Increasing signal to noise in nucleic acid sequencing

Abstract

To provide a method for reducing or preventing phasing in ensemble-based sequencing.SOLUTION: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers comprise an extendable 3 ' end, (b) contacting the plurality with (i) a blocked nucleotide to produce a first subset of primer-template nucleic acid hybrids comprising a blocked nucleotide at the 3 ' end, and (ii) a ternary complex inhibitor to produce a second subset of primer-template nucleic acid hybrids comprising the ternary complex inhibitor, (c) forming ternary complexes each comprising a polymerase, a first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; (d) detecting the ternary complex to identify the nucleotide in the template nucleic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates generally to characterizing nucleic acids and has particular applicability to nucleic acid sequencing. [Background technology]

[0002] Some commercial nucleic acid sequencing technologies are implemented using nucleic acid ensembles. Ensembles are generally generated by amplification techniques that result in localized collections of nucleic acid copies that are manipulated and detected as a whole. Exemplary technologies for creating ensembles include the bridge amplification technology used by the Illumina platform (San Diego, California), the emulsion polymerase chain reaction technology used by the Ion Torrent platform (Thermo Fisher, Waltham, Massachusetts), and the rolling circle technology used by the Complete Genomics platform (BGI, Shenzhen, China). Ensembles offer the advantages of higher signal amplitude compared to single-molecule sequencing technologies, as well as minimized artifacts resulting from stochastic noise when manipulating and detecting single molecules.

[0003] Despite the widespread adoption of ensemble-based sequencing technologies, so-called "phasing" limits read length, overall throughput, and accuracy. Phasing is a phenomenon in which individual molecules within an ensemble lose synchronization with one another. Phasing can manifest as the extension of one or more primer molecules lagging behind other primer molecules in the ensemble or as the extension of one or more primer molecules ahead of other primer molecules in the ensemble. Phasing is detrimental and cumulative. A phasing rate of just 0.5% per cycle results in a cumulative loss of approximately half of the true signal after 120 cycles. This problem is exacerbated by a proportional increase in background noise due to erroneous signals arising from out-of-phase ensemble members. The cumulative loss of signal-to-noise leads to limitations on read length (which reduces sequencing throughput) and increased error, especially in later cycles as noise overwhelms the signal.

[0004] Therefore, there is a need for methods to reduce or prevent phasing in ensemble-based sequencing. The present invention fulfills this need and provides other advantages as well. Summary of the Invention

[0005] The present disclosure provides a method for identifying a nucleotide in a template nucleic acid. The method can include: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3' ends; (b) contacting the plurality with (i) a blocked nucleotide to generate a first subset of primer-template nucleic acid hybrids comprising a blocked nucleotide at their 3' ends, and (ii) a ternary complex inhibitor to generate a second subset of primer-template nucleic acid hybrids comprising a ternary complex inhibitor; (c) forming ternary complexes, each comprising a polymerase, the first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; and (d) detecting the ternary complexes, thereby identifying the nucleotide in the template nucleic acid. Optionally, the blocked nucleotide can be a reversible terminating nucleotide.

[0006] Also provided is a method for identifying nucleotides in a template nucleic acid, comprising: (a) providing a plurality of primer-template nucleic acid hybrids, the primers having extendable 3' ends; (b) incorporating a blocked nucleotide at the 3' end of a first subset of primer-template nucleic acid hybrids; (c) incorporating a ternary complex inhibitor at the 3' end of a second subset of primer-template nucleic acid hybrids; (d) forming ternary complexes, each comprising a polymerase, the first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; and (e) detecting the ternary complexes, thereby identifying the nucleotides in the template nucleic acid. Optionally, the blocked nucleotides can be reversible terminating nucleotides.

[0007] The present disclosure provides a method for sequencing a template nucleic acid, the method including: (a) providing a plurality of primer-template nucleic acid hybrids, the primers having extendable 3' ends; (b) contacting the plurality with (i) a reversible terminating nucleotide to generate a first subset of primer-template nucleic acid hybrids comprising a reversible terminating nucleotide at their 3' ends, and (ii) a ternary complex inhibitor to generate a second subset of primer-template nucleic acid hybrids comprising a ternary complex inhibitor; (c) forming ternary complexes, each comprising a polymerase, the first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; (d) detecting the ternary complexes, thereby identifying nucleotides in the template nucleic acid; (e) deblocking the reversible terminating nucleotides at the 3' ends of the first subset of primer-template nucleic acid hybrids; and (f) repeating steps (b) through (e) to sequence the first subset of template nucleic acids.

[0008] A method for sequencing a template nucleic acid can include: (a) providing a plurality of primer-template nucleic acid hybrids, the primers having extendable 3' ends; (b) incorporating a blocked nucleotide at the 3' end of a primer(s) of a first subset of the primer-template nucleic acid hybrids; (c) incorporating a ternary complex inhibitor at the 3' end of a primer(s) of a second subset of the primer-template nucleic acid hybrids; (d) forming ternary complexes, each comprising a polymerase, the first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; (e) detecting the ternary complexes, thereby identifying the nucleotides in the template nucleic acid; (f) deblocking the reversible terminating nucleotides at the 3' ends of the first subset of primer-template nucleic acid hybrids; and (g) repeating steps (b) through (f) to sequence the first subset of template nucleic acids.

[0009] The present disclosure further provides an apparatus comprising a plurality of primer-template nucleic acid hybrids, wherein a first subset of the primer-template nucleic acid hybrids have a blocked nucleotide at the 3' end of the primer, and a second subset of the primer-template nucleic acid hybrids have a ternary complex inhibitor at the 3' end of the primer. Optionally, the blocked nucleotide can be a reversible terminating nucleotide. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure provides a method for identifying a nucleotide base present at a match position in a primer-template nucleic acid hybrid. The match position is the base located immediately 5' to the template base that hybridizes to the 3' end of the primer. The nucleotide present at the match position can be identified in a test step used to detect a ternary complex formed between the primer-template nucleic acid hybrid, a polymerase, and a nucleotide cognate for the base at the match position. The polymerase functions to pair the cognate nucleotide with the next base on the template. The identity of the base at the match position can be determined by distinguishing the type of nucleotide present in the ternary complex and inferring the template base to which it hybridizes according to Watson-Crick base pairing.

[0011] In certain embodiments, the primer can be incrementally extended to shift the matching position along the template.For example, the sequence of the template can be determined by a series of cycles, in which the primer is extended by a single nucleotide, shifts to the next template position for matching, and checks the new matching position.Incomplete extension can lead to sequencing errors or early termination of the sequencing process.Fading problems also appear, for example, when a group of primer-template nucleic acid hybrids is sequenced as an ensemble.

[0012] The present disclosure provides methods and compositions that may be useful for improving the identification of bases in nucleic acids, for example, in sequencing methods. Improvements can be achieved by including a primer modification process that caps primers that are not subsequently detected. For example, primers that are not extended in a primer extension step can be capped to prevent the unextended primer from contributing to errors in a subsequent testing step. For embodiments in which template nucleotides are identified by testing for ternary complexes, a particularly useful cap is provided by a ternary complex inhibitor. A ternary complex inhibitor can be a moiety that, when attached to a primer, prevents the polymerase and / or cognate nucleotide from participating in the formation or maintenance of a ternary complex at the end of the modified primer. The ternary complex inhibitor can be present at the end of the primer, for example, as a moiety that creates a steric block for one or more components that would otherwise form a ternary complex, or as a moiety that creates a repulsive charge for one or more of the components.

[0013] As one example, inhibition of ternary complex formation can occur when a primer is fully extended such that no template position is accessible for ternary complex formation. Full extension can manifest as the 3' end of the extended primer being annealed to the 5' end of the template. In this configuration, the double-stranded extension product does not contain an unpaired next template base. In an alternative configuration, full extension can manifest as a primer being extended until further extension is prevented by factors in the environment of the extension product. In this configuration, the template may contain an unpaired next template base, but the next template base is inaccessible to the polymerase (and / or the next correct nucleotide) due to the environment surrounding the template and extended primer. For example, the 5' end of the template may be bound to a solid-phase surface, and the 3' end of the extended primer may be too close to the surface for the polymerase to further extend or bind to form a ternary complex. In both of these examples, full extension generated an oligonucleotide moiety that functions as a cap on the extended primer.

[0014] In some configurations, the ternary complex inhibitor moiety can be a first binding partner (e.g., a ligand) that has binding affinity for a second binding partner (e.g., a receptor such as an antibody). Whether or not the first binding partner can inhibit the formation of the ternary complex, inhibition can occur when the second binding partner is bound to the first binding partner. The ternary complex inhibitor can be present at the 3' end of the primer, for example, as a result of extending the 3' end of the primer with a nucleotide analog, which is bound to the first binding partner. In this configuration, the complex between the first binding partner and the second binding partner functions as a cap on the extended primer.

[0015] In an alternative embodiment, a primer modification process can be used to remove primers so that they do not participate in the subsequent detection step. For example, primers that are not extended in the primer extension step can be chemically or enzymatically degraded (e.g., via an exonuclease) so that the unextended primers do not form ternary complexes that contribute to errors in the subsequent testing step.

[0016] Terms used herein will be understood to have their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are listed below.

[0017] As used herein, the term "array" refers to a collection of molecules attached to one or more solid supports such that molecules in one feature can be distinguished from molecules in other features. An array can include different molecules each located in a different addressable feature on the solid support. Alternatively, an array can include separate solid supports that each function as a feature with a different molecule, and the different molecules can be identified according to the position of the solid support on a surface to which the solid support is attached or according to the position of the solid support in a liquid, such as a fluid stream. The molecules in the array can be, for example, nucleotides, nucleic acid primers, nucleic acid templates, or nucleic acid enzymes such as polymerases, ligases, exonucleases, or combinations thereof.

[0018] As used herein, the term "blocking moiety," when used with respect to a nucleotide, refers to a portion of the nucleotide that inhibits or prevents the 3' oxygen of the nucleotide from forming a covalent bond to the next correct nucleotide during a nucleic acid polymerization reaction. The blocking moiety of a "reversible terminating" nucleotide can be removed from the nucleotide analog or otherwise modified to allow the 3' oxygen of the nucleotide to be covalently bonded to the next correct nucleotide. Such blocking moieties are referred to herein as "reversible terminator moieties." Exemplary reversible terminator moieties are described in U.S. Patent Nos. 7,427,673, 7,414,116, 7,057,026, 7,544,794, or 8,034,923, or PCT Publication Nos. WO 91 / 06678 or WO 07 / 123744, each of which is incorporated herein by reference. The nucleotide having a blocking moiety or a reversible terminator moiety may be at the 3' end of a nucleic acid, such as a primer, or may be a monomer that is not covalently bound to a nucleic acid. The blocking moiety does not need to interfere with or prevent ternary complex formation at the 3' end of the nucleic acid to which the blocking moiety is bound. Particularly useful blocking moieties are present at the 3' end of the nucleic acid that participates in the formation of the ternary complex.

[0019] As used herein, the term "catalytic metal ion" refers to a metal ion that promotes phosphodiester bond formation between the 3'-oxygen of a nucleic acid (e.g., a primer) and the phosphate of an incoming nucleotide by a polymerase. A "divalent catalytic metal cation" is a catalytic metal ion having a valency of two. A catalytic metal ion can be present at a concentration that stabilizes the formation of a complex between a polymerase, a nucleotide, and a primed template nucleic acid, referred to as a non-catalytic concentration of the metal ion, as long as phosphodiester bond formation does not occur. A catalytic concentration of a metal ion refers to the amount of metal ion sufficient to allow a polymerase to catalyze the reaction between the 3'-oxygen group of a nucleic acid (e.g., a primer) and the phosphate group of an incoming nucleotide.

[0020] As used herein, the term "consensus sequence" refers to a sequence of nucleotides that is the same in two or more nucleic acid molecules. A sequence that is common to two or more nucleic acids can include all or part of the nucleic acids being compared. A consensus sequence can have a length of at least 5, 10, 25, 50, 100, 250, 500, 1000, or more nucleotides. Alternatively or additionally, the length can be up to 1000, 500, 250, 100, 50, 25, 10, or 5 nucleotides.

[0021] The term "comprising," as used herein, is intended to be open-ended, including not only the recited elements, but also any additional elements.

[0022] As used herein, the term "deblocking" refers to removing or modifying the reversible terminator portion of a nucleotide to make the nucleotide extendible. For example, a nucleotide can be present at the 3' end of a primer, such that deblocking renders the primer extendible. Exemplary deblocking reagents and methods are described in U.S. Patent Nos. 7,427,673, 7,414,116, 7,057,026, 7,544,794, or 8,034,923, or PCT Publication Nos. WO 91 / 06678 or WO 07 / 123744, each of which is incorporated herein by reference.

[0023] As used herein, the term "detecting as an ensemble" refers to detecting a feature of a population of molecules under conditions that do not necessarily distinguish one molecule of the population from other molecules of the population. For example, a population of nucleic acids detected as an ensemble at a feature of an array can result in a distinct feature that is a composite of the nucleic acid features at that feature. The feature can be a luminescence signal that is an average of the emissions from multiple luminophores at the feature of the array.

[0024] As used herein, the term "each," when used in connection with a collection of items, is intended to identify each individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where explicit disclosure or context clearly indicates otherwise.

[0025] As used herein, the term "exogenous," when used in reference to a portion of a molecule, refers to a chemical moiety that is not present in the molecule's natural analog. For example, an exogenous label on a nucleotide is a label that is not present in naturally occurring nucleotides. Similarly, an exogenous label present on a polymerase is not found on the polymerase in its native environment.

[0026] As used herein, the term "extension," when used in reference to a nucleic acid, refers to the process of adding at least one nucleotide to the 3' end of a nucleic acid. The term "polymerase extension," when used in reference to a nucleic acid, refers to a polymerase-catalyzed process of adding at least one nucleotide to the 3' end of a nucleic acid. The nucleotide or oligonucleotide added to a nucleic acid by extension is said to be incorporated into the nucleic acid. Thus, the term "incorporating" can be used to refer to the process of linking a nucleotide or oligonucleotide to the 3' end of a nucleic acid by forming a phosphodiester bond.

[0027] As used herein, the term "extendable," when used in reference to a nucleotide, means that the nucleotide has an oxygen or hydroxyl moiety at the 3' position that can form a covalent bond to the next correct nucleotide if and when the next correct nucleotide is incorporated into a nucleic acid. An extendable nucleotide may be at the 3' position of a primer or may be a monomeric nucleotide. An extendable nucleotide lacks a blocking moiety, such as a reversible terminator moiety.

[0028] As used herein, the term "feature," when used in reference to an array, refers to a location within the array where a particular molecule is present. A feature can include only a single molecule or can include a population of several molecules of the same species (i.e., an ensemble of molecules). Alternatively, a feature can include a population of molecules that are different species (e.g., a population of ternary complexes with different template sequences). The features of an array are typically discrete. Individual features can be adjacent or spaced apart. Arrays useful herein can have features that are, for example, less than 100 microns, 50 microns, 10 microns, 5 microns, 1 micron, or 0.5 microns apart. Alternatively or additionally, arrays can have features that are more than 0.5 microns, 1 micron, 5 microns, 10 microns, 50 microns, or 100 microns apart. Features can each have an area of ​​less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron, or less.

[0029] As used herein, the term "label" refers to a molecule or portion thereof that provides a detectable property. The detectable property can be, for example, an optical signal such as absorption of radiation, fluorescence emission, luminescence emission, fluorescence lifetime, fluorescence polarization, Rayleigh and / or Mie scattering, binding affinity for a ligand or receptor, magnetic properties, electrical properties, charge, mass, radioactivity, etc. Exemplary labels include, but are not limited to, fluorophores, luminophores, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), heavy atoms, radioisotopes, mass labels, charge labels, spin labels, receptors, ligands, etc.

[0030] As used herein, an "artificial vessel" is a human-made or human-modified vessel that functions to separate one chemical process (e.g., a binding event, an incorporation reaction, etc.) from another chemical process or to provide a space in which a chemical process can occur. Non-limiting examples of artificial vessels useful in connection with the techniques of the present disclosure include flow cells, wells of multi-well plates, microscope slides, tubes (e.g., capillaries), and the like. A feature to be examined or detected may be contained within a reaction vessel.

[0031] As used herein, the term "next correct nucleotide" refers to the nucleotide type attached to and / or incorporated at the 3' end of a primer that is complementary to the base of the template strand to which the primer hybridizes. The base of the template strand is called the "next base" and is immediately 5' to the template base that hybridizes to the 3' end of the primer. The next correct nucleotide is called the "cognate" of the next base, and vice versa. Nucleotide cognate nucleotides that interact with each other in a ternary complex or double-stranded nucleic acid are said to "pair" with each other. A nucleotide having a base that is not complementary to the next template base is called an "incorrect," "mismatched," or "non-cognate" nucleotide.

[0032] As used herein, the term "non-catalytic metal ion" refers to a metal ion that, when in the presence of a polymerase enzyme, does not promote the phosphodiester bond formation necessary for the chemical incorporation of a nucleotide into a primer. A non-catalytic metal ion may interact with a polymerase, for example, through competitive binding compared to a catalytic metal ion. Thus, a non-catalytic metal ion can function as an inhibitory metal ion. A "divalent non-catalytic metal ion" is a non-catalytic metal ion having a valence of two. Examples of divalent non-catalytic metal ions include Ca. 2+ , Zn 2+ , Co 2+ , Ni 2+ and Sr 2+ These include, but are not limited to, trivalent Eu 3+ and Tb 3+The ions are non-catalytic metal ions having a valence of three.

[0033] As used herein, the term "nucleotide" can be used to refer to a naturally occurring nucleotide or its analog. Examples include, but are not limited to, nucleotide triphosphates (NTPs), such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), or non-naturally occurring analogs, such as dideoxyribonucleotide triphosphates (ddNTPs), or reversible terminating nucleotide triphosphates (rtNTPs).

[0034] As used herein, the term "oligonucleotide portion" refers to a portion of a nucleic acid comprising at least two adjacent nucleotides. An oligonucleotide portion can comprise, for example, at least 5, 8, 10, 15, 20, 25, 50, 100, or more adjacent nucleotides. Alternatively or additionally, an oligonucleotide portion can comprise up to 100, 50, 25, 20, 15, 10, 8, 5, or 2 adjacent nucleotides. In some embodiments, the oligonucleotide portion added to the primer-template hybrid can have a length equivalent to the length of the template from the next template nucleotide to the 5' end of the template.

[0035] As used herein, the term "polymerase" can refer to nucleic acid synthesizing enzymes, including, but not limited to, DNA polymerases, RNA polymerases, reverse transcriptases, primases, and transferases. Typically, polymerases have one or more active sites where nucleotide binding and / or nucleotide polymerization catalysis can occur. A polymerase can catalyze the polymerization of a nucleotide onto the 3' end of a first strand of a double-stranded nucleic acid molecule. For example, a polymerase can catalyze the addition of the next correct nucleotide to the 3' oxygen group of the first strand of a double-stranded nucleic acid molecule via a phosphodiester bond, thereby covalently incorporating the nucleotide into the first strand of the double-stranded nucleic acid molecule. Optionally, a polymerase need not be capable of incorporating a nucleotide under one or more of the conditions used in the methods described herein. For example, a mutant polymerase may be capable of forming a ternary complex but be unable to catalyze nucleotide incorporation.

[0036] As used herein, the term "primer-template nucleic acid hybrid" or "primer-template hybrid" refers to a nucleic acid having a double-stranded region, where one strand is a primer and the other strand is a template. The double strands can be part of adjacent nucleic acid molecules (e.g., a hairpin structure), or the double strands can be separable molecules that are not covalently linked to each other.

[0037] 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, a primer binds in a configuration that allows replication of the template, for example, via polymerase extension of the primer. A primer can be a first portion of a nucleic acid molecule that binds to a second portion of the nucleic acid molecule, where the first portion is a primer sequence and the second portion is a primer-binding sequence (e.g., a hairpin primer). Alternatively, a primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule having a template sequence. A primer can be composed of DNA, RNA, or an analog thereof. A primer can have an extendable 3' end, a 3' end that is blocked from primer extension, or a 3' end that is capped to prevent or inhibit ternary complex formation.

[0038] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate can optionally take up liquid (e.g., due to porosity), but is typically sufficiently rigid so that the substrate does not substantially expand when it takes up liquid and does not substantially shrink when the liquid is removed by drying. Non-porous solid supports are generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon®, cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, fiber optic bundles, and polymers.

[0039] As used herein, the term "subset" means a collection of one or more things, all of which are included in a larger collection of things. The larger collection of things may be called a "set." A subset may be at least 1, 2, 10, 100, 1 x 10 3 , 1×10 6 , 1×10 9 The components can be, for example, nucleic acids such as a primer-template nucleic acid hybrid described herein, a ternary complex, a polymerase, nucleotides, or other compositions.

[0040] As used herein, the term "ternary complex" refers to an intermolecular association between a polymerase, a double-stranded nucleic acid, and a nucleotide. Typically, the polymerase promotes the interaction between the next correct nucleotide and the template strand of a primed nucleic acid. The next correct nucleotide can interact with the template strand via Watson-Crick hydrogen bonding. The term "stabilized ternary complex" refers to a ternary complex whose existence is promoted or prolonged, or whose disruption is inhibited. Generally, stabilization of a ternary complex prevents the covalent incorporation of a nucleotide component of the ternary complex into the primed nucleic acid component of the ternary complex.

[0041] As used herein, the term "ternary complex inhibitor" refers to a moiety that, when present in a nucleic acid, disrupts or prevents the nucleic acid from binding to a polymerase and / or nucleotide to form a ternary complex. Moieties that create a steric block to ternary complex formation are particularly useful, including, for example, polymerization or ligation products that extend a primer to the end of the template to which it is hybridized. Another example of a steric block is a mismatched nucleotide. Moieties that introduce positive or negative charges that disrupt or prevent ternary complex formation can also be used. The moiety can be a ligand that binds to a receptor that inhibits or prevents ternary complex formation, such as biotin (or an analog thereof) that binds to streptavidin (or an analog thereof), an epitope that binds to an antibody (or a functional fragment thereof), or a carbohydrate that binds to a lectin. Thus, a ternary complex inhibitor can be a ligand (or other) moiety that can bind to a receptor (or other molecule) to form a ligand-receptor complex that inhibits ternary complex formation. Further examples of moieties that can be used as ternary complex inhibitors include base modifications and nucleotide analogs as described in Turcatti et al. Nucl. Acids. Res. 36(4) e25 (2008).

[0042] As used herein, the term "type" is used to identify molecules that share the same chemical structure. For example, a mixture of nucleotides may contain several dCTP molecules. dCTP molecules are understood to be of the same type as each other, but of different types compared to dATP, dGTP, dTTP, etc. Similarly, individual DNA molecules with the same sequence of nucleotides are of the same type, but DNA molecules with different sequences are of different types. The term "type" can also identify moieties that share the same chemical structure. For example, cytosine bases in a template nucleic acid will be understood to be of the same type as each other, regardless of their position in the template sequence.

[0043] The embodiments described and claimed below can be understood in light of the above definitions.

[0044] The present disclosure provides a method for identifying a nucleotide in a template nucleic acid. The method can include: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3' ends; (b) contacting the plurality with (i) a blocked nucleotide to generate a first subset of primer-template nucleic acid hybrids comprising a blocked nucleotide at their 3' ends, and (ii) a ternary complex inhibitor to generate a second subset of primer-template nucleic acid hybrids comprising a ternary complex inhibitor; (c) forming ternary complexes, each comprising a polymerase, the first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; and (d) detecting the ternary complexes, thereby identifying the nucleotide in the template nucleic acid. Optionally, the blocked nucleotide can be a reversible terminating nucleotide.

[0045] The disclosed methods can include a primer modification process in which a nucleotide or other moiety is added to the primer component of a primer-template nucleic acid hybrid. The primer modification process can be used to prepare a primer-template nucleic acid hybrid for a testing process, whereby the next base of the template nucleic acid is detected. Optionally, testing is performed to detect the ternary complex formed between the next base of the primer-template nucleic acid hybrid, a polymerase, and the cognate nucleotide of the next base (i.e., the cognate nucleotide is also referred to as the next correct nucleotide). Further details regarding the testing process are described below.

[0046] Returning to the primer modification process, the modification that causes primer extension by one or more nucleotides can be used to change the position of the template nucleic acid that is next to be inspected.For example, extension can be used to add one nucleotide to primer, thereby shifting the position for ternary complex formation by one template position.Template nucleic acid can be sequenced by using repeated cycles of inspecting ternary complex formation at the end of primer (i.e., at the next template base), and then extending primer to shift the inspection position.

[0047] If the primer cannot be extended during the primer modification process, it can result in reduced accuracy, for example, due to confusion about which position on the template is actually being examined in any given cycle, or, for example, due to reduced read length when extension cannot proceed.When detecting an ensemble of primer-template nucleic acid hybrids, even a relatively small extension inefficiency can result in phasing problems that adversely affect read accuracy and read length.

[0048] The primer modification process used in the methods described herein can provide a means to mitigate against artifacts that would otherwise result from extension failure and inefficiency. In certain embodiments, the primer modification process can include at least two steps: (i) contacting a primer-template nucleic acid hybrid and a polymerase with a reversible terminating nucleotide to generate a first subset of primer-template nucleic acid hybrids containing a reversible terminating nucleotide at their 3' ends, and (ii) contacting the primer-template nucleic acid hybrid and a polymerase with a ternary complex inhibitor to generate a second subset of primer-template nucleic acid hybrids containing a ternary complex inhibitor. Step (i) can provide the advantage of extending the primer so that the first subset of primer-template nucleic acid hybrids can continue to participate in providing sequence information. For example, the reversible terminating nucleotide used in step (i) can be selected to accommodate subsequent formation and testing of a ternary complex for the reversible terminating primer. Step (ii) can provide the advantage of capping primers to prevent the primers from participating in ternary complex formation during the subsequent testing step. This capping can reduce phasing, improve base calling accuracy, and extend sequencing read length. Generally, step (i) is performed before step (ii). However, steps can be performed simultaneously or in reverse order. When performed sequentially, one of the steps can be performed immediately after the other step, one of the steps can start before the previous step is finished, or another step can intervene between the two steps.

[0049] When extension is performed with a reversible terminating nucleotide, particularly useful capping chemistries are selective for the 3' end of the unextended primer compared to the 3' end of the extended primer. For example, the capping chemistries may react with the 3' end of the unblocked primer and be inactive in modifying the blocked primer. In this configuration, capping can function to complete the extension step. For example, chemistries that use enzymes specific for the native 3' end of the primer, including ligases and polymerases, are particularly useful. A capping procedure that is more efficient at modifying the native 3' end of the primer compared to blocked primers is beneficial in many applications.

[0050] In certain embodiments, a ternary complex inhibitor is used to cap a primer. Any of a variety of moieties can be added to a primer to disrupt or prevent subsequent formation of a ternary complex at the 3' end of the primer. For example, a primer can be modified to have an oligonucleotide moiety attached to it. The oligonucleotide moiety can have a length at least as long as the region of the template that was single-stranded before the primer was modified. For example, the oligonucleotide moiety can extend from the 3' end of the primer to the 5' end of the template. In another example, the oligonucleotide moiety can extend from the 3' end of the primer to the 5' end of the double-stranded region adjacent to the template region. In this example, the 5' end of the double-stranded region forms a boundary to the template region. Other boundaries, such as binding proteins, chemical modifications, or attachment points to solid supports, may be present. The oligonucleotide moiety can have a length that prevents ternary complex binding to the template between the primer and the boundary. Ternary complex inhibition can occur when the entire template region is double-stranded, thereby eliminating single-stranded positions where ternary complexes could form.

[0051] Optionally, a primer can be attached to the oligonucleotide moiety as a result of ligase-catalyzed binding of the 5' end of the oligonucleotide to the 3' end of the primer, or as a result of polymerase-catalyzed extension of the primer over a series of nucleotides. Ligases, polymerases, and other enzymes that modify primers can be useful. However, chemical techniques for modifying primers in the manner described herein can also be useful. The oligonucleotide moiety attached to the primer can be composed entirely of natural nucleotides that form natural Watson-Crick base pairs with the template. Alternatively, the oligonucleotide can contain one or more non-natural nucleotide analogs. These analogs can be selected for their ability to base pair with the template, although analogs that do not pair with the template can also be used. Similarly, natural nucleotides can be present in positions in the oligonucleotide moiety that form mismatches that disrupt base pairing between the oligonucleotide and the template. A particularly useful position for mismatches or non-natural nucleotide analogs is at the 3' end of the oligonucleotide moiety, where the oligonucleotide moiety can function to disrupt or prevent subsequent ternary complex formation.

[0052] Another example of a useful moiety that can be added to a primer to disrupt or prevent subsequent formation of a ternary complex at the 3' end of the primer is a single nucleotide (e.g., a natural nucleotide or a non-natural nucleotide analog). For example, a mismatched nucleotide can be attached to the 3' end of the primer to prevent ternary complex formation. Particularly useful mismatches and polymerases that are influenced by their ability to recognize mismatches are described in Kwok et al., Nucleic Acids Res. 18(4):999-1005 (1990), incorporated herein by reference. The mismatched nucleotide can be present at the 3' end of the primer introduced via an oligonucleotide moiety added to the primer. In some embodiments, a series of two or more mismatched nucleotides can be present at or near the 3' end of the primer to achieve inhibition of ternary complex formation. Regardless of whether the nucleotide added to the 3' end of the primer matches the template, the nucleotide can contain an exogenous moiety that functions as a ternary complex inhibitor. The exogenous moiety can have a steric blocking effect, blocking the polymerase, the nucleotide, or both from ternary complex formation. The moiety may have other effects on ternary complex formation, including, but not limited to, charge repulsion of the polymerase or cognate nucleotide, perturbation of the structure of the primer-template nucleic acid hybrid, polarity repulsion to the polymerase or cognate nucleotide, etc.

[0053] Particularly useful moieties include, but are not limited to, biotin or other ligands that can disrupt or prevent ternary complex formation due to their presence at the 3' end or because they interact with streptavidin or other receptors, which prevent ternary complex formation. Other ligand-receptor pairs include, but are not limited to, antibodies (or functional fragments thereof, such as Fab or ScFv) and epitopes, or carbohydrates and lectins, or the binding partners described herein in the context of secondary labels. An advantage of using a ligand-receptor as a primer cap is that ternary complex formation does not need to be inhibited until the ligand is bound to the receptor. For example, a nucleotide bound to the ligand can bind to a polymerase and a primer-template nucleic acid to form a ternary complex, and the nucleotide can be incorporated into the primer, positioning the ligand at the 3' end of the primer. An receptor can then be bound to the ligand at the 3' end of the primer to inhibit ternary complex formation at the 3' end of the primer.

[0054] A primer can be capped with a moiety that inhibits detection of a ternary complex, even if one is present. For example, when detecting ternary complex formation using a luminescent label, the cap can be a quencher that reduces or prevents signal from the luminescent label. As a more specific example, a primer can be capped with a quencher moiety, and a ternary complex can be formed between the capped primer, polymerase, and a luminescent-labeled nucleotide. The luminescent-labeled nucleotide can be prevented from generating a luminescent signal due to the proximity of the quencher and the luminescent label. Another moiety that can be used to inhibit ternary complex detection without necessarily inhibiting ternary complex formation is one member of a FRET pair that can be used in a ternary complex labeled with the other member of the FRET pair. The resulting shift in signal resulting from the FRET phenomenon can effectively reduce the expected signal, thus resulting in apparent inhibition of the signal that would result from the uncapped primer. In the case of FRET, the shifted signal can be detected to identify the presence of a capped primer. In some cases, the shifted FRET signal can be quantified to quantify the capped primer in the sample being tested. When a specific label is used to detect the ternary complex, other moieties that quench or modify the signal from the specific label can be used as capping moieties.

[0055] In some embodiments, a primer modification process can be used to crosslink a polymerase to the 3' end of an unextended primer. Conditions can be used to selectively retain the polymerase at the 3' end of a primer with a natural 3' hydroxyl moiety, while removing the polymerase from a primer with a reversible terminator at its 3' end. The retained polymerase can be crosslinked to the primed template nucleic acid hybrid and inhibited from subsequently binding to nucleotides to form a ternary complex. Inhibition can be the result of denaturing the retained polymerase, crosslinking the polymerase at its nucleotide binding site, or other chemical or photochemical modifications known in the art to inactivate the polymerase.

[0056] A plurality of primer-template nucleic acid hybrids prepared or used in accordance with the teachings herein can include a first subset of species having reversible termination primers and a second subset of species having caps, such as ternary complex inhibitor moieties. The number of primer-template nucleic acid hybrids in the first subset can be less than 99%, 90%, 80%, 70%, 60%, or 51% of the plurality of primer-template nucleic acid hybrids. Alternatively or additionally, the number of primer-template nucleic acid hybrids in the first subset can be at least 50%, 60%, 70%, 80%, 90%, or 99% of the plurality of primer-template nucleic acid hybrids. In some embodiments, the number of primer-template nucleic acid hybrids in the second subset is at most 1%, 10%, 20%, 30%, 40%, 50%, or more of the plurality of primer-template nucleic acid hybrids. Alternatively or additionally, the number of primer-template nucleic acid hybrids in the second subset can be at least 50%, 40%, 30%, 20%, 10%, 1% or less of the plurality of primer-template nucleic acid hybrids. In each of the above examples, the plurality of primer-template nucleic acid hybrids can be attached to a solid support present, for example, in a particular feature of a nucleic acid array.

[0057] In sequencing embodiments, the relative numbers of primer-template nucleic acid hybrids in the first and second subsets may change as sequencing progresses. In such cases, sequencing may be allowed to proceed until a certain threshold population size is reached for one or both subsets. The threshold may be the same as that described above, or the threshold may be different to suit the specific use of the method. Alternatively, sequencing may proceed for a predetermined number of cycles, or until a threshold of a different characteristic is reached, such as obtaining adequate sequence information for identification, or until the signal-to-noise ratio drops to a certain level.

[0058] The first and second subsets of primer-template nucleic acid hybrids may differ in other ways. Typically, the primers of the first subset (e.g., primers extended by the addition of a reversible terminating nucleotide) are the same length as each other. This is generally advantageous for providing a uniform phase when detecting the next base of the template nucleic acid of the ensemble. However, the primers of the second subset (e.g., primers with a ternary complex inhibitor) may differ in length compared to each other. Such a population may occur when species within the population arise from capping in different cycles of the sequencing method. Optionally, the primers of the second subset are shorter than the primers of the first subset. Alternatively, the primers of the second subset may be longer than the primers of the first subset.

[0059] In certain embodiments, the primer modification process can mitigate against artifacts resulting from failed and inefficient extension by removing or degrading unextended primers. For example, the primer modification process can include at least two steps: (i) contacting a primer-template nucleic acid hybrid and a polymerase with a reversible terminating nucleotide to generate a first subset of primer-template nucleic acid hybrids containing a reversible terminating nucleotide at their 3' ends; and (ii) contacting the primer-template nucleic acid hybrids with an agent that removes or degrades the primer, thereby generating a second subset containing template nucleic acids lacking functional primers. Step (i) can provide the advantage of extending the primers so that the first subset of primer-template nucleic acid hybrids can continue to participate in providing sequence information. Step (ii) can provide the advantage of preventing unextended primers from participating in ternary complex formation during subsequent testing steps. The absence of unextended primers can reduce phasing, increase base calling accuracy, and extend sequencing read lengths. Generally, step (i) is performed before step (ii). However, the steps may be performed simultaneously or in reverse order. When performed sequentially, one of the steps may occur immediately after the other, one of the steps may start before the previous step finishes, or another step may intervene between the two steps.

[0060] In certain embodiments, the reagent used to degrade unextended primers is selective for primers lacking a blocking moiety, such as a reversible terminator moiety, present in extended primers. For example, primer degradation can be performed using an exonuclease or chemical agent that is active in degrading primers lacking a blocking moiety at their 3' ends (e.g., primers that have not been extended by adding a blocked nucleotide) but is inhibited by the presence of a blocking moiety on other primers (e.g., primers that have been extended to incorporate a blocked nucleotide at their 3' ends). 3'-5' exonucleases can be particularly useful, examples of which include, but are not limited to, polymerases with 3' exonuclease activity, Exo III or Exo VII. Optionally, the 3' end of the template component of a primed template-nucleic acid hybrid can be blocked, for example, via attachment to a solid support or the presence of a blocking moiety, to prevent undesired degradation of the template. As a further option, the 5' end of the primer and / or template can be blocked to prevent undesired degradation.

[0061] The plurality of template nucleic acids can include a first subset of species hybridized to a reversible termination primer and a second subset of species lacking a primer, e.g., due to removal or degradation of the primer. The number of primer-template nucleic acid hybrids in the first subset can be less than 99%, 90%, 80%, 70%, 60%, or 51% of the plurality of template nucleic acids. Alternatively or additionally, the number of primer-template nucleic acid hybrids in the first subset can be at least 50%, 60%, 70%, 80%, 90%, or 99% of the plurality of template nucleic acids. In some embodiments, the number of template nucleic acids in the second subset is at most 1%, 10%, 20%, 30%, 40%, 50%, or more of the plurality of template nucleic acids. Alternatively or additionally, the number of template nucleic acids in the second subset can be less than 50%, 40%, 30%, 20%, 10%, 1%, or less of the plurality of template nucleic acids. In each of the above examples, multiple template nucleic acids can be attached to a solid support present, for example, in particular features of a nucleic acid array.

[0062] The primer modification process used in the methods described herein does not need to use a labeled polymerase. For example, the polymerase used in the extension step or capping step does not need to be bound (e.g., covalently or otherwise) to an exogenous label. Alternatively, the polymerase used in primer extension or capping can contain an exogenous label, for example, a label used in a previous inspection step.

[0063] Typically, the reversible terminating nucleotide added to primer in the method described herein does not have an exogenous label.This is because the method described herein does not require the detection of extended primer.However, if necessary, one or more types of reversible terminating nucleotide used in the method described herein can be detected, for example, via the exogenous label attached to the nucleotide.Exemplary reversible terminator moieties, the method of incorporating them into primer, and the method of modifying primer for further extension (often referred to as "deblocking") are described in U.S. Patent No. 7,544,794, U.S. Patent No. 7,956,171, U.S. Patent No. 8,034,923, U.S. Patent No. 8,071,755, U.S. Patent No. 8,808,989 or U.S. Patent No. 9,399,798. Further examples are described in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Patent No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, U.S. Patent No. 7,405,281, and US 2008 / 0108082, each of which is incorporated herein by reference.

[0064] Similarly, ternary complex inhibitors or other primer caps added to primers in the methods described herein do not need to have exogenous labels. This is because primer-template nucleic acid hybrids containing caps, such as ternary complex inhibitors, do not need to be detected in the methods described herein. However, if desired, one or more types of ternary complex inhibitors or other caps used in the methods described herein can be detected, for example, via an exogenous label attached to the cap or ternary complex inhibitor.

[0065] The primer capping process can be performed simultaneously with the primer extension process, or the two processes can be separated by other processes described herein. For example, primer capping can occur immediately after primer extension. In another example, inspection can occur between the primer extension process and the primer capping process. In some embodiments, primer capping can occur simultaneously with inspection (e.g., a primer capping reagent can be delivered to the reaction vessel together with the reagent for forming the ternary complex).

[0066] The disclosed methods can include an examination step in which a ternary complex is formed and detected. Method embodiments take advantage of the specificity of the polymerase's ability to form a stabilized ternary complex with a primer-template nucleic acid hybrid and the next correct nucleotide. The next correct nucleotide can non-covalently bind to the stabilized ternary complex and interact with other members of the complex solely through non-covalent interactions. Useful methods and compositions for forming stabilized ternary complexes are described in further detail below and in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or U.S. Patent Application No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), U.S. Patent Application Publication No. 2018 / 0187245 A1 (claiming priority to U.S. Patent Application No. 62 / 440,624), or U.S. Patent Application Publication No. 2018 / 0208983 A1 (claiming the benefit of U.S. Patent Application No. 62 / 450,397), each of which is incorporated herein by reference.

[0067] Typically, testing is performed separately and distinctly from primer modification, e.g., with reagent exchange or washing intervening between testing and modification. Alternatively, in some embodiments, testing and one or more primer modification steps can occur in the same mixture. For example, testing and primer blocking can occur in the same mixture. Alternatively or additionally, testing and primer capping can occur in the same mixture.

[0068] Ternary complexes are formed by the interaction of specific catalytic metal ions (e.g., Mg 2+ In the absence of a catalytic metal ion, a stabilized ternary complex can form between the polymerase, the primer-template nucleic acid hybrid, and the next correct nucleotide; however, in the absence of a catalytic metal ion, chemical addition of the nucleotide is inhibited. Low or absent levels of the catalytic metal ion non-covalently sequester the next correct nucleotide in the stabilized ternary complex. Other methods disclosed herein can also be used to generate stabilized ternary complexes.

[0069] Optionally, a stabilized ternary complex can be formed if the primer of the primer-template nucleic acid hybrid contains a blocking moiety (e.g., a reversible terminator moiety) that prevents enzymatic incorporation of the incoming nucleotide into the primer. The interaction can occur in the presence of a stabilizer, whereby the polymerase-nucleic acid interaction is stabilized in the presence of the next correct nucleotide. The primer of the primer-template nucleic acid hybrid can optionally be either an extendible primer or a primer blocked from extension at its 3' end (e.g., blocking can be achieved by the presence of a reversible terminator moiety on the 3' end of the primer). The primer-template nucleic acid hybrid, polymerase, and cognate nucleotide can form a stabilized ternary complex if the base of the cognate nucleotide is complementary to the next base of the primer-template nucleic acid hybrid.

[0070] As noted above, conditions that support or stabilize the ternary complex can be provided by the presence of a blocking group (e.g., a reversible terminator moiety on the 3' nucleotide of the primer) that prevents enzymatic incorporation of the incoming nucleotide into the primer, or by the absence of catalytic metal ions. Other useful conditions include the presence of ternary complex stabilizers, such as non-catalytic ions (e.g., divalent or trivalent non-catalytic metal ions) that inhibit nucleotide incorporation or polymerization. Non-catalytic metal ions include, but are not limited to, calcium, strontium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, and terbium ions. Optionally, conditions that disfavor or destabilize the binary complex (i.e., the complex between the polymerase and the primed nucleic acid but lacking the cognate nucleotide) can be provided by the presence of one or more monovalent cations and / or glutamate anions. As a further alternative, polymerases engineered to prevent catalytic activity or to prevent the tendency to form binary complexes can be used.

[0071] In certain embodiments, the ternary complex comprises Li + The ternary complex is stabilized by the presence of tetrahydrofuran, betaine, or both. For example, the reagents and techniques described in U.S. Patent Application No. 16 / 355,361 (incorporated herein by reference) can be used. For example, immiscible fluids, including those described in U.S. Patent Application No. 16 / 164,417 (incorporated herein by reference), can be used to stabilize the ternary complex.

[0072] Ternary complex-stabilizing conditions can be further constructed to accentuate differences in polymerase affinity for primer-template nucleic acid hybrids in the presence of different nucleotides, e.g., by destabilizing binary complexes. Optionally, the conditions induce different affinities of the polymerase for the primer-template in the presence of different nucleotides. Exemplary conditions include, but are not limited to, high salt and glutamate ions. For example, salts can be dissolved in aqueous solution to generate monovalent cations, such as monovalent metal cations (e.g., sodium or potassium ions). Optionally, the salt providing the monovalent cations (e.g., monovalent metal cations) further provides glutamate ions. Optionally, the source of glutamate ions can be potassium glutamate. In some cases, potassium glutamate concentrations that can be used to alter polymerase affinity for primer-template hybrids range from 10 mM to 1.6 M potassium glutamate, or any amount between 10 mM and 1.6 M. As noted above, high salt refers to salt concentrations of 50 mM to 1.5 M salt.

[0073] It will be understood that the options described herein for stabilizing the ternary complex are not necessarily mutually exclusive and can instead be used in various combinations. For example, the ternary complex can be stabilized by one or a combination of means, including, but not limited to, cross-linking the polymerase domain; cross-linking the polymerase to the nucleic acid; polymerase mutations that stabilize the ternary complex; allosteric inhibition by small molecules; non-competitive inhibitors, competitive inhibitors, non-competitive inhibitors, the absence of catalytic metal ions, the presence of blocking moieties on the primers, and other means described herein.

[0074] The stabilized ternary complex can contain natural nucleotides, nucleotide analogs, or modified nucleotides, as desired to suit a particular application or configuration of the method. Optionally, the nucleotide analog has a nitrogenous base, a pentose sugar, and a phosphate group, and any portion of the nucleotide can be modified, deleted, and / or substituted compared to the natural nucleotide. The nucleotide analog can be a non-incorporable nucleotide (i.e., a nucleotide that cannot react with the 3' oxygen of the primer to form a covalent bond). Such non-incorporable nucleotides include, for example, monophosphate and diphosphate nucleotides. In another example, the nucleotide can contain a modification(s) to the triphosphate group that renders the nucleotide non-incorporable. Examples of non-incorporable nucleotides are described in U.S. Pat. No. 7,482,120 (incorporated herein by reference). In some embodiments, the non-incorporable nucleotide can be subsequently modified to become incorporable. Non-incorporable nucleotide analogs include, but are not limited to, alpha-phosphate modified nucleotides, alpha-beta nucleotide analogs, beta-phosphate modified nucleotides, beta-gamma nucleotide analogs, gamma-phosphate modified nucleotides, or caged nucleotides. Examples of nucleotide analogs are described in US Pat. No. 8,071,755, which is incorporated herein by reference.

[0075] The nucleotide analogues involved in the stabilized ternary complex can contain a terminator that reversibly prevents subsequent nucleotide incorporation at the 3' end of the primer after the analogue is incorporated into the primer. For example, US Pat. No. 7,544,794 and US Pat. No. 8,034,923 (the disclosures of which are incorporated herein by reference) describe reversible terminators in which the 3'-OH group is replaced with a 3'-ONH2 moiety. Another type of reversible terminator is linked to the nitrogenous base of the nucleotide, as described, for example, in US Pat. No. 8,808,989 (the disclosure of which is incorporated herein by reference). Other reversible terminators that can also be used in connection with the methods described herein include those described in the references cited elsewhere herein or in US Pat. No. 7,956,171, US Pat. No. 8,071,755, and US Pat. No. 9,399,798 (the disclosures of which are incorporated herein by reference). In certain embodiments, reversible terminator moieties can be modified or removed from the primer to allow subsequent nucleotide incorporation by a process known as "deblocking." Compositions and methods for deblocking are provided in the references cited herein in connection with reversible terminators.

[0076] Nucleotide analogs that participate in stabilized ternary complexes may contain ternary complex inhibitors that prevent subsequent ternary complex formation at the 3' end of the primer after the analog is incorporated into the primer. Nucleotide analogs can contain exogenous labels, but nucleotide analogs as used herein do not need to contain a label.

[0077] Alternatively, nucleotide analogs irreversibly prevent incorporation of a nucleotide at the 3' end of the primer into which they are incorporated. Irreversible nucleotide analogs include 2',3'-dideoxynucleotides (ddNTPs, such as ddGTP, ddATP, ddTTP, and ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs that would otherwise participate in polymerase-mediated primer extension. Thus, the 3' position has a hydrogen moiety instead of the natural hydroxyl moiety. Irreversible terminating nucleotides are particularly useful for genotyping applications or other applications where primer extension and sequential detection along a template nucleic acid are undesirable.

[0078] In some embodiments, the nucleotides involved in the formation of the ternary complex may contain an exogenous label. Optionally, the exogenously labeled nucleotide may contain a reversible or irreversible terminator moiety, the exogenously labeled nucleotide may be non-incorporable, the exogenously labeled nucleotide may lack a blocking moiety, the exogenously labeled nucleotide may be incorporable, or the exogenously labeled nucleotide may be both incorporable and non-terminating. Exogenously labeled nucleotides may be particularly useful when used to form a stabilized ternary complex with an unlabeled polymerase. Alternatively, the exogenous label on the nucleotide may provide one partner of a fluorescence resonance energy transfer (FRET) pair, and the exogenous label on the polymerase may provide the second partner of the pair. In this way, FRET detection can be used to identify stabilized ternary complexes containing both partners. Alternatively, the nucleotides involved in the formation of the ternary complex may lack an exogenous label (i.e., the nucleotide may be "unlabeled"). Optionally, the unlabeled nucleotide may include a reversible or irreversible terminator portion, may be non-incorporable, may lack a terminator portion, may be incorporable, or may be both incorporable and non-terminating. Unlabeled nucleotides may be useful when a label on a polymerase is used to detect a stabilized ternary complex. Unlabeled nucleotides may also be useful in the extension step of the methods described herein. It will be understood that the absence of a portion or function of a nucleotide refers to a nucleotide that does not have such a function or portion. It will also be understood that one or more of the functions or portions described herein for a nucleotide or its analog, or otherwise known in the art for a nucleotide or its analog, may be specifically omitted in the methods or compositions described herein.

[0079] Optionally, nucleotides (e.g., natural nucleotides or synthetic nucleotide analogs) are present in the mixture during the formation of the stabilized ternary complex. For example, at least one, two, three, four, or more nucleotide types may be present. Alternatively or additionally, up to four, three, two, or one nucleotide type may be present. Similarly, the one or more nucleotide types present may be complementary to at least one, two, three, or four base types of the template nucleic acid. Alternatively or additionally, the one or more nucleotide types present may be complementary to up to four, three, two, or one base types in the template nucleic acid.

[0080] Any nucleotide modification that does not prevent participation in ternary complex can be used in the methods disclosed herein.Nucleotide can be permanently or temporarily bound to polymerase.Optionally, nucleotide analogue is fused to polymerase, for example, via a covalent linker.Optionally, multiple nucleotide analogues are fused to multiple polymerases, and each nucleotide analogue is fused to a different polymerase.Optionally, the nucleotide present in the stabilized ternary complex is not the means by which the ternary complex is stabilized.Therefore, in the reaction utilizing nucleotide analogue, any of various other ternary complex stabilization methods can be combined.

[0081] In certain embodiments, the primer strand of the primer-template nucleic acid hybrid molecule present in the stabilized ternary complex is not chemically altered by the polymerase present in one or more steps of the method described herein.For example, the primer does not need to be extended by forming a new phosphodiester bond, nor does it need to be shortened by nucleolytic degradation during the step for forming the stabilized ternary complex or the step for detecting the stabilized ternary complex.The primer strand of the primer-template hybrid molecule present in the stabilized ternary complex does not need to be chemically modified, for example, the primer does not need to be modified to include a ternary complex inhibitor moiety or other primer capping moiety.

[0082] Ternary complexes prepared or used in accordance with the present disclosure may optionally include one or more exogenous labels. Labels may be attached to components of the ternary complex (e.g., attached to the polymerase, template nucleic acid, primer, and / or cognate nucleotide) prior to ternary complex formation. Exemplary attachments include covalent or noncovalent attachments, such as those described herein, in the references cited herein, or known in the art. In some embodiments, labeled components are delivered in solution to a solid support bound to unlabeled components, thereby forming a stabilized ternary complex, thereby mobilizing the label to the solid support. Support-bound components can then be detected or identified based on observation of the mobilized label. Exogenous labels, whether used in solution or on a solid support, are useful for detecting a stabilized ternary complex or its individual components during an examination step. Exogenous labels may remain attached to a component after the component separates from the other components that formed the stabilized ternary complex. Exemplary labels, methods for attaching labels, and methods for using labeled components are described in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or U.S. Patent Application No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), U.S. Patent Application No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1), U.S. Patent Application No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 A1), U.S. Patent Application No. 62 / 450,397, or U.S. Patent Application No. 62 / 506,759, each of which is incorporated herein by reference.

[0083] Examples of useful exogenous labels include, but are not limited to, radiolabeled moieties, luminophore moieties, fluorophore moieties, quantum dot moieties, chromophore moieties, enzyme moieties, electromagnetic spin label moieties, nanoparticle light scattering moieties, and any of a variety of other signal-generating moieties known in the art. Suitable enzyme moieties include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Exemplary fluorophore moieties include, but are not limited to, umbelliferone, fluorescein, isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow™, Cascade Blue™, Texas Red™, dansyl chloride, phycoerythrin, phycocyanin, fluorescent lanthanide complexes such as those containing europium and terbium, Cy3, Cy5, Cy7, Alexa dyes, and others known in the art, such as those described in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6th Edition of Molecular Probes Handbook by Richard P. Hoagland.

[0084] Secondary labels can be used in the methods of the present disclosure. A secondary label is a binding moiety that can specifically bind to a partner moiety. For example, a ligand moiety can be attached to a polymerase, nucleic acid, or nucleotide to allow detection via specific affinity for a labeled receptor. Secondary labels need not be detectable by the methods described herein. For example, a secondary label can be a moiety (e.g., a ligand) at the 3' end of a primer that binds to a molecule (e.g., a receptor) such that the bound molecule inhibits ternary complex formation at the 3' end of the labeled primer. Exemplary binding moiety pairs that can be used include, but are not limited to, antigens and immunoglobulins or their active fragments, e.g., FAbs; immunoglobulins and immunoglobulins (or their respective active fragments); avidin and biotin or analogs thereof specific for avidin; streptavidin and biotin or analogs thereof specific for streptavidin; or carbohydrates and lectins. A particularly useful class of epitopes that can be attached to nucleotides or primers are peptides to which antibodies (or functional fragments thereof) can be raised to generate ternary complex inhibitors.

[0085] In some embodiments, the secondary label may be a chemically modifiable moiety. In this embodiment, a label having a reactive functional group may be incorporated into the stabilized ternary complex. The functional group may then be covalently reacted with the primary labeling moiety. Suitable functional groups include, but are not limited to, amino, carboxy, maleimide, oxo, and thiol groups. Functional groups used in click chemistry, as well as related methods for their synthesis and use, may also be useful. Useful click chemistry reagents and methods are described in U.S. Patent Nos. 6,737,236, 7,375,234, 7,427,678, and 7,763,736, each of which is incorporated herein by reference.

[0086] In another embodiment, the ternary complex may lack an exogenous label. For example, the ternary complex and all components involved in the ternary complex (e.g., polymerase, template nucleic acid, primer, and / or cognate nucleotide) may lack one, some, or all of the exogenous labels described herein or in the above-incorporated references. In such embodiments, the ternary complex can be detected based on the inherent properties of the stabilized ternary complex, such as mass, charge, unique optical properties, etc. Exemplary methods for detecting unlabeled ternary complexes are described in co-owned U.S. Patent Application Publication No. 2017 / 0022553 A1, PCT Application No. PCT / US 16 / 68916 (published as WO 2017 / 117243), or U.S. Patent Application Nos. 62 / 375,379 or 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), each of which is incorporated herein by reference.

[0087] Generally, detection can be achieved in an examination step by a method that senses a characteristic property of the ternary complex or the label moiety bound thereto. Exemplary properties that can be the basis for detection include, but are not limited to, mass, conductivity, energy absorption, luminescence, etc. Detection of luminescence can be performed using methods known in the art for nucleic acid arrays. Luminophores can be detected based on any of a variety of luminescence properties, including, for example, emission wavelength, excitation wavelength, fluorescence resonance energy transfer (FRET) intensity, quenching, anisotropy, or lifetime. Other detection techniques that can be used in the methods described herein include, for example, mass spectrometry, which can be used to sense mass; surface plasmon resonance, which can be used to sense binding at a surface; absorbance, which can be used to sense the wavelength of energy absorbed by the label; calorimetry, which can be used to sense a change in temperature due to the presence of the label; electrical conductance or impedance, which can be used to sense the electrical properties of the label, or other known analytical techniques. Examples of reagents and conditions that can be used to create, manipulate, and detect stabilized ternary complexes include those described, for example, in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1, PCT Application No. PCT / US 16 / 68916 (published as WO 2017 / 117243), or U.S. Patent Application No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), U.S. Patent Application No. 15 / 581,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1), U.S. Patent Application No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983), and U.S. Patent Application No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983). Al), U.S. Patent Application No. 62 / 450,397, or U.S. Patent Application No. 62 / 506,759, each of which is incorporated herein by reference.

[0088] Some embodiments of the methods described herein utilize two or more distinguishable signals to distinguish stabilized ternary complexes from one another and / or to distinguish one base type from another in a template nucleic acid. For example, two or more luminophores can be distinguished from one another based on unique optical properties, such as unique wavelengths for excitation or emission. In certain embodiments, the methods can distinguish different stabilized ternary complexes based on differences in emission intensity. For example, a first ternary complex can be detected emitting light at a lower intensity than a second ternary complex. Such intensity scaling (often referred to as "grayscaling") can utilize any distinguishable intensity difference. Exemplary differences include a particular stabilized ternary complex having an intensity that is at most 10%, 25%, 33%, 50%, 66%, or 75% of the intensity of another stabilized ternary complex being detected.

[0089] The difference in intensity may be due, for example, to the use of different luminophores, each with different extinction coefficients (i.e., resulting in different excitation characteristics) and / or different emission quantum yields (i.e., resulting in different emission characteristics). Alternatively, the same luminophore type can be used but present in different amounts. For example, all members of a first population of a ternary complex can be labeled with a particular luminophore, while a second population has only half of its members labeled with the luminophore. In this example, the second population is expected to generate half the signal of the first population. The second population can be generated, for example, by using a mixture of labeled and unlabeled nucleotides (as opposed to a first population containing primarily labeled nucleotides). Similarly, the second population can be generated, for example, by using a mixture of labeled and unlabeled polymerase (as opposed to a first population containing primarily labeled polymerase). In an alternative labeling scheme, a first population of ternary complexes can include polymerase molecules bearing multiple labels that generate a specific luminescent signal, and a second population of ternary complexes can include polymerase molecules each bearing only one of the labels that generate a luminescent signal.

[0090] In some embodiments, the testing step is performed in a manner that assigns the identity of at least one nucleotide type, for example, as described in commonly owned U.S. Patent No. 9,951,385 or U.S. patent application Ser. No. 15 / 922,787 (granted U.S. Patent No. 10,161,003), each of which is incorporated herein by reference. Instead of or in addition to using assignment, the testing step can use disambiguation to identify one or more nucleotide types, for example, as described in commonly owned U.S. Patent No. 9,951,385 or U.S. patent application Ser. No. 15 / 922,787 (granted U.S. Patent No. 10,161,003), each of which is incorporated herein by reference.

[0091] Any of a variety of polymerases can be used in the methods or devices described herein, for example, to form a stabilized ternary complex or to perform primer modification. Polymerases that can be used include naturally occurring polymerases and modified variations thereof, including, but not limited to, mutants, recombinants, fusions, genetically modified forms, chemically modified forms, synthetic forms, and analogs. Naturally occurring polymerases and modified variations thereof are not limited to polymerases capable of catalyzing polymerization reactions. Optionally, the naturally occurring polymerase and / or modified variation thereof has the ability to catalyze polymerization reactions under at least one condition not used during the formation or testing of the stabilized ternary complex. Optionally, the naturally occurring polymerase and / or modified variation involved in the stabilized ternary complex has modified properties, such as 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 the next correct nucleotide, reduced specificity for the next correct nucleotide, reduced catalytic rate, catalytic inactivity, etc. Mutant polymerases include, for example, polymerases in which one or more amino acids are replaced with other amino acids, or one or more amino acids are inserted or deleted.Exemplary polymerase mutants that can be used to form stabilized ternary complexes include, for example, those described in U.S. Patent Application No. 15 / 866,353 (published as U.S. Patent Application Publication No. 2018 / 0155698 A1) or U.S. Patent Application Publication No. 2017 / 0314072, each of which is incorporated herein by reference.

[0092] Modified polymerases include polymerases containing an exogenous labeling moiety (e.g., an exogenous fluorophore) that can be used to detect the polymerase. Optionally, the labeling moiety can be attached after the polymerase has been at least partially purified using protein isolation techniques. For example, the exogenous labeling moiety can be covalently attached to the polymerase using a free sulfhydryl or free amine moiety on the polymerase. This includes covalent attachment to the polymerase via the side chain of a cysteine ​​residue or via a free amino group at the N-terminus. The exogenous labeling moiety can also be attached to the polymerase via protein fusion. Exemplary labeling moieties that can be attached via protein fusion include, for example, green fluorescent protein (GFP), phycobiliproteins (e.g., phycocyanin and phycoerythrin), or wavelength-shifting variants of GFP or phycobiliproteins. In some embodiments, the exogenous label on the polymerase can function as a member of a FRET pair. The other member of the FRET pair can be an exogenous label attached to a nucleotide bound to the polymerase in a stabilized ternary complex. Thus, the stabilized ternary complex can be detected or identified via FRET.

[0093] Alternatively, the polymerase involved in the stabilized ternary complex or used to modify the primer need not be bound to an exogenous label. For example, the polymerase need not be covalently bound to an exogenous label. Instead, the polymerase may lack a label until it associates with a labeled nucleotide and / or labeled nucleic acid (e.g., a labeled primer and / or labeled template).

[0094] Different activities of polymerases can be utilized in the methods described herein. Polymerases can be useful, for example, in primer modification processes, such as primer extension or primer capping, in inspection steps, or in combinations thereof. Various activities can result from structural differences (e.g., through natural activity, mutation, or chemical modification). Nevertheless, polymerases can be obtained from a variety of known sources and applied according to the teachings and recognized polymerase activities described herein. Useful DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and phage DNA polymerases. Bacterial DNA polymerases include E. coli DNA polymerase I, II, III, IV, V, Klenow fragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase, and Sulfolobus solfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, ε, η, ζ, λ, σ, μ, and k, as well as Rev1 polymerase (terminal deoxycytidyl transferase) and terminal deoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerase, PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNA polymerases include Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermusflavusu (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus GB-D polymerase, Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase; Sulfolobus acidocaldarius DNA polymerase, Thermococcus N-7 DNA polymerase, Pyrodictium occultum DNA polymerases include thermostable and / or thermophilic DNA polymerases such as Methanococcus voltae DNA polymerase, Methanococcus thermoautotrophicum DNA polymerase, Methanococcus jannaschii DNA polymerase, Desulfurococcus strain TOK DNA polymerase (D. Tok Pol), Pyrococcus abyssi DNA polymerase, Pyrococcus horikoshii DNA polymerase, Pyrococcus islandicum DNA polymerase, Thermococcus fumicolans DNA polymerase, Aeropyrum pernix DNA polymerase, and heterodimeric DNA polymerase DP1 / DP2. Engineered and modified polymerases are also useful in connection with the disclosed techniques. For example, modified versions of the highly thermophilic marine archaeon Thermococcus sp. 9°N (e.g., New England BioLabsTherminator™ DNA polymerase from Inc., Ipswich, MA) can be used. Still other useful DNA polymerases, including 3PDX polymerase, are disclosed in US 8,703,461, the disclosure of which is incorporated herein by reference.

[0095] Useful RNA polymerases include, but are not limited to, viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kll polymerase, eukaryotic RNA polymerases such as RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymerase V, and archaeal RNA polymerases.

[0096] Another useful type of polymerase is a reverse transcriptase. Exemplary reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastosis virus, and telomerase reverse transcriptase, which maintains telomeres in eukaryotic chromosomes.

[0097] Polymerases with inherent 3'-5' proofreading exonuclease activity may be useful in some embodiments. Polymerases that substantially lack 3'-5' proofreading exonuclease activity may also be useful in some embodiments, e.g., most genotyping and sequencing embodiments. The lack of exonuclease activity may be a wild-type characteristic or a characteristic conferred by a variant or engineered polymerase structure. For example, the exo-minus Klenow fragment is a mutated version of the Klenow fragment that lacks 3'-5' proofreading exonuclease activity. The Klenow fragment and its exo-minus variants may be useful in the methods or compositions described herein.

[0098] The nucleic acid used in the methods or compositions herein can be DNA, such as genomic DNA, synthetic DNA, amplified DNA, or complementary DNA (cDNA). RNA, such as mRNA, ribosomal RNA, or tRNA, can also be used. Nucleic acid analogs can also be used as templates herein. Thus, the template nucleic acid used herein can be derived from a biological source, a synthetic source, or an amplification product. The primer used herein can be DNA, RNA, or an analog thereof.

[0099] Particularly useful nucleic acid templates are genome fragments, each containing a sequence identical to a portion of a genome. A population of genome fragments can cover all or part of the sequence of a particular genome. For example, a population of genome fragments can contain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome's sequence. A genome fragment can have a sequence that is substantially identical to at least about 25, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more contiguous nucleotides of the genome. Alternatively or additionally, a genome fragment can cover 1×10 of a genome. 5 , 1×10 4 , 1×10 3 The genomic fragment may be DNA, RNA, or analogs thereof.

[0100] Exemplary organisms from which nucleic acids can be derived include, for example, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates, humans, or non-human primates; plants such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola, or soybeans; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila melanogaster, mosquitoes, fruit flies, honeybees, or spiders; fish such as zebrafish; reptiles; amphibians such as frogs or Xenopus laevis; dictyostelium discoideum; pneumocystis carinii, Takifugu rubripes, yeast, fungi Sacharamoyces cerevisiae, or Schizosaccharomyces pombe; or plasmodium falciparum. Nucleic acids can also be derived from prokaryotes such as bacteria, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, archaea, viruses such as hepatitis C virus or human immunodeficiency virus, or viroids. Nucleic acids can be obtained from homogenous cultures or populations of the above organisms, or alternatively, from a collection of several different organisms, for example, within a community or ecosystem. Nucleic acids can be isolated using methods known in the art, including, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001), or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.

[0101] Template nucleic acids can be obtained from preparation methods such as genome isolation, genome fragmentation, gene cloning and / or amplification. Templates can be obtained from amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and multiple displacement amplification (MDA). Exemplary methods for isolating, amplifying, and fragmenting nucleic acids to generate templates for analysis on arrays are described in U.S. Patent Nos. 6,355,431 and 9,045,796, each of which is incorporated herein by reference. Amplification can be performed using methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001), or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each of which is incorporated herein by reference.

[0102] Examples of reagents and conditions that can be used for the polymerase-based primer extension step include those described, for example, in co-owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or U.S. Patent Application No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), U.S. Patent Application No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1), or U.S. Patent Application Publication No. 2018 / 0208983 A1 (claiming priority to U.S. Patent Application Nos. 62 / 450,397 and 62 / 506,759). Other useful reagents and conditions for polymerase-based primer extension are described in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, WO 91 / 06678, WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082 A1, each of which is incorporated herein by reference.

[0103] In certain embodiments, reagents used during the primer modification step (e.g., primer extension via the addition of nucleotides or primer capping via the addition of a ternary complex inhibitor moiety) are removed from contact with the primer-template hybrid prior to the step of forming a stabilized ternary complex with the primer-template hybrid. For example, removal of the nucleotide mixture used in the extension step may be desirable if one or more nucleotide types in the mixture interfere with ternary complex formation or detection in a subsequent testing step. Similarly, it may be desirable to remove the polymerase or cofactors used in the primer modification step to prevent undesired catalytic activity during a subsequent testing step. Removal can be followed by a wash step, in which an inert fluid is used to purge the primer-template nucleic acid hybrid of residual components of the reagent mixture used in primer modification.

[0104] Reagent removal or washing procedures can be performed between any of the various steps described herein. Such procedures can be used to remove one or more of the reagents present in the reaction vessel or on the solid support. For example, a reagent removal or washing step can be useful for separating a primer-template nucleic acid hybrid from other reagents that contacted the primer-template nucleic acid hybrid under ternary complex-stabilizing conditions. In certain embodiments, reagent separation is facilitated by binding of the reagent of interest, such as the primer-template hybrid, to the solid support and removal of fluid from contact with the solid support. One or more of the reagents described herein can be bound to a solid support or provided in solution, as needed to suit the particular use of the methods or devices described herein.

[0105] A reagent removal or washing procedure can remove one or more reagents from interfering with the testing of the mixture or from contaminating a second mixture formed on a substrate (or in a container) previously contacted with the first mixture. For example, a primer-template nucleic acid hybrid can be contacted with a polymerase and at least one nucleotide type under ternary complex-stabilizing conditions to form a first mixture, and the first mixture can be tested. Optionally, a wash can be performed before detection to remove reagents not involved in the formation of a stabilized ternary complex. Alternatively, or additionally, a wash can be performed after the detection step to remove one or more components of the first mixture from the primer-template hybrid. The primer-template hybrid can then be contacted with a polymerase and at least one other nucleotide under ternary complex-stabilizing conditions to form a second mixture, and the second mixture can be tested for ternary complex formation. As before, an optional wash can be performed before the second test to remove reagents not involved in the formation of a stabilized ternary complex.

[0106] In certain embodiments, the primer capping step is performed after primer extension. One or more of the reagents present in the primer extension step can be removed before introducing the reagent for the primer capping step. For example, a primer-template nucleic acid hybrid bound to a solid support can be contacted with an extension mixture containing a polymerase(s) and a nucleotide(s), the extension mixture can then be removed from the solid support, and a reagent for capping the primer can then be delivered to the solid support. A wash solution can be delivered to the solid support between the extension process and the capping process. This can help remove residual components of the extension mixture before delivering the capping reagent.

[0107] In alternative embodiments, the reagents for primer extension and primer capping may be contacted simultaneously. For example, a primer-template nucleic acid hybrid bound to a solid support can be contacted with a primer modification mixture containing polymerase(s), nucleotide(s) with a reversible terminator moiety (i.e., for extending the primer), and a ternary complex inhibitor moiety (i.e., for capping unextended primers). The extension and capping reagents need not be delivered to the solid support simultaneously. Rather, the reagents can be delivered sequentially so that they are present simultaneously after cumulative delivery. The extension and capping reagents can be removed before a subsequent step, such as an inspection or deblocking step. Washing can optionally be used to remove residual extension and capping reagents before a subsequent step.

[0108] Nucleotides present in the test step may cause unwanted side reactions, such as nucleotide incorporation reactions, if carried over into a primer modification process, such as a primer extension process or a primer capping process. Therefore, a reagent removal or washing step may be used prior to the primer modification step. Optionally, free nucleotides or other test reagents may be modified or neutralized, for example, by an enzyme such as a phosphatase, by chemical modification, or by physical techniques.

[0109] Certain embodiments of the methods described herein include forming a mixture containing several components. For example, a mixture can be formed between a primer-template nucleic acid hybrid, a polymerase, and one or more nucleotide types. The components of the mixture can be delivered to a container in any desired order, or they can be delivered simultaneously. Furthermore, some components can be mixed together to form a first mixture and then contacted with other components to form a more complex mixture. Taking the step of forming a mixture containing a primer-template nucleic acid hybrid, a polymerase, and multiple different nucleotide types as an example, it will be understood that multiple different nucleotide types can be contacted with each other before contacting with the primer-template nucleic acid hybrid. Alternatively, two or more nucleotide types can be delivered separately to the primer-template hybrid and / or polymerase. Thus, a first nucleotide type can be contacted with the primer-template hybrid before contacting with the second nucleotide type. Alternatively or additionally, a first nucleotide type can be contacted with the polymerase before contacting with the second nucleotide type.

[0110] The stabilized ternary complex, or components capable of forming (i.e., participating in) the formation of a ternary complex, can be attached to a solid support. The solid support can be made from any of a variety of materials used in biochemical analysis. Suitable materials can include glass, polymeric materials, silicon, quartz (fused silica), borofluor glass, silica, silica-based materials, carbon, metals, optical fibers or fiber optic bundles, sapphire, or plastic materials. Materials can be selected based on the properties desired for a particular application. For example, materials that transmit radiation of a desired wavelength are useful for analytical techniques that utilize radiation of that wavelength. Conversely, it may be desirable to select a material that does not transmit radiation of a particular wavelength (e.g., opaque, absorbing, or reflective). Other properties of materials that can be utilized include inertness or reactivity with certain reagents used in downstream processes, such as those described herein, ease of manipulation, or low manufacturing costs.

[0111] Particularly useful solid supports are particles such as beads or microspheres. A population of beads can be used to bind components (e.g., polymerase, template, primer, or nucleotide) capable of forming a population of stabilized ternary complexes or complexes. In some embodiments, it may be useful to use a configuration in which each bead has a single type of component capable of forming a single type of stabilized ternary complex or complex. For example, an individual bead can be bound to a single type of ternary complex, a single type of primer-template nucleic acid hybrid, a single type of primer, a single type of template, a single type of polymerase, or a single type of nucleotide. Alternatively, different types of components need not be separated on each bead. Thus, a single bead can carry multiple different types of ternary complexes, template nucleic acids, primers, primer-template nucleic acid hybrids, and / or nucleotides. The composition of the beads can vary, for example, depending on the type, chemistry, and / or method of binding used. Exemplary bead compositions include solid supports and chemical functionalities imparted thereto for use in protein and nucleic acid capture methods. Such compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, triazoles, carbon graphite, titanium dioxide, latex, or cross-linked dextrans such as Sepharose™, cellulose, nylon, cross-linked micelles and Teflon®, and other materials described in the "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind., which is incorporated herein by reference.

[0112] The shape of particles, such as beads or microspheres, can also accommodate a wide variety of different forms and shapes. For example, particles can be symmetrical (e.g., spherical or cylindrical) or irregular (e.g., controlled pore glass). In addition, particles can be porous, thus increasing the surface area available for capturing the ternary complex or its components. Exemplary sizes of beads used herein can range from nanometers to millimeters, or from about 10 nm to 1 mm.

[0113] In certain embodiments, beads can be arranged or otherwise spatially differentiated.Exemplary bead-based arrays that can be used include, but are not limited to, the BeadChip™ array available from Illumina, Inc. (San Diego, CA), or arrays such as those described in U.S. Patent No. 6,266,459, U.S. Patent No. 6,355,431, U.S. Patent No. 6,770,441, U.S. Patent No. 6,859,570, or U.S. Patent No. 7,622,294, or PCT Publication No. WO 00 / 63437 (each of which is incorporated herein by reference).Beads can be arranged in separate locations, such as wells on solid support, whereby each location contains a single bead. Alternatively, the distinct locations where beads are present can each comprise a plurality of beads, for example, as described in U.S. Patent Application Publication Nos. 2004 / 0263923 A1, 2004 / 0233485 A1, 2004 / 0132205 A1, or 2004 / 0125424 A1, each of which is incorporated herein by reference.

[0114] As can be appreciated from the bead array embodiments described above, the disclosed methods can be performed in a multiplex format, where multiple different types of nucleic acids are detected in parallel using the methods described herein. While it is also possible to process different types of nucleic acids sequentially using one or more steps of the methods described herein, parallel processing can provide cost savings, time savings, and uniformity of conditions. The disclosed devices or methods can be used to detect at least 2, 10, 100, 1x10 3 , 1×104 , 1×10 5 , 1×10 6 , 1×10 9 Alternatively or additionally, the disclosed devices or methods may comprise up to 1 x 10, or more different nucleic acids. 9 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 100, 10, 2, or fewer different nucleic acids. Thus, various reagents or products (e.g., primer-template nucleic acid hybrids or stabilized ternary complexes) described herein as useful in the devices or methods can be multiplexed to have different types or species within these ranges. Different nucleic acids present in the array can be located in different features of the array. Thus, a signal obtained from a feature indicates the particular nucleic acid sequence present in the feature.

[0115] Further examples of commercially available arrays that can be used include, for example, Affymetrix GeneChip™ arrays. According to some embodiments, spotted array substrates can also be used. An exemplary spotted array is the CodeLink™ array available from Amersham Biosciences. Another useful array is one manufactured using inkjet printing methods, such as SurePrint™ technology available from Agilent Technologies.

[0116] Other useful arrays include those used in nucleic acid sequencing applications.For example, the array used to combine amplicons (often referred to as clusters) of genome fragments can be particularly useful.The example of nucleic acid sequencing array that can be used herein includes those described in Bentley et al., Nature 456:53-59 (2008), PCT Publication No. WO 91 / 06678, PCT Publication No. WO 04 / 018497 or PCT Publication No. WO 07 / 123744, US Patent No. 7,057,026, US Patent No. 7,211,414, US Patent No. 7,315,019, US Patent No. 7,329,492 or US Patent No. 7,405,281, or US Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0117] Nucleic acid can be bound to support in a way that provides detection at single molecule level or ensemble level.For example, multiple different nucleic acids can be bound to solid support, so that each stabilized ternary complex formed on one nucleic acid molecule on support can be distinguished from all adjacent ternary complexes formed on nucleic acid molecules on support.In this way, one or more different templates can be bound to solid support in a manner that each single molecule template is physically separated, and single molecules can be detected so as to be distinguished from all other molecules on solid support.

[0118] Alternatively, the disclosed methods can be performed on one or more nucleic acid ensembles, where an ensemble is a population of nucleic acids that share a common template sequence. An ensemble can include, for example, at least 2, 10, 50, 100, 500, 1000, or more nucleic acids that share a common template sequence. Alternatively or additionally, an ensemble can include up to 1000, 500, 100, 50, 10, or 2 nucleic acids that share a common template sequence. The ensembles present in a feature of an array can be clonal, such that substantially all of the nucleic acids in the feature share a common template sequence. However, a feature need not contain a clonal population of nucleic acids. Rather, a feature can include a mixed population of nucleic acids, with a particular template sequence being present in the majority of the nucleic acids. For example, the population of nucleic acids in a particular feature can include at least 51%, 60%, 75%, 90%, 95%, or 99% or more species that share a particular template sequence. A feature having a non-clonal population of nucleic acids can be detected under conditions that allow the population to be detected as an ensemble, whereby the total signal obtained from the feature represents the average of the signals generated by the non-clonal population. As long as contaminating nucleic acids are present as a minority in the feature of interest, the average signal can provide a means of characterizing the majority of the template nucleic acids in the feature.

[0119] Using clustering, one or more ensembles can be attached to a solid support.In this way, an array can have multiple ensembles, and each ensemble is called a cluster or array feature in that format.Clusters can be formed using methods known in the art, such as bridge amplification or emulsion PCR.Useful bridge amplification methods are described, for example, in U.S. Patent No. 5,641,658 or U.S. Patent No. 7,115,400, or U.S. Patent Publication No. 2002 / 0055100 A1, U.S. Patent No. 2004 / 0002090 A1, U.S. Patent No. 2004 / 0096853 A1, U.S. Patent No. 2007 / 0128624 A1, or U.S. Patent No. 2008 / 0009420 A1. Emulsion PCR methods include, for example, those described in Dressman et al., Proc.Natl.Acad.Sci.USA 100:8817-8822 (2003), WO 05 / 010145, or US Patent Publication No. 2005 / 0130173 A1 or US Patent Publication No. 2005 / 0064460 A1, each of which is incorporated herein by reference in its entirety.Another useful method for amplifying nucleic acid on a surface is, for example, rolling circle amplification (RCA), as described in Lizardi et al., Nat.Genet.19:225-232 (1998), or US 2007 / 0099208 A1, each of which is incorporated herein by reference.

[0120] In certain embodiments, the stabilized ternary complex, polymerase, primer, template, primer-template nucleic acid hybrid, or nucleotide is bound to a solid support on the surface of a flow cell or within the flow cell. The flow cell allows for convenient fluid manipulation by moving solutions into and out of a fluid chamber that contacts the support-bound ternary complex. The flow cell also provides for detection of fluid-manipulated components. For example, a detector can be configured to detect a signal from the solid support, such as a signal from a label mobilized to the solid support upon formation of the stabilized ternary complex. Exemplary flow cells that can be used are described, for example, in U.S. Patent Application Publication No. 2010 / 0111768 A1, WO 05 / 065814, or U.S. Patent Application Publication No. 2012 / 0270305 A1, each of which is incorporated herein by reference.

[0121] The present disclosure provides a method for sequencing a template nucleic acid, the method including: (a) providing a plurality of primer-template nucleic acid hybrids, the primers having extendable 3' ends; (b) contacting the plurality with (i) a reversible terminating nucleotide to generate a first subset of primer-template nucleic acid hybrids comprising a reversible terminating nucleotide at their 3' ends, and (ii) a ternary complex inhibitor to generate a second subset of primer-template nucleic acid hybrids comprising a ternary complex inhibitor; (c) forming ternary complexes, each comprising a polymerase, the first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; (d) detecting the ternary complexes, thereby identifying nucleotides in the template nucleic acid; (e) deblocking the reversible terminating nucleotides at the 3' ends of the first subset of primer-template nucleic acid hybrids; and (f) repeating steps (b) through (e) to sequence the first subset of template nucleic acids.

[0122] When included in the methods described herein, the deblocking process can facilitate sequencing of primer-template nucleic acid hybrids. The deblocking process can be used to convert reversible terminating primers into extendable primers. Primer extension can then be used to move the site of ternary complex formation to a different position along the template nucleic acid. Repeated cycles of extension, inspection, and deblocking can be used to reveal the sequence of the template nucleic acid. Each cycle reveals subsequent bases in the template nucleic acid. Exemplary reversible terminator moieties, methods for incorporating them into primers, and methods for modifying primers for further extension (often referred to as "deblocking") are described in U.S. Patent Nos. 7,427,673, 7,414,116, 7,544,794, 7,956,171, 8,034,923, 8,071,755, 8,808,989, or 9,399,798. Further examples are described in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Patent No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, U.S. Patent No. 7,405,281, and US 2008 / 0108082, each of which is incorporated herein by reference.

[0123] Primers modified to disrupt or prevent subsequent formation of the ternary complex do not need to be treated to reverse the modification. Thus, capped primers can remain capped throughout several cycles (and in some cases, all cycles) of a cyclic method, such as a sequencing method. For example, the cyclic process can include a process that deblocks a reversible termination primer, and the capped primer can be inert to the deblocking process.

[0124] The sequencing method can include multiple repetitions of the cycles or steps within a cycle described herein. For example, the inspection and primer modification steps can be repeated multiple times, as can any steps to deblock primers or wash away unwanted reactants or products between various steps. Thus, the primer-template nucleic acid hybrid can be subjected to at least 2, 5, 10, 25, 50, 100, 150, 200, or more repeated cycles of the method described herein. If a shorter read length is desired, fewer cycles can be performed. Thus, the primer-template nucleic acid hybrid can be subjected to a maximum of 200, 150, 100, 50, 25, 10, 5, or 2 cycles of the method described herein.

[0125] In some embodiments, the sequencing method can be carried out in a predetermined number of repeated cycles. Alternatively, the cycle can be repeated until a specific empirically observed state is reached.For example, the cycle can be repeated as long as the signal is above the observable threshold, the noise is below the observable threshold, or the signal-to-noise ratio is above the observable threshold.

[0126] In another example, cycles can be repeated as long as one or more ensembles (e.g., one or more clusters or features in an array) have the desired composition. As one or more ensembles undergo sequencing cycles, their compositions can change. If a single ensemble is considered a set of template nucleic acids, a first subset of templates, each having a primer with a reversible terminating nucleotide at its 3' end, can decrease in number as cycles proceed, and a second subset of template nucleic acids, each having a primer with a ternary complex inhibitor (or each having no primer), can increase in number as cycles proceed. The sequencing method can be continued until the first subset reaches a certain threshold and / or until the second subset reaches a certain threshold. More specifically, cycles can be repeated until the number of primer-template nucleic acid hybrids in the first subset is less than 99%, 90%, 80%, 70%, 60%, or 51% of the plurality of template nucleic acids being detected. Alternatively or additionally, the cycle can be repeated as long as the number of primer-template nucleic acid hybrids in the first subset is at least 50%, 60%, 70%, 80%, 90%, or 99% of the plurality of template nucleic acids being detected. In some embodiments, the cycle can be repeated until the number of template nucleic acids in the second subset is at most 1%, 10%, 20%, 30%, 40%, 50%, or more of the plurality of template nucleic acids being detected. Alternatively or additionally, the cycle can be repeated as long as the number of template nucleic acids in the second subset is at least 50%, 40%, 30%, 20%, 10%, or 1% of the plurality of template nucleic acids being detected.

[0127] It will be understood that not all of the steps described herein need be repeated, nor do the repeated steps need to occur in the same order in each repetition.

[0128] The present disclosure further provides an apparatus comprising a plurality of primer-template nucleic acid hybrids, wherein a first subset of the primer-template nucleic acid hybrids have a blocked nucleotide at the 3' end of the primer, and a second subset of the primer-template nucleic acid hybrids have a ternary complex inhibitor at the 3' end of the primer. Optionally, the blocked nucleotide can be a reversible terminating nucleotide.

[0129] Optionally, a plurality of primer-template nucleic acid hybrids are bound to a solid support within the device of the present disclosure. The solid support can comprise any of the various materials described herein, including, for example, the materials described herein in the context of a nucleic acid array. The plurality of primer-template nucleic acid hybrids can be bound to features of the array, and optionally, the templates bound to the features can have the same sequence. Any of the various reagents described herein can be bound to the solid support in place of, or alternatively in addition to, the primer-template nucleic acid hybrids. In certain embodiments, the device of the present disclosure need not include any type of bound reagent.

[0130] In certain embodiments, the disclosed device includes a container, such as an artificial container. The container can contain multiple primer-template nucleic acid hybrids along with other reagents or reaction products involved in the methods described herein. A particularly useful artificial container is a flow cell, examples of which are described herein above.

[0131] The disclosed device can be a component of a larger system. For example, the system can include (a) a device containing a plurality of primer-template nucleic acid hybrids, wherein a first subset of the primer-template nucleic acid hybrids have a reversible terminating nucleotide at the 3' end of the primer and a second subset of the primer-template nucleic acid hybrids have a ternary complex inhibitor at the 3' end of the primer, and (b) a detector configured to detect the plurality of primer-template nucleic acid hybrids. Optionally, the system can further include (c) a fluidics system including a reservoir containing a polymerase and nucleotides, the reservoir being in fluid communication with the plurality of primer-template nucleic acid hybrids.

[0132] The disclosed systems can be configured to detect nucleic acids, for example, using the methods described herein. For example, the systems can be configured to generate and detect ternary complexes formed between a polymerase and a primer-template nucleic acid hybrid in the presence of nucleotides to identify one or more bases in a template nucleic acid sequence. Optionally, the systems include components and reagents for performing one or more steps described herein, including, but not limited to, forming at least one stabilized ternary complex between a primer-template nucleic acid hybrid, a polymerase, and the next correct nucleotide; detecting the stabilized ternary complex(es); modifying the primer of each primer-template nucleic acid hybrid, for example, via a primer capping or primer extension process; deblocking a reversible terminating primer; and / or identifying a nucleotide, sequence of nucleotides, or sequence of base multiplets present in the template.

[0133] The systems of the present disclosure may include a vessel, solid support, or other device for performing a nucleic acid detection method. For example, the system may include an array, flow cell, multiwell plate, or other convenient device. The device may be removable, allowing it to be placed in or removed from the system. Thus, the system may be configured to process multiple devices (e.g., vessels or solid supports) sequentially or in parallel. The system may include a fluidic component having reservoirs for containing one or more of the reagents described herein (e.g., polymerase, primer, template nucleic acid, nucleotide(s) for ternary complex formation, nucleotides for primer extension, deblocking reagent, ternary complex inhibitor, or a mixture of such components). The fluidic system may be configured to deliver the reagents to the vessel or solid support, for example, via a channel or droplet transfer device (e.g., an electrowetting device). Any of a variety of detection devices can be configured to detect the vessel or solid support with which the reagent interacts. Examples include luminescence detectors, surface plasmon resonance detectors, and others known in the art. Exemplary systems having fluidic and detection components that can be readily modified for use in the systems herein include, but are not limited to, those described in U.S. Patent Application Publication No. 2018 / 0280975 A1 (U.S. Patent Application No. 62 / 481,289, U.S. Patent Nos. 8,241,573, 7,329,860, or 8,039,817, or U.S. Patent Application Publication Nos. 2009 / 0272914 A1 or 2012 / 0270305 A1, each of which is incorporated herein by reference.

[0134] Optionally, the disclosed system further includes a computer processing unit (CPU) configured to operate the system components. The same or a different CPU can interact with the system to acquire, store, and process signals (e.g., signals detected by the methods described herein). In certain embodiments, the CPU can be used to determine the identity of nucleotides present at specific locations in the template nucleic acid from the signals. In some cases, the CPU identifies the sequence of the template nucleotides from the detected signals.

[0135] Useful CPUs include, for example, one or more of personal computer systems, server computer systems, thin clients, thick clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics, network PCs, minicomputer systems, mainframe computer systems, smartphones, or distributed cloud computing environments that include any of the above systems or devices. A CPU can include one or more processors or processing units, a memory architecture that can include RAM and nonvolatile memory. The memory architecture can further include removable / non-removable, volatile / non-volatile computer system storage media. Additionally, the memory architecture may include one or more readers for reading and writing non-removable, non-volatile magnetic media such as hard drives, magnetic disk drives for reading from or writing to removable, non-volatile magnetic disks, and / or optical disk drives for reading from or writing to removable, non-volatile optical disks such as CD-ROMs or DVD-ROMs. A CPU can also include a variety of computer system-readable media. Such media can be any available media accessible to a cloud computing environment, including volatile and non-volatile media, removable and non-removable media, etc.

[0136] The memory architecture may include at least one program product having at least one program module implemented as executable instructions configured to perform one or more steps of the methods described herein. For example, the executable instructions may include an operating system, one or more application programs, other program modules, and program data. Generally, program modules may include routines, programs, objects, components, logic, data structures, etc. that perform particular tasks described herein.

[0137] The components of a CPU may be coupled by an internal bus, which may be implemented as one or more of several types of bus structures, including a memory bus or memory controller, a peripheral bus, a high-speed graphics port, and a processor or local bus using any of a variety of bus architectures. By way of example and not limitation, such architectures include an Industry Standard Architecture (ISA) bus, a MicroChannel Architecture (MCA) bus, an Enhanced ISA (EISA) bus, a Video Electronics Standards Association (VESA) local bus, and a Peripheral Component Interconnect (PCI) bus.

[0138] The CPU can optionally communicate with one or more external devices, such as a keyboard, a pointing device (e.g., a mouse), a display such as a graphical user interface (GUI), or other devices that facilitate use and interaction with the nucleic acid detection system. Similarly, the CPU can communicate with other devices (e.g., via a network card, modem, etc.). Such communication can occur through an I / O interface. Additionally, the CPU of the systems herein can communicate with one or more networks, such as a local area network (LAN), a general wide area network (WAN), and / or a public network (e.g., the Internet) via an appropriate network adapter.

[0139] The present disclosure further provides kits for characterizing nucleic acids. Optionally, the kits can include reagents for performing one or more of the methods described herein. For example, the kits can include reagents for generating a stabilized ternary complex when mixed with one or more primer-template nucleic acid hybrids. In addition to the nucleotide mixture, the kits can include a polymerase capable of forming a stabilized ternary complex and / or a polymerase used in the primer modification step. Other reagents used for primer modification, such as reversible terminating nucleotides or ternary complex inhibitors, can also be included in the kit. Reagents included in the kit, such as the nucleotide(s), polymerase(s), or both, can include exogenous labels, for example, as described herein in the context of various methods.

[0140] Thus, any of the components or items used in carrying out the methods described herein can be usefully packaged into a kit. For example, a kit can be packaged to contain some, many, or all of the components or items used in carrying out the methods described herein. Exemplary components include, for example, labeled nucleotides (e.g., extendible labeled nucleotides), polymerases (labeled or unlabeled), nucleotides having terminator moieties (e.g., unlabeled, reversible terminating nucleotides), deblocking reagents, ternary complex inhibitors, and the like, as described herein and in the references cited herein. Any of such reagents can include, for example, some, many, or all of the buffers, components, and / or items used to perform one or more of the subsequent steps for analysis of primer-template nucleic acid hybrids. A kit need not include primers or template nucleic acids. Rather, the user of the kit can provide one or more primer-template nucleic acid hybrids that are combined by the user with the components of the kit.

[0141] One or more auxiliary reagents can also be included in the kit. Such auxiliary reagents can include any of the reagents exemplified above and / or other types of reagents useful in performing the methods described herein. Instructions can also be included in the kit. The instructions can include, for example, procedures for making any of the components or items used in the methods described herein, performing one or more steps of any of the embodiments of the methods described herein, and / or instructions for performing any of the subsequent analytical steps using primer-template nucleic acid hybrids.

[0142] In certain embodiments, the kit can include a device described herein, such as a flow cell or solid support. Optionally, the kit includes a cartridge having a reservoir for containing the reagent and further having a fluidic component for transferring the reagent from the reservoir to a detection device. For example, the fluidic component can be configured to transfer the reagent to a flow cell where the stabilized ternary complex is detected. An exemplary fluidic cartridge that can be included in the kit (or system) of the present disclosure is described in U.S. Patent Application No. 15 / 922,661 (published as U.S. Patent Application Publication No. 2018 / 0280975 A1), which is incorporated herein by reference. [Example]

[0143] Example I Primer Capping Using Runoff Primer Extension This example demonstrates a technique for improving phasing when extending a population of primers. Phasing is improved by capping unextended primers to prevent the capped primers from forming ternary complexes in a subsequent testing step.

[0144] A FORTEBIO® (Menlo Park, CA) Octet instrument, which uses biolayer interferometry to measure binding reactions on the surface of fiber optic tips, was used in a multiwell plate format. Template nucleic acid was bound to a streptavidin-functionalized tip by incubating it in a well containing a 5'-biotinylated template nucleic acid at 37°C for 5 minutes. Unbound template nucleic acid was removed by contacting the tip with 0.1 M NaOH, blotting the tip dry, and then washing the tip in PRE (50 mM KCl, 50 mM Tricine, pH 8.42, 0.1% Tween-80, 0.1% hydroxylamine, 0.1 mM EDTA). Primers were hybridized to the tip-bound template by incubating in PRE at 37°C for 5 minutes.

[0145] The tip was then subjected to 30 primer extension cycles on an Octet instrument. Each cycle included the following incubation times and steps to transfer the tip to the following reagents: (1) PRE, 5 seconds; (2) RTS (PRE, 5 mM MgCl, 10 U / mL Therminator, 25 μM rtNTP (a reversible terminating deoxynucleotide with an aminooxy reversible terminator moiety at the 3′ position)), 10 seconds; (3) Variable Solution, 30 seconds; (4) ESB (1 M GdSCN, 0.1 mM HEPES, pH 7.5, 0.1% Tween-80, 0.1% hydroxylamine, 2 mM EDTA), 5 seconds; (5) PRE, 5 seconds; (6) CLV (0.25 M sodium acetate, pH 4.8, 0.7 M sodium nitrite), 5 seconds; and (7) PRE, 5 seconds. The Variable Solution contained the following reagents: a) PRE b) PRE+10u / mL Therminator c)PRE+10u / mL Therminator+5mg Mg +2 d)PRE+10u / mL Therminator+5mg Mg +2 +100μM dNTP e)PRE+10u / mL Therminator+5mg Mg+2 +30 μM dNTPs f)PRE+10u / mL Therminator+5mg Mg +2 +10 μM dNTPs g)PRE+10u / mL Therminator+5mg Mg +2 +3 μM dNTPs h)PRE+10u / mL Therminator+5mg Mg +2 +1 μM dNTP

[0146] After the Octet cycle, the primer strand was stripped from the tip-bound template and incubated with capillary electrophoresis (CE) dye. The primer length was then determined using CE analysis. The CE results were analyzed by checking each peak and recording the n and n-1 peak areas. The efficiency of primer extension was evaluated using the following formula: [(cycle number) × (n / n-1 ratio)] ÷ [(cycle number) × (n / n-1 ratio) + 1].

[0147] The results are summarized in Table 1. The extended primers had CE mobilities consistent with their expected length (n = 59 nucleotides). The ratio of n / n-1 was lowest in the control conditions (i.e., a), b), and c)) and increased when nucleotides were present. The efficiency of full-length extension also increased when all components necessary for primer extension were present. [Table 1]

[0148] The results demonstrated that after extension at the reversible terminating nucleotide (in the RTS), further primer extension occurred only when all components required for primer extension (dNTPs, Therminator, and Mg) were present, indicating that an unblocked 3' end remained and the primer could be extended completely through the template region using runoff extension after the RTS.

[0149] Fully extended primers are expected to be unavailable for ternary complex formation during the subsequent testing step, resulting in improved signal-to-noise, longer read lengths, and higher sequencing accuracy.

[0150] Throughout this application, various publications, patents and / or patent applications are referenced, the disclosures of which are incorporated by reference in their entireties into this application.

[0151] A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made. Accordingly, other embodiments are within the scope of the appended claims.

Claims

1. 1. A method for identifying a nucleotide in a template nucleic acid, comprising: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers comprise extendable 3' ends; (b) dividing the plurality into (i) a blocked nucleotide to generate a first subset of primer-template nucleic acid hybrids, each of which contains a blocked nucleotide at its 3' terminus; and (ii) contacting with a ternary complex inhibitor to generate a second subset of primer-template nucleic acid hybrids, each of which contains the ternary complex inhibitor; (c) forming ternary complexes each comprising a polymerase, said first subset of primer-template nucleic acid hybrids, and a cognate nucleotide; (d) detecting the ternary complex, thereby identifying the nucleotide in the template nucleic acid.

2. 2. The method of claim 1, wherein the blocked nucleotide comprises a reversible terminating nucleotide, and the first subset of the primer-template nucleic acid hybrids each comprise a reversible terminating nucleotide at the 3' end.

3. 3. The method of claim 2, further comprising: (e) deblocking the reversible terminating nucleotides at the 3' ends of the primer-template nucleic acid hybrids of the first subset.

4. 4. The method of claim 3, further comprising: (f) repeating steps (b) through (e) to sequence the first subset of template nucleic acids.

5. 5. The method of claim 4, further comprising removing the polymerase and cognate nucleotides from the primer-template nucleic acid hybrids of the first subset prior to step (f).

6. 6. The method of claim 4 or 5, wherein steps (b) through (f) are repeated as long as the number of primer-template nucleic acid hybrids in the first subset exceeds 50% of the plurality of primer-template nucleic acid hybrids.

7. 6. The method of claim 4, wherein steps (b) through (f) are repeated as long as the number of primer-template nucleic acid hybrids in the second subset is less than 50% of the plurality of primer-template nucleic acid hybrids.

8. The method of any one of claims 4 to 7, wherein steps (b) to (f) are repeated at least 100 times.

9. The method of any one of claims 1 to 8, wherein the second subset of primer-template nucleic acids consists essentially of one primer-template nucleic acid hybrid.

10. The method of any one of claims 1 to 8, wherein the second subset of primer-template nucleic acids comprises at least two primer-template nucleic acid hybrids.

11. 11. The method of any one of claims 1 to 10, wherein the second subset of primer-template nucleic acid hybrids comprises less than 1% of the plurality of primer-template nucleic acid hybrids.

12. The method of any one of claims 1 to 11, wherein the plurality of primer-template nucleic acid hybrids are attached to a solid support.

13. 13. The method of claim 12, wherein the plurality of primer-template nucleic acid hybrids are bound to features of an array.

14. 14. The method of claim 13, wherein the plurality of primer-template nucleic acid hybrids bound to the feature comprises at least 100 copies having a common sequence.

15. The method of claim 13 or 14, wherein the primer-template nucleic acid hybrids bound to the features are detected as an ensemble.

16. 13. The method of claim 12, wherein the plurality of primer-template nucleic acid hybrids comprises different templates bound to different features of an array.

17. The method of any one of claims 1 to 16, wherein the ternary complex inhibitor comprises an oligonucleotide moiety.

18. 18. The method of claim 17, wherein the oligonucleotide portion is generated in step (b) by ligating an oligonucleotide to the primer-template nucleic acid hybrid.

19. 19. The method of claim 18, wherein the oligonucleotide portion is generated in step (b) by polymerase-catalyzed extension of the primer-template nucleic acid hybrid.

20. 20. The method of any one of claims 1 to 19, wherein the ternary complex inhibitor comprises a moiety that inhibits binding of a polymerase at the 3' end of the primer.

21. 20. The method of any one of claims 1 to 19, wherein the ternary complex inhibitor comprises a moiety that inhibits the binding of the cognate nucleotide at the 3' end of the primer to a polymerase.

22. The method of any one of claims 1 to 19, wherein the ternary complex inhibitor comprises a mismatched nucleotide at the 3' end of the primer.

23. 23. The method of any one of claims 1 to 22, wherein unreacted blocked nucleotides are separated from the plurality of primer-template nucleic acid hybrids after step (b)(i).

24. 24. The method of claim 23, wherein unreacted blocked nucleotides are separated from the plurality of primer-template nucleic acid hybrids prior to step (b)(ii).

25. 20. The method of any one of claims 1 to 19, wherein step (b)(i) occurs before step (c) and step (b)(ii) occurs after step (d).

26. 1. A device comprising a plurality of primer-template nucleic acid hybrids, wherein a first subset of the primer-template nucleic acid hybrids comprise a blocked nucleotide at the 3' end of the primers, and a second subset of the primer-template nucleic acid hybrids comprise a ternary complex inhibitor at the 3' end of the primers.

27. 27. The device of claim 26, wherein the blocked nucleotides comprise reversible terminating nucleotides.

28. 28. The apparatus of claim 26 or 27, wherein the plurality of primer-template nucleic acid hybrids are attached to a solid support.

29. 30. The device of claim 28, wherein the solid support comprises a nucleic acid array.

30. 30. The apparatus of claim 28 or 29, wherein the plurality of primer-template nucleic acid hybrids are bound to features of the array, and the templates bound to the features comprise the same sequence.

31. The apparatus of any one of claims 26 to 30, wherein the plurality of primer-template nucleic acid hybrids are in an artificial container.

32. 32. The apparatus of claim 31 , wherein the artificial container comprises a flow cell.

33. 33. The apparatus of any one of claims 26 to 32, wherein each of the primer-template nucleic acid hybrids of the first subset is bound to a polymerase and a cognate nucleotide, thereby forming a ternary complex.

34. 34. The device of claim 33, wherein the polymerase is covalently linked to an exogenous label.

35. 34. The device of claim 33, wherein the cognate nucleotide is covalently attached to an exogenous label.

36. 36. The device of any one of claims 33, 34, or 35, wherein the exogenous label comprises a fluorophore.

37. 37. The apparatus of any one of claims 26 to 36, wherein the primers of the first subset of primer-template nucleic acid hybrids are the same length.

38. 38. The apparatus of claim 37, wherein the primers of the second subset of primer-template nucleic acid hybrids differ in length.

39. 39. The apparatus of claim 37 or 38, wherein the primers of the second subset of primer-template nucleic acid hybrids are shorter than the primers of the first subset of primer-template nucleic acid hybrids.

40. 39. The apparatus of claim 37 or 38, wherein the primers of the second subset of primer-template nucleic acid hybrids are longer than the primers of the first subset of primer-template nucleic acid hybrids.

41. 38. The apparatus of claim 37, wherein the primers of the second subset of primer-template nucleic acid hybrids are fully extended.

42. 42. The apparatus of any one of claims 26 to 41, wherein the number of primer-template nucleic acid hybrids in the first subset is less than 50% of the number of primer-template nucleic acid hybrids in the plurality of primer-template nucleic acid hybrids.

43. 42. The apparatus of any one of claims 26 to 41, wherein the number of primer-template nucleic acid hybrids in the second subset is greater than 50% of the number of primer-template nucleic acid hybrids in the plurality of primer-template nucleic acid hybrids.

44. 44. The device of any one of claims 26 to 43, wherein the ternary complex inhibitor comprises an oligonucleotide moiety.

45. 45. The device of any one of claims 26 to 44, wherein the ternary complex inhibitor comprises a moiety that inhibits binding of a polymerase at the 3' end of the primer.

46. 45. The device of any one of claims 26-44, wherein the ternary complex inhibitor comprises a moiety that inhibits binding of the cognate nucleotide at the 3' end of the primer to a polymerase.

47. 45. The device of any one of claims 26 to 44, wherein the ternary complex inhibitor comprises a mismatched nucleotide at the 3' end of the primer.

48. (a) an apparatus according to any one of claims 26 to 47; (b) a detector configured to detect the plurality of primer-template nucleic acid hybrids.

49. 49. The system of claim 48, further comprising: (c) a fluidic system comprising a reservoir containing a polymerase and nucleotides, said reservoir in fluid communication with said plurality of primer-template nucleic acid hybrids.

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