thymidine (T) and the complementary (matching) nucleotide of guanosine (G) is cytosine (C). Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and non- coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence. “Duplex” means at least two oligonucleotides and/or polynucleotides that are fully or partially complementary undergo Watson-Crick type base pairing among all or most of their nucleotides so that a stable complex is formed. Complementary single stranded nucleic acids and/or substantially complementary single stranded nucleic acids can hybridize to each other under hybridization conditions, thereby forming a nucleic acid that is partially or fully double stranded. When referring to a double-stranded polynucleotide including a first strand hybridized to a second strand, it is understood that each of the first strand and the second strand are independently single-stranded polynucleotides. All or a portion of a nucleic acid sequence may be substantially complementary to another nucleic acid sequence, in some embodiments. Attorney Docket No.: 051385-585001WO As referred to herein, “substantially complementary” refers to nucleotide sequences that can hybridize with each other under suitable hybridization conditions. Hybridization conditions can be altered to tolerate varying amounts of sequence mismatch within complementary nucleic acids that are substantially complementary. Substantially complementary portions of nucleic acids that can hybridize to each other can be 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more complementary to each other. In some embodiments substantially complementary portions of nucleic acids that can hybridize to each other are 100% complementary. Nucleic acids, or portions thereof, that are configured to hybridize to each other often comprise nucleic acid sequences that are substantially complementary to each other. [0028] As described herein, the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of nucleotides that complement one another (e.g., about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher complementarity over a specified region). In embodiments, two sequences are complementary when they are completely complementary, having 100% complementarity. In embodiments, sequences in a pair of complementary sequences form portions of a single polynucleotide with non-base-pairing nucleotides (e.g., as in a hairpin or loop structure, with or without an overhang) or portions of separate polynucleotides. In embodiments, one or both sequences in a pair of complementary sequences form portions of longer polynucleotides, which may or may not include additional regions of complementarity. [0029] As used herein, the term “contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. However, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture. The term “contacting” may include Attorney Docket No.: 051385-585001WO allowing two species to react, interact, or physically touch, wherein the two species may be a compound, a protein (e.g., an antibody), or enzyme. [0030] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “strand,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non- limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences. As may be used herein, the terms “nucleic acid oligomer” and “oligonucleotide” are used interchangeably and are intended to include, but are not limited to, nucleic acids having a length of 200 nucleotides or less. In some embodiments, an oligonucleotide is a nucleic acid having a length of 2 to 200 nucleotides, 2 to 150 nucleotides, 5 to 150 nucleotides or 5 to 100 nucleotides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. In some embodiments, an oligonucleotide is a primer configured for extension by a polymerase when the primer is annealed completely or partially to a complementary nucleic acid template. A primer is often a single stranded nucleic acid. In certain embodiments, a primer, or portion thereof, is substantially complementary to a portion of an adapter. In some embodiments, a primer has a length of 200 nucleotides or less. In certain embodiments, a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides. In some embodiments, an oligonucleotide may be immobilized to a solid support. Attorney Docket No.: 051385-585001WO [0031] Two or more associated species are "tethered", "coated”, "attached", or "immobilized" to one another or to a common solid or semisolid support (e.g. a receiving substrate). An association may refer to a relationship, or connection, between two entities. As used herein, an immobilized polynucleotide or an immobilized primer refers to a polynucleotide or a primer that is attached to a solid surface, such as a solid support. The immobilized polynucleotide and/or immobilized primer may be attached covalently (e.g. through a linker) or non-covalently to a solid support. In embodiments, immobilized polynucleotide and/or immobilized primer is covalently attached to a solid support. [0032] As used herein, the terms “polynucleotide primer” and “primer” refers to any polynucleotide molecule that may hybridize to a polynucleotide template, be bound by a polymerase, and be extended in a template-directed process for nucleic acid synthesis. The primer may be a separate polynucleotide from the polynucleotide template, or both may be portions of the same polynucleotide (e.g., as in a hairpin structure having a 3’ end that is extended along another portion of the polynucleotide to extend a double-stranded portion of the hairpin). Primers (e.g., forward or reverse primers) may be attached to a solid support (e.g., a polymer coated solid support). In embodiments, forward primers anneal to the antisense strand of the double-stranded DNA, which runs from the 3’ to 5’ direction. Forward primers, for example, initiate the synthesis of a gene in the 5’ to 3’ direction. In embodiments, reverse primers anneal to the sense strand of the double-stranded DNA, which runs from the 5’ to 3’ direction. Reverse primers, for example, initiate the synthesis of a gene in the 3’ to 5’ direction. A primer can be of any length depending on the particular technique it will be used for. For example, PCR primers are generally between 10 and 40 nucleotides in length. The length and complexity of the nucleic acid fixed onto the nucleic acid template may vary. In some embodiments, a primer has a length of 200 nucleotides or less. In certain embodiments, a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides. A primer typically has a length of 10 to 50 nucleotides. For example, a primer may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer has a length of 18 to 24 nucleotides. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure. The primer permits the addition of a nucleotide residue thereto, or oligonucleotide or polynucleotide synthesis therefrom, under suitable conditions. In an Attorney Docket No.: 051385-585001WO embodiment the primer is a DNA primer, i.e., a primer consisting of, or largely consisting of, deoxyribonucleotide residues. The primers are designed to have a sequence that is the complement of a region of template/target DNA to which the primer hybridizes. The addition of a nucleotide residue to the 3’ end of a primer by formation of a phosphodiester bond results in a DNA extension product. The addition of a nucleotide residue to the 3’ end of the DNA extension product by formation of a phosphodiester bond results in a further DNA extension product. In another embodiment the primer is an RNA primer. In embodiments, a primer is hybridized to a target polynucleotide. A “primer” is complementary to a polynucleotide template, and complexes by hydrogen bonding or hybridization with the template to give a primer/template complex for initiation of synthesis by a polymerase, which is extended by the addition of covalently bonded bases linked at its 3′ end complementary to the template in the process of DNA synthesis. [0033] As used herein, the term “primer binding sequence” or simply “binding sequence” refers to a polynucleotide sequence that is complementary to at least a portion of a primer (e.g., an invasion primer, a sequencing primer, or an amplification primer). Primer binding sequences can be of any suitable length. In embodiments, a primer binding sequence is about or at least about 10, 15, 20, 25, 30, or more nucleotides in length. In embodiments, a primer binding sequence is 10-50, 15-30, or 20-25 nucleotides in length. The primer binding sequence may be selected such that the primer (e.g., sequencing primer) has the preferred characteristics to minimize secondary structure formation or minimize non-specific amplification, for example having a length of about 20-30 nucleotides; approximately 50% GC content, and a Tm of about 55°C to about 65°C. [0034] As used herein, a platform primer is a primer oligonucleotide immobilized or otherwise bound to a solid support (i.e., an immobilized oligonucleotide). Examples of platform primers include P7 and P5 primers, or S1 and S2 sequences, or the reverse complements thereof. A “platform primer binding sequence” refers to a sequence or portion of an oligonucleotide that is capable of binding to a platform primer (e.g., the platform primer binding sequence is complementary to the platform primer). In embodiments, a platform primer binding sequence may form part of an adapter. In embodiments, a platform primer binding sequence is Attorney Docket No.: 051385-585001WO complementary to a platform primer sequence. In embodiments, a platform primer binding sequence is complementary to a primer. [0035] As used herein, the term “capture domain” refers to an oligonucleotide sequence (e.g., an oligonucleotide sequence included in a primer, for example a surface-immobilized capture primer). The capture domain may be any suitable domain capable of hybridizing to RNA or a transcript thereof, such as mRNA. In some embodiments, the capture domain includes a poly- T oligonucleotide. A poly-T oligonucleotide includes a series of consecutive deoxythymidine residues linked by phosphodiester bonds. A poly-T oligonucleotide is capable of hybridizing to the poly-A tail of mRNA. In some embodiments, the capture domain may further include additional sequences to facilitate the capture of a particular RNA (e.g., mRNA) corresponding to select genes or groups of genes. Such a capture primer may be selected or designed based on sequence of the RNA it is desired to capture. Accordingly, the capture primer may be a sequence-specific capture primer as described herein. In some embodiments, the capture domain may target DNA, instead of RNA. In some embodiments, the capture domain may target non- specific or specific DNA sequences. For example, the capture domain may include a nucleic acid sequence to facilitate the capture of a target DNA sequence. The type of target may depend on the specific capture domain used and/or the presence of additional capture moieties on the substrate. For example, capture domains including a poly-dT tail are suited for spatial detection of RNA with poly-A tail. RNA that does not have poly-A tail may be labeled with poly-A before being captured by the substrate. Capture domains including a nucleic acid sequence against a target DNA sequence are useful for spatial detection of DNA. Substrates include a capture primer and an additional capture moiety (e.g. an antibody targeting protein or DNA/RNA probes targeting specific nucleic acid sequence) are useful for multiplex detection of nucleic acid and non-nucleic acid targets. [0036] As used herein, the term “spatial barcode” refers to a known nucleic acid sequence that allows the location of a biological molecule with which the barcode is associated to be resolved. A barcode can be a spatial barcode. The barcode or spatial barcode may be associated with an oligonucleotide as described herein (e.g., a capture oligonucleotide or capture probe). The barcodes can be designed for precision sequence performance, e.g., GC content between 40% and 60%, no homo-polymer runs longer than two, no self-complementary stretches longer than 3, Attorney Docket No.: 051385-585001WO and be comprised of sequences not present in a human genome reference. A barcode sequence can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases. A barcode sequence can be at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases. A barcode sequence can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 bases. An oligonucleotide (e.g., primer or adapter) can include about, more than, less than, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different barcodes. Barcodes can be of sufficient length and include sequences that can be sufficiently different to allow the identification of the spatial position of each biological molecule based on barcode(s) with which each biological molecule is associated. In some cases, each barcode is, for example, four deletions or insertions or substitutions away from any other barcode in an array. The oligonucleotides in each array spot on the barcoded oligonucleotide array can include the same barcode sequence and oligonucleotides in different array spots can include different barcode sequences. The barcode sequence used in one array spot can be different from the barcode sequence in any other array spot. Alternatively, the barcode sequence used in one array spot can be the same as the barcode sequence used in another array spot, as long as the two array spots are not adjacent. Barcode sequences corresponding to particular array spots can be known from the controlled synthesis of the array. Alternatively, barcode sequences corresponding to particular array spots can be known by retrieving and sequencing material from particular array spots. In embodiments, the spatial barcode sequence is indicative of the location of the immobilized capture probe on the solid support to within about 2 μm, about 1 μm, about 0.5 μm, about 0.2 μm, or about 0.1 μm. [0037] The order of elements within a nucleic acid molecule is typically described herein from 5′ to 3′. In the case of a double-stranded molecule, the “top” strand is typically shown from 5′ to 3′, according to convention, and the order of elements is described herein with reference to the top strand. [0038] As used herein, the terms “invasion primer”, “invasion oligonucleotide” and “third polynucleotide” refer to a polynucleotide molecule that may hybridize to a single-stranded nucleic acid sequence of a double-stranded polynucleotide and be extended in a template- directed process (e.g., extended with a polymerase) for nucleic acid synthesis. In embodiments, Attorney Docket No.: 051385-585001WO the invasion primer includes a binding sequence (e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof) and a 5’ tail sequence (i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof). In embodiments, an invasion primer hybridizes at or near the end of the single-stranded nucleic acid sequence (e.g., the 5’ end or the 3’ end), or the invasion primer hybridizes at an internal sequence. Extension of an invasion primer results in the formation of an “invasion strand” complementary to either the first strand or the second strand of the double-stranded polynucleotide. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the sequencing process. In embodiments, the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’- O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof. In embodiments, the invasion primer includes phosphorothioate nucleic acids. In embodiments, the invasion primer includes one or more locked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene- functionalized oligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In embodiments, the invasion primer includes 10 to 15 locked nucleic acids (LNAs). In embodiments, the invasion primer includes a sequence described herein, for example within Table 1. In embodiments, the invasion primer includes one or more phosphorothioates at the 5′ end. In embodiments, the invasion primer includes one or more LNAs at the 5′ end. In embodiments, the invasion primer includes two or more consecutive LNAs at the 3′ end. In embodiments, the invasion primer includes two or more consecutive LNAs at the 5′ end. In embodiments, the invasion primer includes a plurality (e.g., 2 to 10) of synthetic nucleotides (e.g., LNAs) and a plurality (e.g., 2 to 10) canonical or native nucleotides (e.g., dNTPs). In embodiments, the invasion primer includes one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU). In embodiments, the one or more dU nucleobases are at or near the 3’ end of the invasion primer (e.g., within 5 nucleotides of the 3’ end). In embodiments, the one or more dU nucleobases are distributed through the invasion primer. In embodiments, the invasion primer includes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followed by a plurality of synthetic nucleotides (e.g., LNAs), Attorney Docket No.: 051385-585001WO and a plurality (e.g., 2 to 10) of canonical bases. In some embodiments, the invasion primer includes a plurality of canonical bases, wherein the canonical bases terminate (i.e., at the 3′ end) with a deoxyuracil nucleobase (dU). In embodiments, the invasion primer is about 10 to 100 nucleotides in length. In embodiments, the invasion primer is about 15 to about 40 nucleotides in length. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 70°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 75°C to about 85°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is 75°C to 85°C. The invasion primer is capable of hybridizing (i.e., capable of hybridizing under suitable hybridization conditions) to one strand of a double-stranded polynucleotide molecule in a process of strand invasion. The invasion primer may include nucleic acids having a binding affinity greater than the binding affinity of standard or canonical DNA oligonucleotides, such as locked nucleic acids (LNA), peptide nucleic acids (PNAs), 2’-O-methyl RNA:DNA chimeras, minor groove binder probes (MGB), or morpholino probes. In some embodiments, invasion primers can undergo spontaneous strand invasion into dsDNA (e.g., hybridizing to a sequence near the end or terminus of the dsDNA), as is the case for example for PNA invasion primers under low ionic strength conditions, while other invasion primers may need assistance of additives such as DMSO, ethylene glycol, formamide, betaine, or other denaturants that assist strand invasion by inducing more breathability within dsDNA amplicons. In embodiments, the invasion primer may be introduced without a polymerase and allowed to invade and anneal to the complementary region of one strand of a dsDNA molecule, or it may be introduced together with a polymerase for runoff extension. Examples of polymerases that can be used for runoff extension include strand-displacing polymerases such as Bst large fragment, Bst2.0 (New England Biolabs), Bsm DNA polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase or Phi29 polymerase. In embodiments, the invasion primer includes a primer binding sequence and a tail sequence. A “tail sequence” refers to a nucleic acid sequence that is upstream of the primer binding sequence. For example, an invasion primer may include, from 5’ to 3’, the tail sequence and the binding sequence. In embodiments, the tail sequence does not hybridize to the double-stranded polynucleotide (e.g., upon initial contact, the invasion primer hybridizes to the second strand, though it is understood that following extension of the second strand, the tail sequence is complementary to the extended second strand). Attorney Docket No.: 051385-585001WO [0039] As used herein, the term “strand invasion” refers to the displacement of one strand of a double-stranded nucleic acid molecule by a nucleic acid molecule (e.g., single stranded nucleic acid molecule, such as an invasion primer). In embodiments, the nucleic acid molecule includes a nucleotide sequence that is substantially identical to a portion of the displaced strand and can selectively hybridize to the strand complementary to the displaced strand. Strand displacement can occur without degradation of the displaced strands, thus being distinct from exonuclease activity. In embodiments, the displacement occurs as a result of an extension reaction of a primer hybridized to a polynucleotide and a strand displacing enzyme. [0040] As used herein, the term “invasion strand” refers to an extended invasion primer (e.g., an invasion primer that has been hybridized to a first strand of a dsDNA molecule and extended by, for example, a strand-displacing polymerase in runoff extension to generate an invasion strand hybridized to the first strand of the dsDNA molecule). The invasion strand, for example, when hybridized to the first strand of a dsDNA molecule, prevents or blocks hybridization of the second strand of the dsDNA molecule to the first strand. In some embodiments, the invasion strand may be removed (e.g., the invasion strand may be digested with an exonuclease enzyme or denatured and washed away), allowing re-hybridization of the second strand of the dsDNA molecule to the first strand. [0041] As used herein, the term “substrate” refers to a solid support material. The substrate can be non-porous or porous. The substrate can be rigid or flexible. As used herein, the terms “solid support” and “solid surface” refers to discrete solid or semi-solid surface. A solid support may encompass any type of solid, porous, or hollow sphere, ball, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently). A solid support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non- spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. Solid supports may be in the form of discrete particles, which alone does not imply or require any particular shape. The term “particle” means a small body made of a rigid or semi-rigid material. The body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions. As used herein, the term “discrete particles” refers to physically distinct particles Attorney Docket No.: 051385-585001WO having discernible boundaries. The term “particle” does not indicate any particular shape. The shapes and sizes of a collection of particles may be different or about the same (e.g., within a desired range of dimensions, or having a desired average or minimum dimension). A particle may be substantially spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. In embodiments, the particle has the shape of a sphere, cylinder, spherocylinder, or ellipsoid. Discrete particles collected in a container and contacting one another will define a bulk volume containing the particles, and will typically leave some internal fraction of that bulk volume unoccupied by the particles, even when packed closely together. In embodiments, cores and/or core-shell particles are approximately spherical. As used herein the term “spherical” refers to structures which appear substantially or generally of spherical shape to the human eye, and does not require a sphere to a mathematical standard. In other words, “spherical” cores or particles are generally spheroidal in the sense of resembling or approximating to a sphere. In embodiments, the diameter of a spherical core or particle is substantially uniform, e.g., about the same at any point, but may contain imperfections, such as deviations of up to 1, 2, 3, 4, 5 or up to 10%. Because cores or particles may deviate from a perfect sphere, the term “diameter” refers to the longest dimension of a given core or particle. Likewise, polymer shells are not necessarily of perfect uniform thickness all around a given core. Thus, the term “thickness” in relation to a polymer structure (e.g., a shell polymer of a core-shell particle) refers to the average thickness of the polymer layer. [0042] A solid support may further comprise a polymer or hydrogel on the surface to which the primers are attached (e.g., the primers are covalently attached to the polymer, wherein the polymer is in direct contact with the solid support). Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, photopatternable dry film resists, UV-cured adhesives and polymers. The solid supports for some embodiments have at least one surface located within a flow cell. The solid support, or regions thereof, can be substantially flat. The solid support can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts or the like. Attorney Docket No.: 051385-585001WO The term solid support is encompassing of a substrate (e.g., a flow cell) having a surface comprising a polymer coating covalently attached thereto. In embodiments, the solid support is a flow cell. The term “flow cell” as used herein refers to a chamber including a solid surface across which one or more fluid reagents can be flowed. Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008). In certain embodiments a substrate comprises a surface (e.g., a surface of a flow cell, a surface of a tube, a surface of a chip, surface of a particle), for example a metal surface (e.g., steel, gold, silver, aluminum, silicon and copper). In some embodiments a substrate (e.g., a substrate surface) is coated and/or comprises functional groups and/or inert materials. In certain embodiments a substrate comprises a bead, a chip, a capillary, a plate, a membrane, a wafer (e.g., silicon wafers), a comb, or a pin for example. In some embodiments a substrate comprises a bead and/or a nanoparticle. A substrate can be made of a suitable material, non-limiting examples of which include a plastic or a suitable polymer (e.g., polycarbonate, poly(vinyl alcohol), poly(divinylbenzene), polystyrene, polyamide, polyester, polyvinylidene difluoride (PVDF), polyethylene, polyurethane, polypropylene, and the like), borosilicate, silica, nylon, Wang resin, Merrifield resin, metal (e.g., iron, a metal alloy, sepharose, agarose, polyacrylamide, dextran, cellulose and the like or combinations thereof. In some embodiments a substrate comprises a magnetic material (e.g., iron, nickel, cobalt, platinum, aluminum, and the like). In certain embodiments a substrate comprises a magnetic bead (e.g., DYNABEADS®, hematite, AMPure XP). Magnets can be used to purify and/or capture nucleic acids bound to certain substrates (e.g., substrates comprising a metal or magnetic material). [0043] As used herein, the term “polymer” refers to macromolecules having one or more structurally unique repeating units. The repeating units are referred to as “monomers,” which are polymerized for the polymer. Typically, a polymer is formed by monomers linked in a chain- like structure. A polymer formed entirely from a single type of monomer is referred to as a “homopolymer.” A polymer formed from two or more unique repeating structural units may be referred to as a “copolymer.” A polymer may be linear or branched, and may be random, block, polymer brush, hyperbranched polymer, bottlebrush polymer, dendritic polymer, or polymer micelles. The term “polymer” includes homopolymers, copolymers, tripolymers, tetra polymers and other polymeric molecules made from monomeric subunits. Copolymers include alternating Attorney Docket No.: 051385-585001WO copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, linear copolymers and branched copolymers. The term “polymerizable monomer” is used in accordance with its meaning in the art of polymer chemistry and refers to a compound that may covalently bind chemically to other monomer molecules (such as other polymerizable monomers that are the same or different) to form a polymer. [0044] Polymers can be hydrophilic, hydrophobic, or amphiphilic, as known in the art. Thus, “hydrophilic polymers” are substantially miscible with water and include, but are not limited to, polyethylene glycol and the like. “Hydrophobic polymers” are substantially immiscible with water and include, but are not limited to, polyethylene, polypropylene, polybutadiene, polystyrene, polymers disclosed herein, and the like. “Amphiphilic polymers” have both hydrophilic and hydrophobic properties and are typically copolymers having hydrophilic segment(s) and hydrophobic segment(s). Polymers include homopolymers, random copolymers, and block copolymers, as known in the art. The term “homopolymer” refers, in the usual and customary sense, to a polymer having a single monomeric unit. The term “copolymer” refers to a polymer derived from two or more monomeric species. The term “random copolymer” refers to a polymer derived from two or more monomeric species with no preferred ordering of the monomeric species. The term “block copolymer” refers to polymers having two or homopolymer subunits linked by covalent bond. Thus, the term “hydrophobic homopolymer” refers to a homopolymer which is hydrophobic. The term “hydrophobic block copolymer” refers to two or more homopolymer subunits linked by covalent bonds and which is hydrophobic. [0045] As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is substantially insoluble in water, but which is capable of absorbing and retaining large quantities of water to form a substantially stable, often soft and pliable, structure. In embodiments, water can penetrate in between polymer chains of a polymer network, subsequently causing swelling and the formation of a hydrogel. In embodiments, hydrogels are super-absorbent (e.g., containing more than about 90% water) and can be comprised of natural or synthetic polymers. Hydrogels can contain over 99% water and may include natural or synthetic polymers, or a combination thereof. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. A detailed description of suitable hydrogels may be found in published U.S. patent application 2010/0055733, herein incorporated by Attorney Docket No.: 051385-585001WO reference. By “hydrogel subunits” or “hydrogel precursors” is meant hydrophilic monomers, prepolymers, or polymers that can be crosslinked, or “polymerized”, to form a three-dimensional (3D) hydrogel network. In some embodiments, the alternating layers of polymeric gels described herein are hydrogels. Hydrogels may be prepared by cross-linking hydrophilic biopolymers or synthetic polymers. Thus, in some embodiments, the hydrogel may include a crosslinker. As used herein, the term “crosslinker” refers to a molecule that can form a three-dimensional network when reacted with the appropriate base monomers. Examples of the hydrogel polymers, which may include one or more crosslinkers, include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO—PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine), polyethylene glycol (PEG)-thiol, PEG- acrylate, acrylamide, N,N′-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. Thus, for example, a combination may include a polymer and a crosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO). In embodiments, the hydrogel includes chemical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a covalent bond) and may be referred to as a chemical hydrogel. In embodiments, the hydrogel includes physical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a non- covalent bond) and may be referred to as a physical hydrogel. In embodiments, the physical hydrogel include one or more crosslinks including hydrogen bonds, hydrophobic interactions, and/or polymer chain entanglements. Attorney Docket No.: 051385-585001WO [0046] Hydrogels may be prepared by cross-linking hydrophilic biopolymers or synthetic polymers. Thus, in some embodiments, the hydrogel may include a crosslinker. As used herein, the term “crosslinker” refers to a molecule that can form a three-dimensional network when reacted with the appropriate base monomers. Examples of the hydrogel polymers, which may include one or more crosslinkers, include but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO—PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO- PL(G)A copolymers, poly(ethylene imine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N′-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. Thus, for example, a combination may include a polymer and a crosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO). In embodiments, the hydrogel includes chemical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a covalent bond) and may be referred to as a chemical hydrogel. In embodiments, the hydrogel includes physical crosslinks (e.g., intermolecular or intramolecular joining of two or more molecules by a non- covalent bond) and may be referred to as a physical hydrogel. In embodiments, the physical hydrogel include one or more crosslinks including hydrogen bonds, hydrophobic interactions, and/or polymer chain entanglements. [0047] The term “array” as used herein, refers to a container (e.g., a microplate, tube, or flow cell) including a plurality of features (e.g., wells). For example, an array may include a container with a plurality of wells. In embodiments, the array is a microplate. In embodiments, the array is a flow cell. In embodiments, the array is a multiwell container. Attorney Docket No.: 051385-585001WO [0048] The term “microplate,” “microtiter plate,” “multiwell container,” or “multiwell plate” as used herein, refers to a substrate including a surface, the surface including a plurality of chambers or wells separated from each other by interstitial regions on the surface. In embodiments, the microplate has dimensions as provided and described by American National Standards Institute (ANSI) and Society for Laboratory Automation And Screening (SLAS); for example the tolerances and dimensions set forth in ANSI SLAS 1-2004 (R2012); ANSI SLAS 2- 2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSI SLAS 4-2004 (R2012); and ANSI SLAS 6- 2012, which are incorporated herein by reference. The dimensions of the microplate as described herein and the arrangement of the reaction chambers may be compatible with an established format for automated laboratory equipment. In embodiments, the device described herein provides methods for high-throughput screening. High-throughput screening (HTS) refers to a process that uses a combination of modern robotics, data processing and control software, liquid handling devices, and/or sensitive detectors, to efficiently process a large amount of (e.g., thousands, hundreds of thousands, or millions) samples in biochemical, genetic, or pharmacological experiments, either in parallel or in sequence, within a reasonably short period of time (e.g., days). Preferably, the process is amenable to automation, such as robotic simultaneous handling of 96 samples, 384 samples, 1536 samples or more. A typical HTS robot tests up to 100,000 to a few hundred thousand compounds per day. The samples are often in small volumes, such as no more than 1 mL, 500 μl, 200 μl, 100 μl, 50 μl or less. Through this process, one can rapidly identify active compounds, small molecules, antibodies, proteins, or polynucleotides in a cell. [0049] The reaction chambers may be provided as wells, for example an array or microplate may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells. In embodiments, the 96 and 384 wells are arranged in a 2:3 rectangular matrix. In embodiments, the 24 wells are arranged in a 3:8 rectangular matrix. In embodiments, the 48 wells are arranged in a 3:4 rectangular matrix. In embodiments, the reaction chamber is a microscope slide (e.g., a glass slide about 75 mm by about 25 mm). In embodiments the slide is a concavity slide (e.g., the slide includes a depression). In embodiments, the slide includes a coating for enhanced cell adhesion (e.g., poly- L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, or gold). In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 5 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a Attorney Docket No.: 051385-585001WO plurality of 6 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 7 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the microplate is 5 inches by 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the microplate is about 5 inches by about 3.33 inches, and includes a plurality of 8 mm diameter wells. In embodiments, the microplate is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples. [0050] The term “surface” is intended to mean an external part or external layer of a substrate. The surface can be in contact with another material such as a gas, liquid, gel, polymer, organic polymer, second surface of a similar or different material, metal, or coating. The surface, or regions thereof, can be substantially flat. The substrate and/or the surface can have surface features such as wells, pits, channels, ridges, raised regions, pegs, posts or the like. [0051] The term “well” refers to a discrete concave feature in a substrate having a surface opening that is completely surrounded by interstitial region(s) of the surface. Wells can have any of a variety of shapes at their opening in a surface including but not limited to round, elliptical, square, polygonal, or star shaped (i.e., star shaped with any number of vertices). The cross section of a well taken orthogonally with the surface may be curved, square, polygonal, hyperbolic, conical, or angular. The wells of a microplate are available in different shapes, for example F-Bottom: flat bottom; C-Bottom: bottom with minimal rounded edges; V-Bottom: V- shaped bottom; or U-Bottom: U-shaped bottom. In embodiments, the well is substantially square. In embodiments, the well is square. In embodiments, the well is F-bottom. In embodiments, the microplate includes 24 substantially round flat bottom wells. In embodiments, the microplate includes 48 substantially round flat bottom wells. In embodiments, the microplate includes 96 substantially round flat bottom wells. In embodiments, the microplate includes 384 substantially square flat bottom wells. The wells (alternatively referred to as reaction chambers) of a solid support and/or support insert may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 sample wells. In embodiments, the 96 and 384 wells are arranged in a 2:3 rectangular matrix. In embodiments, the 24 wells are arranged in a 3:8 rectangular matrix. In embodiments, the 48 wells are arranged in a 3:4 rectangular matrix. In embodiments, the solid support is a microscope slide (e.g., a glass slide Attorney Docket No.: 051385-585001WO about 75 mm by about 25 mm). In embodiments the slide is a concavity slide (e.g., the slide includes a depression). In embodiments, the slide includes a coating for enhanced cell adhesion (e.g., poly-L-lysine, silanes, carbon nanotubes, polymers, epoxy resins, or gold). In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 5 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 6 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 7 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the solid support is 5 inches by 3.33 inches, and includes a plurality of 7.5 mm diameter wells. In embodiments, the solid support is about 5 inches by about 3.33 inches, and includes a plurality of 8 mm diameter wells. In embodiments, the solid support insert is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples. In embodiments, the solid support is a flat glass or plastic tray in which an array of wells are formed, wherein each well can hold between from a few microliters to hundreds of microliters of fluid reagents and samples. In embodiments, the solid support has a rectangular shape that measures 127.7 mm±0.5 mm in length by 85.4 mm±0.5 mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each well has an average diameter of about 5-7 mm. In embodiments, the solid support has a rectangular shape that measures 127.7 mm±0.5 mm in length by 85.4 mm±0.5 mm in width, and includes 6, 12, 24, 48, or 96 wells, wherein each well has an average diameter of about 6 mm. In embodiments, the solid support includes an array of femtoliter wells, array of nanoliter wells, or array of microliter wells. In embodiments, the wells in an array may all have substantially the same volume. The array of wells may have a volume up to 100 e.g., about 0.1 femtoliter, 1 femtoliter, 10 femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50 microliter, or 100 microliter. [0052] The term “nanowell” refers to a discrete concave feature or depression in a substrate having a surface opening that is completely surrounded by interstitial region(s) of the surface, wherein the diameter of the feature is less than or equal to 1000 nanometers. Attorney Docket No.: 051385-585001WO [0053] The discrete regions (i.e., features, wells) of the microplate may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. In embodiments, the pattern of wells includes concentric circles of regions, spiral patterns, rectilinear patterns, hexagonal patterns, and the like. In embodiments, the pattern of wells is arranged in a rectilinear or hexagonal pattern A regular array of such regions is advantageous for detection and data analysis of signals collected from the arrays during an analysis. These discrete regions are separated by interstitial regions. As used herein, the term “interstitial region” refers to an area in a substrate or on a surface that separates other areas of the substrate or surface. For example, an interstitial region can separate one concave feature of an array from another concave feature of the array. The two regions that are separated from each other can be discrete, lacking contact with each other. In another example, an interstitial region can separate a first portion of a feature from a second portion of a feature. In embodiments the interstitial region is continuous whereas the features are discrete, for example, as is the case for an array of wells in an otherwise continuous surface. The separation provided by an interstitial region can be partial or full separation. In embodiments, interstitial regions have a surface material that differs from the surface material of the wells (e.g., the interstitial region contains a photoresist and the surface of the well is glass). In embodiments, interstitial regions have a surface material that is the same as the surface material of the wells (e.g., both the surface of the interstitial region and the surface of well contain a polymer or copolymer). [0054] As used herein, the terms “cluster” and “colony” are used interchangeably to refer to a discrete site on a solid support that includes a plurality of immobilized polynucleotides and a plurality of immobilized complementary polynucleotides. The term “clustered array” refers to an array formed from such clusters or colonies. In this context the term “array” is not to be understood as requiring an ordered arrangement of clusters. The term “array” is used in accordance with its ordinary meaning in the art, and refers to a population of different molecules that are attached to one or more solid-phase substrates such that the different molecules can be differentiated from each other according to their relative location. An array can include different molecules that are each located at different addressable features on a solid-phase substrate. The molecules of the array can be nucleic acid primers, nucleic acid probes, nucleic acid templates or nucleic acid enzymes such as polymerases or ligases. Arrays useful in the invention can have densities that ranges from about 2 different features to many millions, billions or higher. The Attorney Docket No.: 051385-585001WO density of an array can be from 2 to as many as a billion or more different features per square cm. For example an array can have at least about 100 features/cm2, at least about 1,000 features/cm2, at least about 10,000 features /cm2, at least about 100,000 features /cm2, at least about 10,000,000 features /cm2, at least about 100,000,000 features /cm2, at least about 1,000,000,000 features /cm2, at least about 2,000,000,000 features /cm2 or higher. In embodiments, the arrays have features at any of a variety of densities including, for example, at least about 10 features/cm2, 100 features/cm2, 500 features/cm2, 1,000 features/cm2, 5,000 features/cm2, 10,000 features/cm2, 50,000 features/cm2, 100,000 features/cm2, 1,000,000 features/cm2, 5,000,000 features/cm2, or higher. [0055] Nucleic acids, including e.g., nucleic acids with a phosphorothioate backbone, can include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, e.g., a nucleic acid or polypeptide through covalent, non-covalent or other interactions. By way of example, the nucleic acid can include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide through a covalent, non-covalent or other interaction. [0056] As used herein, the term “template polynucleotide” or “template nucleic acid” refers to any polynucleotide molecule that may be bound by a polymerase and utilized as a template for nucleic acid synthesis. A template polynucleotide may be a target polynucleotide. In general, the term “target polynucleotide” refers to a nucleic acid molecule or polynucleotide in a starting population of nucleic acid molecules having a target sequence whose presence, amount, and/or nucleotide sequence, or changes in one or more of these, are desired to be determined. In general, the term “target sequence” refers to a nucleic acid sequence on a single strand of nucleic acid. The terms “single strand” and “ssDNA” are used in accordance with its plain and ordinary meaning and refer to a single-stranded polynucleotide. The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others. The target sequence may be a target sequence from a sample or a secondary target such as a product of an amplification reaction. A target polynucleotide is not necessarily any single molecule or sequence. For example, a target polynucleotide may be any one of a plurality of target polynucleotides in a reaction, or all polynucleotides in a given reaction, depending on the reaction conditions. For example, in a nucleic acid amplification reaction with random primers, Attorney Docket No.: 051385-585001WO all polynucleotides in a reaction may be amplified. As a further example, a collection of targets may be simultaneously assayed using polynucleotide primers directed to a plurality of targets in a single reaction. As yet another example, all or a subset of polynucleotides in a sample may be modified by the addition of a primer-binding sequence (such as by the ligation of adapters containing the primer binding sequence), rendering each modified polynucleotide a target polynucleotide in a reaction with the corresponding primer polynucleotide(s). In the context of selective sequencing, “target polynucleotide(s)” refers to the subset of polynucleotide(s) to be sequenced from within a starting population of polynucleotides. [0057] In embodiments, a target polynucleotide is a cell-free polynucleotide. In general, the terms “cell-free,” “circulating,” and “extracellular” as applied to polynucleotides (e.g. “cell-free DNA” (cfDNA) and “cell-free RNA” (cfRNA)) are used interchangeably to refer to polynucleotides present in a sample from a subject or portion thereof that can be isolated or otherwise manipulated without applying a lysis step to the sample as originally collected (e.g., as in extraction from cells or viruses). Cell-free polynucleotides are thus unencapsulated or “free” from the cells or viruses from which they originate, even before a sample of the subject is collected. Cell-free polynucleotides may be produced as a byproduct of cell death (e.g. apoptosis or necrosis) or cell shedding, releasing polynucleotides into surrounding body fluids or into circulation. Accordingly, cell-free polynucleotides may be isolated from a non-cellular fraction of blood (e.g. serum or plasma), from other bodily fluids (e.g. urine), or from non-cellular fractions of other types of samples. [0058] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. [0059] As used herein, the terms “analogue” and “analog”, in reference to a chemical compound, refers to compound having a structure similar to that of another one, but differing Attorney Docket No.: 051385-585001WO from it in respect of one or more different atoms, functional groups, or substructures that are replaced with one or more other atoms, functional groups, or substructures. In the context of a nucleotide, a nucleotide analog refers to a compound that, like the nucleotide of which it is an analog, can be incorporated into a nucleic acid molecule (e.g., an extension product) by a suitable polymerase, for example, a DNA polymerase in the context of a nucleotide analogue. The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphorothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see, e.g., see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA)), including those described in U.S. Patent Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, the internucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both. [0060] Other analog nucleic acids include bis-locked nucleic acids (bisLNAs; e.g., including those described in Moreno PMD et al. Nucleic Acids Res.2013; 41(5):3257-73), twisted Attorney Docket No.: 051385-585001WO intercalating nucleic acids (TINAs; e.g., including those described in Doluca O et al. Chembiochem.2011; 12(15):2365-74), bridged nucleic acids (BNAs; e.g., including those described in Soler-Bistue A et al. Molecules.2019; 24(12): 2297), 2’-O-methyl RNA:DNA chimeric nucleic acids (e.g., including those described in Wang S and Kool ET. Nucleic Acids Res.1995; 23(7):1157-1164), minor groove binder (MGB) nucleic acids (e.g., including those described in Kutyavin IV et al. Nucleic Acids Res.2000; 28(2):655-61), morpholino nucleic acids (e.g., including those described in Summerton J and Weller D. Antisense Nucleic Acid Drug Dev.1997; 7(3):187-95), C5-modified pyrimidine nucleic acids (e.g., including those described in Kumar P et al. J. Org. Chem.2014; 79(11): 5047-5061), peptide nucleic acids (PNAs; e.g., including those described in Gupta A et al. J. Biotechnol.2017; 259: 148-59), and/or phosphorothioate nucleotides (e.g., including those described in Eckstein F. Nucleic Acid Ther.2014; 24(6):374-87). [0061] As used herein, a "native" nucleotide is used in accordance with its plain and ordinary meaning and refers to a naturally occurring nucleotide that does not include an exogenous label (e.g., a fluorescent dye, or other label) or chemical modification such as may characterize a nucleotide analog (e.g., a reversible terminating moiety). Examples of native nucleotides useful for carrying out procedures described herein include: dATP (2'-deoxyadenosine-5'-triphosphate); dGTP (2'-deoxyguanosine-5'-triphosphate); dCTP (2'-deoxycytidine-5'-triphosphate); dTTP (2'- deoxythymidine-5'-triphosphate); and dUTP (2'-deoxyuridine-5'-triphosphate). A “canonical” nucleotide is an unmodified nucleotide. [0062] As used herein, the term “modified nucleotide” refers to nucleotide modified in some manner. Typically, a nucleotide contains a single 5-carbon sugar moiety, a single nitrogenous base moiety and 1 to three phosphate moieties. In embodiments, a nucleotide can include a blocking moiety (alternatively referred to herein as a reversible terminator moiety) and/or a label moiety. A blocking moiety on a nucleotide prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide. A blocking moiety on a nucleotide can be reversible, whereby the blocking moiety can be removed or modified to allow the 3' hydroxyl to form a covalent bond with the 5' phosphate of another nucleotide. A blocking moiety can be effectively irreversible under particular conditions used in a method set forth herein. In embodiments, the blocking moiety is attached to the 3’ oxygen of the nucleotide Attorney Docket No.: 051385-585001WO and is independently –NH2, -CN, -CH3, C2-C6 allyl (e.g., -CH2-CH=CH2), methoxyalkyl (e.g., - CH2-O-CH3), or –CH2N3. In embodiments, the blocking moiety is attached to the 3’ oxygen of ,
Figure imgf000036_0001
A label moiety of a nucleotide can be using a spectroscopic method.
Figure imgf000036_0002
Exemplary label moieties are fluorescent labels, mass labels, chemiluminescent labels, electrochemical labels, detectable labels and the like. One or more of the above moieties can be absent from a nucleotide used in the methods and compositions set forth herein. For example, a nucleotide can lack a label moiety or a blocking moiety or both. Examples of nucleotide analogues include, without limitation, 7-deaza-adenine, 7-deaza-guanine, the analogues of deoxynucleotides shown herein, analogues in which a label is attached through a cleavable linker to the 5-position of cytosine or thymine or to the 7-position of deaza-adenine or deaza-guanine, and analogues in which a small chemical moiety is used to cap the OH group at the 3'-position of deoxyribose. Nucleotide analogues and DNA polymerase-based DNA sequencing are also described in U.S. Patent No.6,664,079, which is incorporated herein by reference in its entirety for all purposes. [0063] As used herein, the term “cleavable complement” refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides, wherein the complementary nucleotide or sequence of nucleotides includes a cleavable site, and the cleavable complement also includes a complement to the cleavable site. In embodiments, the cleavable complement of the cleavable site and the cleavable site are cleaved by the same mechanism (e.g., restriction enzyme digestion of the duplexed cleavable site and cleavable complement of the cleavable site). Attorney Docket No.: 051385-585001WO [0064] The term “cleavable linker” or “cleavable moiety” as used herein refers to a divalent or monovalent, respectively, moiety which is capable of being separated (e.g., detached, split, disconnected, hydrolyzed, a stable bond within the moiety is broken) into distinct entities. A cleavable linker is cleavable (e.g., specifically cleavable) in response to external stimuli (e.g., enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents). A chemically cleavable linker refers to a linker which is capable of being split in response to the presence of a chemical (e.g., acid, base, oxidizing agent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite (Na2S2O4), or hydrazine (N2H4)). A chemically cleavable linker is non-enzymatically cleavable. In embodiments, the cleavable linker is cleaved by contacting the cleavable linker with a cleaving agent. In embodiments, the cleaving agent is a phosphine containing reagent (e.g., TCEP or THPP), sodium dithionite (Na2S2O4), weak acid, hydrazine (N2H4), Pd(0), or light-irradiation (e.g., ultraviolet radiation). In embodiments, cleaving includes removing. For clarity, the terms “cleavable linker” and “cleavable site” are different terms with different meanings as used herein. For example, a cleavable linker may include a covalent linker that includes one or more cleavable sites. [0065] A “cleavable site” or “scissile linkage” in the context of a polynucleotide is a site which allows controlled cleavage of the polynucleotide strand (e.g., the linker, the primer, or the polynucleotide) by chemical, enzymatic, or photochemical means known in the art and described herein. A scissile site may refer to the linkage of a nucleotide between two other nucleotides in a nucleotide strand (i.e., an internucleosidic linkage). In embodiments, the scissile linkage can be located at any position within the one or more nucleic acid molecules, including at or near a terminal end (e.g., the 3′ end of an oligonucleotide) or in an interior portion of the one or more nucleic acid molecules. In embodiments, conditions suitable for separating a scissile linkage include a modulating the pH and/or the temperature. In embodiments, a scissile site can include at least one acid-labile linkage. For example, an acid-labile linkage may include a phosphoramidate linkage. In embodiments, a phosphoramidate linkage can be hydrolysable under acidic conditions, including mild acidic conditions such as trifluoroacetic acid and a suitable temperature (e.g., 30°C), or other conditions known in the art, for example Matthias Mag, et al Tetrahedron Letters, Volume 33, Issue 48, 1992, 7319-7322. In embodiments, the Attorney Docket No.: 051385-585001WO scissile site can include at least one photolabile internucleosidic linkage (e.g., o-nitrobenzyl linkages, as described in Walker et al, J. Am. Chem. Soc.1988, 110, 21, 7170–7177), such as o- nitrobenzyloxymethyl or p-nitrobenzyloxymethyl group(s). In embodiments, the scissile site includes at least one uracil nucleobase. In embodiments, a uracil nucleobase can be cleaved with a uracil DNA glycosylase (UDG) or Formamidopyrimidine DNA Glycosylase Fpg. In embodiments, the scissile linkage site includes a sequence-specific nicking site having a nucleotide sequence that is recognized and nicked by a nicking endonuclease enzyme or a uracil DNA glycosylase. [0066] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site blast.ncbi.nlm.nih.gov/Blast.cgi or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the complement of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. [0067] As used herein the term “determine” can be used to refer to the act of ascertaining, establishing or estimating. A determination can be probabilistic. For example, a determination can have an apparent likelihood of at least 50%, 75%, 90%, 95%, 98%, 99%, 99.9% or higher. In some cases, a determination can have an apparent likelihood of 100%. An exemplary determination is a maximum likelihood analysis or report. As used herein, the term “identify,” when used in reference to a thing, can be used to refer to recognition of the thing, distinction of the thing from at least one other thing or categorization of the thing with at least one other thing. Attorney Docket No.: 051385-585001WO The recognition, distinction or categorization can be probabilistic. For example, a thing can be identified with an apparent likelihood of at least 50%, 75%, 90%, 95%, 98%, 99%, 99.9% or higher. A thing can be identified based on a result of a maximum likelihood analysis. In some cases, a thing can be identified with an apparent likelihood of 100%. [0068] As used herein, the term “removable” group, e.g., a label or a blocking group or protecting group, is used in accordance with its plain and ordinary meaning and refers to a chemical group that can be removed from a nucleotide analogue such that a DNA polymerase can extend the nucleic acid (e.g., a primer or extension product) by the incorporation of at least one additional nucleotide. Removal may be by any suitable method, including enzymatic, chemical, or photolytic cleavage. Removal of a removable group, e.g., a blocking group, does not require that the entire removable group be removed, only that a sufficient portion of it be removed such that a DNA polymerase can extend a nucleic acid by incorporation of at least one additional nucleotide using a nucleotide or nucleotide analogue. In general, the conditions under which a removable group is removed are compatible with a process employing the removable group (e.g., an amplification process or sequencing process). [0069] As used herein, the terms “reversible blocking groups” and “reversible terminators” are used in accordance with their plain and ordinary meanings and refer to a blocking moiety located, for example, at the 3' position of the nucleotide and may be a chemically cleavable moiety such as an allyl group, an azidomethyl group or a methoxymethyl group, or may be an enzymatically cleavable group such as a phosphate ester. Non-limiting examples of nucleotide blocking moieties are described in applications WO 2004/018497, U.S. Pat. Nos.7,057,026, 7,541,444, WO 96/07669, U.S. Pat. Nos.5,763,594, 5,808,045, 5,872,244 and 6,232,465 the contents of which are incorporated herein by reference in their entirety. The nucleotides may be labelled or unlabeled. They may be modified with reversible terminators useful in methods provided herein and may be 3'-O-blocked reversible or 3'-unblocked reversible terminators. In nucleotides with 3'-O-blocked reversible terminators, the blocking group –OR [reversible terminating (capping) group] is linked to the oxygen atom of the 3'-OH of the pentose, while the label is linked to the base, which acts as a reporter and can be cleaved. The 3'-O-blocked reversible terminators are known in the art, and may be, for instance, a 3'-ONH2 reversible terminator, a 3'-O-allyl reversible terminator, or a 3'-O-azidomethyl reversible terminator. In Attorney Docket No.: 051385-585001WO embodiments, the reversible terminator moiety ,
Figure imgf000040_0001
,
Figure imgf000040_0002
. The term “allyl” as described herein group (i.e., -CH=CH2), having the
Figure imgf000040_0003
formula . In embodiments, the reversible terminator moiety as described in U.S. 10,738,072, which is incorporated herein by reference
Figure imgf000040_0004
For example, a nucleotide including a reversible terminator moiety may be represented by the formula: , where the nucleobase is adenine or adenine analogue, thymine or thymine analogue, guanine or guanine analogue, or cytosine or cytosine analogue. [0070] In some embodiments, a nucleic acid comprises a molecular identifier or a molecular barcode. As used herein, the term "molecular barcode" (which may be referred to as a "tag", a "barcode", a "molecular identifier", an "identifier sequence" or a “unique molecular identifier” (UMI)) refers to any material (e.g., a nucleotide sequence, a nucleic acid molecule feature) that is capable of distinguishing an individual molecule in a large heterogeneous population of molecules. In embodiments, a barcode is unique in a pool of barcodes that differ from one another in sequence, or is uniquely associated with a particular sample polynucleotide in a pool of sample polynucleotides. In embodiments, every barcode in a pool of adapters is unique, such that sequencing reads comprising the barcode can be identified as originating from a single sample polynucleotide molecule on the basis of the barcode alone. In other embodiments, individual barcode sequences may be used more than once, but adapters comprising the duplicate Attorney Docket No.: 051385-585001WO barcodes are associated with different sequences and/or in different combinations of barcoded adapters, such that sequence reads may still be uniquely distinguished as originating from a single sample polynucleotide molecule on the basis of a barcode and adjacent sequence information (e.g., sample polynucleotide sequence, and/or one or more adjacent barcodes). In embodiments, barcodes are about or at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75 or more nucleotides in length. In embodiments, barcodes are shorter than 20, 15, 10, 9, 8, 7, 6, or 5 nucleotides in length. In embodiments, barcodes are about 10 to about 50 nucleotides in length, such as about 15 to about 40 or about 20 to about 30 nucleotides in length. In a pool of different barcodes, barcodes may have the same or different lengths. In general, barcodes are of sufficient length and include sequences that are sufficiently different to allow the identification of sequencing reads that originate from the same sample polynucleotide molecule. In embodiments, each barcode in a plurality of barcodes differs from every other barcode in the plurality by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions. In some embodiments, substantially degenerate barcodes may be known as random. In some embodiments, a barcode may include a nucleic acid sequence from within a pool of known sequences. In some embodiments, the barcodes may be pre-defined. [0071] In embodiments, a nucleic acid (e.g., an adapter or primer) includes a sample barcode. In general, a “sample barcode” is a nucleotide sequence that is sufficiently different from other sample barcode to allow the identification of the sample source based on sample barcode sequence(s) with which they are associated. In embodiments, a plurality of nucleotides (e.g., all nucleotides from a particular sample source, or sub-sample thereof) are joined to a first sample barcode, while a different plurality of nucleotides (e.g., all nucleotides from a different sample source, or different subsample) are joined to a second sample barcode, thereby associating each plurality of polynucleotides with a different sample barcode indicative of sample source. In embodiments, each sample barcode in a plurality of sample barcodes differs from every other sample barcode in the plurality by at least three nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotide positions. In some embodiments, substantially degenerate sample barcodes may be known as random. In some embodiments, a sample barcode may include a nucleic acid sequence from within a pool of known sequences. In some embodiments, the sample barcodes may be pre-defined. In embodiments, the sample barcode includes about 1 to about 10 nucleotides. In embodiments, the sample barcode includes about 3, 4, 5, 6, 7, 8, 9, or about 10 Attorney Docket No.: 051385-585001WO nucleotides. In embodiments, the sample barcode includes about 3 nucleotides. In embodiments, the sample barcode includes about 5 nucleotides. In embodiments, the sample barcode includes about 7 nucleotides. In embodiments, the sample barcode includes about 10 nucleotides. In embodiments, the sample barcode includes about 6 to about 10 nucleotides. [0072] In some embodiments, the reaction conditions for a plurality of invasion-primer extension cycles includes incubation in a denaturant. As used herein, the terms “denaturant” or plural “denaturants” are used in accordance with their plain and ordinary meanings and refer to an additive or condition that disrupts the base pairing between nucleotides within opposing strands of a double-stranded polynucleotide molecule. The term “denature” and its variants, when used in reference to any double-stranded polynucleotide molecule, or double-stranded polynucleotide sequence, includes any process whereby the base pairing between nucleotides within opposing strands of the double-stranded molecule, or double-stranded sequence, is disrupted. Typically, denaturation includes rendering at least some portion or region of two strands of the double-stranded polynucleotide molecule or sequence single-stranded or partially single-stranded. In some embodiments, denaturation includes separation of at least some portion or region of two strands of the double-stranded polynucleotide molecule or sequence from each other. Typically, the denatured region or portion is then capable of hybridizing to another polynucleotide molecule or sequence. Optionally, there can be “complete” or “total” denaturation of a double-stranded polynucleotide molecule or sequence. Complete denaturation conditions are, for example, conditions that would result in complete separation of a significant fraction (e.g., more than 10%, 20%, 30%, 40% or 50%) of a large plurality of strands from their extended and/or full-length complements. Typically, complete or total denaturation disrupts all of the base pairing between the nucleotides of the two strands with each other. Similarly, a nucleic acid sample is optionally considered fully denatured when more than 80% or 90% of individual molecules of the sample lack any double-strandedness (or lack any hybridization to a complementary strand). [0073] Alternatively, the double-stranded polynucleotide molecule or sequence can be partially or incompletely denatured. A given nucleic acid molecule can be considered partially denatured when a portion of at least one strand of the nucleic acid remains hybridized to a complementary strand, while another portion is in an unhybridized state (even if it is in the presence of a Attorney Docket No.: 051385-585001WO complementary sequence). The unhybridized portion is optionally at least 5, 10, 15, 20, 50, or more nucleotides in length. The hybridized portion is optionally at least 5, 10, 15, 20, 50, or more nucleotides in length. Partial denaturation includes situations where some, but not all, of the nucleotides of one strand or sequence, are based paired with some nucleotides of the other strand or sequence within a double-stranded polynucleotide. In some embodiments, at least 20% but less than 100% of the nucleotide residues of one strand of the partially denatured polynucleotide (or sequence) are not base paired to nucleotide residues within the opposing strand. In embodiments, at least 50% of nucleotide residues within the double-stranded polynucleotide molecule (or double-stranded polynucleotide sequence) are in single-stranded (or unhybridized) from, but less than 20% or 10% of the residues are double-stranded. [0074] Optionally, a nucleic acid sample can be considered to be partially denatured when a substantial fraction of individual nucleic acid molecules of the sample (e.g., above 20%, 30%, 50%, or 70%) are in a partially denatured state. Optionally less than a substantial amount of individual nucleic acid molecules in the sample are fully denatured, e.g., not more than 5%, 10%, 20%, 30% or 50% of the nucleic acid molecules in the sample. Under exemplary conditions at least 50% of the nucleic acid molecules of the sample are partly denatured, but less than 20% or 10% are fully denatured. In other situations, at least 30% of the nucleic acid molecules of the sample are partly denatured, but less than 10% or 5% are fully denatured. Similarly, a nucleic acid sample can be non-denatured when a minority of individual nucleic acid molecules in the sample are partially or completely denatured. [0075] In an embodiment, partially denaturing conditions are achieved by maintaining the duplexes as a suitable temperature range. For example, the nucleic acid is maintained at temperature sufficiently elevated to achieve some heat-denaturation (e.g., above 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C) but not high enough to achieve complete heat-denaturation (e.g., below 95°C or 90°C or 85°C or 80°C or 75°C). In an embodiment the nucleic acid is partially denatured using substantially isothermal conditions. Alternatively, chemical denaturation can be accomplished by contacting the double-stranded polynucleotide to be denatured with appropriate chemical denaturants, such as strong alkalis, strong acids, chaotropic agents, and the like and can include, for example, NaOH, urea, or guanidine-containing compounds. In some embodiments, partial or complete denaturation is achieved by exposure to chemical denaturants such as urea or Attorney Docket No.: 051385-585001WO formamide, with concentrations suitably adjusted, or using high or low pH (e.g., pH between 4-6 or 8-9). In embodiments, the denaturant is a buffered solution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4- methylmorpholine 4-oxide (NMO), or a mixture thereof. In embodiments, the first denaturant is a buffered solution including about 0% to about 50% dimethyl sulfoxide (DMSO); about 0% to about 50% ethylene glycol; about 0% to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof. In an embodiment herein, partial denaturation and/or amplification, including any one or more steps or methods described herein, can be achieved using a recombinase and/or single-stranded binding protein. [0076] In some embodiments, complete or partial denaturation of a double-stranded polynucleotide sequence is accomplished by contacting the double-stranded polynucleotide sequence using appropriate denaturing agents. For example, the double-stranded polynucleotide can be subjected to heat-denaturation (also referred to interchangeably as thermal denaturation) by raising the temperature to a point where the desired level of denaturation is accomplished. In some embodiments, thermal denaturation of a double-stranded polynucleotide, includes adjusting the temperature to achieve complete separation of the two strands of the polynucleotide, such that 90% or greater of the strands are in single-stranded form across their entire length. A completely denatured double-stranded polynucleotide results in a separated first strand and a second strand, each of which is a single-stranded polynucleotide. In some embodiments, complete thermal denaturation of a polynucleotide molecule (or polynucleotide sequence) is accomplished by exposing the polynucleotide molecule (or sequence) to a temperature that is at least 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 50°C, or 100°C, above the calculated or predict melting temperature (Tm) of the polynucleotide molecule or sequence. [0077] In some embodiments, complete or partial denaturation is accomplished by treating the double-stranded polynucleotide sequence to be denatured using a denaturant mixture including an SSB protein (e.g., T4 gp32 protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNA Binding Protein (ET- SSB)), a strand-displacing polymerase (e.g., Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst 2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 Attorney Docket No.: 051385-585001WO polymerase, or a mutant thereof), and one or more crowding agents (poly(ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), dextran, Ficoll (e.g., Ficoll 70 or Ficoll 400), glycerol, or a combination thereof). In embodiments, the crowding agent is poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000), dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, β-lactoglobulin, hemoglobin, bovine serum albumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS). In embodiments, the denaturant mixture including an SSB, a strand-displacing polymerase, and one or more crowding agents does not include a chemical denaturant (e.g., betaine, DMSO, ethylene glycol, formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof). [0078] In some embodiments, a nucleic acid comprises a label. As used herein, the term “label” or “labels” are used in accordance with their plain and ordinary meanings and refer to molecules that can directly or indirectly produce or result in a detectable signal either by themselves or upon interaction with another molecule. Non-limiting examples of detectable labels include fluorescent dyes, biotin, digoxin, haptens, and epitopes. In general, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric, luminescent, bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal. In embodiments, the label is a dye. In embodiments, the dye is a fluorescent dye. Non- limiting examples of dyes, some of which are commercially available, include CF dyes (Biotium, Inc.), Alexa Fluor dyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). In embodiments, a particular nucleotide type is associated with a particular label, such that identifying the label identifies the nucleotide with which it is associated. In embodiments, the label is luciferin that reacts with luciferase to produce a detectable signal in response to one or more bases being incorporated into an elongated complementary strand, such as in pyrosequencing. In embodiment, a nucleotide comprises a label (such as a dye). In embodiments, the label is not associated with any particular nucleotide, but detection of the label identifies whether one or more nucleotides having a known identity were added during an extension step (such as in the case of pyrosequencing). Attorney Docket No.: 051385-585001WO [0079] In embodiments, the detectable label is a fluorescent dye. In embodiments, the detectable label is a fluorescent dye capable of exchanging energy with another fluorescent dye (e.g., fluorescence resonance energy transfer (FRET) chromophores). Examples of detectable agents include imaging agents, including fluorescent and luminescent substances, including, but not limited to, a variety of organic or inorganic small molecules commonly referred to as “dyes,” “labels,” or “indicators.” Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments, the detectable moiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). In embodiments, the detectable moiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). In embodiments, the detectable moiety is a moiety of a derivative of one of the detectable moieties described immediately above, wherein the derivative differs from one of the detectable moieties immediately above by a modification resulting from the conjugation of the detectable moiety to a compound described herein. [0080] The term “cyanine” or “cyanine moiety” as described herein refers to a detectable moiety containing two nitrogen groups separated by a polymethine chain. In embodiments, the cyanine moiety has 3 methine structures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moiety has 5 methine structures (i.e., cyanine 5 or Cy5). In embodiments, the cyanine moiety has 7 methine structures (i.e., cyanine 7 or Cy7). [0081] As used herein, the term “DNA polymerase” and “nucleic acid polymerase” are used in accordance with their plain ordinary meanings and refer to enzymes capable of synthesizing nucleic acid molecules from nucleotides (e.g., deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides to the 3’- end of a DNA strand, one nucleotide at a time. In embodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNA polymerase, Pol µ DNA polymerase, Pol λ DNA polymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g. Therminator γ, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In Attorney Docket No.: 051385-585001WO embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044). In embodiments, the polymerase is an enzyme described in US 2021/0139884. For example, a polymerase catalyzes the addition of a next correct nucleotide to the 3′-OH group of the primer via a phosphodiester bond, thereby chemically incorporating the nucleotide into the primer. Optionally, the polymerase used in the provided methods is a processive polymerase. Optionally, the polymerase used in the provided methods is a distributive polymerase. [0082] As used herein, the term “thermophilic nucleic acid polymerase” refers to a family of DNA polymerases (e.g.,9°NTM) and mutants thereof derived from the DNA polymerase originally isolated from the hyperthermophilic archaea, Thermococcus sp.9 degrees N-7, found in hydrothermal vents at that latitude (East Pacific Rise) (Southworth MW, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member of the family B DNA polymerases. Site-directed mutagenesis of the 3’-5’ exo motif I (Asp-Ile-Glu or DIE) to AIA, AIE, EIE, EID or DIA yielded polymerase with no detectable 3’ exonuclease activity. Mutation to Asp-Ile-Asp (DID) resulted in reduction of 3’-5’ exonuclease specific activity to <1% of wild type, while maintaining other properties of the polymerase including its high strand displacement activity. The sequence AIA (D141A, E143A) was chosen for reducing exonuclease. Subsequent mutagenesis of key amino acids results in an increased ability of the enzyme to incorporate dideoxynucleotides, ribonucleotides and acyclonucleotides (e.g., Therminator II enzyme from New England Biolabs with D141A / E143A / Y409V / A485L mutations); 3’-amino-dNTPs, 3’-azido-dNTPs and other 3’-modified nucleotides (e.g., NEB Therminator III DNA Polymerase with D141A / E143A / L408S / Y409A / P410V mutations, NEB Therminator IX DNA polymerase), or γ-phosphate labeled nucleotides (e.g., Therminator γ: D141A / E143A / W355A / L408W / R460A / Q461S / K464E / D480V / R484W / A485L). Typically, these enzymes do not have 5’-3’ exonuclease activity. Additional information about thermophilic nucleic acid polymerases may be found in (Southworth MW, et al. PNAS. 1996;93(11):5281-5285; Bergen K, et al. ChemBioChem.2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012;2:684; Fuller CW, et al.2016;113(19):5233-5238; Guo J, et al. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(27):9145-9150), which are incorporated herein in their entirety for all purposes. Attorney Docket No.: 051385-585001WO [0083] As used herein, the term “exonuclease activity” is used in accordance with its ordinary meaning in the art, and refers to the removal of a nucleotide from a nucleic acid by an enzyme (e.g. DNA polymerase, a lambda exonuclease, Exo I, Exo III, T5, Exo V, Exo VII or the like). For example, during polymerization, nucleotides are added to the 3’ end of the primer strand. Occasionally a DNA polymerase incorporates an incorrect nucleotide to the 3′-OH terminus of the primer strand, wherein the incorrect nucleotide cannot form a hydrogen bond to the corresponding base in the template strand. Such a nucleotide, added in error, is removed from the primer as a result of the 3′ to 5′ exonuclease activity of the DNA polymerase. In embodiments, exonuclease activity may be referred to as “proofreading.” When referring to 3’-5’ exonuclease activity, it is understood that the DNA polymerase facilitates a hydrolyzing reaction that breaks phosphodiester bonds at the 3’ end of a polynucleotide chain to excise the nucleotide. In embodiments, 3’-5’ exonuclease activity refers to the successive removal of nucleotides in single-stranded DNA in a 3’ → 5’ direction, releasing deoxyribonucleoside 5’-monophosphates one after another. Methods for quantifying exonuclease activity are known in the art, see for example Southworth et al, PNAS Vol 93, 8281-8285 (1996). In embodiments, 5’-3’ exonuclease activity refers to the successive removal of nucleotides in double-stranded DNA in a 5’ → 3’ direction. In embodiments, the 5’-3’ exonuclease is lambda exonuclease. For example, lambda exonuclease catalyzes the removal of 5’ mononucleotides from duplex DNA, with a preference for 5’ phosphorylated double-stranded DNA. In other embodiments, the 5’-3’ exonuclease is E. coli DNA Polymerase I. [0084] As used herein, the term “incorporating” or “chemically incorporating,” when used in reference to a primer and a nucleotide, refers to the process of joining the nucleotide to the primer or extension product thereof by formation of a phosphodiester bond. [0085] As used herein, the term “selective” or “selectivity” or the like of a compound refers to the compound’s ability to discriminate between molecular targets. When used in the context of sequencing, such as in “selectively sequencing,” this term refers to sequencing one or more target polynucleotides from an original starting population of polynucleotides, and not sequencing non-target polynucleotides from the starting population. Typically, selectively sequencing one or more target polynucleotides involves differentially manipulating the target polynucleotides based on known sequence. For example, target polynucleotides may be Attorney Docket No.: 051385-585001WO hybridized to a probe oligonucleotide that may be labeled (such as with a member of a binding pair) or bound to a surface. In embodiments, hybridizing a target polynucleotide to a probe oligonucleotide includes the step of displacing one strand of a double-stranded nucleic acid. Probe-hybridized target polynucleotides may then be separated from non-hybridized polynucleotides, such as by removing probe-bound polynucleotides from the starting population or by washing away polynucleotides that are not bound to a probe. The result is a selected subset of the starting population of polynucleotides, which is then subjected to sequencing, thereby selectively sequencing the one or more target polynucleotides. [0086] As used herein, the terms “specific”, “specifically”, “specificity”, or the like of a compound refers to the agent’s ability to cause a particular action, such as binding, to a particular molecular target with minimal or no action to other proteins in the cell. [0087] As used herein, the terms “bind” and “bound” are used in accordance with their plain and ordinary meanings and refer to an association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be directly bound to one another, e.g., by a covalent bond or non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole- induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). As a further example, two molecules may be bound indirectly to one another by way of direct binding to one or more intermediate molecules, thereby forming a complex. [0088] “Specific binding” is where the binding is selective between two molecules. A particular example of specific binding is that which occurs between an antibody and an antigen. Typically, specific binding can be distinguished from non-specific when the dissociation constant (KD) is less than about 1×10−5 M or less than about 1×10−6 M or 1×10−7 M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical crosslinking, two-hybrid assays and the like. In embodiments, the KD (equilibrium dissociation constant) between two specific binding molecules is less than 10−6 M, less than 10−7 M, less than 10−8 M, less than 10−9 M, less than 10−9 M, less than 10−11 M, or less than about 10−12 M or less.
Figure imgf000049_0001
[0089] As used herein, the term “rolling circle amplification (RCA)” refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., single-stranded DNA Attorney Docket No.: 051385-585001WO circles) via a rolling circle mechanism. Rolling circle amplification reaction is initiated by the hybridization of a primer to a circular, often single-stranded, nucleic acid template. The nucleic acid polymerase then extends the primer that is hybridized to the circular nucleic acid template by continuously progressing around the circular nucleic acid template to replicate the sequence of the nucleic acid template over and over again (rolling circle mechanism). The rolling circle amplification typically produces concatemers comprising tandem repeat units of the circular nucleic acid template sequence. The rolling circle amplification may be a linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCA using a single specific primer), or may be an exponential RCA (ERCA) exhibiting exponential amplification kinetics. Rolling circle amplification may also be performed using multiple primers (multiply primed rolling circle amplification or MPRCA) leading to hyper-branched concatemers. For example, in a double- primed RCA, one primer may be complementary, as in the linear RCA, to the circular nucleic acid template, whereas the other may be complementary to the tandem repeat unit nucleic acid sequences of the RCA product. Consequently, the double-primed RCA may proceed as a chain reaction with exponential (geometric) amplification kinetics featuring a ramifying cascade of multiple-hybridization, primer-extension, and strand-displacement events involving both the primers. This often generates a discrete set of concatemeric, double-stranded nucleic acid amplification products. The rolling circle amplification may be performed in-vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase. RCA may be performed by using any of the DNA polymerases that are known in the art (e.g., a Phi29 DNA polymerase, a Bst DNA polymerase, or SD polymerase). [0090] As used herein, the terms “sequencing”, “sequence determination”, “determining a nucleotide sequence”, and the like include determination of a partial or complete sequence information, including the identification, ordering, or locations of the nucleotides that comprise the polynucleotide being sequenced, and inclusive of the physical processes for generating such sequence information. That is, the term includes sequence comparisons, consensus sequence determination, contig assembly, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of nucleotides in a target polynucleotide. The term also includes the determination of the identification, ordering, and locations of one, two, or three of the four types of nucleotides within a target polynucleotide. In some embodiments, a sequencing process described herein comprises contacting a template and Attorney Docket No.: 051385-585001WO an annealed primer with a suitable polymerase under conditions suitable for polymerase extension and/or sequencing. The sequencing methods are preferably carried out with the target polynucleotide arrayed on a solid substrate. Multiple target polynucleotides can be immobilized on the solid support through linker molecules, or can be attached to particles, e.g., microspheres, which can also be attached to a solid substrate. In embodiments, the solid substrate is in the form of a chip, a bead, a well, a capillary tube, a slide, a wafer, a filter, a fiber, a porous media, or a column. In embodiments, the solid substrate is gold, quartz, silica, plastic, glass, diamond, silver, metal, or polypropylene. In embodiments, the solid substrate is porous. [0091] As used herein, the term “sequencing cycle” is used in accordance with its plain and ordinary meaning and refers to incorporating one or more nucleotides (e.g., nucleotide analogues) to the 3’ end of a polynucleotide with a polymerase, and detecting the one or more nucleotides incorporated. In embodiments, one nucleotide (e.g., a modified nucleotide) is incorporated per sequencing cycle. The sequencing may be accomplished by, for example, sequencing by synthesis, pyrosequencing, and the like. In embodiments, a sequencing cycle includes extending a complementary polynucleotide by incorporating a first nucleotide using a polymerase, wherein the polynucleotide is hybridized to a template nucleic acid, detecting the first nucleotide, and identifying the first nucleotide. An “extension strand” is formed as the one or more nucleotides are incorporated into a complementary polynucleotide hybridized to a template nucleic acid. The extension strand is complementary to the template nucleic acid. In embodiments, to begin a sequencing cycle, one or more differently labeled nucleotides and a DNA polymerase can be introduced. Following nucleotide addition, signals produced (e.g., via excitation and emission of a detectable label) can be detected to determine the identity of the incorporated nucleotide (based on the labels on the nucleotides). Reagents can then be added to remove the 3’ reversible terminator and to remove labels from each incorporated base. Reagents, enzymes, and other substances can be removed between steps by washing. Cycles may include repeating these steps, and the sequence of each cluster is read over the multiple repetitions. [0092] As used herein, the term “sequencing reaction mixture” is used in accordance with its plain and ordinary meaning and refers to an aqueous mixture that contains the reagents necessary to allow a nucleotide or nucleotide analogue to be added (i.e., incorporated) to a DNA strand by a DNA polymerase. As used herein, the term “invasion-reaction mixture” is used in accordance Attorney Docket No.: 051385-585001WO with its plain and ordinary meaning and refers to an aqueous mixture that contains the reagents necessary to allow a nucleotide or nucleotide analogue to be added to a DNA strand by a DNA polymerase that extends the invasion primer. [0093] As used herein, the term “extending”, “extension” or “elongation” is used in accordance with their plain and ordinary meanings and refer to synthesis by a polymerase of a new polynucleotide strand (i.e., an “extension strand”) complementary to a template strand by adding free nucleotides (e.g., dNTPs) from a reaction mixture that are complementary to the template in a 5’-to-3’ direction, including condensing a 5’-phosphate group of a dNTPs with a 3’-hydroxy group at the end of the nascent (elongating) DNA strand. [0094] As used herein, the term “sequencing read” is used in accordance with its plain and ordinary meaning and refers to an inferred sequence of nucleotide bases (or nucleotide base probabilities) corresponding to all or part of a single polynucleotide fragment. A sequencing read may include 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more nucleotide bases. Reads of length 20-40 base pairs (bp) are referred to as ultra-short. Typical sequencers produce read lengths in the range of 100-500 bp. Read length is a factor which can affect the results of biological studies. For example, longer read lengths improve the resolution of de novo genome assembly and detection of structural variants. In embodiments, a sequencing read includes reading a barcode and a template nucleotide sequence. In embodiments, a sequencing read includes reading a template nucleotide sequence. In embodiments, a sequencing read includes reading a barcode and not a template nucleotide sequence. In embodiments, a sequencing read includes a computationally derived string corresponding to the detected label. In some embodiments, a sequencing read may include 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or more nucleotide bases. In embodiments, a sequencing read is a string of characters representing the sequence of nucleotides. In embodiments, the length of a sequencing read corresponds to the length of the target sequence. In embodiments, the length of a sequencing read corresponds to the number of sequencing cycles. A sequencing read may be subjected to initial processing (often termed “pre-processing”) prior to annotation. Pre- processing includes filtering out low-quality sequences, sequence trimming to remove continuous low-quality nucleotides, merging paired-end sequences, or identifying and filtering out PCR repeats using known techniques in the art. The sequenced reads may then be assembled Attorney Docket No.: 051385-585001WO and aligned using bioinformatic algorithms known in the art. A sequencing read may be aligned to a reference sequence. In embodiments, a sequencing read includes a computationally derived string corresponding to the detected complementary nucleotide (e.g., a labeled nucleotide). The sequence reads are optionally stored in an appropriate data structure for further evaluation. In embodiments, a first sequencing reaction can generate a first sequencing read. The first sequencing read can provide the sequence of a first region of the polynucleotide fragment. In embodiments, a second sequencing primer can initiate sequencing at a second location on the nucleic acid template. The second location can be distinct from the first location. In some cases, a 3′ terminal nucleotide of the second primer can hybridize to a location that is more than 5 nucleotides away from a binding site of a 3′ terminal nucleotide of the first primer. The second sequencing reaction can generate a second sequencing read. The second sequencing read can provide the sequence of a second region of the nucleic acid template which is distinct from the first region of the nucleic acid template. In some embodiments, the nucleic acid template is optionally subjected to one or more additional rounds of sequencing using additional sequencing primers, thereby generating additional sequencing reads. In embodiments, a sequencing read is about 25 nucleotide bases. In embodiments, a sequencing read is about 35 nucleotide bases. In embodiments, a sequencing read is about 45 nucleotide bases. In embodiments, a sequencing read is about 55 nucleotide bases. In embodiments, a sequencing read is about 65 nucleotide bases. In embodiments, a sequencing read is about 75 nucleotide bases. In embodiments, a sequencing read is about 85 nucleotide bases. [0095] The term “multiplexing” as used herein refers to an analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid target sequences, can be assayed simultaneously by using the methods and devices as described herein, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic. [0096] “Hybridize” shall mean the annealing of a nucleic acid sequence to another nucleic acid sequence (e.g., one single-stranded nucleic acid (such as a primer) to another nucleic acid) based on the well-understood principle of sequence complementarity. In an embodiment the other Attorney Docket No.: 051385-585001WO nucleic acid is a single-stranded nucleic acid. In some embodiments, one portion of a nucleic acid hybridizes to itself, such as in the formation of a hairpin structure. The propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity. The effect of these parameters on hybridization is described in, for example, Sambrook J., Fritsch E. F., Maniatis T., Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York (1989). As used herein, hybridization of a primer, or of a DNA extension product, respectively, is extendable by creation of a phosphodiester bond with an available nucleotide or nucleotide analogue capable of forming a phosphodiester bond, therewith. For example, hybridization can be performed at a temperature ranging from 15° C to 95° C. In some embodiments, the hybridization is performed at a temperature of about 20° C, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, about 65° C, about 70° C, about 75° C, about 80° C, about 85° C, about 90° C, or about 95° C. In other embodiments, the stringency of the hybridization can be further altered by the addition or removal of components of the buffered solution. [0097] As used herein, “specifically hybridizes” refers to preferential hybridization under hybridization conditions where two nucleic acids, or portions thereof, that are substantially complementary, hybridize to each other and not to other nucleic acids that are not substantially complementary to either of the two nucleic acids. For example, specific hybridization includes the hybridization of a primer or capture nucleic acid to a portion of a target nucleic acid (e.g., a template, or adapter portion of a template) that is substantially complementary to the primer or capture nucleic acid. In some embodiments nucleic acids, or portions thereof, that are configured to specifically hybridize are often about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100% complementary to each other over a contiguous portion of nucleic acid sequence. A specific hybridization discriminates over non- specific hybridization interactions (e.g., two nucleic acids that a not configured to specifically hybridize, e.g., two nucleic acids that are 80% or less, 70% or less, 60% or less or 50% or less complementary) by about 2-fold or more, often about 10-fold or more, and sometimes about 100- fold or more, 1000-fold or more, 10,000-fold or more, 100,000-fold or more, or 1,000,000-fold Attorney Docket No.: 051385-585001WO or more. Two nucleic acid strands that are hybridized to each other can form a duplex which includes a double stranded portion of nucleic acid. [0098] A nucleic acid can be amplified by a suitable method. The term “amplification,” “amplified” or “amplifying” as used herein refers to subjecting a target nucleic acid in a sample to a process that linearly or exponentially generates amplicon nucleic acids having the same or substantially the same (e.g., substantially identical) nucleotide sequence as the target nucleic acid, or segment thereof, and/or a complement thereof (which may be referred to herein as an “amplification product” or “amplification products”). In some embodiments an amplification reaction comprises a suitable thermal stable polymerase. Thermal stable polymerases are known and are stable for prolonged periods of time, at temperature greater than 80° C when compared to common polymerases found in most mammals. In certain embodiments the term “amplification,” “amplified” or “amplifying” refers to a method that comprises a polymerase chain reaction (PCR). Conditions conducive to amplification (i.e., amplification conditions) are known and often comprise at least a suitable polymerase, a suitable template, a suitable primer or set of primers, suitable nucleotides (e.g., dNTPs), a suitable buffer, and application of suitable annealing, hybridization and/or extension times and temperatures. In certain embodiments an amplified product (e.g., an amplicon) can contain one or more additional and/or different nucleotides than the template sequence, or portion thereof, from which the amplicon was generated (e.g., a primer can contain “extra” nucleotides (such as a 5’ portion that does not hybridize to the template), or one or more mismatched bases within a hybridizing portion of the primer). [0099] As used herein, bridge-PCR (bPCR) amplification is a method for solid-phase amplification as exemplified by the disclosures of U.S. Pat. Nos.5,641,658; 7,115,400; and U.S. Patent Publ. No.2008/0009420, each of which is incorporated herein by reference in its entirety. Bridge-PCR involves repeated polymerase chain reaction cycles, cycling between denaturation, annealing, and extension conditions and enables controlled, spatially-localized, amplification, to generate amplification products (e.g., amplicons) immobilized on a solid support in order to form arrays comprised of colonies (or “clusters”) of immobilized nucleic acid molecule. [0100] A nucleic acid can be amplified by a thermocycling method or by an isothermal amplification method. In some embodiments, a rolling circle amplification method is used. In Attorney Docket No.: 051385-585001WO some embodiments, amplification takes place on a solid support (e.g., within a flow cell) where a nucleic acid, nucleic acid library or portion thereof is immobilized. In certain sequencing methods, a nucleic acid library is added to a flow cell and immobilized by hybridization to anchors under suitable conditions. This type of nucleic acid amplification is often referred to as solid phase amplification. In some embodiments of solid phase amplification, all or a portion of the amplified products are synthesized by an extension initiating from an immobilized primer. Solid phase amplification reactions are analogous to standard solution phase amplifications except that at least one of the amplification oligonucleotides (e.g., primers) is immobilized on a solid support. [0101] In some embodiments solid phase amplification comprises a nucleic acid amplification reaction comprising only one species of oligonucleotide primer immobilized to a surface or substrate. In certain embodiments solid phase amplification comprises a plurality of different immobilized oligonucleotide primer species. In some embodiments solid phase amplification may comprise a nucleic acid amplification reaction comprising one species of oligonucleotide primer immobilized on a solid surface and a second different oligonucleotide primer species in solution. Multiple different species of immobilized or solution-based primers can be used. Non- limiting examples of solid phase nucleic acid amplification reactions include interfacial amplification, bridge amplification, emulsion PCR, WildFire amplification (e.g., US patent publication US20130012399), the like or combinations thereof. [0102] As used herein, the term “biomolecule” refers to an agent (e.g., a compound, macromolecule, or small molecule), and the like derived from a biological system (e.g., an organism). The biomolecule may contain multiple individual components that collectively construct the biomolecule, for example, in embodiments, the biomolecule is a polynucleotide wherein the polynucleotide is composed of nucleotide monomers. The biomolecule may be or may include DNA, RNA, organelles, carbohydrates, lipids, proteins, or any combination thereof. These components may be extracellular. In some examples, the biomolecule may be referred to as a clump or aggregate of combinations of components. In some instances, the biomolecule may include one or more constituents of a cell but may not include other constituents of the cell. In embodiments, a biomolecule is a molecule produced by a biological system (e.g., an organism). In embodiments, a biomolecule may be referred to as an analyte. Analytes can be broadly Attorney Docket No.: 051385-585001WO classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In embodiments, the analytes within a cell can be localized to subcellular locations, including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In embodiments, analyte(s) can be peptides or proteins, including antibodies and/or enzymes. In embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein. [0103] As used herein, the term “biological system” refers to a virus, cell, cell derivative, cell nucleus, cell organelle, cell constituent and the like derived from a biological sample. Examples of a cell organelle include, without limitation, a nucleus, endoplasmic reticulum, a ribosome, a Golgi apparatus, an endoplasmic reticulum, a chloroplast, an endocytic vesicle, an exocytic vesicle, a vacuole, and a lysosome. The biological system (e.g., an organism) may contain multiple individual components, such as viruses, cells, cell derivatives, cell nuclei, cell organelles and cell constituents, including combinations of different of these and other components. The biological system may include DNA, RNA, organelles, proteins, or any combination thereof. These components may be extracellular. In some examples, the biological system may be referred to as a clump or aggregate of combinations of components. In some instances, the biological system may include one or more constituents of a cell but may not include other constituents of the cell. An example of such constituents include nucleus or an organelle. A cell may be a live or viable cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix or cultured when including a gel or polymer matrix. A biological system may include a single cell and/or a single nuclei from a cell. Attorney Docket No.: 051385-585001WO [0104] Provided herein are methods and compositions for analyzing a sample (e.g., sequencing nucleic acids within a sample). A sample (e.g., a sample comprising nucleic acid) can be obtained from a suitable subject. A sample can be isolated or obtained directly from a subject or part thereof. In some embodiments, a sample is obtained indirectly from an individual or medical professional. A sample can be any specimen that is isolated or obtained from a subject or part thereof. A sample can be any specimen that is isolated or obtained from multiple subjects. Non-limiting examples of specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof. A fluid or tissue sample from which nucleic acid is extracted may be acellular (e.g., cell-free). Non-limiting examples of tissues include organ tissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder, reproductive organs, intestine, colon, spleen, brain, the like or parts thereof), epithelial tissue, hair, hair follicles, ducts, canals, bone, eye, nose, mouth, throat, ear, nails, the like, parts thereof or combinations thereof. A sample may comprise cells or tissues that are normal, healthy, diseased (e.g., infected), and/or cancerous (e.g., cancer cells). A sample obtained from a subject may comprise cells or cellular material (e.g., nucleic acids) of multiple organisms (e.g., virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasite nucleic acid). [0105] Provided herein are methods, systems, and compositions for analyzing a sample (e.g., sequencing nucleic acids within a sample) in situ. The term “in situ” is used in accordance with its ordinary meaning in the art and refers to a sample surrounded by at least a portion of its native environment, such as may preserve the relative position of two or more elements. For example, an extracted human cell obtained is considered in situ when the cell is retained in its local microenvironment so as to avoid extracting the target (e.g., nucleic acid molecules or proteins) away from their native environment. An in situ sample (e.g., a cell) can be obtained from a suitable subject. An in situ cell sample may refer to a cell and its surrounding milieu, or a tissue. A sample can be isolated or obtained directly from a subject or part thereof. In embodiments, the Attorney Docket No.: 051385-585001WO methods described herein (e.g., sequencing a plurality of target nucleic acids of a cell in situ) are applied to an isolated cell (i.e., a cell not surrounded by least a portion of its native environment). For the avoidance of any doubt, when the method is performed within a cell (e.g., an isolated cell) the method may be considered in situ. In some embodiments, a sample is obtained indirectly from an individual or medical professional. A sample can be any specimen that is isolated or obtained from a subject or part thereof. A sample can be any specimen that is isolated or obtained from multiple subjects. Non-limiting examples of specimens include fluid or tissue from a subject, including, without limitation, blood or a blood product (e.g., serum, plasma, platelets, buffy coats, or the like), umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells, lymphocytes, placental cells, stem cells, bone marrow derived cells, embryo or fetal cells) or parts thereof (e.g., mitochondrial, nucleus, extracts, or the like), urine, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, the like or combinations thereof. Non-limiting examples of tissues include organ tissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder, reproductive organs, intestine, colon, spleen, brain, the like or parts thereof), epithelial tissue, hair, hair follicles, ducts, canals, bone, eye, nose, mouth, throat, ear, nails, the like, parts thereof or combinations thereof. A sample may include cells or tissues that are normal, healthy, diseased (e.g., infected), and/or cancerous (e.g., cancer cells). A sample obtained from a subject may include cells or cellular material (e.g., nucleic acids) of multiple organisms (e.g., virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasite nucleic acid). A sample may include a cell and RNA transcripts. A sample can include nucleic acids obtained from one or more subjects. In some embodiments a sample includes nucleic acid obtained from a single subject. A subject can be any living or non-living organism, including but not limited to a human, non-human animal, plant, bacterium, fungus, virus, or protist. A subject may be any age (e.g., an embryo, a fetus, infant, child, adult). A subject can be of any sex (e.g., male, female, or combination thereof). A subject may be pregnant. In some embodiments, a subject is a mammal. In some embodiments, a subject is a plant. In some embodiments, a subject is a human subject. A subject can be a patient (e.g., a human patient). In some embodiments a subject is suspected of having a genetic variation or a disease or condition associated with a genetic variation. A “tissue section” as used herein refers to a portion of a biological tissue Attorney Docket No.: 051385-585001WO derived from a biological sample, typically from an organism (e.g., a human or animal subject or patient). [0106] As used herein, the term “fresh,” generally in the context of a fresh tissue means that the tissue has recently been obtained from an organism, generally before any subsequent fixation steps, for example, flash freezing or chemical fixation. In embodiments, a fresh tissue is obtained from an organism about 1 second up to about 20 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 second up to about 60 seconds before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 30 seconds up to about 60 seconds before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 20 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 10 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 1 minutes up to about 5 minutes before any fixation steps are performed. In embodiments, a fresh tissue is obtained from an organism about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes before any fixation steps are performed. [0107] As used herein, the term “fix,” refers to formation of covalent bonds, such as crosslinks, between biomolecules or within molecules. The process of fixing tissue samples or biological samples (e.g., cells and nuclei) for example, is called “fixation.” The agent that causes fixation is generally referred to as a “fixative” or “fixing agent.” “Fixed biological samples” (e.g., fixed cells or nuclei) or “fixed tissues” refers to biological samples (e.g., cells or nuclei) or tissues that have been in contact with a fixative under conditions sufficient to allow or result in formation of intra- and inter-molecular crosslinks between biomolecules in the biological sample. Fixation may be reversed and the process of reversing fixation may be referred to as “un-fixing” or “decrosslinking.” Unfixing or decrosslinking refers to breaking or reversing the formation of covalent bonds in biomolecules formed by fixatives. In some examples, the tissue fixed is fresh tissue. In some examples, the tissue fixed may be frozen tissue. In some examples, the tissue fixed may not be dissociated. In some examples, the tissue fixed may be dissociated or partially dissociated (e.g., chopped, cut). In some examples, tissue that has been rapidly frozen Attorney Docket No.: 051385-585001WO and, perhaps, cut or chopped into pieces (e.g., small enough to fit into a tube or container used for fixation) may be used. In some examples, tissue may be dissociated or partially dissociated (e.g., cut, chopped) before or during fixation. In some examples, tissue that is fixed may not be dissociated. The frozen biological tissue can be fixed using a fixing agent, which is suitably an organic fixing agent. Suitable organic fixing agents include without limitation alcohols, ketones, aldehydes (e.g., glutaraldehyde), cross-linking agents, disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), ethylene glycol bis (succinimidyl succinate) (EGS), bis(sulfosuccinimidyl)suberate (BS3) and combinations thereof. A particularly suitable fixing agent is a formaldehyde-based fixing agent such as formalin, which is a mixture of formaldehyde and water. The formalin may include about 1% to about 15% by weight formaldehyde and about 85% to about 99% by weight water, suitable about 2% to about 8% by weight formaldehyde and about 92% to about 98% by weight water, or about 4% by weight formaldehyde and about 96% by weight water. In some examples, tissues may be fixed in 4% paraformaldehyde. Other suitable fixing agents will be appreciated by those of ordinary skill in the art (e.g., International PCT App. No. PCT/US2020/066705, which is incorporated herein by reference in its entirety). [0108] As used herein, the term “permeable” refers to a property of a substance that allows certain materials to pass through the substance. “Permeable” may be used to describe a biological sample, such as a cell or nucleus, in which analytes in the biological sample can leave the biological sample. “Permeabilize” is an action taken to cause, for example, a biological sample (e.g., a cell) to release its analytes. In some examples, permeabilization of a biological sample is accomplished by affecting the integrity (e.g., compromising) of a biological sample membrane (e.g., a cellular or nuclear membrane) such as by application of a protease or other enzyme capable of disturbing a membrane allowing analytes to diffuse out of the biological sample. [0109] As used herein, the term “single biological sample”, such as a single cell or a single nucleus generally refers to a biological sample that is not present in an aggregated form or clump. Single biological samples, such as cells and/or nuclei may be the result of dissociating a tissue sample. Attorney Docket No.: 051385-585001WO [0110] As used herein, the term “disease state” is used in accordance with its plain and ordinary meaning and refers to any abnormal biological or aberrant state of a cell or organism. The presence of a disease state may be identified by the same collection of biological constituents used to determine the cell’s biological state. In general, a disease state will be detrimental to a biological system. A disease state may be a consequence of, inter alia, an environmental pathogen, for example a viral infection (e.g., HIV/AIDS, hepatitis B, hepatitis C, influenza, measles, etc.), a bacterial infection, a parasitic infection, a fungal infection, or infection by some other organism. A disease state may also be the consequence of some other environmental agent, such as a chemical toxin or a chemical carcinogen. As used herein, a disease state further includes genetic disorders wherein one or more copies of a gene is altered or disrupted, thereby affecting its biological function. Exemplary genetic diseases include, but are not limited to polycystic kidney disease, familial multiple endocrine neoplasia type I, neurofibromatoses, Tay-Sachs disease, Huntington's disease, sickle cell anemia, thalassemia, and Down's syndrome, as well as others (see, e.g., The Metabolic and Molecular Bases of Inherited Diseases, 7th ed., McGraw-Hill Inc., New York). Other exemplary diseases include, but are not limited to, cancer, hypertension, Alzheimer's disease, neurodegenerative diseases, and neuropsychiatric disorders such as bipolar affective disorders or paranoid schizophrenic disorders. Disease states are monitored to determine the level or severity (e.g., the stage or progression) of one or more disease states of a subject and, more specifically, detect changes in the biological state of a subject which are correlated to one or more disease states (see, e.g., U.S. Pat. No.6,218,122, which is incorporated by reference herein in its entirety). In embodiments, methods provided herein are also applicable to monitoring the disease state or states of a subject undergoing one or more therapies. Thus, the present disclosure also provides, in some embodiments, methods for determining or monitoring efficacy of a therapy or therapies (i.e., determining a level of therapeutic effect) upon a subject. In embodiments, methods of the present disclosure can be used to assess therapeutic efficacy in a clinical trial, e.g., as an early surrogate marker for success or failure in such a clinical trial. Within eukaryotic cells, there are hundreds to thousands of signaling pathways that are interconnected. For this reason, perturbations in the function of proteins within a cell have numerous effects on other proteins and the transcription of other genes that are connected by primary, secondary, and sometimes tertiary pathways. This extensive interconnection between the function of various proteins means that the alteration of Attorney Docket No.: 051385-585001WO any one protein is likely to result in compensatory changes in a wide number of other proteins. In particular, the partial disruption of even a single protein within a cell, such as by exposure to a drug or by a disease state which modulates the gene copy number (e.g., a genetic mutation), results in characteristic compensatory changes in the transcription of enough other genes that these changes in transcripts can be used to define a “signature” of particular transcript alterations which are related to the disruption of function, e.g., a particular disease state or therapy, even at a stage where changes in protein activity are undetectable. [0111] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may optionally be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. A protein may refer to a protein expressed in a cell. [0112] A polypeptide, or a cell is “recombinant” when it is artificial or engineered, or derived from or contains an artificial or engineered protein or nucleic acid (e.g., non-natural or not wild type). For example, a polynucleotide that is inserted into a vector or any other heterologous location, e.g., in a genome of a recombinant organism, such that it is not associated with nucleotide sequences that normally flank the polynucleotide as it is found in nature is a recombinant polynucleotide. A protein expressed in vitro or in vivo from a recombinant polynucleotide is an example of a recombinant polypeptide. Likewise, a polynucleotide sequence that does not appear in nature, for example a variant of a naturally occurring gene, is recombinant. [0113] As used herein, a “single cell” refers to one cell. Single cells useful in the methods described herein can be obtained from a tissue of interest, or from a biopsy, blood sample, or cell culture. Additionally, cells from specific organs, tissues, tumors, neoplasms, or the like can be obtained and used in the methods described herein. In general, cells from any population can be used in the methods, such as a population of prokaryotic or eukaryotic organisms, including bacteria or yeast. [0114] As used herein, the term “tissue” is used in accordance with its plain and ordinary meaning and refers to an organization of cells in a structure, where the structure generally Attorney Docket No.: 051385-585001WO functions as a unit in an organism (e.g., mammals) and may carry out specific functions. In some examples, cells in a tissue are configured in a mass and may not be free from one another. This disclosure describes methods of obtaining single biological samples (e.g., cells or nuclei) from tissues that can be used in various single biological samples (e.g., single-cell/nucleus) workflows. In some examples, blood cells (e.g., lymphocytes) can be considered a tissue. However, blood cells, like lymphocytes, generally are free from one another in the blood. The methods disclosed herein can be used to process those cells to obtain cells and/or nuclei, although dissociation steps may not be necessary when using those types of tissues. Generally, any type of tissue can be used in the methods described herein. Examples of tissues that may be used in the disclosed methods include, but are not limited to connective, epithelial, muscle and nervous tissue. In some examples, the tissues are from mammals. Tissues that contain any type of cells may be used. For example, tissues from abdomen, bladder, brain, esophagus, heart, intestine, kidney, liver, lung, lymph node, olfactory bulb, ovary, pancreas, skin, spleen, stomach, testicle, and the like. The tissue may be normal or tumor tissue (e.g., malignant). This example is not meant to be limiting. Although the conditions used in the disclosed may not be identical for different types of tissue, the methods may be applied to any tissue. The tissues used in the disclosed methods may be in various states. In some examples, the tissues used in the disclosed methods may be fresh, frozen, or fixed. [0115] The term “cellular component” is used in accordance with its ordinary meaning in the art and refers to any organelle, nucleic acid, protein, or analyte that is found in a prokaryotic, eukaryotic, archaeal, or other organismic cell type. Examples of cellular components (e.g., a component of a cell) include RNA transcripts, proteins, membranes, lipids, and other analytes. In embodiments, a cellular component is a biomolecule. [0116] In some embodiments, a sample comprises nucleic acid, or fragments thereof. A sample can comprise nucleic acids obtained from one or more subjects. In some embodiments a sample comprises nucleic acid obtained from a single subject. In some embodiments, a sample comprises a mixture of nucleic acids. A mixture of nucleic acids can comprise two or more nucleic acid species having different nucleotide sequences, different fragment lengths, different origins (e.g., genomic origins, cell or tissue origins, subject origins, the like or combinations thereof), or combinations thereof. A sample may comprise synthetic nucleic acid. Attorney Docket No.: 051385-585001WO [0117] A subject can be any living or non-living organism, including but not limited to a human, non-human animal, plant, bacterium, fungus, virus or protist. A subject may be any age (e.g., an embryo, a fetus, infant, child, adult). A subject can be of any sex (e.g., male, female, or combination thereof). A subject may be pregnant. In some embodiments, a subject is a mammal. In some embodiments, a subject is a human subject. A subject can be a patient (e.g., a human patient). In some embodiments a subject is suspected of having a genetic variation or a disease or condition associated with a genetic variation. [0118] The term “protein-specific binding agent” refers to an agent to a protein or polypeptide molecule, or portion thereof, capable of selectively binding or interacting with a protein. In embodiments, a protein-specific binding agent specifically binds a particular protein (e.g., a protein antigen or epitope thereof). In embodiments a protein-specific binding agent is an immunoglobulin (IgA, IgD, IgE, IgG, or IgM). Intact immunoglobulins, also known as antibodies, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each, and two heavy (H) chains of approximately 50 kDa each. In embodiments, the protein binding moiety is an antigen-specific antibody. Non-limiting examples of protein-specific binding agent encompassed within the term “antigen-specific antibody” used herein include: (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be recombinantly joined by a synthetic linker, creating a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv)). The most commonly used linker is a 15-residue (Gly4Ser)3 peptide, but other linkers are also known in the art. Single chain antibodies are also intended to be encompassed within the terms “protein-specific binding agent,” of an antibody. The antibody can also be a polyclonal antibody, monoclonal antibody, chimeric antibody, antigen-binding fragment, Fc fragment, single chain antibodies, or any derivatives thereof. In embodiments, the protein- specific binding agent is the antigen-binding site (e.g., fragment antigen-binding (Fab) variable region) of an antibody. The term “antigen-binding site” of an antibody (or simply “antibody Attorney Docket No.: 051385-585001WO portion”), as used herein, refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. [0119] An “antibody” (Ab) is a protein that binds specifically to a particular substance, known as an “antigen” (Ag). An “antibody” or “antigen-binding fragment” is an immunoglobulin that binds a specific “epitope.” The term encompasses polyclonal, monoclonal, and chimeric antibodies. In nature, antibodies are generally produced by lymphocytes in response to immune challenge, such as by infection or immunization. An “antigen” (Ag) is any substance that reacts specifically with antibodies or T lymphocytes (T cells). An antibody may include the entire antibody as well as any antibody fragments capable of binding the antigen or antigenic fragment of interest. Examples include complete antibody molecules, antibody fragments, such as Fab, F(ab')2, CDRs, VL, VH, and any other portion of an antibody which is capable of specifically binding to an antigen. Antibodies used herein are immunospecific for, and therefore specifically and selectively bind to, for example, proteins either detected (e.g., biological targets of interest) or used for detection (e.g., probes containing oligonucleotide barcodes) in the methods and devices as described herein. [0120] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., packaging, buffers, written instructions for performing a method, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits. Attorney Docket No.: 051385-585001WO [0121] The terms “bioconjugate group,” “bioconjugate reactive moiety,” and “bioconjugate reactive group” refer to a chemical moiety which participates in a reaction to form a bioconjugate linker (e.g., covalent linker). Non-limiting examples of bioconjugate groups include –NH2, –COOH, –COOCH3, –N-hydroxysuccinimide, In embodiments, the bioconjugate reactive group may be .
Figure imgf000067_0001
In embodiments, the bioconjugate reactive moiety ,
Figure imgf000067_0002
,
Figure imgf000067_0003
, or -NH2. Additional examples of bioconjugate reactive groups and the
Figure imgf000067_0004
reactive linkers may be found in the Bioconjugate Table below: Bioconjugate reactive group 1 Bioconjugate reactive group 2 (e.g., electrophilic bioconjugate (e.g., nucleophilic bioconjugate Resulting Bioconjugate reactive moiety) reactive moiety) reactive linker activated esters amines/anilines carboxamides acrylamides thiols thioethers acyl azides amines/anilines carboxamides acyl halides amines/anilines carboxamides acyl halides alcohols/phenols esters acyl nitriles alcohols/phenols esters acyl nitriles amines/anilines carboxamides aldehydes amines/anilines imines aldehydes or ketones hydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkyl halides amines/anilines alkyl amines alkyl halides carboxylic acids esters Attorney Docket No.: 051385-585001WO alkyl halides thiols thioethers alkyl halides alcohols/phenols ethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylic acids esters alkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenols esters anhydrides amines/anilines carboxamides aryl halides thiols thiophenols aryl halides amines aryl amines aziridines thiols thioethers boronates glycols boronate esters carbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxides thiols thioethers haloacetamides thiols thioethers haloplatinate amino platinum complex haloplatinate heterocycle platinum complex haloplatinate thiol platinum complex halotriazines amines/anilines aminotriazines halotriazines alcohols/phenols triazinyl ethers halotriazines thiols triazinyl thioethers imido esters amines/anilines amidines isocyanates amines/anilines ureas isocyanates alcohols/phenols urethanes isothiocyanates amines/anilines thioureas maleimides thiols thioethers phosphoramidites alcohols phosphite esters silyl halides alcohols silyl ethers sulfonate esters amines/anilines alkyl amines sulfonate esters thiols thioethers sulfonate esters carboxylic acids esters sulfonate esters alcohols ethers sulfonyl halides amines/anilines sulfonamides sulfonyl halides phenols/alcohols sulfonate esters [0122] As used herein, the term “bioconjugate reactive moiety” and “bioconjugate reactive group” refers to a moiety or group capable of forming a bioconjugate (e.g., covalent linker) as a result of the association between atoms or molecules of bioconjugate reactive groups. The association can be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e.g., –NH2, –COOH, –N-hydroxysuccinimide, or –maleimide) and a second Attorney Docket No.: 051385-585001WO bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate) provided herein can be direct, e.g., by covalent bond or linker (e.g., a first linker of second linker), or indirect, e.g., by non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e., the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol.198, American Chemical Society, Washington, D.C., 1982. In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., –N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., an amine). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., – sulfo–N-hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g., an amine). Useful bioconjugate reactive groups used for bioconjugate chemistries herein include, for example: (a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.; (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an Attorney Docket No.: 051385-585001WO amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom; (d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maleimide groups; (e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized;(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc.; (j) epoxides, which can react with, for example, amines and hydroxyl compounds; (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis; (l) metal silicon oxide bonding; (m) metal bonding to reactive phosphorus groups (e.g., phosphines) to form, for example, phosphate diester bonds.; (n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry; (o) biotin conjugate can react with avidin or strepavidin to form a avidin-biotin complex or streptavidin- biotin complex. [0123] The term “covalent linker” is used in accordance with its ordinary meaning and refers to a divalent moiety which connects at least two moieties to form a molecule. The term “non- covalent linker” is used in accordance with its ordinary meaning and refers to a divalent moiety which includes at least two molecules that are not covalently linked to each other but are capable of interacting with each other via a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bond, hydrogen bond, halogen bond) or van der Waals interactions (e.g., dipole-dipole, dipole- induced dipole, London dispersion). In embodiments, the non-covalent linker is the result of two molecules that are not covalently linked to each other that interact with each other via a non- covalent bond. [0124] The term “adapter” as used herein refers to any oligonucleotide that can be ligated to a nucleic acid molecule, thereby generating nucleic acid products that can be sequenced on a sequencing platform (e.g., an Illumina or Singular Genomics G4™ sequencing platform). In embodiments, adapters include two reverse complementary oligonucleotides forming a double- Attorney Docket No.: 051385-585001WO stranded structure. In embodiments, an adapter includes two oligonucleotides that are complementary at one portion and mismatched at another portion, forming a Y-shaped or fork- shaped adapter that is double stranded at the complementary portion and has two overhangs at the mismatched portion. Since Y-shaped adapters have a complementary, double-stranded region, they can be considered a special form of double-stranded adapters. When this disclosure contrasts Y-shaped adapters and double stranded adapters, the term “double-stranded adapter” or “blunt-ended” is used to refer to an adapter having two strands that are fully complementary, substantially (e.g., more than 90% or 95%) complementary, or partially complementary. In embodiments, adapters include sequences that bind to sequencing primers. In embodiments, adapters include sequences that bind to immobilized oligonucleotides (e.g., P7 and P5 sequences) or reverse complements thereof. In embodiments, the adapter is substantially non- complementary to the 3' end or the 5' end of any target polynucleotide present in the sample. In embodiments, the adapter can include a sequence that is substantially identical, or substantially complementary, to at least a portion of a primer, for example a universal primer. In embodiments, the adapter can include an index sequence (also referred to as barcode or tag) to assist with downstream error correction, identification or sequencing. [0125] As used herein, the term “hairpin adapter” refers to a polynucleotide including a double-stranded stem portion and a single-stranded hairpin loop portion. In some embodiments, an adapter is hairpin adapter. In some embodiments, a hairpin adapter comprises a single nucleic acid strand comprising a stem-loop structure. In some embodiments, a hairpin adapter comprises a nucleic acid having a 5’-end, a 5’-portion, a loop, a 3’-portion and a 3’-end (e.g., arranged in a 5’ to 3’ orientation). In some embodiments, the 5’ portion of a hairpin adapter is annealed and/or hybridized to the 3’ portion of the hairpin adapter, thereby forming a stem portion of the hairpin adapter. In some embodiments, the 5’ portion of a hairpin adapter is substantially complementary to the 3’ portion of the hairpin adapter. In certain embodiments, a hairpin adapter comprises a stem portion (i.e., stem) and a loop, wherein the stem portion is substantially double stranded thereby forming a duplex. In some embodiments, the loop of a hairpin adapter comprises a nucleic acid strand that is not complementary (e.g., not substantially complementary) to itself or to any other portion of the hairpin adapter. In some embodiments, a method herein comprises ligating a first adapter to a first end of a double stranded nucleic acid, and ligating a second adapter to a second end of a double stranded nucleic acid. In some Attorney Docket No.: 051385-585001WO embodiments, the first adapter and the second adapter are different. For example, in certain embodiments, the first adapter and the second adapter may comprise different nucleic acid sequences or different structures. In some embodiments, the first adapter is a Y-adapter and the second adapter is a hairpin adapter. In some embodiments, the first adapter is a hairpin adapter and a second adapter is a hairpin adapter. In certain embodiments, the first adapter and the second adapter may comprise different primer binding sites, different structures, and/or different capture sequences (e.g., a sequence complementary to a capture nucleic acid). In some embodiments, some, all or substantially all of the nucleic acid sequence of a first adapter and a second adapter are the same. In some embodiments, some, all or substantially all of the nucleic acid sequence of a first adapter and a second adapter are substantially different. [0126] In some embodiments, a nucleic acid comprises a capture nucleic acid. A capture nucleic acid refers to a nucleic acid that is attached to a substrate. In some embodiments, a capture nucleic acid comprises a primer. In some embodiments, a capture nucleic acid is a nucleic acid configured to specifically hybridize to a portion of one or more nucleic acid templates (e.g., a template of a library). In some embodiments a capture nucleic acid configured to specifically hybridize to a portion of one or more nucleic acid templates is substantially complementary to a suitable portion of a nucleic acid template, or an amplicon thereof. In some embodiments a capture nucleic acid is configured to specifically hybridize to a portion of an adapter, or a complement thereof. In some embodiments a capture nucleic acid, or portion thereof, is substantially complementary to a portion of an adapter, or a complement thereof. In embodiments, a capture nucleic acid is a probe oligonucleotide. Typically, a probe oligonucleotide is complementary to a target polynucleotide or portion thereof, and further comprises a label (such as a binding moiety) or is attached to a surface, such that hybridization to the probe oligonucleotide permits the selective isolation of probe-bound polynucleotides from unbound polynucleotides in a population. A probe oligonucleotide may or may not also be used as a primer. [0127] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range, and any other stated or unstated intervening value in, or smaller range of values within, that stated range is encompassed within the invention. The upper Attorney Docket No.: 051385-585001WO and lower limits of any such smaller range (within a more broadly recited range) may independently be included in the smaller ranges, or as particular values themselves, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0128] The term “nucleic acid sequencing device” and the like means an integrated system of one or more chambers, ports, and channels that are interconnected and in fluid communication and designed for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions, such as sample introduction, fluid and/or reagent driving means, temperature control, detection systems, data collection and/or integration systems, for the purpose of determining the nucleic acid sequence of a template polynucleotide. Nucleic acid sequencing devices may further include valves, pumps, and specialized functional coatings on interior walls. Nucleic acid sequencing devices may include a receiving unit, or platen, that orients the flow cell such that a maximal surface area of the flow cell is available to be exposed to an optical lens. Other nucleic acid sequencing devices include those provided by Singular Genomics™ (e.g., the G4™ system), Illumina™ (e.g., HiSeq™, MiSeq™, NextSeq™, or NovaSeq™ systems), Life Technologies™ (e.g., ABI PRISM™, or SOLiD™ systems), Pacific Biosciences (e.g., systems using SMRT™ Technology such as the Sequel™ or RS II™ systems), or Qiagen (e.g., Genereader™ system). Nucleic acid sequencing devices may further include fluidic reservoirs (e.g., bottles), valves, pressure sources, pumps, sensors, control systems, valves, pumps, and specialized functional coatings on interior walls. In embodiments, the device includes a plurality of a sequencing reagent reservoirs and a plurality of clustering reagent reservoirs. In embodiments, the clustering reagent reservoir includes amplification reagents (e.g., an aqueous buffer containing enzymes, salts, and nucleotides, denaturants, crowding agents, etc.) In embodiments, the reservoirs include sequencing reagents (such as an aqueous buffer containing enzymes, salts, and nucleotides); a wash solution (an aqueous buffer); a cleave solution (an aqueous buffer containing a cleaving agent, such as a reducing agent); or a cleaning solution (a dilute bleach solution, dilute NaOH solution, dilute HCl solution, dilute antibacterial solution, or water). The fluid of each of the reservoirs can vary. The fluid can be, for example, an aqueous solution which may contain buffers (e.g., saline- sodium citrate (SSC), ascorbic acid, tris(hydroxymethyl)aminomethane or “Tris”), aqueous salts Attorney Docket No.: 051385-585001WO (e.g., KCl or (NH4)2SO4)), nucleotides, polymerases, cleaving agent (e.g., tri-n-butyl-phosphine, triphenyl phosphine and its sulfonated versions (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), and tri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agent scavenger compounds (e.g., 2′- Dithiobisethanamine or 11-Azido-3,6,9- trioxaundecane-1-amine), chelating agents (e.g., EDTA), detergents, surfactants, crowding agents, or stabilizers (e.g., PEG, Tween, BSA). Non- limited examples of reservoirs include cartridges, pouches, vials, containers, and eppendorf tubes. In embodiments, the device is configured to perform fluorescent imaging. In embodiments, the device includes one or more light sources (e.g., one or more lasers). In embodiments, the illuminator or light source is a radiation source (i.e., an origin or generator of propagated electromagnetic energy) providing incident light to the sample. A radiation source can include an illumination source producing electromagnetic radiation in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 390 to 770 nm), or infrared (IR) range (about 0.77 to 25 microns), or other range of the electromagnetic spectrum. In embodiments, the illuminator or light source is a lamp such as an arc lamp or quartz halogen lamp. In embodiments, the illuminator or light source is a coherent light source. In embodiments, the light source is a laser, LED (light emitting diode), a mercury or tungsten lamp, or a super-continuous diode. In embodiments, the light source provides excitation beams having a wavelength between 200 nm to 1500 nm. In embodiments, the laser provides excitation beams having a wavelength of 405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm, 640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm. In embodiments, the illuminator or light source is a light-emitting diode (LED). The LED can be, for example, an Organic Light Emitting Diode (OLED), a Thin Film Electroluminescent Device (TFELD), or a Quantum dot based inorganic organic LED. The LED can include a phosphorescent OLED (PHOLED). In embodiments, the nucleic acid sequencing device includes an imaging system (e.g., an imaging system as described herein). The imaging system capable of exciting one or more of the identifiable labels (e.g., a fluorescent label) linked to a nucleotide and thereafter obtain image data for the identifiable labels. The image data (e.g., detection data) may be analyzed by another component within the device. The imaging system may include a system described herein and may include a fluorescence spectrophotometer including an objective lens and/or a solid-state imaging device. The solid-state imaging device may include a charge coupled device (CCD) and/or a complementary metal oxide semiconductor (CMOS). The system may also include circuitry and Attorney Docket No.: 051385-585001WO processors, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate array (FPGAs), logic circuits, and any other circuit or processor capable of executing functions described herein. The set of instructions may be in the form of a software program. As used herein, the terms “software” and “firmware” are interchangeable, and include any computer program stored in memory for execution by a computer, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory. In embodiments, the device includes a thermal control assembly useful to control the temperature of the reagents. [0129] “Synthetic” agents refer to non-naturally occurring agents, such as enzymes or nucleotides derived or constructed using man-made techniques. Synthetic DNA polymerases refer to non-naturally occurring DNA polymerases such as those constructed by synthetic methods, mutated parent DNA polymerases such as truncated DNA polymerases and fusion DNA polymerases (e.g. U.S. Pat. No.7,541,170). Variants of the parent DNA polymerase have been engineered by mutating residues using site-directed or random mutagenesis methods known in the art. In embodiments, the mutations are in any of Motifs I-VI. The variant is expressed in an expression system such as E. coli by methods known in the art. [0130] The methods and kits of the present disclosure may be applied, mutatis mutandis, to the sequencing of RNA, or to determining the identity of a ribonucleotide. [0131] “GC bias” describes the relationship between GC content and read coverage across a genome. For example, a genomic region of a higher GC content tends to have more (or less) sequencing reads covering that region. As described herein, GC bias can be introduced during amplification of library, cluster amplification, and/or the sequencing reactions. [0132] As used herein, “biological activity” may include the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, may encompass therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Biological activities may be observed in vitro systems designed to test or use such activities. [0133] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not isolated, but the same Attorney Docket No.: 051385-585001WO nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is isolated. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In embodiments, “isolated” refers to a nucleic acid, polynucleotide, polypeptide, protein, or other component that is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, etc.). [0134] As used herein, a “plurality” refers to two or more. [0135] As used herein the terms “automated” and “semi-automated” mean that the operations are performed by system programming or configuration with little or no human interaction once the operations are initiated, or once processes including the operations are initiated. [0136] The terms “cellular component” is used in accordance with its ordinary meaning in the art and refers to any organelle, nucleic acid, protein, or analyte that is found in a prokaryotic, eukaryotic, archaeal, or other organismic cell type. Examples of cellular components (e.g., a component of a cell) include RNA transcripts, proteins, membranes, lipids, and other analytes. [0137] A “gene” refers to a polynucleotide that is capable of conferring biological function after being transcribed and/or translated. Functionally, a genome is subdivided into genes. Each gene is a nucleic acid sequence that encodes an RNA or polypeptide. A gene is transcribed from DNA into RNA, which can either be non-coding (ncRNA) with a direct function, or an intermediate messenger (mRNA) that is then translated into protein. Typically a gene includes multiple sequence elements, such as for example, a coding element (i.e., a sequence that encodes a functional protein), non-coding element, and regulatory element. Each element may be as short as a few bp to 5kb. In embodiments, the gene is the protein coding sequence of RNA. Non- limiting examples of genes include developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1); and enzymes (e.g., ACC synthases Attorney Docket No.: 051385-585001WO and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases). In embodiments, a gene includes at least one mutation associated with a disease or condition mediated by a mutant form of the gene. [0138] The term “genetic locus,” or “locus” as used herein refers to a genome or target polynucleotide, specifically a contiguous subregion or segment of the genome or target polynucleotide. As used herein, genetic locus, or locus, may refer to the position of a nucleotide, a gene, or a portion of a gene in a genome, including mitochondrial DNA, or it may refer to any contiguous portion of genomic sequence whether or not it is within, or associated with, a gene. In one aspect, a genetic locus refers to any portion of genomic sequence, including mitochondrial DNA, from a single nucleotide to a segment of few hundred nucleotides, e.g.100-300, in length. Usually, a particular genetic locus may be identified by its nucleotide sequence, or the nucleotide sequence, or sequences, of one or both adjacent or flanking regions. In another aspect, a genetic locus refers to the expressed nucleic acid product of a gene, such as an RNA molecule or a cDNA copy thereof. [0139] The term “multiplexing" as used herein refers to an analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid target sequences, can be assayed simultaneously by using the methods and devices as described herein, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height), or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic. As used herein, the term “multiplex” is used to refer to an assay in which multiple (i.e. at least two) different biomolecules are assayed at the same time, and more particularly in the same aliquot of the sample, or in the same reaction mixture. In embodiments, more than two Attorney Docket No.: 051385-585001WO different biomolecules are assayed at the same time. In embodiments, at least 2, 4, 6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more biomolecules are detected according to the present method. [0140] As used herein, the term “adjacent,” refers to two nucleotide sequences in a nucleic acid, can refer to nucleotide sequences separated by 0 to about 20 nucleotides, more specifically, in a range of about 1 to about 10 nucleotides, or to sequences that directly abut one another. As those of skill in the art appreciate, two nucleotide sequences that that are to ligated together will generally directly abut one another. [0141] As used herein, the terms “incubate,” and “incubation refer collectively to altering the temperature of an object in a controlled manner such that conditions are sufficient for conducting the desired reaction. Thus, it is envisioned that the terms encompass heating a receptacle (e.g., a microplate) to a desired temperature and maintaining such temperature for a fixed time interval. Also included in the terms is the act of subjecting a receptacle to one or more heating and cooling cycles (i.e., “temperature cycling” or “thermal cycling”). While temperature cycling typically occurs at relatively high rates of change in temperature, the term is not limited thereto, and may encompass any rate of change in temperature. [0142] The term “image” is used according to its ordinary meaning and refers to a representation of all or part of an object. The representation may be an optically detected reproduction. For example, an image can be obtained from fluorescent, luminescent, scatter, or absorption signals. The part of the object that is present in an image can be the surface or other xy plane of the object. Typically, an image is a 2 dimensional representation of a 3 dimensional object. An image may include signals at differing intensities (i.e., signal levels). An image can be provided in a computer readable format or medium. An image is derived from the collection of focus points of light rays coming from an object (e.g., the sample), which may be detected by any image sensor. [0143] As used herein, the term “signal” is intended to include, for example, fluorescent, luminescent, scatter, or absorption impulse or electromagnetic wave transmitted or received. Signals can be detected in the ultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about 391 to 770 nm), infrared (IR) range (about 0.771 to 25 microns), or other range of the electromagnetic spectrum. The term “signal level” refers to an amount or quantity of detected Attorney Docket No.: 051385-585001WO energy or coded information. For example, a signal may be quantified by its intensity, wavelength, energy, frequency, power, luminance, or a combination thereof. Other signals can be quantified according to characteristics such as voltage, current, electric field strength, magnetic field strength, frequency, power, temperature, etc. Absence of signal is understood to be a signal level of zero or a signal level that is not meaningfully distinguished from noise. [0144] The term “xy coordinates” refers to information that specifies location, size, shape, and/or orientation in an xy plane. The information can be, for example, numerical coordinates in a Cartesian system. The coordinates can be provided relative to one or both of the x and y axes or can be provided relative to another location in the xy plane (e.g., a fiducial). The term “xy plane” refers to a 2 dimensional area defined by straight line axes x and y. When used in reference to a detecting apparatus and an object observed by the detector, the xy plane may be specified as being orthogonal to the direction of observation between the detector and object being detected. [0145] As used herein, “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)). [0146] As used herein, “capable of hybridizing” is used in accordance with its ordinary meaning in the art and refers to two oligonucleotides that, under suitable conditions, can form a duplex (e.g., Watson-Crick pairing) which includes a double-stranded portion of nucleic acid. Such conditions, known in the art and described herein, depend upon, for example, the nature of the nucleotide sequence, temperature, and buffer conditions. The stringency of hybridization can be influenced by various parameters, including degree of identity and/or complementarity between the polynucleotides (or any target sequences within the polynucleotides) to be hybridized; melting point of the polynucleotides and/or target sequences to be hybridized, referred to as “Tm”; parameters such as salts, buffers, pH, temperature, GC % content of the Attorney Docket No.: 051385-585001WO polynucleotide and primers, and/or time. Typically, hybridization is favored in lower temperatures and/or increased salt concentrations, as well as reduced concentrations of organic solvents. Some exemplary conditions suitable for hybridization include incubation of the polynucleotides to be hybridized in solutions having sodium salts, such as NaCl, sodium citrate and/or sodium phosphate. In some embodiments, hybridization or wash solutions can include about 10-75% formamide and/or about 0.01- 0.7% sodium dodecyl sulfate (SDS). In some embodiments, a hybridization solution can be a stringent hybridization solution which can include any combination of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt’s solution, 0.1% SDS, and/or 10% dextran sulfate. In some embodiments, the hybridization or washing solution can include BSA (bovine serum albumin). In some embodiments, hybridization or washing can be conducted at a temperature range of about 20-25 °C, or about 25-30 °C, or about 30-35 °C, or about 35-40 °C, or about 40-45 °C, or about 45-50 °C, or about 50-55 °C, or higher. In some embodiments, hybridization or washing can be conducted for a time range of about 1-10 minutes, or about 10-20 minutes, or about 20-30 minutes, or about 30-40 minutes, or about 40-50 minutes, or about 50-60 minutes, or longer. In some embodiments, hybridization or wash conditions can be conducted at a pH range of about 5-10, or about pH 6-9, or about pH 6.5-8, or about pH 6.5-7. [0147] As used herein, the term “upstream” refers to a region in the nucleic acid sequence that is towards the 5’ end of a particular reference point, and the term “downstream” refers to a region in the nucleic acid sequence that is toward the 3’ end of the reference point. [0148] As used herein, the term “feature” refers a site (i.e., a physical location) on a solid support for one or more molecule(s). A feature can contain only a single molecule or it can contain a population of several molecules of the same species (i.e., a cluster). Features of an array are typically discrete. The discrete features can be contiguous, or they can have spaces between each other. An “optically resolvable feature” refers to a feature capable of being distinguished from other features. Optics and sensor resolution has a finite limit as to a resolvable area. The Rayleigh criterion for the diffraction limit to resolution states that two images are just resolvable when the center of the diffraction pattern of one object is directly over the first minimum of the diffraction pattern of the other object. The minimal distance between two resolvable objects, r, is proportional to the wavelength of light and inversely proportional to Attorney Docket No.: 051385-585001WO the numerical aperture (NA). That is, the minimal distance between two resolvable objects is provided as r = 0.61 wavelength / NA. If detecting light in the UV-vis spectrum (about 100 nm to about 900 nm), the remaining mutable variable to increase the resolution is the NA of the objective lens. A lens with a large NA will be able to resolve finer details. For example, a lens with larger NA is capable of detecting more light and so it produces a brighter image. Thus, a large NA lens provides more information to form a clear image, and so its resolving power will be higher. Typical dry objectives have an NA of about 0.80 to about 0.95. Higher NAs may be obtained by increasing the imaging medium refractive index between the object and the objective front lens for example immersing the lens in water (refractive index = 1.33), glycerin (refractive index = 1.47), or immersion oil (refractive index = 1.51). Most oil immersion objectives have a maximum numerical aperture of 1.4, with the typical objectives having an NA ranging from 1.0 to 1.35. [0149] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. II. Compositions, Substrates, & Kits [0150] In an aspect is provided a substrate (e.g., a solid support) including a first polynucleotide attached to the substrate; a second polynucleotide attached to the substrate, wherein the second polynucleotide includes a complementary sequence to the first polynucleotide; and a third polynucleotide (alternatively referred to herein as an invasion primer or extended invasion primer) hybridized to the second polynucleotide. In embodiments, the invasion primer includes a binding sequence (e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof) and a tail sequence (i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof). The binding sequence is also referred to herein as the first region of the third polynucleotide, and the tail sequence is also referred to herein as the second region of the third polynucleotide. In embodiments, the third polynucleotide includes the Attorney Docket No.: 051385-585001WO first region. In embodiments, the third polynucleotide includes the first region and the second region. In embodiments, the third polynucleotide includes, from 5’ to 3’, the tail sequence and the binding sequence (e.g., wherein the binding sequence is downstream from the tail sequence). In embodiments, the third polynucleotide includes three or more regions, wherein the additional regions may or may not be complementary to the first or the second polynucleotide, or a complement thereof. For example, the third polynucleotide may include a third region (e.g., a third region at the 5’ end of the third polynucleotide) that is complementary to a capture oligonucleotide (e.g., an oligonucleotide including a functional group that allows for affinity capture of the third polynucleotide). [0151] In an aspect is provided a composition, the composition including a first invasion strand hybridized to a second strand (e.g., a second polynucleotide) of a double-stranded polynucleotide, wherein the double-stranded polynucleotide includes a first strand (e.g., a first polynucleotide) and the second strand, wherein the first strand and the second strand are both attached to a solid support; and wherein a 5’ end of the first strand is hybridized to a second invasion strand. In embodiments, the first invasion strand includes an invasion primer (e.g., an extended third polynucleotide). In embodiments, the invasion primer includes a binding sequence. In embodiments, the invasion primer includes a binding sequence and a tail sequence (e.g., a 5’ tail sequence). In embodiments, the second invasion strand includes a blocking primer (e.g., an extended third polynucleotide). [0152] In an aspect is provided a cell including a first polynucleotide attached to the cell (e.g., attached to a cellular biomolecule in the cell, such as a cellular compartment, or attached to an exogenous matrix or polymer in the cell); a second polynucleotide attached to the cell, wherein the second polynucleotide includes a complementary sequence to the first polynucleotide; and a third polynucleotide (alternatively referred to herein as an invasion primer or extended invasion primer) hybridized to the second polynucleotide. In embodiments, the invasion primer includes a binding sequence (e.g., a sequence complementary to the first polynucleotide, or complement thereof, or second polynucleotide, or complement thereof) and a 5’ tail sequence (i.e., a sequence that is not complementary to either the first or second polynucleotide, or a complement thereof). The binding sequence is also referred to herein as the first region of the third polynucleotide, and the 5’ tail sequence is also referred to herein as the second region of the third polynucleotide. In Attorney Docket No.: 051385-585001WO embodiments, the third polynucleotide includes the first region. In embodiments, the third polynucleotide includes the first region and the second region. In embodiments, the third polynucleotide includes three or more regions, wherein the additional regions may or may not be complementary to the first or the second polynucleotide, or a complement thereof. For example, the third polynucleotide may include a third region (e.g., a third region at the 5’ end of the third polynucleotide) that is complementary to a capture oligonucleotide (e.g., an oligonucleotide including a functional group that allows for affinity capture of the third polynucleotide). [0153] In embodiments, the cell is attached to a substrate. In embodiments, the cell is attached to the substrate via a bioconjugate reactive moiety. In embodiments, the composition is within a cell or tissue sample. In embodiments, the cell or tissue sample is cleared (e.g., digested) of proteins, lipids, or proteins and lipids. In embodiments, the cell or tissue sample is processed according to a known technique in the art, for example CLARITY (Chung K., et al. Nature 497, 332–337 (2013)), PACT-PARS (Yang Bet al. Cell 158, 945–958 (2014).), CUBIC (Susaki E. A. et al. Cell 157, 726–739 (2014)., 18), ScaleS (Hama H., et al. Nat. Neurosci.18, 1518–1529 (2015)), OPTIClear (Lai H. M., et al. Nat. Commun.9, 1066 (2018)), Ce3D (Li W., et al. Proc. Natl. Acad. Sci. U.S.A.114, E7321–E7330 (2017)), BABB (Dodt H.U. et al. Nat. Methods 4, 331–336 (2007)), iDISCO (Renier N., et al. Cell 159, 896–910 (2014)), uDISCO (Pan C., et al. Nat. Methods 13, 859–867 (2016)), FluoClearBABB (Schwarz M. K., et al. PLOS ONE 10, e0124650 (2015)), Ethanol-ECi (Klingberg A., et al. J. Am. Soc. Nephrol.28, 452–459 (2017)), and PEGASOS (Jing D. et al. Cell Res.28, 803–818 (2018)). [0154] In embodiments, the substrate further includes a plurality of immobilized oligonucleotides (e.g., immobilized primers, such as immobilized forward and immobilized reverse primers) attached to the substrate via a linker. In embodiments, the first and second polynucleotides are covalently attached to the substrate. In embodiments, the 5ʹ end of the first and second polynucleotides contains a functional group that serves to tether the first and second polynucleotides to the substrate (e.g., a bioconjugate linker). Non-limiting examples of covalent attachment include amine-modified polynucleotides reacting with epoxy or isothiocyanate groups on the substrate, succinylated polynucleotides reacting with aminophenyl or aminopropyl functional groups on the substrate, dibenzocycloctyne-modified polynucleotides reacting with azide functional groups on the substrate (or vice versa), trans-cyclooctyne-modified Attorney Docket No.: 051385-585001WO polynucleotides reacting with tetrazine or methyl tetrazine groups on the substrate (or vice versa), disulfide modified polynucleotides reacting with mercapto-functional groups on the substrate, amine-functionalized polynucleotides reacting with carboxylic acid groups on the core via 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) chemistry, thiol- modified polynucleotides attaching to a substrate via a disulfide bond or maleimide linkage, alkyne-modified polynucleotides attaching to a substrate via copper-catalyzed click reactions to azide functional groups on the substrate, and acrydite-modified polynucleotides polymerizing with free acrylic acid monomers on the substrate to form polyacrylamide or reacting with thiol groups on the substrate. In embodiments, the primer is attached to the substrate polymer through electrostatic binding. For example, the negatively charged phosphate backbone of the primer may be bound electrostatically to positively charged monomers in the substrate. In embodiments, the third polynucleotide is not covalently attached to the substrate. [0155] In embodiments, the substrate includes a plurality of first polynucleotides attached to a solid support; a plurality of second polynucleotides attached to a solid support; and a plurality of third polynucleotides hybridized to each of the second polynucleotides. It is understood that when referring to first, second, and third polynucleotides it is in reference to a class of polynucleotide types. For example, the polynucleotides of the first polynucleotides are substantially similar to each other insomuch as they contain substantially identical sequences. [0156] In embodiments, the third polynucleotide, which may also be referred to as the invasion primer and is interchangeable with the third polynucleotide, includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), or combinations thereof. In embodiments, the third polynucleotide includes Bis-locked nucleic acids (bisLNAs). In embodiments, the third polynucleotide includes twisted intercalating nucleic acids (TINAs). In embodiments, the third polynucleotide includes bridged nucleic acids (BNAs). In embodiments, the third polynucleotide includes 2’-O-methyl RNA:DNA chimeric nucleic acids. In embodiments, the third polynucleotide includes minor groove binder (MGB) nucleic acids. In embodiments, the third polynucleotide includes morpholino nucleic acids. Morpholino nucleic acids are synthetic nucleotides that have standard Attorney Docket No.: 051385-585001WO nucleic acid bases (e.g., adenine, guanine, cytosine, and thymine) wherein those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates. Morpholino nucleic acids may be referred to as phosphorodiamidate morpholino oligomers (PMOs). In embodiments, the third polynucleotide includes C5-modified pyrimidine nucleic acids. In embodiments, the third polynucleotide includes peptide nucleic acids (PNAs). In embodiments, the third polynucleotide includes from 5′ to 3′ a plurality of synthetic nucleotides (e.g., LNAs) followed by a plurality (e.g., 2 to 5) canonical or native nucleotides (e.g., dNTPs). In embodiments, the third polynucleotide comprises one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU). In embodiments, the one or more dU nucleobases are at or near the 3’ end of the third polynucleotide (e.g., within 5 nucleotides of the 3’ end). In embodiments, the third polynucleotide includes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followed by a plurality of synthetic nucleotides (e.g., LNAs), and subsequently followed by a plurality (e.g., 2 to 5) of canonical nucleobases. In some embodiments, the third polynucleotide includes a plurality of canonical nucleobases, wherein the canonical nucleobases terminate (i.e., at the 3′ end) with a deoxyuracil nucleobase (dU). [0157] In embodiments, the third polynucleotide includes a plurality of LNAs interspersed throughout the polynucleotide. In embodiments, the third polynucleotide includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the polynucleotide. In embodiments, the entire composition of the third polynucleotide includes less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of LNAs. In embodiments, the entire composition of the third polynucleotide includes up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, or up to about 5% of LNAs. In embodiments, the entire composition of the third polynucleotide includes more than 60%, more than 50%, more than 40%, more than 30%, more than 20%, more than 10%, or more than 5% of LNAs. In embodiments, the entire composition of the third polynucleotide includes about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 60% to about 70% of LNAs. In embodiments, the entire composition of the third polynucleotide includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs. In embodiments, the entire Attorney Docket No.: 051385-585001WO composition of the third polynucleotide includes about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, or up to about 30% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of canonical dNTPs. [0158] In embodiments, the third polynucleotide includes a first region and a second region (e.g., a first region of the polynucleotide that is complementary to a polynucleotide, or complement thereof, and a second region that is not complementary to one or more polynucleotides or complements thereof). In embodiments, the second region of the third polynucleotide is located at a 5’ end of the polynucleotide. In embodiments, the third polynucleotide includes a plurality of LNAs interspersed throughout the first region of the polynucleotide (e.g., the plurality of LNAs are interspersed throughout the region of the third polynucleotide that is complementary to a polynucleotide, or complement thereof, for example, a first or a second polynucleotide or complements thereof, wherein the first and second polynucleotides form a dsDNA complex). In embodiments, the second region of the third polynucleotide does not include LNAs. In embodiments, the third polynucleotide includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the the first region of polynucleotide. In embodiments, the first region of the third polynucleotide includes less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of LNAs. In embodiments, the first region of the third polynucleotide includes up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, or up to about 5% of LNAs. In embodiments, the first region of the third polynucleotide includes more than 60%, more than 50%, more than 40%, more than 30%, more than 20%, more than 10%, or more than 5% of LNAs. In embodiments, the first region of the third polynucleotide includes about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to Attorney Docket No.: 051385-585001WO about 50%, about 50% to about 60%, or about 60% to about 70% of LNAs. In embodiments, the first region of the third polynucleotide includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs. In embodiments, the first region of the third polynucleotide includes about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, or up to about 30% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of canonical dNTPs. [0159] In embodiments, the entire composition of the third polynucleotide includes about 70% of LNAs and about 30% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 65% of LNAs and about 35% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 60% of LNAs and about 40% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 55% of LNAs and about 45% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 50% of LNAs and about 50% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 45% of LNAs and about 55% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 40% of LNAs and about 60% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 35% of LNAs and about 65% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 30% of LNAs and about 70% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 25% of LNAs and about 75% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 20% of LNAs and about 80% of canonical dNTPs. In embodiments, the entire composition of the third Attorney Docket No.: 051385-585001WO polynucleotide includes about 15% of LNAs and about 85% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 10% of LNAs and about 90% of canonical dNTPs. In embodiments, the entire composition of the third polynucleotide includes about 5% of LNAs and about 95% of canonical dNTPs. [0160] In embodiments, the first region of the third polynucleotide includes about 70% of LNAs and about 30% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 65% of LNAs and about 35% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 60% of LNAs and about 40% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 55% of LNAs and about 45% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 50% of LNAs and about 50% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 45% of LNAs and about 55% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 40% of LNAs and about 60% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 35% of LNAs and about 65% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 30% of LNAs and about 70% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 25% of LNAs and about 75% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 20% of LNAs and about 80% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 15% of LNAs and about 85% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 10% of LNAs and about 90% of canonical dNTPs. In embodiments, the first region of the third polynucleotide includes about 5% of LNAs and about 95% of canonical dNTPs. [0161] In embodiments, the third polynucleotide (e.g., the first, second, and/or other region of the third polynucleotide) includes one or more dT nucleobases that are replaced with dU nucleobases. In embodiments, one or both of the first region and the second region of the third polynucleotide includes one or more dT nucleobases that are replaced with dU nucleobases. In embodiments, the third polynucleotide includes a plurality of dT nucleobases that are replaced with dU nucleobases. In embodiments, the third polynucleotide includes all dT nucleobases replaced with dU nucleobases. In embodiments, the third polynucleotide includes dU Attorney Docket No.: 051385-585001WO nucleobases and LNA nucleotides. In embodiments, the third polynucleotide includes dU nucleobases and LNA nucleotides, wherein the LNA nucleotides are not adjacent to the dU nucleobases. [0162] In embodiments, the third polynucleotide (e.g., the first, second, and/or other region of the third polynucleotide) includes a homologous recombination complex including a recombinase bound thereto. In embodiments, the homologous recombination complex further includes a loading factor, a single-stranded binding (SSB) protein, or both. [0163] In embodiments, the substrate includes a silica surface including a polymer coating. In embodiments, the substrate is silica or quartz, such as a microscope slide, having a surface that is uniformly silanized. This may be accomplished using conventional protocols, such as those described in Beattie et al (1995), Molecular Biotechnology, 4: 213. Such a surface is readily treated to permit end-attachment of oligonucleotides (e.g., forward and reverse primers) prior to amplification. In embodiments the substrate surface further includes a polymer coating, which contains functional groups capable of immobilizing primers. In some embodiments, the substrate includes a patterned surface suitable for immobilization of primers in an ordered pattern. A patterned surface refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more of the regions can be features where one or more primers are present. The features can be separated by interstitial regions where capture primers are not present. In some embodiments, the pattern can be an x-y format of features that are in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and/or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and/or interstitial regions. In some embodiments, the primers are randomly distributed upon the substrate. In some embodiments, the primers are distributed on a patterned surface. [0164] In embodiments, the first polynucleotide is immobilized on the substrate via a first linker and the second polynucleotide is immobilized to the substrate via a second linker. The linkers may also include spacer nucleotides. Including spacer nucleotides in the linker puts the polynucleotide in an environment having a greater resemblance to free solution. This can be beneficial, for example, in enzyme-mediated reactions such as sequencing-by-synthesis. It is believed that such reactions suffer less steric hindrance issues that can occur when the polynucleotide is directly attached to the solid support or is attached through a very short linker Attorney Docket No.: 051385-585001WO (e.g., a linker comprising about 1 to 3 carbon atoms). Spacer nucleotides form part of the polynucleotide but do not participate in any reaction carried out on or with the polynucleotide (e.g. a hybridization or amplification reaction). In embodiments, the spacer nucleotides include 1 to 20 nucleotides. In embodiments, the linker includes 10 spacer nucleotides. In embodiments, the linker includes 12 spacer nucleotides. In embodiments, the linker includes 15 spacer nucleotides. It is preferred to use polyT spacers, although other nucleotides and combinations thereof can be used. In embodiments, the linker includes 10, 11, 12, 13, 14, or 15 T spacer nucleotides. In embodiments, the linker includes 12 T spacer nucleotides. Spacer nucleotides are typically included at the 5′ ends of polynucleotides which are attached to a suitable support. Attachment can be achieved via a phosphorothioate present at the 5′ end of the polynucleotide, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, or any other bioconjugate reactive moiety. The linker may be a carbon-containing chain such as those of formula -(CH2)n- wherein “n” is from 1 to about 1000. However, a variety of other linkers may be used so long as the linkers are stable under conditions used in DNA sequencing. In embodiments, the linker includes polyethylene glycol (PEG) having a general formula of -(CH2—CH2—O)m-, wherein m is from about 1 to 500, 1 to 100, or 1 to 12. [0165] In embodiments, the linker, or the immobilized oligonucleotides (e.g., primers) include a cleavable site. In embodiments, the invasion primer (e.g., the first, second, and/or other region of the invasion primer) includes a cleavable site. In embodiments, a cleavable site is a location which allows controlled cleavage of the immobilized polynucleotide strand (e.g., the linker, the primer, or the polynucleotide) by chemical, enzymatic or photochemical means. In embodiments, the cleavable site includes one or more deoxyuracil nucleobases (dUs). [0166] Any suitable enzymatic, chemical, or photochemical cleavage reaction may be used to cleave the cleavable site. The cleavage reaction may result in removal of a part or the whole of the strand being cleaved. Suitable cleavage means include, for example, restriction enzyme digestion, in which case the cleavable site is an appropriate restriction site for the enzyme which directs cleavage of one or both strands of a duplex template; RNase digestion or chemical cleavage of a bond between a deoxyribonucleotide and a ribonucleotide, in which case the cleavable site may include one or more ribonucleotides; chemical reduction of a disulfide linkage with a reducing agent (e.g., THPP or TCEP), in which case the cleavable site should include an Attorney Docket No.: 051385-585001WO appropriate disulfide linkage; chemical cleavage of a diol linkage with periodate, in which case the cleavable site should include a diol linkage; generation of an abasic site and subsequent hydrolysis, etc. In embodiments, the cleavable site is included in the surface immobilized primer (e.g., within the polynucleotide sequence of the primer). In embodiments, the linker, the primer, or the first or second polynucleotide includes a diol linkage which permits cleavage by treatment with periodate (e.g., sodium periodate). It will be appreciated that more than one diol can be included at the cleavable site. One or more diol units may be incorporated into a polynucleotide using standard methods for automated chemical DNA synthesis. Polynucleotide primers including one or more diol linkers can be conveniently prepared by chemical synthesis. The diol linker is cleaved by treatment with any substance which promotes cleavage of the diol (e.g., a diol-cleaving agent). In embodiments, the diol-cleaving agent is periodate, e.g., aqueous sodium periodate (NaIO4). Following treatment with the diol-cleaving agent (e.g., periodate) to cleave the diol, the cleaved product may be treated with a “capping agent” in order to neutralize reactive species generated in the cleavage reaction. Suitable capping agents for this purpose include amines, e.g., ethanolamine or propanolamine. In embodiments, cleavage may be accomplished by using a modified nucleotide as the cleavable site (e.g., uracil, 8oxoG, 5-mC, 5-hmC) that is removed or nicked via a corresponding DNA glycosylase, endonuclease, or combination thereof. [0167] In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) is about 5 to about 25 nucleotides in length. In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) is about 10 to about 40 nucleotides in length. In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) is about 5 to about 100 nucleotides in length. In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) is about 20 to 200 nucleotides in length. In embodiments, each of the plurality of immobilized oligonucleotides (e.g., immobilized primers) about or at least about 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50 or more nucleotides in length. In embodiments, one or more immobilized oligonucleotides include blocking groups at their 3’ ends that prevent polymerase extension. A blocking moiety prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide. In embodiments, the 3’ modification is a 3’-phosphate modification, including a 3’ phosphate moiety, which is removed by a PNK enzyme or a Attorney Docket No.: 051385-585001WO phosphatase enzyme. Alternatively, abasic site cleavage with certain endonucleases (e.g., Endo IV) results in a 3’-OH at the cleavable site from the 3’-diesterase activity. [0168] In embodiments, the immobilized oligonucleotides includes one or more phosphorothioate nucleic acids. In embodiments, the immobilized oligonucleotides includes a plurality of phosphorothioate nucleic acids. In embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100% of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, most of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, all of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, none of the nucleotides in the immobilized oligonucleotides are phosphorothioate nucleic acids. In embodiments, the 5’ end of the immobilized oligonucleotide includes one or more phosphorothioate nucleic acids. In embodiments, the 5’ end of the immobilized oligonucleotide includes between one and five phosphorothioate nucleic acids. [0169] In embodiments, the first and second polynucleotides are each attached to the solid support (i.e., immobilized on the surface of a solid support). The polynucleotide molecules can be fixed to surface by a variety of techniques, including covalent attachment and non-covalent attachment. In embodiments, the polynucleotides are confined to an area of a discrete region (referred to as a cluster). The discrete regions may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. A regular array of such regions is advantageous for detection and data analysis of signals collected from the arrays during an analysis. These discrete regions are separated by interstitial regions. As used herein, the term “interstitial region” refers to an area in a substrate or on a surface that separates other areas of the substrate or surface. For example, an interstitial region can separate one concave feature of an array from another concave feature of the array. The two regions that are separated from each other can be discrete, lacking contact with each other. In another example, an interstitial region can separate a first portion of a feature from a second portion of a feature. In embodiments the interstitial region is continuous whereas the features are discrete, for example, as is the case for an array of wells in an otherwise continuous surface. The separation provided by an interstitial region can be partial or full separation. Interstitial regions will typically have a surface material that differs from the surface material of the features on the Attorney Docket No.: 051385-585001WO surface. For example, features of an array can have polynucleotides that exceeds the amount or concentration present at the interstitial regions. In some embodiments the polynucleotides and/or primers may not be present at the interstitial regions. In embodiments, at least two different primers are attached to the solid support (e.g., a forward and a reverse primer), which facilitates generating multiple amplification products from the first extension product or a complement thereof. [0170] In embodiments of the methods and compositions provided herein, the clusters have a mean or median separation from one another of about 0.5-5 µm. In embodiments, the mean or median separation is about 0.1-10 microns, 0.25-5 microns, 0.5-2 microns, 1 micron, or a number or a range between any two of these values. In embodiments, the mean or median separation is about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 µm or a number or a range between any two of these values. In embodiments, the mean or median separation is about 0.1-10 microns. In embodiments, the mean or median separation is about 0.25-5 microns. In embodiments, the mean or median separation is about 0.5-2 microns. In embodiments, the mean or median separation is about or at least about 0.1 µm. In embodiments, the mean or median separation is about or at least about 0.25 µm. In embodiments, the mean or median separation is about or at least about 0.5 µm. In embodiments, the mean or median separation is about or at least about 1.0 µm. In embodiments, the mean or median separation is about or at least about 2.0 µm. In embodiments, the mean or median separation is about or at least about 5.0 µm. In embodiments, the mean or median separation is about or at least about 10 µm. The mean or median separation may be measured center-to-center (i.e., the center of one cluster to the center of a second cluster). In embodiments of the methods provided herein, the amplicon clusters have a mean or median separation (measured center-to-center) from one another of about 0.5-5 µm. The mean or median separation may be measured edge-to-edge (i.e., the edge of one amplicon cluster to the edge of a second amplicon cluster). In embodiments of the methods provided herein, the amplicon clusters have a mean or median separation (measured edge-to-edge) from one another of about 0.2-5 µm. Attorney Docket No.: 051385-585001WO [0171] In embodiments of the methods provided herein, the amplicon clusters have a mean or median diameter of about 100-2000 nm, or about 200-1000 nm. In embodiments, the mean or median diameter is about 100-3000 nanometers, about 500-2500 nanometers, about 1000-2000 nanometers, or a number or a range between any two of these values. In embodiments, the mean or median diameter is about or at most about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2000 nanometers or a number or a range between any two of these values. In embodiments, the mean or median diameter is about 100-3,000 nanometers. In embodiments, the mean or median diameter is about 100-2,000 nanometers. In embodiments, the mean or median diameter is about 500-2500 nanometers. In embodiments, the mean or median diameter is about 200-1000 nanometers. In embodiments, the mean or median diameter is about 1,000-2,000 nanometers. In embodiments, the mean or median diameter is about or at most about 100 nanometers. In embodiments, the mean or median diameter is about or at most about 200 nanometers. In embodiments, the mean or median diameter is about or at most about 500 nanometers. In embodiments, the mean or median diameter is about or at most about 400 nanometers. In embodiments, the mean or median diameter is about or at most about 500 nanometers. In embodiments, the mean or median diameter is about or at most about 600 nanometers. In embodiments, the mean or median diameter is about or at most about 700 nanometers. In embodiments, the mean or median diameter is about or at most about 1,000 nanometers. In embodiments, the mean or median diameter is about or at most about 2,000 nanometers. In embodiments, the mean or median diameter is about or at most about 2,500 nanometers. In embodiments, the mean or median diameter is about or at most about 3,000 nanometers. [0172] In embodiments of the methods provided herein, each amplicon cluster (e.g., an amplicon cluster having a mean or median diameter of about 100-2000 nm, or about 200-1000 nm) includes about or at least about 100, 500, 1,000, 2,500, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 100 dsDNA molecules. In embodiments, each amplicon cluster includes about 500 dsDNA molecules. In embodiments, each amplicon cluster includes about 1000 dsDNA molecules. In embodiments, each amplicon cluster includes about 500 dsDNA molecules. In embodiments, each amplicon cluster includes about 1,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 2,500 dsDNA molecules. In embodiments, Attorney Docket No.: 051385-585001WO each amplicon cluster includes about 5,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 10,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 20,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 30,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 40,000 dsDNA molecules. In embodiments, each amplicon cluster includes about 50,000 dsDNA molecules. In embodiments, each amplicon cluster includes more than about 50,000 dsDNA molecules. [0173] In embodiments, the substrate is a particle. In embodiments, the substrate is a multiwell container. In embodiments, the substrate is a polymer coated particle or polymer coated planar support. In embodiments, the substrate includes a polymer. In embodiments, the particle includes polymerized units of polyacrylamide (AAm), poly-N-isopropylacrylamide, poly N- isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate, methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N′- bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, glicydyl methacrylate (GMA), hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof. In embodiments, the particle shell includes polymerized units of polyacrylamide (AAm), glicydyl methacrylate (GMA), polyethylene glycol methacrylate (PEGMA), polyethylene glycol methacrylate (PEGMA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof. In embodiments, the particle includes polymerized units of polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate (GMA). In embodiments, the particle includes polymerized units of polyethylene glycol methacrylate (PEGMA) and isocyanatoethyl methacrylate (IEM). In embodiments, the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate, 2-azido-3-hydroxypropyl methacrylate, 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate, Attorney Docket No.: 051385-585001WO 3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or 2-(((2- azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate, 2-azido-3-hydroxypropyl methacrylate, or 2- (((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments, the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate. In embodiments, the particle includes polymerized units of 3-azido-2-hydroxypropyl methacrylate 2-azido-3-hydroxypropyl methacrylate. In embodiments, the particle includes polymerized units of 3-azido-2- hydroxypropyl methacrylate 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. [0174] In an aspect is a kit, wherein the kit includes the substrate as described herein. Generally, the kit includes one or more containers providing a composition and one or more additional reagents (e.g., a buffer suitable for polynucleotide extension). The kit may also include a template nucleic acid (DNA and/or RNA), one or more primer polynucleotides, nucleoside triphosphates (including, e.g., deoxyribonucleotides, ribonucleotides, labeled nucleotides, and/or modified nucleotides), buffers, salts, and/or labels (e.g., fluorophores). [0175] In embodiments, the kit includes a sequencing polymerase, and one or more amplification polymerases. In embodiments, the sequencing polymerase is capable of incorporating modified nucleotides. In embodiments, the polymerase is a DNA polymerase. In embodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNA polymerase, Pol µ DNA polymerase, Pol λ DNA polymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or a thermophilic nucleic acid polymerase (e.g., Therminator γ, 9°N polymerase (exo-), Therminator II, Therminator III, or Therminator IX). In embodiments, the DNA polymerase is a thermophilic nucleic acid polymerase. In embodiments, the DNA polymerase is a modified archaeal DNA polymerase. In embodiments, the polymerase is a reverse transcriptase. In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g., such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO 2020/056044, each of which are incorporated herein by reference for all purposes). In embodiments, the kit includes a strand-displacing polymerase. In embodiments, the kit includes a strand-displacing polymerase, Attorney Docket No.: 051385-585001WO such as a phi29 polymerase, phi29 mutant polymerase or a thermostable phi29 mutant polymerase. [0176] In embodiments, the kit includes a buffered solution. Typically, the buffered solutions contemplated herein are made from a weak acid and its conjugate base or a weak base and its conjugate acid. For example, sodium acetate and acetic acid are buffer agents that can be used to form an acetate buffer. Other examples of buffer agents that can be used to make buffered solutions include, but are not limited to, Tris, bicine, tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, other buffer agents that can be used in enzyme reactions, hybridization reactions, and detection reactions are known in the art. In embodiments, the buffered solution can include Tris. With respect to the embodiments described herein, the pH of the buffered solution can be modulated to permit any of the described reactions. In some embodiments, the buffered solution can have a pH greater than pH 7.0, greater than pH 7.5, greater than pH 8.0, greater than pH 8.5, greater than pH 9.0, greater than pH 9.5, greater than pH 10, greater than pH 10.5, greater than pH 11.0, or greater than pH 11.5. In other embodiments, the buffered solution can have a pH ranging, for example, from about pH 6 to about pH 9, from about pH 8 to about pH 10, or from about pH 7 to about pH 9. In embodiments, the buffered solution can comprise one or more divalent cations. Examples of divalent cations can include, but are not limited to, Mg2+, Mn2+, Zn2+, and Ca2+. In embodiments, the buffered solution can contain one or more divalent cations at a concentration sufficient to permit hybridization of a nucleic acid. [0177] In an aspect is provided a polynucleotide (e.g., an invasion primer). In embodiments, the polynucleotide includes a plurality of LNA nucleotides; one or more cleavable sites, wherein the one or more cleavable sites partition the invasion primer into two or more regions; and a plurality of native nucleotides. [0178] In an aspect is provided a polynucleotide (e.g., an invasion primer). In embodiments, the polynucleotide includes a plurality of LNA nucleotides; one or more dU nucleobases, wherein the one or more dU nucleobases partition the invasion primer into two or more regions; and a plurality of native nucleotides. [0179] In embodiments, the polynucleotide is 20 to 40 nucleotides in length. In embodiments, the polynucleotide is about 10 to 100 nucleotides in length. In embodiments, the polynucleotide is about 15 to about 75 nucleotides in length. In embodiments, the polynucleotide is about 15 to Attorney Docket No.: 051385-585001WO about 90 nucleotides in length. In embodiments, the polynucleotide is about 30 to about 95 nucleotides in length. In embodiments, the polynucleotide is about 20 to about 80 nucleotides in length. In embodiments, the polynucleotide is about 25 to about 75 nucleotides in length. In embodiments, the polynucleotide is about 15 to about 50 nucleotides in length. In embodiments, the polynucleotide is about 10 to about 20 nucleotides in length. In embodiments, the polynucleotide is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length. In embodiments, the polynucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 nucleotides in length. In embodiments, the polynucleotide is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 nucleotides in length. In embodiments, the polynucleotide is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length. In embodiments, the polynucleotide is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or about 60 nucleotides in length. In embodiments, the polynucleotide is about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 nucleotides in length. In embodiments, the polynucleotide is about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or about 80 nucleotides in length. In embodiments, the polynucleotides is about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 nucleotides in length. In embodiments, the polynucleotide is greater than 30 nucleotides in length. In embodiments, the polynucleotide is greater than 40 nucleotides in length. In embodiments, the polynucleotide is greater than 50 nucleotides in length. In embodiments, the polynucleotide is greater than 60 nucleotides in length. In embodiments, the polynucleotide is greater than 70 nucleotides in length. In embodiments, the polynucleotide is greater than 80 nucleotides in length. In embodiments, the polynucleotide is greater than 90 nucleotides in length. In embodiments, the polynucleotide is no less than 15 nucleotides. In embodiments, the polynucleotide is no less than 20 nucleotides. In embodiments, the polynucleotide is about 15 to about 35 nucleotides in length. In embodiments, the polynucleotide is about 15 to about 90 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 25 to about 90 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 35 to about 90 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 25 to about 35 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 25 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, the polynucleotide is about 30 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, the Attorney Docket No.: 051385-585001WO polynucleotide is 30, 31, 32, or 33 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. [0180] In embodiments, the calculated or predicted melting temperature (Tm) of the polynucleotide is about 70°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the polynucleotide is about 80°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the polynucleotide is about 85°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the polynucleotide is about 85°C to about 90°C. In embodiments, the plurality of LNA nucleotides are interspersed throughout the polynucleotide (e.g., throughout the invasion primer). [0181] In embodiments, the one or more dU nucleobases partition the polynucleotide into two or more regions or sequences of nucleotides (e.g., a first plurality of consecutive nucleotides and a second plurality of consecutive nucleotides are separated by the one or more dU nucleobases). In embodiments, the one or more dU nucleobases partition the polynucleotide into a first binding region and a 5’ tail sequence (e.g., a first binding region or first region complementary to a first polynucleotide, a second polynucleotide, or a complement thereof, and a 5’ tail sequence or second region that is not complementary to the first polynucleotide, second polynucleotide, or a complement thereof). In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 30 nucleotides in length, or about 15 to about 45 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length, or about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each at least about 3, 5, 7, 10, 13, or 15 nucleotides in length. In embodiments, each of the two or more regions of consecutive nucleotides is greater than about 15 nucleotides in length. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 50°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 60°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the Attorney Docket No.: 051385-585001WO two or more regions of consecutive nucleotides is about 50°C to about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 60°C. III. Methods [0182] In an aspect is provided a method of sequencing, the method including: hybridizing an invasion primer including a binding sequence and a tail sequence (e.g., annealing an invasion oligonucleotide to the 3’ end of one strand of a double-stranded polynucleotide, wherein a portion of the invasion oligonucleotide, for example the sequence at the 5’ end of the invasion oligonucleotide, does not anneal to the double-stranded polynucleotide) to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand; extending the second strand along the tail sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in the extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. In embodiments, the method further includes removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand. In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. In embodiments, both the first strand and the second strand are both attached to a solid support (e.g., each strand is attached via their 5’ end). [0183] In embodiments, the method includes nicking and/or cleaving the invasion strand to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the invasion primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand. Attorney Docket No.: 051385-585001WO [0184] In an aspect is provided a method of forming a single-stranded polynucleotide attached to a solid support, the method including: contacting a plurality of double-stranded polynucleotides including a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers include a binding sequence and a tail sequence; hybridizing the binding sequence of one of the invasion primers to one of the second strands; and extending the invasion primer hybridized to the second strand with a polymerase (e.g., Bst large fragment (Bst LF) polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, T4 DNA polymerase, T7 DNA polymerase, or a mutant thereof) to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the invasion primer hybridized to the second strand to generate an extended second strand including a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support. In embodiments, the method further includes sequencing the single-stranded polynucleotide. In embodiments, the method further includes removing the invasion strand and sequencing the second strand. [0185] In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on the second strand, and extending the second invasion primer with a polymerase, thereby generating a second invasion strand hybridized to the second strand. In embodiments, the second invasion primer includes a different tail sequence than the first invasion primer. In embodiments, the second invasion primer includes the same tail sequence as the first invasion primer. In embodiments, the invasion primer includes a first primer binding sequence and a second primer binding sequence, wherein the second primer binding sequence may be referred to as the tail sequence. In embodiments, the invasion primer includes multiple copies of the primer binding sequence. [0186] In embodiments, the solid support includes about 100, 500, 1000, 5000, 10000, or more dsDNA molecules in a 2 µm2 area. In embodiments, the solid support includes about 1,000 to about 10,000 dsDNA molecules in a 2 µm2 area. In embodiments, the solid support includes about 1,000 to about 10,000 dsDNA molecules in a 0.5 µm diameter feature. In embodiments, the solid support includes about 1,000 to about 50,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. In embodiments, the solid support includes about Attorney Docket No.: 051385-585001WO 10,000 to about 50,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. In embodiments, the solid support includes about 20,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. In embodiments, the solid support includes about 30,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameter feature. As used herein, a feature may be a wells, pits, channels, ridges, raised regions, pegs, or posts on a solid support. Each feature includes a colony and refers to a discrete site on a solid support that includes a plurality of immobilized polynucleotides. [0187] In embodiments, removing the invasion strand includes digesting (i.e., cleaving internal phosphodiester bonds of a polynucleotide) all or portions thereof of the invasion strand using an exonuclease enzyme. Exonucleases can be active on ssDNA and/or dsDNA, initiate from the 5′ end and/or the 3′ end of polynucleotides, and can also act on RNA polynucleotides. In embodiments, the exonuclease enzyme is a DNA specific exonuclease. In embodiments, the exonuclease catalyzes the removal of nucleotides from linear, and/or nicked double-stranded DNA in the 5' to 3' direction. In embodiments, removing the invasion strand includes contacting the double-stranded polynucleotide with a denaturant (e.g., NaOH or formamide) or increasing the temperature to denature the double-stranded polynucleotide (e.g., increasing the temperature is increased to greater than 90°C) and washing away the invasion strand. In embodiments, removing include washing (e.g., contacting the solid support with a wash buffer) and flowing the un-attached strands away from the solid support. [0188] In embodiments, when hybridized to the second strand, the first invasion strand blocks and/or prevents, and/or reduces rehybridization of the complementary first strand. In embodiments, the invasion primer is not covalently attached to the solid support. In embodiments, the invasion strand, alternatively referred to herein as the third polynucleotide, is not covalently attached to the solid support. In embodiments, the invasion primer is in solution. [0189] In embodiments, the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing an invasion primer to the second strand, and extending the invasion primer, wherein the invasion primer is not covalently attached to the Attorney Docket No.: 051385-585001WO solid support; and generating a first sequencing read by hybridizing one or more sequencing primers to the first strand, and extending the one or more first sequencing primers. In embodiments, the invasion primer does not hybridize at the end of the strand, rather the invasion primer hybridizes about 5 to about 50 nucleotides from the end of the strand. In embodiments, the invasion primer hybridizes about 10 to about 30 nucleotides, about 12 to about 24, or about 15 to about 30 from the end of the strand. In embodiments, the invasion primer hybridizes towards the 5′ end of the strand. In embodiments, the invasion primer hybridizes towards the 3′ end of the strand. In embodiments, the invasion primer hybridizes at the end of the strand (e.g., the invasion primer hybridizes to the last few nucleotides on the strand). In embodiments, a 5’ region (e.g., a tail sequence) of the invasion primer does not hybridize to the end of the strand (e.g., the invasion primer includes a 5’ tail sequence that does not hybridize). [0190] In embodiments, the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing an invasion primer (e.g., a first invasion primer) to the second strand, and extending the first invasion primer (e.g., extending the first invasion primers with a polymerase under strand-displacing conditions); and generating a first sequencing read by hybridizing one or more first sequencing primers to the first strand, and extending the one or more first sequencing primers. In embodiments, when hybridized to the second strand, the first invasion strand blocks and/or prevents rehybridization of the complementary first strand. In embodiments, the first invasion primer is not covalently attached to the solid support. In embodiments, the invasion strand is not covalently attached to the solid support. In embodiments, the invasion strand includes substantially the same sequence as the first strand. [0191] In embodiments, each invasion primer of the one or more invasions primers is complementary to the same sequence (e.g., the same sequence in the first strand or the same sequence in the second strand). In embodiments, each invasion primer of the one or more invasion primers is not complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand). In embodiments, each invasion primer Attorney Docket No.: 051385-585001WO of the one or more invasion primers is complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand). In embodiments, one or more invasions primers is complementary to the same sequence (e.g., the same sequence in the first strand or the same sequence in the second strand). In embodiments, one or more invasion primers is not complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand). In embodiments, one or more invasion primers is complementary to a different sequence (e.g., a different sequence in the first strand or a different sequence in the second strand). [0192] In embodiments, the first strand is covalently attached to the solid support via a first linker and the second strand is covalently attached to the solid support via a second linker. The linker tethering the polynucleotide strands may be any linker capable of localizing nucleic acids to arrays. The linkers may be the same, or the linkers may be different. Solid-supported molecular arrays have been generated previously in a variety of ways, for example, the attachment of biomolecules (e.g., proteins and nucleic acids) to a variety of substrates (e.g., glass, plastics, or metals) underpins modern microarray and biosensor technologies employed for genotyping, gene expression analysis and biological detection. Silica-based substrates are often employed as supports on which molecular arrays are constructed, and functionalized silanes are commonly used to modify glass to permit a click-chemistry enabled linker to tether the biomolecule (e.g., polynucleotide strand). [0193] In embodiments, the method further includes generating a second invasion strand hybridized to the first strand by hybridizing one or more second invasion primers to the first strand, and extending the one or more second invasion primers; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers. In embodiments, the second invasion strand is not covalently attached to the solid support. In embodiments, the method further includes removing the first invasion strand; generating a second invasion strand hybridized to the first strand by hybridizing one or more invasion primers to the first strand, and extending the one or more second invasion primers; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers. In embodiments, the method further includes generating a second invasion Attorney Docket No.: 051385-585001WO strand hybridized to the first strand by hybridizing a second invasion primer to the first strand, and extending the second invasion primer; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers. In embodiments, the second invasion strand is not covalently attached to the solid support. In embodiments, the method further includes removing the first invasion strand; generating a second invasion strand hybridized to the first strand by hybridizing a second invasion primer to the first strand, and extending the second invasion primers; and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand, and extending the one or more second sequencing primers. In embodiments, the method includes sequencing both strands (i.e., the first and the second strand) of the sample double-stranded amplification product. In embodiments, the method includes sequencing both strands (i.e., the first and the second strand) of the template polynucleotide. [0194] In embodiments, the double-stranded amplification product includes common sequences at their 5′ and 3′ ends. In this context the term “common” is interpreted as meaning common to all templates in the library, such as a synthetic amplification primer binding sequence. For example, the double-stranded amplification product may include a first adapter sequence at the 5′ end and a second adapter sequence at the 3′ end. Typically, the first adapter sequence and the second adapter sequence will consist of no more than 100, or no more than 50, or no more than 40 consecutive nucleotides at the 5′ and 3′ ends, respectively, of each strand of each template polynucleotide. The precise length of the two sequences may or may not be identical. The precise sequences of the common regions are generally not material to the invention and may be selected by the user. The common sequences must at least include primer- binding sequences (i.e., regions of complementarity for a primer) which enable specific annealing of primers when the template polynucleotides are in used in a solid-phase amplification reaction. The primer-binding sequences are thus determined by the sequence of the primers to be ultimately used for solid-phase amplification. [0195] In embodiments, generating the invasion strand (i.e., generating the first invasion strand or the second invasion strand) includes hybridizing one or more primers to a common sequence in the double-stranded amplification product. In embodiments, generating the invasion strand (i.e., generating the first invasion strand or the second invasion strand) includes hybridizing one Attorney Docket No.: 051385-585001WO primer to a common sequence in the double-stranded amplification product. In embodiments, generating the invasion strand (i.e., generating the first invasion strand or the second invasion strand) includes hybridizing a primer to at or near the 3′ end of the double-stranded amplification product. In embodiments, generating the invasion strand (i.e., generating the first invasion strand or the second invasion strand) includes hybridizing a primer to at or near the 3′ end of the double-stranded amplification product, wherein the primer is not covalently attached to the solid support (e.g., the primer is in solution prior to hybridization). In embodiments, the invasion primer does not hybridize at the terminus of the strand, rather the invasion primer hybridizes about 10, about 20, about 30, or about 50 nucleotides from the terminus of the strand. In embodiments, the invasion primer hybridizes about 10 to about 30 nucleotides from the terminus of the strand. In embodiments, the invasion primer hybridizes to a common sequence (e.g., a sequence or the complement thereof as described in U.S. Patent Publication 2016/0256846, which is incorporated herein by reference, for example SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 11, or complements thereof, of U.S. Patent Publication 2016/0256846). In embodiments, the invasion primer includes a tail sequence (e.g., a 5’ tail sequence) that does not hybridize to a common sequence. In embodiments, the tail sequence is complementary to a second primer (e.g., a second invasion primer). [0196] In embodiments, the method further includes removing the first strand by cleaving the first strand at a cleavable site, washing away the cleaved strand, and generating a second sequencing read by hybridizing one or more second sequencing primers to the second strand; and extending the one or more second sequencing primers. In embodiments, removing the first strand is optional. The one or more cleavable sites may include a modified nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleavage agent. The cleavable site(s) may be deoxyuracil triphosphate (dUTP), deoxy-8-Oxo- guanine triphosphate (d-8-oxoG), or other modified nucleotide(s), such as those described, for example, in US 2012/0238738, which is incorporated herein by reference for all purposes. In embodiments, the cleavable site includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent. In embodiments, the cleavable site includes one or more ribonucleotides. In embodiments, the cleavable site includes 2 to 5 Attorney Docket No.: 051385-585001WO ribonucleotides. In embodiments, the cleavable site includes one ribonucleotide. In embodiments, the cleavable sites can be cleaved at or near a modified nucleotide or bond by enzymes or chemical reagents, collectively referred to here and in the claims as “cleaving agents.” Examples of cleaving agents include DNA repair enzymes, glycosylases, DNA cleaving endonucleases, or ribonucleases. For example, cleavage at dUTP may be achieved using uracil DNA glycosylase and endonuclease VIII (USER™, NEB, Ipswich, Mass.), as described in U.S. Pat. No.7,435,572. In embodiments, when the modified nucleotide is a ribonucleotide, the cleavable site can be cleaved with an endoribonuclease. In embodiments, cleaving an extension product includes contacting the cleavable site with a cleaving agent, wherein the cleaving agent includes a reducing agent, sodium periodate, RNase, formamidopyrimidine DNA glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase (UDG). In embodiments, the cleaving agent is an endonuclease enzyme such as nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Micrococcal nuclease, Endonuclease II (endo VI, exo III), nuclease BAL-31 or mung bean nuclease. In embodiments, the cleaving agent includes a restriction endonuclease, including, for example a type IIS restriction endonuclease. In embodiments, the cleaving agent is an exonuclease (e.g., RecBCD), restriction nuclease, endoribonuclease, exoribonuclease, or RNase (e.g., RNAse I, II, or III). In embodiments, the cleaving agent is a restriction enzyme. In embodiments, the cleaving agent includes a glycosylase and one or more suitable endonucleases. In embodiments, cleavage is performed under alkaline (e.g., p/’H greater than 8) buffer conditions at between 40°C to 80°C (e.g., 65°C). In embodiments, cleaving includes contacting the cleavable site with a Uracil DNA glycosylase (UDG) enzyme to catalyze the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact, and contacting the abasic site with an endonuclease VIII enzyme to cleave the phosphodiester bond. [0197] In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on the second strand, and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. In embodiments, the second invasion primer does not include a tail sequence. Attorney Docket No.: 051385-585001WO [0198] In embodiments, prior to generating a first invasion strand, the method includes removing immobilized primers that do not contain a first or second strand (i.e., unused primers). Methods of removing immobilized primers can include digestion using an enzyme with exonuclease activity. Removing unused primers may serve to increase the free volume and allow for greater accessibility of the invasion primer. Removal of unused primers may also prevent opportunities for the newly released first strand to rehybridize to an available surface primer, producing a priming site off the available surface primer, thereby facilitating the “reblocking” of the released first strand. In embodiments, prior to generating a first invasion strand, the method includes contacting the immobilized primers with an exonuclease enzyme. [0199] In embodiments, prior to generating a first invasion strand, the method includes blocking the immobilized primers that do not include a first or second strand. In embodiments, the immobilized oligonucleotides include blocking groups at their 3’ ends that prevent polymerase extension. A blocking moiety prevents formation of a covalent bond between the 3' hydroxyl moiety of the nucleotide and the 5' phosphate of another nucleotide. In embodiments, prior to generating a first invasion strand the method includes incubating the amplification products with dideoxynucleotide triphosphates (ddNTPs) to block the 3′-OH of the immobilized oligonucleotides from future extension. In embodiments, prior to generating a first invasion strand, the method includes incorporating a dideoxynucleotide triphosphate (ddNTP) into an immobilized primer. In embodiments, prior to generating a first invasion strand, the method includes contacting the immobilized primer with a polymerase. In embodiments, during generation of a first invasion strand, the method includes contacting the immobilized primer with a polymerase buffer (e.g., incubating the solid support with a buffered solution including a polymerase). [0200] In embodiments, the first strand is cleaved after generating the first sequencing read but before generating the second sequencing read. In embodiments, the first strand is not cleaved after generating the first sequencing read. Cleaving one strand of the double-stranded amplification product may be referred to as linearization. Suitable methods for linearization are known, and described in more detail in application number U.S. Patent Publication 2009/0118128, which is incorporated herein by reference in its entirety. For example, the first strand may be cleaved by exposing the first strand to a mixture containing a glycosylase and one Attorney Docket No.: 051385-585001WO or more suitable endonucleases. In embodiments, the first strand is attached to the surface in a way that allows for selective removal. If the first template strand is removed from the surface, and the partially double-stranded amplification product is denatured, for example by treatment with hydroxide or formamide, then the second strand remains immobilized as a linearized single strand. If one of the surface immobilized primers includes a cleavable site such that it can be cleaved from the surface, (e.g., diol linkage) the resulting partially double-stranded amplification product can be made single-stranded using heat, or chemical denaturing agents, or a combination thereof providing conditions to give a single strand containing a primer hybridization site. [0201] Any suitable enzymatic, chemical, or photochemical cleavage reaction may be used to cleave the cleavable site. The cleavage reaction may result in removal of a part or the whole of the strand being cleaved. Suitable cleavage means include, for example, restriction enzyme digestion, in which case the cleavable site is an appropriate restriction site for the enzyme which directs cleavage of one or both strands of a duplex template; RNase digestion or chemical cleavage of a bond between a deoxyribonucleotide and a ribonucleotide, in which case the cleavable site may include one or more ribonucleotides; chemical reduction of a disulfide linkage with a reducing agent (e.g., THPP or TCEP), in which case the cleavable site should include an appropriate disulfide linkage; chemical cleavage of a diol linkage with periodate, in which case the cleavable site should include a diol linkage; generation of an abasic site and subsequent hydrolysis, etc. In embodiments, the cleavable site is included in the surface immobilized primer (e.g., within the polynucleotide sequence of the primer). In embodiments, one strand of the double-stranded amplification product (or the surface immobilized primer) may include a diol linkage which permits cleavage by treatment with periodate (e.g., sodium periodate). It will be appreciated that more than one diol can be included at the cleavable site. One or more diol units may be incorporated into a polynucleotide using standard methods for automated chemical DNA synthesis. Polynucleotide primers including one or more diol linkers can be conveniently prepared by chemical synthesis. The diol linker is cleaved by treatment with any substance which promotes cleavage of the diol (e.g., a diol-cleaving agent). In embodiments, the diol- cleaving agent is periodate, e.g., aqueous sodium periodate (NaIO4). Following treatment with the diol-cleaving agent (e.g., periodate) to cleave the diol, the cleaved product may be treated with a “capping agent” in order to neutralize reactive species generated in the cleavage reaction. Suitable capping agents for this purpose include amines, e.g., ethanolamine or propanolamine. Attorney Docket No.: 051385-585001WO [0202] In embodiments, the cleavable site is not in the immobilized primer sequence (e.g., within the polynucleotide sequence of the primer). In embodiments, the cleavable site is included in the linking moiety responsible for tethering the primer to the substrate. In embodiments, the cleavable site is a cleavable linker (e.g., a disulfide containing linker that cleaves when exposed to a reducing agent). In embodiments, the cleavable site is a diol linker. [0203] In embodiments, the first strand includes at least one cleavable site. In embodiments, the first linker includes at least one cleavable site. In embodiments, the cleavable site includes deoxyuracil triphosphate (dUTP). The enzyme uracil DNA glycosylase (UDG) may then be used to remove dUTP, generating an abasic site on one strand. The polynucleotide strand including the abasic site may then be cleaved at the abasic site by treatment with endonuclease (e.g EndoIV endonuclease, AP lyase, FPG glycosylase/AP lyase, EndoVIII glycosylase/AP lyase), heat or alkali. In embodiments, the USERTM reagent available from New England Biolabs (NEB catalog #M5508) is used for the creation of a single nucleotide gap at a uracil base in a duplex strand and subsequent cleavage. [0204] In embodiments, the cleavable site includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent. [0205] In embodiments, the cleavable site includes one or more ribonucleotides. In embodiments, the cleavable site includes 2 to 5 ribonucleotides. In embodiments, the cleavable site includes one ribonucleotide. In embodiments, the cleavable site includes more than one ribonucleotide. In embodiments, the cleavable site includes deoxyuracil triphosphate (dUTP) or deoxy-8-oxo-guanine triphosphate (d-8-oxoG). In embodiments, the cleavable site includes two or more deoxyuracil triphosphate (dUTP). In embodiments, the cleavable site includes 2 to 15 dUTPs. In embodiments, the cleavable site includes 2 to 4 dUTPs. [0206] In embodiments, cleaving includes enzymatically cleaving the first strand at the at least one cleavable site (e.g., enzymatically cleaving with an endonuclease). In embodiments, the first strand includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, Attorney Docket No.: 051385-585001WO ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent. [0207] In embodiments, cleaving the first strand includes contacting the cleavable site with a cleaving agent, wherein the cleaving agent includes a reducing agent, sodium periodate, RNase, Formamidopyrimidine DNA Glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase (UDG). In embodiments, the cleaving agent is an endonuclease enzyme such as nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Micrococcal nuclease, Endonuclease II (endo VI, exo III), nuclease BAL-31 or mung bean nuclease. In embodiments, the cleaving agent includes a restriction endonuclease, including, for example a type IIS restriction endonuclease. In embodiments, the cleaving agent is an exonuclease (e.g., RecBCD), restriction nuclease, endoribonuclease, exoribonuclease, or RNase (e.g., RNAse I, II, or III). In embodiments, the cleaving agent is a restriction enzyme. In embodiments, the cleaving agent includes a glycosylase and one or more suitable endonucleases. In embodiments, cleavage is performed under alkaline (e.g., pH greater than 8) buffer conditions at between 40°C to 80°C (e.g., 65°C). [0208] In embodiments, cleaving includes chemically cleaving the first strand at the at least one cleavable site. In embodiments, the first linker includes a diol linker, disulfide linker, photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequence containing a modified or unmodified nucleotide that is specifically recognized by a cleaving agent. [0209] In embodiments, the invasion primer is not covalently attached to the solid support. In embodiments, the invasion primer includes synthetic nucleotides. In embodiments, the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5- modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), or combinations thereof. In embodiments, the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs), or combinations thereof. In embodiments, the invasion primer includes locked nucleic acids (LNAs). In embodiments, the invasion primer includes Bis-locked Attorney Docket No.: 051385-585001WO nucleic acids (bisLNAs). In embodiments, the invasion primer includes twisted intercalating nucleic acids (TINAs). In embodiments, the invasion primer includes bridged nucleic acids (BNAs). In embodiments, the invasion primer includes 2’-O-methyl RNA:DNA chimeric nucleic acids. In embodiments, the invasion primer includes minor groove binder (MGB) nucleic acids. In embodiments, the invasion primer includes morpholino nucleic acids. In embodiments, the invasion primer includes C5-modified pyrimidine nucleic acids. In embodiments, the invasion primer includes peptide nucleic acids (PNAs). In embodiments, the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs), or combinations thereof. In embodiments, the binding sequence of the invasion primer includes synthetic nucleotides (e.g., one or more LNAs), and the tail sequence includes native nucleotides. [0210] In embodiments, the invasion primer includes locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof. In embodiments, the invasion primer includes phosphorothioate nucleic acids. In embodiments, the invasion primer includes one or more locked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalized oligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In embodiments, the invasion primer includes one or more locked nucleic acids (LNAs). In embodiments, the invasion primer includes one or more 2-amino-deoxyadenosine (2- amino-dA). In embodiments, the invasion primer includes one or more trimethoxystilbene- functionalized oligonucleotides (TFOs). In embodiments, the invasion primer includes one or more Pyrene-functionalized oligonucleotides (PFOs). In embodiments, the invasion primer includes one or more peptide nucleic acids (PNAs). In embodiments, the invasion primer includes one or more aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In embodiments, the invasion primer includes 10 to 15 locked nucleic acids (LNAs). In embodiments, the invasion primer includes a sequence described herein, for example within Table 1. In embodiments, the invasion primer includes one or more phosphorothioates at the 5′ end. In embodiments, the invasion primer includes one or more LNAs at the 5′ end. In embodiments, the invasion primer Attorney Docket No.: 051385-585001WO includes two or more consecutive LNAs at the 3′ end. In embodiments, the invasion primer includes two to four consecutive LNAs at the 3′ end. In embodiments, the invasion primer includes two or more consecutive LNAs at the 5′ end. In embodiments, the invasion primer includes two to four consecutive LNAs at the 5′ end. [0211] In embodiments, the invasion primer includes one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 2, 3, 4, 5, or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes a plurality of locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes one locked nucleic acid (LNA) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 2 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 3 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 4 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. In embodiments, the invasion primer includes 5 locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. [0212] In embodiments, the invasion primer includes from 5′ to 3′ a plurality of synthetic nucleotides (e.g., LNAs) followed by a plurality (e.g., 2 to 5) canonical or native nucleotides (e.g., dNTPs). In embodiments, the invasion primer comprises one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU). In embodiments, the one or more dU nucleobases are at or near the 3’ end of the invasion primer (e.g., within 5 nucleotides of the 3’ end). In embodiments, the invasion primer includes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followed by a plurality of synthetic nucleotides (e.g., LNAs), and subsequently followed by a plurality (e.g., 2 to 5) of canonical bases. In some embodiments, the invasion primer includes a plurality of canonical bases, wherein the canonical bases terminate (i.e., at the 3′ end) with a deoxyuracil nucleobase (dU). [0213] In embodiments, the invasion primer includes the sequence provided in Table 1. In embodiments, the first region of the invasion primer (e.g., a first 3’ region of an invasion primer, wherein the invasion primer comprises two or more regions) includes the sequence provided in Table 1. In embodiments, the 5′ end of the sequences provided in Table 1 include one or more Attorney Docket No.: 051385-585001WO phosphorothioate nucleic acids. In embodiments, the binding sequence of the invasion primer includes a sequence provided in Table 1. [0214] Table 1. Invasion primer sequences, from 5’- 3’, wherein the nucleotide contained in brackets indicates an LNA nucleotide. Nucleotide Sequence 5′ to 3′ SEQ ID number
Figure imgf000114_0001
Attorney Docket No.: 051385-585001WO G[T]G[A]C[T]G[G]AG[T]TC[A]GACG[T]GTGC[T]C[T]TCCG[A]TCT SEQ ID NO:10
Figure imgf000115_0001
Attorney Docket No.: 051385-585001WO AG[T]TC[A]GACG[T]GTGC[T]C[T]TCCG[A][T][C] SEQ ID NO:22
Figure imgf000116_0001
Attorney Docket No.: 051385-585001WO AATGATACGGCGACCACCG SEQ ID NO:34
Figure imgf000117_0001
Attorney Docket No.: 051385-585001WO AA[T]GA[T]ACGGC[G]ACCAC[C][G] SEQ ID NO:46
Figure imgf000118_0001
Attorney Docket No.: 051385-585001WO ATCTC[G]TA[T]GC[C]GT[C]TTC[T]GC[T]T[G] SEQ ID NO:58
Figure imgf000119_0001
[0215] In embodiments, the invasion primer includes one or more morpholino nucleic acids. Morpholino nucleic acids are synthetic nucleotides that have standard nucleic acid bases (e.g., adenine, guanine, cytosine, and thymine) wherein those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates. Morpholino nucleic acids may be referred to as phosphorodiamidate morpholino oligomers (PMOs). [0216] In embodiments, the invasion primer includes locked nucleic acids (LNAs). In embodiments, the invasion primer includes LNAs dispersed throughout the primer, wherein about 2 to 5 nucleotides on the 3′ end are canonical dNTPs. In embodiments, the entire composition of the invasion primer includes less than 50%, less than 40%, or less than 30% of LNAs. [0217] In embodiments, the invasion primer includes peptide nucleic acids (PNAs). A PNA is a synthetic nucleic acid analogue wherein the nucleobases are arrayed along a neutral N-(2- aminoethyl)-glycine backbone in place of the negatively charged phosphate backbone of canonical DNA. The unique pseudopeptide backbone is considered to be responsible for dramatically altering the interactions of nucleic acids and proteins with PNA. For example resulting in increased thermostability of PNA hybridization with DNA. It is known that PNA hybridization demonstrates a negative salt dependence wherein lower ionic strength results in increased duplex stability (see, for example, De Costa N. T. S. Heemstra J. M. PLoS One.2013;8:e58670. In embodiments, the invasion primer includes one or more PNAs and anneals to the dsDNA (e.g., the second strand) in a buffer containing less than 200nM NaCl, less than about 100 nM NaCl, or less than about 50 nM NaCl. [0218] In embodiments, the invasion primer includes a plurality of LNAs interspersed throughout the polynucleotide. In embodiments, the invasion primer includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the Attorney Docket No.: 051385-585001WO polynucleotide. In embodiments, the entire composition of the invasion primer includes less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of LNAs. In embodiments, the entire composition of the invasion primer includes up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, or up to about 5% of LNAs. In embodiments, the entire composition of the invasion primer includes more than 60%, more than 50%, more than 40%, more than 30%, more than 20%, more than 10%, or more than 5% of LNAs. In embodiments, the entire composition of the invasion primer includes about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 60% to about 70% of LNAs. In embodiments, the entire composition of the invasion primer includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs. In embodiments, the entire composition of the invasion primer includes about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of canonical dNTPs. In embodiments, the entire composition of the invasion primer includes less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30% of canonical dNTPs. In embodiments, the entire composition of the invasion primer includes up to about 95%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, or up to about 30% of canonical dNTPs. In embodiments, the entire composition of the invasion primer includes more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30% of canonical dNTPs. [0219] In embodiments, the invasion primer includes about 70% of LNAs and about 30% of canonical dNTPs. In embodiments, the invasion primer includes about 65% of LNAs and about 35% of canonical dNTPs. In embodiments, the invasion primer includes about 60% of LNAs and about 40% of canonical dNTPs. In embodiments, the invasion primer includes about 55% of LNAs and about 45% of canonical dNTPs. In embodiments, the invasion primer includes about 50% of LNAs and about 50% of canonical dNTPs. In embodiments, the invasion primer includes about 45% of LNAs and about 55% of canonical dNTPs. In embodiments, the invasion primer includes about 40% of LNAs and about 60% of canonical dNTPs. In embodiments, the invasion primer includes about 35% of LNAs and about 65% of canonical dNTPs. In embodiments, the Attorney Docket No.: 051385-585001WO invasion primer includes about 30% of LNAs and about 70% of canonical dNTPs. In embodiments, the invasion primer includes about 25% of LNAs and about 75% of canonical dNTPs. In embodiments, the invasion primer includes about 20% of LNAs and about 80% of canonical dNTPs. In embodiments, the invasion primer includes about 15% of LNAs and about 85% of canonical dNTPs. In embodiments, the invasion primer includes about 10% of LNAs and about 90% of canonical dNTPs. In embodiments, the invasion primer includes about 5% of LNAs and about 95% of canonical dNTPs. [0220] In embodiments, the invasion primer includes one or more dT nucleobases that are replaced with dU nucleobases. In embodiments, the invasion primer includes a plurality of dT nucleobases that are replaced with dU nucleobases. In embodiments, the invasion primer includes all dT nucleobases replaced with dU nucleobases. In embodiments, the one or more dU nucleobases partition the invasion primer into two or more regions of consecutive nucleotides (e.g., a first plurality of consecutive nucleotides and a second plurality of consecutive nucleotides are separated by the one or more dU nucleobases). In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length, or about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 10 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each about 3 to about 15 nucleotides in length. In embodiments each of the two or more regions of consecutive nucleotides are each at least about 3, 5, 7, 10, 13, or 15 nucleotides in length. In embodiments, each of the two or more regions of consecutive nucleotides is greater than about 15 nucleotides in length. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 50°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 60°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is about 50°C to about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less than about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of each of the two or more regions of consecutive nucleotides is less Attorney Docket No.: 051385-585001WO than about 60°C. In embodiments, the dU and the LNA nucleotides are not adjacent to each other. In embodiments, the dU and the LNA nucleotides are separated by one or more native nucleotides. [0221] In embodiments, the invasion primer is about 10 to 100 nucleotides in length. In embodiments, the invasion primer is about 15 to about 90 nucleotides in length. In embodiments, the invasion primer is about 15 to about 75 nucleotides in length. In embodiments, the invasion primer is about 25 to about 75 nucleotides in length. In embodiments, the invasion primer is about 15 to about 50 nucleotides in length. In embodiments, the invasion primer is about 10 to about 20 nucleotides in length. In embodiments, the invasion primer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length. In embodiments, the invasion primer is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 nucleotides in length. In embodiments, the invasion primer is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 nucleotides in length. In embodiments, the invasion primer is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length. In embodiments, the invasion primer is greater than 30 nucleotides in length. In embodiments, the invasion primer is greater than 40 nucleotides in length. In embodiments, the invasion primer is greater than 50 nucleotides in length. In embodiments, the invasion primer is no less than 20 nucleotides. In embodiments, the invasion primer is about 15 to about 35 nucleotides in length. In embodiments, the invasion primer is about 25 to about 35 nucleotides, wherein 12 to 18 nucleotides are LNA nucleotides. In embodiments, the invasion primer is about 25 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, the invasion primer is about 30 to about 35 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, the invasion primer is 30, 31, 32, or 33 nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. [0222] In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 70°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 80°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 85°C to about 95°C. In embodiments, the calculated or predicted melting temperature (Tm) of the invasion primer is about 85°C to about 90°C. Attorney Docket No.: 051385-585001WO [0223] In an aspect is provided a method of incorporating a sequence, the method including: hybridizing an invasion primer including a first sequence (e.g., a primer binding sequence) and a second sequence (e.g., a tail sequence) to a second strand of a double-stranded polynucleotide and extending the first sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand (e.g., partially hybridized), wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the second sequence of the invasion primer to generate an extended second strand including a complement of the second sequence, thereby incorporating a sequence (i.e., the complement of the second sequence) into the second strand of the double-stranded polynucleotide. In embodiments, the invasion primer hybridizes to a 3’ end of the second strand. [0224] In embodiments, the polymerase extends the first sequence and the second strand simultaneously. In embodiments, the same polymerase (e.g., Bst polymerase) extends the first sequence and the second strand. In embodiments, the method includes contacting the double stranded polynucleotide with amplification reagents (e.g., nucleotides and an enzyme). [0225] In embodiments, the tail sequence includes a barcode sequence. In some embodiments, a barcode includes both a UMI and a spatial barcode. In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode (i.e., non-identifying) sequences. [0226] In embodiments, the binding sequence includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and the tail sequence includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In embodiments, the binding sequence includes 10 to 25 nucleotides and the tail sequence comprises 5 to 25 nucleotides. In embodiments, the binding sequence is greater than the tail sequence (e.g., the binding sequence include more nucleotides than the tail sequence). [0227] In embodiments, the tail sequence includes a capture sequence, wherein the capture sequence is capable of hybridizing to a target polynucleotide. In embodiments, the capture sequence includes a sequence capable of hybridizing to an endogenous region of a target polynucleotide. In embodiments, the tail sequence is 10 to 25 nucleotides. In embodiments, the Attorney Docket No.: 051385-585001WO tail sequence is downstream of the primer binding sequence. In embodiments, the tail sequence is at the 5’ end of the invasion primer. In embodiments, the tail sequence includes 10 to 30 nucleotides. In embodiments, the tail sequence includes 2 to 20 nucleotides. In embodiments, the tail sequence includes 5 to 10 nucleotides. In embodiments, the tail sequence includes an index sequence and a barcode sequence. [0228] As used herein, “endogenous” is used in accordance with its ordinary meaning in the art and refers to an internal origin. For example, an endogenous gene sequence (also referred to herein as an endogenous region) is a polynucleotide sequence found within the original polynucleotide sequence. In embodiments, an endogenous gene sequence is a polynucleotide sequence found within the original polynucleotide sequence in a biological sample. In embodiments, the endogenous region includes a gene or a fragment thereof. In embodiments, the first endogenous region includes a gene or a fragment thereof. In embodiments, the second endogenous region includes a gene or a fragment thereof. In embodiments, both the first endogenous region and the second endogenous region include a gene or a fragment thereof. In embodiments, the endogenous region includes mRNA. In embodiments, the endogenous region includes genomic DNA (e.g., exons, single nucleotide polymorphisms, mutable regions and/or highly conserved regions). In embodiments, the endogenous region includes a genetic locus. In embodiments, the endogenous region includes autosomal DNA and/or mitochondrial DNA. In embodiments, the endogenous region includes a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, or rRNA. In embodiments, an endogenous region may refer to the post hoc ligation (i.e., ligation after the fragmentation) of an exogenous sequence to one of the ends of the target polynucleotide. In embodiments, an endogenous region of a target polynucleotide is a sequence of the target polynucleotide present prior to fragmentation. In embodiments, the endogenous region of a target polynucleotide is not an adapter sequence. In embodiments, the endogenous region of a target polynucleotide does not include a universal priming binding sequence. In embodiments, the endogenous region does not include the P5, P7, or complementary sequences thereof (i.e., P5’ or P7’). The P5 and P7 primers are used on the surface of commercial flow cells for sequencing on various Illumina platforms. The P5 and P7 adapter sequences are described in U.S. Patent Publication No.2011/0059865 A1, which is incorporated herein by reference in its entirety. The terms P5 and P7 may be used when referring to amplification primers, e.g., universal primers. The terms P5’ (P5 prime) and P7’ (P7 Attorney Docket No.: 051385-585001WO prime) refer to the complement of P5 and P7, respectively. In embodiments, an endogenous sequence is not a synthetic sequence. In embodiments, an endogenous sequence is a sequence found in nature. In embodiments, an endogenous sequence is not a synthetic or engineered sequence. [0229] In embodiments, the tail sequence includes a homopolymer sequence. In embodiments, the homopolymer sequence includes consecutive identical nucleotides (e.g., a 5-mer of C nucleotides). In embodiments, the homopolymer sequence includes 10 to 30 consecutive identical nucleotides. In embodiments, the homopolymer sequence includes 2 to 20 consecutive identical nucleotides. In embodiments, the homopolymer sequence includes 5 to 10 consecutive identical nucleotides. In embodiments, the homopolymer sequence includes poly (dA), poly (dT), poly (dC), poly (dG), or poly (dU) nucleotides. In embodiments, the tail sequence includes a poly(dT) sequence. In embodiments, the poly(dT) sequence includes 10 to 30 dT nucleotides. In embodiments, the poly(dT) sequence includes 2 to 20 dT nucleotides. In embodiments, the poly(dT) sequence includes 5 to 10 dT nucleotides. [0230] In some embodiments, the capture sequence includes nucleotides which are functionally or structurally analogous to poly-T and retain the functional property of binding to poly-A. For example, the capture sequence may include a poly-U oligonucleotide. In some embodiments, the capture sequence is nonspecific (e.g., intended to capture all RNAs containing a poly-A tail). In some embodiments, the capture sequence may further include additional sequences, such as random sequences, to facilitate the capture of specific subtypes of RNA. In some embodiments, the capture sequence may further include additional sequences to capture a desired subtype of RNA, such as mRNA or rRNA. In some embodiments, the capture sequence for each primer is the same. In some embodiments, the capture sequence for one or more probes is different from the capture sequence from at least one other probe. Additional embodiments of capture sequence may be found, for example, in PCT Publication No. WO2022/015913 and U.S. Patent Pub. No.2021/0317524, each of which is incorporated herein by reference in its entirety. [0231] The capture sequence can be based on a particular gene sequence or particular motif sequence or common/conserved sequence, that it is designed to capture (i.e., a sequence-specific capture sequence). Thus, in some embodiments, the capture sequence is capable of binding selectively to a desired sub-type or subset of nucleic acid, for example a particular type of RNA, Attorney Docket No.: 051385-585001WO such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of RNA. In a non-limiting example, the capture sequence can be capable of binding selectively to a desired subset of ribonucleic acids, for example, microbiome RNA, such as 16S rRNA. [0232] In some embodiments, a capture sequence includes an “anchor” or “anchoring sequence”, which is a sequence of nucleotides that is designed to ensure that the capture sequence hybridizes to the intended biological analyte. In some embodiments, an anchor sequence includes a sequence of nucleotides, including a 1-mer, 2-mer, 3-mer or longer sequence. In some embodiments, the short sequence is random. For example, a capture sequence including a poly(T) sequence can be designed to capture an mRNA. In such embodiments, an anchoring sequence can include a random 3-mer (e.g., GGG) that helps ensure that the poly(T) capture sequence hybridizes to an mRNA. In some embodiments, an anchoring sequence can be VN, N, or NN. Alternatively, the sequence can be designed using a specific sequence of nucleotides. In some embodiments, the anchor sequence is at the 3′ end of the capture sequence. In some embodiments, the anchor sequence is at the 5′ end of the capture sequence. [0233] In some embodiments, capture sequences of capture probes are blocked prior to contacting the biological sample with the array, and blocking probes are used when the nucleic acid in the biological sample is modified prior to its capture on the array. In some embodiments, the blocking probe is used to block or modify the free 3′ end of the capture sequence. In some embodiments, blocking probes can be hybridized to the capture probes to mask the free 3′ end of the capture sequence, e.g., hairpin probes or partially double stranded probes. In some embodiments, the free 3′ end of the capture sequence can be blocked by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture probes do not include a free 3′ end. Blocking or modifying the capture probes, particularly at the free 3′ end of the capture sequence, prior to contacting the biological sample with the array, prevents modification of the capture probes, e.g., prevents the addition of a poly(A) tail to the free 3′ end of the capture probes. [0234] Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC), 3′ inverted dT, 3′ C3 spacer, 3′Amino, and 3′ phosphorylation. In some embodiments, the nucleic acid in the Attorney Docket No.: 051385-585001WO biological sample can be modified such that it can be captured by the capture sequence. For example, an adaptor sequence (including a binding domain capable of binding to the capture domain of the capture probe) can be added to the end of the nucleic acid, e.g., fragmented genomic DNA. In some embodiments, this is achieved by ligation of the adaptor sequence or extension of the nucleic acid. In some embodiments, an enzyme is used to incorporate additional nucleotides at the end of the nucleic acid sequence, e.g., a poly(A) tail. In some embodiments, the capture probes can be reversibly masked or modified such that the capture sequence of the capture probe does not include a free 3′ end. In some embodiments, the 3′ end is removed, modified, or made inaccessible so that the capture domain is not susceptible to the process used to modify the nucleic acid of the biological sample, e.g., ligation or extension. [0235] In some embodiments, the capture sequence of the capture probe is modified to allow the removal of any modifications of the capture probe that occur during modification of the nucleic acid molecules of the biological sample. In some embodiments, the capture probes can include an additional sequence downstream of the capture sequence, i.e., 3′ to the capture domain, namely a blocking domain. [0236] In some embodiments, the capture sequence of the capture probe can be a non-nucleic acid domain. Examples of suitable capture sequences that are not exclusively nucleic-acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the functionality of any of the capture sequences described herein. [0237] In embodiments, the invasion primer includes, from 5’ to 3’, the capture sequence, optionally the barcode sequence, and the binding sequence. [0238] In embodiments, the double-stranded polynucleotide includes a cleavage domain (i.e., a polynucleotide sequence including a cleavable site). In embodiments, the double-stranded polynucleotide includes one or more cleavable sites. In embodiments, the first strand of the double-stranded polynucleotide includes a cleavable site. [0239] In embodiments, the double-stranded polynucleotide includes a spatial barcode. A “spatial barcode” is a nucleic acid sequence capable of conveying spatial information (e.g., xy coordinates) upon detection. In embodiments, the barcode is associated with a particular location within an array or a particular location on a substrate. Attorney Docket No.: 051385-585001WO [0240] In embodiments, the double-stranded polynucleotide does not include genomic DNA. [0241] In embodiments, the plurality of target polynucleotides do not include a common sequence (e.g., a sequence universal to a substantial majority of the plurality, such as for example a sequence of an adapter). In embodiments, the target polynucleotides do not include a common sequence (e.g., the same sequence within the plurality). In embodiments, the target polynucleotides do not include a synthetic sequence (e.g., a primer binding sequence). In embodiments, the target polynucleotide does not include a universal primer binding sequence (e.g., a polynucleotide sequence that is common to a majority of the target polynucleotides). In embodiments, the target polynucleotide is a fragmented polynucleotide. In embodiments, the target polynucleotide is genomic DNA (gDNA). In embodiments, the target polynucleotide is genomic DNA (gDNA) including a sequence that encodes for a protein. [0242] In embodiments, the method includes generating a first invasion strand by hybridizing a first invasion primer (e.g., an invasion primer that includes one or more PNAs or LNAs) to the first strand. In embodiments, the invasion primer does not hybridize at the end of the strand, rather the invasion primer hybridizes about 5 to about 50 nucleotides from the end of the strand. In embodiments, the invasion primer hybridizes about 10 to about 30 nucleotides, about 12 to about 24, or about 15 to about 30 from the end of the strand. The first invasion primer creates a “bubble” in the duplex (e.g., as depicted in FIGS.4A-4B). A second invasion primer anneals to the second strand (e.g., within the bubble formed by annealing the first invasion primer) and is extended thereby generating a first invasion strand hybridized to the second strand. The first invasion primer may remain during the first sequencing read, or may be removed prior to starting the first sequencing read. In embodiments, the first invasion primer and the second invasion primer are not covalently attached to the solid support. [0243] In embodiments, generating the invasion strand includes a plurality of invasion primer extension cycles, wherein each invasion primer extension cycle includes incorporating one or more nucleotides into the invasion primer. In embodiments, generating the invasion strand includes extending the invasion primer by incorporating one or more nucleotides (e.g., dNTPs) using Bst large fragment (Bst LF) polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, or a mutant thereof. In embodiments, the polymerase extends by incorporating a nucleotide to the 3’ end of the invasion primer. In Attorney Docket No.: 051385-585001WO embodiments, the polymerase extends by incorporating a nucleotide to the 3’ end of an LNA nucleotide of the invasion primer. [0244] In embodiments, generating the invasion strand includes a plurality of invasion-primer extension cycles by incorporating universal nucleobases (e.g., 5-nitroindole and/or inosine nucleobases) into the invasion primer. The blocking strand does not need to be a faithful representation (i.e., an exact copy) of the strand to which the invasion primer is hybridized. In the interest of speed, in embodiments, one or more inosine nucleotides or “universal” nucleotides may be incorporated into the primer to generate a blocking strand. The term “universal nucleotide,” as used herein, refers to a nucleotide analog that is capable of forming a base pair to two or more (e.g., any of the four) natural nucleotide bases (e.g., cytosine (C), guanine (G), adenine (A), or thymine (T)). Thus, any other base may be paired with a universal base analog in a double-stranded polynucleotide. Universal nucleotides may be divided into hydrogen bonding bases and pi-stacking bases. Hydrogen bonding bases form hydrogen bonds with any of the natural nucleobases. The hydrogen bonds formed by hydrogen bonding bases are weaker than the hydrogen bonds between natural nucleobases. Pi-stacking nucleobases are non-hydrogen bonding, hydrophobic, aromatic bases that stabilize duplex polynucleotides by stacking interactions. Examples of hydrogen bonding bases include, but are not limited to, hypoxanthine (inosine), 7-deazahypoxanthine, 2-azahypoxanthine, 2-hydroxypurine, purine, and 4-Amino-1H- pyrazolo [3,4-d]pyrimidine. IExamples of pi-stacking bases include, but are not limited to, nitroimidazole, indole, benzimidazole, 5-fluoroindole, 5-nitroindole, N-indol-5-yl-formamide, isoquinoline, and methylisoquinoline. Examples of universal bases are discussed in Berger et al., Universal Bases for Hybridization, Replication and Chain Termination, Nucleic Acids Research 2000, August 1, 28(15) pp.2911-2914; David Loakes, The Applications of Universal DNA Base Analogs, 29(12) Nucleic Acids Research 2437 (2001); and Feng Liang et al., Universal base analogs and their applications in DNA sequencing technology, 3 RSC Advances 14910-14928 (2013). In embodiments, the invasion strand includes at least a subset of nucleotides that are not universal nucleotides. In embodiments, at least 1% to 10% of the nucleotides in the invasion strand are universal nucleotides. In embodiments, at least 50% of the nucleotides in the invasion strand are not universal nucleotides. Attorney Docket No.: 051385-585001WO [0245] In embodiments, the blocking strand includes universal nucleobases. In embodiments, the invasion strand is generated using an error-prone polymerase, for example Taq, a Y-family member Dpo4, or others known in the art (e.g., Rattray AJ and Strathern JN. Annu Rev Genet. 2003;37:31-66). In embodiments, the blocking strand is not a copy of the strand the invasion primer is hybridized to. In embodiments, the blocking strand does not replicate the exact sequence of the strand to which the invasion primer is hybridized. [0246] In embodiments, generating the invasion strand includes a first plurality of invasion- primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles. In embodiments, generating the invasion strand includes alternating between a first plurality of invasion-primer extension cycles and a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles. In embodiments, the reaction conditions for the first plurality of invasion-primer extension cycles include higher stringency hybridization conditions relative to the second plurality of invasion-primer extension cycles. [0247] In embodiments, the reaction conditions for the first plurality of invasion-primer extension cycles include incubation in a first denaturant. In embodiments, the first denaturant includes additives such as ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine, or tetramethylammonium chloride (TMAC). In embodiments, the first denaturant is a buffered solution including about 0% to about 50% dimethyl sulfoxide (DMSO); about 0% to about 50% ethylene glycol; about 0% to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof. In embodiments, the reaction conditions for the first plurality of invasion-primer extension cycles include incubation in a first denaturant, wherein the first denaturant is a buffered solution including about 15% to about 50% dimethyl sulfoxide (DMSO); about 15% to about 50% ethylene glycol; about 10% to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof. In embodiments, the temperature is between 50°C and about 75°C, inclusive of the endpoints (i.e., the temperature may be 50°C, 52°C, or 75°C, etc.). In embodiments, the temperature is about 50°C to about 75°C. In embodiments, the temperature is Attorney Docket No.: 051385-585001WO about 55°C to about 70°C. In embodiments, the temperature is about 60°C to about 70°C. In embodiments, the temperature is about 55°C to about 68°C. In embodiments, the buffered solution includes 5×SSC. [0248] In embodiments, the reaction conditions for the second plurality of invasion-primer extension cycles include incubation in a second denaturant. In embodiments, the second denaturant includes additives such as ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine, or tetramethylammonium chloride (TMAC), wherein the concentrations of the additives in the second denaturant differ than the concentrations of the additives in the first denaturant. In embodiments, the second denaturant is a buffered solution including about 0 to about 50% dimethyl sulfoxide (DMSO); about 0 to about 50% ethylene glycol; about 0 to about 20% formamide; or about 0 to about 3M betaine, or a mixture thereof. In embodiments, the reaction conditions for the second plurality of invasion-primer extension cycles include incubation in a second denaturant, wherein the second denaturant is a buffered solution including about 0% to about 15% dimethyl sulfoxide (DMSO); about 0 to about 15% ethylene glycol; about 0 to about 10% formamide; or about 0 to about 3M betaine, or a mixture thereof. In embodiments, the temperature is between 50°C and about 75°C, inclusive of the endpoints (i.e., the temperature may be 50°C, 52°C, or 75°C, etc.). In embodiments, the temperature is about 50°C to about 75°C. In embodiments, the temperature is about 55°C to about 70°C. In embodiments, the temperature is about 60°C to about 70°C. In embodiments, the temperature is about 55°C to about 68°C. In embodiments, the buffered solution includes 5×SSC. [0249] In embodiments, the first denaturant is a buffered solution including dimethyl sulfoxide (DMSO); and the second denaturant is a buffered solution including dimethyl sulfoxide (DMSO) and betaine. In embodiments, the first denaturant is a buffered solution including about 25 to about 35% DMSO; and the second denaturant is a buffered solution including about 0 to about 10% DMSO and about 1M to about 4M betaine. In embodiments, the first denaturant is a buffered solution including about 30% DMSO; and the second denaturant is a buffered solution including about 5% DMSO, about 2.5M betaine. [0250] In embodiments, the reaction conditions for the second plurality of invasion-primer extension cycles further includes incubation with a SSB protein. Attorney Docket No.: 051385-585001WO [0251] In embodiments, generating the invasion strand (e.g., the first invasion strand and/or the second invasion strand) comprises contacting the polynucleotide with one or more invasion- reaction mixtures. In embodiments, generating the invasion strand includes contacting the double-stranded amplification product with one or more invasion-reaction mixtures; each of the invasion-reaction mixture including a plurality of invasion primers, a plurality of deoxyribonucleotide triphosphate (dNTPs), and a polymerase. In embodiments, generating the invasion strand includes contacting the double-stranded amplification product with a first invasion-reaction mixture followed by contacting the double-stranded amplification product with a second invasion-reaction mixture; the first invasion-reaction mixture including a plurality of invasion primers and no polymerase; and the second invasion-reaction mixture includes a plurality of deoxyribonucleotide triphosphate (dNTPs) and a polymerase. In embodiments, the polymerase is a strand-displacing polymerase. In embodiments, the strand-displacing polymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, or a mutant thereof. In embodiments, the polymerase is template dependent. In embodiments, the polymerase is not a TdT polymerase. [0252] In embodiments, the polymerase is a strand-displacing or non-strand displacing polymerase. In embodiments, the polymerase is a strand-displacing polymerase. In embodiments, the strand-displacing polymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Phi29 polymerase, or a mutant thereof. In embodiments, the polymerase is Bst DNA Polymerase, Vent (exo-) DNA Polymerase, Pfu DNA polymerase, Taq polymerase, Phusion High-Fidelity DNA Polymerase, Q5 High-Fidelity DNA Polymerase, or mutant of any one of the foregoing. In embodiments, the polymerase is Bst DNA Polymerase, Vent (exo-) DNA Polymerase, Phusion High-Fidelity DNA Polymerase, or Q5 High-Fidelity DNA Polymerase. In embodiments, the polymerase is a Pyrococcus polymerase (e.g., a polymerase described in WO 2018/148723 or WO 2020/056044, each of which are incorporated herein by reference for all purposes). In embodiments, the polymerase is a Bst DNA polymerase (e.g., exonuclease minus Bst), phi29 DNA polymerase, large fragment of Bsu DNA polymerase, and Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, or Deep Vent DNA polymerase. In embodiments, the polymerase is a phi29 DNA Attorney Docket No.: 051385-585001WO polymerase 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). In embodiments, the polymerase is a non-strand displacing polymerase. In embodiments, the non-strand displacing polymerase is T4 DNA polymerase. In embodiments, the non-strand displacing polymerase is T7 DNA polymerase. [0253] In embodiments, each of the plurality of invasion-reaction mixtures include a plurality of invasion primers, a plurality of deoxyribonucleotide triphosphate (dNTPs), a polymerase, or a combination thereof. In embodiments, each of the plurality of invasion-reaction mixtures include a denaturant, single-stranded DNA binding protein (SSB), or both a denaturant and single- stranded DNA binding protein (SSB). In embodiments, each invasion-reaction mixture further includes a denaturant, single-stranded DNA binding protein (SSB), or a combination thereof. In embodiments, each invasion-reaction mixture includes a different amount of a denaturant, single- stranded DNA binding protein (SSB), or a combination thereof. [0254] In embodiments, the denaturant is a buffered solution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4- methylmorpholine 4-oxide (NMO), TMAC, or a mixture thereof. In embodiments, the denaturant is a buffered solution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, or a mixture thereof. [0255] In embodiments, each invasion-reaction mixture includes a denaturant including an SSB, a strand-displacing polymerase, and one or more crowding agents. In embodiments, the denaturant does not include a chemical denaturant (e.g., betaine, DMSO, ethylene glycol, formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof). In embodiments, the SSB in the denaturant is T4 gp32 protein, SSB protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNA Binding Protein (ET- SSB). In embodiments, the strand-displacing polymerase in the denaturant is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Bsm DNA Polymerase, Phi29 polymerase, or a mutant thereof. In embodiments, the crowding agent in the denaturant is poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or Attorney Docket No.: 051385-585001WO PEG 35,000). In embodiments, PEG is present in the denaturant at a concentration of 1% to 25%. In embodiments, PEG is present in the denaturant at a concentration of about 1%, about 5%, about 10%, about 15%, about 20%, or about 25%. In embodiments, the denaturant is a buffered solution including T4 gp32 protein, Bsu polymerase, and 5 to 10% PEG 20,000. In embodiments, the denaturant is a buffered solution including T4 gp32 protein, Bsu polymerase, and 5% PEG 20,000. In embodiments, the denaturant is a buffered solution including T4 gp32 protein, Bsu polymerase, and 10% PEG 20,000. [0256] In embodiments, the SSB is T4 gp32 protein, SSB protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB). In embodiments, the SSB is active (i.e., has measurable activity) at temperatures less than about 72°C. In embodiments, the SSB is active (i.e., has measurable activity) at temperatures about 72°C. In embodiments, the SSB is active (i.e., has measurable activity) at temperatures greater than about 72°C. [0257] In embodiments, the method further includes contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof. [0258] In embodiments, generating the invasion strand includes (i) forming a complex including a portion of the double-stranded amplification product, an invasion primer, and a homologous recombination complex including a recombinase, (ii) releasing the recombinase, and (iii) in a primer extension reaction, extending the invasion primer with a strand-displacing polymerase. In embodiments, the strand-displacing polymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase, Bsm DNA Polymerase, Phi29 polymerase, or a mutant thereof. In embodiments, the recombinase is a T4 UvsX, RecA, RecT, RecO, or Rad51 protein. [0259] In embodiments, the homologous recombination complex further includes a crowding agent. In embodiments, the crowding agent includes poly(ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), bovine serum albumin (BSA), dextran, Ficoll (e.g., Ficoll 70 or Ficoll 400), glycerol, or a combination thereof. In embodiments, the crowding agent is poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, Attorney Docket No.: 051385-585001WO PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000), dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, β-lactoglobulin, hemoglobin, bovine serum albumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS). In embodiments, the crowding agent is PEG 200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000. In embodiments, the crowding agent is PEG 10,000, PEG 20,000, or PEG 35,000. [0260] In embodiments, the homologous recombination complex further includes a loading factor, a single-stranded binding (SSB) protein, or both. In embodiments, the homologous recombination complex includes a single-stranded binding (SSB) protein. In embodiments, the SSB protein is T4 gp32 protein, SSB protein, Extreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB), T7 gene 2.5 SSB protein, Thermococcus kodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, or phi29 SSB protein. [0261] In embodiments, the homologous recombination complex further includes a loading factor. In embodiments, the loading factor includes a T4 UvsY protein. [0262] In embodiments, generating the invasion strand includes thermally cycling between (i) about 72-80°C for about 5 seconds to about 30 seconds (referred to as cycle 1); and (ii) about 60- 70°C for about 30 to 90 seconds (referred to as cycle 2). In embodiments, the method includes a plurality of thermal cycles in a periodic order (e.g., cycle type 1, cycle 2, cycle 1, etc.). In embodiments, generating the invasion strand includes thermally cycling between (i) about 67- 80°C for about 5 seconds to about 30 seconds (referred to as cycle 1); and (ii) about 60-70°C for about 30 to 90 seconds (referred to as cycle 2). In embodiments, the method includes a plurality of thermal cycles in a periodic order (e.g., cycle type 1, cycle 2, cycle 1, etc.). [0263] In embodiments, one or more invasion primers transiently hybridize to the first or second strand. For example, the denaturing conditions in the invasion-reaction mix may be too stringent for the invasion primer to fully and stably hybridize for a significant time, however if a polymerase is present in the invasion-reaction mixture, the polymerase could still extend the invasion primer. In embodiments, generating the first invasion strand includes transient hybridization of one or more invasion primers to the second strand, and extending the one or more invasion strand during their transient hybridization by a polymerase. In embodiments, the invasion primer partially hybridizes (e.g., less than 100% of the invasion primer hybridizes) to Attorney Docket No.: 051385-585001WO the second strand. In embodiments, the invasion primer hybridizes to the second strand and is extended with a polymerase. In embodiments, the invasion primer does not remain fully annealed to the second strand while the polymerase extends the invasion primer. In embodiments, at least three nucleotides of the invasion primer (e.g., the three nucleotides at the 3′ end of the invasion primer) hybridize to the second strand, and in the presence of a strand displacing polymerase the 3′ end of the invasion primer is extended. In embodiments, about 25% to about 90% of the invasion primer hybridizes to the second strand. In embodiments, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 90% of the invasion primer hybridizes to the second strand. [0264] In embodiments, the strand-displacing enzyme is an SD polymerase, Bst large fragment polymerase, or a phi29 polymerase or mutant thereof. In embodiments, the strand-displacing polymerase is phi29 polymerase, phi29 mutant polymerase or a thermostable phi29 mutant polymerase. A “phi polymerase” (or “Φ29 polymerase”) is a DNA polymerase from the Φ29 phage or from one of the related phages that, like Φ29, contain a terminal protein used in the initiation of DNA replication. For example, phi29 polymerases include the B103, GA-1, PZA, Φ15, BS32, M2Y (also known as M2), Nf, G1, Cp-1, PRD1, PZE, SFS, Cp-5, Cp-7, PR4, PR5, PR722, L17, Φ21, and AV-1 DNA polymerases, as well as chimeras thereof. A phi29 mutant DNA polymerase includes one or more mutations relative to naturally-occurring wild-type phi29 DNA polymerases, for example, one or more mutations that alter interaction with and/or incorporation of nucleotide analogs, increase stability, increase read length, enhance accuracy, increase phototolerance, and/or alter another polymerase property, and can include additional alterations or modifications over the wild-type phi29 DNA polymerase, such as one or more deletions, insertions, and/or fusions of additional peptide or protein sequences. Thermostable phi29 mutant polymerases are known in the art, see for example US 2014/0322759, which is incorporated herein by reference for all purposes. For example, a thermostable phi29 mutant polymerase refers to an isolated bacteriophage phi29 DNA polymerase including at least one mutation selected from the group consisting of M8R, V51A, M97T, L123S, G197D, K209E, E221K, E239G, Q497P, K512E, E515A, and F526 (relative to wild type phi29 polymerase). Attorney Docket No.: 051385-585001WO [0265] In embodiments, the template polynucleotide includes genomic DNA, complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), or noncoding RNA (ncRNA). [0266] In embodiments, the template polynucleotide is about 100 to 1000 nucleotides in length. In embodiments, the template polynucleotide is about 500 to 2000 nucleotides in length. In embodiments, the template polynucleotide is about 1000 to 1000 nucleotides in length. In embodiments, the template polynucleotide is about 50 to 500 nucleotides in length. In embodiments, the template polynucleotide is about 500 to 1000 nucleotides in length. In embodiments, the template polynucleotide is about 350 nucleotides in length. In embodiments, the template polynucleotide is about 10, 20, 50, 100, 150, 200, 300, or 500 nucleotides in length. The template polynucleotide molecules can vary length, such as about 100-300 nucleotides long, about 300-500 nucleotides long, or about 500-1000 nucleotides long. In embodiments, the template polynucleotide molecular is about 100-1000 nucleotides, about 150-950 nucleotides, about 200-900 nucleotides, about 250-850 nucleotides, about 300-800 nucleotides, about 350- 750 nucleotides, about 400-700 nucleotides, or about 450-650 nucleotides. In embodiments, the template polynucleotide molecule is about 150 nucleotides. In embodiments, the template polynucleotide is about 100-1000 nucleotides long. In embodiments, the template polynucleotide is about 100-300 nucleotides long. In embodiments, the template polynucleotide is about 300- 500 nucleotides long. In embodiments, the template polynucleotide is about 500-1000 nucleotides long. In embodiments, the template polynucleotide molecule is about 100 nucleotides. In embodiments, the template polynucleotide molecule is about 300 nucleotides. In embodiments, the template polynucleotide molecule is about 500 nucleotides. In embodiments, the template polynucleotide molecule is about 1000 nucleotides. [0267] In embodiments the template polynucleotide (e.g., genomic template DNA) is first treated to form single-stranded linear fragments (e.g., ranging in length from about 50 to about 600 nucleotides). Treatment typically entails fragmentation, such as by chemical fragmentation, enzymatic fragmentation, or mechanical fragmentation, followed by denaturation to produce single-stranded DNA fragments. In embodiments, the template polynucleotide includes an adapter. The adapter may have other functional elements including tagging sequences (i.e., a barcode), attachment sequences, palindromic sequences, restriction sites, sequencing primer Attorney Docket No.: 051385-585001WO binding sites, functionalization sequences, and the like. Barcodes can be of any of a variety of lengths. In embodiments, the primer includes a barcode that is 10-50, 20-30, or 4-12 nucleotides in length. In embodiments, the adapter includes a primer binding sequence that is complementary to at least a portion of a primer (e.g., a sequencing primer). Primer binding sites can be of any suitable length. In embodiments, a primer binding site is about or at least about 10, 15, 20, 25, 30, or more nucleotides in length. In embodiments, a primer binding site is 10-50, 15-30, or 20- 25 nucleotides in length. [0268] In embodiments, the template polynucleotide and the double-stranded amplification products include known adapter sequences on the 5′ and 3′ ends. In embodiments, the template polynucleotide includes known adapter sequences on the 5′ and 3′ ends. In embodiments, the double-stranded amplification products include known adapter sequences on the 5′ and 3′ ends. [0269] In embodiments, prior to hybridizing the invasion primer the method includes amplifying the double-stranded polynucleotides with bridge polymerase chain reaction (bPCR) amplification, solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid- phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR, or combinations of said methods. In embodiments, generating a double-stranded amplification product includes bridge polymerase chain reaction (bPCR) amplification, solid- phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR on particles, or combinations of the methods. In embodiments, generating a double-stranded amplification product includes a bridge polymerase chain reaction amplification. In embodiments, generating a double-stranded amplification product includes a thermal bridge polymerase chain reaction (t- bPCR) amplification. In embodiments, generating a double-stranded amplification product includes a chemical bridge polymerase chain reaction (c-bPCR) amplification. Chemical bridge polymerase chain reactions include fluidically cycling a denaturant (e.g., formamide) and maintaining the temperature within a narrow temperature range (e.g., +/-5°C). In contrast, thermal bridge polymerase chain reactions include thermally cycling between high temperatures (e.g., 85°C-95°C) and low temperatures (e.g., 60°C-70°C). Thermal bridge polymerase chain Attorney Docket No.: 051385-585001WO reactions may also include a denaturant, typically at a much lower concentration than traditional chemical bridge polymerase chain reactions. [0270] In embodiments, the solid support includes a plurality of polynucleotides, wherein each polynucleotide is attached to the solid support at a 5′ end of the polynucleotide. [0271] In embodiments, generating a double-stranded amplification product includes amplifying the template polynucleotide or complement thereof on a solid support including a plurality of primers attached to the solid support, wherein the plurality of primers include a plurality of forward primers with complementarity to the template polynucleotide and a plurality of reverse primers with complementarity to a complement of the template polynucleotide, and the amplifying includes a plurality of cycles of strand denaturation, primer hybridization, and primer extension. [0272] In embodiments, the plurality of strand denaturation cycles are different for one or more cycles, wherein the initial denaturation cycle is maintained at different conditions from the remaining denaturation cycles. For example, in embodiments, the initial denaturation cycle is at about 85°C-95°C for about 1 minute to about 10 minutes, whereas denaturation in the remaining cycles is different (e.g., about 85°C for about 15-30 sec). In embodiments, the initial denaturation is maintained at about 85°C-95°C for about 5 minutes to about 10 minutes. In embodiments, the initial denaturation is maintained at 90°C-95°C for about 1 to 10 minutes. In embodiments, the initial denaturation is maintained at 80°C-85°C for about 1 to 10 minutes. In embodiments, the initial denaturation is maintained at 85°C-90°C for about 1 to 10 minutes. In embodiments, the initial denaturation is maintained at about 85°C-95°C for about 1 minutes to about 10 minutes. In embodiments, the initial denaturation is maintained at about 95°C for about 5 minutes to about 10 minutes. In embodiments, the initial denaturation is maintained at about 85°C-95°C for about 5 minutes to about 10 minutes. [0273] In embodiments, generating a double-stranded amplification product includes a thermal bridge polymerase chain reaction (t-bPCR) amplification. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 30 seconds for annealing/extension of the primer. Attorney Docket No.: 051385-585001WO [0274] In embodiments, the plurality of cycles includes thermally cycling between (i) about 80°C to 90°C for denaturation, and (ii) about 55°C to about 65°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for denaturation, and (ii) about 55°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for denaturation, and (ii) about 65°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) less than 80°C (e.g., 70 to 80°C) for denaturation, and (ii) about 55°C to about 65°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 70°C for denaturation, and (ii) about 65°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 75°C for denaturation, and (ii) about 55°C for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for denaturation, and (ii) about 65°C for annealing/extension of the primer. [0275] In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for less than 1 minute for denaturation, and (ii) about 65°C for about 1 to 2 minutes for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for less than 1 minute for denaturation, and (ii) about 60°C to about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 30 sec for denaturation and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 30 seconds for annealing/extension of the primer. In embodiments, the plurality of cycles includes thermally cycling between (i) about 85°C for about 15-30 sec for denaturation, and (ii) about 65°C for about 1 minute for annealing/extension of the primer. In embodiments, the temperature and duration for the annealing of the primer and the extension of the primer are different. In embodiments, the plurality of cycles includes thermally cycling between (i) about 90°C to 95°C for about 15 to 30 sec for denaturation and (ii) about 55°C to about 65°C for about 30 to 60 Attorney Docket No.: 051385-585001WO seconds for annealing and about 65°C to 70°C for about 30 to 60 seconds for extension of the primer. In embodiments, the plurality of denaturation steps is at a temperature of about 80°C- 95°C. In embodiments, the plurality of denaturation steps is at a temperature of about 80°C- 90°C. In embodiments, the plurality of denaturation steps is at a temperature of about 85°C- 90°C. In embodiments, the plurality of denaturation steps is at a temperature of about 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, or about 90°C. In embodiments, the plurality of denaturation steps is at a temperature of about 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or about 99°C. In embodiments, the plurality of denaturation steps is at a temperature of about 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or about 95°C. In embodiments, the plurality of denaturation steps is at a temperature of about 90°C, 91°C, 92°C, 93°C, 94°C, or about 95°C. In embodiments, the plurality of denaturation steps is at a temperature of about 70°C-85°C. In embodiments, the plurality of denaturation steps is at a temperature of about 70°C-80°C. In embodiments, the plurality of denaturation steps is at a temperature of about 75°C-80°C. In embodiments, the plurality of denaturation steps is at a temperature of about 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or about 80°C. In embodiments, the annealing/extension of the primer cycle is at a temperature of about 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or about 65°C. [0276] In embodiments, amplifying includes incubation in a denaturant. In embodiments, the denaturant is acetic acid, ethylene glycol, hydrochloric acid, nitric acid, formamide, guanidine, sodium salicylate, sodium hydroxide, dimethyl sulfoxide (DMSO), propylene glycol, urea, or a mixture thereof. In embodiments, the denaturant is an additive that lowers a DNA denaturation temperature. In embodiments, the denaturant is betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, 4-methylmorpholine 4-oxide (NMO), or a mixture thereof. In embodiments, the denaturant is betaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidine thiocyanate, or 4-methylmorpholine 4-oxide (NMO). [0277] In embodiments, amplifying includes a plurality of cycles of strand denaturation, primer hybridization, and primer extension. Although each cycle will include each of these three events (denaturation, hybridization, and extension), events within a cycle may or may not be discrete. For example, each step may have different reagents and/or reaction conditions (e.g., Attorney Docket No.: 051385-585001WO temperatures). Alternatively, some steps may proceed without a change in reaction conditions. For example, extension may proceed under the same conditions (e.g., same temperature) as hybridization. After extension, the conditions are changed to start a new cycle with a new denaturation step, thereby amplifying the amplicons. Primer extension products from an earlier cycle may serve as templates for a later amplification cycle. In embodiments, the plurality of cycles is about 5 to about 50 cycles. In embodiments, the plurality of cycles is about 10 to about 45 cycles. In embodiments, the plurality of cycles is about 10 to about 20 cycles. In embodiments, the plurality of cycles is about 20 to about 30 cycles. In embodiments, the plurality of cycles is 10 to 45 cycles. In embodiments, the plurality of cycles is 10 to 20 cycles. In embodiments, the plurality of cycles is 20 to 30 cycles. In embodiments, the plurality of cycles is about 10 to about 45 cycles. In embodiments, the plurality of cycles is about 20 to about 30 cycles. [0278] In embodiments, the double-stranded amplification product is provided in a clustered array. In embodiments, the clustered array includes a plurality of double-stranded amplification products localized to discrete sites on a solid support. In embodiments, the solid support is a bead. In embodiments, the solid support is substantially planar. In embodiments, the solid support is contained within a flow cell. [0279] In embodiments, the method further includes removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on the second strand, and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. [0280] In an aspect is provided a method of sequencing, the method including: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides Attorney Docket No.: 051385-585001WO so as to identify each incorporated nucleotide in the extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. [0281] In embodiments, the sequencing includes sequencing-by-synthesis, sequencing-by- binding, sequencing by ligation, or pyrosequencing. In embodiments, generating a first sequencing read or a second sequencing read includes a sequencing by synthesis process. In embodiments, generating a first sequencing read or a second sequencing read includes a sequencing-by-binding. As used herein, “sequencing-by-binding” refers to a sequencing technique wherein specific binding of a polymerase and cognate nucleotide to a primed template nucleic acid molecule (e.g., blocked primed template nucleic acid molecule) is used for identifying the next correct nucleotide to be incorporated into the primer strand of the primed template nucleic acid molecule. The specific binding interaction need not result in chemical incorporation of the nucleotide into the primer. In some embodiments, the specific binding interaction can precede chemical incorporation of the nucleotide into the primer strand or can precede chemical incorporation of an analogous, next correct nucleotide into the primer. Thus, detection of the next correct nucleotide can take place without incorporation of the next correct nucleotide. As used herein, the “next correct nucleotide” (sometimes referred to as the “cognate” nucleotide) is the nucleotide having a base complementary to the base of the next template nucleotide. The next correct nucleotide will hybridize at the 3′-end of a primer to complement the next template nucleotide. The next correct nucleotide can be, but need not necessarily be, capable of being incorporated at the 3′ end of the primer. For example, the next correct nucleotide can be a member of a ternary complex that will complete an incorporation reaction or, alternatively, the next correct nucleotide can be a member of a stabilized ternary complex that does not catalyze an incorporation reaction. A nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect” (or “non-cognate”) nucleotide. [0282] In embodiments, sequencing includes a plurality of sequencing cycles. In embodiments, sequencing includes 10 to 100 sequencing cycles. In embodiments, sequencing includes 50 to 100 sequencing cycles. In embodiments, sequencing includes 50 to 300 sequencing cycles. In embodiments, sequencing includes 50 to 150 sequencing cycles. In embodiments, sequencing includes at least 10, 20, 3040, or 50 sequencing cycles. In embodiments, sequencing includes at Attorney Docket No.: 051385-585001WO least 10 sequencing cycles. In embodiments, sequencing includes 10 to 20 sequencing cycles. In embodiments, sequencing includes 10, 11, 12, 13, 14, or 15 sequencing cycles. In embodiments, sequencing includes (a) extending a sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue and (b) detecting the label to generate a signal for each incorporated nucleotide or nucleotide analogue. In embodiments, detecting includes two-dimensional (2D) or three-dimensional (3D) fluorescent microscopy. Suitable imaging technologies are known in the art, as exemplified by Larsson et al., Nat. Methods (2010) 7:395-397 and associated supplemental materials, the entire content of which is incorporated by reference herein in its entirety. In embodiments of the methods provided herein, the imaging is accomplished by confocal microscopy. Confocal fluorescence microscopy involves scanning a focused laser beam across the sample, and imaging the emission from the focal point through an appropriately-sized pinhole. This suppresses the unwanted fluorescence from sections at other depths in the sample. In embodiments, the imaging is accomplished by multi-photon microscopy (e.g., two-photon excited fluorescence or two-photon-pumped microscopy). Unlike conventional single-photon emission, multi-photon microscopy can utilize much longer excitation wavelength up to the red or near-infrared spectral region. This lower energy excitation requirement enables the implementation of semiconductor diode lasers as pump sources to significantly enhance the photostability of materials. Scanning a single focal point across the field of view is likely to be too slow for many sequencing applications. To speed up the image acquisition, an array of multiple focal points can be used. The emission from each of these focal points can be imaged onto a detector, and the time information from the scanning mirrors can be translated into image coordinates. Alternatively, the multiple focal points can be used just for the purpose of confining the fluorescence to a narrow axial section, and the emission can be imaged onto an imaging detector, such as a CCD, EMCCD, or s-CMOS detector. A scientific grade CMOS detector offers an optimal combination of sensitivity, readout speed, and low cost. One configuration used for confocal microscopy is spinning disk confocal microscopy. In 2-photon microscopy, the technique of using multiple focal points simultaneously to parallelize the readout has been called Multifocal Two-Photon Microscopy (MTPM). Several techniques for MTPM are available, with applications typically involving imaging in biological tissue. In embodiments of the methods provided herein, the imaging is accomplished by light sheet fluorescence microscopy (LSFM). In embodiments, detecting includes 3D structured illumination (3DSIM). In 3DSIM, patterned light Attorney Docket No.: 051385-585001WO is used for excitation, and fringes in the Moiré pattern generated by interference of the illumination pattern and the sample, are used to reconstruct the source of light in three dimensions. In order to illuminate the entire field, multiple spatial patterns are used to excite the same physical area, which are then digitally processed to reconstruct the final image. See York, Andrew G., et al. “Instant super-resolution imaging in live cells and embryos via analog image processing.” Nature methods 10.11 (2013): 1122-1126 which is incorporated herein by reference. In embodiments, detecting includes selective planar illumination microscopy, light sheet microscopy, emission manipulation, pinhole confocal microscopy, aperture correlation confocal microscopy, volumetric reconstruction from slices, deconvolution microscopy, or aberration- corrected multifocus microscopy. In embodiments, detecting includes digital holographic microscopy (see for example Manoharan, V. N. Frontiers of Engineering: Reports on Leading- edge Engineering from the 2009 Symposium, 2010, 5-12, which is incorporated herein by reference). In embodiments, detecting includes confocal microscopy, light sheet microscopy, or multi-photon microscopy. [0283] In embodiments, the method includes sequencing the first and/or the second strand of a double-stranded amplification product by extending a sequencing primer hybridized thereto. A variety of sequencing methodologies can be used such as sequencing-by-synthesis (SBS), pyrosequencing, sequencing by ligation (SBL), or sequencing by hybridization (SBH). Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res.11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); U.S. Pat. Nos.6,210,891; 6,258,568; and.6,274,320, each of which is incorporated herein by reference in its entirety). In pyrosequencing, released Ppi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated can be detected via light produced by luciferase. In this manner, the sequencing reaction can be monitored via a luminescence detection system. In both SBL and SBH methods, target nucleic acids, and amplicons thereof, that are present at features of an array are subjected to repeated cycles of oligonucleotide delivery and detection. SBL methods, include those described in Shendure et al. Science 309:1728-1732 (2005); U.S. Pat. Nos.5,599,675; and 5,750,341, each of which is incorporated herein by reference in its entirety; and the SBH methodologies are as described in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Attorney Docket No.: 051385-585001WO Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which is incorporated herein by reference in its entirety. [0284] In embodiments, sequencing is performed according to a “sequencing-by-binding” method (see, e.g., U.S. Pat. Pubs. US2017/0022553 and US2019/0048404, each of which is incorporated herein by reference in its entirety), which refers to a sequencing technique wherein specific binding of a polymerase and cognate nucleotide to a primed template nucleic acid molecule (e.g., blocked primed template nucleic acid molecule) is used for identifying the next correct nucleotide to be incorporated into the primer strand of the primed template nucleic acid molecule. The specific binding interaction need not result in chemical incorporation of the nucleotide into the primer. In some embodiments, the specific binding interaction can precede chemical incorporation of the nucleotide into the primer strand or can precede chemical incorporation of an analogous, next correct nucleotide into the primer. Thus, detection of the next correct nucleotide can take place without incorporation of the next correct nucleotide. As used herein, the “next correct nucleotide” (sometimes referred to as the “cognate” nucleotide) is the nucleotide having a base complementary to the base of the next template nucleotide. The next correct nucleotide will hybridize at the 3′-end of a primer to complement the next template nucleotide. The next correct nucleotide can be, but need not necessarily be, capable of being incorporated at the 3′ end of the primer. For example, the next correct nucleotide can be a member of a ternary complex that will complete an incorporation reaction or, alternatively, the next correct nucleotide can be a member of a stabilized ternary complex that does not catalyze an incorporation reaction. A nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect” (or “non-cognate”) nucleotide. [0285] In embodiments, sequencing includes extending a sequencing primer to incorporate a nucleotide containing a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting of steps. In embodiments, the methods include sequencing one or more bases of a target nucleic acid by extending a sequencing primer hybridized to a target nucleic acid (e.g., an amplification product of a target nucleic acid). In embodiments, the sequencing includes sequencing-by-synthesis, sequencing-by-binding, sequencing by ligation, sequencing-by-hybridization, or pyrosequencing, and generates a sequencing read. In embodiments, generating a sequencing read includes Attorney Docket No.: 051385-585001WO executing a plurality of sequencing cycles, each cycle including extending the sequencing primer by incorporating a nucleotide or nucleotide analogue using a polymerase and detecting a characteristic signature indicating that the nucleotide or nucleotide analogue has been incorporated. [0286] In SBS, extension of a nucleic acid primer along a nucleic acid template is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be catalyzed by a polymerase, wherein fluorescently labeled nucleotides are added to a primer (thereby extending the primer) in a template dependent fashion such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template. A plurality of different nucleic acid fragments that have been attached at different locations of an array can be subjected to an SBS technique under conditions where events occurring for different templates can be distinguished due to their location in the array. In embodiments, the sequencing step includes annealing and extending a sequencing primer to incorporate a detectable label that indicates the identity of a nucleotide in the target polynucleotide, detecting the detectable label, and repeating the extending and detecting steps. In embodiments, the methods include sequencing one or more bases of a target nucleic acid by extending a sequencing primer hybridized to a target nucleic acid (e.g., an amplification product produced by the amplification methods described herein). In embodiments, the sequencing step may be accomplished by a sequencing-by-synthesis (SBS) process. In embodiments, sequencing comprises a sequencing by synthesis process, where individual nucleotides are identified iteratively, as they are polymerized to form a growing complementary strand. In embodiments, nucleotides added to a growing complementary strand include both a label and a reversible chain terminator that prevents further extension, such that the nucleotide may be identified by the label before removing the terminator to add and identify a further nucleotide. Such reversible chain terminators include removable 3’ blocking groups, for example as described in U.S. Pat. Nos. 10,738,072, 7,541,444 and 7,057,026. Once such a modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced, there is no free 3′-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides. Once the identity of the base incorporated into the growing chain has been determined, the 3’ block may be removed to allow addition of the next successive nucleotide. By ordering the products derived using these Attorney Docket No.: 051385-585001WO modified nucleotides it is possible to deduce the DNA sequence of the DNA template. Non- limiting examples of suitable labels are described in U.S. Pat. No.8,178,360, U.S. Pat. No. 5,188,934 (4,7-dichlorofluorscein dyes); U.S. Pat. No.5,366,860 (spectrally resolvable rhodamine dyes); U.S. Pat. No.5,847,162 (4,7-dichlororhodamine dyes); U.S. Pat. No.4,318,846 (ether-substituted fluorescein dyes); U.S. Pat. No.5,800,996 (energy transfer dyes); U.S. Pat. No. 5,066,580 (xanthene dyes): U.S. Pat. No.5,688,648 (energy transfer dyes); and the like. [0287] Sequencing includes, for example, detecting a sequence of signals. Examples of sequencing include, but are not limited to, sequencing by synthesis (SBS) processes in which reversibly terminated nucleotides carrying fluorescent dyes are incorporated into a growing strand, complementary to the target strand being sequenced. In embodiments, the nucleotides are labeled with up to four unique fluorescent dyes. In embodiments, the nucleotides are labeled with at least two unique fluorescent dyes. In embodiments, the readout is accomplished by epifluorescence imaging. A variety of sequencing chemistries are available, non-limiting examples of which are described herein. [0288] In embodiments, the method includes determining the nucleic acid sequence of the target polynucleotide. In embodiments, the molecule further includes quantifying the target nucleic acid molecule or amplicons. Methods for quantifying a target polynucleotide or amplicon are well known to one of skilled in the art. For example, during amplification of the target nucleic acid, quantitative techniques such as real-time polymerase chain reaction (RT-PCR) can be used to quantify the copy number of target nucleic acid molecules present in the clonal object as discussed in Logan et al. Real-Time PCR: Current Technology and Applications, Caister Academic Press. (2009). RT-PCR follows the general principle of polymerase chain reaction, however inclusion of detection molecules, such as non-specific fluorescent dyes that intercalate with any double-stranded DNA, or sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary DNA target, allows for the detection of nucleic acid formed during amplification. The rate of detectable molecules is proportional to the copy number of target nucleic acid molecules present in the clonal object. Furthermore, quantifying the target nucleic acid molecule or amplicons can be done following amplification using standard gel electrophoresis and/or Southern blot techniques, which are well known in the art. Attorney Docket No.: 051385-585001WO [0289] In embodiments, the method further includes sequencing the amplification product(s). Sequencing includes, for example, detecting a sequence of signals within the particle. Examples of sequencing include, but are not limited to, sequencing by synthesis (SBS) processes in which reversibly terminated nucleotides carrying fluorescent dyes are incorporated into a growing strand, complementary to the target strand being sequenced. In embodiments, the nucleotides are labeled with up to four unique fluorescent dyes. In embodiments, the readout is accomplished by epifluorescence imaging. A variety of sequencing chemistries are available, non-limiting examples of which are described herein. [0290] In embodiments, the location of each cluster of capture probes (e.g., each cluster of a plurality of immobilized double-stranded polynucleotides including the tail sequence, wherein the tail sequence includes a capture sequence) on the surface of the solid support is determined during manufacture of the substrate itself. For example, the substrate may be manufactured by immobilizing one or more polynucleotides on the surface of the substrate (e.g., by binding to a surface probe on the substrate) and generating clusters of double-stranded polynucleotides (e.g., by bridge amplification), as described herein. The double-stranded polynucleotides may include a spatial barcode, as described above. After cluster generation, the determination of the location of each cluster of double-stranded polynucleotides on the surface may be determined by sequencing the double-stranded polynucleotides on the substrate. In particular, sequencing primers targeting the spatial barcode may be utilized, and the sequence of the spatial barcode may be determined. The sequence of the spatial barcode for each cluster may be assigned to a specific location on the substrate (e.g., an XY coordinate on the substrate). In some embodiments, a high- resolution map of the substrate may be generated based upon the signal detected during sequencing (e.g., the fluorescent signal) and used to assign an XY coordinate to each cluster on the substrate. Either before or after determining the specific location on the substrate for each spatial barcode, the method further includes incorporating a tail sequence into a strand of the double-stranded polynucleotide (e.g., the capture probe). In embodimetheethod further includes hybridizing an invasion primer including a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide includes a first strand hybridized to the second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail Attorney Docket No.: 051385-585001WO sequence of the invasion primer to generate an extended second strand including a complement of the tail sequence, thereby incorporating a sequence (e.g., a capture sequence) into the second strand of the double-stranded polynucleotide. [0291] In some embodiments, the methods for spatial detection of nucleic acid (e.g. RNA) in a tissue sample further include correlating the sequence of the spatial barcode for each sequenced cDNA molecule with the location of the cluster of capture probes on the substrate having the corresponding spatial barcode. The first strand cDNA will contain the same spatial barcode as the capture probe, whereas the second strand cDNA will contain the complement to the spatial barcode of the capture probe. “Corresponding” as used herein covers each of these possibilities, depending on which cDNA strand is sequenced. For instance, if the second strand cDNA is sequenced, the sequence of the second strand cDNA is correlated with the location of the cluster of capture probes on the substrate having the complementary spatial barcode. Alternatively, if the first strand cDNA is sequenced (e.g., no intermittent steps of second strand synthesis and/or amplification are performed prior to sequencing the cDNA), the sequence of the first strand cDNA is correlated with the location of the cluster of capture probes on the substrate having the same spatial barcode. [0292] In embodiments, the plurality of capture probes (e.g., a plurality of immobilized double-stranded polynucleotides including the tail sequence, wherein the tail sequence includes a capture sequence) is arranged in clusters on the surface of the substrate, each cluster including multiple capture probes. In embodiments, each capture probe in a cluster includes the same spatial barcode. Additionally, in embodiments, the spatial barcode for each cluster is unique. For example, cluster A contains probes including spatial barcode A, cluster B contains probes including spatial barcode B, cluster C contains probes including spatial barcode C, etc. [0293] In some embodiments, the methods further include generating cDNA molecules from the bound RNA molecules. The cDNA generated is considered to be indicative of the RNA present in a cell at the time in which a tissue sample was taken. Therefore, cDNA represents all or some of the genes that were expressed in the cell at the time the tissue sample was taken. The capture probe acts as a primer for reverse transcription, such that the sequence of the capture probe is incorporated into the sequence of the first strand cDNA molecule along with the sequence complementary to the captured RNA strand. Accordingly, the spatial barcode of the Attorney Docket No.: 051385-585001WO capture probe is incorporated into the sequence of the first strand cDNA molecule. Generating cDNA molecules from the bound RNA molecules may be performed by any suitable method. For example, generating cDNA molecules from the bound RNA molecules may be performed by addition of a reverse transcriptase to facilitate reverse transcription of the RNA (e.g., mRNA) to generate a complementary or copy DNA (i.e., cDNA). The cDNA resulting from the reverse transcription of RNA is referred to herein as “first strand cDNA”. First strand cDNA synthesis (e.g., reverse transcription) may be performed directly on the substrate. [0294] In some embodiments, the reverse transcription reaction includes a reverse transcriptase, dNTPs and a suitable buffer. The reaction mixture may include other components, such as RNase inhibitor(s). Each dNTP is typically present in an amount ranging from about 10 to 5000 mM, usually from about 20 to 1000 mM. Any suitable reverse transcriptase enzyme may be used. Suitable enzymes include: M-MLV, MuLV, AMV, HIV, ArrayScript™, MultiScribe™, ThermoScript™, and Superscript® I, II, and III enzymes. The reverse transcriptase reaction may be carried out at any suitable temperature, which is dependent on the properties of the enzyme. Typically, reverse transcriptase reactions are performed between 37-55°C, although temperatures outside of this range may also be appropriate. The reaction time may be as little as 1, 2, 3, 4 or 5 minutes or as much as 48 hours. Typically, the reaction is carried out for between 3-12 hours, although other suitable reaction times (e.g., overnight) may be used. [0295] In some embodiments, a strand complementary to the first strand cDNA may be synthesized. The strand complementary to the first strand cDNA is referred to herein as “second strand cDNA”. The term “cDNA” as used herein is used in the broadest sense and refers to any cDNA, including first strand cDNA and second strand cDNA. In some embodiments, “generating cDNA” includes performing second strand synthesis (e.g., following the reverse transcription reaction) to generate second strand cDNA. In some embodiments, second strand cDNA synthesis may occur without increasing the number of copies of the second strand cDNA (e.g., without amplifying the second strand). In other embodiments, second strand cDNA may be synthesized and amplified, resulting in multiple copies of the second strand. Second strand cDNA synthesis, if performed, may be performed on the substrate (e.g., while the cDNA is immobilized on the substrate). Alternatively, the first strand cDNA may be released from the substrate and second strand cDNA synthesis may be performed in solution. The second strand Attorney Docket No.: 051385-585001WO cDNA includes a complement of the capture probe and therefore includes a complement of the spatial barcode sequence of the capture probe. The second strand cDNA may be amplified using a suitable primer or combination of primers upstream of the complement to the spatial barcode sequence, such that the complement of the spatial barcode sequence is presence in each amplified second strand cDNA. In some embodiments, second strand cDNA synthesis is performed using random primers. For example, the first strand cDNA may be incubated with random primers, such as hexamer primers, and a DNA polymerase, under conditions sufficient for synthesis of the complementary DNA strand (e.g., second strand cDNA) to form. [0296] In some embodiments, the second strand cDNA may be isolated, purified and amplified following synthesis. For example, the second strand cDNA may be synthesized by a suitable method as described above (e.g., using random primers). In some embodiments, the secondary strand cDNA may be isolated through DNA denaturation in any solutions with high pH and/or organic solutions that can denature the DNA. In some embodiments, the secondary strand cDNA may be isolated through heat denaturation. The isolated second strand may be purified, and then amplified by PCR. Primers for PCR amplification of the second strand cDNA may be any suitable primers, including primers targeting the additional features (e.g., primer binding sites, sequencing barcodes, unique molecular identifiers) added to the second strand cDNA. Any suitable number of isolation, amplification, and purification steps may be performed to generate the final library of cDNA prior to sequencing. In some embodiments, the capture probes used for the initial capture of RNA (e.g., mRNA) may contain one or more additional features (e.g., additional to the spatial barcode and capture domain) that facilitate sequencing library preparation. For example, the capture probes may contain a sequencing handle (e.g., sequencing barcode). Therefore, the complement of the sequencing barcode will be present in the cDNA. Accordingly, cDNA generated by the methods described herein may include two distinct sequencing barcodes. For example, the cDNA may include sequencing barcode(s) compatible with a Singular GenomicsTM or IlluminaTM sequencing platform. In some embodiments, the cDNA includes sequencing barcode(s), a spatial barcode, and/or a unique molecular identifier. These additional features may facilitate library preparation, sequencing, and spatial detection of RNA by the methods described herein. Attorney Docket No.: 051385-585001WO [0297] In some embodiments, the generated cDNA may be sequenced with no intervening treatment steps prior to sequencing. For example, in tissue samples that include large amounts of RNA, generating the cDNA may yield a sufficient amount of cDNA such that it may be sequenced directly. In other embodiments, it may be desirable to generate double stranded cDNA and/or generate multiple copies of the DNA prior to sequencing. Such methods may be performed while the cDNA is bound to the substrate, or the cDNA may be released from the substrate and subsequently treated to generate double stranded copies and/or amplify the DNA. In some embodiments, it may be desirable to generate double stranded DNA without increasing the number of double stranded DNA molecules. In other embodiments, it may be desirable to generate double stranded DNA and generate multiple copies of the second strand. For example, one or multiple amplification reactions may be conducted to generate multiple copies of single stranded or double stranded DNA. [0298] In some embodiments, generation of cDNA (e.g., by reverse transcription of the RNA bound to the capture probes) may take place on the substrate and the generated cDNA maybe released from the substrate prior to subsequent treatment steps. For example, the cDNA may be generated on the substrate and the generated DNA may be released from the substrate and collected in a tube. Subsequent steps (e.g., second strand cDNA synthesis, amplification, sequencing, etc.) may be performed in solution. In some embodiments, RNA may be removed prior to subsequent treatment of the cDNA strand. For example, RNA may be removed using an RNA digesting enzyme (e.g., RNase). In some embodiments, no specific RNA removal step is necessary, as RNA will degrade naturally and/or removal of the tissue from the substrate is sufficient for RNA removal. [0299] In some embodiments, the methods for spatial detection of nucleic acid (e.g. RNA) in a tissue sample further include sequencing the cDNA molecules. The cDNA molecules may be sequenced on the substrate or may be released and collected into a suitable device (e.g., a tube) prior to sequencing. Sequencing may be performed by any suitable method. [0300] In some embodiments, the full length of the cDNA molecules may be sequenced. In some embodiments, less than the full length of the cDNA molecules may be sequenced. The claimed methods are not limited to sequencing the entire length of each cDNA molecule. For example, the first 100 nucleotides from each end of the cDNA molecules may be sequenced and Attorney Docket No.: 051385-585001WO used to identify the gene expressed. In some embodiments, sequencing may be performed to determine the sequence of the spatial barcode and at least about 20 bases of RNA transcript specific sequence data. For example, the sequencing may be performed to determine the sequence of the spatial barcode and at least 10, 25, 30, 35, 40, 45, 50 bases of RNA transcript specific sequence data. Additional bases of RNA transcript specific sequence data may be obtained. For example, the sequencing may be performed to determine the sequence of the spatial barcode and at least 50, 60, 70, 80, 90, or 100 bases of RNA transcript specific data. [0301] In embodiments, the methods for spatial detection of nucleic acid (e.g. RNA) in a tissue sample further include imaging the tissue after contacting the tissue with the substrate (e.g., after immobilizing the tissue on the substrate). Imaging the tissue may assist in the determination of the spatial location of RNA molecules within the tissue sample. In embodiments, imaging the tissue is performed before generating cDNA. In embodiments, imaging the tissue is performed after generating cDNA. In embodiments, prior to imaging, the method further includes permeabilizing the immobilized tissue section. In embodiments, prior to imaging, the method does not include permeabilizing the immobilized tissue section. In embodiments, prior to imaging, the method further includes contacting the immobilized tissue section with one or more imaging reagents or stains. In embodiments, following permeabilization, the tissue section is contacted with one or more imaging reagents or stains. In embodiments, the tissue section is contacted with one or more imaging reagents or stains without permeabilization. In embodiments, the imaging reagents or stains include hematoxylin and eosin (H&E) staining reagents. In embodiments, the imaging includes phase-contrast microscopy, bright-field microscopy, Nomarski differential-interference-contrast microscopy, dark field microscopy, electron microscopy, or cryo-electron microscopy. In embodiments, the imaging reagents or stains include phase-contrast microscopy, bright-field microscopy, Nomarski differential- interference-contrast microscopy, or dark field microscopy imaging reagents. In embodiments, the light transmittance of the sample is measured. For example, light transmittance may be measured with a visible near-infrared optical fiber spectrometer, wherein a circular spot of light (e.g., diameter, 5 mm) is irradiated on the central part a sample and the transmitted light is collected using an optical sensor. Attorney Docket No.: 051385-585001WO [0302] In embodiments, the imaging reagents or stains include electron microscopy (e.g., transmission electron microscopy or scanning electron microscopy) or cryo-electron microscopy imaging reagents. Examples of electron microscopy contrast agents may include one or more heavy metals (e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium) and/or antibodies bound to one or more types of heavy metals (e.g., gold particles, colloidal gold particles, uranium, lead, platinum, and/or osmium). For example, immunogold labels that may be used to contact the tissue section include may include different antibodies bound to gold particles of different sizes to image different molecules of interest. Optionally, the method may include contacting the tissue section with heavy metals. Heavy metals that may be used to stain additional features of interest and/or provide contrast between different structures in the tissue section may include uranium, lead, platinum, and/or osmium (see, e.g., U.S. Pat. Pubs. 2019/0355550 and 2013/0344500, each of which is incorporated herein by reference in its entirety). [0303] In embodiments, the tissue section includes a tissue portion or a cell (e.g. plurality of cells such as blood cells). In embodiments, the tissue section includes one or more cells. [0304] In embodiments, the tissue section is embedded in an embedding material including paraffin wax, polyepoxide polymer, polyacrylic polymer, agar, gelatin, celloidin, cryogel, optimal cutting temperature (OCT) compositions, glycols, or a combination thereof. In embodiments, the tissue section is embedded in an embedding material including paraffin wax. In embodiments, the OCT composition includes about 10% polyvinyl alcohol and about 4% polyethylene glycol. In embodiments, the OCT composition includes sucrose (e.g., 30% sucrose). In embodiments, the OCT composition is Tissue Freezing Medium (TFM) available from Leica Microsystems, Catalog #14020108926. [0305] In embodiments, the tissue is immobilized to the substrate (e.g., the solid support including one or more clusters of the first plurality of capture probes and the second plurality of capture probes) by covalently binding the tissue to one or more bioconjugate reactive moieties of the substrate. In embodiments, the tissue is immobilized to the substrate by non-covalently binding the tissue to the substrate. For non-covalent binding, the tissue sections attach to the substrate surface due to surface interactions, such as Van der Waal forces, electrostatic forces, Attorney Docket No.: 051385-585001WO hydrophobic interactions and hydrogen bonds. The physical adsorption efficiency can be enhanced by treating the material with air plasma to increase its hydrophilicity. [0306] In embodiments, the substrate includes a functionalized glass surface or a functionalized plastic surface. In embodiments, the functionalized glass surface includes (3- aminopropyl)triethoxysilane (APTES), (3-Aminopropyl)trimethoxysilane (APTMS), γ- Aminopropylsilatrane (APS), N-(6-aminohexyl)aminomethyltriethoxysilane (AHAMTES), polyethylenimine (PEI), 5,6-epoxyhexyltriethoxysilane, or triethoxysilylbutyraldehyde, or a combination thereof. [0307] Tissue sections include tissue or organ samples obtained from a subject, e.g., a mammal. In certain embodiments, the subject is diagnosed with a disease or disorder, such as a cancerous tumor, or considered at risk of having or developing the disease or disorder. Tissue sections may also be obtained from healthy donors, e.g., as normal control samples. In certain embodiments, both a disease tissue (e.g., a tumor tissue) sample and a normal sample are obtained from the same subject. In particular embodiments, the tissue section is obtained from a patient, e.g., a mammal such as a human. In other embodiments, a tissue section is obtained from an animal model of disease. Various animal models of disease are known and available in the art. Particular animal models of cancer include but are not limited to xenograft, syngeneic, and PDx models, e.g., in mice or rats. Animal models may also include human cells, cancerous or otherwise, introduced into animal models wherein tumor properties, progress, and treatment may be assessed. In vitro 3D tissue arrangements, organoids, and stem or iPS-cell-derived 3D compositions are also relevant models, and these may include human or other animal cells, for example. [0308] In embodiments, the tissue section includes a tissue or a cell. Biological tissue samples suitable for use with the methods and systems described herein generally include any type of tissue samples collected from living or dead subjects, such as, for example, tumor tissue and autopsy samples. Tissue samples may be collected and processed using the methods and systems described herein and subjected to microscopic analysis immediately following processing, or may be preserved and subjected to microscopic analysis at a future time, e.g., after storage for an extended period of time. In some embodiments, the methods described herein may be used to preserve tissue samples in a stable, accessible and fully intact form for future analysis. For Attorney Docket No.: 051385-585001WO example, tissue samples, such as, e.g., human tumor tissue samples, may be processed as described herein and cleared to remove a plurality of cellular components, such as, e.g., lipids, and then stored for future analysis. In some embodiments, the methods and systems described herein may be used to analyze a fresh tissue section. In some embodiments, the methods and systems described herein may be used to analyze a previously-preserved (e.g., previously fixed) or stored tissue section (e.g., tissue sample). For example, in some embodiments a previously- preserved tissue sample that has not been subjected to a sample preparation process described herein may be processed and analyzed as described herein. In particular methods, a tissue sample is frozen prior to being processed as described herein. [0309] In certain embodiments, tissue sections are tumor tissue samples. Tumor samples may contain only tumor cells, or they may contain both tumor cells and non-tumor cells. In particular embodiments, a tissue section includes only non-tumor cells. In particular embodiments, the tumor is a solid tumor. In particular embodiments, the tissue section is obtained from or includes an adrenal cortical cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumor, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, head or neck cancer, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, myelodysplasia syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal or squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment, is a tissue section obtained from a subject diagnosed with or suspected of having any of these tumors or cancers. Attorney Docket No.: 051385-585001WO [0310] The methods of the invention can be used to characterize a cancer or metastasis thereof, including without limitation, a carcinoma, a sarcoma, a lymphoma or leukemia, a germ cell tumor, a blastoma, or other cancers. Carcinomas include without limitation epithelial neoplasms, squamous cell neoplasms squamous cell carcinoma, basal cell neoplasms basal cell carcinoma, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas (glands), adenoma, adenocarcinoma, linitis plastica insulinoma, glucagonoma, gastrinoma, vipoma, cholangiocarcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, carcinoid tumor of appendix, prolactinoma, oncocytoma, hurthle cell adenoma, renal cell carcinoma, grawitz tumor, multiple endocrine adenomas, endometrioid adenoma, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic, mucinous and serous neoplasms, cystadenoma, pseudomyxoma peritonei, ductal, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, warthin's tumor, thymoma, specialized gonadal neoplasms, sex cord stromal tumor, thecoma, granulosa cell tumor, arrhenoblastoma, sertoli leydig cell tumor, glomus tumors, paraganglioma, pheochromocytoma, glomus tumor, nevi and melanomas, melanocytic nevus, malignant melanoma, melanoma, nodular melanoma, dysplastic nevus, lentigo maligna melanoma, superficial spreading melanoma, and malignant acral lentiginous melanoma. Sarcoma includes without limitation Askin's tumor, botryodies, chondrosarcoma, Ewing's sarcoma, malignant hemangio endothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcomas including: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovialsarcoma. Lymphoma and leukemia include without limitation chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as waldenstrom macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases, extranodal marginal zone B cell lymphoma, also called malt lymphoma, nodal marginal zone B cell lymphoma (nmzl), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, burkitt lymphoma/leukemia, Attorney Docket No.: 051385-585001WO T cell prolymphocytic leukemia, T cell large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides/sezary syndrome, primary cutaneous CD30-positive T cell lymphoproliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, classical hodgkin lymphomas (nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte depleted or not depleted), and nodular lymphocyte- predominant hodgkin lymphoma. Germ cell tumors include without limitation germinoma, dysgerminoma, seminoma, nongerminomatous germ cell tumor, embryonal carcinoma, endodermal sinus turmor, choriocarcinoma, teratoma, polyembryoma, and gonadoblastoma. Blastoma includes without limitation nephroblastoma, medulloblastoma, and retinoblastoma. Other cancers include without limitation labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid carcinoma), renal carcinoma, kidney parenchyma carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumors, gall bladder carcinoma, bronchial carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma. [0311] In a further embodiment, the cancer under analysis may be a lung cancer including non- small cell lung cancer and small cell lung cancer (including small cell carcinoma (oat cell cancer), mixed small cell/large cell carcinoma, and combined small cell carcinoma), colon cancer, breast cancer, prostate cancer, liver cancer, pancreas cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, or a solid tumor. Attorney Docket No.: 051385-585001WO [0312] Tissue sections may be obtained from a subject by any means known and available in the art. In particular embodiments, a tissue section, e.g., a tumor tissue sample, is obtained from a subject by fine needle aspiration, core needle biopsy, stereotactic core needle biopsy, vacuum- assisted core biopsy, or surgical biopsy. In particular embodiments, the surgical biopsy is an incisional biopsy, which removes only part of the suspicious area. In other embodiments, the surgical biopsy is an excisional biopsy, which removes the entire diseased tissue (e.g., tumor) or abnormal area. In particular embodiments, an excisional tumor tissue sample is obtained from a tumor that has been excised with the intent to “cure” a patient in the case of early stage disease, wherein in other embodiments, the excisional tumor tissue sample is obtained from an excised bulk of primary tumor in later stage disease. Tumor tissue samples may include primary tumor tissue, metastastic tumor tissue and/or secondary tumor tissue. Tumor tissue samples may be cell cultures, e.g., cultures of tumor-derived cell lines. In certain embodiments, a tissue section is a cell line, e.g., a cell pellet of a cultured cell line, such as a tumor cell line. In particular embodiments, the cell line or cell pellet is frozen or was previously frozen. Such cell lines and pellets are useful, e.g., as positive or negative controls for imaging with various reagents. Tumor tissue samples may also be xenograft tumors, e.g., tumors obtained from animals administered with tumor cells, e.g., a human tumor cell line. In certain embodiments, a first tumor tissue sample from a subject is a primary tumor tissue sample obtained during an initial surgery intended to remove the entire tumor, and a second tumor tissue sample is obtained from the same subject is a metastatic tumor tissue sample or a secondary tumor tissue sample obtained during a later surgery. [0313] Tissue sections, e.g., tumor tissue samples, may be obtained surgically or using a laparoscope. A tissue section may be a tissue sample obtained from any part of the body to examine it for disease or injury, e.g., presence of cancer tissue or cells, or the extent or characteristics thereof. In particular embodiments, the tissue section includes abdominal tissue, bone, bone marrow, breast tissue, endometrial tissue, kidney tissue, liver tissue, lung or chest tissue, lymph node, nerve tissue, skin, testicular tissue, head or neck tissue, or thyroid tissue. In certain embodiments, the tissue is obtained from brain, breast, skin, bone, joint, skeletal muscle, smooth muscle, red bone marrow, thymus, lymphatic vessel, thoracic duct, spleen, lymph node, nasal cavity, pharynx, larynx, trachea, bronchus, lung, oral cavity, esophagus, liver, stomach, small intestine, large intestine, rectum, anus, spinal cord, nerve, pineal gland, pituitary gland, Attorney Docket No.: 051385-585001WO thyroid gland, thymus, adrenal gland, pancreas, ovary, testis, heart, blood vessel, kidney, uterus, urinary bladder, urethra, prostate gland, penis, prostate, testis, scrotum, ductus deferens, mammary glands, ovary, uterus, vagina, or uterine tube. [0314] In particular embodiments, a tissue section has a size greater than sections typically examined by traditional pathology thin section or immunohistochemical analysis, which are typically in the range of 4-10 microns thick. In certain embodiments, a tissue section is greater than 20 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, greater than 500 microns, greater than 1 mm, greater than 2 mm, greater than 5 mm, greater than 10 mm or greater than 20 mm in thickness and/or length. In particular embodiments, the tissue section has a length and/or a thickness between 20 microns and 20 mm, between 20 microns and 10 mm, or between 50 microns and 1 mm. In certain embodiments, a tissue section is a cubic sample with each side greater than 10 microns, greater than 20 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, greater than 500 microns, greater than 1 mm, greater than 2 mm, greater than 5 mm, greater than 10 mm, or greater than 2 mm in thickness and/or length. In some embodiments, a tissue section is thinner, e.g., from about 4-10 or 4-20 microns in thickness. [0315] RNA, including mRNA, is highly susceptible to degradation upon exposure to one or more RNAses. RNAses are present in a wide range of locations, including water, many reagents, laboratory equipment and surfaces, skin, and mucous membranes. Working with RNA often requires preparing an RNAse-free environment and materials, as well as taking precautions to avoid introducing RNAses into an RNAse-free environment. These precautions include, but are not limited to, cleaning surfaces with an RNAse cleaning product (e.g., RNASEZAP™ and other commercially available products or 0.5% sodium dodecyl sulfate [SDS] followed by 3% H2O2); using a designated workspace, materials, and equipment (e.g., pipets, pipet tips); using barrier tips; baking designated glassware (e.g., 300° C. for 2 hours) prior to use; treating enzymes, reagents, and other solutions (e.g., with diethyl pyrocarbonate [DEPC] or dimethyl pyrocarbonate [DMPC]) or using commercially available, certified RNAse-free water or solutions, or ultrafiltered water (e.g., for Tris-based solutions); including an RNAse inhibitor while avoiding temperatures or denaturing conditions that could deactivate the inhibitor); and wearing clean gloves (while avoiding contaminated surfaces) and a clean lab coat. Attorney Docket No.: 051385-585001WO [0316] In embodiments, the tissue section forms part of a tissue in situ. In embodiments, the tissue section includes one or more prokaryotic cells. In embodiments, the tissue section includes one or more eukaryotic cells. In embodiments, the tissue section includes a bacterial cell (e.g., a bacterial cell or bacterial spore), a fungal cell (e.g., a fungal spore), a plant cell, or a mammalian cell. In embodiments, the tissue section includes a stem cell. In embodiments, the stem cell is an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, or an induced pluripotent stem cell. In embodiments, the tissue section includes an endothelial cell, muscle cell, myocardial, smooth muscle cell, skeletal muscle cell, mesenchymal cell, epithelial cell; hematopoietic cell, such as lymphocytes, including T cell, e.g., (Th1 T cell, Th2 T cell, ThO T cell, cytotoxic T cell); B cell, pre-B cell; monocytes; dendritic cell; neutrophils; or a macrophage. In embodiments, the tissue section includes a stem cell, an immune cell, a cancer cell (e.g., a circulating tumor cell or cancer stem cell), a viral-host cell, or a cell that selectively binds to a desired target. In embodiments, the cell includes a T cell receptor gene sequence, a B cell receptor gene sequence, or an immunoglobulin gene sequence. In embodiments, the cell includes a Toll-like receptor (TLR) gene sequence. In embodiments, the cell includes a gene sequence corresponding to an immunoglobulin light chain polypeptide and a gene sequence corresponding to an immunoglobulin heavy chain polypeptide. In embodiments, the tissue section includes a genetically modified cell. In embodiments, the tissue section includes a circulating tumor cell or cancer stem cell. [0317] In embodiments, the tissue section includes a prokaryotic cell. In embodiments, the tissue section includes a bacterial cell. In embodiments, the bacterial cell is a Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, or Bifidobacterium cell. In embodiments, the bacterial cell is a Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, Enterococcus faecalis, Escherichia coli, Enterobacter sp., Klebsiella sp., Bifidobacterium bifidum, Staphylococcus aureus, Lactobacillus, Clostridium perfringens, Proteus mirabilis, Clostridium tetani, Clostridium septicum, Pseudomonas aeruginosa, Salmonella enterica, Faecalibacterium prausnitzii, Peptostreptococcus sp., or Peptococcus sp. cell. In embodiments, tissue section includes a fungal cell. In embodiments, the fungal cell is a Candida, Saccharomyces, Aspergillus, Penicillium, Rhodotorula, Trametes, Pleospora, Sclerotinia, Bullera, or a Galactomyces cell. Attorney Docket No.: 051385-585001WO [0318] In embodiments, the tissue section includes a viral-host cell. A “viral-host cell” is used in accordance with its ordinary meaning in virology and refers to a cell that is infected with a viral genome (e.g., viral DNA or viral RNA). The cell, prior to infection with a viral genome, can be any cell that is susceptible to viral entry. In embodiments, the viral-host cell is a lytic viral-host cell. In embodiments, the viral-host cell is capable of producing viral protein. In embodiments, the viral-host cell is a lysogenic viral-host cell. In embodiments, the cell is a viral- host cell including a viral nucleic acid sequence, wherein the viral nucleic acid sequence is from a Hepadnaviridae, Adenoviridae, Herpesviridae, Poxviridae, Parvoviridae, Reoviridae, Coronaviridae, Retroviridae virus. [0319] In embodiments, the tissue section includes an adherent cell (e.g., epithelial cell, endothelial cell, or neural cell). Adherent cells are usually derived from tissues of organs and attach to a substrate (e.g., epithelial cells adhere to an extracellular matrix coated substrate via transmembrane adhesion protein complexes). Adherent cells typically require a substrate, e.g., tissue culture plastic, which may be coated with extracellular matrix (e.g., collagen and laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation. In embodiments, the tissue section includes a neuronal cell, an endothelial cell, epithelial cell, germ cell, plasma cell, a muscle cell, peripheral blood mononuclear cell (PBMC), a myocardial cell, or a retina cell. In embodiments, the tissue section includes a suspension cell (e.g., a cell free-floating in the culture medium, such a lymphoblast or hepatocyte). In embodiments, the tissue section includes a glial cell (e.g., astrocyte, radial glia), pericyte, or stem cell (e.g., a neural stem cell). In embodiments, the tissue section includes a neuronal cell. In embodiments, the tissue section includes an endothelial cell. In embodiments, the tissue section includes an epithelial cell. In embodiments, the tissue section includes a germ cell. In embodiments, the tissue section includes a plasma cell. In embodiments, the tissue section includes a muscle cell. In embodiments, the tissue section includes a peripheral blood mononuclear cell (PBMC). In embodiments, the tissue section includes a myocardial cell. In embodiments, the tissue section includes a retina cell. In embodiments, the tissue section includes a lymphoblast. In embodiments, the tissue section includes a hepatocyte. In embodiments, the tissue section includes a glial cell. In embodiments, the tissue section includes an astrocyte. In embodiments, the tissue section includes a radial glia. In embodiments, the tissue Attorney Docket No.: 051385-585001WO section includes a pericyte. In embodiments, the tissue section includes a stem cell. In embodiments, the tissue section includes a neural stem cell. [0320] In embodiments, the tissue section includes a cell bound to a known antigen. In embodiments, the cell is a cell that selectively binds to a desired target, wherein the target is an antibody, or antigen binding fragment, an aptamer, affimer, non-immunoglobulin scaffold, small molecule, or genetic modifying agent. In embodiments, the cell is a leukocyte (i.e., a white-blood cell). In embodiments, leukocyte is a granulocyte (neutrophil, eosinophil, or basophil), monocyte, or lymphocyte (T cells and B cells). In embodiments, the cell is a lymphocyte. In embodiments, the cell is a T cell, an NK cell, or a B cell. [0321] In embodiments, the tissue section includes an immune cell. In embodiments, the immune cell is a granulocyte, a mast cell, a monocyte, a neutrophil, a dendritic cell, or a natural killer (NK) cell. In embodiments, the immune cell is an adaptive cell, such as a T cell, NK cell, or a B cell. In embodiments, the cell includes a T cell receptor gene sequence, a B cell receptor gene sequence, or an immunoglobulin gene sequence. In embodiments, the immune cell is a granulocyte. In embodiments, the immune cell is a mast cell. In embodiments, the immune cell is a monocyte. In embodiments, the immune cell is a neutrophil. In embodiments, the immune cell is a dendritic cell. In embodiments, the immune cell is a natural killer (NK) cell. In embodiments, the immune cell is a T cell. In embodiments, the immune cell is a B cell. In embodiments, the cell includes a T cell receptor gene sequence. In embodiments, the cell includes a B cell receptor gene sequence. In embodiments, the cell includes an immunoglobulin gene sequence. In embodiments, the plurality of target nucleic acids includes non-contiguous regions of a nucleic acid molecule. In embodiments, the non-contiguous regions include regions of a VDJ recombination of a B cell or T cell. [0322] In embodiments, the tissue section includes a cancer cell. In embodiments, the cancer is lung cancer, colorectal cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, lymphoma, leukemia, or a cancer associated with aberrant K-Ras, aberrant APC, aberrant Smad4, aberrant p53, or aberrant TGFβ. In embodiments, the cancer cell includes a ERBB2, KRAS, TP53, PIK3CA, or FGFR2 gene. In embodiments, the cancer cell includes a cancer-associated gene (e.g., an oncogene associated with kinases and genes involved in DNA repair) or a cancer-associated biomarker. A “biomarker” is a substance that is associated with a Attorney Docket No.: 051385-585001WO particular characteristic, such as a disease or condition. A change in the levels of a biomarker may correlate with the risk or progression of a disease or with the susceptibility of the disease to a given treatment. In embodiments, the cancer is Acute Myeloid Leukemia, Adrenocortical Carcinoma, Bladder Urothelial Carcinoma, Breast Ductal Carcinoma, Breast Lobular Carcinoma, Cervical Carcinoma, Cholangiocarcinoma, Colorectal Adenocarcinoma, Esophageal Carcinoma, Gastric Adenocarcinoma, Glioblastoma Multiforme, Head and Neck Squamous Cell Carcinoma, Hepatocellular Carcinoma, Kidney Chromophobe Carcinoma, Kidney Clear Cell Carcinoma, Kidney Papillary Cell Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma, Lung Squamous Cell Carcinoma, Mesothelioma, Ovarian Serous Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Paraganglioma & Pheochromocytoma, Prostate Adenocarcinoma, Sarcoma, Skin Cutaneous Melanoma, Testicular Germ Cell Cancer, Thymoma, Thyroid Papillary Carcinoma, Uterine Carcinosarcoma, Uterine Corpus Endometrioid Carcinoma, or Uveal Melanoma. In embodiments, the cancer-associated gene is a nucleic acid sequence identified within The Cancer Genome Atlas Program, accessible at www.cancer.gov/tcga. [0323] In embodiments, the cancer-associated biomarker is MDC, NME-2, KGF, PlGF, Flt- 3L, HGF, MCP1, SAT-1, MIP-1-b, GCLM, OPG, TNF RII, VEGF-D, ITAC, MMP-10, GPI, PPP2R4, AKR1B1, Amy1A, MIP-1b, P-Cadherin, or EPO. In embodiments, the cancer- associated gene is a AKT1, AKT2, AKT3, ALK, AR, ARAF, ARID1A, ATM, ATR, ATRX, AXL, BAP1, BRAF, BRCA1, BRCA2, BTK, CBL, CCND1, CCND2, CCND3, CCNE1, CDK12, CDK2, CDK4, CDK6, CDKN1B, CDKN2A, CDKN2B, CHEK1, CHEK2, CREBBP, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ERCC2, ERG, ESR1, ETV1, ETV4, ETV5, EZH2, FANCA, FANCD2, FANCI, FBXW7, FGF19, FGF3, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FOXL2, GATA2, GNA11, GNAQ, GNAS, H3F3A, HIST1H3B, HNF1A, HRAS, IDH1, IDH2, IGF1R, JAK1, JAK2, JAK3, KDR, KIT, KNSTRN, KRAS, MAGOH, MAP2K1, MAP2K2, MAP2K4, MAPK1, MAX, MDM2, MDM4, MED12, MET, MLH1, MRE11A, MSH2, MSH6, MTOR, MYB, MYBL1, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NRAS, NRG1, NTRK1, NTRK2, NTRK3, NUTM1, PALB2, PDGFRA, PDGFRB, PIK3CA, PIK3CB, PIK3R1, PMS2, POLE, PPARG, PPP2R1A, PRKACA, PRKACB, PTCH1, PTEN, PTPN11, RAC1, RAD50, RAD51, RAD51B, RAD51C, RAD51D, RAF1, RB1, RELA, RET, RHEB, RHOA, RICTOR, RNF43, ROS1, RSPO2, RSPO3, SETD2, SF3B1, SLX4, SMAD4, SMARCA4, SMARCB1, Attorney Docket No.: 051385-585001WO SMO, SPOP, SRC, STAT3, STK11, TERT, TOP1, TP53, TSC1, TSC2, U2AF1, or XPO1 gene. In embodiments, the cancer-associated gene is a ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, or VHL gene. In embodiments, the tissue section includes a cell (e.g., a T cell) within a tumor. In embodiments, the tissue section includes a non-allogenic cell (i.e., native cell to the subject) within a tumor. In embodiments, the tissue section includes a tumor infiltrating lymphocyte (TIL). In embodiments, the tissue section includes an allogenic cell. In embodiments, the tissue section includes a circulating tumor cell. [0324] In embodiments, the tissue section is obtained from a subject (e.g., human or animal tissue). Once obtained, the tissue section is placed in an artificial environment in plastic or glass containers supported with specialized medium containing essential nutrients and growth factors to support proliferation. In embodiments, the tissue section is permeabilized and immobilized to a solid support surface. In embodiments, the tissue section is permeabilized and immobilized to an array (i.e., to discrete locations arranged in an array). In embodiments, the tissue section is immobilized to a solid support surface. In embodiments, the surface includes a patterned surface (e.g., suitable for immobilization of a plurality of cells in an ordered pattern. The discrete regions of the ordered pattern may have defined locations in a regular array, which may correspond to a rectilinear pattern, circular pattern, hexagonal pattern, or the like. These discrete regions are separated by interstitial regions. As used herein, the term “interstitial region” refers to an area in a substrate or on a surface that separates other areas of the substrate or surface. In embodiments, a plurality of cells are immobilized on a patterned surface that have a mean or median separation from one another of about 10-20 µm. In embodiments, a plurality of cells are immobilized on a patterned surface that have a mean or median separation from one another of about 10-20; 10-50; or 100 µm. In embodiments, a plurality of cells are arrayed on a substrate. In embodiments, a plurality of cells are immobilized in a 96-well microplate having a mean or median well-to-well spacing of about 8 mm to about 12 mm (e.g., about 9 mm). In embodiments, a plurality of cells are immobilized in a 384-well microplate having a mean or median well-to-well spacing of about 3 mm to about 6 mm (e.g., about 4.5 mm). Attorney Docket No.: 051385-585001WO [0325] In embodiments, the tissue section is attached to the receiving substrate via a bioconjugate reactive linker. In embodiments, the tissue section is attached to the substrate via a specific binding reagent. In embodiments, the specific binding reagent includes an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer. In embodiments, the specific binding reagent includes an antibody, or antigen binding fragment, an aptamer, affimer, or non-immunoglobulin scaffold. In embodiments, the specific binding reagent is a peptide, a cell penetrating peptide, an aptamer, a DNA aptamer, an RNA aptamer, an antibody, an antibody fragment, a light chain antibody fragment, a single-chain variable fragment (scFv), a lipid, a lipid derivative, a phospholipid, a fatty acid, a triglyceride, a glycerolipid, a glycerophospholipid, a sphingolipid, a saccharolipid, a polyketide, a polylysine, polyethyleneimine, diethylaminoethyl (DEAE)-dextran, cholesterol, or a sterol moiety. Substrates may be prepared for selective capture of particular cells of the tissue section. For example, a substrate containing a plurality of bioconjugate reactive moieties or a plurality of specific binding reagents, optionally in an ordered pattern, contacts a plurality of cells of the tissue section. Only cells of the tissue section containing complementary bioconjugate reactive moieties or complementary specific binding reagents are capable of reacting, and thus adhering, to the substrate. [0326] In embodiments, the methods are performed in situ in tissue sections that have been prepared according to methodologies known in the art. Methods for permeabilization and fixation of cells and tissue samples are known in the art, as exemplified by Cremer et al., The Nucleus: Volume 1: Nuclei and Subnuclear Components, R. Hancock (ed.) 2008; and Larsson et al., Nat. Methods (2010) 7:395-397, the content of each of which is incorporated herein by reference in its entirety. In embodiments, the tissue section is cleared (e.g., digested) of proteins, lipids, or proteins and lipids. [0327] In embodiments, the tissue section is exposed to paraformaldehyde (i.e., by contacting the cell with paraformaldehyde). Any suitable permeabilization and fixation technologies can be used for making the cell available for the detection methods provided herein. In embodiments the method includes affixing single cells or tissues to a transparent substrate. Exemplary tissue include those from skin tissue, muscle tissue, bone tissue, organ tissue and the like. In embodiments, the method includes immobilizing the tissue section in situ to a substrate and Attorney Docket No.: 051385-585001WO permeabilized for delivering probes, enzymes, nucleotides and other components required in the reactions. In embodiments, the tissue section includes many cells from a tissue section in which the original spatial relationships of the cells are retained. In embodiments, the tissue section in situ is within a Formalin-Fixed Paraffin-Embedded (FFPE) sample. In embodiments, the tissue section is subjected to paraffin removal methods, such as methods involving incubation with a hydrocarbon solvent, such as xylene or hexane, followed by two or more washes with decreasing concentrations of an alcohol, such as ethanol. The tissue section may be rehydrated in a buffer, such as PBS, TBS or MOPs. In embodiments, the FFPE sample is incubated with xylene and washed using ethanol to remove the embedding wax, followed by treatment with Proteinase K to permeabilized the tissue. In embodiments, the tissue section is fixed with a chemical fixing agent. In embodiments, the chemical fixing agent is formaldehyde or glutaraldehyde. In embodiments, the chemical fixing agent is glyoxal or dioxolane. In embodiments, the chemical fixing agent includes one or more of ethanol, methanol, 2-propanol, acetone, and glyoxal. In embodiments, the chemical fixing agent includes formalin, Greenfix®, Greenfix® Plus, UPM, CyMol®, HOPE®, CytoSkelFixTM, F-Solv®, FineFIX®, RCL2/KINFix, UMFIX, Glyo-Fixx®, Histochoice®, or PAXgene®. In embodiments, the tissue section is fixed within a synthetic three- dimensional matrix (e.g., polymeric material). In embodiments, the synthetic matrix includes polymeric-crosslinking material. In embodiments, the material includes polyacrylamide, poly- ethylene glycol (PEG), poly(acrylate-co-acrylic acid) (PAA), or Poly(N-isopropylacrylamide) (NIPAM). [0328] In embodiments, the fixed tissue may be frozen tissue. The frozen biological tissue can be fixed using a fixing agent, which is suitably an organic fixing agent. In some embodiments, the fixing agent can be chilled and can be at a temperature of about 0° C to about 100° C, suitably about zero to about 50° C, or about 1° C to about 50° C. The fixing agent can be chilled by placing it over a bed of ice to maintain its temperature as close to 0° C as possible. The frozen biological tissue can be treated with the fixing agent using any suitable technique, suitably by immersing it in the fixing agent for a period of time. Depending on the type and size of the biological tissue sample, the treatment time can range from about 5 minutes to about 60 minutes, suitably about 10 minutes to about 30 minutes, or about 15 minutes to about 25 minutes, or about 20 minutes. In some embodiments, treatment time may be overnight. During fixing, the snap- Attorney Docket No.: 051385-585001WO frozen tissue will thaw but will suitably remain at a low temperature due to the low temperature environment of the fixing agent. [0329] In some embodiments, the type/identity of a fixation agent, the amount/concentration of a fixation agent, the temperature at which it is used, the duration for which it is used, and the like, may be empirically determined or titrated. These parameters, and others, may need to be varied to obtain optimal results for different tissues, for different organisms, or for different days on which an experiment is performed. Insufficient fixation (e.g., too little fixing agent, too low temperature, too short duration) may not, for example, stabilize/preserve the cells/organelles/analytes of tissues. Excess fixation (e.g., too much fixing agent, too high temperature, too long duration) may result in the single biological samples (e.g., cells/nuclei) obtained from the methods not yielding good results in single biological sample (e.g., single-cell or single nucleus) workflows or assays in which the biological samples (e.g., cells or nuclei) are used. Generally, the quality of data obtained in these workflows/assays may be a good measure of the extent of the fixation process. [0330] In some embodiments, the fixative can be diluted in a buffer, e.g., saline, phosphate buffer (PB), phosphate buffered saline (PBS), citric acid buffer, potassium phosphate buffer, etc., usually at a concentration of about 1-10%, e.g.1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%, for example, 4% paraformaldehyde/0.1 M phosphate buffer; 2% paraformaldehyde/0.2% picric acid/0.1 M phosphate buffer; 4% paraformaldehyde/0.2% periodate/1.2% lysine in 0.1 M phosphate buffer; 4% paraformaldehyde/0.05% glutaraldehyde in phosphate buffer; etc. The type of fixative used and the duration of exposure to the fixative can depend on the sensitivity of the molecules of interest in the tissue section to denaturation by the fixative, and can be readily determined using conventional histochemical or immunohistochemical techniques, for example as described in Buchwalow and Bocker. Immunohistochemistry: Basics and Methods. Springer- Verlag Berlin Heidelberg 2010. [0331] During the fixing, the biological tissue sample can be periodically cut into successively smaller segments while it is submerged in the fixation solution, to facilitate perfusion and fixation of the biological tissue sample by the organic fixing agent. For example, the tissue sample may have an initial length, width and/or diameter of about 0.25 cm to about 1.5 cm or may be initially cut into segments having such suitable dimensions. After a first periodic interval, Attorney Docket No.: 051385-585001WO the tissue sample or segments can be cut into smaller segments, and the smaller segments can remain immersed in the fixing agent. This process can be repeated after a second periodic interval, after a third periodic interval, after a fourth periodic interval, and so on. The periodic intervals can range from about 1 to about 10 minutes, or about 2 to about 8 minutes, or about 4 to about 6 minutes. The sum of the periodic intervals can equal the entire fixing time and can range from about 5 to about 60 minutes, or about 10 to about 30 minutes, or about 15 to about 25 minutes, for example. The resulting fixed tissue segments can have a length, width and/or diameter in a range of less than 1 mm to about 10 mm, by way of example. In some embodiments, the tissue is not cut into smaller segments during fixation. In some embodiments, this may be performed prior to fixation. In some embodiments, this may be performed after fixation. [0332] Once the biological tissue segments have been sufficiently fixed, the fixation process may be stopped and/or the tissue may be removed from the fixation and the tissue may be washed. Generally, fixation is stopped to cease additional activity of the fixative on the tissue. Fixation may also be stopped so that any subsequent biochemical reactions performed on the tissue (e.g., enzymatic cell dissociation) can function. In some embodiments, the tissue segments may be treated or contacted with a quenching medium to quench the fixation. The term “quenching” means to stop the fixation reaction, i.e., the chemical interactions that cause the fixation. Quenching the fixation can be accomplished by immersing the fixed tissue segments in a suitable quenching medium. The fixation quenching medium can be chilled and can have a temperature of about 0° C to about 100° C, or about 1° C to about 50° C. In embodiments, the quenching medium is a phosphate buffer solution (PBS). One suitable phosphate buffer solution is 1×PBS, available from Sigma Aldrich Corp.1×PBS has a pH of about 7.4 and the following composition in water: NaCl—137 mM, KCl—2.7 mM, Na2HPO4—10 mM, KH2PO4—1.8 mM. In one embodiment, the phosphate buffer solution can be combined with fetal bovine serum (FBS) to aid in quenching the fixation reaction. FBS is the liquid fraction of clotted blood from fetal calves, depleted of cells, fibrin and clotting factors, but containing many nutritional and macromolecular factors essential for cell growth. Bovine serum albumin (BSA) is the major component of FBS. The fetal bovine serum can be combined with the phosphate buffer solution at a concentration of about 1% to about 25% by weight FBS and about 75% to about 99% by weight PBS, suitably about 5% to about 15% by weight FBS and about 85% to about 95% by Attorney Docket No.: 051385-585001WO weight PBS, or about 10% by weight FBS and about 90% by weight PBS. In another embodiment, a solution of concentrated ethanol in water can be used instead of the PBS in the quenching medium. The ethanol solution can contain about 50% to about 90% by weight ethanol, or about 55% to about 85% by weight ethanol, or about 60% to about 80% by weight ethanol, or about 70% by weight ethanol. In some embodiments, fixation may be quenched using a quenching solution that does not contain serum. In some examples, Tris-based buffers may be used. In some examples, PBS + 50 mM Tris pH 8.0 + 0.02% BSA (RNAse free) + 0.1 U/ul of RNAse Inhibitor may be used. In some examples, the tissue may be removed from the fixative and washed using a quenching solution or biological buffer. [0333] In embodiments, the tissue section is lysed to release nucleic acid or other materials from the cells. For example, the tissue section may be lysed using reagents (e.g., a surfactant such as Triton-X or SDS, an enzyme such as lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase, proteinase K, etc.) or a physical lysing mechanism a physical condition (e.g., ultrasound, ultraviolet light, mechanical agitation, etc.). The cells may release, for instance, DNA, RNA, mRNA, proteins, or enzymes. The cells may arise from any suitable source. For instance, the cells may be any cells for which nucleic acid from the cells is desired to be studied or sequenced, etc., and may include one, or more than one, cell type. The cells may be for example, from a specific population of cells, such as from a certain organ or tissue (e.g., cardiac cells, immune cells, muscle cells, cancer cells, etc.), cells from a specific individual or species (e.g., human cells, mouse cells, bacteria, etc.), cells from different organisms, cells from a naturally-occurring sample (e.g., pond water, soil, etc.), or the like. In some cases, the cells may be dissociated from tissue. In embodiments, the method does not include dissociating the cell from the tissue or the cellular microenvironment. In embodiments, the method does not include lysing the tissue section. [0334] In embodiments, a permeabilization solution can contain additional reagents or a biological sample may be treated with additional reagents in order to optimize biological sample permeabilization. In some embodiments, an additional reagent is an RNA protectant. As used herein, the term “RNA protectant” typically refers to a reagent that protects RNA from RNA nucleases (e.g., RNases). Any appropriate RNA protectant that protects RNA from degradation can be used. A non-limiting example of an RNA protectant includes organic solvents (e.g., at Attorney Docket No.: 051385-585001WO least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% v/v organic solvent), which includes ethanol, methanol, propan-2-ol, acetone, trichloroacetic acid, propanol, polyethylene glycol, acetic acid, or a combination thereof. In embodiments, the RNA protectant includes ethanol, methanol and/or propan-2-ol, or a combination thereof. In embodiments, the RNA protectant includes RNAlater ICE (ThermoFisher Scientific). In embodiments, the RNA protectant includes a salt. The salt may include ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, potassium chloride, potassium sulfate, sodium chloride, sodium acetate, sodium sulfate, zinc chloride, zinc acetate and zinc sulfate. In some embodiments, the biological sample is treated with one or more RNA protectants before, contemporaneously with, or after permeabilization. [0335] In embodiments, the method includes imaging the immobilized tissue section. In embodiments, the method further includes an imaging modality, immunofluorescence (IF), or immunohistochemistry modality (e.g., immunostaining). In embodiments, the method includes ER staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the endoplasmic reticula), Golgi staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the Golgi), F-actin staining (e.g., contacting the tissue section with a phalloidin-conjugated dye that binds to actin filaments), lysosomal staining (e.g., contacting the tissue section with a cell-permeable dye that accumulates in the lysosome via the lysosome pH gradient), mitochondrial staining (e.g., contacting the tissue section with a cell-permeable dye which localizes to the mitochondria), nucleolar staining, or plasma membrane staining. For example, the method includes live cell imaging (e.g., obtaining images of the tissue section) prior to or during fixing, immobilizing, and permeabilizing the tissue section. Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, commonly detected and examined with the light microscope. Known IHC modalities may be used, such as the protocols described in Magaki, S., Hojat, S. A., Wei, B., So, A., & Yong, W. H. (2019). Methods in molecular biology (Clifton, N.J.), 1897, 289–298, which is incorporated herein by reference. In embodiments, the additional imaging modality includes bright field microscopy, phase contrast microscopy, Nomarski differential-interference-contrast microscopy, or dark field microscopy. In Attorney Docket No.: 051385-585001WO embodiments, the method further includes determining the cell morphology of the tissue section (e.g., the cell boundary or cell shape) using known methods in the art. For example, to determining the cell boundary includes comparing the pixel values of an image to a single intensity threshold, which may be determined quickly using histogram-based approaches as described in Carpenter, A. et al Genome Biology 7, R100 (2006) and Arce, S., Sci Rep 3, 2266 (2013)). By “microscopic analysis” is meant the analysis of a specimen using techniques that provide for the visualization of aspects of a specimen that cannot be seen with the unaided eye, i.e., that are not within the resolution range of the normal human eye. Such techniques may include, without limitation, optical microscopy, e.g., bright field, oblique illumination, dark field, phase contrast, differential interference contrast, interference reflection, epifluorescence, confocal microscopy, CLARITY-optimized light sheet microscopy (COLM), light field microscopy, tissue expansion microscopy, etc., laser microscopy, such as, two photon microscopy, electron microscopy, and scanning probe microscopy. By “preparing a biological specimen for microscopic analysis” is generally meant rendering the specimen suitable for microscopic analysis at an unlimited depth within the specimen. In embodiments, the immobilized tissue section is imaged using “optical sectioning” techniques, such as laser scanning confocal microscopes, laser scanning 2-Photon microscopy, parallelized confocal (i.e. spinning disk), computational image deconvolution methods, and light sheet approaches. Optical sectioning microscopy methods provide information about single planes of a volume by minimizing contributions from other parts of the volume and do so without physical sectioning. The resulting “stack” of such optically sectioned images, represents a full reconstruction of the 3-dimensional features of a tissue volume. A typical confocal microscope includes a 10×/0.5 objective (dry; working distance, 2.0 mm) and/or a 20×/0.8 objective (dry; working distance, 0.55 mm), with a s z-step interval of 1 to 5 μm. A typical light sheet fluorescence microscope includes an sCMOS camera, a 2×/0.5 objective lens, and zoom microscope body (magnification range of ×0.63 to ×6.3). For entire scanning of whole samples, the z-step interval is 5 or 10 μm, and for image acquisition in the regions of interest, an interval in the range of 2 to 5 μm may be used. [0336] To microscopically visualize tissue sections prepared by the subject methods, in some embodiments the tissue section is embedded in a mounting medium. Mounting medium is typically selected based on its suitability for the reagents used to visualize the cellular Attorney Docket No.: 051385-585001WO biomolecules, the refractive index of the tissue section, and the microscopic analysis to be performed. For example, for phase-contrast work, the refractive index of the mounting medium should be different from the refractive index of the specimen, whereas for bright-field work the refractive indexes should be similar. As another example, for epifluorescence work, a mounting medium should be selected that reduces fading, photobleaching or quenching during microscopy or storage. In certain embodiments, a mounting medium or mounting solution may be selected to enhance or increase the optical clarity of the cleared tissue specimen. Nonlimiting examples of suitable mounting media that may be used include glycerol, CC/Mount™, Fluoromount™ Fluoroshield™, ImmunHistoMount™, Vectashield™, Permount™, Acrytol™, CureMount™, FocusClear™, or equivalents thereof. [0337] The biological targets or molecules to be detected can be any biological molecules including but not limited to proteins, nucleic acids, lipids, carbohydrates, ions, or multicomponent complexes containing any of the above. Examples of subcellular targets include organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. Exemplary nucleic acid targets can include genomic DNA of various conformations (e.g., A-DNA, B-DNA, Z-DNA), mitochondria DNA (mtDNA), mRNA, tRNA, rRNA, hRNA, miRNA, and piRNA. For example, following immobilization on the receiving substrate, the sections may be fixed with methanol, permeabilized with 0.025% Triton in PBS solution, and stained with primary antibodies directed against vimentin (fibroblasts) and macrophages, followed by secondary antibody labeling (e.g., Alexa-594 conjugated secondary antibodies). Additional counterstaining may be performed, for example using 4,6-diamidino-2-phenylindole (DAPI) mounting media to counterstain nuclei. [0338] In embodiments, the collection of information (e.g., sequencing information and cell morphology) is referred to as a signature. The term “signature” may encompass any gene or genes, protein or proteins, or epigenetic element(s) whose expression profile or whose occurrence is associated with a specific cell type, subtype, or cell state of a specific cell type or subtype within a population of cells. It is to be understood that also when referring to proteins (e.g., differentially expressed proteins), such may fall within the definition of “gene” signature. Levels of expression or activity or prevalence may be compared between different cells in order to characterize or identify for instance signatures specific for cell (sub)populations. Increased or Attorney Docket No.: 051385-585001WO decreased expression or activity of signature genes may be compared between different cells in order to characterize or identify for instance specific cell (sub)populations. [0339] In embodiments, the methods described herein may further include constructing a 3- dimensional pattern of abundance, expression, and/or activity of each target from spatial patterns of abundance, expression, and/or activity of each target of multiple samples. In embodiments, the multiple samples can be consecutive tissue sections of a 3-dimensional tissue sample. [0340] In embodiments, the method further includes digesting the tissue section by contacting the sample-carrier construct with an endopeptidase. In embodiments, the endopeptidase is pepsin. [0341] In embodiments, the method further includes removing the embedding material from the sample. For example, if the embedding material is paraffin wax, the embedding material is removed by contacting the sample-carrier construct with a hydrocarbon solvent, such as xylene or hexane, followed by two or more washes with decreasing concentrations of an alcohol, such as ethanol. [0342] In embodiments, the method includes measuring changes in the amount of biomaterial present in a well relative to a control (e.g., a sample obtained at a different time point or exposed to alternate conditions). As used herein, “biomaterial” refers to any biological material produced by an organism. In some embodiments, biomaterial includes secretions, extracellular matrix, proteins, lipids, organelles, membranes, cells, portions thereof, and combinations thereof. In some embodiments, cellular material includes secretions, extracellular matrix, proteins, lipids, organelles, membranes, cells, portions thereof, and combinations thereof. In some embodiments, biomaterial includes viruses. In some embodiments, the biomaterial is a replicating virus and thus includes virus infected cells. [0343] In embodiments, additional methods may be performed to further characterize the sample. For example, in addition to sequencing, the method includes protein analysis, lipid analysis, metabolite analysis (e.g., glucose analysis), or measuring the transcriptomic profile, gene expression activity, genomic profile, protein expression activity, proteomic profile, protein interaction activity, cellular receptor expression activity, lipid profile, lipid activity, carbohydrate profile, microvesicle activity, glucose activity, and combinations thereof. Attorney Docket No.: 051385-585001WO [0344] In some embodiments, determining the spatial location of RNA molecules within a tissue sample includes correlating the location of the cluster of capture probes (e.g., the cluster including the first plurality of immobilized capture probes and second plurality of immobilized capture probes) on the substrate with a corresponding location within the tissue sample. In some embodiments, the spatial location of the RNA molecules in the tissue sample may allow identification of a single cell expressing the RNA molecules. [0345] In some embodiments, the methods described herein may include each of the following steps (in no particular order): a. providing a solid support as described herein (e.g., a solid support including clusters including a plurality of immobilized double-stranded polynucleotides; b. determining the sequence of the spatial barcode for at least one polynucleotide in each cluster on the solid support; c. assigning each cluster a location (e.g., XY coordinate) on the solid support based upon the sequence of the spatial barcode; d. hybridizing an invasion primer including a capture sequence to a strand of the the double-stranded polynucleotide and incorporating the capture sequence into the strand; e. contacting the solid support with a tissue sample and allowing RNA molecules in the tissue sample to bind to the capture probes; f. imaging the tissue sample while the sample is bound to the solid support; g. generating cDNA molecules from the RNA molecules bound to the capture probes; h. determining the sequence of the spatial barcode for the cDNA molecules and correlating this sequence with the location of a corresponding cluster on the substrate (e.g., cluster of capture probes containing the corresponding spatial barcode); i. correlating the location of the corresponding cluster of capture probes on the solid support with a corresponding location within the tissue sample, thus identifying the spatial location of RNA (e.g., gene) expression in the sample. [0346] Sequencing of the cDNA molecules enables determination of gene expression in the tissue sample, as cDNA is considered indicative of RNA expression in the tissue at the time it was isolated. Accordingly, determining the location within the tissue to which the sequence of the spatial barcode for the cDNA molecules corresponds allows for localized, spatial detection of RNA expression in the tissue sample. In some embodiments, the methods described herein have a high enough resolution to enable determination of gene expression in a single cell. [0347] In some embodiments, the methods may further include analyzing the tissue sample for the presence of one or more additional targets, such as targets bound to the additional capture Attorney Docket No.: 051385-585001WO moieties on the substrate. For example, the methods may further include determining whether the tissue sample additionally contains one or more proteins of interest, which may be detected by an antibody conjugated capture moiety on the substrate. In some embodiments, the location of the additional capture moieties on the substrate may be known and thus used to determine the corresponding location of the additional target in the tissue sample. For example, the location of the additional capture moieties on the substrate may be known based upon the location of the cluster of capture probes in which the additional capture moieties are integrated. [0348] Flow cells provide a convenient format for housing an array of clusters produced by the methods described herein, in particular when subjected to an SBS or other detection technique that involves repeated delivery of reagents in cycles. For example, to initiate a first SBS cycle, one or more labeled nucleotides and a DNA polymerase in a buffer, can be flowed into/through a flow cell that houses an array of clusters. The clusters of an array where primer extension causes a labeled nucleotide to be incorporated can then be detected. Optionally, the nucleotides can further include a reversible termination moiety that temporarily halts further primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent (e.g., a reducing agent) is delivered to remove the moiety. Thus, for embodiments that use reversible termination, a deblocking reagent (e.g., a reducing agent) can be delivered to the flow cell (before, during, or after detection occurs). Washes can be carried out between the various delivery steps as needed. The cycle can then be repeated N times to extend the primer by N nucleotides, thereby detecting a sequence of length N. Example SBS procedures, fluidic systems and detection platforms that can be readily adapted for use with an array produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), US Patent Publication 2018/0274024, WO 2017/205336, US Patent Publication 2018/0258472, each of which are incorporated herein in their entirety for all purposes. [0349] Use of the sequencing method outlined above is a non-limiting example, as essentially any sequencing methodology which relies on successive incorporation of nucleotides into a polynucleotide chain can be used. Suitable alternative techniques include, for example, Attorney Docket No.: 051385-585001WO pyrosequencing methods, FISSEQ (fluorescent in situ sequencing), MPSS (massively parallel signature sequencing), or sequencing by ligation-based methods. [0350] In embodiments, generating a sequencing read includes determining the identity of the nucleotides in the template polynucleotide (or complement thereof). In embodiments, a sequencing read, e.g., a first sequencing read or a second sequencing read, includes determining the identity of a portion (e.g., 1, 2, 5, 10, 20, 50 nucleotides) of the total template polynucleotide. In embodiments the first sequencing read determines the identity of 5-10 nucleotides and the second sequencing read determines the identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides). In embodiments the first sequencing read determines the identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides) and the second sequencing read determines the identity of 5-10 nucleotides. In embodiments, following the generation of a sequencing read, subsequent extension is performed using a plurality of standard (e.g., non-modified) dNTPs until the complementary strand is copied. In other embodiments, following the generation of a sequencing read, subsequent extension is performed using a plurality of dideoxy nucleotide triphosphates (ddNTPs) to prevent further extension of the first sequencing read product during a second sequencing read. In embodiments, following the identification of at least 5-10 (e.g., 11 to 200 nucleotides, or up to 1000 nucleotides), subsequent extension is performed using a plurality of standard (e.g., non-modified) dNTPs until the complementary strand is copied. In embodiments, following the identification of at least 5-10 (e.g., 11 to 200 nucleotides, or up to 1000 nucleotides), subsequent extension is performed using a plurality of dideoxy nucleotide triphosphates (ddNTPs) to prevent further extension of the sequencing read product. [0351] In embodiments, following the generation of a first sequencing read, the cleavable site located within the invasion primer is cleaved, thereby exposing a free 5’ phosphate in the invasion strand. In embodiments, following the generation of a first sequencing read, the cleavable site located within the invasion primer is cleaved with a cleaving agent, thereby exposing a free 5’ phosphate in the invasion strand, and the invasion strand is removed (e.g., enzymatically digested using an exonuclease enzyme). In embodiments, following generation of a first sequencing read, the invasion strand is cleaved at one or more cleavable sites. In embodiments, following cleavage at one or more cleavable sites, the extension product of the invasion primer (i.e., the invasion strand) is removed under suitable non-aggressive conditions Attorney Docket No.: 051385-585001WO (e.g., degraded or denatured under conditions that leave the complementary strand intact, and optionally still hybridized to at least a portion of the invasion primer). In embodiments, the cleavable site is a dU. In embodiments, the cleaving agent includes a glycosylase and one or more suitable endonucleases. In embodiments, cleavage is performed under alkaline (e.g., pH greater than 8) buffer conditions at between 40°C to 80°C. In embodiments, degradation of the invasion strand is enzymatic degradation. In embodiments, degradation of the invasion strand is accomplished with a 5’ to 3’ exonuclease. In embodiments, the 5’ to 3’ exonuclease is lambda exonuclease, or a mutant thereof. In embodiments, following the degradation of the invasion strand, the cleaved invasion primer subsequently initiates a second sequencing read. In embodiments, the second sequencing read is generated without removal of the first sequencing read. In embodiments, the invasion primer (or a portion thereof) is the sequencing primer. [0352] In embodiments, the sequencing method relies on the use of modified nucleotides that can act as reversible reaction terminators. Once the modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3’-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides. Once the identity of the base incorporated into the growing chain has been determined, the 3’ reversible terminator may be removed to allow addition of the next successive nucleotide. These such reactions can be done in a single experiment if each of the modified nucleotides has attached a different label, known to correspond to the particular base, to facilitate discrimination between the bases added at each incorporation step. Alternatively, a separate reaction may be carried out containing each of the modified nucleotides separately. [0353] In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3′ end of the extension strand and hybridizing a second sequencing primer to the second strand and incorporating one or more nucleotides into the second sequencing primer with a polymerase to create a second extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said second extension strand. In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the second extension strand; removing the invasion strand; hybridizing Attorney Docket No.: 051385-585001WO a third sequencing primer to the first strand and incorporating one or more nucleotides into the third sequencing primer with a polymerase to create a third extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said third extension strand. In embodiments, the method includes terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the third extension strand; and hybridizing a fourth sequencing primer to the first strand and incorporating one or more nucleotides into the fourth sequencing primer with a polymerase to create a fourth extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said fourth extension strand. In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3′ end of the extension strand; hybridizing a second sequencing primer to the second strand and incorporating one or more nucleotides into the second sequencing primer with a polymerase to create a second extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said second extension strand; terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the second extension strand; removing the invasion strand; hybridizing a third sequencing primer to the first strand and incorporating one or more nucleotides into the third sequencing primer with a polymerase to create a third extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said third extension strand; terminating extension by incorporating one or more unmodified dNTPs or one or more ddNTPs into the 3′ end of the third extension strand; and hybridizing a fourth sequencing primer to the first strand and incorporating one or more nucleotides into the fourth sequencing primer with a polymerase to create a fourth extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said fourth extension strand. [0354] In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs and/or one or more ddNTPs into the 3′ end of the extension strand. In embodiments, the method further includes terminating extension by incorporating one or more unmodified dNTPs. In embodiments, the method further includes terminating extension by incorporating one or more ddNTPs into the 3′ end of the extension strand. Attorney Docket No.: 051385-585001WO [0355] In embodiments, the method further includes hybridizing a second sequencing primer to the second strand and incorporating one or more nucleotides (e.g., labeled nucleotides) with a polymerase into the second sequencing primer to create a second extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said second extension strand. In embodiments, the nucleotides are modified nucleotides including a label and a reversible terminator, as described herein. [0356] The modified nucleotides may carry a label (e.g., a fluorescent label) to facilitate their detection. Each nucleotide type may carry a different fluorescent label. However, the detectable label need not be a fluorescent label. Any label can be used which allows the detection of an incorporated nucleotide. One method for detecting fluorescently labeled nucleotides includes using laser light of a wavelength specific for the labeled nucleotides, or the use of other suitable sources of illumination. The fluorescence from the label on the nucleotide may be detected (e.g., by a CCD camera, CMOS camera, or other suitable detection means). [0357] In embodiments, the methods of sequencing a nucleic acid include extending a complementary polynucleotide (e.g., a primer) that is hybridized to the nucleic acid by incorporating a first nucleotide. In embodiments, the method includes a buffer exchange or wash step (e.g., between each sequencing cycle). In embodiments, the methods of sequencing a nucleic acid include a sequencing solution. The sequencing solution includes (a) an adenine nucleotide, or analog thereof; (b) (i) a thymine nucleotide, or analog thereof, or (ii) a uracil nucleotide, or analog thereof; (c) a cytosine nucleotide, or analog thereof; and (d) a guanine nucleotide, or analog thereof. [0358] In certain embodiments, the sequencing methods provided herein comprises sequencing both strands of a double-stranded nucleic acid with an error rate of 5 x 10-5 or less, 1 x 10-5 or less, 5 x 10-6 or less, 1 x 10-6 or less, 5 x 10-7 or less, 1 x 10-7 or less, 5 x 10-8 or less, or 1 x 10-8 or less. In certain embodiments, the sequencing methods provided herein comprises sequencing both strands of a double-stranded nucleic acid with an error rate of 5 x 10-5 to 1 x 10-8, 1 x 10-5 to 1 x 10-8, 5 x 10-5 to 1 x 10-7, 1 x 10-5 to 1 x 10-7, 5 x 10-6 to 1 x 10-8, or 1 x 10-6 to 1 x 10-8. In certain embodiments, the sequencing methods provided herein comprises sequencing both strands of a double-stranded nucleic acid with an error rate of 1 x 10-7 to 1 x 10-8. Attorney Docket No.: 051385-585001WO [0359] In an aspect is provided a method of reducing GC bias in a plurality of sequencing reads, the method including sequencing a template polynucleotide to generate a plurality of sequencing reads as described herein. In embodiments, the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, as described herein, for example wherein generating the first invasion strand includes a first plurality of invasion-primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles and extending the one or more invasion primers; generating a first sequencing read by hybridizing one or more sequencing primers to the first strand, and extending the one or more first sequencing primers. In embodiments, the invasion primer is not covalently attached to the solid support. [0360] In embodiments, the method includes: generating a double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) the double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to a solid support; generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, wherein generating the invasion strand comprises alternating between a first plurality of invasion-primer extension cycles and a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles and extending the one or more invasion primers; generating a first sequencing read by hybridizing one or more sequencing primers to the first strand, and extending the one or more first sequencing primers. [0361] In embodiments, generating the invasion strand includes a first plurality of invasion- primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different than the second plurality of invasion-primer extension cycles. In embodiments, the Attorney Docket No.: 051385-585001WO method further includes a third plurality of invasion-primer extension cycles, wherein the reaction conditions for the third plurality of invasion-primer extension cycles are optionally different than the first or second plurality of invasion-primer extension cycles. In embodiments, the method further includes a third plurality of invasion-primer extension cycles, wherein the reaction conditions for the third plurality of invasion-primer extension cycles are the same as the first plurality of invasion-primer extension cycles. [0362] In an aspect is provided a method of generating a template for nucleic acid sequencing reaction. In embodiments, the method includes providing a solid support including a plurality of immobilized oligonucleotide primers attached to the solid support via a linker, wherein the plurality of oligonucleotide primers include a plurality of forward primers and a plurality of reverse primers, amplifying a template nucleic acid by using the oligonucleotide primers attached to the solid support to generate a plurality of double-stranded amplification products, each double-stranded amplification product including a first strand hybridized to a second strand, wherein (i) each double-stranded amplification product includes the template polynucleotide or complement thereof, and (ii) the first strand and second strand are both attached to the solid support; and generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, and extending the one or more invasion primers; thereby generating a template nucleic acid for a nucleic acid sequencing reaction. In embodiments, the method further includes hybridizing one or more sequencing primers to the first strand. In embodiments, the method includes generating a cluster of ssDNA templates. In embodiments, the invasion primer is not covalently attached to the solid support. In embodiments, the invasion strand is not covalently attached to the solid support. [0363] In another aspect is provided a method including: amplifying a template nucleic acid by contacting the template nucleic acid with a plurality of oligonucleotide primers attached to a solid support to generate a plurality of double-stranded amplification products, each double- stranded amplification product including a first strand hybridized to a second strand, wherein the first strand and second strand are both attached to the solid support; and generating a first invasion strand hybridized to the second strand by hybridizing one or more invasion primers to the second strand, and extending the one or more invasion primers to produce a single-stranded first strand. In embodiments, the invasion primer is not covalently attached to the solid support. Attorney Docket No.: 051385-585001WO [0364] In an aspect is provided a method of removing a polynucleotide hybridized to a first strand, wherein the polynucleotide includes one or more of cleavable sites. In embodiments, the method includes fragmenting a polynucleotide in the presence of a plurality of dsDNA polynucleotides. In embodiments, the method includes contacting the polynucleotide with a cleaving agent thereby fragmenting the polynucleotide and generating two or more fragments. In embodiments, the method includes denaturing the fragments (e.g., contacting the fragments with a chemical denaturant, increasing the temperature, or a combination thereof). In embodiments, the method includes digesting the fragments (e.g., contacting the fragments with one or more exonuclease enzymes). In embodiments, the method includes modulating the temperature to be at or below the calculated or predicted melting temperature (Tm) of the fragments (e.g., about 0°C to about 65°C). In embodiments, the method includes modulating the temperature to be at about 50°C to about 65°C. [0365] In embodiments, the first strand is covalently attached to a solid support. In embodiments, the polynucleotide, alternatively referred to herein as the third polynucleotide and/or the invasion primer, is not attached to a solid support. In embodiments, the first strand is attached to a solid support, wherein the solid support includes a plurality of double-stranded polynucleotides. In embodiments, the first strand is in a colony of double-stranded polynucleotides. In embodiments, the solid support includes a second strand hybridized to a sequenced strand, wherein the sequenced strand includes one or more sequenced nucleotides. In embodiments, the sequenced nucleotides include a scar remnant (e.g., an alkynyl moiety attached to the nucleobase). In embodiments, the nucleotides have the , wherein B
Figure imgf000184_0001
is a nucleobase, R1 is the scar remnant, and ” is the attachment point to the remainder of the sequenced strand polynucleotide.
Figure imgf000184_0002
Attorney Docket No.: 051385-585001WO [0366] In embodiments, B is a divalent nucleobase. In ,
Figure imgf000185_0001
,
Figure imgf000185_0002
- - a or or substituted or unsubstituted heteroalkyl. In embodiments, R1 is hydrogen. In embodiments, R1 is -OH. In embodiments, R1 is -NH. In embodiments, R1 is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. In embodiments, R1 is a substituted or unsubstituted alkenyl. In embodiments, R1 is a substituted or unsubstituted alkynyl. In embodiments, R1 is a substituted or unsubstituted heteroalkenyl. In embodiments, R1 is a substituted or unsubstituted heteroalkynyl. In embodiments, R1 is a substituted (e.g., substituted with a substituent group, size-limited substituent group, or lower substituent group) or unsubstituted alkyl or substituted Attorney Docket No.: 051385-585001WO (e.g., substituted with a substituent group, size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl. In embodiments, R1 is substituted with an oxo or -OH. In embodiments, R1 is substituted with an oxo and -OH. [0368] In embodiments, R1 is an oxo-substituted heteroalkyl (e.g., 2 to 10 membered heteroalkyl, 2 to 8 membered heteroalkyl, or 4 to 8 membered heteroalkyl). In embodiments, R1 is an oxo-substituted heteroalkenyl (e.g., 2 to 10 membered heteroalkenyl, 2 to 8 membered heteroalkenyl, or 4 to 8 membered heteroalkenyl). In embodiments, R1 is an oxo-substituted heteroalkynyl (e.g., 2 to 10 membered heteroalkynyl, 2 to 8 membered heteroalkynyl, or 4 to 8 membered heteroalkynyl). In embodiments, R1 is an oxo-substituted 10 membered heteroalkynyl. In embodiments, R1 is an oxo-substituted 9 membered heteroalkynyl. In embodiments, R1 is an oxo-substituted 8 membered heteroalkynyl. In embodiments, R1 is an oxo-substituted 7 membered heteroalkynyl. In embodiments, R1 is an oxo-substituted 6 membered heteroalkynyl. [0369] In embodiments, the one or more nucleotides including a scar remnant include a ,
Figure imgf000186_0001
, Attorney Docket No.: 051385-585001WO ,
Figure imgf000187_0001
the fragments is about 50°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of the fragments is about 60°C to about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of the fragments is about 50°C to about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of the fragments is less than about 75°C. In embodiments, the calculated or predicted melting temperature (Tm) of the fragments is less than about 65°C. In embodiments, the calculated or predicted melting temperature (Tm) of the fragments is less than about 60°C. In embodiments, two or more fragments are generated. In embodiments, three or more fragments are generated. In embodiments, four or more fragments are generated. In embodiments, at least three fragments are generated. In embodiments, four fragments are generated. [0371] In embodiments, the fragments are 3-10 nucleotides in length. In embodiments, the fragments are 3-15 nucleotides in length. In embodiments, the fragments are 5 to 20 nucleotides in length. In embodiments, the fragments are 4 to 6 nucleotides in length. EXAMPLES Attorney Docket No.: 051385-585001WO Example 1. Sequencing of two strands of the same polynucleotide with efficient strand invasion [0372] Before a target nucleic acid is sequenced, some degree of DNA pre-processing and converting it to a library molecule is typically required. For example, these steps may involve fragmenting input polynucleotides into an appropriate platform-specific size range, followed by an end-polishing step to generate blunt-ended DNA fragments. Common nucleic acid sequences (referred to as adapter sequences) on the 3′ and 5′ ends are then ligated to these fragments. A functional library molecule typically includes the target molecule with specific adapter sequences added to the 3′ and 5′ ends, e.g., Illumina’s P5 and P7 adapters/primers, to ensure compatibility with the underlying flow cell, so it may be amplified appropriately. For example, common platform primers include 5’-AATGATACGGCGACCACCG (P5) (SEQ ID NO:60), or the complement thereof, and 5’-CAAGCAGAAGACGGCATACGA (P7) (SEQ ID NO:61), or the complement thereof. An example of an adapter ligation protocol includes phosphorylated template oligos at the 5′ end using a T4 polynucleotide kinase in 1x T4 ligase buffer for 30 minutes at 37 °C in a thermocycler. The kinase is then denatured (e.g., by heating) and the oligo reaction mixture is slowly cooled to 20 °C (e.g., by slowly changing the temperature by 0.1 °C every 2 seconds). [0373] Current SBS platforms require clonal amplification of the initial template library molecules to create clusters (i.e., polonies), each containing 100s to 10,000s of forward and reverse copies of an initial template library molecule, to increase the signal-to-noise ratio because the systems are not sensitive enough to detect the extension of one base at the individual DNA template molecule level. Standard amplification methods employed in commercial sequencing devices (e.g., solid-phase bridge amplification) typically amplify a template using surface immobilized primers to produce a plurality of double-stranded nucleic acid molecules, wherein at least one strand of each double-stranded nucleic acid molecule is attached to the solid support at its 5′ ends. A common method of doing solid-phase amplification involves bridge amplification methodologies (referred to as bridge PCR) as exemplified by the disclosures of U.S. Pat. Nos.5,641,658; 7,115,400; 7,790,418; U.S. Patent Publ. No.2008/0009420, each of which is incorporated herein by reference in its entirety. In sum, bridge amplification methods allow amplification products (e.g., amplicons) to be immobilized on a solid support in order to form arrays comprised of colonies (or “clusters”) of immobilized nucleic acid molecules. Each Attorney Docket No.: 051385-585001WO cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. The products of solid-phase amplification reactions are referred to as “bridged” structures when formed by annealed pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at the 5′ end, preferably via a covalent attachment. During bridge PCR, additional chemical additives may be included in the reaction mixture, in which the DNA strands are denatured by flowing a denaturant such as formamide or NaOH over the DNA, which chemically denatures complementary strands. This is followed by washing out the denaturant and reintroducing a polymerase in buffer conditions that allow primer annealing and extension. [0374] Sequencing two strands of the sample dsDNA template, referred to as paired-end, paired-strand, linked-strand, or dual-read sequencing, is a powerful technique to improve sequencing accuracy and is commonly performed in next-generation sequencing (NGS) workflows. Sequencing by synthesis (SBS) is a common implementation of NGS and paired-end sequencing is typically performed on monoclonal clusters generated by a clonal amplification process. For example, nucleic acid libraries that have common nucleic acid sequences (referred to as adapter sequences) on the 3′ and 5′ ends of every library molecule are delivered into a flow cell. Within the flow cell are nucleic acid sequences (referred to as primers) that are complementary to one or both of the adapter sequences of the library molecules. The primers may be immobilized to a solid support (e.g., a flow cell or a bead); a solid support encompasses any type of solid, porous, or hollow sphere, ball, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently). After hybridization of the adapter region of a library nucleic acid molecule to the immobilized oligonucleotides (i.e., primers) on the solid phase, a polymerase will make an initial copy of the library nucleic acid molecule by extending the primer. The complement of the initial library molecule is now attached to a solid support, and the initial library nucleic acid molecules can either be removed from the flow cell, or can stay present during subsequent steps, depending on which clonal amplification method is used. Next, spatially localized amplification of the initial single seed molecule will occur by means of a solid-phase clonal amplification process. Examples of clonal amplification techniques include, but are not limited to, bridge PCR, solid-phase rolling circle amplification (RCA), solid- Attorney Docket No.: 051385-585001WO phase exponential rolling circle amplification, solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, emulsion PCR on particles (beads), or combinations of the aforementioned methods. Optionally, during clonal amplification, additional solution-phase primers can be supplemented in the flow cell for enabling or accelerating amplification. [0375] It is typical for solid-phase clonal amplification to generate monoclonal clusters that each consist of many double-stranded DNA (dsDNA) copies (10s to 100,000s) of the initially seeded library nucleic acid molecule. In SBS workflows, clusters of dsDNA are difficult to sequence effectively with high accuracy and read length, especially as miniaturization pushes the clusters to become more densely arranged on a solid support. To initiate an SBS sequencing reaction, a sequencing primer needs to hybridize to a single-stranded region in the dsDNA and be extended by a polymerase. Individual strands in dsDNA clusters are difficult to access for hybridization of sequencing primers. Additionally, the polymerases used during SBS to incorporate 3’ reversibly terminated nucleotides (dNTPs) or native dNTPs (for example in pyrosequencing) typically do not have strand-displacement capabilities, and so even if one is successful in incorporating sequencing primers into dsDNA molecules, it is still challenging to extend said sequencing primers when the vast majority of DNA molecules are in dsDNA format. [0376] Due to these constraints, dsDNA amplicons in clusters are typically processed into single-stranded DNA (ssDNA), sometimes referred to as linearization, by a variety of methods. The dsDNA structures may be linearized by cleavage of one or both strands with a restriction endonuclease or by cleavage of one strand with a nicking endonuclease. Other methods of cleavage can be used as an alternative to restriction enzymes or nicking enzymes, including chemical cleavage (e.g., cleavage of a diol linkage with periodate), cleavage of abasic sites by cleavage with endonuclease, by exposure to heat or alkali, cleavage of ribonucleotides incorporated into amplification products otherwise comprised of deoxyribonucleotides, photochemical cleavage, or cleavage of a peptide linker. Alternatively, the primers may be attached to the solid support with a cleavable linker, such that upon exposure to a cleaving agent, all or a portion of the primer is removed from the surface. For example, one linearization method requires one or both of the immobilized primers to have a cleavable site, such as a uracil, diol, 8- oxoG, disulfide, photocleavable moieties, an RNA base or an endonuclease cleaving site. After Attorney Docket No.: 051385-585001WO the solid phase clonal amplification process is complete, one of the two species of solid phase primers (either forward or reverse) can be cleaved (chemically, enzymatically or optically), followed by a denaturation step to remove the cleaved molecules. This transforms the dsDNA molecules into ssDNA molecules within the cluster and provides a region available for hybridization of a sequencing primer to initiate a sequencing reaction. The monoclonal clusters can proceed to any necessary post-processing steps such as blocking of free 3’ ends, removal of select amplicons, or hybridization of a sequencing primer. [0377] In conventional workflows, once ssDNA molecules are generated a first sequencing read is performed by hybridizing a first sequencing primer to a complementary region (e.g., a region within the adapter portion) of the ssDNA molecule. In the presence of an enzyme (e.g., a DNA polymerase), nucleotides (e.g., labeled nucleotides) are incorporated and detected such that the identity of the incorporated nucleotides allows for the identification of the first strand. When the first read is complete (i.e., the first strand is read to a sufficient length with sufficient accuracy) the second strand that was initially cleaved during linearization must be regenerated prior to starting the second read. This can be done by additional amplification steps, such as additional rounds of bridge PCR or another amplification process. Following an additional amplification step after the first sequencing read, the second strand may then be sequenced. All of these steps add complexity and time to the DNA sequencing workflow and can also introduce additional errors made by the polymerase used during solid phase amplification. Highly accurate sequencing methods would greatly benefit from novel methods that bypass the need for additional amplification steps between the two sequencing reads of conventional paired-end sequencing workflows. [0378] In accordance with various embodiments, the methods disclosed herein permit reading of the original first and second strands (e.g., the first and second strand of the amplicons), reducing the time, reagents, expense, and risk of polymerase error inherent in previous methods. Importantly, methods described herein prevent the need for additional solid phase amplification between the two sequencing reads. In embodiments, methods disclosed herein utilize strand invasion using invasion primers into dsDNA amplicons bound to a solid phase, followed by polymerase extension of the invasion primers. Strand invasion into dsDNA can be challenging in general, but can be particularly challenging in dense monoclonal clusters of dsDNA where DNA Attorney Docket No.: 051385-585001WO molecules are packed tightly together in a spatially localized fashion on a solid phase. Because the local concentration of full-length complementary strands is very high, insertion of a traditional primer oligonucleotide is thermodynamically unfavorable. [0379] The invasion primers are oligonucleotide sequences that binds to one strand of the dsDNA molecule in the cluster. For example, the invasion primer may bind to a portion of the common adapter sequence of only the forward, or only the reverse amplicons in clusters. These invasion oligonucleotides may include nucleic acids having a binding affinity higher than the binding affinity of standard or canonical DNA oligonucleotides, such as locked nucleic acids (LNA), peptide nucleic acids (PNAs), 2’-O-methyl RNA:DNA chimeras, minor groove binder probes (MGB), or morpholino probes. The invasion primers are introduced into a flow cell that contains monoclonal dsDNA clusters generated using a known amplification method or an amplification method described herein. Some of these invasion primers can undergo spontaneous strand invasion into dsDNA, as is the case for example for PNA invasion primers under low ionic strength conditions, while other invasion primers may need assistance of additives such as DMSO, ethylene glycol, formamide, betaine, or other denaturants that assist strand invasion by inducing more breathability within dsDNA amplicons. For example, such additives may include a buffered solution containing about 0 to about 50% DMSO, about 0 to about 50% ethylene glycol, about 0 to about 20% formamide, or about 0 to about 3M betaine. In order to achieve sufficient “breathability” within dsDNA amplicons that are bound to a solid phase, it is helpful to include additives that can assist the “fraying” of the dsDNA molecules, particularly at the 5’ and 3’ ends. [0380] The invasion oligonucleotide can be introduced without a polymerase and allowed to invade and anneal to the complementary region, or it may be introduced together with a polymerase for runoff extension. Examples of polymerases that can be used for runoff extension are strand-displacing polymerases such as Bst large fragment, Bst2.0 (New England Biolabs), Bsm DNA polymerase, Bsu polymerase, SD polymerase, Vent exo- polymerase or Phi29 polymerase. In certain experiments, it is preferable to introduce the invasion oligonucleotide (e.g., a 15-75 bp invasion primer) together with a polymerase in the same reaction mixture. Because of the close physical proximity of the forward and reverse strands of the dsDNA molecules within a cluster, the hybridization of the invasion oligo to one of the DNA strands is Attorney Docket No.: 051385-585001WO often transient, and can be outcompeted easily by the reannealing of the full-length forward and reverse strands of the dsDNA molecules. To efficiently extend the invasion oligos that transiently hybridize, it is useful to have the polymerase within the same reaction mixture such that the polymerase can immediately extend the invasion oligo during the transient hybridizations that occur. For example, we have previously found (see, e.g., U.S. Pat. Application No.17/666,458, which is incorporated herein by reference in its entirety) that particular reaction conditions (e.g., 30% DMSO and in the presence of Bst LF polymerase and dNTPs) can enable efficient invasion and runoff extension of the invasion oligo. [0381] An example of the strand invasion and runoff method outlined above was executed on dsDNA clusters that were generated with bridge PCR, as described in U.S. Pat. Application No. 17/666,458. A Salmonella genomic dsDNA library with an average library molecule size of approximately 350 bp was generated by using standard library preparation techniques. The dsDNA genomic library was introduced into a proprietary flow cell at a 1 pM concentration in presence of 5x SSC buffer and 30% ethylene glycol. The flow cell was heated to 95°C to denature the dsDNA library molecules, followed by cooling the flow cell to 45°C to allow the denatured library molecules to bind to the immobilized primers on the surface of the flow cell. A strand displacing (SD) polymerase (with SD polymerase buffer, 3 mM MgCl2, and 0.2 mM of each dNTP) was subsequently introduced into the flow cell and heated to 60°C for 10 min to make an initial first copy of the library molecules that were hybridized to the flow cell primers. The initial library molecules were subsequently removed from the flow cell by flushing 0.1M NaOH through the lanes. This was followed by 45 bridge PCR cycles with a Bst LF polymerase and formamide as a chemical denaturant, which were cyclically introduced into the flow cell. A positive control lane was subsequently treated with USER enzyme mix which cleaved the forward amplicons which contained a uracil base and were formed by extending the forward flow cell primers from the flow cell surface. A second lane of the flow cell did not go through cleavage protocols and instead went through a strand invasion and runoff protocol, according to the following steps. [0382] For strand invasion and runoff, dsDNA clusters immobilized in a lane of the flow cell are exposed to a reagent mix that contains a plurality of LNA invasion oligos (at 1 uM concentration) capable of invading and hybridizing to a portion of the common adapter sequence, Attorney Docket No.: 051385-585001WO 0.56 units/uL of Bst polymerase, 30% DMSO, and 0.2 mM of each dNTP. This reaction mix is incubated at 65°C for 5 min, followed by flowing in fresh reagent mix. A total of four distinct 5 min incubations with fresh invasion-reagent mixtures containing the LNA invasion oligonucleotides, Bst polymerase, DMSO and dNTPs are performed. Subsequently, the 3’ end of the ssDNA molecule of the cleaved positive control lane and the single-stranded fragment in the lanes that went through strand invasion and runoff is probed with a FAM-labeled DNA probe. For the strand invasion and runoff conditions, fluorescent signal is expected if strand invasion and runoff is successful, since non-treated dsDNA clusters do not allow hybridization of the labeled DNA probe due to inaccessibility of the region to which the FAM-labeled probe would hybridize. [0383] The methods described herein provide for an alternate strand invasion and runoff protocol with enhanced invasion strand efficiency. As illustrated in FIGS.2A-2B illustrate an embodiment of strand invasion including an invasion primer with a 5’ tail sequence. The hashed box represents a polymer scaffold that is anchored to a solid support, such as glass or silicon support. FIG.2A illustrates two dsDNA duplex strands, each duplex having a first strand hybridized to a second strand, and each strand is attached to the solid support. An invasion primer including a binding sequence (e.g., a sequence complementary to a region within one of the strands of the dsDNA) and a 5’ tail sequence (wherein the tail sequence is not complementary to the dsDNA) is introduced, wherein the binding sequence of the elongated invasion primer hybridizes to one of the strands. An invasion mixture as described above may be used to introduce and hybridize the elongated invasion primer to the dsDNA. Following invasion, extension of the elongated invasion primer using an extension reaction mixture as described herein generates a blocking strand hybridized to one of the strands of the dsDNA (e.g., a blocking strand, also referred to herein as an invasion strand, is now hybridized to the second strand, for example, resulting in a single-stranded first strand that is accessible to a primer). To reduce the probability of the second strand re-annealing to the first strand, the second strand is extended along the elongated invasion primer to incorporate the complement of the 5’ tail sequence, thereby modifying the 3’ end of the second strand such that it is no longer complementary to the 5’ end of the first strand. FIG.2B illustrates an additional embodiment of strand invasion, wherein after generating an extended second strand as shown in FIG.2A (and after generating a first sequencing read), the blocking strand is removed (e.g., removed via Attorney Docket No.: 051385-585001WO exonuclease digestion). Following removal of the blocking strand, the first strand may reanneal to the second strand, but the incorporated tail sequence at the 3’ end of the second strand is free, allowing for annealing of a primer (e.g., a second invasion primer), followed by extension with a polymerase (e.g., a strand-displacing polymerase) thereby generating a second blocking strand hybridized to the extended second strand. Such an approach, as illustrated in FIG.2B, may be useful, for example, if the reaction conditions (e.g., buffer composition, such as high denaturant concentration) for hybridization of the invasion primer are not equally optimal for extension of the invasion primer, or the invasion primer melts off during invasion or is outcompeted by residual surface primers. In such a case, the invasion/blocking strand/primer may be removed by exonuclease digestion, for example, and then a new invasion primer hybrized to the incorporated tail sequence at the 3’ end of the second strand. This then allows for optimal strand-displacing extension conditions to be used for generating the second blocking strand. [0384] FIG.3 illustrates an alternate embodiment of strand invasion that may be used, for example, in combination with any of the strand invasion embodiments described herein. As illustrated, a bridged dsDNA complex is present on a solid support, wherein the first strand and the second strand of the dsDNA are immobilized on the solid support. Following invasion by an invasion oligonucleotide and strand synthesis along the second strand, for example as illustrated in FIG.1, a second oligonucleotide primer (e.g., a second competitive oligonucleotide) is annealed to the 5’ end of the immobilized first strand and extended towards the solid support, generating a competitor strand at the 5’ end of the immobilized first strand that may reduce reannealing of a portion of the second strand (prevent the 3’ end of the second strand from reannealing to the 5’ end of the first strand). Following extension of the blocking primer, the 5’ end of the immobilized first strand is double-stranded, and no longer able to anneal to the 3’ end of the second strand, for example. By reducing the likelihood that the first and second strands of the immobilized dsDNA will reanneal after strand invasion and hybridization of the invasion primer, the probability that one or more sequencing primers may be introduced and extended successfully may be increased, resulting in a higher efficiency of paired-end sequencing according to the methods herein. [0385] As described above, the amplification methods produced clusters of oligonucleotides for sequencing. The initiation point for the first sequencing reaction was provided by annealing a Attorney Docket No.: 051385-585001WO sequencing primer complementary to a region within one of the strands. In the presence of an enzyme (e.g., a DNA polymerase), nucleotides (e.g., labeled nucleotides) are incorporated and detected such that the identity of the incorporated nucleotides allows for the identification of the first strand. Thus, the first sequencing reaction may include hybridizing a sequencing primer to a region of an amplification product, sequentially incorporating one or more nucleotides into a polynucleotide strand complementary to the region of amplified template strand to be sequenced, identifying the base present in one or more of the incorporated nucleotide(s) and thereby determining the sequence of a region of the template strand. Note, the second sequenced strand is present while sequencing the first strand, albeit the second strand is hybridized to the invasion strand. Example 2. PNAs within invasion primers [0386] Peptide nucleic acids (PNAs) can be used as invasion oligonucleotides in another example of this invention; see the schematic illustrated in FIGS.4A-4B. Peptide nucleic acids consist of a pseudopeptide backbone, which has been shown to be capable of invading dsDNA. MiniPEG-γPNAs are particularly beneficial because they have better water solubility (Bahal et al. ChemBioChem, 13(1), 56–60). PNAs typically do not have a 3′-OH that is extendible by a DNA polymerase, though one can consider PNA-DNA chimeras that have 3-7 bp of canonical DNA nucleotides at the 3′ end of the oligonucleotide to be extendable by a DNA polymerase. MiniPEG-γPNAs can be designed to invade into dsDNA clusters by targeting a sequence region in the common adapter sequence of all clusters. PNAs can be designed for strand invasion into any part of the common adapter sequences, but targeting near the 5′ end of one of the amplicons is beneficial because it renders the complementary strand available for hybridization of another oligonucleotide, as shown in FIG.4A. A second invasion oligonucleotide that hybridizes on the “liberated” ssDNA fragment opposite of the PNA invasion site can then be extended by a strand- displacing DNA polymerase. As a result, one of the two strands of every dsDNA duplex has now been rendered into a ssDNA fragment that can be sequenced by hybridizing a sequencing primer followed a plurality of sequencing reactions. Example 3: Recombinase-assisted invasion [0387] Another possibility for enabling strand invasion into dsDNA molecules in monoclonal clusters is by using a recombinase enzyme that enables the insertion of a DNA oligonucleotide Attorney Docket No.: 051385-585001WO complementary to part of the common adapter sequence; see FIGS.5A-5B. A reagent mixture consisting of an invasion oligonucleotide, a recombinase, and necessary cofactors for forming a pre-synaptic filament (i.e. an oligonucleotide complexed with recombinase enzymes) is flowed into the flow cell that contains dsDNA clusters. The pre-synaptic filaments search the dsDNA molecules in monoclonal clusters until homology is found, after which the invasion oligonucleotide is inserted into the dsDNA to form a D-loop. After strand invasion, a strand- displacing polymerase can be introduced that extends the invasion oligonucleotide, thereby rendering the opposite strand of the original dsDNA duplex into a single-stranded form. The ssDNA molecule that is generated is then available for hybridization of a sequencing primer and the subsequent start of a first sequencing read. Examples of recombinases include, but are not limited to, T4 UvsX (and possibly its cofactor UvsY, and single-stranded binding protein gp32), Rad51, and RecA. The recombinase can be present in the same reaction mix as the strand- displacing polymerase, or the strand-displacing polymerase can be introduced after strand invasion with the recombinase has been done first. Example 4. Additional approaches to sequencing two strands of the same polynucleotide [0388] Sequencing two strands of the sample dsDNA template is a powerful technique to improve sequencing accuracy and is commonly performed in next-generation sequencing (NGS) workflows. Sequencing by synthesis (SBS) is a common implementation of NGS and paired-end sequencing is typically performed on monoclonal clusters generated by a clonal amplification process. It is typical for solid-phase clonal amplification to generate monoclonal clusters that each consist of many double-stranded DNA (dsDNA) copies (10s to 100,000s) of the initially seeded library nucleic acid molecule. In SBS workflows, clusters of dsDNA are difficult to sequence effectively with high accuracy and read length, especially as miniaturization pushes the clusters to become more densely arranged on a solid support. To initiate an SBS sequencing reaction, a sequencing primer needs to hybridize to a single-stranded region in the dsDNA and be extended by a polymerase. Individual strands in dsDNA clusters are difficult to access for hybridization of sequencing primers. Additionally, the polymerases used during SBS to incorporate 3’ reversibly terminated nucleotides (dNTPs) or native dNTPs (for example in pyrosequencing) typically do not have strand-displacement capabilities, and so even if one is Attorney Docket No.: 051385-585001WO successful in incorporating sequencing primers into dsDNA molecules, it is still challenging to extend said sequencing primers when the vast majority of DNA molecules are in dsDNA format. [0389] Due to these constraints, dsDNA amplicons in clusters are typically processed into single-stranded DNA (ssDNA), sometimes referred to as linearization, by a variety of methods. The dsDNA structures may be linearized by cleavage of one or both strands with a restriction endonuclease or by cleavage of one strand with a nicking endonuclease. Other methods of cleavage can be used as an alternative to restriction enzymes or nicking enzymes, including chemical cleavage (e.g., cleavage of a diol linkage with periodate), cleavage of abasic sites by cleavage with endonuclease, by exposure to heat or alkali, cleavage of ribonucleotides incorporated into amplification products otherwise comprised of deoxyribonucleotides, photochemical cleavage, or cleavage of a peptide linker. Alternatively, the primers may be attached to the solid support with a cleavable linker, such that upon exposure to a cleaving agent, all or a portion of the primer is removed from the surface. For example, one linearization method requires one or both of the immobilized primers to have a cleavable site, such as a uracil, diol, 8- oxoG, disulfide, photocleavable moieties, an RNA base or an endonuclease cleaving site. After the solid phase clonal amplification process is complete, one of the two species of solid phase primers (either forward or reverse) can be cleaved (chemically, enzymatically or optically), followed by a denaturation step to remove the cleaved molecules. This transforms the dsDNA molecules into ssDNA molecules within the cluster and provides a region available for hybridization of a sequencing primer to initiate a sequencing reaction. The monoclonal clusters can proceed to any necessary post-processing steps such as blocking of free 3’-OH ends, removal of select amplicons, or hybridization of a sequencing primer. [0390] In conventional workflows, once ssDNA molecules are generated a first sequencing read is performed by hybridizing a first sequencing primer to a complementary region (e.g., a region within the adapter portion) of the ssDNA molecule. In the presence of an enzyme (e.g., a DNA polymerase), nucleotides (e.g., labeled nucleotides) are incorporated and detected such that the identity of the incorporated nucleotides allows for the identification of the first strand. When the first read is complete (i.e., the first strand is read to a sufficient length with sufficient accuracy) the second strand that was initially cleaved during linearization must be regenerated prior to starting the second read. This can be done by additional amplification steps, such as Attorney Docket No.: 051385-585001WO additional rounds of bridge PCR or another amplification process. Following an additional amplification step after the first sequencing read, the second strand may then be sequenced. All of these steps add complexity and time to the DNA sequencing workflow and can also introduce additional errors made by the polymerase used during solid phase amplification. Highly accurate sequencing methods would greatly benefit from novel methods that bypass the need for additional amplification steps between the two sequencing reads of conventional paired-end sequencing workflows. [0391] In accordance with various embodiments, the methods disclosed herein permit reading of the original first and second strands (e.g., the first and second strand of the amplicons), reducing the time, reagents, expense, and risk of polymerase error inherent in previous methods. Importantly, methods described herein prevent the need for additional solid phase amplification between the two sequencing reads. In embodiments, methods disclosed herein utilize strand invasion using invasion primers into dsDNA amplicons bound to a solid phase, followed by polymerase extension of the invasion primers. Strand invasion into dsDNA can be challenging in general, but can be particularly challenging in dense monoclonal clusters of dsDNA where DNA molecules are packed tightly together in a spatially localized fashion on a solid phase. Because the local concentration of full-length complementary strands is very high, insertion of a traditional primer oligonucleotide is thermodynamically unfavorable. [0392] The invasion primers are oligonucleotide sequences that binds to one strand of the dsDNA molecule in the cluster. For example, the invasion primer may bind to a portion of the common adapter sequence of only the forward, or only the reverse amplicons in clusters. These invasion oligonucleotides may include nucleic acids having a binding affinity higher than the binding affinity of standard or canonical DNA oligonucleotides, such as locked nucleic acids (LNA), peptide nucleic acids (PNAs), 2’-O-methyl RNA:DNA chimeras, minor groove binder probes (MGB), or morpholino probes. The invasion primers may include one or more deoxyuracils(dUs). The invasion primers may include one or more phosphorothioate groups. The invasion primers are introduced into a flow cell that contains monoclonal dsDNA clusters generated using a known amplification method or an amplification method described herein. Some of these invasion primers can undergo spontaneous strand invasion into dsDNA, as is the case for example for PNA invasion primers under low ionic strength conditions, while other Attorney Docket No.: 051385-585001WO invasion primers may need assistance of additives such as DMSO, ethylene glycol, formamide, betaine, or other denaturants or additives that assist strand invasion by inducing more breathability within dsDNA amplicons. For example, such additives may include a buffered solution containing about 0 to about 50% DMSO, about 0 to about 50% ethylene glycol, about 0 to about 20% formamide, or about 0 to about 3M betaine. In order to achieve sufficient “breathability” within dsDNA amplicons that are bound to a solid phase, it is helpful to include additives that can assist the “fraying” of the dsDNA molecules, particularly at the 5’ and 3’ ends. [0393] As described herein and in Example 1, the invasion oligonucleotide can be introduced without a polymerase and allowed to invade and anneal to the complementary region, or it may be introduced together with a polymerase for runoff extension. In certain experiments, it is preferable to introduce the invasion oligonucleotide (e.g., a 15-75 bp invasion primer) together with a polymerase in the same reaction mixture. Because of the close physical proximity of the forward and reverse strands of the dsDNA molecules within a cluster, the hybridization of the invasion oligo to one of the DNA strands is often transient, and can be outcompeted easily by the reannealing of the full-length forward and reverse strands of the dsDNA molecules. To efficiently extend the invasion oligos that transiently hybridize, it is useful to have the polymerase within the same reaction mixture such that the polymerase can immediately extend the invasion oligo during the transient hybridizations that occur. [0394] The initiation point for the first sequencing reaction is provided by annealing a sequencing primer complementary to a region within one of the strands. In the presence of an enzyme (e.g., a DNA polymerase), nucleotides (e.g., labeled nucleotides) are incorporated and detected such that the identity of the incorporated nucleotides allows for the identification of the first strand. Thus, the first sequencing reaction may include hybridizing a sequencing primer to a region of an amplification product, sequentially incorporating one or more nucleotides into a polynucleotide strand complementary to the region of amplified template strand to be sequenced, identifying the base present in one or more of the incorporated nucleotide(s) and thereby determining the sequence of a region of the template strand. Note, the second sequenced strand is present while sequencing the first strand, albeit the second strand is hybridized to the invasion strand. Attorney Docket No.: 051385-585001WO [0395] Additional embodiments of methods of paired-strand sequencing by strand invasion of an invasion primer are disclosed herein. FIG.6A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion primer includes one or more 5’ phosphorothioate groups (e.g., 3-5 phosphorothioate linking groups) to protect from exonuclease digestion. In embodiments, the invasion primer further includes a cleavable site (e.g., a 3’ deoxyuracil triphosphate (dUTP)). After runoff extension of the invasion oligonucleotide has been completed, one strand of the initial dsDNA molecule is now single-stranded and available for a first sequencing read, as shown in FIG.6B. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process. The sequenced strand may optionally further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.6B. Subsequently, the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicked at the dU), leaving behind a 5’-phosphate in the extended part of the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.6C- 6D. In some embodiments, the invasion primer is treated with a 3’ phosphatase (for example Endonuclease IV or PNK) to generate a 3’ hydroxyl group prior to sequencing. [0396] In a modified embodiment of the above method, once runoff extension of the invasion oligonucleotide has been completed, one strand of the initial dsDNA molecule is now single- stranded and available for a first sequencing read, as shown in FIG.7B. This renders one of the two strands of the original dsDNA amplicon available for hybridization of a sequencing primer to initiate the SBS process. The sequenced strand may further be extended with natural dNTPs after sequencing the first read to complete the extension of the sequenced strand, as illustrated in FIG.7B, thereby preventing any rehybridization of any non-sequenced amplicon to the complement. Subsequently, the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicked at the dU and removed), leaving behind a 5’-phosphate in the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.7C-7D. Alternatively, the sequenced strand may further be extended with a one or more ddNTPs to prevent further extension, as illustrated in FIG.8B. Subsequently, the 3’ end of the invasion primer may be cleaved at a cleavable site (e.g., nicked at the dU and removed), leaving behind a 5’-phosphate in Attorney Docket No.: 051385-585001WO the invasion strand that can subsequently be degraded with a 5’ to 3’ exonuclease, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS. 8C-8D. In some embodiments, the invasion primer is treated with a 3’ phosphatase (for example Endonuclease IV or PNK) to generate a 3’ hydroxyl group prior to sequencing. Advantageously, neither of these two embodiments require the removal of the first sequenced strand, further reducing cost and time required for high-accuracy paired-strand sequencing. [0397] As an alternative to digesting away the invasion strand prior to sequencing the second strand, internal cleavable sites (e.g., cleavable internucleosidic bonds) may be introduced into the invasion strand. As in the methods shown supra, FIG.9A illustrates an embodiment wherein the invasion primer is annealed to the 3′ end of one of the strands. In the embodiment depicted in FIG.9A, the invasion primer includes one or more phosphorothioate nucleic acids at the 5′ end to protect from exonuclease digestion, and a cleavable site at the 3′ end (e.g., one or more deoxyuracil nucleobases). Runoff extension of the invasion oligonucleotide is then performed with an amplification mixture that provides cleavable sites (e.g., a mixture of dUTP, dATP, dGTP, and dCTP nucleotides) leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.9B. The sequenced strand may optionally further be cleaved at a cleavable site (represented as ‘X’) and removed, thus leaving the complementary strand available for sequencing, as illustrated in FIG.9B. Subsequently, the invasion strand may be cleaved at internal cleavable sites (e.g., cleaved at the dU sites), leaving behind small, low Tm fragments (e.g., melting temperatures in the range of 0°C to about 60°C) that may be thermally denatured away, as shown in FIG.9C. Additionally, this cleavage and denaturation step exposes the 3’ end of the invasion oligo, allowing for the invasion primer to serve as a sequencing primer for the second strand, as illustrated in FIGS.9C-9D. In some embodiments, the invasion primer is treated with a 3’ phosphatase (for example Endonuclease IV or PNK) to generate a 3’ hydroxyl group prior to sequencing. [0398] As an additional alternative to digesting away the invasion strand prior to sequencing the second strand, internal cleavable sites (e.g., cleavable internucleosidic bonds) may be introduced into the invasion strand. Following cleavage, the small products annealed to the second strand may then be digested away. As in the methods shown supra, FIG.10A illustrates an invasion primer annealed to the 3′ end of one of the strands. In embodiments, the invasion Attorney Docket No.: 051385-585001WO primer includes one or more phosphorothioate group(s) towards the 5′ end to protect the invasion primer from 5’ to 3’ exonuclease digestion. In embodiments, the invasion primer also includes a cleavable site (also referred to herein as a scissile linkage). For example, as depicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towards the 3’ end of the invasion oligo. Runoff extension of the invasion oligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNA molecule single-stranded and available for a first sequencing read, as shown in FIG.10B. Once the first sequencing read has been obtained, the 3’ end of the first sequencing read is capped by ddNTP incorporation. A second sequencing read is then obtained by annealing and extending a second sequencing primer 3’ of the terminated first sequencing read. Subsequently, a ddNTP is incorporated into the 3’ end of the second sequencing read, and thereafter the invasion strand may be nicked at internal scissile sites (e.g., resulting from amplification with the dUTP), leaving behind small fragments with exposed 5’ ends that may be removed under suitable conditions, for example, by lambda exonuclease digestion, as shown in FIGS.10C-10D. This cleavage and removal step exposes the 3’ end of the second strand, making it available for a third sequencing read, as shown in FIG.10E. Once the third sequencing read has been obtained, the 3’ end of the third sequencing read is capped by ddNTP incorporation. A fourth sequencing read is then obtained by annealing and extending a fourth sequencing primer 3’ of the terminated third sequencing read, as illustrated in FIG.10E. Example 5. Spatially barcoded arrays [0399] The position of any given cell, relative to its neighbors and non-cellular structures, can provide useful information for defining cellular phenotype, cell state, and ultimately cell and tissue function. Location can determine the signals to which cells are exposed. While endocrine signals act at macroscopic scales, many other types of signals act upon neighboring cells via cell- cell interactions or via soluble signals acting in the vicinity. One form these signals can take is of cell surface-bound protein receptors and ligand pairs, the mRNA for which can be detected by transcriptomics (see, e.g., Armingol E et al. Nat. Rev. Genet.2021; 22(2): 71-88). The expansion of protein–protein interaction databases and recent advances in RNA sequencing technologies have enabled routine analyses of intercellular signaling from gene expression measurements of bulk and single-cell data sets. Single-cell RNA sequencing (scRNA-seq) has greatly advanced our understanding of cellular heterogeneity by profiling individual cell transcriptomes. However, cell dissociation from the tissue structure causes a loss of spatial information, which hinders the Attorney Docket No.: 051385-585001WO identification of intercellular communication networks and global transcriptional patterns present in the tissue architecture. To overcome this limitation, novel transcriptomic platforms that preserve spatial information have been actively developed. Significant achievements in imaging technologies have enabled in situ targeted transcriptomic profiling in single cells at single molecule resolution (see, e.g., Lee J et al. BMB Rep.2022; 55(3): 113-124). [0400] Standard immunohistochemistry and RNA in situ hybridization (ISH), for example, can examine only one or a handful of target molecular species at a time; therefore, the amount of information obtained from a single experimental session is limited. To overcome this, emerging spatial transcriptomics techniques aim to examine all genes expressed from the genome from a single histological slide (see, e.g., Asp M et al. Bioessays.2020; 42(10): e1900221). There are three major methodologies to experimentally implement spatial transcriptomics. First, the sequential in situ hybridization method, often combined with combinatorial multiplexing, can increase the number of RNA species that can be detected from a single histological section. Second, in situ sequencing can identify RNA sequences from the tissue through fluorescence- based direct sequencing. Finally, spatial barcoding methods associate RNA sequences and their spatial locations by capturing tissue RNA using a spatially barcoded oligonucleotide array. [0401] There are a growing number of in situ capture technologies being developed and commercialized to perform such examinations on a single histological slide. For example, one iteration of the 10X Genomics Visium platform includes the use of glass slides containing marked 6.5 × 6.5 mm areas where thin tissue sections are placed and imaged. Each area contains 5000 printed regions of barcoded mRNA capture probes with the dimensions of 55 µm in diameter and a center-to-center distance of 100 µm. Tissue is permeabilized and mRNAs are hybridized to the barcoded capture probes directly underneath. cDNA synthesis connects the spatial barcode and the captured mRNA, and sequencing reads are later overlaid with the captured tissue image. Another example includes NanoString’s GeoMX Digital Spatial Profiler, wherein a slide-fixed tissue section is first exposed to a pre-known panel of barcoded hybridization probes, after which regions of interest (ROIs) are manually selected. The ROIs are then subjected to UV light and target specific barcodes are cleaved off and digital counting performed with the nCounter system. Attorney Docket No.: 051385-585001WO [0402] The techniques for spatial transcriptomics with barcoded oligonucleotide capture arrays described, however, have limitations in the spatial resolution of up to 55-100 µm due to the physical size of capturing spots. To resolve the issue of low spatial resolution in capture-based sequencing methodology, bead-based capturing sequencing was developed. In 2019, Rodriques et al. developed Slide-seq (see, e.g., Rodriques SG et al. Science.2019; 363(6434): 1463-67) for higher cellular resolution genome-wide analysis using DNA-barcoded 10 μm beads onto a rubber-coated glass slide. In Slide-seq, each bead’s distinct barcoded sequence is determined via SBL (sequencing-by-ligation) methods. Using the beads, the resolution comparable to the size of an individual cell was achieved by NGS of barcoded RNA-seq libraries and successfully applied to the hippocampus area in mice. Additionally, high-definition spatial transcriptomics (HDST) was developed as another effort to accomplish higher resolution for barcoded transcripts (see, e.g., Vickovic S et al. Nat. Methods.2019; 16(10): 987-990). HDST randomly deposits barcoded poly(d)T oligonucleotides into a 2 μm well with bead array-based fabrication. Mouse brain and primary breast cancers were profiled with 25× higher resolution than that of Slide-seq using this method, resulting in a reconstruction of the spatial architecture of mouse brain and primary breast cancer tissues. [0403] Cho et al. developed a high-resolution spatial barcoding technology, Seq-Scope, with a 0.5-0.8 μm center-to-center resolution (see, e.g., Cho CS et al. Cell.2021; 184(13): 3559-3572). This technique was developed based on the NGS technology that utilizes randomly barcoded single-molecule oligonucleotides with a transcriptome capture capacity of approximately 4,700 UMIs/cell on average, comparable to conventional scRNA-seq methods. This technical improvement enabled visualizing the histological organization of the transcriptome architecture in liver tissues at subcellular level. More recently, another high- resolution spatial barcoding technology, SpaTial Enhanced Re-solution Omics-sequencing (Stereo-seq) was introduced with a center-to-center resolution of 500-715 nm (see, Chen A et al. Cell.2022; 185(10): 1777-1792). This method applies microfluidic channels perpendicularly for unique pair-wise barcoding on each spot of the tissue section. Stereo-seq can capture up to 133,775 UMIs per 100 μm diameter bin, and was capable of profiling a whole mouse embryo. Stereo-seq is currently undergoing commercial development by BGI as its STOmics platform, currently in early access. Attorney Docket No.: 051385-585001WO [0404] While most of these techniques are designed for fresh frozen tissues stored below the temperature at which mRNAs degrade, some methods such as Visium FFPE are compatible with tissues that are fixed with formalin and embedded in paraffin wax, although this requires extra steps to prepare the tissue for profiling and a different, gene-specific probe-set (although all genes in the genome are nonetheless profiled). Independently developed techniques to adapt Visium reagents to FFPE-preserved tissues are also available but it is unclear whether these are in active commercial development (see, e.g., Gracia Villacampa E et al. Cell Genomics.2021; 1(3): 100065). [0405] A common approach for in situ spatial transcriptomic analysis of mRNAs is to use a capture array including poly-d(T) capture probes which then hybridize to polyadenylated RNA species. The Seq-Scope method, for example, relies on the targeting of poly-A tails of mRNA molecules for capture onto a physical array of spatially barcoded RNA-capture molecules, and a spatial map of barcodes where each barcoded sequence is associated with a spatial coordinate. In this system, the RNA-capture molecules include a poly-d(T) capture domain for associating with the mRNA molecules. The poly-d(T) approach introduces several limitations, including, for example, the inability to target RNA species which are not poly-A tailed (e.g., non- polyadenylated), for example, histone mRNAs, lncRNAs, rRNAs, and snRNAs (see, e.g., Yang L et al. Genome Biol.2011; 12(2): R16, which is incorporated herein by reference in its entirety). Additionally, biases may be introduced towards polyadenylated RNA molecules due to the varying degree of lengths of poly-A tails present. For example, a recent study identified a population of mRNAs (>10% of genes’ mRNAs) that were inconsistently captured by poly(A) selection due to highly variable poly(A) tails (see, Viscardi MJ and Arribere JA. BMC Genomics.2022; 23:530, which is incorporated herein by reference in its entirety). Variable tail length genes (genes with mRNAs exhibiting a large difference in mean poly(A) tail lengths in poly(A)-selected samples compared to unselected samples) were found to be preferentially lost upon poly(A) selection, indicating that poly(A) selection may cause gene-specific recovery biases, and also added unnecessary noise to sample replicates. Recently, a method for performing in situ polyadenylation of non-polyadenylated RNAs followed by poly(A) selection and sequencing was described (see, McKellar DW et al. bioRxiv.2022; 2022.03.20.488964), although the biases described supra for poly(A) selection may still need to be considered with such an approach. Attorney Docket No.: 051385-585001WO [0406] The methods described herein provide a solution for specifically targeting a broad range of RNA species in situ regardless of polyadenylation status, for example, or length of poly(A) tails. This approach offers users an opportunity to customize the tissue nucleic acid capture arrays of existing and future spatial transcriptomic platforms towards a large number of nucleic acid types. For example, the methods described herein include hybridizing an invasion primer including a binding sequence and a tail sequence to a strand of a double-stranded nucleic acid molecule immobilized on a solid support (e.g., a spatial array), wherein the tail sequence of the invasion primer includes a capture sequence capable of hybridizing to an endogenous region of a target polynucleotide of a cell in situ, and extending the strand along the tail sequence to generate an extended strand including the capture sequence. The methods described herein may be used with any of the spatial transcriptomics platforms described or referenced supra. For example, the Seq-Scope method described in Cho et al. used capture probes including an oligo- dT tail for targeting mRNA molecules in cells. One could instead use the invasion primers as described herein to incorporate capture sequences into immobilized double-stranded polynucleotides, wherein the capture sequences target an endogenous region of the target polynucleotide, thereby avoiding any biases and underrepresentation of mRNA species, while also targeting non-polyadenylated RNA types using the same spatially barcode array. It is envisioned that the capture sequence may instead include an oligo-dT sequence for capture of polyadenylated mRNA species. [0407] Briefly, a plurality of polynucleotides each including a spatial barcode and a cleavage domain are immobilized on a solid support. The polynucleotides include a platform primer binding sequence and are bound to the solid support by interactions with a corresponding platform primer attached to the solid support. The polynucleotides are then amplified, for example, by bridge amplification, resulting in the generation of multiple clusters including immobilized pluralities of polynucleotides on the surface of the solid support. The resulting substrate includes millions of clusters, each cluster containing pluralities of immobilized polynucleotides including the same spatial barcode. An invasion primer including a binding sequence and a tail sequence is then hybridized to one strand of each double-stranded polynucleotide immobilized on the solid support (e.g., the spatial array), wherein the tail sequence of the invasion primer includes, for example, a capture sequence capable of hybridizing to an endogenous region of a target polynucleotide of a cell in situ. The strand of the double- Attorney Docket No.: 051385-585001WO stranded polynucleotide hybridized to the invasion primer is then extended along the tail sequence to generate an extended strand including the capture sequence. The tail sequence may also include a barcode sequence, or a primer binding sequence. The invasion primer may be blocked (e.g., blocked with a non-extendible 3’ blocking moiety) such that it is not extended. In some embodiments, the invasion primer is extended to generate an invasion strand hybridized to the strand of the double-stranded polynucleotide. Once the extended strand including the capture sequence is generated, the invasion strand may be removed (e.g., removed by denaturation or by exonuclease digestion). Sequencing is performed to determine the sequence of the spatial barcodes for each cluster. The sequence is then used to assign each cluster to a specific location on the solid support. [0408] After determining the spatial barcode sequences of each cluster, the solid support is then further processed (e.g., removing unused platform primer oligonucleotides and other non- specific ssDNA) to generate a spatial barcode array that can capture target nucleic acids (e.g., mRNAs) released from a tissue section or plurality of cells, as described in PCT Pub. No. WO2022/015913, which is incorporated by reference herein in its entirety. The quality of the clustered array may be inspected by staining with a DNA dye, such as SYBR Gold. [0409] Tissue, for example OCT-mounted fresh frozen tissue, is sectioned in a cryostat using methods known in the art. The tissue sections are then placed on the spatial capture array and fixed, for example using 4% formaldehyde. The tissues may then be hematoxylin and eosin stained using methods known in the art and imaged under a light microscope. To release RNAs from the fixed tissues, the tissues are treated, for example, with 0.2U/uL collagenase I at 37 °C 20 min, and then with l mg/mL pepsin in 0.1M HC1 at 37 °C 10 min, as previously described (see, Salmen F et al. Nat. Protoc.2018; 13(11): 2501-34, which is incorporated herein by reference in its entirety). [0410] The tissue is then washed with a reverse-transcription buffer including, for example, Maxima 5x RT Buffer (Cat. No. EP0751, Thermofisher) and RNase Inhibitor (Cat. No.30281, Lucigen). Subsequently, reverse transcription is performed by incubating the tissue-attached spatial barcode array in an RT buffered reaction solution containing, for example, Maxima 5x RT Buffer (Cat. No. EP0751, Thermofisher), 20% Ficoll PM-400 (Cat. No. F4375-10G, Sigma), dNTPs (Cat. No. N0477L, NEB), RNase Inhibitor (Cat. No.30281, Lucigen), Maxima H- RTase Attorney Docket No.: 051385-585001WO (Cat. No. EP0751, Thermofisher), and Actinomycin D (500ng/pl, Cat. No. A1410, Sigma- Aldrich). The RT reaction solution is then incubated at 42 °C overnight. [0411] The following day, the RT solution is removed and the tissue is submerged in an exonuclease I cocktail (1U Exo I enzyme (Cat. No. M2903, NEB) in IX Exo I buffer) and incubated at 37 °C for 45 min, to eliminate DNA that did not hybridize with mRNA, for example. Then the cocktail is removed, and the tissues are submerged in lx tissue digestion buffer (100 mM Tris pH 8.0, 100 mM NaCl, 2% SDS, 5 mM EDTA, 16 U/mL Proteinase K (Cat. No. P8107S, NEB) and incubated at 37 °C for 40 min. After tissue digestion, the array is washed with water 3 times, 0. IN NaOH 3 times (each with 5 min incubation at room temperature), 0.1M Tris (pH7.5) 3 times (each with a brief wash), and then water 3 times (each with brief wash). This will eliminate all mRNA from the spatially barcoded array. [0412] After the washing steps, second strand synthesis is performed by adding to the tissue a second strand synthesis mix including a sequencing primer-conjugated random primer, dNTP mix (Cat. No. N0477, NEB), and Klenow Fragment (exonuclease-deficient; Cat. No. M0212, NEB). Then the spatially barcoded array is incubated at 37 °C for 2 hr in a humidity-controlled chamber. After second strand synthesis, the array is washed with water to remove all DNAs that are taken off from the array, so that each spatial barcode molecule corresponds to each single copy of second strand. Then the array is treated with 0.1 N NaOH to elute the second strand. The elution step is duplicated and combined with the first elution. The second strand product elution is then neutralized by mixing with 3 M potassium acetate, pH 5.5. The neutralized secondary strand product is then subjected to AMPure XP purification (Cat. No. A63881, Beckman Coulter) using 1.8X bead/sample ratio, according to the manufacturer’s protocol. The final elution is performed using water. Sequencing libraries are then prepared according to manufacturer’s protocols. The libraries are subjected to, for example, paired-end (e.g., 100- 150bp) sequencing processes on, for example, a Singular Genomics™ sequencer (e.g., the G4™ system) or Illumina™ sequencer (e.g., HiSeq™, MiSeq™, NextSeq™, or NovaSeq™ systems). A plurality of sequencing cycles then occur, wherein each cycle includes extension and detection of an incorporated nucleotide. The entire array may be selectively sequenced by choosing the appropriate initiator, i.e., the appropriate sequencing primer. Attorney Docket No.: 051385-585001WO P-EMBODIMENTS [0413] The present disclosure provides the following illustrative embodiments. [0414] Embodiment P1. A method of sequencing, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; extending the second strand along the tail sequence of said invasion primer to generate an extended second strand comprising a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. [0415] Embodiment P2. The method of Embodiment P1, further comprising removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand. [0416] Embodiment P3. The method of Embodiment P1, further comprising removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. [0417] Embodiment P4. The method of Embodiment P1, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand. [0418] Embodiment P5. A method of forming a single-stranded polynucleotide attached to a solid support, said method comprising: contacting a plurality of double-stranded polynucleotides comprising a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers comprise a binding sequence and a tail sequence; hybridizing the binding sequence of one of said invasion primers to one of said second strands; Attorney Docket No.: 051385-585001WO and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the invasion primer hybridized to the second strand to generate an extended second strand comprising a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support. [0419] Embodiment P6. The method of Embodiment P5, further comprising sequencing the single-stranded polynucleotide. [0420] Embodiment P7. The method of Embodiment P5 or Embodiment P6, further comprising removing the invasion strand and sequencing the second strand. [0421] Embodiment P8. The method of any one of Embodiment P5 to Embodiment P7, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand. [0422] Embodiment P9. The method of Embodiment P1, comprising nicking the invasion strand to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the invasion primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand. [0423] Embodiment P10. The method of any one of Embodiment P2 to Embodiment P9, wherein removing the invasion strand comprises digesting the invasion strand using an exonuclease enzyme. [0424] Embodiment P11. The method of any one of Embodiment P1 to Embodiment P7, wherein the first strand is covalently attached to the solid support via a first linker and the second strand is covalently attached to the solid support via a second linker. [0425] Embodiment P12. The method of any one of Embodiment P1 to Embodiment P9, wherein the double-stranded polynucleotides comprise known adapter sequences on 5′ and 3′ ends. [0426] Embodiment P13. The method of any one of Embodiment P1 to Embodiment P10, wherein the invasion primer comprises locked nucleic acids (LNAs), Bis-locked nucleic acids Attorney Docket No.: 051385-585001WO (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O- methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof. [0427] Embodiment P14. The method of any one of Embodiment P1 to Embodiment P11, wherein the invasion primer is about 15 to about 90 nucleotides in length. [0428] Embodiment P15. The method of any one of Embodiment P1 to Embodiment P12, wherein the invasion primer comprises one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. [0429] Embodiment P16. The method of any one of Embodiment P1 to Embodiment P13, further comprising contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof. [0430] Embodiment P17. The method of any one of Embodiment P1 to Embodiment P14, wherein generating the invasion strand comprises contacting the polynucleotide with a buffered solution comprising dimethyl sulfoxide (DMSO), betaine, or a combination of dimethyl sulfoxide (DMSO) and betaine. [0431] Embodiment P18. The method of any one of Embodiment P1 to Embodiment P15, wherein prior to hybridizing the invasion primer the method comprises amplifying the double- stranded polynucleotides with bridge polymerase chain reaction (bPCR) amplification, solid- phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR, or combinations of said methods. [0432] Embodiment P19. A method of incorporating a sequence, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail Attorney Docket No.: 051385-585001WO sequence of said invasion primer to generate an extended second strand comprising a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double-stranded polynucleotide. [0433] Embodiment P20. The method of any one of Embodiment P1 to Embodiment P19, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence. [0434] Embodiment P21. A method of sequencing, said method comprising: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. NUMBERED EMBODIMENTS [0435] Embodiment 1. A method of incorporating a sequence, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand and said second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail sequence of said invasion primer with a polymerase to generate an extended second strand comprising a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double-stranded polynucleotide. [0436] Embodiment 2. The method of Embodiment 1, wherein the tail sequence comprises a barcode sequence. Attorney Docket No.: 051385-585001WO [0437] Embodiment 3. The method of Embodiment 1 or 2, wherein the tail sequence comprises a capture sequence, wherein the capture sequence is capable of hybridizing to a target polynucleotide. [0438] Embodiment 4. The method of Embodiment 3, wherein the capture sequence comprises a sequence capable of hybridizing to an endogenous sequence of a target polynucleotide. [0439] Embodiment 5. The method of Embodiment 1 or 2, wherein the tail sequence comprises a poly(dT) sequence. [0440] Embodiment 6. The method of any one of Embodiments 1 to 4, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence, optionally a barcode sequence, and the binding sequence. [0441] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the double-stranded polynucleotide comprises a cleavable site. [0442] Embodiment 8. The method of any one of Embodiments 1 to 7, wherein the binding sequence comprises 10 to 25 nucleotides and the tail sequence comprises 5 to 25 nucleotides. [0443] Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the double-stranded polynucleotide does not comprise genomic DNA. [0444] Embodiment 10. The method of any one of Embodiments 1 to 9, further comprising sequencing the first strand. [0445] Embodiment 11. The method of any one of Embodiments 1 to 9, further comprising hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. [0446] Embodiment 12. The method of Embodiments 10 or 11, further comprising removing the invasion strand and sequencing the second strand. Attorney Docket No.: 051385-585001WO [0447] Embodiment 13. A method of sequencing, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase, thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; extending the second strand along the tail sequence of said invasion primer to generate an extended second strand comprising a complement of the tail sequence; hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. [0448] Embodiment 14. The method of Embodiment 13, further comprising removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand. [0449] Embodiment 15. The method of Embodiment 13, further comprising removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. [0450] Embodiment 16. The method of Embodiment 13, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand. [0451] Embodiment 17. A method of forming a single-stranded polynucleotide attached to a solid support, said method comprising: contacting a plurality of double-stranded polynucleotides comprising a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers comprise a binding sequence and a tail sequence; hybridizing the binding sequence of one of said invasion primers to one of said second strands; and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the invasion primer hybridized to the second strand to generate an extended second Attorney Docket No.: 051385-585001WO strand comprising a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support. [0452] Embodiment 18. The method of Embodiment 17, further comprising sequencing the single-stranded polynucleotide. [0453] Embodiment 19. The method of Embodiment 17 or 18, further comprising removing the invasion strand and sequencing the second strand. [0454] Embodiment 20. The method of any one of Embodiments 17 to 19, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand. [0455] Embodiment 21. The method of Embodiment 13, comprising nicking the invasion strand to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the invasion primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand. [0456] Embodiment 22. The method of any one of Embodiments 14 to 21, wherein removing the invasion strand comprises digesting the invasion strand using an exonuclease enzyme. [0457] Embodiment 23. The method of any one of Embodiments 1 to 19, wherein the first strand is covalently attached to the solid support via a first linker and the second strand is covalently attached to the solid support via a second linker. [0458] Embodiment 24. The method of any one of Embodiments 1 to 21, wherein the double-stranded polynucleotides comprise known adapter sequences on 5′ and 3′ ends. [0459] Embodiment 25. The method of any one of Embodiments 1 to 22, wherein the invasion primer comprises locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof. Attorney Docket No.: 051385-585001WO [0460] Embodiment 26. The method of any one of Embodiments 1 to 23, wherein the invasion primer is about 15 to about 90 nucleotides in length. [0461] Embodiment 27. The method of any one of Embodiments 1 to 24, wherein the invasion primer comprises one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. [0462] Embodiment 28. The method of any one of Embodiments 1 to 25, further comprising contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof. [0463] Embodiment 29. The method of any one of Embodiments 1 to 26, wherein generating the invasion strand comprises contacting the polynucleotide with a buffered solution comprising dimethyl sulfoxide (DMSO), betaine, or a combination of dimethyl sulfoxide (DMSO) and betaine. [0464] Embodiment 30. The method of any one of Embodiments 1 to 27, wherein prior to hybridizing the invasion primer the method comprises amplifying the double-stranded polynucleotides with bridge polymerase chain reaction (bPCR) amplification, solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid- phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR, or combinations of said methods. [0465] Embodiment 31. The method of any one of Embodiments 1 to 30, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence. [0466] Embodiment 32. A method of sequencing, said method comprising: hybridizing an invasion primer to a 3′ end of a second strand of a double-stranded polynucleotide and extending the invasion primer with a polymerase, thereby generating a first invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; hybridizing a blocking primer to a 5′ end of the first strand and extending the blocking primer with a polymerase, thereby generating a second invasion strand; hybridizing a sequencing primer to a 3′ end of the first strand; and incorporating one or more nucleotides into the sequencing primer Attorney Docket No.: 051385-585001WO with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. [0467] The Embodiment of any one of claims 1 to 30, prior to hybridizing an invasion primer, said method comprises amplifying a nucleic acid molecule thereby forming a plurality of double- stranded polynucleotides immobilized to the solid support.

Claims

Attorney Docket No.: 051385-585001WO WHAT IS CLAIMED IS: 1. A method of incorporating a sequence, said method comprising: hybridizing an invasion primer comprising a binding sequence and a tail sequence to a second strand of a double-stranded polynucleotide and extending the binding sequence with a polymerase thereby generating an invasion strand, wherein the double-stranded polynucleotide comprises a first strand hybridized to said second strand, wherein the first strand and the second strand are both attached to a solid support; and extending the second strand along the tail sequence of said invasion primer with a polymerase to generate an extended second strand comprising a complement of the tail sequence, thereby incorporating a sequence into the second strand of the double-stranded polynucleotide. 2. The method of claim 1, wherein the tail sequence comprises a barcode sequence. 3. The method of claim 1, wherein the tail sequence comprises a capture sequence, wherein the capture sequence is capable of hybridizing to a target polynucleotide. 4. The method of claim 3, wherein the capture sequence comprises a sequence capable of hybridizing to an endogenous sequence of a target polynucleotide. 5. The method of claim 1, wherein the tail sequence comprises a poly(dT) sequence. 6. The method of claim 1, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence, optionally a barcode sequence, and the binding sequence. 7. The method of claim 1, wherein the double-stranded polynucleotide comprises a cleavable site. 8. The method of claim 1, wherein the binding sequence comprises 10 to 25 nucleotides and the tail sequence comprises 5 to 25 nucleotides. 9. The method of claim 1, wherein the double-stranded polynucleotide does not comprise genomic DNA. Attorney Docket No.: 051385-585001WO 10. The method of claim 1, further comprising sequencing the first strand. 11. The method of claim 1, further comprising hybridizing a sequencing primer to the first strand and incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand, thereby sequencing the first strand of the double-stranded polynucleotide. 12. The method of claim 11, further comprising removing the invasion strand and sequencing the second strand. 13. The method of claim 1, further comprising removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand. 14. The method of claim 11, further comprising removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand. 15. The method of claim 15, further comprising sequencing the second strand. 16. A method of forming a single-stranded polynucleotide attached to a solid support, said method comprising: contacting a plurality of double-stranded polynucleotides comprising a first strand hybridized to a second strand with a plurality of invasion primers, wherein the first strand and the second strand are attached to the solid support, and wherein each of the invasion primers comprise a binding sequence and a tail sequence; hybridizing the binding sequence of one of said invasion primers to one of said second strands; and extending the invasion primer hybridized to the second strand with a polymerase to generate an invasion strand, displacing the first strand, and extending the second strand along the tail sequence of the invasion primer hybridized to the second strand to generate an extended second strand comprising a complement of the tail sequence, thereby forming a single-stranded polynucleotide attached to the solid support. Attorney Docket No.: 051385-585001WO 17. The method of claim 16, further comprising sequencing the single-stranded polynucleotide. 18. The method of claim 16, further comprising removing the invasion strand and sequencing the second strand. 19. The method of claim 16, further comprising removing the invasion strand and hybridizing a second invasion primer to the complement of the tail sequence on said second strand, and extending said second invasion primer with a polymerase, thereby generating a second invasion strand. 20. The method of claim 12, wherein removing the invasion strand comprises digesting the invasion strand using an exonuclease enzyme. 21. The method of claim 1, wherein the first strand is covalently attached to the solid support via a first linker and the second strand is covalently attached to the solid support via a second linker. 22. The method of claim 1, wherein the double-stranded polynucleotides comprise known adapter sequences on 5′ and 3′ ends. 23. The method of claim 1, wherein the invasion primer comprises locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2’-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof. 24. The method of claim 1, wherein the invasion primer is about 15 to about 90 nucleotides in length. Attorney Docket No.: 051385-585001WO 25. The method of claim 1, wherein the invasion primer comprises one or more locked nucleic acids (LNAs) at the 3′ end of the invasion primer sequence. 26. The method of claim 1, further comprising contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof. 27. The method of claim 1, wherein generating the invasion strand comprises contacting the polynucleotide with a buffered solution comprising dimethyl sulfoxide (DMSO), betaine, or a combination of dimethyl sulfoxide (DMSO) and betaine. 28. The method of claim 1, wherein prior to hybridizing the invasion primer the method comprises amplifying the double-stranded polynucleotides with bridge polymerase chain reaction (bPCR) amplification, solid-phase rolling circle amplification (RCA), solid-phase exponential rolling circle amplification (eRCA), solid-phase recombinase polymerase amplification (RPA), solid-phase helicase dependent amplification (HDA), template walking amplification, or emulsion PCR, or combinations of said methods. 29. The method of claim 1, wherein the invasion primer comprises, from 5’ to 3’, the tail sequence and the binding sequence. 30. The method of claim 1, prior to hybridizing an invasion primer, said method comprises amplifying a nucleic acid molecule thereby forming a plurality of double-stranded polynucleotides immobilized to the solid support.
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