WO2023081485A1

WO2023081485A1 - Stepwise sequencing of a polynucleotide with a homogenous reaction mixture

Info

Publication number: WO2023081485A1
Application number: PCT/US2022/049169
Authority: WO
Inventors: Min-Jui Richard Shen; Andrew Sparks; Pinar IYIDOGAN; Jeremiah Hanes
Original assignee: Pacific Biosciences Of California, Inc.
Priority date: 2021-11-08
Filing date: 2022-11-07
Publication date: 2023-05-11

Abstract

Provided includes methods, compositions and system for stepwise sequencing of nucleic acids with a homogenous reaction mixture.

Description

STEPWISE SEQUENCING OF A POLYNUCLEOTIDE WITH A HOMOGENOUS

REACTION MIXTURE

BACKGROUND

Field

[0001] The present application generally relates to molecular biology and more specifically to amplification and sequencing of nucleic acids.

Description of the Related Art

[0002] All current stepwise cyclic next generation sequencing (NGS) systems require flows of different reagents to determine the sequence of a polynucleotide. These different reagents perform specific steps of the cyclic reaction. For example, in a sequencing-by-binding system, incorporation reagent, exam reagent, a nucleotide strip buffer and a cleavage reagent all are flowed into a flow cell sequentially to perform specific processes. The flows and the reagent exchanges needed can be wasteful since many of the reactants are not fully consumed and more reagents are flowed than is necessary to fill a flow cell due to mixing during reagent exchanges. There is a need for sequencing methods that are less demanding on reagent consumption.

SUMMARY

[0003] Provided herein include methods, compositions, kits and systems for performing stepwise sequencing of nucleic acids with a homogenous reaction mixture.

[0004] Disclosed herein include methods of identifying a nucleotide in a primed- template nucleic acid. The method, in some embodiments, comprises:

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a first polymerase, a primed-template nucleic acid, reversibly 3 ’-blocked nucleotides, fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides are incapable of being covalently incorporated into the primed-template nucleic acid, optionally an unblocking enzyme, and a second polymerase;

(b) subjecting the vessel to a first condition to incorporate a reversibly 3 ’-blocked nucleotide into the primed-template nucleic acid by the first polymerase;

(c) subjecting the vessel to a second condition to form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the second polymerase and (iii) a fluorescently-labeled nucleotide bound at the base position of the primer-template nucleic acid 3’ adjacent to the incorporated reversibly 3 ’-blocked nucleotide, wherein the first polymerase is active in the first condition and inactive in the second condition, and wherein the second polymerase is capable of binding to fluorescently-labeled nucleotides in the second condition and not the first condition;

(d) imaging the ternary complex in the second condition to identify the bound fluorescently-labeled nucleotide; and

(e) deblocking the incorporated reversibly 3 ’-blocked nucleotide in the second condition by the unblocking enzyme, light exposure or both, wherein the unblocking enzyme is inactive in the first condition and active in the second condition. In some embodiments, steps (a) to (e) or steps (b) to (e) are conducted iteratively. In some embodiments, the second condition comprises light exposure (e.g., UV exposure). The first condition, in some embodiments, does not comprise light exposure.

[0005] The method of identifying a nucleotide in a primed-template nucleic acid, in some embodiments, can comprise

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a polymerase, a primed-template nucleic acid, reversibly 3 ’-blocked nucleotides, fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides are incapable of being covalently incorporated into the primed-template nucleic acid, and optionally an unblocking enzyme;

(b) subjecting the vessel to a first condition to incorporate a reversibly 3 ’-blocked nucleotide to the primed-template nucleic acid and form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the polymerase and (iii) a fluorescently-labeled nucleotide bound at the base position of the primer-template nucleic acid ‘ adjacent to the incorporated reversibly 3 ’-blocked nucleotide;

(c) imaging the ternary complex in the first condition to identify the type of the fluorescently-labeled nucleotide bound; and

(d) subjecting the vessel to a second condition to deblock the incorporated reversibly 3’- blocked nucleotide by the unblocking enzyme, light exposure or both, and wherein the polymerase is active in the first condition and inactive in the second condition, and wherein the unblocking enzyme is inactive in the first condition and active in the second condition. Steps (a) to (d) or steps (b) to (d) of the method, in some embodiments, are conducted iteratively. In some embodiments, the second condition comprises light exposure (e.g., UV exposure). The first condition, in some embodiments, does not comprise light exposure.

[0006] The method of identifying a nucleotide in a primed-template nucleic acid, in some embodiments, can comprise:

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a polymerase, a primed-template nucleic acid, fluorescently labeled reversibly 3 ’-blocked nucleotides, and optionally an unblocking enzyme;

(b) subjecting the vessel to a first condition to incorporate a fluorescently labeled reversibly 3 ’-blocked nucleotide to the primed-template nucleic acid to form a fluorescently labeled primed-template nucleic acid;

(c) imaging the fluorescently labeled primed-template nucleic acid in the first condition to identify the type of the fluorescently-labeled nucleotide bound; and

(d) subjecting the vessel to a second condition to deblock the incorporated fluorescently labeled 3 ’-blocked nucleotide by the unblocking enzyme, light exposure or both; and wherein the polymerase is active in the first condition and inactive in the second condition, and wherein the unblocking enzyme is inactive in the first condition and active in the second condition. In some embodiments, steps (a) to (d) or steps (b) to (d) of the method are conducted iteratively. In some embodiments, the second condition comprises light exposure (e.g., UV exposure). The first condition, in some embodiments, does not comprise light exposure.

[0007] Disclosed herein include methods for sequencing at least part of a nucleic acid template. The method, in some embodiments, comprises: incubating a sequencing composition and the template at a first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a first position; incubating the sequencing composition and the template at a second temperature to remove a 3’ blocking group from a first incorporated base of a sequencing strand; returning the sequencing composition and the template to the first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a second position; and returning the sequencing composition and the template to the second temperature to remove a 3’ blocking group from a second incorporated base of a sequencing strand, wherein the method is performed in a single volume without addition of exogenous reagents between assaying for fluorescence indicative of a labeled base paired to a template base at the first position and assaying for fluorescence indicative of a labeled base paired to a template base at the second position. The 3’ blocking group can be removed by light exposure (e.g., UV exposure), an unblocking enzyme, or both.

[0008] Disclosed herein also includes compositions for conducting stepwise sequencing in one single reaction mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 illustrates a non-limiting embodiment for the sequencing method described herein. Two polymerases are used for identifying a nucleotide in a primed-template nucleic acid.

[0010] FIG. 2 illustrates a non-limiting embodiment for the sequencing method described herein. A polymerase capable of incorporating reversibly 3’-blocked nucleotides to a primed-template nucleic acid and binding to fluorescently-labeled nucleotides at high temperatures is used for identifying a nucleotide in a primed-template nucleic acid.

[0011] FIG. 3 illustrates a non-limiting embodiment for the sequencing method described herein. A polymerase capable of incorporating reversibly 3’-blocked nucleotides to a primed-template nucleic acid and binding to fluorescently -lab eled nucleotides at low temperatures is used for identifying a nucleotide in a primed-template nucleic acid.

[0012] FIG. 4 illustrates a non-limiting embodiment for the preparation and capture of library members for sequencing.

DETAILED DESCRIPTION

[0013] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology. Definitions

[0014] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0015] As used herein, the term “immobilized,” when used in reference to a molecule, refers to direct or indirect, covalent or non-covalent attachment of the molecule to a surface such as a surface of a solid support. In some configurations, covalent attachment may be preferred, but generally all that is required is that the molecules (e.g. nucleic acids) remain immobilized or attached to the surface under the conditions in which surface retention is intended.

[0016] As used herein, the term “nucleotide” refers to a native nucleotide or analog thereof. Examples include, but are not limited to, nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), or non-natural analogs thereof such as dideoxyribonucleotide triphosphates (ddNTPs) or reversibly terminated nucleotide triphosphates (rtNTPs).

[0017] As used herein, the term “polymerase” refers to a nucleic acid synthesizing enzyme, including but not limited to, DNA polymerase, RNA polymerase, reverse transcriptase, primase and transferase. Typically, the polymerase has one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization may occur. The polymerase may catalyze the polymerization of nucleotides to the 3’ end of the first strand of the double stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of a next correct nucleotide to the 3’ oxygen moiety of the first strand of the double stranded nucleic acid molecule via a phosphodiester bond, thereby covalently incorporating the nucleotide to the first strand of the double stranded nucleic acid molecule. Optionally, a polymerase need not be capable of nucleotide incorporation under one or more conditions used in a method set forth herein. For example, a mutant polymerase may be capable of forming a ternary complex but incapable of catalyzing nucleotide incorporation.

[0018] As used herein, the term “primer” refers to a nucleic acid having a sequence that binds to a nucleic acid at or near a template sequence. Generally, the primer binds in a configuration that allows replication of the template, for example, via polymerase extension of the primer. The primer can be a first portion of a nucleic acid molecule that binds to a second portion of the nucleic acid molecule, the first portion being a primer sequence and the second portion being a primer binding sequence (e.g. a hairpin primer). Alternatively, the primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule having the template sequence. A primer can consist of DNA, RNA or analogs thereof. A primer can have an extendible 3’ end or a 3’ end that is blocked from primer extension.

[0019] As used herein, the term “vessel” refers to a container that functions to isolate one chemical process (e.g., a binding event; an incorporation reaction; etc.) from another, or to provide a space in which a chemical process can take place. Examples of vessels useful in connection with the disclosed technique include, but are not limited to, flow cells, wells of a multiwell plate; microscope slides; tubes (e.g., capillary tubes); droplets, vesicles, test tubes, trays, centrifuge tubes, features in an array, tubing, channels in a substrate etc.

[0020] As used herein, the term “thermostable” refers to a property of an enzyme, such that the enzyme is active at elevated temperatures including, for example, temperatures at which DNA duplexes denature in a given fluid. The enzyme can be, for example, a polymerase, a phosphatase or an esterase. “Active” means the ability of the polymerase to form a stabilized ternary complex or to catalyze primer extension reactions. Elevated temperatures as used herein can refer to the range of about 40 °C to about 65 °C, whereas non-elevated temperatures as used herein can refer to the range of about 15 °C to about 30 °C.

[0021] As used herein, the term “blocking moiety,” when used in reference to a nucleotide, refers to a part of the nucleotide that inhibits or prevents the 3’ oxygen of the nucleotide from forming a covalent linkage to a next correct nucleotide during a nucleic acid polymerization reaction. The blocking moiety of a “reversibly terminated” nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3 ’-oxygen of the nucleotide to covalently link to a next correct nucleotide. Such a blocking moiety is referred to herein as a “reversible terminator moiety.” Exemplary reversible terminator moieties are set forth in U.S. Pat Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or PCT publications WO 91/06678 published on May 16, 1991 or WO 07/123744 published on November 1, 2007, each of which is incorporated herein by reference. A nucleotide that has a blocking moiety or reversible terminator moiety can be a subunit at the 3’ end of a nucleic acid, such as a primer, or the nucleotide can be a monomeric molecule that is not covalently attached to a nucleic acid. A particularly useful blocking moiety will be present at the 3’ end of a nucleic acid that participates in formation of a stabilized ternary complex.

[0022] As used herein, the term “deblock” refers to removal or modification of a reversible terminator moiety of a nucleotide to render the nucleotide extendable. For example, the nucleotide can be present at the 3’ end of a primer such that deblocking renders the primer extendable. Exemplary deblocking reagents and methods are set forth in U.S. Pat Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or PCT publications WO 91/06678 published on May 16, 1991 or WO 07/123744 published on November 1, 2007, each of which is incorporated herein by reference.

[0023] As used herein, the term “incorporating” refers to the process of joining a nucleotide or oligonucleotide to the 3’ end or 5’ end of a nucleic acid by formation of a phosphodiester bond.

[0024] As used herein, the term “flow cell” refers to a vessel that includes one or more channels that have a detection zone. The detection zone can be coupled to a detector such that a reaction occurring in the vessel can be observed. For example, a flow cell can contain primed- template nucleic acid molecules tethered to a solid support, to which nucleotides and ancillary reagents are iteratively applied and washed away. The detection zone can include a transparent material that permits the sample to be imaged after a desired reaction occurs. For example, a flow cell can include a slide, such as a glass or plastic slide, containing small fluidic channels through which polymerases, dNTPs and buffers can be pumped. The channels, such as a glass or plastic slide of the channels, can be decorated with one or more primed-template nucleic acid molecules to be sequenced. As the subject methods to stepwise sequencing in a homogenous reaction mixture obviates the need for flowing reagents in every cycle, the primed-template nucleic acids could be distributed across, and imaged in, three dimensions. This can increase throughput in a single flow cell. For example, primed-template nucleic acid molecules can be distributed along a 3D scaffold (e.g., a porous, fibrous or permeable biomaterial that permits transport of fluids therein) that would still allow the initial introductions of the nucleic acid molecules and any other reagents described herein. If the primed-template nucleic acid is a concatemer, such as a rolling circle amplification product, its diffusion rate may be sufficiently low to allow the template nucleic acid to be unbound to any solid support and instead tracked in solution across multiple sequencing cycles. An external imaging system can be positioned to detect the molecules at a detection zone. Exemplary flow cells, methods for their manufacture and methods for their use (including imaging) are described in US Pat. App. Publ. Nos. 2010/0111768 Al published on May 6, 2010; or 2012-0270305 Al published on October 5, 2012; 2012-0014837 published January 12, 2012; or WO 05/065814 published on July 21, 2005, each of which is incorporated by reference herein. The imaging system may include a chip integrated within the flow cell. For example, an optode array chip integrated with the flow cell may comprises an array of optical detectors (e.g., photodiodes) and optionally further comprises one or more optical elements (e.g., lenses, filters, gratings, mirrors, prisms, refractive material, waveguides, and the like). Alternatively, the imaging system may comprise a fluorescent microscope, such as n epi-fluorescent microscope, configured to illuminate the flow cell at one or more excitation wavelengths and detect one or more emission wavelengths (e.g., wavelengths emitted by fluorescently labeled nucleotides). If the primed- template nucleic acids are distributed across three dimensions in the flow cell, 3D imaging can be performed with a wide-field microscope, such as by imaging across multiple focal planes, or can be performed by certain scanning microscopes such as a confocal microscope.

[0025] As used herein, the term “label” refers to a molecule, or moiety thereof, that provides a detectable characteristic. The detectable characteristic can be, for example, an optical signal such as absorbance of radiation, fluorescence emission, luminescence emission, fluorescence lifetime, luminescence lifetime, fluorescence polarization, luminescence polarization or the like; Rayleigh and/or Mie scattering; binding affinity for a ligand or receptor; magnetic properties; electrical properties; charge; mass; radioactivity or the like. Exemplary labels include, without limitation, a fluorophore, luminophore, chromophore, nanoparticle (e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive isotope, mass label, charge label, spin label, receptor, ligand, or the like.

[0026] As used herein, the term “polymerase” refers to a nucleic acid synthesizing enzyme, including but not limited to, DNA polymerase, RNA polymerase, reverse transcriptase, primase and transferase. Typically, the polymerase has one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. The polymerase can catalyze the polymerization of nucleotides to the 3’ end of the first strand of the double stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of a next correct nucleotide to the 3’ oxygen moiety of the first strand of the double stranded nucleic acid molecule via a phosphodiester bond, thereby covalently incorporating the nucleotide to the first strand of the double stranded nucleic acid molecule. Optionally, a polymerase need not be capable of nucleotide incorporation under one or more conditions used in a method set forth herein. For example, a mutant polymerase can be capable of forming a ternary complex but incapable of catalyzing nucleotide incorporation.

[0027] As used herein, the term “primed-template nucleic acid” or “primed-template” refers to a nucleic acid having a double stranded region such that one of the strands functions as a primer and the other strand functions as a template. The two strands can be parts of a contiguous nucleic acid molecule (e.g., a hairpin structure) or the two strands can be separable molecules that are not covalently attached to each other.

[0028] As used herein, the term “ternary complex” refers to an intermolecular association between a polymerase, a double stranded nucleic acid (e.g., a partially double stranded nucleic acid) and a nucleotide. The polymerase can facilitate interaction between a next correct nucleotide and a template strand of the primed nucleic acid. A next correct nucleotide can interact with the template strand via Watson-Crick hydrogen bonding. The term “stabilized ternary complex” means a ternary complex having promoted or prolonged existence or a ternary complex for which disruption has been inhibited. Generally, stabilization of the ternary complex prevents covalent incorporation of the nucleotide component of the ternary complex into the primed nucleic acid component of the ternary complex.

[0029] Disclosed herein include methods, compositions, kits and systems for performing all of the cyclic steps of stepwise sequencing in one homogenous reaction mixture. The control of the step-wise sequencing of the polynucleotide can be done, for example, via temperature cycling. The methods, compositions and systems disclosed herein can be used in nucleic acid sequencing, for example in sequencing-by-binding (SBB) or in sequencing-by- synthesis (SBS).

Homogenous Reaction Mixture for Sequencing

[0030] Disclosed herein include methods, compositions, kits and systems for performing all of the cyclic steps of sequencing in one homogenous reaction mixture. The control of the stepwise polynucleotide sequencing can be achieved by, for example, temperature cycling.

[0031] In some embodiments, the homogenous reaction mixture can include a first polymerase (e.g., DNA polymerase), an unblocking enzyme, fluorescently labeled nucleotides that cannot be incorporated into a primed-template polynucleotide, reversibly 3 ’-blocked nucleotides that can be incorporated into a primed-template polynucleotide, and a second polymerase. The homogenous reaction mixture can further include a primed-template nucleic acid. The fluorescently-labeled nucleotides can comprise fluorescently-labeled thiol nucleotides. In some embodiments, the fluorescently-labeled thiol nucleotides comprise fluorescently-labeled thiodiphosphate nucleotides, fluorescently-labeled thiotriphosphate nucleotides, or a mixture thereof. For example, the first polymerase can be active in a first condition (e.g., high temperatures) and inactive in a second condition (e.g., low temperatures); the second polymerase can be capable of binding to the fluorescently labeled nucleotides that cannot be incorporated into a primed-template polynucleotide in the second condition and not the first condition; and the unblocking enzyme can be inactive in the first condition and active in the second condition. In some embodiments, the first polymerase can be active at high temperatures, for example a temperature of about 40 °C to 65 °C or higher, including 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, or a number or a range between any two of these values. Non-limiting examples of DNA polymerase that are active at high temperatures (e.g., the first DNA polymerase) include Family B DNA polymerases, including but not limited to variants of 9°N DNA polymerase. In some embodiments, the first polymerase is one of the engineered polymerases described in US Patent Application Publication No. 20210139867, including K02, or a variant thereof. In some embodiments, the first polymerase is Therminator DNA Polymerase. Non-limiting examples of the second polymerase include Family A DNA polymerases, including but not limited to KlenTaq DNA polymerase or a variant thereof, Bst DNA polymerase or a variant thereof, or Bsu DNA polymerase or a variant thereof. It is advantageous for the unblocking enzyme described herein to be thermolabile in some embodiments. The term “unblocking enzyme” is used herein to refer to enzymes capable of deblock reversible 3 ’-blocked nucleotides. Non-limiting examples of unblocking enzyme include polynucleotide phosphatase and esterase. In some embodiments, the reversibly 3 ’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase (including but not limited to T4 polynucleotide phosphatase/kinase (PNK) or a variant thereof). In some embodiments, the reversibly 3 ’-blocked nucleotides comprise 3’- esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase. In some embodiments, the second polymerase is catalytically inactive so that the fluorescently-labeled nucleotides cannot be incorporated into the primed- template nucleic acid.

[0032] The first polymerase, the second polymerase and the unblocking enzyme can be inactivated under a condition via various mechanisms. For example, one or more of the first polymerase, the second polymerase and the unblocking enzyme can be temperature sensitive enzymes that have enzymatic activity in a first condition but not a second condition. As another example, one or more of the first polymerase, the second polymerase, and the unblocking enzyme can be temperature sensitive enzymes that have no enzymatic activity but have binding activity in a first condition but not a second condition. As yet another example, one or more of the first polymerase, the second polymerase, and the unblocking enzyme can be temperature sensitive enzymes that have no enzymatic activity nor binding activity in a first condition but not a second condition. One or the more of the first polymerase, the second polymerase, and the unblocking enzyme can be inactivated by an aptamer, an antibody, or a combination thereof. For example, the homogenous reaction mixture can comprise a first aptamer or a first antibody capable of inactivating the first polymerase in the second condition. In some embodiments, the reaction mixture comprises a second aptamer or a second antibody capable of inactivating the unblocking enzyme in the first condition. In some embodiments, the reaction mixture comprises a third aptamer or a third antibody capable of inactivating the second polymerase in the first condition. One or more of the first polymerase, the second polymerase and the unblocking enzyme can also be a chemically reversibly modified enzyme that is active in one condition not in another condition. For example, the first polymerase can be a chemically reversibly modified polymerase that is active in the first condition and inactive in the second condition. In some embodiments, the unblocking enzyme is a chemically reversibly modified unblocking enzyme that is inactive in the first condition and active in the second condition. For example, the unblocking enzyme can be renaturable and/or active at low temperature, for example a temperature of about 15 °C to 30 °C, including 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, or a number or a range between any two of these values. In some embodiments, the unblocking enzyme is renaturable and/or active at a temperature of, or of about, 25 °C.

[0033] In some embodiments, the homogenous reaction mixture can include a polymerase (e.g., DNA polymerase), an unblocking enzyme, fluorescently labeled nucleotides that cannot be incorporated into a primed-template polynucleotide, and reversibly 3 ’-blocked nucleotides that can be incorporated into a primed-template polynucleotide. The homogenous reaction mixture can further include a primed-template nucleic acid. The fluorescently-labeled nucleotides can comprise fluorescently-labeled thiol nucleotides. In some embodiments, the fluorescently-labeled thiol nucleotides comprise fluorescently-labeled thiodiphosphate nucleotides, fluorescently-labeled thiotriphosphate nucleotides, or a mixture thereof. The control of the stepwise polynucleotide sequencing can be achieved by, for example, temperature cycling. It can be advantageous for the DNA polymerase and the unblocking enzyme to be active at different conditions (e.g., different temperature or temperature ranges) so that incorporation of a reversibly 3 ’-blocked nucleotide to the primed-template nucleic acid is conducted at a condition different from the temperature under which the binding of the fluorescently labeled nucleotides to the primer-template nucleic acid occurs. For example, the polymerase can be active in the first condition and inactive in the second condition, and the unblocking enzyme is inactive in the first condition and active in the second condition. In some embodiments, the polymerase is active in high temperatures (for example a temperature of about 40 °C to 65 °C or higher, including 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, or a number or a range between any two of these values) and the unblocking enzyme is active in low temperatures (for example a temperature of about 15 °C to 30 °C, including 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, or a number or a range between any two of these values). The polymerase can be a Family B DNA polymerase, including but not limited to variants of 9°N DNA polymerase. In some embodiments, the polymerase is one of the engineered polymerases described in US Patent Application Publication No. 20210139867, including K02, or a variant thereof. The reversibly 3 ’-blocked nucleotides can comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase (e.g., T4 polynucleotide phosphatase/kinase (PNK) or a variant thereof). In some embodiments, reversibly 3 ’-blocked nucleotides comprise 3’-esterified dNTPs, the polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

[0034] In some embodiments, the first condition is low temperatures (e.g., a temperature of about 15 °C to 30 °C, including 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, or a number or a range between any two of these values) and the second condition is high temperatures (e.g., a temperature of about 40 °C to 65 °C or higher, including 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, or a number or a range between any two of these values). Thus, the polymerase in these embodiments is active at low temperatures and the unblocking enzyme in these embodiments is active in high temperatures. For example, the polymerase can be a Family A or Family B DNA polymerase, including but not limited to Klenow or a variant thereof, or Phi29 polymerase or a variant thereof. In some embodiments, the reversibly 3 ’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase (e.g., Thermophilic PNK RM378 or a variant thereof). In some embodiments, the reversibly 3 ’-blocked nucleotides comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

[0035] In some embodiments, the homogenous reaction mixture can include a polymerase (e.g., DNA polymerase), fluorescently-labeled and reversibly 3 ’-blocked nucleotides; and an unblocking enzyme. The homogenous reaction mixture can further include a primed- template nucleic acid. The control of the stepwise polynucleotide sequencing can be achieved by, for example, temperature cycling. For example, the polymerase can be active in the first condition and inactive in the second condition, and the unblocking enzyme is inactive in the first condition and active in the second condition. In some embodiments, the polymerase is active in high temperatures (for example a temperature of about 40 °C to 65 °C or higher, including 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, or a number or a range between any two of these values) and the unblocking enzyme is active in low temperatures (for example a temperature of about 15 °C to 30 °C, including 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, or a number or a range between any two of these values).

[0036] It can be advantageous that the thermostable DNA polymerase has low processivity or is nonprocessive. As used herein, the term “processivity”, when used in reference to a polymerase, is a measure of the number of nucleotides that the polymerase can incorporate into a nascent primer prior to dissociation of the polymerase from the template to which the primer is hybridized. Accordingly, processivity of a polymerase can be measured as the number of nucleotides being added to a primer in a single polymerase-DNA binding event. DNA polymerase’s processivity is typically influenced by the rate of nucleic acid synthesis rate, as well as the affinity of the polymerase for its substrates (e.g., primed-template nucleic add and nucleotide). In some embodiments, the DNA polymerase in the homogenous reaction mixture described herein has a processivity that is, or is about, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or a number or a range between any two of these values, lower than the processivity of a wildtype Family B DNA polymerase, a wildtype Family A DNA polymerase 9°N DNA polymerase, or Phi29 polymerase. In some embodiments, the processivity of the DNA polymerase in the homogenous reaction mixture described herein is, is about, or is at most about, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or a number or a range between any two of these values, of the processivity of a wildtype Family B DNA polymerase, a wildtype Family A DNA polymerase 9°N DNA polymerase, or Phi29 polymerase. In some embodiments, the DNA polymerase in the homogenous reaction mixture described herein is nonprocessive. The polymerase (e.g., DNA polymerase) suitable for use in the homogenous reaction mixture described herein can be thermostable.

[0037] It is advantageous for the unblocking enzyme described herein to be thermolabile in some embodiments. The term “unblocking enzyme” is used herein to refer to enzymes capable of deblock reversible 3 ’-blocked nucleotides. Non-limiting examples of unblocking enzyme include polynucleotide phosphatase and esterase. In some embodiments, the unblocking enzyme is renaturable and/or active at low temperature, for example a temperature of about 15 °C to 30 °C, including 15 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, or a number or a range between any two of these values. In some embodiments, the unblocking enzyme is renaturable and/or active at low temperature, for example a temperature of, or of about, 25 °C. In some embodiments, the unblocking enzyme is renaturable and/or active at high temperature, for example a temperature of about 40 °C to 65 °C, including 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 55 °C, 60 °C, 65 °C, or a number or a range between any two of these values. In some embodiments, the unblocking enzyme is renaturable and/or active at low temperature, for example a temperature of, or of about, 45 °C. In some embodiments, the unblocking enzyme is optional. For example, the reversibly 3’-blocked nucleotides can comprise dNTPs having a 3’ photolabile protecting group, which can be deblocked by, for example, light exposure (e.g., UV exposure, photolysis). For example, the reversibly 3 ’-blocked nucleotides can be, or comprise, 3’-O-(2-nitrobenzyl)- 2’deoxyribonucleoside triphosphates (NB-dNTPs), 3’-O-(4,5-dimethoxy-2-nitrobenzyl)-2’- deoxyribonucleoside triphosphates (DMNB-dNTPs), or a combination thereof. In some embodiments, the second condition comprises light exposure, and optionally the light exposure is UV exposure. In some embodiments, the first condition does not comprise light exposure. It can be advantageous, in some embodiments, to focus the light exposure to a cluster region of the vessel. The focusing can be achieved, for example, using zero-mode waveguides (ZMWs). Sequencing Using A Homogenous Reaction Mixture

[0038] Disclosed herein include sequencing methods for performing stepwise sequencing of nucleic acids with a homogenous reaction mixture, which are less demanding on reagent consumption than currently available techniques.

[0039] The methods disclosed herein can be used in various sequencing platforms, including but not limited to, sequencing-by-synthesis or sequencing-by-binding (sometimes collectively referred to as sequencing-by-incorporation chemistries), pH-based sequencing, sequencing by polymerase monitoring, sequencing by hybridization, and other methods of massively parallel sequencing or next-generation sequencing. In some embodiments, the sequencing is carried out as described in US Pat. No. 10,077,470, which is incorporated by reference herein in its entirety. Suitable surfaces for carrying out sequencing include, but are not limited to, a planar substrate, a hydrogel, a nanohole array, a microparticle, or nanoparticle.

[0040] Sequencing by binding has been described, for example, in US Pat. Nos. 10,443,098 and 10,246,744, and US Pat. App. Pub. No. 2018,0044727; the content of each is incorporated herein by reference in its entirety. Briefly, in SBB, the polymerase undergoes conformational transitions between open and closed conformations during discrete steps of a reaction. In one step, the polymerase binds to a primed template nucleic acid to form a binary complex, also referred to herein as the pre-insertion conformation. In a subsequent step, an incoming nucleotide is bound and the polymerase fingers close, forming a pre-chemistry conformation comprising the polymerase, primed template nucleic acid and labeled nucleotide; wherein the bound labeled nucleotide has not been incorporated. This step, also referred to herein as an examination step, is followed by removal of the labeled nucleotide without incorporation, and is then followed by de-blocking of the extended strand 3’ end so as to render it suitable for extension. Unlabeled, 3’ blocked nucleotides are then added, followed by a chemical incorporation step wherein a phosphodiester bond is formed with concomitant pyrophosphate cleavage from the nucleotide (nucleotide incorporation), to form an extension strand that has been extended by one base and that is not competent for further extension without modification. Unincorporated blocked extension bases are removed and labeled bases added, so that they can from ternary complexes at positions where they base pair with the template. These ternary complexes are assayed for fluorescence or other output to determine the identity of the paired base, and then the process is repeated through removal of the labeled base, chemical modification of the extending strand to reveal a 3’ OH, and contacting with a population of 3’ blocked, unlabeled nucleotides for another single base extension.

[0041] SBS generally involves the enzymatic extension of a nascent primer through the iterative addition of nucleotides against a template strand to which the primer is hybridized. SBS differs from SBB, above, in that labeled nucleotides are incorporated into the extending strand, assayed and then the label is removed or deactivated, and the 3’ block removed, to iteratively sequence a template. In SBB, a labeled base is not incorporated into an extending strand. Rather, ternary complex formation is assayed, usually for the presence of a labeled base but sometimes for the presence of a labeled polymerase or other feature, after which point the complex is disassembled and a 3’ blocked, unlabeled base is used to extend the primer strand. Briefly, SBS can be initiated by contacting target nucleic acids, attached to sites in a flow cell, with one or more labeled nucleotides, DNA polymerase, etc. Those sites where a primer is extended using the target nucleic acid as template will incorporate a labeled nucleotide that can be detected. Detection can include scanning using an apparatus or method set forth herein. Optionally, the labeled nucleotides can further include a reversible termination property that terminates 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 is delivered to remove the moiety. Thus, for embodiments that use reversible termination, a deblocking reagent can be delivered to the vessel (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can be performed n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, reagents and detection components that can be readily adapted for use with a method, system or apparatus of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281, and US Pat. App. Pub. No. 2008/0108082 Al, each of which is incorporated herein by reference.

[0042] FIG. 1 illustrates a non-limiting embodiment of the sequencing method described herein in an SBB system. Two polymerases are used in this workflow for identifying a nucleotide in a primed-template nucleic acid. In the workflow, a reaction mixture (e.g., a reaction mixture provided in a vessel) is provided which comprises: a first polymerase, a primed-template nucleic acid, reversibly 3 ’-blocked nucleotides, fluorescently-labeled nucleotides incapable of being covalently incorporated into the primed-template nucleic acid (e.g., fluorescently-labeled alpha thiol dNTPs or dNDPs), an unblocking enzyme (e.g., a polynucleotide phosphatase operable on polynucleotides but not nucleotides), and a second polymerase. As shown in FIG. 1, the first polymerase can be a Family B polymerase, for example Therminator polymerase. The first polymerase is capable of incorporating reversibly 3 ’-blocked nucleotides into the primed-template nucleic acid at a first condition (e.g., high temperatures) but not a second condition (e.g., low temperatures). The second polymerase can be a Family A polymerase, for example KlenTaq. The second polymerase is incapable of incorporating reversibly 3 ’-blocked nucleotides into the primed-template nucleic acid at neither the first condition nor the second condition, and is capable of binding fluorescently-labeled nucleotides and allows imaging of the ternary complex comprising the unincorporated but bound fluorescently-labeled nucleotide at the second condition (e.g., the low temperatures). The reaction mixture can be subjected to a first condition to incorporate a reversibly 3 ’-blocked nucleotide into the primed-template nucleic acid by the first polymerase, and then be subjected to a second condition to form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the second polymerase and (iii) a fluorescently-labeled nucleotide bound at the base position of the primer-template nucleic acid 3’ adjacent to the incorporated reversibly 3 ’-blocked nucleotide. The ternary complex is imaged in the second condition to identify the bound fluorescently-labeled nucleotide, and the incorporated reversibly 3 ’-blocked nucleotide is deblocked in the second condition by the unblocking enzyme which is inactive in the first condition and active in the second condition. Steps (a) to (e) of the workflow/method can be conducted iteratively so that multiple nucleotides of the template polynucleotide can be identified. In the embodiment illustrated in FIG. 1, the first condition can comprise a temperature of about 40 °C to 65 °C, and the second condition can comprise a temperature of about 15 °C to 30 °C. In some embodiments, the reversibly 3 ’-blocked nucleotides can comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme can be an esterase.

[0043] FIG. 2 illustrates a non-limiting embodiment of the sequencing method described herein in an SBB system. One polymerase is used in this workflow for identifying a nucleotide in a primed-template nucleic acid. In the workflow, a reaction mixture (e.g., a reaction mixture provided in a vessel) is provided which comprises: a polymerase, a primed-template nucleic acid, reversibly 3 ’-blocked nucleotides, fluorescently-labeled nucleotides incapable of being covalently incorporated into the primed-template nucleic acid (e.g., fluorescently-labeled alpha thiol dNTPs or dNDPs), and an unblocking enzyme (e.g., a polynucleotide phosphatase operable on polynucleotides but not nucleotides). As shown in FIG. 2, the polymerase can be a Family B polymerase, for example K02 polymerase. The polymerase is capable of incorporating reversibly 3 ’-blocked nucleotides into the primed-template nucleic acid and binding the fluorescently-labeled nucleotides at a first condition (e.g., high temperatures) but not a second condition (e.g., low temperatures). It can be advantageous that the polymerase can survive temperature cycling. The reaction mixture can be subjected to a first condition to incorporate a reversibly 3 ’-blocked nucleotide to the primed-template nucleic acid and form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the polymerase and (iii) a fluorescently- labeled thiol nucleotide bound at the base position of the primer-template nucleic acid 3’ adjacent to the incorporated reversibly 3 ’-blocked nucleotide. The ternary complex is imaged in the first condition to identify the type of the fluorescently-labeled thiol nucleotide bound, and the incorporated reversibly 3 ’-blocked nucleotide is deblocked in the second condition by the unblocking enzyme which is inactive in the first condition and active in the second condition. Steps (a) to (d) of the workflow/method can be conducted iteratively so that multiple nucleotides of the template polynucleotide can be identified. In the embodiment illustrated in FIG. 2, the first condition can comprise a temperature of about 40 °C to 65 °C, and the second condition can comprise a temperature of about 15 °C to 30 °C. In some embodiments, the reversibly 3’-blocked nucleotides can comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme can be an esterase. In some embodiments, the polynucleotide phosphatase is a T4 PNK or a variant thereof.

[0044] FIG. 3 illustrates a non-limiting embodiment of the sequencing method described herein in an SBB system. One polymerase is used in this workflow for identifying a nucleotide in a primed-template nucleic acid. In the workflow, a reaction mixture (e.g., a reaction mixture provided in a vessel) is provided, where the reaction mixture comprises: a polymerase, a primed-template nucleic acid, reversibly 3 ’-blocked nucleotides, fluorescently-labeled nucleotides incapable of being covalently incorporated into the primed-template nucleic acid (e.g., fluorescently-labeled alpha thiol dNTPs or dNDPs), and an unblocking enzyme (e.g., a polynucleotide phosphatase operable on polynucleotides but not nucleotides). As shown in FIG. 3, the polymerase can be a Family B polymerase, for example K02 polymerase. The polymerase is capable of incorporating reversibly 3 ’-blocked nucleotides into the primed-template nucleic acid and binding the fluorescently-labeled nucleotides at a first condition (e.g., high temperatures) but not a second condition (e.g., low temperatures). It can be advantageous that the polymerase can survive temperature cycling. The reaction mixture can be subjected to a first condition to incorporate a reversibly 3 ’-blocked nucleotide to the primed-template nucleic acid and form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the polymerase and (iii) a fluorescently-labeled thiol nucleotide bound at the base position of the primer-template nucleic acid 3’ adjacent to the incorporated reversibly 3 ’-blocked nucleotide. The ternary complex is imaged in the first condition to identify the type of the fluorescently-labeled thiol nucleotide bound, and the incorporated reversibly 3 ’-blocked nucleotide is deblocked in the second condition by the unblocking enzyme which is inactive in the first condition and active in the second condition. Steps (a) to (d) of the workflow/method can be conducted iteratively so that multiple nucleotides of the template polynucleotide can be identified. In the embodiment illustrated in FIG. 3, the first condition can comprise a temperature of about 15 °C to 30 °C, and the second condition can comprise a temperature of about 40 °C to 65 °C. In some embodiments, the reversibly 3’- blocked nucleotides can comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme can be an esterase. In some embodiments, the polymerase is Klenow or Phi29 polymerase or a variant thereof. In some embodiments, the polynucleotide phosphatase is Thermophilic PNK RM378 or a variant thereof.

[0045] The methods described herein can also be used in other sequencing platform, for example SBS, to identify one or more nucleotides in a primed-template nucleic acid using a polymerase that is active in the first condition and inactive in the second condition, and an unblocking enzyme that is inactive in the first condition and active in the second condition. The method can, in some embodiments, comprise: providing a reaction mixture (e.g., a reaction mixture in a vessel) comprising: a polymerase, a primed-template nucleic acid, fluorescently labeled reversibly 3 ’-blocked nucleotides, and an unblocking enzyme. The reaction mixture can be subjected to a first condition to incorporate a fluorescently labeled 3 ’-blocked nucleotide to the primed-template nucleic acid to form a fluorescently labeled primed-template nucleic acid, and the fluorescently labeled primed-template nucleic acid can be imaged in the first condition to identify the type of the fluorescently-labeled nucleotide bound. Then the reaction mixture (or the vessel containing the reaction mixture) can be subjected to a second condition to deblock the incorporated fluorescently labeled 3 ’-blocked nucleotide by the unblocking enzyme. Steps (a) to (d) of the workflow/method can be conducted iteratively so that multiple nucleotides of the template polynucleotide can be identified.

[0046] Also disclosed herein include methods of sequencing at least part of a nucleic acid template. The method, in some embodiments, can comprise: incubating a sequencing composition and the template at a first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a first position; incubating the sequencing composition and the template at a second temperature to remove a 3 ’ blocking group from a first incorporated base of a sequencing strand; returning the sequencing composition and the template to the first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a second position; and returning the sequencing composition and the template to the second temperature to remove a 3’ blocking group from a second incorporated base of a sequencing strand, wherein the method is performed in a single volume without addition of exogenous reagents between assaying for fluorescence indicative of a labeled base paired to a template base at the first position and assaying for fluorescence indicative of a labeled base paired to a template base at the second position. The method can further comprise: returning the sequencing composition and the template to the first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a third position; and returning the sequencing composition and the template to the second temperature to remove a 3’ blocking group from a third incorporated base of a sequencing strand, wherein the method is performed in a single volume without addition of exogenous reagents between assaying for fluorescence indicative of a labeled base paired to a template base at the first position and assaying for fluorescence indicative of a labeled base paired to a template base at the third position.

[0047] The assaying for fluorescence indicative of a labeled base paired to a template base at a first position comprises assaying for a nucleotide can comprise a fluorophore incorporated into a ternary complex. The nucleotide comprising a fluorophore may not comprise a blocked 3’ end. In some embodiments, the nucleotide comprising a fluorophore is not covalently bound to the sequencing strand.

[0048] The assaying for fluorescence indicative of a labeled base paired to a template base at a first position comprises assaying for a nucleotide can comprise a fluorophore incorporated into the sequencing strand. The nucleotide can comprise a fluorophore comprises a blocked 3’ end. The method can further comprise removing the fluorophore subsequent to the assaying for fluorescence. In some embodiments, the method comprises washing the template and the sequencing strand to replace the sequencing composition only after at least two cycles of assaying for fluorescence indicative of a labeled base paired to a template base. In some embodiments, the method washing the template and the sequencing strand to replace the sequencing composition only after at least three cycles of assaying for fluorescence indicative of a labeled base paired to a template base. In some embodiments, the method comprises performing at least 7 additional cycles of assaying for fluorescence indicative of a labeled base paired to a template base and removing a 3’ blocking group.

[0049] Multiple consecutive base positions of the template can be determined without replacing the sequencing composition. The sequencing composition can comprises any one of the reaction mixtures disclosed herein. The sequencing composition can comprise a fluorescently labeled, 3’ blocked nucleotide, a fluorescently labeled, 3’ unblocked nucleotide, excised former 3 ’blocking moi eties, or any combination thereof. The excised former 3’ blocking moieties can be present in at least a 2x excess relative to template-sequencing strand double stranded complexes, or can be present in at least a 2x excess relative to sequencing strand unblocked free 3’ ends, or both.

[0050] The method of sequencing at least part of a nucleic acid template can be performed in a flow cell. The method of sequencing at least part of a nucleic acid template can be performed in contact with a thermocycling heat block. The method of sequencing at least part of a nucleic acid template can be performed in a single file emulsion of droplets. In some embodiments, the single file emulsion of droplets flow repeatedly through a circular course or through a serpentine course, wherein one side of the serpentine is at a first condition (e.g., the first temperature) and the other side of the serpentine course is at a second condition (e.g., the second temperature). The number of serpentine turns can define the number of cycles (bases) that is sequenced. A detector can be used at each turn to determine the sequence in the droplet.

[0051] In any of the methods, systems and compositions disclosed herein, when the reagent(s) (e.g., one or more of the first polymerase, the second polymerase, unblocking enzyme, reversibly 3 ’-blocked nucleotides, fluorescently-labeled nucleotides, and fluorescently-labeled and reversibly 3 ’-blocked nucleotides) become exhausted or fall below a desirable or threshold amount/concentration during the sequencing process, new reagent(s) can be added into (e.g., flowed into) the sequencing reaction/vessel to enable sequencing to continue. In some embodiments, zero mode wave guides (ZMWs) can be used in the methods and systems described herein. The ZMW guides can, for example, help to reduce background signal from the freely floating fluorescently labeled thiodiphosphate nucleotides in solution.

[0052] Any of a variety of polymerases can be used in a method or composition set forth herein, for example, to form a polymerase-nucleic acid complex or to carry out primer extension. Polymerases that can be used include naturally occurring polymerases and modified variations thereof, including, but not limited to, mutants, recombinants, fusions, genetic modifications, chemical modifications, synthetics, and analogs. Naturally occurring polymerases and modified variations thereof are not limited to polymerases that have the ability to catalyze a polymerization reaction. Optionally, the naturally occurring and/or modified variations thereof have the ability to catalyze a polymerization reaction in at least one condition that is not used during formation or examination of a stabilized ternary complex. Optionally, the naturally occurring and/or modified variations that participate in polymerase-nucleic acid complexes have modified properties, for example, enhanced binding affinity to nucleic acids, reduced binding affinity to nucleic acids, enhanced binding affinity to nucleotides, reduced binding affinity to nucleotides, enhanced specificity for next correct nucleotides, reduced specificity for next correct nucleotides, reduced catalysis rates, catalytic inactivity etc. Mutant polymerases include, for example, polymerases wherein one or more amino acids are replaced with other amino acids, or insertions or deletions of one or more amino acids. Exemplary polymerase mutants that can be used to form a stabilized ternary complex include, for example, those set forth in U.S. Patent Application Publication No. 2020/0087637 Al or U.S. Patent Nos. 10,584,379 or 10,597,643, each of which is incorporated herein by reference.

[0053] Systems disclosed herein for nucleic acid amplification, detection and/or sequencing can include a vessel, solid support or other apparatus for carrying out a nucleic acid amplification, detection and/or sequencing. For example, the system can include an array, flow cell, multi-well plate, test tube, channel in a substrate, collection of droplets or vesicles, tray, centrifuge tube, tubing or other convenient apparatus. The apparatus can be removable, thereby allowing it to be placed into or removed from the system. As such, a system can be configured to process a plurality of apparatus (e.g. vessels or solid supports) sequentially or in parallel. The system can include a fluidic component configured to deliver one or more reagents (e.g., one or more reagents in a solution) to a vessel or solid support, for example, via channels or droplet transfer apparatus (e.g. electrowetting apparatus). Any of a variety of detection apparatus can be configured to detect the vessel or solid support where reagents interact. Exemplary systems having fluidic and detection components those set forth in US Pat. App. Pub. No. 2018/0280975A1; U.S. Pat. Nos. 8,241,573; 7,329,860 or 8,039,817; or US Pat. App. Pub. Nos. 2009/0272914 Al or 2012/0270305 Al, each of which is incorporated herein by reference.

[0054] In certain aspects, nucleic acid amplification is performed in a vessel prior to sequencing in the vesell. For example, bridge amplification or rolling circle amplification may be used to create clusters on a solid surface in the vessel. Such clustering methods are described in US Pub. Nos. 20060024711, PCT publication WO 21/20564 and US Pat. No. US7,790,418, each of which are incorporated herein by reference. Such clusters may have known “adaptor” regions and unkown “insert” regions. The insert region may be from a sample, and in certain embodiments may comprise genomic DNA or cDNA. The adaptor regions may include one or more primer binding sites at which a sequencing primer may bind, and may optionally further include one or more barcode sequences (e.g., one or more sample barcodes and/or degenerate molecular barcodes). The homogeneous sequencing described herein may comprise sequencing of clusters in which multiple copies of the same sequencing primer and insert within a cluster together provide an amplified signal.

[0055] In other aspects, the homogeneous sequencing described herein may comprise single molecule sequencing, such as sequencing of a single copy of an insert from a single sequencing primer. Single molecule sequencing my improve detection by using waveguide illumination, a ZMW detection region, and/or integrated detection such as is described in US Pat. No. 8,471,230 which is incorporated herein by reference. Alternatively or in addition, stepwise sequeincg, such as stepwise homogenous sequencing, may be used to increase exposure time for each step in comparasion to single molecule real time sequencing. For example, a cycle of detecting and extending a single base may take more than 5 seconds, more than 10 seconds, more than 30 seconds, or more than 60 seconds, in a stepwise single molecule sequencing reaction.

[0056] Aspects include reagents and methods of library preparation for stepwise sequeincg, such as sequencing in a homogeneous reaction mixture, stepwise single molecule sequencing, or stepwise single molecule sequencing in a homogeneous reaction mixture. Library preparation may include attaching adaptors to inserts, such as by ligation, and may further include amplification and/or attachment of library members to a surface for sequencing. US Pat. Nos. 6,372,434, 8,153,375, and 8,288,097 describe suitable adaptors and each of which is incorporated herein by reference.

[0057] In certain aspects, adaptors may each comprise a double stranded region that is ligated to ends of a double stranded insert to form a library member. Such ligation may be facilitated by end repair and/or generation of sticky ends (e.g., by a restriction digest or a tailing enzyme such as A-tailing). Such adaptors may further include one or more single stranded regions, such as a hairpin or two unique ends comprising “mismatched” regions that do not hybridize to each other, for example as shown in FIG. 4. Mismatched adaptors provide unique ends when library members are denatured (e.g., displaced by amplifiaiton and/or melted by an increase in temperature). In specific embodiments, a first unique end may comprise a single stranded capture sequence for attachment to a solid support for sequencing and a second unique end may comprise a hairpin region that provides a 3’ end that may be extended in a sequencing reaction. A single stranded DNA region of the second unique end, or the double stranded region, may comprise one or more barcodes, such as a sample barcode and/or a degenerate molecular barcode. The capture sequences may be matched to sample barcodes to allow for index matching and/or duplex sequencing. In certain aspects, polymerase may be bound to the hairpin in solution but prevented from extension, such as through the presence of a non-catalytic ion (e.g., calcium or strontium) and/or absence of a catalytic ion (e.g., absence of magnesium). Library members may be captured without any prior amplification. Alternatively, polymerase may be added after capture of the first unique end on a solid support. In certain aspects, the solid support may be in a ZMW. Capture may be by hybridization of a capture sequence as described above, or may be by binding to a moiety at the 5’ end of the first unique end, such as biotin-streptavidin binding or click chemistry. In certain aspects, sequencing continues through the insert such that the polymerase used for sequencing displaces the hybridization of the capture sequence, allowing that region of the solid support to be used in sequencing of another library member.

[0058] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0059] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0060] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

[0061] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0062] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0063] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying a nucleotide in a primed-template nucleic acid, comprising:

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a first polymerase, a primed-template nucleic acid, reversibly 3 '-blocked nucleotides, fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides are incapable of being covalently incorporated into the primed-template nucleic acid, an unblocking enzyme, and a second polymerase;

(c) subjecting the vessel to a second condition to form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the second polymerase and (iii) a fluorescently -labeled nucleotide bound at the base position of the primer-template nucleic acid 3’ adjacent to the incorporated reversibly 3 ’-blocked nucleotide, wherein the first polymerase is active in the first condition and inactive in the second condition, and wherein the second polymerase is capable of binding to fluorescently-labeled nucleotides in the second condition and not the first condition;

(e) deblocking the incorporated reversibly 3 ’-blocked nucleotide in the second condition by the unblocking enzyme, wherein the unblocking enzyme is inactive in the first condition and active in the second condition.

2. The method claim 1, comprising conducting steps (b) to (e) iteratively.

3. The method of any one of claims 1-2, wherein the first condition comprises a temperature of about 40 °C to 65 °C, and the second condition comprises a temperature of about 15 °C to 30 °C.

4. The method of any one of claims 1-3, wherein the first condition comprises a temperature of about 45 °C and the second condition comprise a temperature of about 25 °C.

5. The method of any one of claims 1-4, wherein one or more of the first polymerase, the second polymerase and the unblocking enzyme are thermostable.

-25-

6. The method of any one of claims 1-5, wherein the first polymerase, the second polymerase, or both, is nonprocessive.

7. The method of any one of claims 1-6, wherein the first polymerase is a Family B DNA polymerase.

8. The method of claim 7, wherein the first polymerase is a variant of 9°N DNA polymerase, and optionally the variant of 9°N DNA polymerase is K02.

9. The method of claim 7, wherein the first polymerase is Therminator DNA Polymerase.

10. The method of any one of claims 1-9, wherein the second polymerase is Family A DNA polymerase.

11. The method of claim 10, wherein the second polymerase is KlenTaq DNA polymerase or a variant thereof, Bst DNA polymerase or a variant thereof, or Bsu DNA polymerase or a variant thereof.

12. The method of any one of claims 1-11, wherein the reversibly 3’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase.

13. The method of claim 12, wherein the polynucleotide phosphatase is T4 polynucleotide phosphatase/kinase (PNK) or a variant thereof.

14. The method of any one of claims 1-11, wherein the reversibly 3’-blocked nucleotides comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

15. The method of any one of claims 1-11, wherein the reversibly 3 ’-blocked nucleotides comprises dNTPs having a 3’ photolabile protecting group.

16. The method of any one of claims 1-11, wherein the reversibly 3 ’-blocked nucleotides comprises 3’-O-(2-nitrobenzyl)-2’deoxyribonucleoside triphosphates (NB-dNTPs) and 3’-O- (4,5-dimethoxy-2-nitrobenzyl)-2’-deoxyribonucleoside triphosphates (DMNB-dNTPs).

17. The method of any one of claims 1-11, 15, and 16, wherein the second condition comprises light exposure, and optionally the light exposure is UV exposure.

18. The method of any one of claims 1-11, 15, and 16, wherein the first condition does not comprise light exposure.

19. The method of claim 17, wherein the light exposure is focused to a cluster region of the vessel, and optionally using zero-mode waveguides (ZMWs).

20. The method of any one of claims 1-19, wherein the reaction mixture comprises a first aptamer or a first antibody capable of inactivating the first polymerase in the second condition.

21. The method of any one of claims 1-20, wherein the reaction mixture comprises a second aptamer or a second antibody capable of inactivating the unblocking enzyme in the first condition.

22. The method of any one of claims 1-21, wherein the reaction mixture comprises a third aptamer or a third antibody capable of inactivating the second polymerase in the first condition.

23. The method of any one of claims 1-19, wherein the first polymerase is a chemically reversibly modified polymerase that is active in the first condition and inactive in the second condition.

24. The method of any one of claims l-20and 22-23, wherein the unblocking enzyme is a chemically reversibly modified unblocking enzyme that is inactive in the first condition and active in the second condition.

25. A method of identifying a nucleotide in a primed-template nucleic acid, comprising

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a polymerase, a primed-template nucleic acid, reversibly 3 '-blocked nucleotides, fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides are incapable of being covalently incorporated into the primed-template nucleic acid, and an unblocking enzyme;

(b) subjecting the vessel to a first condition to incorporate a reversibly 3 '-blocked nucleotide to the primed-template nucleic acid and form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the polymerase and (iii) a fluorescently-labeled nucleotide bound at the base position of the primer-template nucleic acid 3 ’ adj acent to the incorporated reversibly 3 '-blocked nucleotide;

(d) subjecting the vessel to a second condition to deblock the incorporated reversibly 3 ’-blocked nucleotide by the unblocking enzyme; and wherein the polymerase is active in the first condition and inactive in the second condition, and wherein the unblocking enzyme is inactive in the first condition and active in the second condition.

26. The method of claim 25, comprising conducting steps (b) to (d) iteratively.

27. The method of any one of claims 25-26, wherein the first condition comprises a temperature of about 40 °C to 65 °C, and the second condition comprises a temperature of about 15 °C to 30 °C.

28. The method of claim 27, wherein the first condition comprises a temperature of about 45 °C and the second condition comprise a temperature of about 25 °C.

29. The method of any one of claims 27-28, wherein the polymerase is a Family B DNA polymerase.

30. The method of claim 29, wherein the polymerase is a variant of 9°N DNA polymerase.

31. The method of any one of claims 25-30, wherein the reversibly 3’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase.

32. The method of claim 31, wherein the polynucleotide phosphatase is T4 polynucleotide phosphatase/kinase (PNK) or a variant thereof.

33. The method of any one of claims 25-29, wherein the reversibly 3’-blocked nucleotides comprise 3’-esterified dNTPs, the polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

34. The method of any one of claims 25-26, wherein the first condition comprises a temperature of about 15 °C to 30 °C, and the second condition comprises a temperature of about 40 °C to 65 °C, and.

35. The method of claim 34, wherein the first condition comprises a temperature of about 25 °C and the second condition comprise a temperature of about 45 °C.

36. The method of any one of claims 34-35, wherein the polymerase is a Family A or Family B DNA polymerase.

37. The method of claim 36, wherein the polymerase is Klenow or a variant thereof, or Phi29 polymerase or a variant thereof.

38. The method of any one of claims 34-37, wherein the reversibly 3’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase.

39. The method of claim 38, wherein the polynucleotide phosphatase is Thermophilic PNK RM378 or a variant thereof.

40. The method of any one of claims 34-37, wherein the reversibly 3’-blocked nucleotides comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

41. The method of any one of claims 25-40, wherein the fluorescently-labeled

-28- nucleotides comprise fluorescently-labeled thiol nucleotides.

42. The method of any one of claims 25-41, wherein the polymerase, the unblocking enzyme, or both, are thermostable.

43. The method of any one of claims 25-42, wherein the polymerase is nonprocessive.

44. The method of any one of claims 25-43, wherein the reversibly 3’-blocked nucleotides comprises dNTPs having a 3’ photolabile protecting group.

45. The method of any one of claims 25-44, wherein the reversibly 3 ’-blocked nucleotides comprises 3’-O-(2-nitrobenzyl)-2’deoxyribonucleoside triphosphates (NB-dNTPs) and 3’-O- (4,5-dimethoxy-2-nitrobenzyl)-2’-deoxyribonucleoside triphosphates (DMNB-dNTPs).

46. The method of any one of claims 25-45, wherein the second condition comprises light exposure, and optionally the light exposure is UV exposure.

47. The method of any one of claims 25-45, wherein the first condition does not comprise light exposure.

48. The method of claim 47, wherein the light exposure is focused to a cluster region of the vessel, and optionally using zero-mode waveguides (ZMWs).

49. The method of any one of claims 25-48, wherein the reaction mixture comprises a first aptamer or a first antibody capable of inactivating the polymerase in the second condition.

50. The method of any one of claims 25-49, wherein the reaction mixture comprises a second aptamer or a second antibody capable of inactivating the unblocking enzyme in the first condition.

51. The method of any one of claims 25-50, wherein the polymerase is a chemically reversibly modified polymerase that is active in the first condition and inactive in the second condition.

52. The method of any one of claims 25-49 and 51, wherein the unblocking enzyme is a chemically reversibly modified unblocking enzyme that is inactive in the first condition and active in the second condition.

53. The method of any one of claims 1-52, wherein the fluorescently-labeled nucleotides comprise fluorescently-labeled thiodiphosphate nucleotides, fluorescently-labeled thiotriphosphate nucleotides, or a mixture thereof

54. The method of any one of claims 1-53, wherein the vessel is a flow cell.

55. A composition, comprising: a first polymerase; a primed-template nucleic acid; reversibly 3 '-blocked nucleotides; fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides

-29- are incapable of being covalently incorporated into a primed template nucleic acid; an unblocking enzyme; and a second polymerase that is different from the first polymerase.

56. A composition, comprising: a polymerase; a primed-template nucleic acid; reversibly 3 '-blocked nucleotides; fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides are incapable of being covalently incorporated into a primed template nucleic acid; and an unblocking enzyme.

57. The composition of any one of claims 55-56, wherein the fluorescently-labeled nucleotides comprise fluorescently-labeled thiol nucleotides.

58. A composition, comprising: a polymerase; a primed-template nucleic acid; fluorescently-labeled and reversibly 3 '-blocked nucleotides; and an unblocking enzyme.

59. The composition of any one of claims 55-58, wherein the reversibly 3’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase.

60. The composition of any one of claims 55-58, wherein the reversibly 3’-blocked nucleotides comprise 3’-esterified dNTPs, and the unblocking enzyme is an esterase.

61. A method of sequencing at least part of a nucleic acid template, the method comprising incubating a sequencing composition and the template at a first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a first position; incubating the sequencing composition and the template at a second temperature to remove a 3’ blocking group from a first incorporated base of a sequencing strand; returning the sequencing composition and the template to the first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a second position; and returning the sequencing composition and the template to the second temperature to remove a 3’ blocking group from a second incorporated base of a sequencing strand, wherein the method is performed in a single volume without addition of exogenous

-30- reagents between assaying for fluorescence indicative of a labeled base paired to a template base at the first position and assaying for fluorescence indicative of a labeled base paired to a template base at the second position.

62. The method of claim 61, comprising returning the sequencing composition and the template to the first temperature; assaying for fluorescence indicative of a labeled base paired to a template base at a third position; and returning the sequencing composition and the template to the second temperature to remove a 3’ blocking group from a third incorporated base of a sequencing strand, wherein the method is performed in a single volume without addition of exogenous reagents between assaying for fluorescence indicative of a labeled base paired to a template base at the first position and assaying for fluorescence indicative of a labeled base paired to a template base at the third position.

63. The method of claim 61, wherein the assaying for fluorescence indicative of a labeled base paired to a template base at a first position comprises assaying for a nucleotide comprising a fluorophore incorporated into a ternary complex.

64. The method of claim 63, wherein the nucleotide comprising a fluorophore does not comprise a blocked 3’ end.

65. The method of claim 63, wherein the nucleotide comprising a fluorophore is not covalently bound to the sequencing strand.

66. The method of claim 61, wherein the assaying for fluorescence indicative of a labeled base paired to a template base at a first position comprises assaying for a nucleotide comprising a fluorophore incorporated into the sequencing strand.

67. The method of claim 66, wherein the nucleotide comprising a fluorophore comprises a blocked 3’ end.

68. The method of claim 66, comprising removing the fluorophore subsequent to the assaying for fluorescence.

69. The method of claim 61, comprising washing the template and the sequencing strand to replace the sequencing composition only after at least two cycles of assaying for fluorescence indicative of a labeled base paired to a template base.

70. The method of claim 62, comprising washing the template and the sequencing strand to replace the sequencing composition only after at least three cycles of assaying for fluorescence indicative of a labeled base paired to a template base.

71. The method of claim 62, comprising performing at least 7 additional cycles of assaying for fluorescence indicative of a labeled base paired to a template base and removing a 3’

-31- blocking group.

72. The method of any one of claims 61 - 68 or 71, wherein multiple consecutive base positions of the template are determined without replacing the sequencing composition.

73. The method of any one of claims 61 - 72, wherein the sequencing composition comprises a composition of any one of claims 55 - 60.

74. The method of any one of claims 61 - 72, wherein the sequencing composition comprises a fluorescently labeled, 3’ blocked nucleotide.

75. The method of any one of claims 61 - 72, wherein the sequencing composition comprises a fluorescently labeled, 3’ unblocked nucleotide.

76. The method of any one of claims 61 - 75, wherein the composition comprises excised former 3 ’blocking moi eties.

77. The method of claim 76, wherein the excised former 3 ’blocking moi eties are present in at least a 2x excess relative to template-sequencing strand double stranded complexes.

78. The method of claim 76, wherein the excised former 3 ’blocking moieties are present in at least a 2x excess relative to sequencing strand unblocked free 3’ ends.

79. The method of any one of claims 61 - 78, wherein the method is performed in a flow cell.

80. The method of any one of claims 61 - 78, wherein the method is performed in contact with a thermocycling heat block.

81. The method of any one of claims 61 - 78, wherein the method is performed in a single file emulsion of droplets.

82. The method of claim 81, wherein the single file emulsion of droplets flow repeatedly through a circular course or through a serpentine course, wherein one side of the serpentine is at the first temperature and the other side of the serpentine course is at a second temperature.

83. The method of any one of claims 61-82, wherein the first temperature is about 40 °C to 65 °C, and the second temperature is about 15 °C to 30 °C.

84. The method of claim 83, wherein the first temperature is about 45 °C and the second condition comprise a temperature of about 25 °C.

85. The method of any one of claims 61-82, wherein the first temperature is about 15 °C to 30 °C, and the second temperature is about 40 °C to 65 °C.

86. The method of claim 85, wherein the first temperature is about 25 °C and the second condition comprise a temperature of about 45 °C.

87. The method of any one of claims 61-86, wherein the sequence composition comprises:

-32- a polymerase; a primed-template nucleic acid; reversibly 3 '-blocked nucleotides; fluorescently-labeled nucleotides, wherein the fluorescently-labeled nucleotides are incapable of being covalently incorporated into a primed template nucleic acid; and an unblocking enzyme.

88. The method of any one of claims 61-86, wherein the sequence composition comprises: a polymerase; a primed-template nucleic acid; fluorescently-labeled and reversibly 3 '-blocked nucleotides; and an unblocking enzyme.

89. The method of any one of claims 61-88, wherein the duration of a cycle of assaying for fluorescence indicative of a labeled base paired to a template base is shorter.

90. A method of identifying a nucleotide in a primed-template nucleic acid, comprising

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a polymerase, a primed-template nucleic acid, fluorescently labeled reversibly 3 '-blocked nucleotides, and an unblocking enzyme;

(b) subjecting the vessel to a first condition to incorporate a fluorescently labeled 3 '-blocked nucleotide to the primed-template nucleic acid to form a fluorescently labeled primed-template nucleic acid;

(d) subjecting the vessel to a second condition to deblock the incorporated fluorescently labeled 3 '-blocked nucleotide by the unblocking enzyme; and wherein the polymerase is active in the first condition and inactive in the second condition, and wherein the unblocking enzyme is inactive in the first condition and active in the second condition.

91. The method of claim 90, comprising conducting steps (a) to (d) iteratively.

92. The method of any one of claims 90-91, wherein the first condition comprises a temperature of about 40 °C to 65 °C, and the second condition comprises a temperature of about

-33- 15 °C to 30 °C.

93. The method of claim 92, wherein the first condition comprises a temperature of about 45 °C and the second condition comprise a temperature of about 25 °C.

94. The method of any one of claims 92-93, wherein the polymerase is a Family B DNA polymerase, and optionally the Family B DNA polymerase is a variant of 9°N DNA polymerase.

95. The method of any one of claims 90-94, wherein the reversibly 3 ’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase.

96. The method of claim 95, wherein the polynucleotide phosphatase is T4 polynucleotide phosphatase/kinase (PNK) or a variant thereof.

97. The method of any one of claims 90-94, wherein the reversibly 3 ’-blocked nucleotides comprise 3’-esterified dNTPs, the polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

98. The method of any one of claims 90-91, wherein the first condition comprises a temperature of about 15 °C to 30 °C, and the second condition comprises a temperature of about 40 °C to 65 °C, and.

99. The method of claim 98, wherein the first condition comprises a temperature of about 25 °C and the second condition comprise a temperature of about 45 °C.

100. The method of any one of claims 98-99, wherein the polymerase is a Family A or Family B DNA polymerase.

101. The method of claim 100, wherein the polymerase is KI enow or a variant thereof, or Phi29 polymerase or a variant thereof.

102. The method of any one of claims 98-101, wherein the reversibly 3 ’-blocked nucleotides comprise 3’ phosphate-dNTPs and the unblocking enzyme is a polynucleotide phosphatase.

103. The method of claim 102, wherein the polynucleotide phosphatase is Thermophilic PNK RM378 or a variant thereof.

104. The method of any one of claims 98-103, wherein the reversibly 3’-blocked nucleotides comprise 3’-esterified dNTPs, the first polymerase has no esterase activity in the first condition, and the unblocking enzyme is an esterase.

105. The method of any one of claims 90-104, wherein the polymerase, the unblocking enzyme, or both, are thermostable.

106. The method of any one of claims 90-105, wherein the polymerase is nonprocessive.

107. The method of any one of claims 90-106, wherein the reaction mixture comprises

-34- a first aptamer or a first antibody capable of inactivating the polymerase in the second condition.

108. The method of any one of claims 90-107, wherein the reaction mixture comprises a second aptamer or a second antibody capable of inactivating the unblocking enzyme in the first condition.

109. The method of any one of claims 90-106, wherein the polymerase is a chemically reversibly modified polymerase that is active in the first condition and inactive in the second condition.

110. The method of any one of claims 90-107 and 109, wherein the unblocking enzyme is a chemically reversibly modified unblocking enzyme that is inactive in the first condition and active in the second condition.

111. The method of any one of claims 90-110, wherein the fluorescently-labeled nucleotides comprise fluorescently-labeled thiodiphosphate nucleotides, fluorescently-labeled thiotriphosphate nucleotides, or a mixture thereof

112. The method of any one of claims 90-111, wherein the vessel is a flow cell.

113. A method of identifying a nucleotide in a primed-template nucleic acid, comprising:

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a polymerase that is active in a first condition and inactive in a second condition, a primed-template nucleic acid, labeled nucleotides that are incapable of being covalently incorporated into the primed-template nucleic acid, reversibly 3 '-blocked nucleotides, and an unblocking enzyme that is inactive in the first condition and active in the second condition;

(b) subjecting the vessel to a first condition to incorporate a reversibly 3 '-blocked nucleotide to the primed-template nucleic acid and form a ternary complex comprising (i) the primed-template nucleic acid, (ii) the polymerase and (iii) a labeled nucleotide bound at the base position of the primer-template nucleic acid 3’ adjacent to the incorporated reversibly 3 '-blocked nucleotide;

(c) identifying the type of the labeled nucleotide bound in the ternary complex in the first condition; and

(d) subjecting the vessel to a second condition to deblock the incorporated reversibly 3 ’-blocked nucleotide by the unblocking enzyme.

114. The method of claim 113, comprising conducting steps (b) to (d) iteratively.

-35-

115. The method of any one of claims 113-114, wherein the method is performed in the vessel without addition of exogenous reagents between step (b) and step (d).

116. A method of identifying a nucleotide in a primed-template nucleic acid, comprising:

(a) providing a vessel comprising a reaction mixture, wherein the reaction mixture comprises: a polymerase, a primed-template nucleic acid, fluorescently labeled reversibly 3 ’-blocked nucleotides, and an unblocking enzyme;

(d) subjecting the vessel to a second condition to deblock the incorporated fluorescently labeled 3 ’-blocked nucleotide by the unblocking enzyme; wherein the polymerase is active in the first condition and inactive in the second condition, and wherein the unblocking enzyme is inactive in the first condition and active in the second condition.

-36-