Continuous formation of ternary complex species

Abstract

A method for identifying a nucleotide in a primed template nucleic acid, comprising the steps of: (a) providing a vessel having a primed template nucleic acid, a polymerase, and a nucleotide cognate of a first base type; (b) checking the vessel for a stable ternary complex comprising the polymerase and the nucleotide cognate of the first base type bound at a base position of the primed template nucleic acid; (c) delivering a nucleotide cognate of a second base type to the vessel, whereby the vessel retains the primed template nucleic acid and the polymerase from step (b); (d) checking the vessel for a stable ternary complex comprising the polymerase and the nucleotide cognate of the second base type bound at the base position of the primed template nucleic acid; and (e) identifying the type of nucleotide at the base position of the primed template nucleic acid.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims the rights of U.S. Provisional Application No. 62 / 702,468, filed July 24, 2018, which is incorporated herein by reference in its entirety. Background of the Invention

[0004] This disclosure generally relates to the detection of nucleic acids and has specific applicability to nucleic acid sequencing technology.

[0005] Accurate sequencing of the template nucleic acid strand is crucial for molecular diagnostics. Identifying single nucleotide bases at known positions from surrogates serves as the basis for analyzing single nucleotide polymorphisms (i.e., "SNPs"). SNPs, in turn, can be used to determine an individual's phenotype, such as susceptibility to disease or a predisposition to having desired traits. Detecting genetic variants in a patient can indicate the effectiveness of certain drug treatments or the risk of adverse side effects when using certain drugs.

[0006] Commercially available nucleic acid sequencing platforms have greatly increased our understanding of the genetic basis of operable traits. Improvements in sequencing biochemistry and detection hardware continue to emerge. However, many platforms only achieve relatively short reads. Massive parallel processing allows for the generation of many short reads, which can then be woven together to assemble larger genome sequences. For example, millions of reads, each only a few hundred nucleotides long, can be assembled together to produce a human genome approximately 3 billion nucleotides long. By increasing sequencing read length, the time and resources required to achieve massively parallel processing of DNA and high-throughput assembly of data can be reduced. This invention addresses this need and also provides related advantages. Summary of the Invention

[0007] This disclosure provides a method for identifying nucleotides in an induced template nucleic acid. The method may include the following steps: (a) providing a container having an induced template nucleic acid, a polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound at a base position of the induced template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the induced template nucleic acid and polymerase from step (b); (d) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound at a base position of the induced template nucleic acid; and (e) identifying the type of nucleotide at the base position of the induced template nucleic acid. Optionally, step (c) includes removing the nucleotide homolog of the first base type from the container and delivering the nucleotide homolog of the second base type to the container, whereby the container retains the induced template nucleic acid and polymerase from step (b). As an alternative to this option, it is not necessary to remove the nucleotide homolog of the first base type; and alternatively, the container may retain the nucleotide homolog of the first base type from steps (c) and (d).

[0008] In some embodiments, a method for identifying nucleotides in an induced template nucleic acid may include the following steps: (a) providing a container having an induced template nucleic acid, a polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound to a base position of the induced template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the induced template nucleic acid and polymerase from step (b); and (d) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound to a base position of the induced template nucleic acid; (c) delivering a nucleotide homolog of the second base type to the container, wherein the container retains the induced template nucleic acid and polymerase from step (b); and (d) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound to a base position of the induced template nucleic acid; A stable ternary complex is present, comprising a polymerase and a nucleotide homolog of a second base type bound at a base position of the initiated template nucleic acid; (e) identifying the type of nucleotide at the base position of the initiated template nucleic acid; (f) delivering a nucleotide homolog of a third base type to the container, whereby the container retains the initiated template nucleic acid and polymerase from step (b); and (g) checking the container for the presence of a stable ternary complex comprising a polymerase and a nucleotide homolog of a third base type bound at a base position of the initiated template nucleic acid. Optionally, the method further comprises the steps of: (h) delivering a nucleotide homolog of a fourth base type to the container, whereby the container retains the initiated template nucleic acid and polymerase from step (b); and (i) checking the container for the presence of a stable ternary complex comprising a polymerase and a nucleotide homolog of a fourth base type bound at a base position of the initiated template nucleic acid.

[0009] This disclosure provides a method for sequencing an initiated template nucleic acid, comprising the following steps: (a) providing a container having an initiated template nucleic acid, a first polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the first polymerase and a nucleotide homolog of the first base type bound to a base position of the initiated template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the initiated template nucleic acid and the first polymerase from step (b); (d) checking the container for the presence of a stable ternary complex comprising the first polymerase and a nucleotide homolog of the second base type bound to a base position of the initiated template nucleic acid; (e) identifying the type of nucleotide at the base position of the initiated template nucleic acid; and (f) delivering a nucleotide homolog of a third base type to the container, whereby the container retains the nucleotide homolog of step (b). (b) Initiating template nucleic acid and a first polymerase; (g) Checking the container for a stable ternary complex comprising the first polymerase and a nucleotide homolog of a third base type bound to a base position of the initiated template nucleic acid; (h) Delivering a nucleotide homolog of a fourth base type to the container, whereby the container retains the initiated template nucleic acid and the first polymerase from step (b); (i) Checking the container for a stable ternary complex comprising the first polymerase and a nucleotide homolog of a fourth base type bound to a base position of the initiated template nucleic acid; (j) Adding a nucleotide to a primer of the initiated template nucleic acid, whereby the container contains an extended initiated template nucleic acid; (k) Delivering a second polymerase and a nucleotide homolog of the first base type to the container; and (l) Repeating steps (b) to (i) using the extended initiated template instead of the initiated template nucleic acid and using the second polymerase instead of the first polymerase. The first polymerase may be a polymerase of the same type as the first type, or the first polymerase and the second polymerase may be polymerases of different types.

[0010] A method for sequencing an initiated template nucleic acid may include the following steps: (a) providing a container having an initiated template nucleic acid, a first polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the first polymerase and a nucleotide homolog of the first base type bound to a base position of the initiated template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the initiated template nucleic acid and the first polymerase from step (b); and (d) checking the container for the presence of a stable ternary complex. The complex, the ternary complex comprising a first polymerase and a nucleotide homolog of a second base type bound to a base position of the priming template nucleic acid; (e) identifying the type of nucleotide at the base position of the priming template nucleic acid; (f) adding the nucleotide to a primer of the priming template nucleic acid, thereby containing an extended priming template nucleic acid; (g) delivering the second polymerase and the nucleotide homolog of the first base type to the container; and (h) repeating steps (b) to (e) using the extended priming template instead of the priming template nucleic acid and using the second polymerase instead of the first polymerase. The first polymerase may be a polymerase of the same type as the first type, or the first polymerase and the second polymerase may be polymerases of different types.

[0011] This disclosure also provides a method for identifying nucleotides in an initiated template nucleic acid, comprising the steps of: (a) providing an array of initiated template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a polymerase, a nucleotide homolog of a first base type, and the initiated template nucleic acid in the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the initiated template nucleic acid and polymerase in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, step (d) includes removing the nucleotide homolog of the first base type from the array, and then repeating steps (b) and (c) for the nucleotide homolog of the second base type, thereby retaining the initiated template nucleic acid and polymerase in the array. As an alternative to this option, the nucleotide homolog of the first base type does not need to be removed; instead, the nucleotide homolog of the first base type may be retained with the array in step (d).

[0012] In some embodiments, a method for identifying nucleotides in an induced template nucleic acid may include the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and polymerase in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, step (d) is performed as follows: repeating steps (b) and (c) for nucleotide homologs of the second base type, and then repeating steps (b) and (c) for nucleotide homologs of the third base type. Alternatively, step (d) may be performed as follows: repeat steps (b) and (c) for nucleotide homologs of the second base type, then repeat steps (b) and (c) for nucleotide homologs of the third base type, and then repeat steps (b) and (c) for nucleotide homologs of the fourth base type.

[0013] A method for sequencing induced template nucleic acids is also provided, comprising the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a first polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for a nucleotide homolog of a second base type, then repeating steps (b) and (c) for a nucleotide homolog of a third base type, then repeating steps (b) and (c) for a nucleotide homolog of a fourth base type, thereby retaining the induced template nucleic acid and the first polymerase in the array; (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c); (f) adding nucleotides to primers for each induced template nucleic acid, thereby including an extended induced template nucleic acid in the array; and (g) repeating steps (b) to (e) using the extended induced template in place of the induced template nucleic acid and using the second polymerase in place of the first polymerase. The first polymerase can be the same type of polymerase as the first type, or the first polymerase and the second polymerase can be polymerases of different types.

[0014] A method for sequencing induced template nucleic acids may include the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a first polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and the first polymerase in the array; (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c); (f) adding nucleotides to primers for each induced template nucleic acid, thereby including an extended induced template nucleic acid in the array; and (g) repeating steps (b) through (e) using the extended induced template nucleic acid instead of the induced template nucleic acid and using a second polymerase instead of the first polymerase. The first polymerase may be a polymerase of the same type as the first type, or the first polymerase and the second polymerase may be polymerases of different types.

[0015] This disclosure also provides a method for identifying nucleotides in an initiated template nucleic acid, comprising the steps of: (a) providing an array of initiated template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the initiated template nucleic acid of the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the initiated template nucleic acid and the polymerase of the multiple polymerases in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, step (d) includes removing the multiple nucleotide homologs of the first base type from the array, and then repeating steps (b) and (c) for nucleotide homologs of the second base type, thereby retaining the initiated template nucleic acid and the polymerase of the multiple polymerases in the array. As an alternative to this option, nucleotide homologs of the first base type do not need to be removed; and alternatively, nucleotides in multiple nucleotide homologs of the first base type can be retained together with the array in step (d).

[0016] A method for identifying nucleotides in an induced template nucleic acid is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and the polymerase of the multiple polymerases in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, step (d) is performed as follows: repeating steps (b) and (c) for nucleotide homologs of the second base type, and then repeating steps (b) and (c) for nucleotide homologs of the third base type. Alternatively, step (d) may be performed as follows: repeat steps (b) and (c) for nucleotide homologs of the second base type, then repeat steps (b) and (c) for nucleotide homologs of the third base type, and then repeat steps (b) and (c) for nucleotide homologs of the fourth base type.

[0017] A method for sequencing induced template nucleic acids is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; and (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, and then for nucleotide homologs of a third base type. (b) and (c) are repeated for nucleotide homologs, and then (b) and (c) are repeated for nucleotide homologs of the fourth base type, thereby retaining the initiating template nucleic acid and the polymerases in the array in the array; (e) the type of nucleotide present in each stable ternary complex detected in step (c) is identified; (f) a nucleotide is added to the primer of each initiating template nucleic acid, thereby the array includes the extended initiating template nucleic acid; and (g) steps (b) to (e) are repeated using the extended initiating template instead of the initiating template nucleic acid and using the multiple second polymerases instead of the multiple polymerases.

[0018] A method for sequencing induced template nucleic acids may include the following steps: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, whereby the induced template nucleic acid of the array and the polymerase of the multiple polymerases are retained in the array; (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c); (f) adding nucleotides to primers for each induced template nucleic acid, whereby the array comprises extended induced template nucleic acids; and (g) repeating steps (b) to (e) using extended induced templates instead of induced template nucleic acids and using multiple second polymerases instead of multiple polymerases.

[0019] A method for identifying nucleotides in an induced template nucleic acid is also provided, comprising the following steps: (a) providing an array of induced template nucleic acids; (b) delivering multiple nucleotide homologs of a first base type and multiple polymerases to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array comprising nucleotide homologs of the first base type; (d) delivering multiple nucleotide homologs of a second base type to the array in the presence of the polymerase from step (b) to form stable ternary complexes, each ternary complex comprising a polymerase of the polymerase from step (b), a nucleotide of the multiple nucleotide homologs of the second base type, and the induced template nucleic acid of the array; (e) detecting stable ternary complexes in the array comprising nucleotide homologs of the second base type; and (f) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, the method further includes repeating steps (d) and (e) by replacing the second base type nucleotide homolog with a nucleotide homolog of a third base type. Alternatively, the method may further include repeating steps (d) and (e) by replacing the second base type nucleotide homolog with a nucleotide homolog of a fourth base type. In another option, the method may include the steps of: (g) adding a nucleotide to the primer of each priming template nucleic acid, whereby the array comprises an extended priming template nucleic acid; and (h) repeating steps (b) to (f) by replacing the priming template nucleic acid with an extended priming template. Attached Figure Description

[0020] Figure 1 The diagram shows a comparison of the “on” signal intensity (corresponding to the binding of homologous nucleotides) and the “off” signal intensity (corresponding to the binding of non-homologous nucleotides) of the sequencing cycle, which includes a step between inspection steps to wash the induced template nucleic acid to replace the polymerase and nucleotides.

[0021] Figure 2 The diagram shows a comparison of the "on" and "off" signal intensities of a sequencing cycle, which includes a step of washing the induced template nucleic acid to replace nucleotides between inspection steps within the sequencing cycle, wherein a polymerase is added prior to the first inspection step.

[0022] Figure 3 The diagram shows a comparison of the "on" and "off" signal intensities of a sequencing cycle, which includes a step of washing the initiated template nucleic acid with salt and ethanol between inspection steps within the sequencing cycle, wherein polymerase is added before the first inspection step. Detailed Implementation

[0023] This disclosure provides a method for identifying nucleotides in an initiated template nucleic acid. The identification of nucleotides is based on the formation of a ternary complex comprising the initiated template nucleic acid, a polymerase bound to the template at the 3' end of the primer, and a homologous nucleotide bound to the polymerase to pair with a nucleotide in the template adjacent to the 3' end of the primer. The ability of various different nucleotide types to form the ternary complex can be assessed. The nucleotide type observed to participate in the formation of the ternary complex can be identified as a homologous nucleotide at the queried template position. Based on this observation and known nucleotide pairing rules (i.e., adenine pairs with thymine or uracil, cytosine pairs with guanine), the nucleotide type at the template position can be inferred.

[0024] A useful method for querying template nucleic acids involves delivering a polymerase and a first-type nucleotide to a fixed nucleic acid, examining a solid support that recruits the ternary complex components to the fixed nucleic acid, removing the polymerase and nucleotides from the fixed solid support, and then repeating the cycle for different types of nucleotides. While this method can be used to characterize nucleic acids, the delivery and removal of reagents from the solid support can be time-consuming. Furthermore, this substitution cycle consumes a relatively large amount of polymerase, which can be a production-expensive reagent.

[0025] This disclosure provides a method for sequentially delivering different nucleotide types and then removing them from a container from which a ternary complex is to be formed and examined. In this mode, a first nucleotide type is delivered to the reaction container and then removed from the container before a second nucleotide type is delivered to the container. Nucleotide homologs can be removed from the container under conditions that dissociate the nucleotide from the ternary complex, thereby separating the nucleotide from the initiating template nucleic acid without causing significant removal by the polymerase. Another nucleotide can then be delivered to the initiating template nucleic acid. If the polymerase is substantially not removed due to the presence of the initiating template nucleic acid, it is not necessary to deliver more polymerase. This saves the time and resources that would otherwise be spent preparing more polymerase.

[0026] In other embodiments, different nucleotide types can be sequentially delivered to a container containing one or more initiating template nucleic acids under conditions suitable for forming ternary complexes. For example, a first nucleotide type can be delivered to a container containing an array of initiating template nucleic acids, followed by the delivery of a second nucleotide type, allowing both nucleotide types to accumulate in the container. Thus, two types of ternary complexes (each containing one of two different nucleotide types) can accumulate in the array. Optionally, at least two, three, or four different nucleotide types can accumulate in the array to form at least two, three, or four different types of ternary complexes on the array. When the method is carried out in a pattern of sequentially delivering different nucleotide types to the reaction container to allow different nucleotides to accumulate, the container can be checked for ternary complexes after each delivery. In some embodiments, such as when each nucleotide type is distinguishably labeled, a single check can be performed after all nucleotides have been delivered.

[0027] In certain implementations, a primer extension step may be added to advance to the next template location for subsequent examination. Detecting a series of locations within the template region can be used to determine the nucleotide sequence of that region. As illustrated in the Examples section below, when used for binding... TM During sequencing, the above implementation scheme surprisingly provided improved sequencing accuracy and read length.

[0028] While the above embodiments are illustrated with reference to the delivery of a single type of nucleotide in each step, it should be understood that multiple nucleotide types can be delivered in one or more steps. For example, different markers attached to each type of nucleotide can be used to distinguish the nucleotides. Mixtures of nucleotides can be different from each other, such that the net result of different deliveries and checks is the generation of a series of signals encoding a specific nucleotide type. Exemplary encoding schemes and mixtures of nucleotides that can be used to generate said codes are set forth in U.S. Patent Application No. 9,951,385 and Serial No. 15 / 922,787 (now granted U.S. Patent No. 10,161,003), each of which is incorporated herein by reference.

[0029] Unless otherwise stated, the terms used herein shall be understood to have their common meaning in the relevant field. Several terms used herein and their meanings are as follows.

[0030] As used herein, the term "array" refers to a group of molecules attached to one or more solid supports such that molecules at one feature are distinguishable from molecules at other features. An array may include different molecules, each located at a different addressable feature on a solid support. Alternatively, an array may include individual solid supports, each serving to carry the properties of different molecules, wherein the different molecules can be identified by their position on the surface to which the solid supports are attached, or by their position in a liquid such as a fluid flow. The molecules in an array may be, for example, nucleotides, nucleic acid primers, nucleic acid templates, initiating template nucleic acids, or nucleases such as polymerases, ligases, exonucleases, or combinations thereof.

[0031] As used herein, the term "closing portion," when referring to a nucleotide, means the portion of the nucleotide that inhibits or prevents the 3' oxygen of the nucleotide from forming a covalent bond with the next correct nucleotide during the nucleic acid polymerization process. The closing portion of a "reversibly terminated" nucleotide may be removed from a nucleotide analogue or otherwise modified to allow the 3'-oxygen of the nucleotide to covalently link with the next correct nucleotide. Such a closing portion is referred to herein as a "reversible terminator portion." Exemplary reversible terminator portions are described in U.S. Patent Nos. 7,427,673, 7,414,116, 7,057,026, 7,544,794, or 8,034,923, or PCT Publications WO91 / 06678 or WO 07 / 123744 (each of which is incorporated herein by reference). A nucleotide having a closing portion or a reversible terminator portion may be located at the 3' end of a nucleic acid (such as a primer) or may be a monomer not covalently attached to a nucleic acid. A particularly useful blocking region will be located at the 3' end of the nucleic acid involved in forming the ternary complex.

[0032] As used herein, the term "catalytic metal ion" refers to a metal ion that promotes the formation of a phosphodiester bond between the 3'-oxygen group of a nucleic acid (e.g., a primer) and the phosphate group of the introduced nucleotide by polymerase. A "divalent catalytic metal cation" is a catalytic metal ion that is divalent. Catalytic metal ions can exist at a concentration that stabilizes the complex formed between the polymerase, the nucleotide, and the initiated template nucleic acid (this concentration, referred to as the non-catalytic concentration of the metal ion, is maintained as long as phosphodiester bond formation does not occur). The catalytic concentration of a metal ion is the amount of metal ion sufficient to enable the polymerase to catalyze the reaction between the 3'-oxygen group of the nucleic acid (e.g., a primer) and the phosphate group of the introduced nucleotide.

[0033] As used in this article, the term "binary complex" refers to the intermolecular association between the polymerase and the initiated template nucleic acid, excluding nucleotide molecules, such as the next correct nucleotide of the initiated template nucleic acid.

[0034] The term "includes" is open-ended in this document and includes not only the listed elements but also any additional elements.

[0035] As used herein, the term "unblocking" refers to the removal or modification of the reversible terminator portion of a nucleotide to make the nucleotide extendable. For example, a nucleotide may be present at the 3' end of a primer, such that unblocking makes the primer extendable. Exemplary unblocking 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 Publications WO 91 / 06678 or WO 07 / 123744, each of which is incorporated herein by reference.

[0036] As used herein, the term "each" is intended to identify an individual item in a collection when used to refer to a set of items, but does not necessarily refer to every item in the collection. Exceptions may occur if explicitly stated otherwise or if the context clearly indicates otherwise.

[0037] As used herein, the term "exogenous," when referring to a part of a molecule, means a chemical part that is not present in the molecule's natural analogues. For example, an exogenous marker of a nucleotide is a marker that is not present on naturally occurring nucleotides. Similarly, an exogenous marker present on a polymerase is not present on the polymerase in its natural environment.

[0038] As used herein, the term "extension," when referring to nucleic acids, means the process of adding at least one nucleotide to the 3' end of a nucleic acid. When referring to nucleic acids, the term "polymerase extension" refers to the polymerase-catalyzed process of adding at least one nucleotide to the 3' end of a nucleic acid. Nucleotides or oligonucleotides added to nucleic acids through extension are considered to be incorporated into the nucleic acid. Therefore, the term "incorporation" can be used to refer to the process of attaching a nucleotide or oligonucleotide to the 3' end of a nucleic acid by forming a phosphodiester bond.

[0039] As used herein, the term "extensible," when referring to a nucleotide, means that the nucleotide has an oxygen or hydroxyl moiety at the 3' position and, if incorporated into a nucleic acid, can form a covalent bond with the next correct nucleotide. An extensible nucleotide can be located at the 3' position of a primer, or it can be a monomeric nucleotide. Extensible nucleotides lack a closing moiety, such as a reversible terminator.

[0040] As used herein, the term "feature," when referring to an array, means a position within an array in which a particular molecule is present. A feature may contain only a single molecule, or it may contain a group of several molecules of the same kind (i.e., an ensemble of molecules). Alternatively, a feature may include groups of molecules of different kinds (e.g., a group of ternary complexes with different template sequences). The features of an array are typically discrete. Discrete features may be continuous or spaced apart from each other. Arrays useful herein may have features spaced, for example, less than 100 micrometers, 50 micrometers, 10 micrometers, 5 micrometers, 1 micrometer, or 0.5 micrometers apart. Optionally or additionally, an array may have features spaced greater than 0.5 micrometers, 1 micrometer, 5 micrometers, 10 micrometers, 50 micrometers, or 100 micrometers apart. The area of ​​each feature may be less than 1 square millimeter, 500 square micrometers, 100 square micrometers, 25 square micrometers, 1 square micrometer, or less.

[0041] As used herein, a "flow cell" is a reaction chamber comprising one or more channels that direct fluid to a detection zone. The detection zone may be coupled to a detector, enabling observation of the reaction occurring within the reaction chamber. For example, a flow cell may contain a template nucleic acid molecule tethered to a solid support, through which nucleotides and auxiliary reagents may be repeatedly applied and washed away. The flow cell may contain a transparent material to allow imaging of the sample after the desired reaction has occurred. For example, a flow cell may comprise a glass or plastic slide containing small fluid channels through which polymerase, dNTPs, and buffer solutions may be pumped. The glass or plastic within the channels may be modified with one or more template nucleic acid molecules to be sequenced. An external imaging system may be placed to detect molecules in the detection zone. Exemplary flow cells, methods of their manufacture, and methods of their use are described in U.S. Patent Application Publication No. 2010 / 0111768 A1 or No. 2012-0270305 A1, or WO 05 / 065814, each of which is incorporated herein by reference.

[0042] As used herein, the term "label" refers to a molecule or portion thereof that provides a detectable characteristic. Detectable characteristics can be, for example, optical signals such as radiation absorptivity, fluorescence emission, luminescence emission, fluorescence lifetime, fluorescence polarization, etc.; Rayleigh and / or Mie scattering; binding affinity to a ligand or acceptor; magnetism; electrical properties; charge; mass; radioactivity, etc. Exemplary labels include, but are not limited to, fluorophores, luminescent organisms, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive isotopes, mass labels, charge labels, spin labels, acceptors, ligands, etc.

[0043] As used herein, the term "next correct nucleotide" refers to the type of nucleotide that will bind to and / or be incorporated into the 3' end of a primer to complement a base in the template strand that hybridizes with the primer. The base in the template strand is called the "next base," and is exactly the 5' of the base in the template that hybridizes with the 3' end of the primer. The next correct nucleotide can be called a "homolog" of the next base, and vice versa. Homologous nucleotides that interact with each other in a ternary complex or double-stranded nucleic acid are called "paired." According to the Watson-Crick pairing rule, adenine (A) pairs with thymine (T) or uracil (U), and cytosine (C) pairs with guanine (G). Nucleotides with a base that is not complementary to the next template base are called "incorrect," "mismatched," or "non-homologous" nucleotides.

[0044] As used herein, the term "non-catalytic metal ion" refers to a metal ion that, in the presence of polymerase, does not promote the formation of phosphodiester bonds required for the chemical incorporation of nucleotides into primers. Non-catalytic metal ions can interact with polymerases, for example, through competitive binding compared to catalytic metal ions. Therefore, non-catalytic metal ions can function as repressive metal ions. "Divalent non-catalytic metal ion" refers to a non-catalytic metal ion with a valence of 2. Examples of divalent repressive 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+ The ion is a non-catalytic metal ion with a trivalent oxidation state.

[0045] As used herein, the term "nucleotide" may refer to natural nucleotides or their analogues. Examples include, but are not limited to, nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs) or their non-natural analogues such as dideoxyribonucleotide triphosphates (ddNTPs) or reversibly terminated nucleotide triphosphates (rtNTPs).

[0046] As used herein, the term "polymerase" can refer to nucleic acid synthases, including but not limited to DNA polymerases, RNA polymerases, reverse transcriptases, primases, and transferases. Typically, a polymerase has one or more catalytic active sites thereon where nucleotide binding and / or nucleotide polymerization can occur. A polymerase can catalyze the polymerization of a nucleotide to the 3' end of the first strand of a double-stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of the next correct nucleotide to the 3' oxygen group of the first strand of a double-stranded nucleic acid molecule via a phosphodiester bond, thereby covalently incorporating a nucleotide into the first strand of the double-stranded nucleic acid molecule. Optionally, under one or more conditions used in the methods described herein, the polymerase does not need to be able to incorporate nucleotides. For example, a mutant polymerase may be able to form a ternary complex but cannot catalyze nucleotide incorporation.

[0047] As used herein, the term "initiated template nucleic acid" or "initiated template" refers to a nucleic acid having a double-stranded region such that one strand serves as a primer and the other as a template. The two strands can be part of a continuous nucleic acid molecule (e.g., a hairpin structure), or the two strands can be separable molecules that are not covalently linked to each other.

[0048] 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, primers bind in a conformation that allows template replication (e.g., extension by a primer polymerase). A primer can be a first part of a nucleic acid molecule that binds to a second part of that molecule, the first part being the primer sequence and the second part 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. Primers can consist of DNA, RNA, or analogues thereof. Primers can have an extendable 3' end or a blocked 3' end that prevents primer extension.

[0049] As used herein, the term "solid carrier" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate may optionally be able to absorb liquids (e.g., due to its porosity), but typically has sufficient rigidity such that the substrate does not substantially expand when absorbing liquids and does not substantially shrink when the liquid is removed by drying. Non-porous solid carriers are typically impermeable to liquids or gases. Exemplary solid carriers 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, polybutene, polyurethane, Teflon, etc.). TM Materials include cycloolefins, polyimides, nylon, ceramics, resins, Zeonor, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass, fiber bundles, and polymers.

[0050] As used herein, the term "ternary complex" refers to the intermolecular association between a polymerase, a double-stranded nucleic acid, and a nucleotide. Typically, the polymerase facilitates the interaction between the next correct nucleotide and the template strand of the initiating nucleic acid. The next correct nucleotide interacts with the template strand via Worson-Crick hydrogen bonds. The term "stable ternary complex" refers to a ternary complex that promotes or prolongs its presence or has been inhibited from being disrupted. Generally, stabilization of the ternary complex prevents the nucleotide component of the ternary complex from covalently incorporating into the initiating nucleic acid component of the ternary complex.

[0051] As used herein, the term "type" is used to identify molecules that have the same chemical structure. For example, a mixture of nucleotides may include several dCTP molecules. dCTP molecules will be understood as nucleotides of the same type as each other, but different types of nucleotides compared to dATP, dGTP, dTTP, etc. Similarly, individual DNA molecules with the same nucleotide sequence are of the same type, while DNA molecules with different sequences are of different types. The term "type" can also identify portions that have the same chemical structure. For example, cytosine bases in a template nucleic acid will be understood as bases of the same type as each other, regardless of their position in the template sequence.

[0052] As used herein, a “container” is a container used to isolate a chemical process (e.g., a binding event; an incorporation reaction; etc.) from each other, or to provide space in which a chemical process may occur. Non-limiting examples of containers used in conjunction with the disclosed techniques include: flow cells, pores in a multi-well plate, microscope slides, tubes (e.g., capillaries), microdroplets, vesicles, test tubes, trays, centrifuge tubes, features in an array, conduits, channels in a substrate, etc. As used herein, an “artificial container” is an artificial or modified container used to isolate a chemical process (e.g., a binding event; an incorporation reaction; etc.) from each other, or to provide space in which a chemical process may occur.

[0053] Based on the above definitions, the embodiments described below and exemplified in the claims can be understood.

[0054] This disclosure provides a method for identifying nucleotides in an induced template nucleic acid. The method may include the following steps: (a) providing a container having an induced template nucleic acid, a polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound at a base position of the induced template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the induced template nucleic acid and polymerase from step (b); (d) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound at a base position of the induced template nucleic acid; and (e) identifying the type of nucleotide at the base position of the induced template nucleic acid.

[0055] A method for identifying nucleotides in an induced template nucleic acid is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a polymerase, a nucleotide homolog of a first base type, and an induced template nucleic acid in the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for a nucleotide homolog of a second base type, thereby retaining the induced template nucleic acid and polymerase in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0056] This disclosure also provides a method for identifying nucleotides in an induced template nucleic acid, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting the stabilized ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and the polymerase of the multiple polymerases in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0057] The methods disclosed herein may include a check step for detecting ternary complexes. Embodiments of the methods utilize the specificity of polymerases capable of forming stable ternary complexes with an initiating template nucleic acid and the next correct nucleotide. The next correct nucleotide may non-covalently bind to the stable ternary complex, interacting only with other members of the complex through non-covalent interactions. Useful methods and compositions for forming stable ternary complexes are set forth in more detail in the following and in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), U.S. Patent Application Publication No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1 and claiming priority to U.S. Patent Application Publication No. 62 / 440,624), or U.S. Patent Application Publication No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 A1 and claiming priority to U.S. Patent Application Publication No. 62 / 450,397), each of which is incorporated herein by reference.

[0058] Typically, for example, the testing and primer extension are performed separately and discretely because reagent exchange or washing can interfere with testing and extension. Alternatively, in some embodiments, the testing and primer extension steps can be performed in the same mixture.

[0059] Although in some catalytic metal ions (e.g., Mg) 2+In the absence of a catalytic metal ion, a ternary complex can form between the polymerase, the initiated template nucleic acid, 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 lead to non-covalent chelation of the next correct nucleotide in a stable ternary complex. Other methods disclosed herein can also be used to generate stable ternary complexes.

[0060] Optionally, a stable ternary complex can be formed when the primer of the initiating template nucleic acid contains a blocking portion (e.g., a reversible terminator portion), which prevents the enzymatic incorporation of the introduced nucleotide into the primer. The interaction can occur in the presence of a stabilizer, thereby stabilizing the polymerase-nucleic acid interaction in the presence of the next correct nucleotide. The primer of the initiating template nucleic acid can optionally be an extendable primer or a primer whose extension is prevented at its 3' end (e.g., blocking can be achieved by the presence of a reversible terminator portion at the 3' end of the primer). The initiating template nucleic acid, polymerase, and homologous nucleotide can form a stable ternary complex when the base of the next correct nucleotide is complementary to the next base of the initiating template nucleic acid.

[0061] As described above, conditions favorable to or stabilizing ternary complexes can be provided by the presence of blocking groups (e.g., reversible terminator moieties on the 3' nucleotide of a primer) or by the absence of catalytic metal ions that prevent the enzymatic incorporation of introduced nucleotides into the primer. Other useful conditions include the presence of ternary complex stabilizers such as non-catalytic ions (e.g., divalent or trivalent non-catalytic metal ions) that inhibit nucleotide incorporation or polymerization. Non-catalytic metal ions include, but are not limited to, calcium, strontium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, and terbium ions. Optionally, the presence of one or more monovalent cations and / or glutamate anions provides conditions unfavorable to binary complexes (i.e., complexes between polymerases and initiated nucleic acids, but lacking homologous nucleotides) or destabilizing said binary complexes. Alternatively, polymerases engineered to inhibit catalytic activity or prevent the formation of binary complexes can be used.

[0062] The ternary complex stabilization conditions can be further formulated, for example, by disrupting the stability of the binary complex, to enhance the polymerase’s affinity for the initiated template nucleic acid. Optionally, the conditions result in different polymerase affinities for the primer-template in the presence of different nucleotides. For example, the conditions include, but are not limited to, high salt concentrations and glutamate ions. For example, the salt can be dissolved in an aqueous solution to produce a monovalent cation, such as a monovalent metal cation (e.g., sodium or potassium ions). Optionally, the salt providing the monovalent cation (e.g., a monovalent metal cation) further provides glutamate ions. Optionally, the glutamate ion source can be potassium glutamate. In some cases, the concentration of potassium glutamate used to alter the polymerase affinity of the primer-template hybrid can be extended from 10 mM to 1.6 M, or any amount between 10 mM and 1.6 M. As mentioned above, high salt concentration refers to a salt concentration of 50 mM to 1.5 M.

[0063] It should be understood that the options described herein for stabilizing ternary complexes are not mutually exclusive, but can be used in various combinations. For example, ternary complexes can be stabilized by a combination of one or more means, including but not limited to cross-linking of polymerase domains, cross-linking of polymerase with nucleic acids, polymerase mutations stabilizing ternary complexes, allosteric inhibition by small molecules, uncompetitive inhibitors, competitive inhibitors, noncompetitive inhibitors, absence of catalytic metal ions, presence of blocking moieties on primers, and other means described herein. In the specific configurations of the methods or compositions described herein, the polymerase is not covalently attached to other components of the ternary complex in which the polymerase participates. Furthermore, the polymerase does not need to be covalently attached to any solid material, such as a substrate for a nucleic acid array. Instead, the polymerase can diffuse freely in solution without its non-covalent affinity for components of the ternary complex attached to a substrate (such as a nucleic acid array).

[0064] Stable ternary complexes may include, as needed, native nucleotides, nucleotide analogs, or modified nucleotides to suit the specific application or configuration of the method. Optionally, the nucleotide analog has a nitrogenous base, a pentose sugar, and a phosphate group, wherein any portion of the nucleotide may be modified, removed, and / or substituted compared to the native nucleotide. The nucleotide analog may be a non-incorporable nucleotide (i.e., a nucleotide that cannot react with the 3' oxygen of a primer to form a covalent bond). Such non-incorporable nucleotides include, for example, monophosphate and diphosphate nucleotides. In another example, the nucleotide may contain one or more modifications at the 5' position (e.g., at the triphosphate group) to make the nucleotide non-incorporable. Examples of non-incorporable nucleotides can be found in U.S. Patent No. 7,482,120 (which is incorporated herein by reference). In some embodiments, the non-incorporable nucleotide may subsequently be modified to be incorporable. Non-incorporable nucleotide analogues include, but are not limited to, α-phosphate-modified nucleotides, α-β nucleotide analogues, β-phosphate-modified nucleotides, β-γ nucleotide analogues, γ-phosphate-modified nucleotides, nucleotides having a 5' thiophosphate moiety, or cage-like nucleotides. Examples of nucleotide analogues are described in U.S. Patent No. 8,071,755 (which is incorporated herein by reference).

[0065] Nucleotide analogs involved in stabilizing ternary complexes may include terminators that reversibly prevent subsequent incorporation of a nucleotide at the 3' end of the primer after the analog has been incorporated into the primer. For example, US 7,544,794 and US 8,034,923 (the disclosures of which are incorporated herein by reference) describe reversible terminators in which the 3'-OH group is partially substituted by 3'-ONH2. Another type of reversible terminator is linked to a nitrogenous base of a nucleotide as described, for example, in US 8,808,989 (the disclosure of which is incorporated herein by reference). Other reversible terminators that can be similarly used in conjunction with the methods described herein include those described in the azidomethyl moiety or in other references cited herein or in US 7,956,171, US 8,071,755, and US 9,399,798 (the disclosures of which are incorporated herein by reference). In some implementations, the reverse terminator moiety can be modified or removed from the primer in a process known as "unblocking," thereby allowing subsequent nucleotide incorporation. In the context of reversible terminators, compositions and methods for unblocking are described in the references cited herein.

[0066] Alternatively, nucleotide analogs irreversibly prevent the incorporation of nucleotides into the 3' end of primers in which they have already 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 would normally participate in polymerase-mediated primer extension. Therefore, the 3' position has a hydrogen moiety instead of the natural hydroxyl moiety. Irreversible terminating nucleotides are particularly useful for genotyping applications or other applications where primer extension is not required or sequential detection along template nucleic acids is not desired.

[0067] In some embodiments, the nucleotides involved in forming the ternary complex may include exogenous markers such as luminescent organisms. Optionally, the exogenously labeled nucleotides may include reversible or irreversible terminator moieties, may be non-incorporated, may lack a blocking moieties, may be incorporated, or may be both incorporated and non-incorporated. Exogenously labeled nucleotides can be particularly useful when used to form stable ternary complexes with unlabeled polymerases. For example, the marker may produce the luminescence detected in the methods described herein. Alternatively, the exogenous marker on the nucleotide may provide one of the couplers in a fluorescence resonance energy transfer (FRET) pair, and the exogenous marker on the polymerase may provide a second coupler. Thus, FRET detection can be used to identify stable ternary complexes containing both couplers. Alternatively, the nucleotides involved in forming the ternary complex may lack an exogenous marker (i.e., the nucleotides may be “unlabeled”). Optionally, the unlabeled nucleotide may include a reversible or irreversible terminator motif; the unlabeled nucleotide may be non-incorporable; the unlabeled nucleotide may lack a terminator motif; the unlabeled nucleotide may be incorporable; or the unlabeled nucleotide may be incorporable and non-terminant. Unlabeled nucleotides can be useful when a label on the polymerase is used to detect stable ternary complexes. Unlabeled nucleotides can also be used in extension steps of the methods described herein. It should be understood that the absence of a portion or function of a nucleotide means that the nucleotide does not possess such a function or portion. It should also be understood that one or more functions or portions of the nucleotide or similar substances described herein, or originally known in the art, may be explicitly omitted in the methods or compositions described herein.

[0068] Optionally, during the formation of a stable ternary complex, the mixture contains nucleotides (e.g., native nucleotides or synthetic nucleotide analogs). For example, at least one, two, three, four, or more nucleotide types may be present. Optionally or additionally, up to four, three, two, or one nucleotide type may be present. Similarly, one or more nucleotide types present may be complementary to at least one, two, three, or four base types in the template nucleic acid. Optionally or additionally, one or more nucleotide types present may be complementary to up to four, three, two, or one base type in the template nucleic acid.

[0069] Any nucleotide modification that does not prevent participation in the ternary complex can be used in the methods disclosed herein. Nucleotides can bind to polymerases permanently or transiently. Optionally, nucleotide analogs are fused to polymerases, for example, via covalent linkers. Optionally, multiple nucleotide analogs are fused to multiple polymerases, wherein each nucleotide analog is fused to a different polymerase. Optionally, the nucleotides present in the stable ternary complex are not a means of stabilizing the ternary complex. Therefore, any of a variety of other methods for stabilizing ternary complexes can be combined in a reaction utilizing nucleotide analogs.

[0070] In certain embodiments, the primer strands of the template nucleic acid molecule initiated in the stable ternary complex are not chemically altered by the present polymerase in one or more steps of the method described herein. For example, the primers do not need to be extended by forming new phosphodiester bonds, nor do they need to be shortened by nucleolytic degradation in the steps of forming the stable ternary complex and in the steps of detecting the stable ternary complex.

[0071] Ternary complexes prepared or used according to this disclosure may optionally include one or more exogenous markers. Prior to the formation of the ternary complex, the marker may be attached to a component of the ternary complex (e.g., to a polymerase, template nucleic acid, primer, and / or homologous nucleotide). Exemplary attachments include covalent or non-covalent attachments, such as those set forth herein, in the references cited herein, or known in the art. In some embodiments, the labeled component is delivered in solution to a solid support attached to an unlabeled component, thereby recruiting the marker to the solid support by forming a stable ternary complex. Therefore, the component attached to the support can be detected or identified based on observation of the recruited marker. Whether used in solution or on a solid support, the exogenous marker can be used to detect a stable ternary complex or its individual components during an inspection step. The exogenous marker may remain attached to the component after dissociation from other components that have formed a stable ternary complex. Exemplary labels, methods for attaching marks, and methods for using components of marks are set forth in more detail in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or U.S. Patent Application Serial Nos. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1), 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 A1), 62 / 450,397, and 62 / 506,759, each of which is incorporated herein by reference.

[0072] Examples of useful exogenous labels include, but are not limited to, any of the following: radiolabeled portions, luminescent portions, fluorophore portions, quantum dot portions, chromophore portions, enzyme portions, electromagnetic spin-labeled portions, nanoparticle light-scattering portions, and various other signal-generating portions known in the art. Suitable enzyme portions include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Exemplary fluorophore portions include, but are not limited to, umbelliferone, luciferin, isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein and its wavelength-shifted variants, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, and Lucifer Yellow. TM Cascade Blue TM Texas Red TM , dye, Dyes, 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 those in Principles of Fluorescence Spectroscopy Joseph R. Lakowicz (ed.), Plenum Pub Corp, 2nd edition (July 1999) and Richard P. Hoagland Molecular Probes Handbook The dyes described in the 6th edition.

[0073] A second marker can be used in the methods of this disclosure. The second marker is a binding moiety that can specifically bind to a ligand portion. For example, the ligand portion can be linked to a polymerase, nucleic acid, or nucleotide to allow detection of specific affinity for the labeled receptor via said ligand. Exemplary binding moieties that can be used include, but are not limited to, antigens and immunoglobulins or their active fragments, such as FAb; immunoglobulins and immunoglobulins (or their respective active fragments); avidin and biotin, or analogs thereof specific to avidin; streptavidin and biotin, or analogs thereof specific to streptavidin; complementary oligonucleotides; or carbohydrates and lectins.

[0074] In some embodiments, the second labeling moiety can be a chemically modifiable portion. In this embodiment, a labeling moiety having reactive functional groups can be incorporated into a stable ternary complex. Subsequently, the functional groups can covalently react with the primary labeling moiety. Suitable functional groups include, but are not limited to, amino, carboxyl, maleimide, oxo, and thiol groups.

[0075] In alternative embodiments, the ternary complex may lack an exogenous marker. For example, the ternary complex and all components involved in the ternary complex (e.g., polymerase, template nucleic acid, primers, and / or homologous nucleotides) may lack one, several, or all of the exogenous markers described herein or in the references incorporated above. In such embodiments, the ternary complex may be detected based on the inherent properties of the stable ternary complex, such as mass, charge, inherent optical properties, etc. Exemplary methods for detecting unlabeled ternary complexes are set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1, 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 A1), each of which is incorporated herein by reference.

[0076] Typically, detection can be performed during the inspection step by sensing the inherent properties of the ternary complex or the labeled portion attached thereto. Exemplary properties upon which detection may be based include, but are not limited to, mass, conductivity, energy absorption, luminescence, etc. Detection of luminescence can be performed using methods known in the art related to nucleic acid arrays. The luminescent material can be detected based on any of a variety of luminescent properties, including, for example, emission wavelength, excitation wavelength, fluorescence resonance energy transfer (FRET) intensity, quenching, anisotropy, or lifetime. Other detection techniques that can be used in the methods described herein include, for example, mass spectrometry for sensing mass; surface plasmon resonance for sensing binding on a surface; absorbance at a wavelength for sensing the energy absorbed by the label; calorimetry for sensing temperature changes due to the presence of the label; conductivity or impedance for sensing the electrical properties of the 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, those set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1; PCT Application Serial No. PCT / US16 / 68916; or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245 A1); No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983 A1); No. 62 / 450,397, or No. 62 / 506,759, each of which is incorporated herein by reference.

[0077] Some embodiments of the methods described herein utilize two or more distinguishable signals to differentiate stable ternary complexes from each other and / or to differentiate one base type from another in a template nucleic acid. For example, two or more emitting sources can be differentiated from each other based on unique optical properties, such as unique excitation wavelengths or unique emission wavelengths. In a particular embodiment, the method can differentiate different stable ternary complexes based on differences in emission intensity. For example, a first ternary complex can be detected under conditions where a first ternary complex emits at a lower intensity than a second ternary complex. This intensity scaling (sometimes referred to as “grayscale scaling”) can utilize any distinguishable intensity difference. Exemplary differences include a particular stable ternary complex having an intensity that is at most 10%, 25%, 33%, 50%, 66%, or 75% higher than that of another stable ternary complex to be detected.

[0078] Intensity differences can be generated by using different luminescent emitters, for example, each emitter having a different extinction coefficient (i.e., resulting in different excitation characteristics) and / or different emission quantum yields (i.e., resulting in different emission characteristics). Alternatively, the same type of luminescent emitter can be used, but it can be present in different amounts. For example, all members of a first ternary complex group can be labeled with a specific luminescent emitter, while only half of the members of a second group are labeled with said luminescent emitter. In this example, the second group is expected to produce half the signal of the first group. The second group can be generated, for example, by using a mixture of labeled and unlabeled nucleotides (as opposed to the first group, which mainly contains labeled nucleotides). Similarly, the second group can be generated, for example, by using a mixture of labeled and unlabeled polymerases (as opposed to the first group, which mainly contains labeled polymerases). In alternative labeling schemes, the first ternary complex group may include polymerase molecules having multiple labels that produce a specific emission signal, and the second ternary complex group may include polymerase molecules each having only one of the labels that produce an emission signal.

[0079] In some embodiments, the examination step is performed in a manner that presumes the identity of at least one nucleotide type, for example, as described in commonly owned U.S. Patent No. 9,951,385 or U.S. Patent Application Sequence No. 15 / 922,787 (granted U.S. Patent No. 10,161,003), each of which is incorporated herein by reference. As an alternative or supplement to the use of interpolation, the examination step may use disambiguation to identify one or more nucleotide types, for example, as set forth in commonly owned U.S. Patent No. 9,951,385 or U.S. Patent Application Sequence No. 15 / 922,787 (granted U.S. Patent No. 10,161,003) (each of which is incorporated herein by reference).

[0080] The method disclosed herein can be carried out in a mode in which different nucleotide types are delivered sequentially and then removed from the container from which the ternary complex is to be formed and examined. In this mode, a first nucleotide type is delivered to the reaction vessel and then removed from the vessel before a second nucleotide type is delivered. When the nucleotides are removed, the polymerase can remain in the vessel. Thus, the polymerase can be initially delivered to the flow cell to create conditions that promote the formation of the ternary complex with the first nucleotide, and new polymerase can be added (but not necessarily) in subsequent deliveries to promote the formation of the ternary complex with subsequently delivered nucleotides.

[0081] Therefore, a method for identifying nucleotides in an induced template nucleic acid may include the following steps: (a) providing a container having an induced template nucleic acid, a polymerase, and a nucleotide homolog of a first base type; (b) checking the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound at a base position of the induced template nucleic acid; (c) removing the nucleotide homolog of the first base type from the container and delivering a nucleotide homolog of a second base type to the container, whereby the container retains the induced template nucleic acid and polymerase from step (b); (d) checking the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound at a base position of the induced template nucleic acid; and (e) identifying the type of nucleotide at the base position of the induced template nucleic acid.

[0082] Furthermore, a method for identifying nucleotides in an induced template nucleic acid may include the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting stable ternary complexes in the array; (d) removing the nucleotide homolog of the first base type from the array, and then repeating steps (b) and (c) for the nucleotide homolog of the second base type, thereby retaining the induced template nucleic acid and polymerase in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0083] In addition, a method for identifying nucleotides in an induced template nucleic acid is provided, comprising the following steps: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; (d) removing the nucleotide homologs of the first base type from the array, and then repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and the polymerase of the multiple polymerases in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0084] Nucleotide homologs can be removed from the container under conditions that dissociate the nucleotide from the priming template nucleic acid, thereby separating the nucleotide from the priming template nucleic acid without causing significant removal of the polymerase. For example, the dissociated nucleotides can be removed by moving the fluid away from the priming template nucleic acid, decanting the fluid away from the priming template nucleic acid, or separating the solid carrier attached to the priming template nucleic acid from the fluid. Another nucleotide (usually, but not always, a different type of nucleotide than the one previously removed) can then be delivered to the priming template nucleic acid. If the polymerase is essentially not removed due to the presence of the priming template nucleic acid, no additional polymerase needs to be delivered. This saves the time and resources that would otherwise be spent preparing more polymerase.

[0085] Nucleotides can be removed from the ternary complex using any of a variety of techniques without substantially removing the initiating template nucleic acid and polymerase. For example, both the initiating template nucleic acid and polymerase can be immobilized on a solid support such that disruption of the equilibrium conditions underlying the formation of the ternary complex will cause the nucleotides to dissociate into the solution and away from the immobilized components. Separation of the fluid from the immobilized components will cause the nucleotides to separate from the initiating template nucleic acid and polymerase. Simply reducing the concentration of unbound nucleotides in the fluid (e.g., by removing unbound nucleotides from the fluid surrounding the polymerase and the initiating template nucleic acid) will induce dissociation by shifting the binding equilibrium. As an alternative or supplement to reducing the nucleotide concentration, chemical or physical conditions that disrupt the non-covalent forces binding the components of the ternary complex can be used to dissociate the nucleotides from the ternary complex. Exemplary conditions will be described in further detail below.

[0086] Relatively long and flexible joints are particularly useful when immobilizing two components capable of participating in a ternary complex. This length and flexibility allow the two components to associate and dissociate when positioned on a solid support. Exemplary joints include, but are not limited to, those comprising polyethylene glycol (PEG), nucleic acids, peptide nucleic acids, peptides, polypropylene glycol, polyethylene, polypropylene, polyamide, polyester, etc. Exemplary joints and the reactive groups used for their attachment are described in Krishnamurthy et al., (2007) J. Am. Chem. Soc., 129:1312-1320 and U.S. Patent Application Publication No. 2016 / 0032379 A1, each of which is incorporated herein by reference.

[0087] In another exemplary embodiment, only one component of the ternary complex is immobilized. For example, the initiating template nucleic acid or polymerase can be immobilized on a solid support. Immobilization can be mediated by adapters (such as those described above) or by chemicals used to attach the analyte to an array described herein or in conjunction with an array cited in references herein. In such embodiments, the ternary complex can be dissociated using chemical or physical conditions that selectively dissociate nucleotides from the polymerase and the initiating template nucleic acid while maintaining association between the polymerase and the initiating template nucleic acid. This association can be used to retain the polymerase and the initiating template nucleic acid as long as either component of the pair is immobilized on a solid support. The nucleotides can then be removed by separating the nucleotide-containing fluid from the solid support. A single component of the ternary complex can be immobilized using adapters described above in the context of immobilizing two components. Other immobilization portions can also be used, regardless of whether they have the flexibility or length of the adapters exemplified above.

[0088] Physical conditions that can be used in the methods described herein to dissociate nucleotides from ternary complexes include, for example, raising the temperature to a range above the physiological range, thereby causing selective dissociation of nucleotides from the ternary complex, or an electric current that attracts charged nucleotide species away from the polymerase and nucleic acid. Nucleotides with physically manipulated portions include chromophores responsive to optical tweezers or optical trapping, ferromagnets or magnets responsive to magnetic manipulation, or double bonds that can be photoisomerized from a high-affinity binding state to a low-affinity binding state (or vice versa). In certain embodiments, physical conditions are selected to maintain polymerase binding to nucleic acids while dissociating nucleotides from both the polymerase and nucleic acids.

[0089] Chemical conditions that can be used to dissociate nucleotides from ternary complexes include, for example, high salt concentrations. Useful high salt conditions include, for example, salt concentrations of at least 50 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, or higher. Alternatively or supplemented, salt concentrations may be up to 500 mM, 400 mM, 300 mM, 200 mM, 150 mM, 100 mM, 50 mM, or lower. Useful salts include, but are not limited to, KCl, NaCl, guanidine thiocyanate, and other salts used in biochemical reactions.

[0090] Another useful chemical condition for dissociating nucleotides from ternary complexes is the presence of an organic solvent in an aqueous solution of at least 10% (v / v), 20% (v / v), 30% (v / v), 40% (v / v), 50% (v / v), or more. Optionally or additionally, the organic solvent may be present in an aqueous solution of no more than 50% (v / v), 40% (v / v), 30% (v / v), 20% (v / v), 10% (v / v), or less. Particularly useful organic solvents are alcohols, which may optionally be present in an aqueous solution of at least 10% (v / v), 20% (v / v), 30% (v / v), 40% (v / v), 50% (v / v), or more. Optionally or additionally, the alcohol may be present in an amount not exceeding 50% (v / v), 40% (v / v), 30% (v / v), 20% (v / v), 10% (v / v), or less in aqueous solution. Ethanol, methanol, isopropanol, glycols, and 1,3-butanediol are particularly useful alcohols. Other polar solvents may also be used, such as polar protic organic solvents (e.g., buffered organic acids) and polar aprotic organic solvents (e.g., DMSO, DMF). Typically, the organic solvent (e.g., alcohol) is miscible with the aqueous solution or is present in an amount soluble in the aqueous solution. In certain embodiments, both the salt and the organic solvent (e.g., alcohol) are present, for example, in the amounts described above.

[0091] Other useful chemical conditions for dissociating nucleotides from ternary complexes are pH values ​​outside the physiological range (e.g., at or below pH 6, 5, or 4; at or above pH 8, 9, or 10). Other reagents that may be used include, but are not limited to, redox agents such as dithiothreitol, glutathione, or 2-mercaptoethanol; detergents such as anionic, cationic, or zwitterionic detergents; or proteins that bind to nucleotides (e.g., proteins that compete with polymerases for nucleotide binding). The chemical conditions described herein for dissociating nucleotides from ternary complexes can be used in various combinations (e.g., aqueous solutions may have pH values ​​outside the physiological range and may also contain miscible organic solvents). Alternatively, one or more chemical conditions for dissociating nucleotides from ternary complexes may be combined with physical conditions for dissociating nucleotides from ternary complexes.

[0092] When the method is performed in a pattern of sequentially delivering different types of nucleotides to and then removing them from the reaction vessel, the ternary complex in the vessel can be examined after each delivery. In this pattern, different types of ternary complexes (i.e., ternary complexes containing different types of nucleotides) will form after each delivery. Ternary complexes formed in previous deliveries of other types of nucleotides will dissociate because the other types of nucleotides have been removed. Therefore, based on the expectation that one type of ternary complex will dominate in each examination, ternary complexes formed by each type of nucleotide can be identified. For example, when detecting ternary complexes based on the recruitment of labeled polymerases or labeled nucleotides to the initiating template nucleic acid in the array, the array feature with the highest signal can be identified as the feature where a ternary complex has formed. The type of ternary complex formed at each feature (i.e., the type of nucleotides present in the ternary complex) can be deduced from knowing which nucleotides were delivered prior to the examination step.

[0093] In this mode, there is no need to distinguish different types of ternary complexes using unique markers. Instead, different types of ternary complexes can be distinguished based on time information about when they form and which nucleotide type was delivered to induce formation. Different types of ternary complexes can be distinguishably labeled if desired. For example, each nucleotide type can have a marker that produces a signal distinct from all other nucleotide types used. Distinguishing markers can provide an advantage in increasing detection speed because a single check can be performed after the delivery of multiple different types of nucleotides. Time savings can be achieved by simultaneously delivering two or more distinguishably labeled nucleotide types in the method described herein. If desired, even when using distinguishable markers to identify different types of ternary complexes, checks can be performed after each nucleotide delivery.

[0094] The method disclosed herein can be carried out in a mode by sequentially delivering different nucleotide types into a container in which a ternary complex will be formed and examined. In this mode, a first nucleotide type can be delivered to the reaction container, followed by a second nucleotide type, allowing both nucleotide types to accumulate in the container. Multiple different types of ternary complexes can accumulate in the container when it contains various different initiating template nucleic acids, such as arrays or other multiplex forms. A polymerase may initially be added to create conditions that promote the formation of a ternary complex with the first nucleotide. New polymerases may (but not necessarily) be added in subsequent deliveries to promote the formation of a ternary complex with subsequently delivered nucleotides.

[0095] Therefore, a method for identifying nucleotides in an induced template nucleic acid may include the following steps: (a) providing a container having an induced template nucleic acid, a polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound at a base position of the induced template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the nucleotide homolog of the first base type, the induced template nucleic acid, and the polymerase from step (b); (d) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound at a base position of the induced template nucleic acid; and (e) identifying the type of nucleotide at the base position of the induced template nucleic acid.

[0096] A method for identifying nucleotides in an induced template nucleic acid may further include the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for a nucleotide homolog of a second base type, thereby retaining the induced template nucleic acid, the nucleotide homolog of the first base type, and the polymerase in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0097] A method for identifying nucleotides in an induced template nucleic acid may optionally include the following steps: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, whereby the induced template nucleic acid of the array, the nucleotide of the multiple nucleotides, and the polymerase of the multiple polymerases remain in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0098] When the method is performed in a pattern of sequentially delivering different types of nucleotides to a reaction vessel to allow for the accumulation of different nucleotides, the ternary complexes in the vessel can be examined after each delivery. In this pattern, different types of ternary complexes (i.e., ternary complexes containing different types of nucleotides) will be formed after each delivery. Ternary complexes formed in previously delivered other types of nucleotides will also be present in the vessel. Therefore, from one examination to the next, ternary complexes formed by each type of nucleotide can be identified based on the presence of newly formed ternary complexes. For example, when detecting ternary complexes based on the recruitment of labeled polymerases or labeled nucleotides to the initiating template nucleic acid in an array, an array feature with an enhanced signal intensity compared to the signal intensity detected for an array feature in a previous examination can be identified as a feature where new ternary complexes have formed. The type of ternary complex formed at each feature (i.e., the type of nucleotides present in the ternary complex) can be deduced from knowing which nucleotides were delivered prior to the examination step in which a new ternary complex signal appeared.

[0099] Therefore, it is not necessary to distinguish different types of ternary complexes using unique markers. Instead, different types of ternary complexes can be distinguished based on time information about when they form and which nucleotide type is delivered to induce the formation of the ternary complex. Different types of ternary complexes can be distinguishably labeled if desired. For example, two or more nucleotide types can have markers that produce signals that distinguish them from each other. In some embodiments, all nucleotide types can be distinguished based on unique markers. Thus, a marker can distinguish nucleotides that pair with one type of nucleotide in the template from nucleotides that pair with all other nucleotide types in the template. Distinguishing markers can provide the advantage of increased detection speed because a single check can be performed after all nucleotides have been delivered sequentially. If desired, even when using distinguishable markers to identify different types of ternary complexes, the check can be performed after each nucleotide delivery.

[0100] Multiple nucleotide delivery and inspection steps can be performed at a given location in the induced template nucleic acid. In sequencing implementations, multiple inspection steps can be performed in a subroutine during a single sequencing cycle prior to primer extension to proceed to the next sequencing cycle.

[0101] Therefore, this disclosure provides a method for identifying nucleotides in a template nucleic acid that has been initiated. The method may include the following steps: (a) providing a container having an initiating template nucleic acid, a polymerase, and a nucleotide homolog of a first base type; (b) checking the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound at a base position of the initiating template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the initiating template nucleic acid and the polymerase from step (b); (d) checking the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound at a base position of the initiating template nucleic acid; (e) identifying the type of nucleotide at a base position of the initiating template nucleic acid; (f) delivering a nucleotide homolog of a third base type to the container, whereby the container retains the initiating template nucleic acid and the polymerase from step (b); and (g) checking the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the third base type bound at a base position of the initiating template nucleic acid. Optionally, the method further includes the steps of: (h) delivering a fourth-base type nucleotide homolog to the container, whereby the container retains the induced template nucleic acid and polymerase from step (b); and (i) checking the container for the presence of a stable ternary complex comprising the polymerase and a fourth-base type nucleotide homolog bound to a base position of the induced template nucleic acid.

[0102] A method for identifying nucleotides in an induced template nucleic acid is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and polymerase in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, step (d) is performed as follows: repeating steps (b) and (c) for nucleotide homologs of the second base type, and then repeating steps (b) and (c) for nucleotide homologs of the third base type. Alternatively, step (d) may be performed as follows: repeat steps (b) and (c) for nucleotide homologs of the second base type, then repeat steps (b) and (c) for nucleotide homologs of the third base type, and then repeat steps (b) and (c) for nucleotide homologs of the fourth base type.

[0103] A method for identifying nucleotides in an induced template nucleic acid is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and the polymerase of the multiple polymerases in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c). Optionally, step (d) is performed as follows: repeating steps (b) and (c) for nucleotide homologs of the second base type, and then repeating steps (b) and (c) for nucleotide homologs of the third base type. Alternatively, step (d) may be performed as follows: repeat steps (b) and (c) for nucleotide homologs of the second base type, then repeat steps (b) and (c) for nucleotide homologs of the third base type, and then repeat steps (b) and (c) for nucleotide homologs of the fourth base type.

[0104] For ease of explanation, the methods of this disclosure are illustrated herein by way of example formed in the presence of one or more stable ternary complexes in the presence of nucleotide homologs of a single base type. It should be understood that one or more ternary complexes can be formed in the presence of one or more nucleotide homologs of only one base type, for example, in the presence of only a single type of nucleotide or in the presence of one or more nucleotide types that are homologs of the same base type. Alternatively, ternary complexes can be formed in the presence of a mixture of nucleotide types, which are homologs of more than one base type intended to be present in the template nucleic acid. For example, the nucleotide types present in a particular step of the methods described herein may be at least two, three, or four different base types of homologs intended to be present in the template nucleic acid. Optionally or additionally, the nucleotide types present in a particular step of the methods described herein may be up to four, three, or two different base types of homologs. Different nucleotide types may be mixed with each other before being delivered to a container in which an initiating template nucleic acid is generated. In other embodiments, different nucleotide types may be sequentially delivered to a container in which an initiating template nucleic acid is generated. Thus, different nucleotides will accumulate to produce a reaction mixture in which different types of nucleotides coexist with the initiating template nucleic acid.

[0105] Therefore, this disclosure provides a method for identifying nucleotides in an induced template nucleic acid, comprising the steps of: (a) providing a container having an induced template nucleic acid, a polymerase, a nucleotide homolog of a first base type, and a nucleotide homolog of a third base type; (b) checking the container for the presence of a stable ternary complex comprising the polymerase and (i) a nucleotide homolog of the first base type bound to a base position of the induced template nucleic acid or (ii) a nucleotide homolog of the third base type bound to a base position of the induced template nucleic acid; (c) delivering a nucleotide homolog of the second base type to the container, whereby the container retains the induced template nucleic acid and the polymerase from step (b); (d) checking the container for the presence of a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound to a base position of the induced template nucleic acid; and (e) identifying the type of nucleotide at the base position of the induced template nucleic acid. Optionally, step (c) further includes delivering a fourth base type nucleotide homolog to the container, and step (d) includes checking the container for a stable ternary complex comprising the polymerase and (i) a second base type nucleotide homolog bound to a base position of the induced template nucleic acid or (ii) a fourth base type nucleotide homolog bound to a base position of the induced template nucleic acid.

[0106] Furthermore, this disclosure provides a method for identifying nucleotides in an induced template nucleic acid, comprising the following steps: (a) providing an array of induced template nucleic acids; (b) forming a stable ternary complex comprising a polymerase, the induced template nucleic acid in the array, and a nucleotide homolog of a first base type, and forming a stable ternary complex comprising a polymerase, the induced template nucleic acid in the array, and a nucleotide homolog of a third base type; (c) detecting a stable ternary complex in the array comprising nucleotide homologs of the first and third base types; (d) repeating steps (b) and (c) for a nucleotide homolog of a second base type, wherein the induced template nucleic acid and polymerase are retained in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0107] A method for identifying nucleotides in an induced template nucleic acid is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases, multiple nucleotide homologs of a first base type, and multiple nucleotide homologs of a third base type to the array to form a stable ternary complex comprising a polymerase of the multiple polymerases, the induced template nucleic acid of the array, and nucleotides of the multiple nucleotide homologs of the first or third base type; (c) detecting stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, whereby the induced template nucleic acid of the array and the polymerase of the multiple polymerases remain in the array; and (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c).

[0108] The methods disclosed herein may include steps of modifying primers (e.g., extending primers by adding one or more nucleotides). In a particular embodiment, the nucleotides added to the primer will include a reversible terminator portion. The reversible terminator portion provides non-limiting benefits such as preventing more than one nucleotide from being added to the primer during extension and stabilizing the formation of a ternary complex at the 3' end of the primer during inspection.

[0109] Typically, the nucleotides used in the methods described herein, such as reversibly terminated nucleotides, do not have exogenous markers. This is because the methods described herein do not require detection of extension primers. However, if desired, one or more types of reversibly terminated nucleotides used in the methods described herein can be detected, for example, by an exogenous marker attached to the nucleotide.

[0110] The primer extension process or the process of forming a ternary complex does not require the use of a labeled polymerase. For example, it is not necessary to attach the polymerase used in the extension step to an exogenous label (e.g., covalently or otherwise). Alternatively, the polymerase used for primer extension may include an exogenous label, such as a label used in a previous inspection step.

[0111] Examples of reagents and conditions that can be used in polymerase-based primer extension steps include, for example, those set forth in commonly owned U.S. Patent Application Publication No. 2017 / 0022553 A1 or U.S. Patent Application Serial No. 15 / 677,870 (published as U.S. Patent Application Publication No. 2018 / 0044727 A1), No. 15 / 851,383 (published as U.S. Patent Application Publication No. 2018 / 0187245A1), No. 62 / 450,397, or No. 62 / 506,759, each of which is incorporated herein by reference. Exemplary reversible terminator portions, methods of incorporating them into primers, and methods of modifying primers for further extension (commonly referred to as "unblocking") 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. Further examples are illustrated in Bentley et al., Nature 456:53-59 (2008), WO04 / 018497; U.S. Patent Nos. 7,057,026, WO 91 / 06678, WO 07 / 123744, 7,329,492, 7,211,414, 7,315,019, 7,405,281, and US 2008 / 0108082 (each of which is incorporated herein by reference).

[0112] In certain embodiments, reagents used in primer extension are removed from contact with the initiated template nucleic acid prior to the step of forming a stable ternary complex with the primer-template hybrid. For example, removing the nucleotide mixture used in the extension step may be necessary when one or more types of nucleotides in the mixture would interfere with the formation or detection of the ternary complex in subsequent assay steps. Similarly, it may be necessary to remove polymerases or cofactors used in the primer modification step to prevent unwanted catalytic activity in subsequent assay steps. Following removal, a washing step may be performed, where an inert fluid is used to remove residual components of the reagent mixture used for primer modification from the primer-template hybrid.

[0113] Reagent removal or washing procedures may be performed between the various steps described herein. Such procedures can be used to remove one or more reagents present in the reaction vessel or on a solid support. For example, a reagent removal or washing step can be used to separate a primer-template hybrid from other reagents that have been in contact with the primer-template hybrid under conditions of a stable ternary complex. In a particular embodiment, reagent separation is facilitated by attaching a target reagent, such as an induced template nucleic acid, to a solid support and removing fluids in contact with the solid support. One or more reagents described herein may be attached to a solid support or provided in solution as needed, to suit the specific application of the method or apparatus described herein.

[0114] Reagent removal or washing procedures can be used to remove one or more reagents so as not to interfere with the examination of the ternary complex or to prevent contamination of a second ternary complex to be formed on (or in) a substrate that has previously been contacted with the reagents used to form the first ternary complex. For example, under conditions that stabilize the ternary complex, an initiating template nucleic acid can be contacted with a polymerase and at least one type of nucleotide to form a first mixture, and the first mixture or its product can be examined. However, reagent removal and washing need not be performed between the steps or processes described herein. For example, it may be necessary to avoid removing one or more reagents between examination steps. As further detailed elsewhere herein, when different ternary complex species are formed sequentially, it is not necessary to remove or wash away the polymerase or nucleotides used to form the first ternary complex species when forming the second ternary complex species.

[0115] Optionally, washing may be performed before detection to remove reagents that do not participate in the formation of a stable ternary complex. Optionally or additionally, washing may be performed after the detection step to remove one or more components of the first mixture from the primer-template hybrid. Then, under conditions stable for the ternary complex, the initiated template nucleic acid may be contacted with a polymerase and at least one other nucleotide to form a second mixture, and the formation of the ternary complex in the second mixture may be examined. As previously described, optional washing may be performed before the second detection to remove reagents that do not participate in the formation of a stable ternary complex.

[0116] If nucleotides present in the testing step are introduced into the primer extension process, they may cause unwanted side reactions, such as nucleotide incorporation. Therefore, a reagent removal or washing step can be performed before the primer extension step. Optionally, free nucleotides or other testing reagents can be modified or disabled, for example by enzymes such as phosphatases, by chemical modification, or by physical techniques.

[0117] This disclosure provides a method for sequencing an initiated template nucleic acid. The method may include the following steps: (a) providing a container having an initiated template nucleic acid, a first polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the first polymerase and a nucleotide homolog of the first base type bound to a base position of the initiated template nucleic acid; (c) delivering a nucleotide homolog of a second base type to the container, whereby the container retains the initiated template nucleic acid and the first polymerase from step (b); (d) checking the container for the presence of a stable ternary complex comprising the first polymerase and a nucleotide homolog of the second base type bound to a base position of the initiated template nucleic acid; (e) identifying the type of nucleotide at the base position of the initiated template nucleic acid; and (f) delivering a nucleotide homolog of a third base type to the container, whereby the container retains the initiated template nucleic acid and the first polymerase from step (b). (b) A polymerase; (g) Checking the container for a stable ternary complex comprising a first polymerase and a nucleotide homolog of a third base type bound to a base position of the priming template nucleic acid; (h) Delivering a nucleotide homolog of a fourth base type to the container, whereby the container retains the priming template nucleic acid and the first polymerase from step (b); (i) Checking the container for a stable ternary complex comprising a first polymerase and a nucleotide homolog of a fourth base type bound to a base position of the priming template nucleic acid; (j) Adding a nucleotide to a primer of the priming template nucleic acid, whereby the container contains an extended priming template nucleic acid; (k) Delivering a second polymerase and a nucleotide homolog of the first base type to the container; and (l) Repeating steps (b) to (i) using the extended priming template instead of the priming template nucleic acid and using the second polymerase instead of the first polymerase. The first polymerase may be a polymerase of the same type as the first type, or the first and second polymerases may be polymerases of different types.

[0118] A method for sequencing an initiated template nucleic acid is also provided, comprising the following steps: (a) providing a container having an initiated template nucleic acid, a first polymerase, and a nucleotide homolog of a first base type; (b) checking the container for the presence of a stable ternary complex comprising the first polymerase and a nucleotide homolog of the first base type bound to a base position of the initiated template nucleic acid; (c) delivering a nucleotide homolog of a second base type into the container, whereby the container retains the initiated template nucleic acid and the first polymerase from step (b); and (d) checking the container for the presence of a stable ternary complex. A ternary complex comprising a first polymerase and a nucleotide homolog of a second base type bound to a base position of the priming template nucleic acid; (e) identifying the type of nucleotide at the base position of the priming template nucleic acid; (f) adding the nucleotide to a primer of the priming template nucleic acid, thereby containing an extended priming template nucleic acid; (g) delivering the second polymerase and the nucleotide homolog of the first base type to the container; and (h) repeating steps (b) to (e) using the extended priming template instead of the priming template nucleic acid and using the second polymerase instead of the first polymerase. The first polymerase may be a polymerase of the same type as the first type, or the first polymerase and the second polymerase may be polymerases of different types.

[0119] A method for sequencing induced template nucleic acids is also provided, comprising the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a first polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for a nucleotide homolog of a second base type, then repeating steps (b) and (c) for a nucleotide homolog of a third base type, then repeating steps (b) and (c) for a nucleotide homolog of a fourth base type, thereby retaining the induced template nucleic acid and the first polymerase in the array; (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c); (f) adding nucleotides to primers for each induced template nucleic acid, thereby including an extended induced template nucleic acid in the array; and (g) repeating steps (b) to (e) using the extended induced template in place of the induced template nucleic acid and using the second polymerase in place of the first polymerase. The first polymerase can be the same type of polymerase as the first type, or the first polymerase and the second polymerase can be polymerases of different types.

[0120] A method for sequencing induced template nucleic acids may include the following steps: (a) providing an array of induced template nucleic acids; (b) forming stable ternary complexes, each ternary complex comprising a first polymerase, a nucleotide homolog of a first base type, and the induced template nucleic acid in the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, thereby retaining the induced template nucleic acid and the first polymerase in the array; (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c); (f) adding nucleotides to primers for each induced template nucleic acid, thereby including an extended induced template nucleic acid in the array; and (g) repeating steps (b) through (e) using the extended induced template nucleic acid instead of the induced template nucleic acid and using a second polymerase instead of the first polymerase. The first polymerase may be a polymerase of the same type as the first type, or the first polymerase and the second polymerase may be polymerases of different types.

[0121] A method for sequencing induced template nucleic acids is also provided, comprising the steps of: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting stable ternary complexes in the array; and (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, and then for nucleotide homologs of a third base type. (b) and (c) are repeated for nucleotide homologs, and then (b) and (c) are repeated for nucleotide homologs of the fourth base type, thereby retaining the initiating template nucleic acid and the polymerases in the array in the array; (e) the type of nucleotide present in each stable ternary complex detected in step (c) is identified; (f) a nucleotide is added to the primer of each initiating template nucleic acid, thereby the array contains the extended initiating template nucleic acid; and (g) steps (b) to (e) are repeated using the extended initiating template instead of the initiating template nucleic acid and using the multiple second polymerases instead of the multiple polymerases.

[0122] A method for sequencing induced template nucleic acids may include the following steps: (a) providing an array of induced template nucleic acids; (b) delivering multiple polymerases and multiple nucleotide homologs of a first base type to the array to form stable ternary complexes, each ternary complex comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and the induced template nucleic acid of the array; (c) detecting the stable ternary complexes in the array; (d) repeating steps (b) and (c) for nucleotide homologs of a second base type, whereby the induced template nucleic acid of the array and the polymerase of the multiple polymerases are retained in the array; (e) identifying the type of nucleotide present in each stable ternary complex detected in step (c); (f) adding nucleotides to primers for each induced template nucleic acid, whereby the array contains extended induced template nucleic acids; and (g) repeating steps (b) to (e) using extended induced templates instead of induced template nucleic acids and using multiple second polymerases instead of multiple polymerases.

[0123] When included in the methods described herein, the unblocking process facilitates the sequencing of the initiated template nucleic acid. The unblocking process can be used to convert a reversibly terminated primer into an extendable primer. Primer extension can then be used to move the ternary complex formation site along the template nucleic acid to different locations. Repeated cycles of extension, checking, and unblocking can be used to reveal the sequence of the template nucleic acid. Each cycle reveals subsequent bases in the template nucleic acid. Exemplary reversible terminator portions, methods of incorporating them into primers, and methods of modifying primers for further extension (commonly referred to as "unblocking") are described in U.S. Patents 7,427,673, 7,414,116, 7,544,794, 7,956,171, 8,034,923, 8,071,755, 8,808,989, or 9,399,798. Further examples are illustrated 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.

[0124] Sequencing methods may include multiple repetitions of the cycles described herein or steps within cycles. For example, a cycle including inspection and primer extension steps may be repeated multiple times. Optionally, the cycle may also include a step of unblocking primers, or a step of washing away unused reactants or used products between steps. Thus, the induced template nucleic acid may undergo at least 2, 5, 10, 25, 50, 100, 150, 200 or more repeated cycles of the methods described herein. Fewer cycles may be performed when a shorter read length is sufficient. Thus, the induced template nucleic acid may undergo up to 200, 150, 100, 50, 25, 10, 5 or 2 cycles of the methods described herein.

[0125] In some implementations, the sequencing method can be repeated a predetermined number of times. Alternatively, these cycles can be repeated until a specific empirical observation state 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] Although this document illustrates embodiments of the present disclosure using repeating cyclic sequencing reactions, the cycles do not need to be repeated, nor do they need to include a primer extension step. For example, genotyping can be performed by examining individual nucleotide positions in a template nucleic acid by forming a stable ternary complex. Genotyping can be performed using sequential delivery and / or accumulation of nucleotide homologs of different base types. Examples of genotyping techniques that can be modified to employ the nucleotide delivery methods described herein include those described in commonly owned U.S. Patent No. 9,932,631 (which is incorporated herein by reference).

[0127] Any of a variety of polymerases can be used in the methods or apparatus described herein, for example, to form stable ternary complexes or to perform primer extension. Usable polymerases include naturally occurring polymerases and their modified variants, including but not limited to mutants, recombinants, fusions, genetically modified forms, chemically modified forms, compounds, and analogs. Naturally occurring polymerases and their modified variants are not limited to polymerases capable of catalyzing polymerization reactions. Optionally, their naturally occurring variants and / or modified variants have the ability to catalyze polymerization reactions under at least one condition not used during the formation or inspection of the stable ternary complex. Optionally, the naturally occurring variants and / or modified variants involved in the stable ternary complex have modified properties, such as 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, for example, polymerases in which one or more amino acids are substituted by other amino acids or by the insertion or deletion of one or more amino acids. Exemplary polymerase mutants that can be used to form stable ternary complexes include, for example, wild-type and mutant polymerases set forth in U.S. Patent Application Serial No. 15 / 866,353 (published as U.S. Patent Application Publication No. 2018 / 0155698A1) or U.S. Patent Application Publication No. 2017 / 0314072, each of which is incorporated herein by reference.

[0128] The modified polymerase includes a polymerase containing an exogenous labeled moiety (e.g., an exogenous fluorophore) that can be used to detect the polymerase. Optionally, the labeled moiety can be attached after the polymerase has been at least partially purified using protein separation techniques. For example, the exogenous labeled moiety can be covalently linked to the polymerase using the free thiol or free amine moiety of the polymerase. This may involve covalently linking the polymerase via a side chain of a cysteine ​​residue or via a free amino group at the N-terminus. The exogenous labeled moiety can also be attached to the polymerase via protein fusion. Exemplary labeled moieties that can be attached via protein fusion include, for example, 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.

[0129] Alternatively, polymerases involved in stabilizing ternary complexes or used for extending primers do not require attachment to exogenous labels. For example, polymerases do not require covalent attachment to exogenous labels. Instead, polymerases may be unlabeled until they bind to labeled nucleotides and / or labeled nucleic acids (e.g., labeled primers and / or labeled templates).

[0130] Different activities of polymerases can be utilized in the methods described herein. Polymerases can be used, for example, in primer extension steps, inspection steps, or combinations thereof. Different activities can arise from structural differences (e.g., through natural activity, mutation, or chemical modification). However, polymerases are available from a variety of known sources and can be used in accordance with the teachings and recognized polymerase activities described herein. Useful DNA polymerases include, but are not limited to, bacterial DNA polymerases, eukaryotic DNA polymerases, archaea DNA polymerases, viral DNA polymerases, and bacteriophage DNA polymerases. Bacterial DNA polymerases include E. coli DNA polymerases I, II, and III, IV, and V, the Klenow fragment of E. coli DNA polymerase, Clostridium tridium stercorarium (Cst) DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase, and Sso (Sulphurophytum) DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases α, β, γ, δ, ε, η, ζ, λ, σ, μ, and κ, as well as Rev1 polymerase (terminal deoxycytidine transferase) and terminal deoxynucleotide 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, Cp1 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 Turbo Pfu DNA polymerase, *Thermococcus litoralis* (Tli) DNA polymerase, and a certain GB-D (Pyrococcus sp.) of the genus *Pyrococcus*.GB-D) polymerase, Thermotoga maritima (Tm a) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase, a certain Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase, Sulfolobus acidocaldarius DNA polymerase, a certain Thermococcus sp. go N-7 (Go N-7) DNA polymerase, Pyrodictiu moccultum DNA polymerase, Methanococcus voltae DNA DNA polymerases including A, *Methanococcus thermoautotrophicum* DNA polymerase, *Methanococcus jannaschii* DNA polymerase, *Desulfurococcus* strain TOK DNA polymerase (D. TokPol); *Pyrococcus horikoshii* DNA polymerase, *Pyrococcus islandicum* DNA polymerase, *Thermococcus fumicolans* DNA polymerase, *Aero pyrum pernix* DNA polymerase, and heterodimeric DNA polymerases DP1 / DP2. Engineered and modified polymerases can also be used in conjunction with the disclosed techniques. For example, a modified form of the extreme thermophilic marine archaea *Thercoccus* species 9°N can be used (e.g., Therminator DNA polymerase from New England BioLabs Inc.; Ipswich, MA). Other useful DNA polymerases, including 3PDX polymerase, are disclosed in US 8,703,461 (the disclosure of which is incorporated herein by reference).

[0131] 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.

[0132] Another useful type of polymerase is reverse transcriptase. Exemplary reverse transcriptases include, but are not limited to, HIV-1 reverse transcriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse transcriptase from human immunodeficiency virus type 2, M-MLV reverse transcriptase from Moloney murine leukemia virus, AMV reverse transcriptase from avian myeloblastoma virus, and telomerase reverse transcriptase for maintaining eukaryotic chromosome telomeres.

[0133] For some implementations, polymerases with intrinsic 3'-5' proof-of-care exonuclease activity can be useful. Polymerases that substantially lack 3'-5' proof-of-care exonuclease activity are also useful in some implementations, for example, in most genotyping and sequencing implementations. The absence of exonuclease activity can be a wild-type characteristic or a characteristic conferred by a variant or engineered polymerase structure. For example, the exominus Klenow fragment is a mutant form of the Klenow fragment that lacks 3'-5' proof-of-care exonuclease activity. The Klenow fragment and its exominus variants can be used in the methods or compositions described herein.

[0134] Stable ternary complexes, or components capable of forming ternary complexes, can be attached to a solid support. The solid support can be made from any of a variety of materials used in analytical biochemistry. Suitable materials may include glass, polymers, silicon, fused silica, borofloat glass, silica, silicon-based materials, carbon, metals, optical fibers or fiber bundles, sapphire, or plastics. Materials can be selected based on the properties required for a specific application. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques utilizing that wavelength. Conversely, materials that do not transmit radiation at a certain wavelength may need to be selected (e.g., opaque, absorptive, or reflective). Other usable material properties include inertness or reactivity to certain reagents used in downstream processes, ease of handling, or low manufacturing cost.

[0135] Particularly useful solid carriers are particles, such as beads or microspheres. Bead populations can be used for the attachment of stable ternary complex populations or components capable of forming complexes (e.g., polymerases, templates, primers, or nucleotides). In some embodiments, it may be useful to use a configuration in which each bead has a single type of stable ternary complex or a single type of component capable of forming complexes. For example, a single bead can be attached to a single type of ternary complex, a single type of initiating template nucleic acid, 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 do not need to be separated on a bead-by-bead basis. In this way, a single bead can carry multiple different types of ternary complexes, template nucleic acids, primers, initiating template nucleic acids, 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 carriers for protein and nucleic acid capture methods and chemical functionalities imparted to them. Such compositions include, for example, plastics, ceramics, glass, polystyrene, melamine, methylstyrene, acrylic polymers, paramagnetic materials, thorium oxide sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran, such as Sepharose. TM Cellulose, nylon, cross-linked micelles and Teflon TM Other materials described in the "Microsphere Detection Guide" of Bangs Laboratories, Fishers Ind (which is incorporated herein by reference).

[0136] The geometry of particles such as beads or microspheres can also correspond to a variety of different forms and shapes. For example, particles can be symmetrical (e.g., spherical or cylindrical) or irregular (e.g., controlled-pore glass). Additionally, particles can be porous, thus increasing the surface area available for trapping ternary composites or their components. Exemplary sizes of the beads used herein can range from nanometers to millimeters or from about 10 nm to 1 mm.

[0137] In certain implementations, the beads may be arranged in a specific pattern or otherwise spatially differentiated. Exemplary bead-based arrays that may be used include, but are not limited to, BeadChips available from Illumina, Inc. (San Diego, CA). 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 / 0263923A1, 2004 / 0233485A1, 2004 / 0132205A1, or 2004 / 0125424A1 (each of which is incorporated herein by reference).

[0138] 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. 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., induced template nucleic acids 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 a feature will indicate the specific nucleic acid sequence present at that feature.

[0139] 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, commercially available from Amersham Biosciences. TMArrays. Another useful array method is to use inkjet printing, such as SurePrint, which is commercially available from Agilent Technologies. TM An array manufactured using technology.

[0140] Other useful arrays include those used for nucleic acid sequencing applications. For example, arrays for attaching amplicon (often called clusters) to genomic fragments can be particularly useful. Examples of nucleic acid sequencing arrays that can be used in this paper include those described in the following literature: Bentley et al., Nature 456:53-59 (2008), PCT Publication Nos. WO 91 / 06678, WO04 / 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.

[0141] Nucleic acids can be attached to vectors in a manner that provides detection at the single-molecule or ensemble level. For example, multiple different nucleic acids can be attached to a solid vector in such a way that a single, stable ternary complex formed on a single nucleic acid molecule on the vector can be distinguished from all adjacent ternary complexes formed on the nucleic acid molecules on the vector. Similarly, one or more different templates can be attached to a solid vector in a manner that physically isolates and detects each single-molecule template in a way that resolves the single molecule from all other molecules on the solid vector.

[0142] Alternatively, the methods of this disclosure can be applied to one or more nucleic acid ensembles, an ensemble being a population of nucleic acids having a common template sequence. An ensemble may include, for example, at least 2, 10, 50, 100, 500, 1000, or more nucleic acids having a common template sequence. Optionally or additionally, an ensemble may include up to 1000, 500, 100, 50, 10, or 2 nucleic acids having the same template sequence. An ensemble present at a feature of the array may be cloned, such that substantially all nucleic acids at the feature have a common template sequence. However, the feature does not need to contain a clonal population of nucleic acids. Instead, the feature may include a mixed population of nucleic acids in which a particular template sequence is present in the majority of the nucleic acids. For example, a population of nucleic acids at a particular feature may include at least 51%, 60%, 75%, 90%, 95%, or 99% or more of the species having the particular template sequence. Features of non-clonal populations of nucleic acids can be detected under conditions that allow the population to be detected as an ensemble, thereby obtaining a total signal from the feature representing the average value of the signal generated by the non-clonal population. As long as the contaminating nucleic acids are present in the minority at the target feature, the average signal can provide a means of characterizing the majority of the template nucleic acids at that feature.

[0143] Clustering methods can be used to attach one or more ensembles to a solid vector. Thus, an array can have multiple ensembles, each referred to as a cluster or array feature in this format. Clusters can be formed using methods known in the art, such as bridging amplification or emulsion PCR. Useful bridging amplification methods are described, for example, in U.S. Patent Nos. 5,641,658 or 7,115,400, or U.S. Patent Publications 2002 / 0055100A1, 2004 / 0002090A1, 2004 / 0096853A1, 2007 / 0128624A1, or 2008 / 0009420A1. Emulsion PCR methods include, for example, those described in the following literature: Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05 / 010145, or US Patent Publication No. 2005 / 0130173A1 or No. 2005 / 0064460A1, 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), as described, for example, in Lizardi et al., Nat. Genet. 19:225-232 (1998) or US 2007 / 0099208A1 (each of which is incorporated herein by reference).

[0144] In certain embodiments, a stable ternary complex, polymerase, primers, template, and initiated template nucleic acid or nucleotide are attached to a solid support on or within a flow cell surface. The flow cell allows for convenient fluid manipulation by moving a solution into and out of a fluid chamber that contacts the ternary complex bound to the support. The flow cell also provides for the detection of the fluidly manipulated components. For example, a detector may be positioned to detect signals from the solid support, such as signals from markers recruited to the solid support due to the formation of the stable ternary complex. Exemplary flow cells that may be used are described, for example, in U.S. Patent Application Publication No. 2010 / 0111768A1, WO05 / 065814, or U.S. Patent Application Publication No. 2012 / 0270305A1 (each of which is incorporated herein by reference).

[0145] The nucleic acids used in the methods or compositions described herein can be DNA, such as genomic DNA, synthetic DNA, amplified DNA, complementary DNA (cDNA), etc. RNA, such as mRNA, ribosomal RNA, tRNA, etc., can also be used. Nucleic acid analogs can also be used as templates in this paper. Therefore, the template nucleic acids used herein can be derived from biological sources, synthetic sources, or amplification products. The primers used herein can be DNA, RNA, or analogs thereof.

[0146] 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%, or 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.

[0147] Exemplary organisms from which nucleic acids may be derived include, for example, those derived 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 soybeans; 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 African clawed frogs; Dictyostelium discoideum; fungi such as Pneumocystis carinii, Takifugu rubripes, yeasts, Saccharomyces cerevisiae, or Schizosaccharomyces cerevisiae; or Plasmodium falciparum. Nucleic acids can also originate from prokaryotes, such as bacteria: *Escherichia coli*, *Staphylococci*, or *Mycoplasma pneumoniae*; archaea; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Nucleic acids can originate from homogeneous cultures or populations of the above-mentioned organisms, or optionally from a collection of several different organisms, for example, 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, 3rd Edition, Cold Spring Harbor Laboratory, New York (2001)* or *Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998)* (each of which is incorporated herein by reference).

[0148] Template nucleic acids can be obtained through preparation methods such as genome isolation, genome fragmentation, gene cloning, and / or amplification. Templates can be obtained through amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and multiple displacement amplification (MDA). Exemplary methods for isolating, amplifying, and fragmenting nucleic acids to produce templates for array analysis are described 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 described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, New York (2001) or Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998) (each of which is incorporated herein by reference).

[0149] Optionally, in the apparatus of this disclosure, multiple initiating template nucleic acids are attached to a solid support. The solid support may include any of the various materials described herein, including, for example, those described herein in the context of nucleic acid arrays. Multiple initiating template nucleic acids may be attached to features of the array, and optionally, the templates attached to the features may have the same sequence. Any of the various reagents described herein may be attached to the solid support instead of the initiating template nucleic acid, or alternatively, any of the various reagents described herein may be attached to the solid support in addition to the attached initiating template nucleic acid. In certain embodiments, the apparatus of the present invention does not require attachment to any type of reagent.

[0150] In certain embodiments, the apparatus of this disclosure includes containers, such as artificial containers. The containers may contain a variety of initiating template nucleic acids and other reagents or reaction products involved in the methods described herein. Particularly useful artificial containers are flow cells, examples of which have been described above.

[0151] The system disclosed herein can be configured for, for example, the detection of nucleic acids using the methods described herein. For example, the system can be configured to generate and detect a reaction in the presence of nucleotides that produces a ternary complex formed between a polymerase and an initiated template nucleic acid to identify one or more bases in the template nucleic acid sequence. Optionally, the system includes components and reagents for performing one or more steps described herein, including but not limited to: forming at least one stable ternary complex between the initiated template nucleic acid, polymerase, and the next correct nucleotide; detecting one or more stable ternary complexes; extending primers for each primer-template hybrid; unblocking reversibly terminated primers; and / or identifying nucleotides or nucleotide sequences in the template.

[0152] The systems disclosed herein may include containers, solid carriers, or other devices for performing nucleic acid detection methods. For example, the systems may include arrays, flow cells, multiwell plates, test tubes, channels in a substrate, collections of droplets or vesicles, trays, centrifuge tubes, tubing, or other convenient devices. Devices may be removable, allowing them to be placed into or removed from the system. Thus, the system can be configured to process multiple devices (e.g., containers or solid carriers) continuously or in parallel. The systems may include fluid components having containers for containing one or more reagents described herein (e.g., polymerases for forming ternary complexes, primers, template nucleic acids, one or more nucleotides, nucleotides for primer extension, unblocking reagents, ternary complex inhibitors, or mixtures of such components). The fluid system may be configured to deliver reagents to containers or solid carriers, for example, via channels or droplet transfer devices (e.g., electrowetting devices). Any of a variety of detection devices may be configured to detect containers or solid carriers interacting with reagents. Examples include luminescent detectors, surface plasmon resonance detectors, and other detectors known in the art. Exemplary systems having fluid and detection components that can be readily modified for use in the systems described herein include, but are not limited to, those described in U.S. Patent Application Publication No. 2018 / 0280975A1 (which claims priority to U.S. Patent Application 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 / 0272914A1 or 2012 / 0270305A1 (each of which is incorporated herein by reference).

[0153] Optionally, the system disclosed herein also includes a computer processing unit (CPU) configured as an operating system component. The same or different CPUs can interact with the system to acquire, store, and process signals (e.g., signals detected in the methods described herein). In certain embodiments, the CPU can be used to determine the identity of nucleotides present at specific locations in the template nucleic acid based on signals. In some cases, the CPU will identify the nucleotide sequence of the template based on the detected signals.

[0154] Useful CPUs may include one or more of the following: personal computer systems, server computer systems, thin clients, fat clients, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics, network PCs, minicomputer systems, mainframe computers, smartphones, and distributed cloud computing environments that include any of the aforementioned systems or devices. A CPU may include one or more processors or processing units and a memory architecture that may include RAM and non-volatile memory. The memory architecture may also include removable / non-removable, volatile / non-volatile computer system storage media. Additionally, the memory architecture may include one or more readers for reading from and writing to non-removable, non-volatile magnetic media, such as hard disk drives, disk drives for reading from and writing to removable, non-volatile disks, and / or optical disk drives for reading from and writing to removable, non-volatile optical disks, such as CD-ROMs or DVD-ROMs. A CPU may also include various computer system-readable media. This media may be any available media accessible in a cloud computing environment, such as volatile and non-volatile media, as well as removable and non-removable media.

[0155] A memory architecture may include at least one program product having at least one program module implemented as executable instructions, configured to perform one or more steps of the methods described herein. For example, executable instructions may include an operating system, one or more application programs, other program modules, and program data. Typically, program modules may include routines, programs, objects, components, logic, data structures, etc., that perform the specific tasks described herein.

[0156] CPU components can be coupled via an internal bus, which can be implemented as any one or more of several types of bus architectures, 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 the Industry Standard Architecture (ISA) bus, the Micro Channel Architecture (MCA) bus, the Enhanced ISA (EISA) bus, the Video Electronics Standards Association (VESA) local bus, and the Peripheral Component Interconnect (PCI) bus.

[0157] The CPU can optionally communicate with one or more external devices such as a keyboard, pointing device (e.g., mouse), display (e.g., 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., via a network card, Bluetooth). TM (e.g., WiFi modems, etc.). This communication can be performed via I / O interfaces. Furthermore, the CPU of the system described herein can communicate with one or more networks, such as local area networks (LANs), wide area networks (WANs), and / or public networks (e.g., the Internet), via suitable network adapters.

[0158] Example I

[0159] Through Binding TM Efficient methods for delivering nucleotides during sequencing

[0160] This embodiment describes the use of Binding TM The sequencing procedure involves sequentially delivering different types of nucleotides to an array of template nucleic acids in the presence of polymerase to form ternary complexes. Each delivery is followed by an examination to distinguish one type of ternary complex from another. The results shown here indicate that altering the reagent delivery or washing steps leads to improvements such as shorter cycle times, reduced reagent consumption, and improved sequencing results.

[0161] The following describes the preparation of flow cells containing the template nucleic acid as a starter. Template nucleic acid strands synthesized in 12 PCR reactions using 5'-biotinylated primers were individually bound to magnetic beads coated with streptoacidin. This resulted in a population of 12 bead types, each containing a homogeneous set of template strands. The beads used in the procedure were functionalized with 1 mM NHS-PEG4-TCO in phosphate-buffered saline (PBS). Next, the beads with the immobilized template strands were flown through the surface of a flow cell containing tetrazine-functionalized aminosilane. The mixture was incubated for 1 hour to allow the modified beads to covalently attach to the functionalized surface within the flow cell. Sequencing primers were then introduced into the flow cell and hybridized with the immobilized template strands.

[0162] Sequencing is performed through repeated cycles. The sequencing cycle is initiated by incorporating a reversible terminator nucleotide at the 3' end of the hybridized sequencing primers to generate a closed set of template nucleic acid molecules. This is achieved by delivering RTS (containing: 50 mM Tricine pH 8.4, 0.1% Tween-80, and 40 U / ml Therminator) to the flow cell. TMThe procedure was performed using polymerase, 5 mM MgCl2, 0.1% hydroxylamine, 50 mM KCl, 0.1% Tween-80, 0.1 mM EDTA, and 200 nM of unlabeled reversibly terminated nucleotide analogs dATP, dGTP, dCTP, and dTTP. The reversible terminator nucleotides used in this illustrative procedure comprise a 3'-ONH2 reversible terminator moiety. A description of this reversible terminator nucleotide can be found in U.S. Patent No. 7,544,794, the disclosure of which is incorporated herein by reference. The flow cell was then washed with ESB solution (1 M guanidine thiocyanate, 60 mM HEPES, 0.1% Tween-80, 0.1% hydroxylamine, and 2 mM EDTA) and then with PRE solution (50 mM Tricine pH 8.4, 50 mM KCl, 0.1% Tween-80, 0.1% hydroxylamine, and 0.1 mM EDTA).

[0163] The loop then continues using a check subroutine, in which four different nucleotides are sequentially delivered to the flow cell. In the ternary complex formation and detection steps, a reversible terminator portion on the 3' nucleotide of the primer strand excludes nucleotide incorporation. Under standard conditions, one of the four differently labeled nucleotides is delivered to EXAM solution (400 nM for each of Cy5-dNTP, Cy5-dGTP, or Cy5-dCTP; or 800 nM for Cy5-dTTP), 1 mg MgCl2, and a Therminator in 20 U / ml IMG solution. TM The ternary complex was placed in a flow cell containing polymerase and then washed with IMG solution (20 mM Tricine pH 7.0, 1 M betaine, 50 mM LiCl, 0.1% Tween-80, 50 mM KCl, 10 mM ammonium sulfate, 0.1% hydroxylamine, and 0.1 mM EDTA). Cy5-dNTP nucleotides are described in U.S. Patent Application Sequence No. 15 / 873,343 (published as U.S. Patent Application Publication No. 2018 / 0208983A1, which is incorporated herein by reference). The flow cell was imaged by fluorescence microscopy to detect the ternary complex retained in the IMG solution. After imaging, the flow cell was washed with ESB solution and then with PRE solution. The steps of the subroutine were repeated individually for each of the four nucleotide types. As illustrated below in the context of the figures, the examination subroutine was modified in several experiments.

[0164] Following the check subroutine, the sequencing cycle continues, during which the reversible terminator portion of the primers is removed by treating the flow cell with a solution containing 0.25 M sodium acetate and 0.7 M sodium nitrite (titrated to pH 4.8 with acetic acid). The flow cell is then washed in PRE solution to remove sodium acetate and sodium nitrite. The sequencing process then returns to the sequencing cycle initiation step.

[0165] Figure 1 This demonstrates the binding achieved using the standard conditions described above. TM The sequencer signal intensity is compared with the sequencing cycle. Independent traces of the "on" intensity and "off" intensity detected for each nucleotide type are shown. For each bead in each cycle, the nucleotide type producing the highest signal is identified as the "on" signal, and the other three nucleotide types are identified as the "off" signal. The "on" signal for each nucleotide type is averaged across all bead types detected in a given cycle, and an average intensity map is plotted over 100 cycles to obtain each "on" signal trace shown in the figure. A similar averaging of the signal intensity for all bead types is performed on a cycle-by-cycle basis to obtain... Figure 1 The intensity trace of "off" is shown.

[0166] The signal attenuation of the "open" trace was evaluated by fitting the trace to a curve defined by the following formula:

[0167] I = I0e-(n / τ) (Equation 1)

[0168] Where I is the signal intensity, n is the cycle number, and τ is the cycle length at which the signal is approximately 37% of I0 (the initial signal intensity). A higher τ indicates a slower signal decay rate, which is generally preferred for increasing read length and sequencing accuracy, while a faster signal decay rate is characterized by a lower τ value. The goodness of fit is calculated as the coefficient of determination R. 2 Higher R 2 The value is associated with a decrease in signal intensity variance due to variability from the sequence background, while an increase in the adverse effects of the sequence background leads to a lower R value. 2 value. Figure 1 The standard trajectory shown has an average τ value of 37 and an average R value of 0.88. 2 Value (average value taken on all four nucleotide types of open traces).

[0169] Experiments were conducted to test the effect of different concentrations of NaCl in the washing solution used between imaging steps in the inspection routine. Specifically, the ESB was replaced with a salt solution between imaging steps in the inspection subroutine. Salt concentrations tested were 1 M GdSCN (standard wash), no salt, 64 mM NaCl, 160 mM NaCl, 400 mM NaCl, 1 M NaCl, and 2.5 M NaCl. Results showed that using lower salt washes (e.g., 0 mM, 64 mM, or 160 mM salt) resulted in higher “on” signal intensity compared to using higher salt washes (400 mM NaCl, 1 M NaCl, 2.5 M NaCl, and 1 M GdSCN). Lower salt washes also resulted in smaller changes in signal intensity compared to standard washes.

[0170] Figure 2 Showing the effect of Binding TM The diagram showing the signal intensity of the sequencing cycle compared to the sequencing sequence was presented in the Binding diagram. TM The protocol did not include polymerase in the EXAM solution delivered during the inspection subroutine. Instead, the polymerase was retained in the flow cell from the previous RTS delivery. Surprisingly, the polymerase was retained throughout multiple reagent deliveries and imaging steps in the sequencing cycle. Furthermore, retaining the polymerase resulted in smaller changes in signal intensity compared to the standard procedure. However, the "off" intensity was higher when the polymerase was retained, especially for the C and T traces, compared to standard conditions.

[0171] Figure 3 Showing the effect of Binding TM The diagram showing the signal intensity of the sequencing cycle compared to the sequencing sequence was presented in the Binding diagram. TM The standard conditions were adjusted by replacing ESB and PRE washing with an eluent containing salt and ethanol. Specifically, the eluent contained 50 mM Tricine pH 8.4, 150 mM KCl, 0.1% Tween-80, 0.1% hydroxylamine, and 0.1 mM EDTA, along with 25% ethanol. Furthermore, under the modified conditions, polymerase was absent from any EXAM solution delivered in the examination subroutine. Instead, the polymerase was retained in the flow cell from the previous RTS delivery. As previously stated, the results indicate that polymerase was retained throughout the multiple reagent deliveries and imaging steps performed in the examination subroutine, and that retaining the polymerase resulted in less change in signal intensity compared to the standard procedure. However, the use of ethanol with high salt resulted in a reduced “off” intensity, compared to… Figure 2 The results were somewhat improved compared to the previous ones.

[0172] exist Figure 3 The sequencing protocol was run for 150 cycles under the aforementioned conditions. The run's τ=53 value indicates that, compared to... Figure 1 Compared to the standard condition τ=37 plotted in the diagram, the signal attenuation is improved. The use of salt / ethanol washing solution in the inspection subroutine also resulted in... Figure 1 Standard conditions (R) 2 Compared to (=0.88), the improved run reduces sequence background artifacts (R0.88). 2 =0.94).

[0173] The sequencing protocol was performed for 100 cycles, with standard conditions adjusted by omitting ESB and PRE washes between imaging steps. Furthermore, under modified conditions, polymerase was absent from any EXAM solutions delivered during the inspection subroutine. Instead, polymerase was retained in the flow cell from the previous RTS delivery. Therefore, different types of ternary complexes (i.e., ternary complexes bound with different types of homologous nucleotides) accumulated during the inspection subroutine. In this case, the "on" intensity was identified as the bead exhibiting the greatest increase in signal intensity from one image to the next, although within a specific cycle. Figure 1 Compared to the standard condition (τ=37), the signal attenuates faster under the improved condition (τ=27). However, compared to... Figure 1 conditions (R) 2 Compared to 0.88, for the improved conditions, R = 0.97 2 The value represents an improvement, indicating a reduction in sequence background artifacts as the ternary complex accumulates on the inspection subroutine. These results suggest that the accumulation of the ternary complex provides very good sequencing results.

[0174] Throughout this application, references have been made to various publications, patents, and / or patent applications. The disclosures of these documents are incorporated herein by reference in their entirety.

[0175] Many embodiments have been described. However, it should be understood that various modifications can be made. Therefore, other embodiments are within the scope of the appended claims.

Claims

1. A method for identifying nucleotides in a template nucleic acid, the method comprising: (a) Provides an array of template nucleic acids that trigger the reaction; (b) Delivering multiple nucleotide homologs of the first base type and multiple polymerases to the array to form a stable ternary complex, each of the ternary complexes comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and a template nucleic acid initiated in the array; (c) Detecting the stable ternary complex in the array that includes the nucleotide homolog of the first base type; (d) Remove the nucleotide homolog of the first base type, thereby retaining the initiated template nucleic acid and the polymerase in the array; (e) In the presence of the polymerase from step (b), multiple nucleotide homologs of the second base type are delivered to the array to form a stable ternary complex, each of the ternary complexes comprising a polymerase from the polymerase from step (b), a nucleotide from the multiple nucleotide homologs of the second base type, and a priming template nucleic acid in the array, wherein the second base type is different from the first base type; (f) Detecting the stable ternary complex in the array that includes nucleotide homologs of the second base type; and (g) Identify the type of nucleotide present in each of the stable ternary complexes in steps (c) and (f) and at the corresponding base position of the initiated template nucleic acid.

2. The method of claim 1, further comprising repeating steps (e) and (f) using a nucleotide homolog of a third base type instead of the nucleotide homolog of the second base type.

3. The method of claim 2, further comprising repeating steps (e) and (f) using a nucleotide homolog of a fourth base type instead of the nucleotide homolog of the third base type.

4. The method of claim 1, wherein the nucleotide homologs of the first base type are removed by washing the array with an aqueous solution containing at least 10% to at most 50% ethanol.

5. The method of claim 1, wherein the unbound nucleotide homologs of the first base type are removed in step (d), and the bound nucleotide homologs of the first base type are retained in the array in step (e).

6. The method of claim 1, wherein the nucleotide homolog of the first base type contains an exogenous marker, and the nucleotide homolog of the second base type contains an exogenous marker.

7. The method of claim 6, wherein the exogenous marker of the nucleotide homolog of the first base type produces a signal different from the signal produced by the exogenous marker of the nucleotide homolog of the second base type.

8. The method of claim 7, wherein step (f) further includes distinguishing different signals from different exogenous markers.

9. The method of claim 6, wherein the signal generated by the exogenous marker on the nucleotide homolog of the first base type is the same as the signal generated by the exogenous marker on the nucleotide homolog of the second base type.

10. The method of claim 1, further comprising: (h) Adding nucleotides to the primer of each of the initiated template nucleic acids, thereby concluding the array with extended initiated template nucleic acids; and (i) Repeat steps (b) to (g) using an extended priming template instead of the priming template nucleic acid.

11. The method of claim 10, wherein the second polymerase adds the nucleotide to the primer of each of the initiated template nucleic acids, and wherein the polymerase and the second polymerase are the same type of polymerase.

12. The method of claim 10, wherein the primer comprises a reversible terminator portion, and wherein step (h) comprises unblocking the primer and adding the nucleotide to the unblocked primer of each of the initiated template nucleic acids, thereby the array comprising extended initiated template nucleic acids.

13. A method for identifying nucleotides in a template nucleic acid that has been initiated, comprising: (a) Provide a container containing the template nucleic acid that initiates the reaction, polymerase, and nucleotide homologs of the first base type; (b) Check the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the first base type bound to the base position of the initiated template nucleic acid; (c) Remove the nucleotide homolog of the first base type from the container and deliver the nucleotide homolog of the second base type into the container, whereby the container retains the template nucleic acid initiated in step (b) and the polymerase, wherein the second base type is different from the first base type; (d) Check the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the second base type bound to the base position of the initiated template nucleic acid; as well as (e) Identify the type of nucleotide present in each of the stable ternary complexes of steps (c) and (d) at the corresponding base position of the initiated template nucleic acid.

14. The method of claim 13, wherein the nucleotide homolog of the first base type is removed by washing the container with an aqueous solution containing at least 10% to at most 50% ethanol.

15. The method of claim 13, wherein step (c) comprises removing unbound nucleotide homologs of the first base type, and wherein the container also retains the bound nucleotide homologs of the first base type from steps (c) and (d).

16. The method of claim 13, wherein the initiated template nucleic acid is immobilized on a solid support.

17. The method of claim 16, wherein the priming template nucleic acid is one of a variety of different priming template nucleic acids immobilized in an array, and wherein the method is performed to identify the type of nucleotide at the base position in each of the different priming template nucleic acids.

18. The method of claim 13, wherein the nucleotide homolog of the first base type comprises an exogenous marker, and the nucleotide homolog of the second base type comprises an exogenous marker.

19. The method of claim 18, wherein the exogenous marker of the nucleotide homolog of the first base type is different from the exogenous marker of the nucleotide homolog of the second base type.

20. The method of claim 19, wherein step (d) further includes distinguishing signals from different exogenous markers.

21. The method of claim 19, wherein the exogenous marker on the nucleotide homolog of the first base type produces the same signal as the exogenous marker on the nucleotide homolog of the second base type.

22. The method of any one of claims 13 to 21, wherein the method further comprises (f) Remove nucleotide homologs of the second base type; (g) Delivering a nucleotide homolog of the third base type into the container, whereby the container retains the template nucleic acid initiated in step (b) and the polymerase; and (h) Check the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the third base type bound to the base position of the initiated template nucleic acid.

23. The method of claim 22, further comprising: (i) Remove nucleotide homologs of the third base type; (j) Delivering a fourth-base-type nucleotide homolog into the container, whereby the container retains the template nucleic acid initiated in step (b) and the polymerase; and (k) Check the container for a stable ternary complex comprising the polymerase and a nucleotide homolog of the fourth base type bound to the base position of the initiated template nucleic acid.

24. The method of claim 23, further comprising: (l) Adding a nucleotide to the primer of the initiated template nucleic acid, thereby the container containing the extended initiated template nucleic acid; (m) Delivering the second polymerase and the nucleotide homolog of the first base type into the container; as well as (n) Repeat steps (b) to (j) using the extended priming template instead of the priming template nucleic acid and using the second polymerase instead of the polymerase.

25. The method of claim 13, wherein the container in step (a) further comprises a nucleotide homolog of a third base type, and wherein step (b) comprises checking the container for a stable ternary complex comprising the polymerase and (1) the nucleotide homolog of the first base type bound to the base position of the induced template nucleic acid or (2) the nucleotide homolog of the third base type bound to the base position of the induced template nucleic acid.

26. The method of claim 25, wherein the nucleotide homolog of the first base type comprises an exogenous marker, and the nucleotide homolog of the third base type comprises an exogenous marker.

27. The method of claim 26, wherein the exogenous marker of the nucleotide homolog of the first base type is different from the exogenous marker of the nucleotide homolog of the third base type.

28. The method of claim 27, wherein step (b) further comprises distinguishing signals from different exogenous markers.

29. The method of claim 26, wherein the exogenous marker on the nucleotide homolog of the first base type produces the same signal as the exogenous marker on the nucleotide homolog of the third base type.

30. The method of claim 29, wherein step (b) includes detecting the signal.

31. The method of claim 13, wherein step (c) further comprises delivering a fourth base type nucleotide homolog to the container, and wherein step (d) comprises checking the container for the presence of a stable ternary complex comprising the polymerase and (1) the second base type nucleotide homolog bound to the base position of the induced template nucleic acid or (2) the fourth base type nucleotide homolog bound to the base position of the induced template nucleic acid.

32. The method of claim 31, wherein the nucleotide homolog of the second base type contains an exogenous marker, and the nucleotide homolog of the fourth base type contains an exogenous marker.

33. The method of claim 32, wherein the exogenous marker on the nucleotide homolog of the second base type is different from the exogenous marker on the nucleotide homolog of the fourth base type.

34. The method of claim 33, wherein step (b) further comprises distinguishing signals from different exogenous markers.

35. The method of claim 32, wherein the exogenous marker on the nucleotide homolog of the second base type produces the same signal as the exogenous marker on the nucleotide homolog of the fourth base type.

36. The method of claim 35, wherein step (b) includes detecting the signal.

37. The method of claim 13, wherein the container is selected from the group consisting of: flow cells, pores in a porous plate, droplets, vesicles, test tubes, trays, centrifuge tubes, pipes, and channels in a substrate.

38. The method of claim 13, further comprising: (f) Adding a nucleotide to the primer of the initiated template nucleic acid, thereby the container containing the extended initiated template nucleic acid; (g) Delivering the second polymerase and the nucleotide homolog of the first base type into the container; as well as (h) Repeat steps (b) to (e) using the extended priming template instead of the priming template nucleic acid and using the second polymerase instead of the polymerase.

39. The method of claim 38, wherein the polymerase and the second polymerase are the same type of polymerase.

40. The method of claim 38, wherein the primer comprises a reversible terminator portion, and wherein step (f) comprises unblocking the primer and adding the nucleotide to the unblocked primer, thereby the container comprising an extended, initiated template nucleic acid.

41. The method of claim 40, wherein the nucleotide added to the primer comprises a reversible terminator portion, thereby the extended primer comprises a reversible terminator portion.

42. A method for identifying nucleotides in a template nucleic acid, the method comprising: (a) Provides an array of template nucleic acids that trigger the reaction; (b) Forming a stable ternary complex, each of the ternary complexes comprising a polymerase, a nucleotide homolog of the first base type, and a template nucleic acid initiated in the array; (c) Detect the stable ternary complex in the array; (d) Removing nucleotide homologs of the first base type from the array, and then repeating steps (b) and (c) for nucleotide homologs of the second base type, thereby retaining the initiated template nucleic acid and the polymerase in the array, wherein the second base type is different from the first base type; and (e) Identify the type of nucleotide present in each of the stable ternary complexes of steps (c) and (d) at the corresponding base position of the initiated template nucleic acid.

43. The method of claim 42, wherein step (d) comprises repeating steps (b) and (c) on the nucleotide homolog of the second base type, and then repeating steps (b) and (c) on the nucleotide homolog of the third base type, wherein the initiated template nucleic acid and the polymerase are retained in the array.

44. The method of claim 43, wherein step (d) comprises repeating steps (b) and (c) on the nucleotide homolog of the second base type, then repeating steps (b) and (c) on the nucleotide homolog of the third base type, and then repeating steps (b) and (c) on the nucleotide homolog of the fourth base type, wherein the initiated template nucleic acid and the polymerase are retained in the array.

45. The method of claim 42, wherein the nucleotide homologs of the first base type are removed by washing the array with an aqueous solution containing at least 10% to at most 50% ethanol.

46. ​​The method of claim 42, wherein unbound nucleotide homologs of the first base type are removed, and bound nucleotide homologs of the first base type are retained in the array in step (d).

47. The method of claim 42, wherein the nucleotide homolog of the first base type comprises an exogenous marker, and the nucleotide homolog of the second base type comprises an exogenous marker.

48. The method of claim 47, wherein the exogenous marker of the nucleotide homolog of the first base type is different from the exogenous marker of the nucleotide homolog of the second base type.

49. The method of claim 47, wherein step (c) further comprises distinguishing signals from different exogenous markers.

50. The method of claim 48, wherein the exogenous marker on the nucleotide homolog of the first base type produces the same signal as the exogenous marker on the nucleotide homolog of the second base type.

51. The method of claim 50, wherein step (c) includes detecting the signal.

52. The method of claim 43, wherein step (b) comprises forming a stable ternary complex, each of the ternary complexes comprising a polymerase, an initiating template nucleic acid in the array, and a nucleotide homolog of the first base type or the third base type.

53. The method of claim 44, wherein step (d) comprises repeating steps (b) and (c) on the nucleotide homolog of the second base type and the nucleotide homolog of the fourth base type, thereby retaining the initiated template nucleic acid and the polymerase in the array.

54. The method of claim 52, wherein the nucleotide homolog of the first base type contains an exogenous marker, and the nucleotide homolog of the third base type contains an exogenous marker.

55. The method of claim 54, wherein the exogenous marker on the nucleotide homolog of the first base type is different from the exogenous marker on the nucleotide homolog of the third base type.

56. The method of claim 55, wherein step (c) further includes distinguishing signals from different exogenous markers.

57. The method of claim 54, wherein the exogenous marker on the nucleotide homolog of the first base type produces the same signal as the exogenous marker on the nucleotide homolog of the third base type.

58. The method of claim 57, wherein step (c) includes detecting the signal.

59. The method of any one of claims 42 to 57, wherein the method further comprises (f) Adding nucleotides to the primers in each of the initiated template nucleic acids, thereby concluding the array with extended initiated template nucleic acids; and (g) Repeat steps (b) to (e) using the extended priming template instead of the priming template nucleic acid and using a second polymerase instead of the polymerase.

60. The method of claim 59, wherein the polymerase and the second polymerase are the same type of polymerase.

61. The method of claim 59, wherein the primer comprises a reversible terminator portion, and wherein step (f) comprises unblocking the primer and adding the nucleotide to the unblocked primer, thereby the array comprising an extended, induced template nucleic acid.

62. The method of claim 61, wherein the nucleotide added to the primer comprises a reversible terminator portion, thereby the extended primer comprises a reversible terminator portion.

63. A method for identifying nucleotides in a template nucleic acid, the method comprising: (a) Provides an array of template nucleic acids that trigger the reaction; (b) Delivering multiple polymerases and multiple nucleotide homologs of the first base type to the array to form a stable ternary complex, each of the ternary complexes comprising a polymerase of the multiple polymerases, a nucleotide of the multiple nucleotide homologs of the first base type, and a template nucleic acid initiated by the array; (c) Detect the stable ternary complex in the array; (d) Removing multiple nucleotide homologs of the first base type from the array, and then repeating steps (b) and (c) for nucleotide homologs of the second base type, thereby retaining the template nucleic acid of the array and the polymerase of the multiple polymerases in the array, wherein the second base type is different from the first base type; and (e) Identify the type of nucleotide present in each of the stable ternary complexes of steps (c) and (d) at the corresponding base position of the initiated template nucleic acid.

64. The method of claim 63, wherein step (d) comprises repeating steps (b) and (c) on the nucleotide homolog of the second base type, and then repeating steps (b) and (c) on the nucleotide homolog of the third base type, thereby retaining the template nucleic acid of the array and the polymerase of the plurality of polymerases in the array.

65. The method of claim 64, wherein step (d) comprises repeating steps (b) and (c) for the nucleotide homolog of the second base type, then repeating steps (b) and (c) for the nucleotide homolog of the third base type, and then repeating steps (b) and (c) for the nucleotide homolog of the fourth base type, thereby retaining the template nucleic acid of the array and the polymerase of the plurality of polymerases in the array.

66. The method of claim 63, wherein unbound nucleotides in the polynucleotide homologs of the first base type are removed, and bound nucleotides in the polynucleotide homologs of the first base type are retained in the array in step (d).

67. The method of claim 63, wherein step (b) comprises delivering multiple polymerases, multiple nucleotide homologs of a first base type and multiple nucleotide homologs of a third base type to the array to form a stable ternary complex, each of the ternary complexes comprising a polymerase of the multiple polymerases, a template nucleic acid initiated by the array, and (1) a nucleotide of the multiple nucleotide homologs of the first base type or (2) a nucleotide of the multiple nucleotide homologs of the third base type.

68. The method of claim 65, wherein step (d) comprises repeating steps (b) and (c) on the nucleotide homolog of the second base type and the nucleotide homolog of the fourth base type, thereby retaining the template nucleic acid of the array and the polymerase of the plurality of polymerases in the array.

69. The method of any one of claims 63 to 68, wherein the method further comprises (f) Adding nucleotides to the primers of each of the initiated template nucleic acids, thereby concluding the array with extended initiated template nucleic acids; and (g) Repeat steps (b) to (e) using the extended priming template instead of the priming template nucleic acid and using multiple second polymerases instead of the multiple polymerases.

70. The method of claim 69, wherein the primer comprises a reversible terminator portion, and wherein step (f) comprises unblocking the primer and adding the nucleotide to the unblocked primer, thereby the array comprising an extended, initiated template nucleic acid.

71. The method of claim 70, wherein the nucleotide added to the primer comprises a reversible terminator portion, thereby the extended primer comprises a reversible terminator portion.

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