Increased signal-to-noise ratio in nucleic acid sequencing

Abstract

A method for identifying a nucleotide in a template nucleic acid by: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3' ends; (b) contacting the plurality of primer-template nucleic acid hybrids 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 a ternary complex inhibitor; (c) forming ternary complexes each comprising a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; and (d) detecting the ternary complexes, thereby identifying a nucleotide in the template nucleic acid.

Description

BACKGROUND

[0001] The present disclosure relates generally to the characterization of nucleic acids, and has particular applicability to nucleic acid sequencing.

[0002] Several commercial nucleic acid sequencing technologies use an ensemble of nucleic acids. The ensemble is typically produced by an amplification technique that produces a local collection of nucleic acid copies that are manipulated and detected as a whole. Exemplary techniques for creating an ensemble include the bridge amplification technique used by the Illumina platform (San Diego, CA), the emulsion polymerase chain reaction technique used by the Ion Torrent platform (Thermo Fisher, Waltham MA), and the rolling circle technique used by the Complete Genomics platform (BGI, Shenzhen China). Compared to single molecule sequencing technologies, ensembles provide the benefit of higher signal amplitude and minimize artifacts produced by random 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 the phenomenon of individual molecules within an ensemble falling out of synchronization with one another. Phasing can manifest as the extension of one or more primer molecules falling behind the extension of another primer molecule in the ensemble, or as the extension of one or more primer molecules falling ahead of the extension of another primer molecule in the ensemble. Phasing is detrimental and cumulative. A phasing rate of only 0.5% per cycle results in a cumulative loss of about half of the true signal after 120 cycles. The problem is exacerbated by a proportional increase in background noise caused by spurious signals produced by out-of-phase ensemble members. The cumulative loss of signal-to-noise ratio results in a limitation on read length (which in turn results in a decrease in sequencing throughput) and an increase in error (especially for later cycles) because the noise overwhelms the signal.

[0004] Accordingly, there is a need for methods that reduce or prevent phasing in ensemble-based sequencing. The present invention satisfies this need and provides other benefits as well. SUMMARY

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

[0006] Also provided are methods for identifying nucleotides in a template nucleic acid comprising the steps of: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3' ends; (b) incorporating blocked nucleotides at the 3' end of one or more primers of a first subset of primer-template nucleic acid hybrids; (c) incorporating ternary complex inhibitors at the 3' end of one or more primers of a second subset of primer-template nucleic acid hybrids; (d) forming ternary complexes each comprising a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; and (e) detecting the ternary complexes, thereby identifying nucleotides in the template nucleic acid. Optionally, the blocked nucleotides can be reversible terminated nucleotides.

[0007] The present disclosure provides methods for sequencing a template nucleic acid. The methods can comprise the steps of: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3' ends; (b) contacting the plurality of primers with: (i) reversible terminated nucleotides to produce a first subset of primer-template nucleic acid hybrids comprising reversible terminated nucleotides at the 3' end, and (ii) ternary complex inhibitors to produce a second subset of primer-template nucleic acid hybrids comprising ternary complex inhibitors; (c) forming ternary complexes each comprising a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; (d) detecting the ternary complexes, thereby identifying nucleotides in the template nucleic acid; (e) deblocking the reversible terminated nucleotides at the 3' end of the primer-template nucleic acid hybrids in the first subset; and (f) repeating steps (b) through (e) to sequence the template nucleic acid in the first subset.

[0008] Methods for sequencing a template nucleic acid can include the steps of: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3' ends; (b) incorporating a blocked nucleotide at the 3' end of one or more primers of a first subset of primer-template nucleic acid hybrids; (c) incorporating a ternary complex inhibitor at the 3' end of one or more primers of a second subset of primer-template nucleic acid hybrids; (d) forming ternary complexes each comprising a polymerase, a primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; (e) detecting the ternary complexes, thereby identifying a nucleotide in the template nucleic acid; (f) unblocking the reversibly terminated nucleotide at the 3' end of the primer-template nucleic acid hybrid in the first subset; and (g) repeating steps (b) through (f) to sequence the template nucleic acid in the first subset.

[0009] The present disclosure also provides a device comprising a plurality of primer-template nucleic acid hybrids, wherein a first subset of primer-template nucleic acid hybrids has a blocked nucleotide at the 3' end of the primer, and wherein a second subset of primer-template nucleic acid hybrids has a ternary complex inhibitor at the 3' end of the primer. Optionally, the blocked nucleotide can be a reversibly terminated nucleotide. DETAILED DESCRIPTION

[0010] The present disclosure provides methods for identifying the nucleotide base present at an interrogation position in a primer-template nucleic acid hybrid. The position interrogated is the base immediately 5' of the base in the template that is hybridized to the 3' end of the primer. The nucleotide present at the interrogation position can be identified in an inspection step for ternary complexes formed between a primer-template nucleic acid hybrid, a polymerase, and a nucleotide cognate of the base at the interrogation position. The function of the polymerase is to pair the cognate nucleotide with the next base of the template. By distinguishing the type of nucleotide present in the ternary complex, and inferring the template base with which it is hybridized according to Watson Crick base pairing, the identity of the base at the interrogation position can be determined.

[0011] In particular embodiments, the primers can be extended incrementally, so as to move the interrogation position along the template. For example, the sequence of a template can be determined by a series of cycles in which the primers are extended by a single nucleotide to move to the next position of the template to be interrogated and an inspection is performed at the new interrogation position. Incomplete extension can lead to sequencing errors or premature termination of the sequencing process. Phasing problems can also arise when a population of primer-template nucleic acid hybrids is sequenced as an ensemble.

[0012] This disclosure provides methods and compositions that can be used, for example, to improve the identification of bases in nucleic acids in sequencing methods. Improvements can be achieved by including primer modification methods that will not cap primers subsequently detected. For example, unextended primers in a primer extension step can be capped so that the unextended primers do not cause errors in subsequent inspection steps. For embodiments where template nucleotides are identified by inspecting ternary complexes, capping is provided by ternary complex inhibitors. A ternary complex inhibitor can be a portion that, when linked to a primer, prevents polymerase and / or homologous nucleotides from participating in the formation or maintenance of a ternary complex at the modified primer terminus. The ternary complex inhibitor can be present at the terminus of the primer, for example, as a portion that creates steric hindrance to one or more components (otherwise, these components would form a ternary complex), or as a portion that generates a charge repelling one or more components.

[0013] For example, complete primer extension can lead to inhibition of ternary complex formation when no template site is available for its formation. Complete extension can manifest as the 3' end of the extending primer annealing 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, complete extension can manifest as the primer being extended until further extension is prevented by factors in the extension product environment. In this configuration, the template may contain an unpaired next template base, but the polymerase cannot access the next template base (and / or access the next correct nucleotide) due to the environment surrounding the template and the extending primer. For example, the 5' end of the template can be attached to a solid surface such that the 3' end of the extending primer is so close to the surface that not only is further extension by the polymerase prevented, but also its binding to form the ternary complex. In both examples, complete extension produces an oligonucleotide moiety that functions as a cap on the extending primer.

[0014] In some configurations, the ternary complex inhibitor portion 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). Inhibition occurs when the second binding partner binds to the first binding partner, regardless of whether the first binding partner inhibits ternary complex formation. The ternary complex inhibitor can be located at the 3' end of a primer, for example, as a result of extending the 3' end of a primer with a nucleotide analog linked to the first binding partner. In this configuration, the complex between the first and second binding partners acts as a cap on the extended primer.

[0015] In alternative embodiments, primer modification methods can be used to remove primers so that they do not participate in subsequent detection steps. For example, primers that are not extended in the primer extension step can be chemically or enzymatically degraded (e.g., by exonucleases) so that the non-extended primers do not form triplexes that would lead to false positives in subsequent inspection steps.

[0016] Unless otherwise indicated, the terms used herein are to be understood in the ordinary sense of the associated terminology. Several terms used herein are defined below.

[0017] As used herein, the term "array" refers to a population of molecules attached to one or more solid supports such that molecules at one feature can be distinguished from molecules at other features. An array can include different molecules, each located at a different addressable feature on a solid support. Alternatively, an array can include separate solid supports, each serving the function of bearing a different characteristic of a molecule, where the different molecules can be identified according to the location of the solid support on a surface to which the solid support is attached, or according to the location of the solid support in a liquid such as a fluid stream. The molecules of an 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 in reference to a nucleotide, means a moiety of a nucleotide that inhibits or prevents the 3' oxygen of the nucleotide from forming a covalent linkage with the next correct nucleotide during the course of a nucleic acid polymerization reaction. The blocking moiety of a "reversible terminator" nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3'-oxygen of the nucleotide to covalently link with the next correct nucleotide. Such blocking moieties are referred to herein as "reversible terminator moieties." Exemplary reversible terminator moieties are set forth in U.S. Patent Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794; or 8,034,923; or PCT publications WO 91 / 06678 or WO 07 / 123744, each of which is incorporated herein by reference. Nucleotides having a blocking moiety or a reversible terminator moiety can be located at the 3' terminus of a nucleic acid, such as a primer, or can be monomers that are not covalently attached to a nucleic acid. The blocking moiety need not hinder or exclude the formation of a triplex at the 3' terminus of a nucleic acid to which the blocking moiety is attached. Particularly useful blocking moieties will be present at the 3' terminus of a nucleic acid that participates in the formation of a triplex.

[0019] As used herein, the term "catalytic metal ion" refers to a metal ion that facilitates the formation of a phosphodiester bond 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 that has a valence of two. A catalytic metal ion can be present at a concentration that stabilizes the complex formation between a polymerase, a nucleotide, and a primed template nucleic acid (as long as no phosphodiester bond formation is occurring, which is referred to as a non-catalytic concentration of the metal ion). A catalytic concentration of a metal ion refers to an amount of a metal ion sufficient to catalyze the reaction between the 3'-oxygen of a nucleic acid (e.g., a primer) and the phosphate group of an incoming nucleotide by a polymerase.

[0020] As used herein, the term "common sequence" means a nucleotide sequence that is identical for two or more nucleic acid molecules. The common sequence of two or more nucleic acids can include all or a portion of the nucleic acids being compared. The common sequence can be at least 5, 10, 25, 50, 100, 250, 500, 1000, or more nucleotides in length. Alternatively or additionally, the length can be at most 1000, 500, 250, 100, 50, 25, 10, or 5 nucleotides.

[0021] The term "comprising" is used herein to mean including whatever follows the term the use of the term "comprising" means "including at least" so that the list of elements following the term is an inclusive list, for example a composition "comprising" elements A, B, and C is a composition that includes at least A, B, and C, and can also include one or more other

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

[0023] As used herein, the term "ensemble detection" means detecting a characteristic of a population of molecules under conditions that do not necessitate distinguishing one molecule in the population from other molecules in the population. For example, a population of nucleic acids detected as an ensemble at a feature of an array can produce an apparent characteristic that is a combination of the characteristics of the nucleic acids at the feature. The characteristic can be a luminescent signal that is an average of the luminescence from a plurality of luminescers at the feature of the array.

[0024] As used herein, the term "each", when used in reference to a set of items, is intended to mean every individual item in the set, but not necessarily every item in the set. Exceptions to this can occur if explicitly disclosed or the context clearly indicates otherwise.

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

[0026] As used herein, the term "extension," when used in reference to a nucleic acid, means 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 the polymerase-catalyzed process of adding at least one nucleotide to the 3' end of a nucleic acid. A nucleotide or oligonucleotide that is added to a nucleic acid by extension is considered to be incorporated into the nucleic acid. Thus, the term "incorporation" 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 and is capable of forming a covalent bond with the next correct nucleotide if and when incorporated into a nucleic acid. An extendable nucleotide can be at the 3' position of a primer, or it can 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, means a location in the array in which a particular molecule is present. A feature can contain only a single molecule, or it can contain a population of several molecules of the same species (i.e., an ensemble of molecules). Alternatively, a feature can include a population of different species of molecules (e.g., a population of ternary complexes having different template sequences). Features of an array are typically discrete. Discrete features can be contiguous, or they can have spacing between one another. Arrays useful herein can have features, for example, that are spaced 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 spaced more than 0.5 microns, 1 micron, 5 microns, 10 microns, 50 microns, or 100 microns apart. The area of each feature can be less than 1 mm2, 500 microns2, 100 microns2, 25 microns2, 1 micron2, or less.

[0029] As used herein, the term "label" refers to a molecule or portion thereof that provides a detectable characteristic. The detectable characteristic can be, for example, an optical signal, such as absorbance of radiation, fluorescence emission, luminescence emission, fluorescence lifetime, fluorescence polarization, etc.; Rayleigh and / or Mie scattering; binding affinity for a ligand or receptor; magnetism; 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, a "fabricated container" is a man-made or artificially modified container that functions to isolate 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 fabricated containers used in conjunction with the disclosed technology include: a flow cell, a well of a multi-well plate; a microscope slide; a tube (e.g., a capillary tube); etc. The features to be interrogated or detected can be contained within the reaction container.

[0031] As used herein, the term "next correct nucleotide" refers to the type of nucleotide that will bind and / or be incorporated at the 3' end of a primer to complement the base in the template strand that is hybridized to the primer. The base in the template strand is referred to as the "next base," and is immediately 5' of the base in the template that is hybridized to the 3' end of the primer. The next correct nucleotide can be referred to as the "cognate" of the next base, and vice versa. Cognate nucleotides that interact with each other in a ternary complex or double-stranded nucleic acid are said to be "paired" to each other. A nucleotide having a base that is not complementary to the next template base is referred to as an "incorrect," "mismatched," or "non-cognate" nucleotide.

[0032] As used herein, the term "non-catalytic metal ion" refers to a metal ion that, when present with a polymerase, does not promote the phosphodiester bond formation required for chemical incorporation of a nucleotide into a primer. A non-catalytic metal ion can interact with a polymerase, for example, by competing for binding compared to a catalytic metal ion. Thus, a non-catalytic metal ion can act as an inhibitory metal ion. A "divalent non-catalytic metal ion" is a non-catalytic metal ion having a valence of 2. Examples of divalent non-catalytic metal ions include, but are not limited to, Ca 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Sr 2+ . Trivalent Eu 3+ , and Tb 3+ ions are non-catalytic metal ions having a valence of 3.

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

[0034] As used herein, the term "oligonucleotide moiety" refers to a nucleic acid moiety comprising at least 2 contiguous nucleotides. An oligonucleotide moiety can comprise, for example, at least 5, 8, 10, 15, 20, 25, 50, 100, or more contiguous nucleotides. Alternatively or additionally, an oligonucleotide moiety can comprise at most 100, 50, 25, 20, 15, 10, 8, 5, or 2 contiguous nucleotides. In some embodiments, the length of an oligonucleotide moiety added to a primer-template hybrid can be equal 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 be used to refer to a nucleic acid synthesis enzyme, including but not limited to a DNA polymerase, an RNA polymerase, a reverse transcriptase, a primase, and a transferase. Typically, a polymerase has one or more active sites on which nucleotide binding and / or catalysis of nucleotide polymerization can occur. A polymerase can catalyze the polymerization of a nucleotide to the 3' end of a first strand of a double-stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of the next correct nucleotide to the 3' oxy group of the first strand of a double-stranded nucleic acid molecule through a phosphodiester linkage, thereby covalently incorporating the nucleotide into the first strand of the double-stranded nucleic acid molecule. Optionally, a polymerase need not be able to incorporate nucleotides under one or more conditions used in the methods set forth herein. For example, a mutant polymerase can be able to form a ternary complex, but not 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 such that one strand is a primer and the other strand is a template. The two strands can be part of a contiguous nucleic acid molecule (e.g., a hairpin structure), or the two 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, the primer binds in a configuration that permits replication of the template (e.g., by 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, the first portion being the primer sequence and the second portion being the 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 analogs thereof. A primer can have an extendable 3' end, a 3' end blocked from primer extension, or a 3' end capped to hinder or exclude ternary complex formation.

[0038] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate can be non-porous or porous. The substrate can optionally be capable of absorbing liquid (e.g., due to porosity), but generally has sufficient rigidity such that the substrate does not substantially expand when liquid is absorbed and does not substantially contract when 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 acrylic resins, polystyrene, and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM , cyclic olefins, polyimides, etc.), nylon, ceramic, resin, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metal, inorganic glass, optical fiber bundles, and polymers.

[0039] As used herein, the term "subgroup" denotes a collection of one or more things, all of which are contained in a larger collection of things. The larger collection of things can be referred to as a "group." A subgroup can contain at least 1, 2, 10, 100, 1 x 10 3 , 1 x 10 6 , 1 x 10 9 thing. The things can be, for example, nucleic acids, such as primer-template nucleic acid hybrids, ternary complexes, polymerases, nucleotides, or other compositions set forth herein.

[0040] As used herein, the term "ternary complex" refers to an intermolecular association between a polymerase, a double-stranded nucleic acid, and a nucleotide. Generally, the polymerase facilitates an interaction between the next correct nucleotide and the template strand of the primed nucleic acid. The next correct nucleotide can interact with the template strand through Watson-Crick hydrogen bonding. The term "stable ternary complex" refers to a ternary complex that is facilitated or prolonged in existence or has been inhibited from destruction. Generally, stabilization of a ternary complex prevents covalent incorporation of the 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 impedes or excludes the formation of a ternary complex of a nucleic acid with a polymerase and / or a nucleotide when present in the nucleic acid. Moieties that create a steric block to ternary complex formation are particularly useful, including, for example, a polymerization or ligation product that extends a primer to the end of the template to which the primer is hybridized. Another example of a steric block is a mismatched nucleotide. Moieties can also serve to introduce a positive or negative charge that impedes or prevents the formation of a ternary complex. Moieties can be ligands that bind to receptors that impede or prevent the formation of a ternary complex, such as biotin (or analogs thereof) that bind to streptavidin (or analogs thereof), epitopes that bind to antibodies (or functional fragments thereof), carbohydrates that bind to lectins, and the like. Thus, a ternary complex inhibitor can be a ligand (or other) moiety that is capable of binding to a receptor (or other molecule) to form a ligand-receptor complex that inhibits the formation of a ternary complex. Other examples of moieties that can be used as ternary complex inhibitors include the base modifications and nucleotide analogs 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 have the same chemical structure. For example, a mixture of nucleotides can include several dCTP molecules. The dCTP molecules would be understood to be the same type as each other, but a different type than dATP, dGTP, dTTP, and the like. Likewise, individual DNA molecules that are identical in nucleotide sequence are the same type, whereas DNA molecules that differ in sequence are different types. The term "type" can also identify moieties that have the same chemical structure. For example, cytosine bases in a template nucleic acid would be understood to be the same type of base as each other, independent of their position in the template sequence.

[0043] In accordance with the above definitions, the embodiments set forth below and exemplified in the claims can be understood.

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

[0045] The methods of the present disclosure can include a primer modification method whereby a nucleotide or other moiety is added to the primer component of the primer-template nucleic acid hybrid. The primer modification method can be used to prepare the primer-template nucleic acid hybrid for the inspection process whereby the next base of the template nucleic acid is detected. Optionally, the inspection is performed to detect a ternary complex formed between the next base of the primer-template nucleic acid hybrid, the 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 inspection process are set forth below.

[0046] Returning to the primer modification method, the modification that results in the extension of the primer by one or more nucleotides can be used to change the position of the template nucleic acid that will be inspected at the next step. For example, the extension can be used to add one nucleotide to the primer, thereby moving the position at which the ternary complex will be formed by one template position. The template nucleic acid can be sequenced using repeated cycles that inspect for ternary complex formation at the end of the primer (i.e., at the next template base), and then extend the primer to move the inspection position.

[0047] Failure to extend the primer in the primer modification method can result in a loss of accuracy, for example, due to confusion about which position of the template is actually being inspected at any given cycle, or a reduction in read length when extension cannot proceed, for example. When detecting an ensemble of primer-template nucleic acid hybrids, even relatively small extension inefficiencies can result in phasing problems that will adversely affect read accuracy and read length.

[0048] The primer modification method used in the methods set forth herein can provide a means to mitigate artifacts that would otherwise arise from extension failure and inefficiency. In particular embodiments, the primer modification method can include at least two steps: (i) contacting the primer-template nucleic acid hybrid and polymerase with a reversibly terminated nucleotide to produce a first subset of primer-template nucleic acid hybrids comprising a reversibly terminated nucleotide at the 3' end, and (ii) contacting the primer-template nucleic acid hybrid and polymerase with a ternary complex inhibitor to produce a second subset of primer-template nucleic acid hybrids comprising a ternary complex inhibitor. Step (i) can provide for extension of the primers, such that the first subset of primer-template nucleic acid hybrids can continue to participate in the benefits of providing sequence information. For example, the reversibly terminated nucleotide used in step (i) can be selected to accommodate subsequent formation and inspection of the ternary complex of reversibly terminated primers. Step (ii) can provide the benefit of capping the primers to prevent their participation in the formation of ternary complexes in subsequent inspection steps. Such capping can reduce phasing, improve accuracy of base calling, and extend read lengths of sequencing. Typically, step (i) will be performed prior to step (ii). However, the steps can be performed simultaneously, or can be performed in the reverse order. When performed consecutively, one of the steps can immediately follow the other, one of the steps can begin prior to the completion of the previous step, or another step can intervene between the two steps.

[0049] When extending with reversibly terminated nucleotides, particularly useful capping chemistries are selective for the 3' end of non-extended primers compared to the 3' end of extended primers. For example, the capping chemistry can be reactive toward the 3' end of non-blocked primers, while being inert toward modified blocked primers. In this configuration, capping can be used to clean up the extension step. Chemistry using enzymes specific for the natural 3' end of primers is particularly useful, including, for example, ligases and polymerases. A capping procedure that is more efficient at modifying the natural 3' primer end compared to blocked primers is beneficial for many applications.

[0050] In particular embodiments, primers are capped using a ternary complex inhibitor. Any of a variety of moieties can be added to the primer to hinder or prevent the subsequent formation of a ternary complex at the 3' end of the primer. For example, the primer can be modified to have an oligonucleotide moiety attached to it. The oligonucleotide moiety can be at least as long as the region of the template that was single-stranded prior to modification of the primer. 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 a double-stranded region adjacent to the template region. In this example, the 5' end of the double-stranded region forms a boundary of the template region. Other boundaries can be present, such as bound proteins, chemical modifications, points of attachment to a solid support, etc. The length of the oligonucleotide moiety can prevent the ternary complex from binding to the template between the primer and the boundary. When the entire template region is double-stranded, ternary complex inhibition can result, removing the single-stranded location where the ternary complex can form.

[0051] Optionally, the primer can be attached to the oligonucleotide moiety as a result of a ligation of the 5' end of the oligonucleotide to the 3' end of the primer catalyzed by a ligase or as a result of an extension of the primer with a series of nucleotides catalyzed by a polymerase. Ligases, polymerases, and other enzymes that modify primers can be useful. However, chemical techniques can also be used to modify primers in the methods set forth herein. The oligonucleotide moiety attached to the primer can consist of completely natural nucleotides that form natural Watson-Crick base pairs with the template. Alternatively, the oligonucleotide can include one or more non-natural nucleotide analogs. These analogs can be selected for their ability to form base pairs with the template, but analogs that do not pair with the template can also be used. Similarly, natural nucleotides can be present in the oligonucleotide moiety in positions that form mismatches that disrupt base pairing between the oligonucleotide and the template. One particularly useful position for a mismatch or non-natural nucleotide analog is at the 3' end of the oligonucleotide moiety, where it can act to hinder or exclude the subsequent formation of a ternary complex.

[0052] Another example of a moiety that can be added to a primer to hinder or prevent the 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 a primer to prevent the formation of a ternary complex. Particularly useful mismatches and polymerases whose ability to recognize mismatches is affected are set forth in Kwok et al., Nucleic Acids Res. 18(4):999-1005 (1990), which is incorporated by reference. The mismatched nucleotide can be present at the 3' end of the primer, having been introduced by an oligonucleotide portion 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 effect inhibition of ternary complex formation. Whether or not the nucleotide added to the 3' end of the primer matches the template, the nucleotide can comprise an exogenous moiety that functions as a ternary complex inhibitor. The exogenous moiety can have a steric blocking effect, thereby blocking the polymerase, the nucleotide, or both from forming a ternary complex. The moiety can have other effects on the formation of a ternary complex, including but not limited to charge repulsion of the polymerase or cognate nucleotide, perturbation of the primer-template nucleic acid hybrid structure, repulsion of the polymerase or cognate nucleotide by polarity, and the like.

[0053] Particularly useful moieties include, but are not limited to, biotin or other ligands that can hinder or prevent the formation of a ternary complex either because they are present at the 3' end or because of their interaction with streptavidin or other receptors, which in turn prevents the formation of a ternary complex. Other ligand-receptor pairs include, but are not limited to, an antibody (or a functional fragment thereof, such as a Fab or ScFv) and an epitope; or a carbohydrate and a lectin; or binding partners set forth herein in the context of a second label. An advantageous aspect of using a ligand-receptor as a primer cap is that the ligand does not need to inhibit the formation of a ternary complex before binding to the receptor. For example, a nucleotide attached to a ligand can bind to the polymerase and primer-template nucleic acid, forming a ternary complex, the nucleotide can be incorporated into the primer, leaving the ligand at the 3' end of the primer. The receptor can then bind to the ligand at the 3' end of the primer to inhibit the formation of a ternary complex at the 3' end of the primer.

[0054] The primer can be capped with a moiety that inhibits detection of the ternary complex, even in the presence of the ternary complex. For example, when a luminescent label is used to detect ternary complex formation, the cap can be a quencher that reduces or prevents signal from the luminescent label. By way of a more specific example, the primer can be capped with a quencher moiety, and a ternary complex can form between the capped primer, the polymerase, and the luminescently labeled nucleotide. The luminescently labeled nucleotide can be prevented from producing a luminescent signal due to the proximity of the quencher and the luminescent label. Another moiety that can be used to inhibit detection of the ternary complex without necessarily inhibiting formation of the ternary complex is a member of a FRET pair that can be used to label the ternary complex with the other member of the FRET pair. The signal shift caused by the FRET phenomenon can effectively reduce the expected signal, resulting in a significant inhibition of the signal that would otherwise be produced by the uncapped primer. In the case of FRET, the shifted signal can be detected to determine the presence of the capped primer. In some cases, the shifted FRET signal can be quantified to quantify the capped primer in the sample being tested. Other moieties that quench or modify the signal from a particular label can be used as capping moieties when that particular label is to be used to detect the ternary complex.

[0055] In some embodiments, the primer modification method can be used to crosslink the polymerase to the 3' end of the non-extended primer. The conditions can be used to selectively retain the polymerase at the 3' end of a primer having a native 3' hydroxyl moiety, while removing the polymerase from a primer having a reversible terminator at the 3' end. The retained polymerase can be crosslinked to the primed-template nucleic acid hybrid and inhibit its subsequent binding to nucleotides to form a ternary complex. The inhibition can be the result of denaturing the retained polymerase, crosslinking at the nucleotide binding site of the polymerase, or other chemical or photochemical modifications known in the art to inactivate the polymerase.

[0056] The plurality of primer-template nucleic acid hybrids prepared or used according to the teachings herein can include a first subset of species of primers with a reversible termination and a second subset of species of primers with a cap such as a triplex inhibitor moiety. 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, for example, present at a particular feature of a nucleic acid array.

[0057] In sequencing embodiments, the relative number of primer-template nucleic acid hybrids in the first and second subsets can change as sequencing proceeds. In such cases, sequencing can be allowed to continue until one or both subsets reaches a particular threshold population size. The threshold can be the same as those set forth above, or the threshold can be different to accommodate the particular use of the method. Alternatively, sequencing can continue for a predetermined number of cycles, or until a threshold of a different characteristic is reached, such as sufficient sequence information is acquired for identification, or until the signal-to-noise ratio falls to a particular level.

[0058] The primer-template nucleic acid hybrids of the first and second subsets can differ in other ways. Typically, the primers of the first subset (e.g., primers extended by addition of a reversibly terminated nucleotide) will have the same length as one another. This is generally advantageous to provide a uniform phase when detecting the next base of a template nucleic acid in the ensemble. However, the primers of the second subset (e.g., primers with a triplex inhibitor) can differ in length from one another. This population can arise when the species in 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 can be longer than the primers of the first subset.

[0059] In particular embodiments, primer modification methods can reduce artifacts caused by failed and inefficient extension by removing or degrading non-extended primers. For example, primer modification methods can include at least two steps: (i) contacting primer-template nucleic acid hybrids and polymerase with reversibly terminated nucleotides to produce a first subset of primer-template nucleic acid hybrids comprising reversibly terminated nucleotides at the 3' end, and (ii) contacting primer-template nucleic acid hybrids with a reagent that removes or degrades primers, thereby producing a second subset comprising template nucleic acids that lack functional primers. Step (i) can provide extended primers, such that the first subset of primer-template nucleic acid hybrids can continue to participate in the benefits of providing sequence information. Step (ii) can provide the benefit of preventing non-extended primers from participating in the formation of ternary complexes in subsequent inspection steps. The absence of non-extended primers can reduce phasing, improve the accuracy of base calling, and extend read lengths. Typically, step (i) will be performed prior to step (ii). However, the steps can be performed simultaneously, or in the reverse order. When performed consecutively, one of the steps can immediately follow the other, one of the steps can begin before the previous step ends, or another step can intervene between the two steps.

[0060] In particular embodiments, the reagent used to degrade non-extended primers is selective for primers that lack 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 reagent that is active at degrading primers that lack a blocking moiety at the 3' end (e.g., primers that have not been extended by the addition of a blocked nucleotide), but is inhibited on other primers (e.g., primers that have been extended to incorporate a blocked nucleotide at the 3' end) due to the presence of the blocking moiety. A 3' to 5' exonuclease can be particularly useful, examples of which include but are not limited to a polymerase with 3' exonuclease activity, Exo III, or Exo VII. Optionally, the 3' end of the template component of a primer-template nucleic acid hybrid can be blocked, e.g., by attachment to a solid support or by the presence of a blocking moiety, to prevent unwanted template degradation. Alternatively, the 5' end of the primer and / or template can be blocked to prevent unwanted degradation.

[0061] The plurality of template nucleic acids can include species that are hybridized to the reversibly terminated primers of the first subset and species that do not have a primer (e.g., because the primer has been removed or degraded) of the second subset. The number of primer-template nucleic acid hybridizers 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 hybridizers 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, the plurality of template nucleic acids can be attached to a solid support, e.g., present at a particular feature of a nucleic acid array.

[0062] The primer modification methods used in the methods set forth herein do not require the use of a labeled polymerase. For example, the polymerase used in the extension step or used in the capping step does not need to be attached (e.g., covalently or otherwise) to an exogenous label. Alternatively, the polymerase used for primer extension or capping can comprise an exogenous label, e.g., the label used in the previous inspection step.

[0063] Typically, the reversibly terminated nucleotides added to the primers in the methods set forth herein do not have an exogenous label. This is because there is no need to detect the extended primers in the methods set forth herein. However, if desired, one or more types of reversibly terminated nucleotides used in the methods set forth herein can be detected, e.g., by an exogenous label attached to the nucleotide. Exemplary reversible terminator moieties, methods of incorporating them into primers, and methods of modifying the primers for further extension (often referred to as “deblocking”) are set forth in U.S. Patent Nos. 7,544,794; 7,956,171; 8,034,923; 8,071,755; 8,808,989; or 9,399,798. Additional examples are set forth 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, the ternary complex inhibitor or other primer cap added to the methods set forth herein need not have an exogenous label. This is because the primer-template nucleic acid hybrid comprising the cap, such as the ternary complex inhibitor, need not be detected in the methods set forth herein. However, if desired, one or more types of ternary complex inhibitor or other cap used in the methods set forth herein can be detected, for example, by an exogenous label attached to the cap or ternary complex inhibitor.

[0065] The primer capping process can be performed concurrently with the primer extension process, or the two processes can be separated by other processes set forth herein. For example, primer capping can be performed immediately after primer extension. In another example, an inspection can be performed between the primer extension process and the primer capping process. In some embodiments, primer capping can be performed concurrently with the inspection (e.g., primer capping reagents can be delivered to the reaction vessel along with reagents used to form the ternary complex).

[0066] The methods of the present disclosure can include an inspection step in which a ternary complex is formed and detected. Embodiments of the methods take advantage of the specificity with which a polymerase can utilize to form a stable ternary complex with a primer-template nucleic acid and a next correct nucleotide. The next correct nucleotide can non-covalently bind to the stable ternary complex, interacting with the additional members of the complex only through non-covalent interactions. Useful methods and compositions for forming stable ternary complexes are set forth in more detail below and in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 Al or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 Al); U.S. Patent Application Publication No. 2018 / 0187245 Al (which claims priority to U.S. Patent Application Serial No. 62 / 440,624) or U.S. Patent Application Publication No. 2018 / 0208983 Al (which claims priority to U.S. Patent Application Serial No. 62 / 450,397), each of which is incorporated herein by reference.

[0067] Generally, the inspection is performed separately and discontinuously from the primer modification, for example, because reagent exchange or washing interferes with the inspection and modification. Alternatively, in some embodiments, the inspection and one or more primer modification steps can be performed in the same mixture. For example, the inspection and primer blocking can be performed in the same mixture. Alternatively or additionally, the inspection and primer blocking can be performed in the same mixture.

[0068] While in certain catalytic metal ions (e.g., Mg 2+In the absence of the polymerase, the primer-template nucleic acid hybrid, and the next correct nucleotide, a ternary complex can form between the polymerase, the primer-template nucleic acid hybrid, and the next correct nucleotide, but the chemical addition of the nucleotide is inhibited in the absence of the catalytic metal ion. Low or insufficient levels of the catalytic metal ion can result in non-covalent sequestration of the next correct nucleotide in the stable ternary complex. Other methods disclosed herein can also be used to generate the stable ternary complex.

[0069] Optionally, a stable ternary complex can form when the primer of the primer-template nucleic acid hybrid comprises a blocking moiety (e.g., a reversible terminator moiety) that prevents the enzymatic incorporation of an incoming nucleotide into the primer. The interaction can occur in the presence of a stabilizing agent, such that 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 an extendable primer, or a primer that is blocked from extension at its 3’ end (e.g., blocking can be achieved by the presence of a reversible terminator moiety at the 3’ end of the primer). The primer-template nucleic acid hybrid, the polymerase, and the cognate nucleotide can form a stable ternary complex when the base of the cognate nucleotide is complementary to the next base of the primer-template nucleic acid hybrid.

[0070] As described above, conditions that favor 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 the enzymatic incorporation of an incoming nucleotide into the primer, or by the absence of a catalytic metal ion. Other useful conditions include the presence of a ternary complex stabilizing agent such as a non-catalytic metal ion (e.g., a divalent or trivalent non-catalytic metal ion) that inhibits 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, the presence of one or more monovalent cations and / or glutamate anions provides conditions that are unfavorable for or destabilize a binary complex (i.e., a complex between the polymerase and the primed nucleic acid, but lacking the cognate nucleotide). As an additional alternative, a polymerase can be used that is engineered to prevent catalytic activity or to prevent a propensity for binary complex formation.

[0071] In particular embodiments, the ternary complex is stabilized by the presence of Li + betaine, or both. For example, the reagents and techniques set forth in U.S. Patent Application Serial No. 16 / 355,361 (which is incorporated by reference herein) can be used. Immiscible fluids can also be used to stabilize the ternary complex, including, for example, the immiscible fluids set forth in U.S. Patent Application Serial No. 16 / 164,417 (which is incorporated by reference herein).

[0072] The ternary complex stabilizing conditions can be further configured to enhance the difference in polymerase affinity for the primer-template nucleic acid hybrid in the presence of different nucleotides, for example, by disrupting the stability of the binary complex. Optionally, the conditions result in different polymerase affinities for the primer-template hybrid in the presence of different nucleotides. For example, the conditions include, but are not limited to, high salt and glutamate ions. For example, the salt can be dissolved in an aqueous solution to produce monovalent cations, such as monovalent metal cations (e.g., sodium or potassium ions). Optionally, the salt that provides 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, the concentration of potassium glutamate that can be used to alter the polymerase affinity for the primer-template hybrid extends from 10 mM to 1.6 M potassium glutamate, or any amount between 10 mM and 1.6 M. As described above, high salt refers to a salt concentration of 50 mM to 1.5 M salt.

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

[0074] The stabilized ternary complex can include natural nucleotides, nucleotide analogs, or modified nucleotides as desired to suit the particular application or configuration of the method. Optionally, the nucleotide analogs have a nitrogenous base, a five-carbon sugar, and a phosphate group, wherein any of the portions of the nucleotide can be modified, removed, and / or replaced as compared to a natural nucleotide. The nucleotide analogs can be non-incorporable nucleotides (i.e., nucleotides that cannot react with the 3’ oxygen of the primer to form a covalent linkage). Such non-incorporable nucleotides include, for example, monophosphate and diphosphate nucleotides. In another example, the nucleotide can include one or more modifications to the triphosphate group such that the nucleotide is non-incorporable. Examples of non-incorporable nucleotides can be found in U.S. Patent No. 7,482,120, which is incorporated by reference herein. In some embodiments, the non-incorporable nucleotides can be subsequently modified to be 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 cage nucleotides. Examples of nucleotide analogs are described in U.S. Patent No. 8,071,755, which is incorporated by reference herein.

[0075] Nucleotide analogs that participate in stable ternary complexes can include terminators that reversibly prevent subsequent nucleotide incorporation at the 3' end of the primer after the analog has been incorporated into the primer. For example, U.S. 7,544,794 and U.S. 8,034,923 (the disclosures of which are incorporated herein by reference) describe reversible terminators in which the 3'-OH group is replaced by a 3'-ONH2 moiety. Another type of reversible terminator is linked to the nitrogenous base of the nucleotide as set forth, for example, in U.S. 8,808,989 (the disclosure of which is incorporated herein by reference). Other reversible terminators that can be similarly used in connection with the methods described herein include those described in references cited elsewhere herein or in U.S. 7,956,171, U.S. 8,071,755, and U.S. 9,399,798 (the disclosures of which are incorporated herein by reference). In certain embodiments, the reverse terminator moiety can be modified or removed from the primer in a process called "deblocking" to allow for subsequent nucleotide incorporation. Compositions and methods for deblocking are set forth in the references cited herein in the context of reversible terminators.

[0076] Nucleotide analogs that participate in stable ternary complexes can include ternary complex inhibitors that prevent the subsequent formation of a ternary complex at the 3' end of the primer after the analog has been incorporated into the primer. Nucleotide analogs can include exogenous labels, but the nucleotide analogs used herein need not include a label.

[0077] Alternatively, nucleotide analogs irreversibly prevent nucleotide incorporation at the 3'-end of the primer into which they have been incorporated. Irreversible nucleotide analogs include 2',3'-dideoxynucleotides (ddNTPs, such as ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH group of dNTPs that otherwise participate in polymerase-mediated primer extension. Thus, the 3' position has a hydrogen moiety instead of the natural hydroxyl moiety. Irreversibly terminated nucleotides are particularly useful for genotyping applications or other applications where primer extension or sequential detection along a template nucleic acid is not desired.

[0078] In some embodiments, the nucleotides involved in forming the ternary complex can include an exogenous label. Optionally, the exogenously labeled nucleotides can include a reversible or irreversible terminator moiety, the exogenously labeled nucleotides can be non-incorporable, the exogenously labeled nucleotides can lack a blocking moiety, the exogenously labeled nucleotides can be incorporable, or the exogenously labeled nucleotides can be both incorporable and non-terminating. Exogenously labeled nucleotides can be particularly useful when used to form a stable ternary complex with a non-labeled polymerase. Alternatively, an exogenous label on the nucleotide can provide one partner in a fluorescence resonance energy transfer (FRET) pair, and an exogenous label on the polymerase can provide the second partner in the pair. Thus, FRET detection can be used to identify a stable ternary complex comprising both partners. Alternatively, the nucleotides involved in forming the ternary complex can lack an exogenous label (i.e., the nucleotides can be "non-labeled"). Optionally, the non-labeled nucleotides can include a reversible or irreversible terminator moiety, the non-labeled nucleotides can be non-incorporable, the non-labeled nucleotides can lack a terminator moiety, the non-labeled nucleotides can be incorporable, or the non-labeled nucleotides can be both incorporable and non-terminating. Non-labeled nucleotides can be useful when a label on the polymerase is used to detect a stable ternary complex. Non-labeled nucleotides can also be used in the extension step of the methods set forth herein. It will be understood that a reference to the absence of a moiety or function of a nucleotide means that the nucleotide does not have such a function or moiety. It will also be understood that a reference to a nucleotide or its analogs in the methods or compositions set forth herein can explicitly omit one or more functions or moieties set forth herein or otherwise known in the art with respect to a nucleotide or its analogs.

[0079] Optionally, during the formation of the stable ternary complex, nucleotides (e.g., natural nucleotides or synthetic nucleotide analogs) are present in the mixture. For example, at least 1, 2, 3, 4, or more nucleotide types can be present. Alternatively or additionally, at most 4, 3, 2, or 1 nucleotide types can be present. Similarly, the nucleotide type(s) present can be complementary to at least 1, 2, 3, or 4 base types in the template nucleic acid. Alternatively or additionally, the nucleotide type(s) present can be complementary to at most 4, 3, 2, or 1 base types in the template nucleic acid.

[0080] Any nucleotide modification that does not prevent participation in a ternary complex can be used in the methods disclosed herein. The nucleotides can be permanently or transiently bound to the polymerase. Optionally, the nucleotide analogs are fused to the polymerase, e.g., by a covalent linker. Optionally, a plurality of nucleotide analogs are fused to a plurality of polymerases, wherein each nucleotide analog is fused to a different polymerase. Optionally, the nucleotides present in the stable ternary complex are not the means by which the ternary complex is stabilized. Thus, any of a variety of other ternary complex stabilization methods can be combined in a reaction that utilizes nucleotide analogs.

[0081] In particular embodiments, the primer strand of the primer-template nucleic acid hybrid molecule present in the stable ternary complex is not chemically altered by the presence of the polymerase during one or more steps of the methods set forth herein. For example, the primer need not be extended by formation of new phosphodiester bonds, nor need it be shortened by exonucleolytic degradation during the step of forming the stable ternary complex and during the step of detecting the stable ternary complex. The primer strand of the primer-template hybrid molecule present in the stable ternary complex need not be chemically modified, e.g., the primer need not be modified to contain a ternary complex inhibitor moiety or other primer capping moiety.

[0082] A ternary complex prepared or used according to the present disclosure can optionally comprise one or more exogenous labels. Labels can be attached to components of the ternary complex (e.g., to the polymerase, template nucleic acid, primer, and / or cognate nucleotides) prior to formation of the ternary complex. Exemplary attachments include covalent or non-covalent attachments, such as those set forth herein, in references cited herein, or known in the art. In some embodiments, a labeled component is delivered in solution to a solid support to which an unlabeled component is attached, thereby recruiting the label to the solid support by formation of a stable ternary complex. Thus, the component attached to the support can be detected or identified based on observation of the recruited label. Whether used in solution phase or on a solid support, exogenous labels can be used to detect the stable ternary complex or individual components thereof during an interrogation step. Exogenous labels can remain attached to a component after the component dissociates from other components with which a stable ternary complex has been formed. Exemplary labels, methods for attaching labels, and methods for using labeled components are set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 Al or U.S. Patent Application Serial Nos. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 Al), 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 Al), 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 Al), 62 / 450,397, or 62 / 506,759, each of which is incorporated by reference herein.

[0083] Examples of useful exogenous labels include, but are not limited to, any of a radioactively labeled moiety, a luminophore moiety, a fluorophore moiety, a quantum dot moiety, a chromophore moiety, an enzyme moiety, an electromagnetic spin label moiety, a nanoparticle light scattering moiety, and various 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, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow TM , Cascade Blue TM , Texas Red TM , dansyl chloride, phycoerythrin, phycocyanin, fluorescent lanthanide complexes such as those including europium and terbium, Cy3, Cy5, Cy7, Alexa dyes, and other dyes known in the art, such as in Principles of Fluorescence SpectroscopyJoseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and Molecular Probes Handbook those described in the 6th edition of G. Gauglitz and K. Moeller's

[0084] A second label can be used in the methods of the disclosure. The second 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 by specific affinity to a labeled receptor. Detection of the secondary label is not required in the methods described herein. For example, the secondary label can be a moiety (e.g., a ligand) located at the 3' end of a primer and bound to a molecule (e.g., a receptor) such that the bound molecule inhibits formation of a ternary complex at the 3' end of the labeled primer. Exemplary binding moiety pairs that can be used include, but are not limited to, an antigen and an immunoglobulin or active fragment thereof, such as a FAb; an immunoglobulin and an immunoglobulin (or active fragment, respectively); an avidin and a biotin, or an analog thereof specific for avidin; a streptavidin and a biotin, or an analog thereof specific for streptavidin; or a carbohydrate and a lectin. One particularly useful class of epitopes that can be attached to a nucleotide or primer is a peptide for which an antibody (or functional fragment thereof) can be raised to create a ternary complex inhibitor.

[0085] In some embodiments, the second label can be a chemically modifiable moiety. In this embodiment, a label having a reactive functional group can be incorporated into a stable ternary complex. Subsequently, the functional group can be covalently reacted with a primary label moiety. Suitable functional groups include, but are not limited to, an amino group, a carboxyl group, a maleimide group, an oxo group, and a thiol group. Functional groups for click chemistry and related methods of their synthesis and use can also be useful. Useful click chemistry reagents and methods are set forth 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 alternative embodiments, the ternary complex can lack exogenous labels. For example, the ternary complex and all components involved in the ternary complex (e.g., polymerase, template nucleic acid, primer, and / or cognate nucleotides) can lack one, several, or all exogenous labels described herein or described in the references incorporated above. In such embodiments, the ternary complex can be detected based on intrinsic properties of the stable ternary complex, such as mass, charge, intrinsic optical properties, etc. Exemplary methods for detecting non-labeled ternary complexes are set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 Al, PCT Application Serial No. PCT / US16 / 68916 (published as WO 2017 / 117243), or U.S. Patent Application Serial Nos. 62 / 375,379 or 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 Al), each of which is incorporated herein by reference.

[0087] Generally, detection can be achieved in the interrogation step by methods that sense an intrinsic property of the ternary complex or a labeled moiety attached thereto. Exemplary properties upon which detection can be based include, but are not limited to, mass, electrical conductivity, energy absorption, luminescence, etc. Detection of luminescence can be performed using methods known in the art in connection with nucleic acid arrays. Luminescers 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 set forth herein include, for example, mass spectrometry that can be used to sense mass; surface plasmon resonance that can be used to sense binding at a surface; absorbance that can be used to sense the wavelength of energy absorbed by a label; calorimetry that can be used to sense a change in temperature due to the presence of a label; electrical conductivity or impedance that can be used to sense an electrical property of a label, or other known analytical techniques. Examples of reagents and conditions that can be used to generate, manipulate, and detect stable ternary complexes include, for example, set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 Al; PCT Application Serial No. PCT / US16 / 68916 (published as WO 2017 / 117243), or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 Al), U.S. Patent Application Serial No. 15 / 581,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 Al); U.S. Patent Application Serial No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 Al); U.S. Patent Application Serial No. 62 / 450,397, or U.S. Patent Application Serial No. 62 / 506,759), each of which is incorporated herein by reference.

[0088] Some embodiments of the methods set forth herein utilize two or more distinguishable signals to distinguish between stable ternary complexes and / or to distinguish between one base type and another base type in a template nucleic acid. For example, two or more luminescers can be distinguished from one another based on unique optical properties, such as unique excitation wavelengths or unique emission wavelengths. In particular embodiments, the methods can distinguish between different stable ternary complexes based on differences in luminescence intensity. For example, a first ternary complex can be detected under conditions in which it emits a lower intensity than a second ternary complex. Such intensity scaling (sometimes referred to as “gray scale scaling”) can utilize any distinguishable difference in intensity. Exemplary differences include a particular stable ternary complex having an intensity that is at most 10%, 25%, 33%, 50%, 66%, or 75% of the intensity of another stable ternary complex to be detected.

[0089] For example, the intensity difference can be produced using different light emitters, each having a different extinction coefficient (i.e., resulting in different excitation characteristics) and / or different light emission quantum yield (i.e., resulting in different emission characteristics). Alternatively, the same light emitter type can be used, but it can be present in different amounts. For example, all members of a first population of ternary complexes can be labeled with a particular light emitter, while only half of the members of a second population are labeled with the light emitter. In this example, the second population is expected to produce half the signal of the first population. The second population can be produced, for example, by using a mixture of labeled nucleotides and unlabeled nucleotides (as opposed to the first population, which primarily contains labeled nucleotides). Similarly, the second population can be produced, for example, by using a mixture of labeled polymerases and unlabeled polymerases (as opposed to the first population, which primarily contains labeled polymerases). In an alternative labeling scheme, a first population of ternary complexes can include polymerase molecules having a plurality of labels that produce a particular light emission signal, and a second population of ternary complexes can include polymerase molecules that each have only one of the labels that produce a light emission signal.

[0090] In some embodiments, the inspection step is performed in a manner to infer the identity of at least one nucleotide type, e.g., as set forth in commonly owned U.S. Patent No. 9,951,385 or U.S. Patent Application Serial No. 15 / 922,787 (issued as U.S. Patent No. 10,161,003) (each of which is incorporated herein by reference). As an alternative or supplement to the use of imputation, the inspection step can use disambiguation to identify one or more nucleotide types, e.g., as set forth in commonly owned U.S. Patent No. 9,951,385 or U.S. Patent Application Serial No. 15 / 922,787 (issued as 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 set forth herein, e.g., to form a stable ternary complex or to perform primer modification. Polymerases that can be used include naturally occurring polymerases and modified variants thereof, including but not limited to mutants, recombinants, fusions, genetically modified, chemically modified, synthetics, and analogs. Naturally occurring polymerases and modified variants thereof are not limited to polymerases having the ability to catalyze polymerization reactions. Optionally, naturally occurring and / or modified variants thereof have the ability to catalyze polymerization reactions under at least one condition that is not used during formation of a stable ternary complex or inspection. Optionally, naturally occurring and / or modified variants that participate in a stable ternary complex have improved properties, e.g., enhanced binding affinity for nucleic acids, reduced binding affinity for nucleic acids, enhanced binding affinity for nucleotides, reduced binding affinity for nucleotides, enhanced specificity for the next correct nucleotide, reduced specificity for the next correct nucleotide, reduced catalytic rate, catalytic inactivation, etc. Mutant polymerases include, e.g., polymerases in which one or more amino acids are replaced with other amino acids or an insertion or deletion of one or more amino acids. Exemplary polymerase mutants that can be used to form a stable ternary complex include, e.g., those set forth in U.S. Patent Application Serial No. 15 / 866,353 (published as U.S. Patent Application Publication No. 2018 / 0155698 Al) or U.S. Patent Application Publication No. 2017 / 0314072, each of which is incorporated herein by reference.

[0092] Modified polymerases include polymerases that contain 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 separation techniques. For example, the exogenous labeling moiety can be covalently linked to the polymerase using a free thiol or a free amine moiety of the polymerase. This can involve a covalent linkage to the polymerase through the side chain of a cysteine residue or through a free amino group at the N-terminus. The exogenous labeling moiety can also be attached to the polymerase through a protein fusion. Exemplary labeling moieties that can be attached through a protein fusion include, e.g., green fluorescent protein (GFP), phycobiliproteins (e.g., phycocyanin and phycoerythrin), or wavelength-shifted 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 that binds to the polymerase in a stable ternary complex. Thus, the stable ternary complex can be detected or identified by FRET.

[0093] Alternatively, the polymerase participating in the stable ternary complex or used to modify the primer need not be attached to an exogenous label. For example, the polymerase need not be covalently attached to an exogenous label. Rather, the polymerase can be free of any label until it binds to a labeled nucleotide and / or a labeled nucleic acid (e.g., a labeled primer and / or a labeled template).

[0094] Different activities of polymerases can be utilized in the methods set forth herein. Polymerases can be used, for example, in primer modification methods, such as primer extension steps or primer capping steps, inspection steps, or combinations thereof. Different activities can arise from differences in structure (e.g., by natural activity, mutation, or chemical modification). However, polymerases can be obtained from a variety of known sources and applied according to the teachings set forth herein and accepted polymerase activities. Useful DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA polymerases, and bacteriophage DNA polymerases. Bacterial DNA polymerases include E. coli DNA polymerases I, II, and III, IV, and 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 a, b, g, d, e, h, z, l, s, m, and k, as well as Revl 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 thermostable and / or thermophilic DNA polymerases, such as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase and TurboPfu DNA polymerase, Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp.GB-D) polymerases, Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase, Sulfolobus acidocaldarius DNA polymerase, Thermococcus sp. go N-7 DNA polymerase, Pyrodictium occultum DNA polymerase, Methanococcus voltae DNA polymerase, Methanococcus thermoautotrophicum DNA polymerase, Methanococcus jannaschii DNA polymerase, Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus furiosus 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 can also be used in conjunction with the disclosed technology. For example, a modified version of the extreme thermophilic marine archaeon Thermococcus sp. 9°N (e.g., Therminator DNA polymerase from New England BioLabs Inc.; Ipswich, MA) can be used. Other useful DNA polymerases, including 3PDX polymerase, are disclosed in U.S. 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 class of polymerases is reverse transcriptases. 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 that maintains telomeres of eukaryotic chromosomes.

[0097] For some embodiments, a polymerase with intrinsic 3'-5' proofreading exonuclease activity can be useful. Polymerases that are essentially lacking 3'-5' proofreading exonuclease activity are also useful in some embodiments, for example, in most genotyping and sequencing embodiments. The absence of exonuclease activity can be a wild-type property or can be a property conferred by a variant or engineered polymerase structure. For example, an exo minus Klenow fragment is a mutant form of the Klenow fragment that lacks 3'-5' proofreading exonuclease activity. Klenow fragments and their exo minus variants can be used in the methods or compositions set forth herein.

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

[0099] A particularly useful nucleic acid template is a genome fragment, each comprising a sequence identical to a portion of the genome. A population of genome fragments can cover all or part of a particular genome. For example, a population of genome fragments may include at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the genome's sequence. Genome fragments may have a sequence substantially identical to, for example, at least about 25, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more consecutive nucleotides of the genome. Optionally or additionally, genome fragments may have a sequence not exceeding 1 x 10^6 nucleotides of the genome. 5 1 x 10 4 1 x 10 3 A genome segment may consist of 1, 800, 600, 400, 200, 100, 75, 50, or 25 consecutive nucleotides that are substantially identical. The genome segment may be DNA, RNA, or an analogue thereof.

[0100] Exemplary organisms from which nucleic acids can be derived include, for example, those from 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, maize, sorghum, oats, wheat, rice, Brassica, or soybean; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, bees, or spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or Xenopus laevis; dictyostelium discoideum; fungi, such as Pneumocystis carinii, Takifugu rubripes, yeast, Saccharomyces cerevisiae, or Schizosaccharomyces pombe; or Plasmodium falciparum. Nucleic acids can also be derived from prokaryotes, such as bacteria: Escherichia coli, staphylococci, or Mycoplasma pneumoniae; archae; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Nucleic acids can be derived from a homogeneous culture or population of the above organisms, or alternatively from a collection of several different organisms, e.g., in 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, 3rded., 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] Templates nucleic acids can be obtained by preparation methods such as genomic isolation, genomic fragmentation, genetic cloning, and / or amplification. Templates can be obtained by amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), and the like. Exemplary methods for isolating, amplifying, and fragmenting nucleic acids to produce templates for array analysis are set forth in U.S. Patent Nos. 6,355,431 or 9,045,796 (each of which is incorporated herein by reference). Amplification can also be performed using methods set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., 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, for example, those set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 Al or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 Al), U.S. Patent Application Serial No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 Al), or U.S. Patent Application Publication No. 2018 / 0208983 Al (which claims priority to U.S. Patent Application Serial Nos. 62 / 450,397 and 62 / 506,759) (each of which is incorporated herein by reference). Other useful reagents and conditions for polymerase-based primer extension are set forth 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 Al (each of which is incorporated herein by reference).

[0103] In particular embodiments, reagents used in a primer modification step (e.g., capping of a primer by addition of a nucleotide extension primer or by addition of a ternary complex inhibitor moiety) are removed from contact with a primer-template hybrid prior to a step in which a stable ternary complex is formed with the primer-template hybrid. For example, removal of a nucleotide mixture used in an extension step can be desirable when one or more types of nucleotides in the mixture would interfere with formation or detection of ternary complexes in a subsequent inspection step. Similarly, it can be desirable to remove a polymerase or cofactor used in a primer modification step in order to prevent unwanted catalytic activity in a subsequent detection step. Removal can be followed by a washing step in which an inert fluid is used to clear the primer-template hybrid of residual components of the reagent mixture used for primer modification.

[0104] Reagent removal and washing procedures can be performed between any of the various steps set forth herein. Such procedures can be used to remove one or more reagents present in a reaction vessel or on a solid support. For example, a reagent removal or washing step can be used to separate an isolated primer-template hybrid from other reagents that were in contact with the primer-template hybrid under ternary complex stabilizing conditions. In particular embodiments, separation of a reagent is facilitated by attachment of the reagent of interest (such as a primer-template hybrid) to a solid support and removal of fluid from contact with the solid support. One or more reagents set forth herein can be attached to a solid support or provided in solution as needed to suit the particular use of a method or device described herein.

[0105] A reagent removal or washing procedure can be used to remove one or more reagents from interfering with inspection of a mixture or from contaminating a second mixture that will be formed on a substrate (or in a vessel) that was previously in contact with a first mixture. For example, a primer-template nucleic acid hybrid can be contacted with a polymerase and at least one type of nucleotide to form a first mixture under ternary complex stabilizing conditions, and the first mixture can be inspected. Optionally, a wash can be performed prior to detection in order to remove reagents that did not participate in formation of stable ternary complexes. Alternatively or additionally, a wash can be performed after a detection step in order to remove one or more components of the first mixture from the primer-template hybrid. Then, the primer-template hybrid can be contacted with a polymerase and at least one other type of nucleotide to form a second mixture under ternary complex stabilizing conditions, and the second mixture can be inspected for ternary complex formation. As before, an optional wash can be performed prior to a second inspection in order to remove reagents that did not participate in formation of stable ternary complexes.

[0106] In particular embodiments, the primer capping step is performed after primer extension. One or more reagents present in the primer extension step can be removed prior to the introduction of reagents for the primer capping step. For example, the primer-template nucleic acid hybrid attached to the solid support can be contacted with an extension mix comprising one or more polymerases and one or more nucleotides, and then the extension mix can be removed from the solid support, and then reagents for capping the primers can 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 helps to remove residual components of the extension mix prior to the delivery of the capping reagents.

[0107] In alternative embodiments, reagents for primer extension and primer capping can be contacted with each other simultaneously. For example, the primer-template nucleic acid hybrid attached to the solid support can be contacted with a primer modification mix comprising one or more polymerases, one or more nucleotides having a reversible terminator moiety (i.e., for extending the primers) and a ternary complex inhibitor moiety (i.e., for capping non-extended primers). The extension reagents and capping reagents need not be delivered to the solid support simultaneously. Rather, the reagents can be delivered in a serial fashion such that they are present simultaneously after a cumulative delivery is performed. The extension reagents and capping reagents can be removed prior to a subsequent step, such as an inspection or deblocking step. A wash can optionally be employed to remove residual extension reagents and capping reagents prior to the subsequent step.

[0108] If nucleotides present in the inspection step are carried over into a primer modification process, such as a primer extension process or a primer capping process, unwanted side reactions can result, such as nucleotide incorporation reactions. Thus, a reagent removal or wash step can be employed prior to the primer modification step. Optionally, free nucleotides or other inspection reagents can be modified or disabled, e.g., by enzymes such as phosphatases, by chemical modification, or by physical techniques.

[0109] Particular embodiments of the methods set forth herein include a step of forming a mixture comprising 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 the vessel in any desired order, or they can be delivered simultaneously. Further, some components can be mixed with each other to form a first mixture, which is then contacted with other components to form a more complex mixture. By way of example, the step of forming a mixture comprising a primer-template nucleic acid hybrid, a polymerase, and a plurality of different nucleotide types, it is understood that different nucleotide types of the plurality can be contacted with each other prior to contact with the primer-template nucleic acid hybrid. Alternatively, two or more nucleotide types can be delivered separately to the primer-template hybrid and / or the polymerase. Thus, a first nucleotide type can be contacted with the primer-template hybrid prior to contact with a second nucleotide type. Alternatively or additionally, a first nucleotide type can be contacted with the polymerase prior to contact with a second nucleotide type.

[0110] The stable ternary complex, or components capable of forming (i.e., participating in the formation of) the ternary complex, can be attached to a solid support. The solid support can be made of any of a variety of materials used in biochemical analysis. Suitable materials can include glass, polymeric materials, silicon, quartz (fused silica), borofloat glass, silica, silicon-based materials, carbon, metals, optical fibers or fiber bundles, sapphire, or plastic materials. The material can be selected according to the properties desired for the particular use. For example, materials transparent to the wavelength of radiation desired for the analysis technique are useful for techniques utilizing radiation at that wavelength. Conversely, it can be desirable to select a material that is opaque, absorbing, or reflective to radiation at a certain wavelength. Other properties of the materials that can be utilized are inertness or reactivity to certain reagents used in downstream processes, such as those set forth herein, or ease of handling, or low cost of manufacture.

[0111] Particularly useful solid supports are particles, such as beads or microspheres. Populations of beads can be used for attachment of populations of stable ternary complexes or components capable of forming complexes (e.g., polymerases, templates, primers, or nucleotides). In some embodiments, it can be useful to use configurations in which each bead has a single type of stable ternary complex or a single type of component capable of forming a complex. For example, a single bead can be attached 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 segregated bead by bead. As such, 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 format, chemistry, and / or attachment method to be used. Exemplary bead compositions include solid supports and the chemical functionalities imparted thereto for protein and nucleic acid capture methods. Such compositions include, for example, plastic, ceramic, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran, such as Sepharose TM , cellulose, nylon, cross-linked micelles, and Teflon TM , and other materials set forth in Bangs Laboratories, Fishers Ind’s “Microsphere Detection Guide,” which is incorporated herein by reference.

[0112] The geometry of the particles, such as beads or microspheres, can also correspond to a variety of different forms and shapes. For example, the particles can be in a symmetrical shape (e.g., spherical or cylindrical) or an irregular shape (e.g., controlled-porosity glass). Additionally, the particles can be porous, thus increasing the surface area available for capture of ternary complexes or components thereof. Exemplary sizes of beads used herein can range from nanometers to millimeters or from about 10 nm to 1 mm.

[0113] In particular embodiments, beads can be arranged, or otherwise spatially distinguished. Exemplary bead-based arrays that can be used include, but are not limited to, BeadChip TMArrays, or arrays such as those described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, or 7,622,294, or PCT Publication No. WO 00 / 63437 (each of which is incorporated herein by reference). The beads can be located at discrete locations on the solid support, such as pores, whereby each location accommodates a single bead. Alternatively, the discrete locations where the beads are located can each comprise multiple beads, as described, for example, in U.S. Patent Application Publications 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 above bead array embodiments, the methods of this disclosure can be performed in multiple forms, thereby enabling the parallel detection of multiple different types of nucleic acids in the methods described herein. Although one or more steps of the methods described herein can also be used to process different types of nucleic acids sequentially, parallel processing can save costs, save time, and provide consistency of conditions. The apparatus or methods of this disclosure may include at least 2, 10, 100, or 1x10^6 nucleic acids. 3 Seed, 1x10 4 Seed, 1x10 5 Seed, 1x10 6 Seed, 1x10 9 One or more different nucleic acids. Optionally or additionally, the apparatus or method of this disclosure may include up to 1 x 103 9 Seed, 1x10 6 Seed, 1x10 5 Seed, 1x10 4 Seed, 1x10 3 There can be one, 100, 10, 2, or fewer different nucleic acids. Therefore, the various reagents or products (e.g., primer-template nucleic acid hybrids or stable ternary complexes) described herein that can be used in the apparatus or method can be multiplexed to have different types or kinds within these ranges. Different nucleic acids present in the array can be located at different features of the array. Therefore, the signal obtained from the feature will indicate the specific nucleic acid sequence present at the feature.

[0115] Other examples of commercially available arrays that can be used include, for example, the Affymetrix GeneChip. TM Arrays. Spotted arrays can also be used depending on the implementation. A typical spotted array is the CodeLink array, available from Amersham Biosciences. TMarrays. Another useful array is one that uses an inkjet printing method, such as the SurePrint TM arrays manufactured using nanofabrication techniques.

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

[0117] Nucleic acids can be attached to a support in a manner that provides detection at the single molecule level or ensemble level. For example, a plurality of different nucleic acids can be attached to a solid support in such a manner that a single stable ternary complex formed on one nucleic acid molecule on the support can be distinguished from all adjacent ternary complexes formed on nucleic acid molecules on the support. In this way, one or more different templates can be attached to a solid support in a format that physically isolates and detects each single molecule template in a manner that resolves the single molecule from all other molecules on the solid support.

[0118] Alternatively, the methods of the disclosure can be performed on one or more nucleic acid ensembles, an ensemble being a population of nucleic acids having a common template sequence. An ensemble can include, for example, at least 2, 10, 50, 100, 500, 1000, or more nucleic acids having a common template sequence. Alternatively or additionally, an ensemble can include at most 1000, 500, 100, 50, 10, or 2 nucleic acids having a common template sequence. An ensemble present at a feature of an array can be clonal, such that substantially all of the nucleic acids at the feature have a common template sequence. However, a feature need not comprise a clonal population of nucleic acids. Instead, a feature can comprise a mixed population of nucleic acids, in which a particular template sequence is present in a majority of the nucleic acids. For example, a population of nucleic acids having a particular feature can include at least 51%, 60%, 75%, 90%, 95%, or 99% or more of the species having 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 an average of the signal produced by the non-clonal population. As long as contaminating nucleic acids are present as a minority at a feature of interest, the average signal can provide a means of characterizing the majority template nucleic acids at that feature.

[0119] Clustering methods can be used to attach one or more ensembles to a solid support. As such, an array can have multiple ensembles, each of which is referred to in the format as a cluster or array feature. 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 Nos. 5,641,658 or 7,115,400, or U.S. Patent Publication Nos. 2002 / 0055100 Al, 2004 / 0002090 Al, 2004 / 0096853 Al, 2007 / 0128624 Al, or 2008 / 0009420 Al. 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 U.S. Patent Publication Nos. 2005 / 0130173 Al or 2005 / 0064460 Al, each of which is incorporated herein by reference in its entirety. Another useful method for amplifying nucleic acids on a surface is rolling circle amplification (RCA), for example, as described in Lizardi et al., Nat. Genet. 19:225-232 (1998) or US 2007 / 0099208 Al, each of which is incorporated herein by reference.

[0120] In particular embodiments, the stable ternary complex, polymerase, primer, template, primer-template nucleic acid hybrid, or nucleotide is attached to a surface of a flow cell or a solid support in a flow cell. A flow cell allows for convenient fluidic manipulation by fluidic chambers that contact the ternary complex-bound support by solution in and out. A flow cell also provides for detection of fluidically manipulated components. For example, a detector can be positioned to detect a signal from the solid support, such as from a label recruited to the solid support by formation of a stable ternary complex. Exemplary flow cells that can be used are described in, e.g., U.S. Patent Application Publication No. 2010 / 0111768 Al, WO 05 / 065814, or U.S. Patent Application Publication No. 2012 / 0270305 Al, each of which is incorporated herein by reference.

[0121] The present disclosure provides methods for sequencing a template nucleic acid. The methods can include the steps of: (a) providing a plurality of primer-template nucleic acid hybrids, wherein the primers have extendable 3’ ends; (b) contacting the plurality of primer-template nucleic acid hybrids with: (i) reversibly terminated nucleotides to produce a first subset of primer-template nucleic acid hybrids comprising reversibly terminated nucleotides 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 primer-template nucleic acid hybrid of the first subset, and a cognate nucleotide; (d) detecting the ternary complexes, thereby identifying a nucleotide in the template nucleic acid; (e) deblocking the reversibly terminated nucleotide at the 3’ end of the primer-template nucleic acid hybrid in the first subset; and (f) repeating steps (b) through (e) to sequence the template nucleic acid in the first subset.

[0122] When included in the methods set forth herein, the deblocking process can facilitate sequencing of the primer-template nucleic acid hybrid. The deblocking process can be used to convert the reversibly terminated primer to an extendable primer. Primer extension can then be used to move the ternary complex-shaped site along the template nucleic acid to a different location. Repeated cycles of extension, interrogation, and deblocking can be used to reveal the sequence of the template nucleic acid. Each cycle reveals a subsequent base in the template nucleic acid. Exemplary reversible terminator moieties, methods of incorporating them into primers, and methods of modifying primers for further extension (often referred to as “deblocking”) are set forth 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. Other examples are set forth 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 impede or exclude subsequent formation of ternary complexes need not be treated to reverse the modification. Thus, capped primers can remain capped through several cycles (and in some cases, all cycles) of a cycling method such as a sequencing method. For example, the cycling process can include a process of deblocking the reversibly terminated primers, and the capped primers can be inert to the deblocking process.

[0124] The sequencing method can include multiple repeated cycles or steps within a cycle as set forth herein. For example, the interrogation and primer modification steps can be repeated multiple times, and optional steps of deblocking or washing away unwanted reactants or products between the various steps can also be repeated multiple times. Thus, the primer-template nucleic acid hybrid can undergo at least 2, 5, 10, 25, 50, 100, 150, 200, or more repeated cycles of the method set forth herein. Fewer cycles can be performed when a shorter read length is desired. Thus, the primer-template nucleic acid hybrid can undergo at most 200, 150, 100, 50, 25, 10, 5, or 2 cycles of the method set forth herein.

[0125] In some embodiments, the sequencing method can be repeated for a predetermined number of cycles. Alternatively, the cycles can be repeated until a particular empirically observed condition is reached. For example, the cycles can be repeated as long as the signal is above an observable threshold, the noise is below an observable threshold, or the signal-to-noise ratio is above an observable threshold.

[0126] In another example, the cycles can be repeated as long as one or more ensembles (e.g., one or more clusters or features in an array) have a desired composition. As one or more ensembles proceed through the sequencing cycles, their composition can change. Viewing a single ensemble as a group of template nucleic acids, each having a first subset of templates with primers having a reversible terminated nucleotide at the 3' end, the number of the first subset can decrease as the cycles proceed, and each having a second subset of template nucleic acids with primers having a ternary complex inhibitor (or each without a primer) can increase as the cycles proceed. The sequencing method can continue until the first subset reaches a particular threshold and / or until the second subset reaches a particular threshold. More specifically, the 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 detected. Alternatively or additionally, the cycles 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 detected. In some embodiments, the cycles 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 detected. Alternatively or additionally, the cycles 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 detected.

[0127] It will be appreciated that not all of the steps set forth herein need to be repeated, nor do the repeated steps need to occur in the same order each time they are repeated.

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

[0129] Optionally, a plurality of primer-template nucleic acid hybridizers are attached to a solid support in a device of the disclosure. The solid support can comprise any of the various materials set forth herein, including, for example, the materials set forth herein in the context of a nucleic acid array. The plurality of primer-template nucleic acid hybridizers can be attached to features of the array, and optionally, the templates attached to the features can have the same sequence. Any of the various reagents set forth herein can be attached to the solid support instead of, or in addition to, the primer-template nucleic acid hybridizers. In particular embodiments, a device of the disclosure need not include any type of attachment reagent.

[0130] In particular embodiments, a device of the disclosure comprises a container, such as a manufactured container. The container can contain a plurality of primer-template nucleic acid hybridizers, as well as other reagents or reaction products involved in the methods set forth herein. A particularly useful manufactured container is a flow cell, examples of which are set forth above.

[0131] A device of the disclosure can be a component of a larger system. For example, the system can comprise (a) a device comprising a plurality of primer-template nucleic acid hybridizers, wherein a first subset of primer-template nucleic acid hybridizers has a reversibly terminated nucleotide at the 3' end of the primer, and wherein a second subset of primer-template nucleic acid hybridizers has 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 hybridizers. Optionally, the system can further comprise (c) a fluidic system comprising a reservoir containing a polymerase and nucleotides, wherein the reservoir is in fluid communication with the plurality of primer-template nucleic acid hybridizers.

[0132] A system of the disclosure can be configured to detect nucleic acids, for example, using the methods set forth herein. For example, a system can be configured to generate and detect ternary complexes formed between a polymerase and a primer-template nucleic acid hybridizer in the presence of a nucleotide to identify one or more bases in a sequence of a template nucleic acid. Optionally, the system comprises components and reagents for performing one or more steps set forth herein, including, but not limited to, forming at least one stable ternary complex between a primer-template nucleic acid hybridizer, a polymerase, and a next correct nucleotide; detecting the stable ternary complex(es); modifying (e.g., by primer capping or primer extension process) the primer of each primer-template hybrid; deblocking the reversibly terminated primer; and / or identifying a nucleotide, a sequence of nucleotides, or a series of base multiplets present in a template.

[0133] The systems of the present disclosure can include vessels, solid supports, or other devices for carrying out nucleic acid detection methods. For example, the systems can include arrays, flow cells, multi-well plates, or other convenient devices. The devices can be removable, allowing them to be placed into and removed from the system. In this way, the system can be configured to process multiple devices (e.g., vessels or solid supports) sequentially or in parallel. The system can include a fluidic assembly having vessels for containing one or more of the reagents set forth herein (e.g., polymerase, primers, template nucleic acids, one or more nucleotides, nucleotides for primer extension, deblocking reagents, ternary complex inhibitors, or mixtures of such components for forming ternary complexes). The fluidic system can be configured to deliver reagents to the vessels or solid supports, e.g., via channels or droplet transfer devices (e.g., electrowetting devices). Any of a variety of detection devices can be configured to detect vessels or solid supports that have interacted with reagents. Examples include luminescence detectors, surface plasmon resonance detectors, and other detectors known in the art. Exemplary systems having fluidic and detection components that can be readily modified for use herein include, but are not limited to, those described in U.S. Patent Application Publication No. 2018 / 0280975 Al (which claims priority to U.S. Patent Serial 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 Al or 2012 / 0270305 Al (each of which is incorporated herein by reference).

[0134] Optionally, the systems of the present disclosure further include a computer processing unit (CPU) configured to operate the components of the system. The same or different CPUs can interact with the system to acquire, store, and process signals (e.g., signals detected in the methods set forth herein). In particular embodiments, the CPU can be used to determine the identity of a nucleotide present at a particular location in a template nucleic acid from a signal. In some cases, the CPU will identify the nucleotide sequence of a template from the detected signals.

[0135] Useful CPUs can include one or more of a personal computer system, a server computer system, a thin client, a fat client, a handheld or laptop device, a multiprocessor system, a microprocessor-based system, a set top box, programmable consumer electronics, network PC, minicomputer system, mainframe computer system, a smart phone, and distributed cloud computing environments that include any of the above systems or devices, and the like. The CPU can include one or more processors or processing units, a memory architecture that can include RAM and non-volatile memory. The memory architecture can also include removable / non-removable, volatile / non-volatile computer system storage media. Additionally, the memory architecture can include one or more readers for reading from and writing to non-removable, non-volatile magnetic media, such as a hard disk drive, a disk drive that reads from and writes to a removable, non-volatile disk, and / or an optical disk drive that reads from or writes to a removable, non-volatile media, such as a CD-ROM or DVD-ROM. The CPU can also include various computer system readable media. Such media can be any available media that is accessible by a cloud computing environment, such as volatile and non-volatile media, and removable and non-removable media.

[0136] The memory architecture can include at least one program product having at least one program module that is implemented in executable instructions. The program module can include, for example, an operating system, one or more application programs, other program modules, and program data. Generally, a program module can include routines, programs, objects, components, logic, data structures, and so on that perform particular tasks or implement particular abstract data types.

[0137] The components of the CPU can be coupled by an internal bus, which can be implemented as any one or more of several types of bus structures, including a memory bus or memory controller, a peripheral bus, an accelerated 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 Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and 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 interaction with the nucleic acid detection system. Similarly, the CPU can communicate with other devices (e.g., through a network card, a modem, etc.). Such communication can be facilitated through the I / O interface. In addition, the CPU of the systems herein can communicate with one or more networks, such as a local area network (LAN), a wide area network (WAN), and / or a public network (e.g., the Internet) through an appropriate network adapter.

[0139] The present disclosure also provides kits for characterizing nucleic acids. Optionally, the kits can include reagents for carrying out one or more of the methods set forth herein. For example, the kits can include reagents for generating stable ternary complexes when mixed with one or more primer-template nucleic acid hybrids. In addition to a mixture of nucleotides, the kits can also include a polymerase capable of forming stable ternary complexes and / or a polymerase for a primer modification step. Other reagents for primer modification, such as reversibly terminated nucleotides or ternary complex inhibitors, can also be included in the kits. The reagents included in the kits, such as one or more nucleotides, one or more polymerases, or both, can include exogenous labels, e.g., as set forth herein in the context of the various methods.

[0140] Accordingly, any of the components or articles for carrying out the methods set forth herein can be effectively packaged into a kit. For example, the kits can be packaged to include some, many, or all of the components or articles for carrying out the methods set forth herein. Exemplary components include, e.g., labeled nucleotides (e.g., extendable labeled nucleotides), polymerases (labeled or unlabeled); nucleotides with terminator moieties (e.g., unlabeled, reversibly terminated nucleotides), deblocking reagents, ternary complex inhibitors, etc., as set forth herein and in the references cited herein. Any such reagents can include, e.g., some, many, or all of the buffers, components, and / or articles for carrying out one or more subsequent steps for analyzing the primed template nucleic acids. The kits need not include primers or template nucleic acids. Rather, the user of the kit can provide one or more primer-template nucleic acid hybrids by the user combining the components of the kit.

[0141] One or more ancillary reagents can also be included in the kits. Such ancillary reagents can include any of the reagents exemplified above and / or other types of reagents that can be used to carry out the methods set forth herein. Instructions can also be included in the kits. The instructions can include, e.g., procedures for preparing any of the components or articles used in the methods set forth herein, performing one or more steps of any of the embodiments of the methods set forth herein, and / or instructions for using the primer-template nucleic acid hybrids for performing any subsequent analysis steps.

[0142] In particular embodiments, a kit can include a device described herein, such as a flow cell or a solid support. Optionally, the kit includes a cartridge having a container for holding reagents, and also having a fluidic component for transferring reagents from the container to a detection instrument. For example, the fluidic component can be configured to transfer reagents to a flow cell in which the stable ternary complex is detected. An exemplary fluidic cartridge that can be included in a kit (or system) of the present disclosure is described in U.S. Patent Application Serial No. 15 / 922,661 (published as U.S. Patent Application Publication No. 2018 / 0280975 Al, which is incorporated herein by reference).

[0143] Example I

[0144] Primer capping using run-off primer extension

[0145] This example demonstrates a technique to improve phasing when amplifying a population of primers. Phasing is improved by capping non-extended primers to prevent capped primers from forming ternary complexes in subsequent inspection steps.

[0146] Use of a multi-well plate format to measure binding reactions on the surface of fiber optic tips using biolayer interferometry (Menlo Park, CA) Octet instrument. Template nucleic acid was attached to streptavidin-functionalized tips by incubating the tips in wells containing 5’ biotinylated template nucleic acid at 37°C for 5 minutes. Unbound template nucleic acid was removed by contacting the tips with 0.1 M NaOH, aspirating the tips, and then washing the tips 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 template bound to the tips by incubating in PRE at 37°C for 5 minutes.

[0147] The tips were then subjected to 30 cycles of primer extension on the Octet instrument. Each cycle comprised the following steps: transfer of the tips to the following reagents for the following times: (1) in PRE for 5 seconds; (2) in RTS (PRE, 5mM MgCI2, 10U / mL Therminator, 25μM rtNTP (reversibly terminated deoxynucleotides with aminooxy reversible terminator moiety at the 3’ position)) for 10 seconds; (3) in a variable solution for 30 seconds; (4) in ESB (1M GdSCN, 0.1 mM HEPES pH 7.5, 0.1% Tween-80, 0.1% hydroxylamine, 2mM EDTA) for 5 seconds; (5) in PRE for 5 seconds; (6) in CLV (0.25M sodium acetate pH 4.8, 0.7M sodium nitrite) for 5 seconds; (7) in PRE for 5 seconds. The variable solution comprised the following reagents:

[0148] a) PRE

[0149] b) PRE + 10u / m Therminator

[0150] c) PRE + 10u / ml Therminator + 5mg Mg +2

[0151] d) PRE + 10u / ml Therminator + 5mg Mg +2 + 100μM dNTP

[0152] e) PRE + 10u / ml Therminator + 5mg Mg +2 + 30μM dNTP

[0153] f) PRE + 10u / ml Therminator + 5mg Mg +2 + 10μM dNTP

[0154] g) PRE + 10u / ml Therminator + 5mg Mg +2 + 3μM dNTP

[0155] h) PRE + 10u / ml Therminator + 5mg Mg +2 + 1μM dNTP

[0156] After Octet cycling, the primer strand was stripped from the template attached to the tip and incubated with capillary electrophoresis (CE) dye. The length of the primer was then determined using CE analysis. The CE results were analyzed by examining each peak and recording the n and n-1 peak areas. The efficiency of primer extension was evaluated using the following formula: [((cycle number) x (n / n-1 ratio)] ÷ [(cycle number) x (n / n-1 ratio) + 1].

[0157] The results are summarized in Table 1. The CE mobility of the extended primer was consistent with the expected length (n = 59 nucleotides). The n / n-1 ratio was lowest under 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 required for primer extension were present.

[0158] Table 1 Results of CE

[0159]

[0160] The results show that after extension using reversibly terminated nucleotides (in the RTS), further primer extension only occurs when all components required for primer extension (dNTPs, Therminator, and Mg) are present. This indicates that the unblocked 3’ end is preserved, and that the post-RTS over-flow extension can be used to fully extend the primer through the template region.

[0161] The fully extended primer is not expected to be available for ternary complex formation in the subsequent inspection step, resulting in improved signal-to-noise, longer read lengths, and higher sequencing accuracy.

[0162] Throughout this application, various publications, patents and / or patent applications have been referenced. The disclosures of these documents in respect of the materials described therein are hereby incorporated by reference in their entirety.

[0163] A number of implementations have been described. Nevertheless, it will be understood that various modifications can be made. Accordingly, other implementations are within the scope of the following claims.

Claims

1. A flow cell comprising a plurality of primer-template nucleic acid hybrids and a solid support, wherein the plurality of primer-template nucleic acid hybrids are attached to the solid support; wherein a first subset of primer-template nucleic acid hybrids comprises a blocked nucleotide at the 3' end of the primer, and wherein a second subset of primer-template nucleic acid hybrids comprises a ternary complex inhibitor at the 3' end of the primer, wherein the primer-template nucleic acid hybrids in the first subset each bind to a polymerase and a cognate nucleotide, thereby forming a ternary complex; wherein the blocked nucleotide is a reversibly terminated nucleotide; wherein the cognate nucleotide is a nucleotide that is complementary to the next base of the template nucleic acid; and wherein the ternary complex inhibitor is (i) an oligonucleotide moiety, (ii) a moiety that inhibits binding of a polymerase to the 3' end of the primer, (iii) a moiety that inhibits binding of a cognate nucleotide to the 3' end of the primer to a polymerase, or (iv) a mismatched nucleotide at the 3' end of the primer.

2. The flow cell of claim 1, wherein the solid support comprises a nucleic acid array.

3. The flow cell of claim 2, wherein the plurality of primer-template nucleic acid hybrids are attached to features of the array, and wherein the templates attached to the features comprise identical sequences.

4. The flow cell of any one of claims 1 to 3, wherein the plurality of primer-template nucleic acid hybrids are present in a manufactured vessel.

5. The flow cell of claim 4, wherein the manufactured vessel comprises a flow cell.

6. The flow cell of claim 1, wherein the polymerase is covalently attached to an exogenous label.

7. The flow cell of claim 1, wherein the cognate nucleotide is covalently attached to an exogenous label.

8. The flow cell of claim 6 or 7, wherein the exogenous label comprises a fluorophore.

9. The flow cell of any one of claims 1 to 8, wherein the primers of the first subset of primer-template nucleic acid hybrids are the same length.

10. The flow cell of claim 9, wherein the primers of the second subset of primer-template nucleic acid hybrids are different lengths.

11. The flow cell of claim 9 or 10, 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.

12. The flow cell of claim 9 or 10, 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.

13. The flow cell of claim 9, wherein the primers of the second subset of primer-template nucleic acid hybrids are fully extended.

14. The flow cell of any one of claims 1 to 13, 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.

15. The flow cell of any one of claims 1 to 13, wherein the number of primer- template nucleic acid hybrid 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.

16. A system comprising: (a) the flow cell of any one of claims 1 to 15; and (b) a detector configured to detect the plurality of primer-template nucleic acid hybrids.

17. The system of claim 16, further comprising: (c) a fluidic system comprising a reservoir containing a polymerase and nucleotides, wherein the reservoir is in fluid communication with the plurality of primer-template nucleic acid hybrids.

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