Step-by-step sequencing by unlabeled reversible terminators or natural nucleotides

Abstract

The present invention provides compositions and methods for nucleic acid sequencing and other applications. In sequencing by synthesis, an unlabeled reversible terminator is incorporated in each cycle by a polymerase and then labeled after incorporation by binding a directly or indirectly labeled antibody or other affinity reagent to the reversible terminator.

Description

This application is a divisional application of the invention patent application with application number 201880005910.5, application date January 4, 2018, applicants are Shenzhen BGI Intelligent Manufacturing Technology Co., Ltd. and Shenzhen BGI Life Sciences Institute, and the invention name is "Step-by-step sequencing by non-labeled reversible terminators or natural nucleotides". CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 442,263, filed January 4, 2017, and U.S. Provisional Application No. 62 / 490,511, filed April 26, 2017. The entire contents of these applications are incorporated herein by reference for all purposes. Background Art

[0002] The demand for low-cost, high-throughput methods for nucleic acid sequencing and resequencing has led to the development of "massively parallel sequencing" (MPS) technology. A commonly used method for DNA sequencing is called "sequencing by synthesis" (SBS), as disclosed in Ronaghi et al., Science, 281:363-365, 1998; Li et al., Proc. Natl. Acad. Sci. USA, 100:414-419, 2003; Metzker, Nat Rev Genet. 11:31-46, 2010; Ju et al., Proc. Natl. Acad. Sci. USA 103:19635-19640, 2006; Bentley et al., Nature 456:53-59, 2008; and in U.S. Patent Nos. 6,210,891, 6,828,100, 6,833,246, and 6,911,345 and U.S. Patent Publication No. 2016 / 0130647.

[0003] SBS requires the controlled (i.e., one at a time) incorporation of the correct complementary nucleotide relative to the oligonucleotide being sequenced. This allows accurate sequencing by adding nucleotides in multiple cycles because each nucleotide residue is sequenced one at a time, preventing uncontrolled serial incorporation. In one approach, reversible terminator nucleotides (RTs) are used to determine the sequence of the DNA template. In the most commonly used SBS approach, each RT contains a modified nucleotide that includes (1) a blocking group that ensures that only a single base is added to the 3' end of the growing DNA copy strand by the DNA polymerase, and (2) a fluorescent tag that can be detected by a camera. In the most common SBS method, the template and sequencing primer are immobilized on a solid support and the support is exposed to each of four DNA nucleotide analogs, each analog containing a different fluorophore attached to the nitrogenous base by a cleavable linker and a 3'-O-azidomethyl group at the 3'-OH position of the deoxyribose sugar, and a DNA polymerase. Only the correct complementary base anneals to the target and is subsequently incorporated at the 3' end of the primer. Unincorporated nucleotides are washed away and the solid support is imaged. TCEP (tris(2-carboxyethyl)phosphine) is introduced to cleave the linker and release the fluorophore and remove the 3'-O-azidomethyl group, thereby regenerating the 3'-OH. The cycle can then be repeated ((Bentley et al., Nature 456, 53-59, 2008). A different fluorescent color label is used for each of the four bases so that in each sequencing cycle, the identity of the incorporated RT can be identified by its color.

[0004] Despite its widespread use, SBS still needs improvement. For example, current SBS methods require expensive reversibly terminated dNTPs (RTs), in which a label (e.g., a dye) on the base is attached to a cleavable linker, resulting in a) chemical scarring on the incorporated base after label cleavage, b) low incorporation efficiency, c) quenching, d) termination of excited dye-induced extension, and reduced signal in each sequencing cycle. Summary of the Invention

[0005] The present invention relates to methods and compositions for nucleic acid analysis and sequencing. Disclosed herein is a method for sequencing by sequencing (SBS) in which the last incorporated nucleotide base is identified by binding an affinity reagent (e.g., an antibody, aptamer, affimer, knottin, etc.) that recognizes a base, a sugar, a cleavable blocking group, or a combination of these components in the last incorporated nucleotide. Binding is directly or indirectly associated with the generation of a detectable signal.

[0006] According to one embodiment, the invention provides the method for adopting non-labeled reversible terminator (NLRT) nucleotide to order-check.Reversible terminator (RT) nucleotide is the deoxynucleotide triphosphate (dNTP) or dNTP analogue of modification, and it contains removable blocking group, and described blocking group guarantees that only single base can be added to the 3 ' end of growing DNA copy chain by DNA polymerase.As is well known, dNTP (2 '-deoxynucleoside triphosphate) is mixed into the 3 ' end of growing chain and relates to the release of pyrophosphate, and when dNTP is mixed into DNA chain, the part mixed is monophosphate nucleotide (or more precisely, the nucleotide monomer connected with one or two adjacent nucleotide monomers by phosphodiester bond).Reversible terminator (RT) nucleotide is the deoxynucleotide triphosphate (dNTP) or dNTP analogue of modification, and it contains removable blocking group, and described blocking group guarantees that only single base can be added to the 3 ' end of growing DNA copy chain by DNA polymerase.Non-labeled RT nucleotide does not contain detectable label. In each sequencing cycle, nucleotides or nucleotide analogs are incorporated by polymerase, thereby extending the 3' end of the DNA copy chain by one base, and washing out unincorporated nucleotides or nucleotide analogs. An affinity reagent is introduced, which specifically recognizes and binds to the epitope of the newly incorporated nucleotides or nucleotide analogs. After the image is taken, the blocking group and the labeled affinity reagent are removed from DNA, thereby allowing the next sequencing cycle to begin. In some embodiments, the epitope identified by the affinity reagent is formed by the nucleoside itself (i.e., alkali sugaring) or nucleoside and 3' blocking groups incorporated. In some embodiments, the epitope identified by the affinity reagent is formed by a reversible terminator itself, a reversible terminator combined with deoxyribose, or a reversible terminator and a core base or a core base and deoxyribose combination.

[0007] According to one such embodiment, the invention provides a method for sequencing a nucleic acid, the method comprising: (a) contacting a nucleic acid template comprising the nucleic acid, a nucleic acid primer complementary to a portion of the template, a polymerase, and an unlabeled RT of Formula I under conditions wherein the primer is extended to incorporate the unlabeled RT into a sequence complementary to the nucleic acid template, thereby producing an unlabeled extension product comprising the incorporated RT: wherein: R1 is a 3'-O reversible blocking group; R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R3 comprises or consists of one or more phosphates; (b) contacting the unlabeled extension product with the affinity reagent under conditions in which the affinity reagent specifically binds to the incorporated RT to produce a labeled extension product comprising the RT; (c) detecting binding of the affinity reagent, and (d) identifying the nucleotide incorporated into the labeled extension product to identify at least a portion of the sequence of the extension product, and thereby identifying at least a portion of the sequence of the template nucleic acid.

[0008] In dNTP analogs typically used for sequencing by synthesis, the nucleobase is conjugated to a cleavable linker that connects the base to a detectable label, such as a fluorophore. See, for example, U.S. Patent Publication No. 2002 / 0227131. In contrast, in the dNTP analogs of the present invention, R2 is typically not a nucleobase conjugated to a dye or other detectable label via a linker.

[0009] According to another embodiment, the method further comprises (d) removing the reversible blocking group from the RT to generate a 3'-OH; and (e) removing the affinity reagent from the RT.

[0010] According to another embodiment, the method further comprises repeating the steps of the method one or more times, ie performing a plurality of sequencing cycles, wherein at least a portion of the sequence of the nucleic acid template is determined.

[0011] According to another embodiment, the method comprises removing the reversible blocking group and the affinity reagent in the same reaction.

[0012] According to another embodiment, the method comprises removing the affinity reagent without removing the reversible blocking group and re-probing with a different affinity reagent.

[0013] In this method, the affinity agent may include an antibody (including binding fragments of an antibody, single-chain antibodies, bispecific antibodies, etc.), an aptamer, a knottin, an affimer, or any other known agent that binds to the incorporated NLRT with suitable specificity and affinity. In one embodiment, the affinity agent is an antibody. In another embodiment, the affinity agent is an antibody comprising a detectable label, wherein the detectable label is a fluorescent label.

[0014] According to one embodiment, R1 is selected from allyl, azidomethyl, aminoalkoxy, 2-cyanoethyl, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heteroalkenyl, unsubstituted heteroalkenyl, substituted heteroalkynyl, unsubstituted heteroalkynyl, allenyl, cis-cyanovinyl, trans-cyanovinyl, cis-cyanofluorovinyl, trans-cyanofluorovinyl, cis-trifluoromethylvinyl, trans-trifluoromethylvinyl, biscyanovinyl , bisfluorovinyl, cis-propenyl, trans-propenyl, nitrovinyl, acetylvinyl, methylcarbonylvinyl, amidovinyl, methylsulfonylvinyl, methylsulfonylethyl, methylimidate, methylhydroxamate (formhydroxymate), vinylvinyl, vinylsubstituted vinyl, cyanovinyl, nitrovinyl, amidoethylenyl, amino, cyanovinyl, cyanoethyl, alkoxy, acyl, methoxymethyl, aminooxy, carbonyl, nitrobenzyl, coumarinyl and nitronaphthyl.

[0015] According to another embodiment, R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G) and thymine (T).

[0016] According to another embodiment, R3 consists of or comprises one or more phosphate esters.

[0017] The term non-labeled reversible terminator (NLRT) may refer to the triphosphate form of the nucleotide analog, or may refer to an incorporated NLRT.

[0018] According to another embodiment of the present invention, a method for sequencing a nucleic acid is provided, comprising: (a) providing a DNA array, the DNA array comprising (i) a plurality of template DNA molecules, each template DNA molecule comprising a fragment of the nucleic acid, wherein each of the plurality of template DNA molecules is attached at one position of the array, (b) contacting the DNA array with a nucleic acid primer, a polymerase, and an unlabeled RT of Formula I, under conditions in which the primers are extended to incorporate an unlabeled RT into a sequence complementary to at least some of the plurality of the template DNA molecules, thereby generating an unlabeled extension product comprising the RT; the nucleic acid primer is complementary to a portion of each of the template DNA molecules; wherein: R1 is a 3'-O reversible blocking group; R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R3 consists of or comprises one or more phosphates; (c) contacting the unlabeled extension product with the affinity reagent comprising a detectable label under conditions in which the affinity reagent specifically binds to the RT to produce a labeled extension product comprising the RT; and (d) identifying the RT in the labeled extension product to identify at least a portion of the sequence of the nucleic acid.

[0019] According to one embodiment of the present invention, the method comprises: (b) contacting the DNA array with a nucleic acid primer, a polymerase, and a set of unlabeled RTs of Formula I under conditions where primers are extended to incorporate unlabeled RTs into sequences complementary to at least some of a plurality of template DNA molecules, thereby producing unlabeled extension products comprising the RTs, wherein the nucleic acid primers are complementary to a portion of each of the template DNA molecules, and the set of unlabeled RTs of Formula I comprises a first RT in which R2 is A, a second RT in which R2 is T, a third RT in which R2 is C, and a fourth RT in which R2 is G; and (c) contacting the unlabeled extension products with the affinity reagent set under conditions where the affinity reagent set specifically binds to the incorporated RTs. The method comprises contacting a plurality of affinity reagents with a plurality of affinity reagents to produce a labeled extension product comprising the RT, wherein: (i) the affinity reagent set includes a first affinity reagent that specifically binds to the first RT, a second affinity reagent that specifically binds to the second RT, a third affinity reagent that specifically binds to the third RT, and optionally, a fourth affinity reagent that specifically binds to the fourth RT; (ii) each of the first affinity reagent, the second affinity reagent, and the third affinity reagent comprises a detectable label; (d) the RT in the labeled extension product is identified by identifying the label of the affinity reagent bound to the RT at its respective position on the array to identify at least a portion of the sequence of the nucleic acid (e.g., one base per cycle). According to a related embodiment, each of the first affinity reagent, the second affinity reagent, the third affinity reagent, and the fourth affinity reagent comprises a detectable label. According to another related embodiment, each of the first affinity reagent, the second affinity reagent, and the third affinity reagent comprises a different detectable label. According to another related embodiment, each of the first affinity reagent, the second affinity reagent, and the third affinity reagent comprises a different amount of the same label (e.g., the same fluorophore) to produce signals of different intensities. According to another embodiment, the affinity reagent bound to the incorporated RT is not directly labeled, but is indirectly labeled using a secondary affinity reagent.

[0020] According to another embodiment of the present invention, a DNA array is provided. The array comprises: a plurality of template DNA molecules, each DNA molecule attached at a position of the array; a complementary DNA sequence that base pairs with a portion of the template DNA molecules at the plurality of positions, wherein the complementary DNA sequence comprises an incorporated RT at its 3' end; and an affinity reagent that specifically attaches to at least some of the RTs, the affinity reagent comprising a detectable label for identifying the RTs to which the affinity reagent is attached.

[0021] According to another embodiment of the present invention, a kit is provided, comprising: (a) unlabeled RT of Formula I: wherein: R1 is a 3'-O reversible blocking group; R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R3 consists of or comprises one or more phosphates; (b) a labeled affinity reagent that specifically binds to one of the RTs; and (c) packaging for the RTs and the affinity reagent. According to another embodiment, such a kit comprises: a plurality of RTs, wherein each RT comprises a different nucleobase; and a plurality of affinity reagents, wherein each affinity reagent specifically binds to one of the RTs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flow chart illustrating an example of the sequencing method of the present invention.

[0023] Figure 2 It is an explanation Figure 1 An example of the antibody staining procedure is shown in the flow chart.

[0024] Figure 3 An example showing the NLRT structure: Figure 3 A3'-O-azidomethyl-2'-deoxyguanine; Figure 3 B 3'-O-amino-2'-deoxyguanine; Figure 3 C 3'-O-cyanoethylene-2'-deoxyguanine; Figure 3 D 3'-O-phosphate; Figure 3 E: 3'-ethyldisulfide-methylene-2'-deoxythymidine.

[0025] Figure 4 Various blocking groups that can be used in the practice of the present invention are described. Figure 4 middle, Indicates the point of attachment of the molecule to the rest of the structure.

[0026] Figure 5The synthesis of an active ester of 3'-O-azidomethyl-2'-deoxyguanine (G4) is described.

[0027] Figure 6 The synthesis of 3'-O-azidomethyl-2'-activated ester (C8) is described.

[0028] Figure 7 The synthesis of an active ester of 3'-O-azidomethyl-2'-deoxyadenine (A12) is described.

[0029] Figure 8 The synthesis of an active ester of 3'-O-azidomethyl-2'-deoxythymine (T16) is described.

[0030] Figure 9 Conjugation of 3'-O-azidomethyl-dC-NHS ester to BSA, KHL, and agarose resin is described (using 3'-O-azidomethyl-2'-deoxycytidine) for use as an immunogen, titer monitor, and substrate for affinity purification.

[0031] Figure 10A and 10B Rho is shown for 5 and 10 sequencing cycles using three labeled RTs and one NLRT.

[0032] Figure 10C and 10D Shown are the signal-to-noise ratios (SNRs) for 5 and 10 sequencing cycles using three labeled RTs and one NLRT.

[0033] Figure 11A and 11B Illustrate sequencing data metrics obtained using the BGISEQ-1000 DNA sequencer, where unlabeled 3'-azidomethyl-dGTP was detected by anti-3'-azidomethyl-dG rabbit primary antibody and anti-rabbit AF647 fragment secondary antibody for 50 cycles of sequencing by synthesis.

[0034] Figure 12A and 12B Illustrated are the results of 25 sequencing cycles of E. coli genomic DNA on a BGISEQ-500 instrument using fluorescently directly labeled anti-azidomethyl-base antibodies. DETAILED DESCRIPTION 1. Overview

[0035] In certain aspects, the present invention provides methods and compositions for carrying out synthetic sequencing (SBS) or combined probe anchor sequencing (cPAS) of nucleic acid using unlabeled reversible terminator nucleotides. In one approach, SBS is carried out by generating fixed single-stranded template DNA at a position on an array. In most approaches, each fixed single-stranded template DNA is located at a position with a large number of copies (e.g., amplicons) of similar sequences. For example, bridge PCR can be used to generate template sequence clusters at positions on an array (Illumina), or rolling circle replication can be used to generate single-stranded concatemers, or DNA nanoballs (DNBs), which have many copies of template sequences (Complete Genomics, Inc.). SBS is carried out by hybridizing one or more primers to template DNA and extending the primers to generate extension primers or growing DNA chains (GDS). Extension primers refer to adding ("incorporating") nucleotides at the 3' end of the primer DNA chain while hybridizing it to the template. The nucleotides incorporated at the 3' end are complementary to the corresponding nucleotides of the primers, so that the nucleotide sequence of the template can be determined by determining the identity of the incorporated nucleotides in each sequencing cycle.

[0036] In a prior art approach, labeled nucleotide analogs are incorporated into GDS. Typically, the labeled nucleotide analogs comprise blocking groups that ensure that only single nucleotides can be incorporated into each step, and a dye (typically a fluorescent dye) is attached to the nucleotide via a cleavable linker. Each sequencing cycle involves incorporating labeled nucleotide analogs into one end of the GDS, detecting the incorporated labeled nucleotide analog label, removing the label from the incorporated nucleotide analog, and removing the blocking group from the incorporated nucleotide analog to allow incorporation of newly labeled nucleotide analogs. In contrast, the present invention does not require labeled nucleotide analogs comprising dyes attached to bases or sugars via a cleavable linker.

[0037] In the alternative approach described in U.S. Patent Publication No. US2017 / 0240961 (which is incorporated herein by reference), when incorporated, the nucleotide analog comprises an affinity tag attached to the nucleotide via a joint. The affinity tag is a member of a specific binding pair (SBP). In one approach, the affinity tag is biotin. After incorporation, the incorporated nucleotide is exposed to an affinity reagent comprising a second member (e.g., streptavidin) of the SBP and a detectably labeled tag. The detectable label is detected to identify the incorporated nucleotide. After detection, the incorporated nucleotide analog-affinity reagent complex is processed to cleave the joint and release the detectable label. In one approach, the affinity tag is an antigen and the affinity reagent is a fluorescently labeled antibody that specifically binds to the antigen. On the contrary, the present invention does not require an affinity tag, and in some aspects, an affinity reagent is used instead of an affinity tag, the affinity reagent binding nucleobase, a sugar unit (moiety), a cleavable blocking group or a combination thereof.

[0038] According to one aspect of the method disclosed herein, a non-labeled reversible terminator, i.e., a nucleotide analog containing a reversible terminator or a blocking group ( N on- L abeled R eversible T The GDS is prepared by a DNA polymerase (DNA polymerase) and then exposed to an affinity reagent (e.g., an antibody) that specifically binds to the incorporated NLRT (a "binding event"). After the binding event is detected, the affinity reagent is removed. In one approach, a nucleotide analog comprising a reversible blocking group is incorporated at the 3' end of the GDS, and after the binding event is detected, the reversible blocking group and the affinity reagent are optionally removed in the same step. In this approach, each sequencing cycle comprises: (i) incorporation of the NLRT comprising a blocking group by a DNA polymerase, followed by washing away the unincorporated NLRT; (ii) contacting the incorporated nucleotide analog with a labeled affinity reagent that recognizes and specifically binds to the incorporated NLRT; (iii) detecting binding of the affinity reagent; (iv) removing the blocking group in a manner that allows the incorporation of additional nucleotide analogs (e.g., generating a hydroxyl group at the 3' position of the deoxyribose unit), and (v) removing the affinity reagent. This step may be followed by a new cycle or cycles in which new nucleotide analogs are incorporated and detected. Affinity reagents (e.g., antibodies) can be directly labeled (e.g., fluorescently labeled antibodies) or can be detected indirectly (e.g., by binding to labeled anti-affinity reagents or secondary affinity reagents). Thus, it should be understood that a "labeled affinity reagent" can be directly labeled, for example, by conjugation to a fluorophore; or indirectly labeled.

[0039] In another approach, a nucleotide analog containing a reversible blocking group is incorporated at the 3' end of the GDS, and after the binding event is detected, the reversible blocking group and the affinity reagent are removed. In this approach, each sequencing cycle includes: (i) incorporation of a NLRT containing a blocking group by a DNA polymerase, optionally followed by washing away unincorporated NLRT; (ii) removal of the blocking group in a manner that regenerates a hydroxyl (OH) group at the 3' position of the deoxyribonucleotide; (iii) removal of the blocking group in a manner that allows incorporation of additional nucleotide analogs (e.g., generating a hydroxyl group at the 3' position of the deoxyribose unit), contacting the incorporated nucleotide analog with a labeled affinity reagent that recognizes and specifically binds to the incorporated NLRT; (iii) detecting binding of the affinity reagent; and (v) removal of the affinity reagent. This step can be followed by one or more new cycles in which new nucleotide analogs are incorporated and detected. The affinity reagent (e.g., an antibody) can be directly labeled (e.g., a fluorescently labeled antibody) or can be detected indirectly (e.g., by binding to a labeled anti-affinity reagent or secondary affinity reagent). Thus, it will be understood that a "labeled affinity reagent" may be directly labeled, for example, by conjugation to a fluorophore; or indirectly labeled.

[0040] SBS relates to two or more primer extension cycles, wherein nucleotides are incorporated at the 3' ends of the extension primers. The present invention utilizes affinity reagents, such as antibodies, to (i) detect the nucleotides incorporated at the 3' ends of the extension primers ("3' terminal nucleotides") and (ii) identify the core bases of the 3' terminal nucleotides and distinguish one from another core base (e.g., distinguishing A from G). It is not desirable to be bound by a specific mechanism, but this is possible because every kind of affinity reagent is designed to distinguish other "interior" nucleotides of the 3' terminal nucleotides from extension primers, even when the 3' terminal nucleotides and the inner nucleotides comprise identical core bases. Every kind of affinity reagent (or in some cases, the combination of affinity reagents) is also designed to detect the properties of the 3' terminal nucleotides of the core bases associated with the 3' terminal nucleotides. Many strategies, methods, and materials are provided to perform these and other steps. This section provides an overview, wherein many variations are omitted, and should not be considered as limiting in any way.

[0041] In some approaches, nucleotides with 3' reversible terminator units are used to carry out the SBS reaction of the present invention. In these approaches, based on the presence of the reversible terminator unit, the 3' terminal nucleotides incorporated are different from the internal nucleotides. Therefore, the affinity reagent bound to the reversible terminator unit in the extension primer binds to (and therefore detects) the 3' terminal nucleotides, thereby distinguishing them from the internal nucleotides. In different approaches, based on the presence of free 3'-OH (hydroxyl) groups not present on the internal nucleotides, the 3' terminal nucleotides incorporated are different from the internal nucleotides. Therefore, the affinity reagent bound to the free 3'-OH group in the extension primer binds to the 3' terminal nucleotides, binds to (and therefore detects) the 3' terminal nucleotides, thereby distinguishing them from the internal nucleotides. In some approaches, free 3'-OH groups are generated by the reversible terminator in the nucleotide analogs incorporated by cleavage. In another approach, free 3'-OH groups are produced by incorporating nucleotides (e.g., naturally occurring nucleotides) that do not comprise a reversible terminator unit. In additional approaches that can be combined with either of the two approaches described above, the incorporated 3' terminal nucleotide differs from the internal nucleotides based on other structural differences in the 3' terminal nucleotide, including but not limited to: greater accessibility of the deoxyribose sugar of the 3' terminal nucleotide to the affinity reagent relative to the deoxyribose sugars of the internal nucleotides; greater accessibility of the affinity reagent to the nucleobase of the 3' terminal nucleotide relative to the accessibility of the affinity reagent to the deoxyribose sugars of the internal nucleotides, and other molecular and conformational differences between the 3' terminal nucleotide and the internal nucleotides.

[0042] Thus, in one aspect of the invention, and as described in the Examples below, affinity reagents are used to detect these structural differences between the 3' terminal nucleotide and the other nucleotides of the extended primer.

[0043] Also provided are many strategies, methods and materials for detecting the properties of the 3' terminal nucleotides of the core bases of the 3' terminal nucleotides of the identification. In one approach, naturally occurring nucleotides or nucleotide analogs comprising naturally occurring core bases (such as A, T, C and G) are used for sequencing reactions and incorporated into primer extension products. An affinity reagent that specifically binds a core base (such as A) and distinguishes this core base from other core bases (such as T, C and G) that it does not bind is used to identify the core base of the 3' terminal nucleotide. In another approach, nucleotide analogs comprising modified (i.e. non-naturally occurring) core bases are used for sequencing reactions and incorporated into primer extension products. An affinity reagent specifically binds a modified core base (such as, modified A) and distinguishes this modified core base from other modified or natural core bases. The affinity reagent that specifically binds to the modified core base usually recognizes modification so that the combination with the modified core base is different from the combination with the naturally occurring core base in the absence of modification. For example, an affinity reagent in conjunction with an adenosine analog, wherein position 7 (N 7) is replaced by a methylated carbon (see Structure XV below), may not bind to the naturally occurring (unmodified) adenosine nucleobase, or may bind less willingly. Without wishing to be bound by a particular mechanism, it is believed that the affinity reagent that specifically recognizes the modified unit (in this case, the modified nucleobase) achieves this effect by binding to the modified feature (in this case, the portion of the modified adenosine that contains the methylated carbon). In other words, the affinity reagent binds to the epitope that contains the methylated carbon. It should be understood that the affinity reagent also binds to other parts of the incorporated nucleotide.

[0044] In another approach, the nucleotide (reversible terminator nucleotide) with 3 ' reversible blocking groups is mixed in the primer extension product. In each sequencing cycle, the blocking group is removed so that the nucleotide only last mixed of the primer extension product comprises the blocking group. In this approach, the affinity reagent in conjunction with the blocking group is used. In this approach, at least two nucleotide analogs (that is, they have different core bases) used in the sequencing reaction comprise different blocking groups. For illustration, by using the first blocking group (for example, 3 '-O-azidomethyl) for the nucleotide comprising adenine or an adenine analog, by using the second different blocking groups (for example, 3 '-O-cyanoethylene) for the nucleotide comprising guanine or a guanine analog, or the like, the specificity of the affinity reagent will identify the associated core base. For example, when extending as described above, if the 3 ' terminal nucleotide is identified by the 3 '-O-cyanoethylene specific affinity reagent, this indicates that the associated core base is guanine or a guanine analog, and the template base in this position is cytosine. In a variation of this approach, blocking groups that differ only in a small feature can be used, and the affinity reagent binds to an epitope that includes the small distinguishing feature.

[0045] As described below, in one aspect of the invention, an affinity reagent (for example, an affinity reagent with a specific blocking group and a specific core base of a specific modification) is used to identify and specifically bind nucleotides or nucleotide analogs based on a combination of structural features. In this respect, for the properties identified by specific affinity reagents, nucleotides or nucleotide analogs are designed and / or selected. In some cases, the affinity reagents in conjunction with multiple structural features have the advantage of being more strongly and more specifically combined with affinity reagents. Table A below is a non-exhaustive set of examples of structural differences, which can be identified by affinity reagents to distinguish between nucleotides with different core bases (column 2) and can be combined by affinity reagents to provide the unit in the last nucleotide incorporated for providing enough binding efficiencies; and / or based on those features distinguishing between the last incorporated nucleotides and internal nucleotide districts (column 3). Table A

[0046] As discussed in detail below, the portion of the incorporated nucleotide analog to which the labeled affinity reagent binds can include, for example, but not limited to, a nucleobase and a blocking group, or a combination of a nucleobase and / or a blocking group with a sugar unit of the nucleotide analog. See Table A, below. Binding of the labeled affinity reagent can depend on the position of the target nucleotide, for example, thereby distinguishing between a nucleotide analog having a blocking group at the 3' end of the GDS and a similar nucleotide analog (lacking a blocking group) located in or within the GDS. Binding of the labeled affinity reagent can also depend on the nucleobase itself, such that an affinity reagent binds to one target NLRT incorporated at the end of the GDS (e.g., NLRT-A) at one position on the array but does not bind to another NLRT incorporated at the end of the GDS (e.g., NLRT-C, -T, or -G) at a different position on the array.

[0047] The present invention has advantages over other SBS methods. The removal of the labeled affinity reagent does not leave a chemical "scar," which is caused by the group still attached to the dNTP after the linker is cleaved. This is advantageous because this "scar" may reduce the efficiency of dNTP incorporation by polymerase. In addition, in this approach, the affinity reagent can include multiple fluorescent units, and according to a common method, a stronger signal than a single fluorescent dye attached to the dNTP is provided. This approach can also cause less light damage because lower excitation power or shorter exposure time can be used. It is expected that the approach disclosed herein allows longer readings (e.g., longer than 500 bases or longer than 1000 bases) and / or more accurate readings (e.g., with less than one error in 2000 bases or less than one error in 5000 bases) of 50, 100 or 200 bases. The compositions and methods of the present invention can also be more economical than the labeled reversible terminator (RT) method commonly used for SBS. Unlabeled RT costs less than labeled RT. In standard SBS using labeled RT, high concentrations of labeled RT are used to drive complete RT incorporation, and most of the labeled RT (70-99% or more) is washed away without being incorporated by the polymerase. Using lower-cost unlabeled RT reduces this cost. Furthermore, in the labeling step of the present invention (wherein a labeled affinity reagent is used), it may be sufficient that only a small percentage of the target template is bound by the affinity reagent (which has multiple effectively labeled binding agents with multiple labeled molecules, or even a single molecule of binding agent), and even 30% may be sufficient; labeling (e.g., about 5%, or about 10%, or less than about 15%, less than about 20%, less than about 25%, or less than about 30%) to obtain sufficient imaging signal, particularly when the affinity reagent effectively binds to the target dNTP and contains multiple labeled molecules. Higher levels of binding may be preferred if the affinity reagent carries only a single labeled molecule (e.g., 70% or more). 2. Definitions and Terminology

[0048] As used herein, in the context of nucleotide analogs, the terms "unlabeled" and "non-labeled" are used interchangeably.

[0049] As used herein, unless otherwise apparent from the context, "non-labeled reversible terminator [nucleotide]," "NLRT," "reversible terminator nucleotide," "reversible terminator," "RT," and the like are used to refer to a sequencing reagent comprising a nucleobase or analog, a deoxyribose sugar or analog, and a cleavable blocking group. The non-labeled reversible terminator nucleotide can refer to a dNTP (i.e., a substrate for a polymerase) or a reversible terminator nucleotide that is initially incorporated into a primer extension product at the 3' end, as well as into the "inner" portion of the primer extension product after additional incorporation cycles (if any).

[0050] As used herein, "dNTPs" include naturally occurring deoxyribonucleotide triphosphates and analogs thereof, including analogs having 3'-O cleavable blocking groups.

[0051] As used herein, in the context of cleavable blocking groups of nucleotide analogs, the designation 3'-O-" is sometimes implied rather than explicit. For example, the terms "azidomethyl" and "3'-O-azidomethyl" are interchangeable, as will be apparent from the context.

[0052] "Amplicon" means the product of a polynucleotide amplification reaction, i.e., a population of polynucleotides replicated from one or more starting sequences. Amplicon can be produced by a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification, rolling circle amplification, and similar reactions (see, e.g., U.S. Patent Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159; 5,210,015; 6,174,670; 5,399,491; 6,287,824 and 5,854,033; and U.S. Publication No. 2006 / 0024711).

[0053] As used herein, "antigen" refers to a compound that can be specifically bound by an antibody. Some antigens are immunogens (see Janeway et al., Immunobiology, 5th Edition, 2001, Garland Publishing). Some antigens are haptens, which are recognized by antibodies but do not elicit an immune response unless conjugated to a protein. Exemplary antigens include NLRTs, reversible terminator blocking groups, dNTPs, polypeptides, small molecules, lipids, or nucleic acids.

[0054] "Array" or "microarray" means a solid support (or a collection of solid supports, such as beads) having a surface (preferably, but not limited to, a flat or substantially flat surface) that carries a collection of sites containing nucleic acids such that each site of the collection is spatially defined and does not overlap with other sites of the array; that is, the sites are spatially discrete. An array or microarray may also include non-planar interrogable structures having a surface such as beads or wells. The oligonucleotides or polynucleotides of the array may be covalently bound to the solid support, or it may be non-covalently bound. Traditional microarray technology is reviewed in, for example, Schena, Ed. (2000), Microarrays: A Practical Approach (IRL Press, Oxford). As used herein, a "random array" or "random microarray" refers to a microarray in which the identity of the oligonucleotides or polynucleotides cannot be discerned based on their position, at least initially, but can be determined by specific biochemical detection techniques for the array. See, e.g., U.S. Patent Nos. 6,396,995; 6,544,732; 6,401,267; and 7,070,927; PCT Publication Nos. WO 2006 / 073504 and 2005 / 082098; and U.S. Patent Publication Nos. 2007 / 0207482 and 2007 / 0087362.

[0055] The terms "reversible," "removable," and "cleavable" when referring to blocking groups have the same meaning.

[0056] The term "reversible blocking group" of a reversible terminator nucleotide may also be referred to as a "removable blocking group," "cleavable linker," "blocking unit," "blocking group," "reversible terminator blocking group," and the like. A reversible blocking group is a chemical unit that is attached to a nucleotide sugar (e.g., deoxyribose) typically at the 3'-O position of the sugar unit, which prevents the addition of nucleotides by polymerases at that position. A reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), a chemical reaction, heat, light, or the like to provide a hydroxyl group at the 3'-position of a nucleoside or nucleotide so that the addition of nucleotides by a polymerase can occur.

[0057] " derivative " or " analogue " means such compound or molecule, its core structure is identical or very similar to the core structure of the parent compound, but it has chemical or physical modification, such as different or additional side groups, or 2 ' and / or 3 ' blocking groups, which allow derivative nucleotides or nucleosides to be connected to another molecule. For example, the base can be a deazapurine. Derivatives should be able to carry out Watson-Crick pairing. " Derivative " and " analogue " also refer to synthetic nucleotides or nucleoside derivatives with modified base units and / or modified sugar units. Such derivatives and analogs are discussed in, for example, Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584,1990. Nucleotide analogs can also comprise modified phosphodiester linkages (linkage), including phosphorothioate, phosphorodithioate, alkyl-phosphate, phosphoramidite and phosphoramidate linkages. Analogs should be able to carry out Watson-Crick base pairing. For example, deoxyadenosine analogs include didanosine (ddI) and vidarabine, and adenosine analogs include BCX4430; deoxycytidine analogs include cytarabine, gemcitabine, emtricitabine (FTC), lamivudine (3TC) and zalcitabine (ddC); guanosine and deoxyguanosine analogs include abacavir, acyclovir and entecavir; thymidine and deoxythymidine analogs include stavudine (d4T), telbivudine and zidovudine (azidothymidine, or AZT); and deoxyuridine analogs include idoxuridine and trifluridine. As used herein, "derivatives," "analogs," and "modified" are used interchangeably and are encompassed by the terms "nucleotide" and "nucleoside" as defined herein.

[0058] "Incorporation" means becoming part of a nucleic acid molecule. In SBS, incorporation of RT occurs when a polymerase adds RT to a growing DNA chain by forming a phosphodiester or modified phosphodiester bond between the 3' position of the pentose sugar of one nucleotide (i.e., the 3' nucleotide on the DNA chain) and the 5' position of the pentose sugar on the adjacent nucleotide.

[0059] In the context of labeled affinity reagents, "label" means any atom or molecule that can be used to provide a detectable and / or quantifiable signal. Suitable labels include radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. In some embodiments, the detection label is a molecule containing a charged group (e.g., a molecule containing a cationic group or a molecule containing an anionic group), a fluorescent molecule (e.g., a fluorescent dye), a fluorescent molecule, or a metal. Optionally, the detection label is a fluorescent label. The fluorescent label can be any label that can emit light in an unquenched form (e.g., when not quenched by another agent). When excited by an appropriate excitation wavelength, the fluorescent unit emits light energy (i.e., fluoresces) at a specific emission wavelength. When the fluorescent unit and the quencher unit are in close proximity, the light energy emitted by the fluorescent unit is absorbed by the quencher unit. In some embodiments, the fluorescent dye is fluorescein, rhodamine, phenoxazine, acridine, coumarin, or a derivative thereof. In some embodiments, the fluorescent dye is carboxyfluorescein. Other examples of suitable fluorescent dyes include Alexa Fluor Fluorescent dyes are commercially available under the FLASH® product line (Life Technologies, Carlsbad, CA). Alternatively, non-fluorescent labels can be used, including but not limited to redox labels, reduction tags, thio- or thiol-containing molecules, substituted or unsubstituted alkyl groups, fluorescent proteins, non-fluorescent dyes, and photoproteins.

[0060] "Nucleobase" refers to a nitrogenous base that can base pair with a complementary nitrogenous base of a template nucleic acid. Exemplary nucleobases include adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), inosine (I), and derivatives thereof. References to thymine herein should be understood as references to uracil as well, unless otherwise apparent from the context. As used herein, the terms "nucleobase," "nitrogenous base," and "base" are used interchangeably.

[0061] As used herein, "naturally occurring nucleobase" means adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). In some cases, naturally occurring nucleobases refer to A, C, G, and T (naturally occurring bases found in DNA).

[0062] "Nucleotides" are composed of a nucleobase, a sugar, and one or more phosphate groups. They are the monomeric units of a nucleic acid sequence. In RNA, the sugar is ribose, while in DNA, the sugar is deoxyribose, a sugar that lacks the hydroxyl group present in ribose. Nitrogenous bases are derivatives of purines or pyrimidines. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) [or, in the case of RNA, uracil (U)]. The C-1 atom of the deoxyribose sugar is bound to the N-1 of a pyrimidine or the N-9 of a purine. Nucleotides are also phosphates or nucleosides, in which esterification occurs at the hydroxyl group attached to the C-5 of the sugar. Nucleotides are typically monophosphates, diphosphates, or triphosphates. "Nucleosides" are structurally similar to nucleotides, but do not include the phosphate unit. Common abbreviations include "dNTP" for deoxynucleotide triphosphates.

[0063] "Nucleic acid" means a polymer of nucleotide monomers. As used herein, the term can refer to single-stranded or double-stranded forms. The monomers constituting nucleic acids and oligonucleotides can specifically bind to natural polynucleotides by means of a regular pattern of monomer-to-monomer interactions (e.g., Watson-Crick type base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, etc.) to form a duplex or triplex form. Such monomers and their internucleoside linkages can be naturally occurring or can be analogs thereof, such as naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs can include peptide nucleic acids, locked nucleic acids, phosphorothioate internucleoside linkages, bases containing linking groups that allow labeling to attach, such as fluorophores or haptens, etc. The size of nucleic acids is typically several monomeric units (e.g., 5-40, when they are typically referred to as "oligonucleotides") or even hundreds of thousands or more monomeric units. Whenever a nucleic acid or oligonucleotide is represented by a sequence of letters (uppercase or lowercase), for example, "ATGCCTG," it is understood that the nucleotides are in 5' to 3' order from left to right, and that "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents thymidine, "I" represents deoxyinosine, and "U" represents uridine, unless otherwise indicated or obvious from the context. Unless otherwise indicated, the nomenclature and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Typically, nucleic acids comprise natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA, or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, such as modified bases, sugars, or internucleoside linkages. For those skilled in the art, when the enzyme has specific oligonucleotide or nucleic acid substrate requirements for activity (e.g., single-stranded DNA, RNA / DNA duplexes, etc.), the selection of suitable oligonucleotide or nucleic acid substrate compositions is within the knowledge of ordinary technicians, especially under the guidance of papers [e.g., Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989)] and similar references.

[0064] "Primer" means a natural or synthetic oligonucleotide that, after forming a duplex with a polynucleotide template, can serve as a starting point for nucleic acid synthesis and extend from its 3' end along the template to form an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Typically, primers are extended by DNA polymerase. Primers typically have a length of 9 to 40 nucleotides, or in some embodiments, 14 to 36 nucleotides.

[0065] "Polynucleotide" is used interchangeably with the term "nucleic acid" to refer to DNA, RNA, and hybrid and synthetic nucleic acids, and can be single-stranded or double-stranded. "Oligonucleotide" is a short polynucleotide between about 6 and about 300 nucleotides in length. "Complementary polynucleotide" refers to a polynucleotide that is complementary to a target nucleic acid.

[0066] "Solid support" and "support" are used interchangeably and refer to a material or group of materials having one or more rigid or semi-rigid surfaces. A microarray typically comprises at least one planar solid support, such as a microscope slide.

[0067] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" refers to one agent or a mixture of agents, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art.

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the devices, compositions, formulations, and methods that are described in the publications and that may be used in connection with the present invention.

[0069] Where a range of values is provided, it is understood that unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0070] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described to avoid obscuring the present invention.

[0071] While the invention has been described primarily with reference to certain embodiments, it is also contemplated that other embodiments will become apparent to those skilled in the art upon reading this disclosure, and such embodiments are intended to be encompassed within the methods of the invention.

[0072] Unless otherwise indicated, the practice of the present invention can adopt the conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and immunology, and these techniques and descriptions are within the technical scope of this area. These conventional techniques include polymer array synthesis, hybridization, connection (ligation) and hybridization detection using labels. By reference to the examples below, a specific description of suitable technology can be obtained. However, other equivalent traditional procedures can also be used, of course. These general techniques and descriptions can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, NY, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., WH Freeman Pub., New York, NY, and Berg et al. (2002) Biochemistry, 5th Ed., WH Freeman Pub., New York, NY, all of which are incorporated herein by reference in their entirety for all purposes. 3. Nucleotides and nucleotide analogs

[0073] In various embodiments, SBS according to the present invention can use non-labeled reversible terminators ("NLRTs") (e.g., nucleotide analogs with blocking groups), non-labeled naturally occurring nucleotides (e.g., dATP, dTTP, dCTP, and dGTP), or non-labeled nucleotide analogs that do not contain blocking groups. 3.1 Non-labeled reversible terminator (NLRT)

[0074] The unlabeled reversible terminators ("NLRTs") of the present invention are nucleotide analogs that contain a removable blocking group at the 3'-OH position of the deoxyribose sugar. Although many reversible terminators have been described, and reversible terminators are widely used in SBS, the unlabeled reversible terminators used in accordance with the present invention are different from those used commercially because they are unlabeled and because they are used in conjunction with the affinity reagents described below. In one aspect, the NLRTs of the present invention are unlabeled. In one embodiment, unlabeled means that the NLRT does not contain a fluorescent dye. In one embodiment, unlabeled means that the NLRT does not contain a chemiluminescent dye. In one embodiment, unlabeled means that the NLRT does not contain a luminescent unit.

[0075] In some embodiments, prior to incorporation of the NLRT into a DNA strand, the exemplary NLRT has the following structure: wherein R1 is a 3'-O reversible blocking group, R2 is or comprises a nucleobase; and R3 comprises at least one phosphate group or an analog thereof.

[0076] After incorporation of the NLRT into the DNA strand, the reversible blocking group R1 can be removed. After incorporation of the analog at the 3' end of the DNA strand, the blocking group is removed to produce a 3'-OH. Any reversible blocking group can be used. Exemplary reversible blocking groups are described below.

[0077] The nucleobase R2 can be, for example, adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or inosine (I), or an analog thereof. NLRTs can be referred to by the nucleobase; for example, an NLRT with an A nucleobase is referred to as NLRT-A. Accordingly, the corresponding NLRTs are referred to herein as "NLRT-A," "NLRT-C," "NLRT-G," "NLRT-T," "NLRT-U," and "NLRT-I," respectively. NLRT-T and NLRT-C can be referred to as NLRT-pyrimidines. NLRT-G and NLRT-A can be referred to as NLRT-purines.

[0078] The nucleobase R2 can be any nucleobase or nucleobase analog (e.g., an analog of adenine, cytosine, guanine, thymine, uracil, or inosine). For example, naturally occurring nucleobases can be modified to increase the immune response to the analog when raising antibodies, or to increase the specificity of the antibody to a particular nucleobase.

[0079] R3 can be 1-10 phosphate or phosphate analog groups.Phosphate analogs include phosphorothioate (PS), wherein the phosphorothioate bond replaces the non-bridging oxygen in the phosphate backbone of DNA with a sulfur atom, or any other suitable phosphate analog known in the art.In some cases, R3 can be 1-10 phosphate groups.In some cases, R3 can be 3-12 phosphate groups.In some cases, nucleotide analogs are nucleoside triphosphates.

[0080] In certain embodiments, R1 of Formula I has a MW of less than 184, typically less than 174, typically less than 164, typically less than 154, typically less than 144, typically less than 134, typically less than 124, typically less than 114, typically less than 104, typically less than 94, and sometimes less than 84. When conjugated to a larger carrier molecule such as KLH, R1 can act as a hapten and elicit an immune response.

[0081] It will be understood that unincorporated NLRT nucleotide analogs are suitable as substrates for enzymes with DNA polymerase activity and can be incorporated into the DNA chain at the 3' end. For example, the reversible blocking group has a size and structure that allows the NLRT to be a substrate for at least some DNA polymerases. Incorporation of the NLRT can be accomplished by a terminal transferase, a polymerase, or a reverse transcriptase. Any DNA polymerase used for sequencing can be used, including, for example, a DNA polymerase from the genus Thermococcus, such as 9°N or mutants thereof, including A485L, including the double mutants Y409V and A485L. As is known in the art, polymerases are highly discriminatory with respect to the nature of the 3' blocking group. Therefore, it is generally necessary to mutate the polymerase protein to promote efficient incorporation. Exemplary DNA polymerases and methods that can be used in the present invention include those described in Chen, C., 2014, "DNA Polymerases Drive DNA Sequencing-By-Synthesis Technologies: Both Past and Present" Frontiers in Microbiology, Vol. 5, Article 305, Pinheiro, V. et al. 2012 "Polymerase Engineering: From PCR and Sequencing to Synthetic Biology" Protein Engineering Handbook: Volume 3: 279-302; International Patent Publication Nos. WO 2005 / 024010 and WO 2006 / 120433, each of which is incorporated by reference for all purposes. In some cases, the polymerase is a DNA polymerase from Thermococcus, such as 9°N or a mutant thereof, including A485L, including the double mutants Y409V and A485L. Other examples include E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T7 or T5 bacteriophage DNA polymerase, HIV reverse transcriptase; Phi29 polymerase, and Bst DNA polymerase.

[0082] It will be appreciated that modifications to the blocking groups should not interfere with the reversible terminator function. That is, they should be cleavable to generate 3'-OH deoxyribonucleotides.

[0083] In one embodiment, prior to incorporation of RT into a DNA strand, RT has the following structure II. Structure II wherein R1 is a 3'-O reversible blocking group, R4 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U); and R3 comprises at least one (e.g., 1-10) phosphate. In some cases, R3 is a triphosphate.

[0084] In one embodiment, after incorporation of RT into a DNA strand, RT has the following structure III. Structure III wherein R1 is a 3'-O-reversible blocking group, R2 is a nucleoside base, such as adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U) or inosine (I) or an analog thereof, and X is a polynucleotide (e.g., GDS) comprising 10-1000 nucleosides linked by phosphate-sugar bonds (e.g., a phosphodiester bond linking the 3' carbon atom of one nucleoside sugar molecule to the 5' carbon atom of another nucleoside sugar molecule).

[0085] In another embodiment, after incorporation and removal of the reversible blocking group, RT has structure IV: Structure IV R6 is H and R7 is a polynucleotide comprising 10-1000 nucleosides linked by phosphate-sugar bonds (eg, GDS) as defined above, or is R3 as defined above.

[0086] In certain embodiments of Structures I, III, and IV, R2 is a nucleobase analog (e.g., an analog of A, T, G, C, U) whose modification does not alter the binding specificity of that base (i.e., an A analog binds to T, a T analog binds to A, etc., (ii) but may render the analog more immunogenic than the naturally occurring base. In some embodiments, the modification may comprise the addition of a group comprising no more than 3 carbons. The added group is not removed from the nucleoside when it is incorporated into the GDS, such that the GDS comprises multiple nucleotides comprising the modification. In such embodiments, the affinity reagent binds to the terminal nucleotide analog comprising the modification, but binds to internal nucleotides with much lower affinity using the modification.

[0087] In applications where there is more than one terminal nucleotide at a given end (e.g., the 3' end), various methods can be used to block the end of interest, such as by different blocking groups or by attaching a "contaminating" end to the support. For example, for DNB sequencing, there may be other 3' ends in addition to the 3' end used for sequencing. In PCR clusters generated by bridge PCR, the sequencing template is attached via the 5' end, so the 3' end of the template cannot be extended or modified by RT to prevent binding to the molecular binders described herein. 3.2 Reversible terminator blocking group

[0088] The NLRTs used in the present invention may include any suitable blocking group. In some embodiments, a suitable blocking group is one that can be removed by chemical or enzymatic treatment to generate a 3'-OH group. The chemical treatment should not significantly degrade the template or primer extension strand. Various molecular units of 3' blocking groups of reversible terminators have been described, such as 3'-O-allyl groups (Ju et al., Proc. Natl. Acad. Sci. USA 103: 19635-19640, 2006), 3'-O-azidomethyl-dNTP (Guo et al., Proc. Natl Acad. Sci. USA 105, 9145-9150, 2008), aminoalkoxy groups (Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29: 879-895, 2010) and 3'-O-(2-cyanoethyl) groups (Knapp et al., Chem. Eur. J., 17, 2903-2915, 2011). Exemplary RT blocking groups include -O-azidomethyl and -O-cyanovinyl. Other exemplary RT blocking groups are described in Figure 3 and 4 The figures are shown for purposes of illustration and not limitation.

[0089] In other embodiments, R of Formula I (above) is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, or substituted or unsubstituted heteroalkynyl. In some examples, R can be selected from allenyl, cis-cyanovinyl, trans-cyanovinyl, cis-cyanofluorovinyl, trans-cyanofluorovinyl, cis-trifluoromethylvinyl, trans-trifluoromethylvinyl, biscyanovinyl, bisfluorovinyl, cis-propenyl, trans-propenyl, nitrovinyl, acetylvinyl, methylcarbonylvinyl, amidoethyl, methylsulfonylvinyl, methylsulfonylethyl, methylimidate, methylhydroxamate, vinylvinyl, vinylvinyl, cyanovinyl, nitrovinyl, amidovinyl, 3-oxobut-1-ynyl, and 3-methoxy-3-oxoprop-1-ynyl.

[0090] A variety of 3'-O reversible blocking groups (R1 in Formula I) can be used in the practice of the present invention. According to one embodiment of the method of the present invention, R1 is selected from allyl, azidomethyl, aminoalkoxy, 2-cyanoethyl, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heteroalkenyl, unsubstituted heteroalkenyl, substituted heteroalkynyl, unsubstituted heteroalkynyl, allyl, cis-cyanovinyl, trans-cyanovinyl, cis-cyanofluorovinyl, trans-cyanofluorovinyl, cis-trifluoromethylethyl

[0065] The following examples include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 113, 114, 125, 136, 137, 148, 159, 119, 129, 130

[0091] As used herein, the terms "alkyl," "alkenyl," and "alkynyl" include straight and branched monovalent substituents. Examples include methyl, ethyl, isobutyl, 3-butynyl, and the like. The range of these groups useful in the compounds and methods described herein includes C1-C 10 Alkyl, C2-C 10 Alkenyl and C2-C 10 Alkynyl. Additional scopes of these groups that can be used in the compounds and methods described herein include C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C4 alkyl, C2-C4 alkenyl, and C2-C4 alkynyl.

[0092] "Heteroalkyl," "heteroalkenyl," and "heteroalkynyl" are similarly defined as alkyl, alkenyl, and alkynyl, but may contain O, S, or N heteroatoms, or combinations thereof, within the backbone. The range of these groups useful in the compounds and methods described herein includes C1-C 10 Heteroalkyl, C2-C 10 Heteroalkenyl and C2-C 10 Additional scopes of these groups that can be used in the compounds and methods described herein include C1-C8 heteroalkyl, C2-C8 heteroalkenyl, C2-C8 heteroalkynyl, C1-C6 heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, C1-C4 heteroalkyl, C2-C4 heteroalkenyl, and C2-C4 heteroalkynyl.

[0093] As used herein, the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl molecules can be substituted or unsubstituted. As used herein, the term substituted includes adding an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl group at a position attached to the main chain of an alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl group, for example, replacing hydrogen with one of these molecules. Examples of substituents include, but are not limited to, hydroxyl, halogen (e.g., F, Br, Cl or I) and carboxyl. On the contrary, as used herein, the term unsubstituted means that an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl group has complete hydrogen, i.e., comparable to its saturation level, without substitution, for example, linear butane (-(CH2)3-CH3).

[0094] In other embodiments, the reversible blocking group is an amino-containing blocking group (e.g., NH2-). See Hutter et al., 2010, Nucleosides Nucleotides Nucleic Acids 29(11) (incorporated herein by reference), which describes exemplary amino-containing reversible blocking groups. In some embodiments, the reversible blocking group is an allyl-containing blocking group (e.g., CH2=CHCH2-). In some embodiments, the reversible blocking group comprises a cyano group (e.g., a cyanovinyl or cyanoethyl group). In some embodiments, the reversible blocking group is an azide-containing blocking group (e.g., N3-). In some embodiments, the reversible blocking group is an azidomethyl (N3CH2-). In some embodiments, the reversible blocking group is an alkoxy-containing blocking group (e.g., CH3CH2O-). In some embodiments, the reversible blocking group contains a polyethylene glycol (PEG) unit having one or more ethylene glycol moieties. In some embodiments, the reversible blocking group is a substituted or unsubstituted alkyl group (i.e., a substituted or unsubstituted hydrocarbon). In some embodiments, the reversible blocking group is an acyl group. See, U.S. Patent No. 6,232,465, which is incorporated herein by reference. In some embodiments, the reversible blocking group is or contains a methoxymethyl group. In some embodiments, the reversible blocking group is or contains an aminooxy group (H2NO-). In some embodiments, the reversible blocking group is or contains a carbonyl group (O=CH-). In some embodiments, the reversible blocking group comprises an ester or phosphate group.

[0095] In some embodiments, the reversible blocking group is a nitrobenzyl group (C6H4(NO2)-CH2-). In some embodiments, the reversible blocking group is a coumarin group (i.e., containing a coumarin unit or a derivative thereof), wherein, for example, any one of the CH carbons of the coumarin-based reversible blocking group is covalently attached to the 3'-O of the nucleotide analog.

[0096] In some embodiments, the reversible blocking group is nitronaphthyl (ie, contains a nitronaphthyl unit or a derivative thereof), wherein, for example, either of the CH carbons of the nitronaphthyl reversible blocking group is covalently attached to the 3'-O of the nucleoside analog.

[0097] In some embodiments, the reversible blocking group is selected from: wherein R3 and R4 are H or alkyl, and R5 is alkyl, cycloalkyl, alkenyl, cycloalkenyl, and benzyl. In certain embodiments, the determination of R3-R5 is subject to the MW constraints described herein (e.g., see Section 3.2.1).

[0098] Other reversible blocking groups suitable for use in the present invention are those described in the literature as blocking groups for labeled reversible terminators. Generally, any suitable reversible blocking group for sequencing by synthesis can be used in the practice of the present invention. 3.2.1 Properties of reversible terminator blocking groups and nucleotides containing them

[0099] Preferably, for sequencing applications, the blocking group of the RT is removable under reaction conditions that do not interfere with the integrity of the DNA being sequenced. An ideal blocking group will exhibit long-term stability, be efficiently incorporated by the polymerase, result in complete blocking of secondary or further incorporation, and be removable under mild conditions, preferably aqueous conditions, that do not damage the polynucleotide structure.

[0100] In certain embodiments of the invention, the blocking group (including the 3' oxygen atom of the deoxyribose) has a molecular weight (MW) of less than 200, typically less than 190, typically less than 180, typically less than 170, typically less than 160, typically less than 150, typically less than 140, typically less than 130, typically less than 120, typically less than 110, and sometimes less than 100. In other words, in certain embodiments, R3 of Formula I has a MW of less than 184, typically less than 174, typically less than 164, typically less than 154, typically less than 144, typically less than 134, typically less than 124, typically less than 114, typically less than 104, typically less than 94, and sometimes less than 84.

[0101] The molecular weight of deoxyribose monophosphate nucleotides ranges from about 307 to 322 (dAMP 331.2, dCMP 307.2, dGMP 347.2, and dTMP 322.2). In certain embodiments, the NLRT unit, when incorporated into a GDS (i.e., excluding the pyrophosphates of the dNTPs), has a molecular weight of less than 550, typically less than 540, typically less than 530, typically less than 520, typically less than 510, typically less than 500, typically less than 490, typically less than 480, typically less than 470, and sometimes less than 460. 3.3 Containing phosphate units

[0102] In some embodiments, the R3 unit comprises one or more phosphate and / or phosphate analog units. In some embodiments, the R3 unit may have the following structure (Structure V), wherein n=0 to 12 (typically 0, 1, 3, 4, 5, or 6) and X is H or any structure compatible with incorporation by a polymerase in a primer extension reaction. For example, X can be an alkyl group or any of the various linkers described in the art. See, for example, U.S. Patent No. 9,702,001, which is incorporated herein by reference. It should be understood that in the process of incorporating the reversible terminator into the GDS, the X unit (all except α phosphate) is removed from the nucleotide so that X is not present in the incorporated reversible terminator deoxyribonucleotide. In certain embodiments, X can be a detectable label or affinity tag, provided that the affinity reagent of the present invention does not bind to the X unit, or discriminates the reversible terminator based on the presence, absence, or structure of the X unit, and does not exist in the incorporated reversible terminator deoxyribonucleotide. 3.4 NLRT group

[0103] In some approaches, SBS sequencing according to the present invention comprises contacting a sequencing array with a plurality of NLRTs (e.g., NLRT-A, NLRT-T, NLRT-C, and NLRT-G). The contacting can be performed sequentially, one NLRT at a time. Alternatively, four NLRTs can be contacted with the sequencing array simultaneously, most commonly as a mixture of the four NLRTs. The four NLRTs together constitute an "NLRT set." The NLRTs of the NLRT set can be packaged as a mixture or can be packaged as a kit that includes each different NLRT in a separate container. Each base can be included in an equal proportion or can be included in unequal amounts in the mixture of the four NLRTs.

[0104] In one embodiment, each NLRT in the NLRT set comprises the same blocking group (e.g., azidomethyl). In one embodiment, the NLRTs in the NLRT set comprise different blocking groups (e.g., NLRT-A comprises azidomethyl, and NLRT-T comprises cyanoethylene; or NLRT-A and NLRT-G comprise azidomethyl, and NLRT-C and NLRT-T comprise cyanoethylene). If different blocking groups are used, such blocking groups are optionally selected so that different blocking groups can be removed by the same treatment. Alternatively, the blocking groups can be removed by different treatments, optionally at different times. In one embodiment, one or more NLRTs in a group comprise modified (non-naturally occurring) nucleobases.

[0105] The NLRTs described herein can be provided or used in the form of a mixture. For example, the mixture can contain two, three, or four (or more) structurally distinct NLRTs. The structurally distinct NLRTs can differ in their respective nucleobases. For example, the mixture can contain four structurally distinct NLRTs, each comprising one of the four natural DNA nucleobases (i.e., adenine, cytosine, guanine, and thymine) or a derivative thereof.

[0106] For sequencing purposes, the different NLRTs in the NLRT set can be packaged separately and then mixed on the sequencer itself (e.g., before delivery to the flow cell) or can be packaged together (i.e., pre-mixed). A kit comprising an NLRT set (wherein the different NLRTs are packaged in separate containers or as a mixture in the same container) can be provided. 3.5 Nucleobase analogs with groups that improve affinity reagent binding

[0107] In one embodiment, the nucleobase includes a non-removable chemical group that, when present at the 3' end of the growing DNA strand (i.e., as the last base incorporated), increases the specificity or affinity of the affinity reagent for the nucleobase, but is not recognized or accessible to the affinity reagent in nucleotides within the primer extension product. In one approach, the modification is recognized or bound by the affinity reagent, but with lower affinity or less efficiency relative to the same modification in the 3' terminal nucleotide.

[0108] By way of illustration and not limitation, examples of such modified nucleobases include: R6, R7, R8, and R9: can be the same or different and are each selected from H, I, Br, F, Structures XIX-XXVIII, or any group that does not interfere with base pairing. Note that R9 is the methyl group in thymidine in Structure XVIII. In some cases, modifications have the added benefit of increasing the antigenicity of the nucleotide.

[0109] The molecular weights of naturally occurring nucleobases are: adenine 135; guanine 151, thymine 126, and cytosine 111. In some embodiments, the nucleobase analogs have a molecular weight that is no more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 Da greater than that of the natural bases. 3.6 Unblocked dNTPs

[0110] In one embodiment, natural dNTPs (e.g., dATP, dGTP, dCTP, or dTTP) or dNTP analogs without 3'-O-blocking groups are used for sequencing. In some embodiments, nucleotides are incorporated one at a time during sequencing (e.g., in pyrophosphate sequencing), or are incorporated by polymerase, which pauses after a base is incorporated. Exemplary methods are described in the literature (see, e.g., Ju et al., 2006, Proc. Natl. Acad. Sci. USA 103: 19635-40, 2006; Guo, Proc. Natl Acad. Sci. USA 105, 9145-50, 2008, and Ronaghi et al., Science, 281: 363-365, 1998), which can be modified for the present invention by removing the label and / or joint connecting the label to RT. In some approaches, dNTPs with different nucleobases are added and incorporated sequentially (e.g., A, then G, etc.) The nucleobases are typically imaged individually before the next dNTP is added. 3.7 Deoxyribose analogs

[0111] In some embodiments of the present invention, the sugar (deoxyribose) unit is modified. For example, using an affinity reagent that recognizes both the blocking group and the sugar unit, an NLRT having the nucleobase adenine, the blocking group azidomethyl, and the sugar deoxyribose can be distinguished from an NLRT having the nucleobase cytosine, the blocking group azidomethyl, and the sugar-modified deoxyribose. 3.8 Nucleotides without 3'-O reversible terminators

[0112] In a different aspect that can be used in several applications, nucleotides with non-removable (i.e., non-cleavable) 3' blocking groups are used instead of NLRTs. In one approach, after detection with an affinity reagent, the last incorporated base is removed and its place is filled with a similar nucleotide but with a cleavable blocking group (Koziolkiewicz et al., FEBS Lett. 434:77-82, 1998).

[0113] The examples given above include reversible blocking groups attached to the nucleotide via the 3'-O of the deoxyribose sugar unit. The present invention also includes NLRTs with reversible and irreversible blocking groups attached to the 2'-O of the deoxyribose sugar. These embodiments can be used for single-base detection (single or a few base primer extensions), monitoring gaps and nicks in DNA, and other detection methods. Therefore, one of ordinary skill in the art will be able to apply the methods and information herein to NLRTs with 2' rather than 3' blocking groups. 4. Affinity reagents

[0114] The present invention utilizes an affinity reagent that specifically binds to the NLRT at the 3' end of the GDS, e.g., after incorporation by a polymerase into the end of the growing DNA strand during SBS. In one embodiment, the affinity reagent binds to the NLRT of Structure III. In one embodiment, the affinity reagent binds to the NLRT of Structure IV. 4.1 Common affinity reagents

[0115] In one aspect, the present invention relates to an affinity reagent for detecting the presence or absence of an NLRT incorporated at the 3' end of a nucleic acid. An affinity reagent is a molecule or macromolecule that specifically binds to an NLRT based on the structural characteristics of the incorporated NLRT. For example, the affinity reagent can specifically bind to an NLRT having, for example, a specific base and / or a specific reversible blocking group. To illustrate, an example of an affinity reagent is a monoclonal antibody (mAb) that binds with high affinity to an NLRT incorporated at the 3' end of a DNA chain when the NLRT comprises the nucleobase adenosine and an azidomethyl reversible blocking group, but does not bind with high affinity to an NLRT incorporated at the 3' end of a DNA chain when the NLRT comprises the nucleobase adenosine but has a 3' hydroxyl group instead of an azidomethyl reversible blocking group, and does not bind with high affinity to an NLRT incorporated at the 3' end of a DNA chain when the NLRT comprises the nucleobase cytosine, guanine, or thymine (each with or without an azidomethyl reversible blocking group). Affinity reagents can be labeled directly or indirectly.

[0116] "Specificity" is the degree to which an affinity reagent discriminates between different molecules (e.g., NLRTs), as measured, for example, by the relative binding affinities of the affinity reagent for the molecules. With respect to the affinity reagents of the present invention, the affinity reagent should have a substantially higher affinity for one NLRT (its target RT) than for other NLRTs (e.g., the affinity reagent binds to a C nucleoside analog but not to A, T, or G). Furthermore, when incorporated by a polymerase at the 3' end of a growing DNA strand, the affinity reagent binds to the target nucleoside analog at the end of the polynucleotide but does not bind to nucleotide bases at other positions on the DNA strand. If there are multiple template polynucleotides (e.g., an array of template polynucleotides), wherein the 3'-end of the GDS includes NLRT-A, NLRT-T, NLRT-C, and NLRT-G (e.g., in an array), then the affinity reagent has specificity for a particular NLRT (e.g., NLRT-A), and the affinity reagent preferentially binds to NLRT-A under the reaction conditions used in SBS sequencing. As used herein, "preferential binding" of an affinity agent to a first structure as compared to a second structure means that the affinity agent binds to the first structure but does not bind to the second structure or binds to the second structure less strongly (i.e., with lower affinity) or less efficiently.

[0117] In the context of affinity agent binding to an incorporated NLRT, the terms "specifically bind," "specifically binds," and the like refer to the preferential association of the affinity agent with a particular NLRT (e.g., NLRT-A with a 3'-O azido group) compared to an NLRT with a different nucleobase (NLRT-T, NLRT-C, or NLRT-G), a different blocking group, or no blocking group (e.g., deoxyadenosine with a 3'-OH). Specific binding between an affinity agent and an NLRT sometimes means at least 10 -6 M -1 The affinity (i.e., the dissociation constant K d Measured with less than 10 -6 M -1 Affinity of the value). Greater than 10 -8 M -1 An affinity of 1% to 1% is preferred. Specific binding can be determined using any binding (e.g., antibody binding) assay known in the art, including Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunohistochemistry, and detection of fluorescently labeled affinity reagents bound to the target NLRT in a sequencing reaction. As discussed below, the specificity of binding can be determined by positive and negative binding assays.

[0118] The specific binding interaction between an affinity agent (e.g., an antibody) and an incorporated reversible terminator deoxyribonucleotide can be described in various ways, including by reference to a portion or unit of the incorporated reversible terminator deoxyribonucleotide that is responsible for specificity. An analogy is helpful here: imagine a protein with two domains (Domain 1 and Domain 2). Two different antibodies can specifically bind to this protein. However, they may recognize different epitopes. For example, one antibody may bind to an epitope in Domain 1, while a second antibody may bind to an epitope in Domain 2. In this hypothetical scenario, if a modification is made in Domain 1, this may affect the binding of the primary antibody to the protein without altering the binding of the second antibody. In this case, the binding of the primary antibody to the protein can be said to be "dependent" on Domain 1, meaning that changes in Domain 1 (e.g., changes in the amino acid sequence) will alter the binding properties of Antibody 1 (e.g., abolish binding, increase binding affinity, decrease binding affinity, etc.). Equivalently, Domain 1 can be said to be "responsible" for the binding of Antibody 1. In the case of an incorporated reversible terminator deoxyribonucleotide, the specificity of binding may be due to the structural features of one unit (e.g., a blocking group) and not be affected by other units (e.g., a nucleobase) or by the structure of other units. Alternatively, the specificity of binding may be due to structural features of multiple units (e.g., a nucleobase and a blocking group), etc. When binding of an affinity agent to an incorporated reversible terminator deoxyribonucleotide requires the presence of specific structural features of a unit, the binding of the affinity agent may be "specific for" or "based on" the presence or absence of a unit having those structural features. Equivalently, a unit having those structural features may be "responsible for" binding by the affinity agent, or binding of the affinity agent may be "dependent on" the presence of a unit having those structural features.

[0119] It should also be noted that "specificity" can depend on the context. For example, imagine an affinity reagent that binds A and A' but not B, C, or D. In a reaction or sample containing A, A', B, and C, the affinity reagent may bind to both A and A' and thus might not be considered to "specifically bind" A. However, in a reaction or sample containing A, B, C, and D, the affinity reagent would only bind A and, in that context, would be said to specifically bind A. In another example, in a sample containing A, A', B, and C, the affinity reagent may bind to A and A' with different affinities or efficiencies, allowing for the distinction between binding to A and binding to A' based on this.

[0120] Another related term is "discriminate" (or sometimes "discriminate"). An affinity reagent that binds to an incorporated reversible terminator deoxyribonucleotide only when a particular blocking group (e.g., an azidomethyl group) is present, but that binds to incorporated reversible terminator deoxyribonucleotides having an azidomethyl blocking group regardless of the nucleobase present, can be said to "discriminate" between incorporated reversible terminator deoxyribonucleotides having and not having an azidomethyl blocking group, or more broadly, to "discriminate based on the blocking group."

[0121] The specificity of an affinity reagent is a result of the process used to prepare it. For example, reagents that recognize an azidomethyl blocking unit can be empirically tested using positive and negative binding assays. To illustrate, in one approach, the reagent is an antibody that binds to the NLRT based on the presence of an O-azidomethyl blocking unit. In one approach, an azidomethyl conjugated to keyhole limpet hemocyanin is used to raise antibodies against the hapten O-azidomethyl. The desired antibody can be selected for binding to 3'-O-azidomethyl-2'-deoxyguanine, but antagonizing binding to other deoxyguanine nucleotides [such as 3'-O-2-(cyanoethoxy)methyl-2'-deoxyguanine; 3'-O-(2-nitrobenzyl)-2'-deoxyguanine; and 3'-O-allyl-2'-deoxyguanine]; and antagonizing binding to other azidomethyl NLRTs (such as 3'-O-azidomethyl-2'-deoxyadenosine; 3'-O-azidomethyl-2'-deoxycytosine; and 3'-O-azidomethyl-2'-deoxythymidine).

[0122] The nature of the antibody-hapten interaction can also be determined using methods known in the art, such as those described in: Al Qaraghuli, 2015, “Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction” BMC Biotechnology, 15(1) p.1; Britta et al., 2005, “Generation of hapten-specific recombinant antibodies: Antibody phage display technology: A review” Vet Med. 50:231-52; Charlton et al., 2002. “Isolation of anti-hapten specific antibody fragments from combinatorial libraries” Methods Mol Biol. 178:159-71; and Hongtao et al., 2014, “Molecular Modeling Application on Hapten Epitope Prediction: An Enantioselective Immunoassay for Ofloxacin Optical Isomers” J. Agric. Food Chem. 62(31) pp 7804-7812. It will be understood that describing an affinity agent as binding to certain units (e.g., a nucleobase and a sugar unit) does not exclude binding to other parts of the incorporated nucleotide. For example, an affinity agent that binds to a nucleobase and a sugar unit may also bind to a blocking group.

[0123] Examples of useful affinity reagents include antibodies (including binding fragments of antibodies, single-chain antibodies, bispecific antibodies, etc.), aptamers, knottins, affimers, labeled dNTPs that form single-base triple helices, guanine nucleotide binding proteins (G-proteins), or any other known reagent that binds to the incorporated NLRT with appropriate specificity and affinity.

[0124] Affinity reagents can specifically recognize a nucleobase, a sugar (e.g., deoxyribose), a blocking group, or any other unit or combination thereof in a target NLRT. In one approach, an affinity reagent recognizes an epitope that includes a blocking group. In another approach, an affinity reagent recognizes an epitope that includes a nucleobase. In another approach, an affinity reagent recognizes an epitope that includes a nucleobase and a blocking group. It should be understood that even if an affinity reagent does not contact a unit, the unit can determine the position of other units. For example, for an affinity reagent that discriminates NLRTs based on a nucleobase and a 3' blocking group, a deoxyribose unit is required to position the nucleobase and the 3' blocking group for recognition.

[0125] In the case of an affinity reagent that is an antibody, specific binding can be determined using any antibody binding assay known in the art, including Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, or column chromatography. In one approach, specific binding is demonstrated using an ELISA-type assay. For example, serum antibodies raised against 3'-O-azidomethyl-dC are titrated continuously against 3'-O-azidomethyl-dC (positive specificity assay) and nucleotides such as 3'-O-azidomethyl-dG or -dA or 3'-OH-dC (negative specificity assay).

[0126] In some embodiments, the base-specific binding of an affinity reagent to its target nucleoside is 2-100 times greater than the binding to other nucleosides or analogs. In some embodiments, the base-specific binding of an affinity reagent to its target nucleoside is at least 10 times greater than the binding to other nucleosides, or at least 30 times, or at least 100 times.

[0127] Preferably, the antibody binds to a specific base at a concentration of less than 100 pM, or less than 1 nM, or less than 10 nM, or less than 1 μM.

[0128] Affinity reagents with the desired specificity can be selected using methods known in the art. For example, affinity reagents such as antibodies can be identified, selected, or purified by multiple rounds of positive selection (i.e., binding to the target molecule) and negative selection (i.e., not binding to molecules that are not the target molecule).

[0129] The affinity reagent can bind both dNTPs in solution and the corresponding nucleotide incorporated at the 3' end of the primer extension product. In some embodiments, the affinity reagent does not bind to unincorporated NLRTs (e.g., NLRTs in solution) or binds with significantly lower specificity. However, generally, binding of the affinity reagent to unincorporated NLRTs does not occur during sequencing because the unincorporated NLRTs are removed (washed away) before the affinity reagent is introduced. Alternatively, the complex formed by the affinity reagent bound to the NLRT is removed (washed away) before imaging.

[0130] In one approach, an affinity reagent specifically binds to a nucleobase and discriminates between different bases (e.g., A, T, G, C) based in part on the presence or absence of a 3'-OH group. In this approach, the affinity reagent distinguishes the nucleotide at the 3' end of the GDS from the 3'-OH of incorporated nucleotides from within the GDS (not the 3' end). In some cases, affinity reagents that recognize a specific nucleobase also discriminate between the presence or absence of a 3'-OH group, thereby identifying an incorporated NLRT as having a 3'-terminal nucleotide of a specific nucleobase.

[0131] In one approach, an affinity reagent recognizes an epitope containing a blocking group but is base insensitive. For example, given four RT blocking groups [A. azidomethyl, B. 2-(cyanoethoxy)methyl, C. 3'-O-(2-nitrobenzyl), and D. 3'-O-allyl], an affinity reagent can be prepared that discriminates against these four blocking groups. To illustrate, given the deoxyguanine analogs labeled A through D below, an affinity reagent can be selected that recognizes only one NLRT but not the other three. A. 3'-O-azidomethyl-2'-deoxyguanine B. 3'-O-2-(Cyanoethoxy)methyl-2'-deoxyguanine C. 3'-O-(2-nitrobenzyl)-2'-deoxyguanine D. 3'-O-allyl-2'-deoxyguanine

[0132] In some embodiments, the affinity reagent selected does not discriminate between nucleotides having different nucleobases, as long as the nucleotides have the same blocking group. For example, an affinity reagent that recognizes B (3'-O-2-(cyanoethoxy)methyl-2'-deoxyguanine) described above may also recognize 3'-O-2-(cyanoethoxy)methyl-2'-deoxyadenine; 3'-O-2-(cyanoethoxy)methyl-2'-deoxythymine; and 3'-O-2-(cyanoethoxy)methyl-2'-deoxycytosine.

[0133] In light of the present disclosure, it is within the skill of one of ordinary skill in the art to generate affinity reagents (e.g., monoclonal antibodies) that differentially recognize RT blocking groups. In one approach, antibodies are raised against the hapten O-azidomethyl (e.g., -O-azidomethyl or azidomethyl conjugated to keyhole limpet hemocyanin) and positively and negatively screened for binding to the 3'-O-azidomethyl-2'-dNM nucleotide (where N is one of A, T, G, or C) at the 3' end of the GDS TP, but not to the 3'-OX-2'-dNM, where OX is a different blocking group present in the sequencing reaction. It should be appreciated that in other embodiments, the hapten can be a deoxyribose with a 3'-O blocking group, a nucleotide (e.g., a monophosphate or triphosphate with a 3'-O blocking group), etc., as long as the selection process identifies an affinity reagent with the desired specificity.

[0134] Although the above examples describe embodiments in which the four nucleotides have very different structurally distinct blocking groups (e.g., azidomethyl versus 2-(cyanoethoxy)methyl), in some embodiments of the present invention, there are only minor differences between the blocking groups bound by different affinity reagents. For example, a hydrogen atom in a blocking group can be replaced by a fluorine atom or a methyl group to generate three related blocking groups [blocking group, F-substituted blocking group, methyl-substituted blocking group] that can be distinguished by the affinity reagent set.

[0135] In some embodiments of the invention, sequencing is performed using four NLRTs, each having a 3'-O-blocking group, wherein two or more, alternatively three or more, alternatively all four, of the blocking groups are structurally similar in the following sense: (1) they have the same number of atoms or differ in the number of atoms by no more than a small number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); (2) the molecular formula of the blocking group units differs by 1 to 10 atoms (e.g., there are 3 differences: a single H is replaced by CH3; H is replaced by F, and O is replaced by S), such as 1 atom, 2 atoms, 3 atoms, 4 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, or 10 atoms. In these and other embodiments, the blocking group units can have any of the properties described above in the section entitled "Properties of Reversible Terminator Blocking Groups and Nucleotides Containing Them."

[0136] In some embodiments, the affinity reagent binds to the NLRT (eg, 3'-O-azidomethyl-2'-deoxyguanine) but does not bind to the corresponding unblocked nucleotide (eg, 3'-OH-2'-deoxyguanine).

[0137] In one embodiment, the affinity reagent binds to the NLRT (eg, 3'-O-azidomethyl-2'-deoxyguanine) but dissociates from the nucleotide analog upon treatment [eg, upon treatment with TCEP (tris(2-carboxyethyl)phosphine)] to remove the blocking group.

[0138] An affinity reagent that specifically recognizes NLRT-A is referred to as anti-A. An affinity reagent that specifically recognizes NLRT-T is referred to as anti-T. An affinity reagent that specifically recognizes NLRT-G is referred to as anti-G. An affinity reagent that specifically recognizes NLRT-C is referred to as anti-C. An affinity reagent that specifically recognizes NLRT-U is referred to as anti-U. Although this nomenclature is similar to the nomenclature used to describe the specificity of immunoglobulins, the use of this term in the present invention is not intended to indicate that the affinity reagent must be an antibody. As described.

[0139] The affinity reagent can be directly labeled. Alternatively, the affinity reagent can be an unlabeled primary affinity reagent detectable using a labeled secondary affinity reagent. For example, an unlabeled primary affinity reagent that specifically binds to the NLRT can be detected using a labeled secondary affinity reagent that binds to the primary affinity reagent (e.g., a labeled antibody that binds to the primary affinity reagent). See Section 4.5 below. 4.2 Exemplary affinity reagents

[0140] In some embodiments, the affinity agent is an antibody.Any method known in the art for producing antibodies can be used. 4.2.1 Antibody

[0141] As used herein, "antibody" means immunoglobulin molecules or compositions (e.g., monoclonal and polyclonal antibodies), as well as genetically engineered forms such as chimeric, humanized and human antibodies, heterologous conjugate antibodies (e.g., bispecific antibodies), and antibody fragments. Antibodies can be produced from recombinant sources and / or in animals (including but not limited to transgenic animals). As used herein, the term "antibody" includes "antibody fragments", including but not limited to Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, miniantibodies, nanoantibodies, diabodies, and multimers thereof, as well as bispecific antibody fragments. Conventional techniques can be used to fragment antibodies. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimer, miniantibody, diabody, bispecific antibody fragment and other fragments can also be synthesized by recombinant technology.Antibody can be any useful isotype, including IgM and IgG, such as IgG1, IgG2, IgG3 and IgG4.In some embodiments, affinity reagent is miniantibody.Miniantibody is an engineered antibody construct, which is composed of the variable heavy chain (VH) and variable light chain (VL) domains of the natural antibody fused with the hinge region and CH3 domains of immunoglobulin molecules.Therefore, miniantibody is a small version of the complete antibody encoded in a single protein chain, which retains antigen binding region, CH3 domains to allow assembly into divalent molecules and allow antibody hinges to adapt to the dimerization by disulfide bonding.Single domain antibodies (sdAb) can also be used.Single domain antibodies or NANOBODY (Ablynx) are approximate antibody fragments with single monomer variable antibody domains. Single-domain antibodies selectively bind to specific antigens and are smaller than conventional antibodies (MW 12-15 kDa). 4.2.1.1 Antibody production

[0142] Methods for culturing polyclonal antibodies are known and can be used to generate NLRT-specific antibodies. One approach is described in Example 2 below. According to one method for culturing polyclonal antibodies specific for a particular NLRT (e.g., NLRT-A), rabbits are injected with NLRT-A (conjugated to an immunogen) to raise antibodies, and the antibodies are selected so as not to bind to: the lack of a blocking group (e.g., having a 3'-OH) and the identical structure of other NLRTs (NLRT-T, NLRT-G, and NLRT-C). Thus, the polyclonal antibodies generated recognize the specific NLRT incorporated at the 3' end of the growing DNA chain at a specific position on the sequencing array, rather than the same nucleoside at other internal positions of the growing chain or other NLRTs that may be incorporated at other positions on the array. (The polyclonal antibodies can also recognize unincorporated NLRT-A, but the unincorporated NLRT is washed away before the incorporated NLRT is detected using a labeled affinity reagent).

[0143] It should be recognized that, depending on the researcher's needs, it is not always necessary to raise antibodies against the entire NLRT. For example, if an antibody specific for a blocking group is desired, the hapten can be a deoxyribose with a 3'-O-blocking group (i.e., no nucleobase) or only a 3'-O-blocking group. In some embodiments, antibodies are raised against a polynucleotide having the NLRT of interest at its 3' end. In some embodiments, antibodies are raised against a polynucleotide annealed to a template molecule.

[0144] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from animals immunized with an immunogen containing NLRT, and fused with myeloma cells by standard somatic cell fusion methods to immortalize these cells and harvest hybridoma cells. These techniques are well known in the art [e.g., the hybridoma technology initially developed by Kohler and Milstein (Kohler and Milstein Nature 256:495-497, 1975), and other techniques, such as human B-cell hybridoma technology (Kozbor et al., 1983, Immunol. Today 4:72), EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., 1986, Methods Enzymol, 121:140-67), and screening of combinatorial antibody libraries (Huse et al., 1989, Science 246:1275)]. Hybridoma cells can be immunochemically screened to produce antibodies that specifically react with a particular RT, and monoclonal antibodies can be isolated.

[0145] Specific antibodies or antibody fragments reactive to specific antigens or molecules can also be generated by screening expression libraries encoding immunoglobulin genes or portions thereof that are expressed in bacteria with cell surface components. For example, complete Fab fragments, VH regions, and FV regions can be expressed in bacteria using phage expression libraries (see, e.g., Ward et al., Nature 341:544-546, 1989; Huse et al. Science 246:1275, 1989; and McCafferty et al. Nature 348:552-554, 1990).

[0146] In addition, antibodies specific for the target NLRT can be readily isolated by screening antibody phage display libraries. For example, antibody phage libraries can be optionally screened using antibody fragments to identify antibody fragments specific for the target NLRT. Methods for screening antibody phage libraries are well known in the art.

[0147] Anti-NLRT antibodies can also be produced in cell-free systems. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44, 2009; Spirin, Trends Biotechnol. 22:538-45, 2004; and Endo et al., Biotechnol. Adv. 21:695-713, 2003. 4.2.1.2 Antibody purification

[0148] Anti-NLRT antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides. See Section 8.4 below. Numerous methods for purifying polypeptides are known in the art. Affinity purification of anti-NLRT antibodies from polyclonal antisera is described in Example 2 below. 4.2.1.3 Antibody labeling

[0149] Antibodies can be labeled using any method known in the art. Methods for linking antibodies and other affinity reagents to reporter molecules (e.g., signal-generating proteins including enzymes and fluorescent / luminescent proteins) are well known in the art (Wild, The Immunoassay Handbook, 4 th ed.; Elsevier: Amsterdam, the Netherlands, 2013; Kobayashi and Oyama, Analyst 136:642-651, 2011). 4.2.2 Aptamer

[0150] Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers can be classified as: (a) DNA or RNA or XNA aptamers, which consist of a (usually short) oligonucleotide chain; and (b) peptide aptamers, which consist of one (or more) short variable peptide domains attached at both ends to a protein scaffold.

[0151] Nucleic acid aptamers are nucleic acid species that have been engineered to bind to various molecular targets (e.g., NLRTs) through repeated rounds of in vitro selection or, equivalently, SELEX (systematic evolution of ligands by exponential enrichment). For example, aptamers with affinity for target NLRTs can be selected from large oligonucleotide libraries by SELEX, an iterative process in which non-binding aptamers are discarded and aptamers that bind to the proposed target are expanded. The initial positive selection round is sometimes followed by negative selection. This improves the selectivity of the resulting aptamer candidates. In this process, the target NLRT is fixed on an affinity column. An aptamer library is applied and allowed to bind. Weak binders are washed off, and PCR is used to elute and amplify the bound aptamers. The amplified aptamer pool is then reapplied to the target. The process is repeated multiple times under increasingly stringent conditions until an aptamer with the desired selectivity and affinity is obtained. See, for example, Jayasena, et al., Clinical Chemistry 45:1628-1650, 1999. Peptide aptamer selection can be performed using different systems (including the yeast two-hybrid system). Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies (e.g., mRNA display, ribosome display, bacterial display, and yeast display). These experimental procedures are also called biopanning. See, for example, Reverdatto et al., 2015, Curr. Top. Med. Chem. 15: 1082-1101. 4.2.3 Affimer

[0152] Affimers are small (12-14 kDa) highly stable proteins that bind to their target molecules with specificity and affinity similar to antibodies. These proteins have a common tertiary structure of α-helices located on an anti-parallel β-sheet. Affimer proteins display two peptide loops and an N-terminal sequence, both of which can be randomized to bind to the desired target protein with high affinity and specificity in a manner similar to monoclonal antibodies. Stabilizing the two peptides by a protein scaffold limits the possible conformations that the peptides can adopt, thereby increasing binding affinity and specificity compared to free peptide libraries.

[0153] Affimers specific for NLRTs can be selected by using phage display libraries that are screened to identify Affimer proteins with high specific binding and high binding affinity (e.g., in the nM range) to the target NLRT. Many different labels, tags, and fusion proteins, such as fluorophores, have been conjugated to Affimer proteins for various applications. See, for example, U.S. Patent Nos. 8,481,491, 8,063,019, and WO 2009 / 136182, which are incorporated herein by reference. See also Crawford et al., Brief Funct. Genomic Proteomic, 2:72-79, 2003. 4.2.4 Knottin

[0154] "Knottin" or "inhibitor cystine knot" (ICK) is a protein structural motif containing three disulfide bridges. Two disulfides, together with the polypeptide segment between them, form a ring through which a third disulfide bond (linking the third and sixth cysteines in the sequence) passes, thereby forming a knot. Protein engineering can be used to introduce new binding epitopes into natural knottins, and knottins have been engineered to target a wide range of targets. One approach to producing knottins specific for NLRTs is to create and screen knottin libraries using yeast surface display and fluorescence-activated cell sorting. For information on generating knottins that are selective and highly affinity for target NLRTs and labeling such knottins for use in conjunction with the present invention, see, e.g., Kintzing and Cochran, Curr. Opin. Chem. Biol. 34:143-150, 2016; Moore et al., Drug Discovery Today: Technologies 9(1):e3-e11, 2012; and Moore and Cochran, Meth. Enzymol. 503:223-51, 2012. 4.3 Labeled affinity reagent

[0155] Labeled affinity reagents can be used to sequence template nucleic acids by a variety of methods. They can also be used in a variety of applications other than sequencing, as will be apparent to those skilled in the art. Any method of labeling antibodies and other affinity reagents of the present invention can be used. 4.3.1 Fluorescent detectable labels

[0156] Affinity reagents for use in the practice of the present invention, including antibodies, aptamers, Affimers, Knottins, and other affinity reagents described herein, can be detectably labeled. For example, affinity reagents described herein can be detectably labeled with fluorescent dyes or fluorophores. "Fluorescent dye" means a fluorophore (a compound that absorbs light energy of a specific wavelength and re-emits light of a longer wavelength). Fluorescent dyes typically have a maximum molar extinction coefficient at a wavelength of about 300 nm to about 1,000 nm or at least about 5,000, more preferably at least about 10,000, and most preferably at least about 50,000 cm-1 m-1, and a quantum yield of at least about 0.05, preferably at least about 0.1, more preferably at least about 0.5, and most preferably from about 0.1 to about 1.

[0157] There is a wealth of practical guidance in the literature for selecting appropriate detectable labels to attach to affinity reagents, as exemplified by the following references: Grimm et al., Prog. Mol. Biol. Transl. Sci. 113:1-34, 2013; Oushiki et al., Anal. Chem. 84:4404-4410, 2012; Medintz & Hildebrandt, eds., 2013, "FRET- Resonance Energy Transfer: from theory to applications," (John Wiley & Sons); etc. The literature also includes references providing lists of fluorescent molecules and their relevant optical properties for selecting fluorophores or reporter-quencher pairs, such as Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 2005); etc. In addition, there is extensive guidance in the literature for derivatizing reporter molecules for covalent attachment via common reactive groups that can be added to RT or its parts, as exemplified in Ullman et al., U.S. Patent No. 3,996,345; Khanna et al., U.S. Patent No. 4,351,760; etc. Each of the above publications is incorporated herein by reference in its entirety for all purposes.

[0158] Exemplary fluorescent dyes include, but are not limited to, acridine dyes, cyanine dyes, fluorescent dyes, oxazine dyes, phenanthridinium dyes, and rhodamine dyes. Exemplary fluorescent dyes include, but are not limited to, fluorescein, FITC, Texas Red, ROX, Cy3, Alexa Fluor dyes (e.g., Alexa Fluor 647 or 488), ATTO dyes (e.g., ATTO 532 or 655), and Cy5. Exemplary fluorescent dyes may further include dyes used in or compatible with two-channel or four-channel SBS chemistries and workflows.

[0159] Exemplary labeling molecules can be selected from xanthene dyes (including fluorescein) and rhodamine dyes. Many suitable forms of these compounds are generally commercially available, and they have substituents on their phenyl units that can be used as sites for attachment to affinity reagents. Another group of fluorescent compounds are naphthylamines, which have amino groups in the α or β positions. These naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate, and 2-p-toluidinyl-6-naphthalenesulfonate. Other labels include 3-phenyl-7-isocyanate coumarin; acridines, such as 9-isothiocyanate acridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazolyl; stilbenes; pyrenes; and the like.

[0160] In some embodiments, the label is selected from fluorescein and rhodamine dyes. These dyes and appropriate methods of attachment are described in many references, such as Khanna et al. (as described above); Marshall, Histochemical J., 7:299-303 (1975); Menchen et al., U.S. Patent No. 5,188,934; Menchen et al., European Patent Application No. 87310256.0; and Bergot et al., International Application No. PCT / US90 / 05565. Fluorophores that can be used as detectable labels for affinity reagents or nucleoside analogs include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, Vic TM , Liz TM Tamra TM 、5-Fam TM 、6-Fam TM , 6-HEX, CAL Fluor Green 520, CAL Fluor Gold 540, CALFluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 615, CALFluor Red 635, and Texas Red (Molecular Probes).

[0161] By judicious choice of labels, analyses can be performed in which different labels are excited and / or detected at different wavelengths in a single reaction. See, for example, Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 1972; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (2005). 4.3.2 Enzyme-labeled affinity reagents

[0162] In one approach, an affinity reagent (e.g., an antibody or Affirmer) is enzyme-labeled, and in the presence of a substrate, an enzyme associated with the affinity reagent of the primer extension product produces a detectable signal. For example, enzymes include but are not limited to peroxidase, phosphatase, luciferase, etc. In one approach, the enzyme is a peroxidase. In one approach, an affinity reagent (e.g., an antibody or Affimer) is directly enzyme-labeled. In one approach, for example, peroxidase [e.g., horseradish peroxidase (HRP)] or phosphatase (e.g., alkaline phosphatase) is used to label antibodies or other affinity reagents (Beyzavi et al., Annals Clin Biochem 24:145-152,1987). In one approach, an affinity reagent is coupled to luciferase or other proteins that can be used to produce chemiluminescent signals (or are part of their fusion protein). In another approach, affinity reagents can be coupled / fused to enzyme systems selected to generate non-optical signals, such as pH changes in protons, which can be detected, for example, by ion semiconductor sequencing (e.g., Ion Torrent sequencer; Life Technologies Corporation, Grand Island, NY). The use of enzyme-labeled affinity reagents has certain advantages, including high sensitivity due to signal amplification and the ability to customize sequencing methods for various instruments. Enzyme reporter systems are reviewed in Rashidian et al., Bioconjugate Chem. 24: 1277-1294, 2013. 4.3.3 Antibody Fusion Affinity Reagents

[0163] In addition, fusion methods can be used in which recombinant antibody fragments, such as single-chain Fv fragments (scFv), are directly linked to reporter proteins (Skerra and Plückthun, Science 240:1038-1041, 1988; Bird et al., Science 242:423-426, 1988; Huston et al., Methods Enzymol 203:46-88, 1991; Ahmad et al., Clin. Dev. Immunol. 2012:1, 2012). For example, photoproteins with bioluminescent properties, such as luciferase and aequorin, can be used as reporter proteins in fusion proteins with, for example, antibody fragments, epitope peptides, and streptavidin (Oyama et al., Anal Chem 87:12387-12395, 2015; Wang et al., Anal Chim Acta 435:255-263, 2001; Desai et al., Anal Biochem 294:132-140, 2001; Inouye et al., Biosci Biotechnol Biochem 75:568-571, 2011). 4.4 Indirect and direct detection methods

[0164] The affinity reagent can be directly labeled (e.g., by conjugation to a label, e.g., via a covalent bond, to a fluorophore) or indirectly labeled, e.g., by binding to a labeled secondary affinity reagent that binds to a primary affinity reagent that is directly bound to an extension primer having a 3' NLRT. The unlabeled primary affinity reagent binds to the target nucleotide, and the labeled secondary affinity reagent (e.g., an antibody, aptamer, affimer, or knottin) binds to the primary affinity reagent. In some approaches, the primary and / or secondary affinity reagent is an antibody. For example, in one approach, the affinity reagent is a "primary" antibody (e.g., a rabbit anti-NLRT-C antibody), and the secondary binding agent is a labeled anti-primary antibody (e.g., a dye-labeled goat anti-rabbit antibody). In some approaches, the use of a secondary affinity reagent provides advantageous signal amplification.

[0165] In the case of indirect detection, the assay comprises two distinct parts: first, an incubation period of time (usually one hour) with unlabeled primary antibodies during which the antibody binds to the antigen (assuming, of course, that the antigen is present). Excess unbound primary antibodies are then washed off and labeled secondary reagents are added. After incubation for a period of time (another hour), excess secondary reagents are washed off, and the amount of label associated with the primary antibody (i.e., indirectly via the secondary reagent) is quantified. If the antigen is present, the labeling typically results in the production of a colored substance or an increase in the amount of light emitted at a specific wavelength. In the absence of the antigen, there is no binding of the primary antibody, and no binding of the secondary reagent, and therefore no signal. With direct detection, the previous covalent attachment of the label to the primary antibody means that only a single incubation step for the antigen and only one round of washing steps are required, whereas indirect detection requires two rounds of incubation and washing steps. 4.4.1 Secondary Antibody Specificity

[0166] Primary antibodies and secondary antibodies can be selected to distinguish multiple antigens (e.g., to distinguish RT-A, RT-C, RT-G, and RT-T from each other). Unlabeled primary antibodies (typically monoclonal antibodies or engineered antibodies) may have different isotypes and / or sequences with different species characteristics (e.g., polyclonal antibodies or corresponding monoclonal antibodies or other affinity reagents cultured in different animals). In this case, the labeled secondary (i.e., anti-primary) antibody for each antigen is specific for the appropriate isotype or species sequence. For example, primary antibodies of isotypes IgG1, IgG2a, IgG2b, and IgG3 can be used together with isotype-specific secondary antibodies. 4.4.2 Pre-combined primary and secondary antibodies

[0167] Primary antibodies and secondary antibodies or other agents can be added to the sequencing array sequentially, or they can be pre-combined under conditions where the secondary antibody binds to the primary antibody and is added to the array as a complex. Figure 2 and Example 7. 4.5 Single-color, two-color, three-color, or four-color sequencing

[0168] Sequencing using the methods of the present invention can be two-color, three-color, or four-color sequencing. In one approach (four-color sequencing), each affinity reagent is directly or indirectly labeled with a different detectable label (e.g., a fluorescent dye) or a combination of labels that produce a unique signal. It should be understood that when two or more dyes (or other labels) are used to identify a single antigen, it is possible (but not necessary) to label a single affinity reagent molecule with two (or all) dyes or other labels. Conversely, a portion (e.g., 50%) of the affinity reagent molecules specific for a single antigen can be labeled with one dye, and another portion (e.g., 50%) of the affinity reagent molecules specific for a single antigen can be labeled with another dye.

[0169] According to one such method, an array is provided that includes a single-stranded nucleic acid template disposed at a position on a surface. Sequencing is performed by extension or SBS such that the identity of a nucleotide at a detection position in the nucleic acid template is determined over multiple sequencing cycles by: (i) binding (or incorporating) an unlabeled complementary nucleotide (NLRT) to (or incorporating) a nucleotide at the detection position, (ii) labeling the NLRT by binding it to a directly or indirectly labeled affinity reagent that specifically binds to such NLRT; and (iii) detecting the presence or absence of a signal associated with the complementary NLRT at the detection position, the signal (e.g., a fluorescent signal) being generated by the label; wherein (1) detection of a first signal but not a second signal at the detection position identifies the complementary NLRT as being selected from the group consisting of NLRT-A, NLRT-T, NLRT-G, and NLRT-C; and (2) detection of a second signal but not the first signal at the detection position identifies the complementary NLRT as being selected from the group consisting of NLRT-A, NLRT-T, NLRT-G, and NLRT-C. The LRT is identified as a NLRT selected from NLRT-A, NLRT-T, NLRT-G or NLRT-C that is different from the NLRT selected in (1); (3) detection of both the first signal and the second signal at the detection position identifies the complementary NLRT as a NLRT selected from NLRT-A, NLRT-T, NLRT-G and NLRT-C that is different from the nucleotide selected in (1) and (2); and (4) absence of the first signal and the second signal at the said position identifies the complementary NLRT as a NLRT selected from NLRT-A, NLRT-T, NLRT-G and NLRT-C that is different from the nucleotide selected in (1), (2) and (3); and (iii) inferring the identity of the nucleotide at the detection position in the nucleic acid template based on the identity of the complementary NLRT.

[0170] Another such method includes providing a plurality of nucleic acid templates, each nucleic acid template comprising a primer binding site, and a target nucleic acid sequence adjacent to the primer binding site; performing sequencing reactions on the plurality of different nucleic acid templates by hybridizing a primer to the primer binding site and extending a single primer by one nucleotide per cycle using a set of NLRTs and corresponding affinity reagents in one or more cycles of sequencing by synthesis, the set of NLRTs and corresponding affinity reagents being, for example: (i) a first NLRT and a first affinity reagent that specifically binds to the first NLRT and comprises a first label; (ii) a second NLRT and a second affinity reagent that specifically binds to the second NLRT and comprises a second label; (iii) a third NLRT and a third affinity reagent that specifically binds to the third NLRT and comprises a first label and a second label; and (iv) a fourth NLRT and a fourth affinity reagent that specifically binds to the fourth NLRT and comprises neither the first label nor the second label, wherein the first label and the second label are distinguishable from each other; and determining the identity of the NLRT at the detection position by detecting the presence or absence of the first label and the presence or absence of the second label in each cycle of sequencing by synthesis to determine the target nucleic acid sequence. An alternative to the above approach is to use a mixture of third affinity reagents that specifically bind to the third NLRT, some of which contain the first label and some of which contain the second label (eg, an equal mixture).

[0171] In a single-color sequencing method, the affinity reagent includes a detectable label that is present at a distinguishable intensity. For example, according to one such embodiment, the method includes providing a plurality of nucleic acid templates, each nucleic acid template comprising a primer binding site and a target nucleic acid sequence adjacent to the primer binding site; performing a sequencing reaction on the plurality of different nucleic acid templates by hybridizing a primer to the primer binding site and extending a single primer by one nucleotide per cycle in one or more sequencing-by-synthesis cycles using a set of NLRTs and corresponding affinity reagents, such as: (i) a first NLRT and a first affinity reagent that specifically binds to the first NLRT and comprises a first intensity label; (ii) a second NLRT and a second affinity reagent that specifically binds to the first NLRT and comprises a first intensity label; The method further comprises the steps of: (i) a first affinity reagent comprising a second affinity reagent that binds to a second NLRT and comprises a label of a second intensity; (ii) a third NLRT and a third affinity reagent that specifically binds to the third NLRT and comprises a label of a third intensity; and (iii) a fourth NLRT and a fourth affinity reagent that specifically binds to the fourth NLRT and comprises an unlabeled fourth affinity reagent (or, alternatively, the affinity reagent set comprises only the first affinity reagent, the second affinity reagent, and the third affinity reagent but does not comprise the fourth affinity reagent that binds to the fourth NLRT); and in each cycle of synthesis sequencing, determining the identity of the NLRT at the detection position by detecting the presence and intensity (or absence) of the label to determine the target nucleic acid sequence.

[0172] In another approach, affinity reagents are used that are labeled with one or the same number of molecules of a single dye, but that discriminate between the four NLRTs due to different binding efficiencies (i.e., the average amount of affinity reagent that binds to a single spot on the array, e.g., 10% of all copies of the target DNA molecule for NLRT-A, 30% for NLRT-T, and 60% for NLRT-C, and 0% or almost no detectable binding for NLRT-G). In one approach, the targets have the same blocking group, and affinity reagents are selected that have different affinities for their targets. In another approach, the blocking groups can be modified with small chemical changes to tune the binding efficiency of the same affinity reagent, thereby generating base-specific signal levels. For example, an unmodified blocking group may produce the highest signal (100% signal), a blocking group with modification 1 may produce a lower level of signal (e.g., 50%), a blocking group with modification 2 may produce an even lower or even less signal (25%), and so on.

[0173] In a related approach, two different blocking groups (azidomethyl and cyanoethoxymethyl) and one chemical variant of each (azidomethyl-prime and cyanoethoxymethyl-prime) are used, along with two antibodies for dual-color sequencing (2 colors x 2 intensities). The following is used for illustration: Azidomethyl-dA Affinity Agent 1, Color 1, Low Intensity (0-40%) Azidomethyl-prime-dC affinity reagent 1, color 1, high intensity (60-100%) Cyanoethoxymethyl-dG affinity reagent 2, color 2, low intensity (0-40%) Cyanoethoxymethyl-prime-dT affinity reagent 2, color 2, high intensity (60-100%)

[0174] In one embodiment, Affinity 1, Color 1, Low Intensity has a signal intensity close to zero, and Affinity 2, Color 2, Low Intensity has a higher signal intensity (25-40%).

[0175] In a related approach embodiment, dual-color sequencing can be performed, where a single nucleotide is used as a mixture of nucleotides, a portion of which is labeled with one blocking group and the remainder with another blocking group. The following is used for illustration: Azidomethyl-dA blocking group 1 Cyanoethoxymethyl-dG blocking group 2 Azidomethyl-prime-dC 70% are nucleotides with blocking group 1 and 30% A mixture of nucleotides with blocking groups 2 Cyanoethoxymethyl-prime-dT 30% is a nucleotide with blocking group 1 and 70% A mixture of nucleotides with blocking groups 2

[0176] In another approach, only one affinity reagent is used. A mixture of nucleotides with varying ratios of blocking groups recognized by the affinity reagent is used to generate distinguishable signal levels. The remainder of the mixture contains one blocking group but no corresponding affinity reagent. The following is for illustration: dA 0% blocking group 1, 100% blocking group 2 dG 25% blocking group 1, 75% blocking group 2 dC 50% blocking group 1, 50% blocking group 2 dT 100% Blocking Group 1, 0% Blocking Group 2

[0177] In another embodiment, the antibody can recognize two bases (nucleotide dimers) where the downstream base is modified by the addition of a cleavable or non-cleavable group.

[0178] In another embodiment, the last incorporated base is identified by binding two affinity reagents in the following combination: one affinity reagent specifically recognizes and binds to the nucleobase, while the second affinity reagent specifically recognizes and binds to the blocking group. The identity of the terminal base can only be determined when the two affinity reagents are bound and / or in close spatial proximity, for example, when the two affinity reagents include a FRET donor-acceptor pair as their respective "labels." Alternatively, binding of one of the affinity reagents may result in a conformational change that allows or enhances binding of the second affinity reagent.

[0179] Nucleoside analogs as described herein can be used in a variety of sequencing methods. For example, analogs can be used in single-label (sometimes referred to as "no label"), double-label, triple-label or quadruple-label sequencing methods, wherein the unlabeled analog is paired with an affinity reagent that is directly or indirectly labeled according to a single-label, double-label, triple-label or quadruple-label scheme.

[0180] Exemplary single-label sequencing methods include but are not limited to such methods, wherein continuous delivery of nucleoside analogs with different core bases (for example, A, C, G, T) and by detecting the presence or absence of the identical signal or label for each different core base to detect incorporation. Therefore, single-label method is sometimes referred to as monochrome method, because for all core bases, detection signal and / or label are identical, even if it may be different in terms of the intensity (or presence) for each nucleoside analog. For example, by detecting the pyrophosphate cracked from nucleoside pyrophosphate, it is possible to detect that the template-guided polymerization mediated by DNA polymerase is incorporated into a primer. Pyrophosphate can be detected using a coupled assay, wherein ATP sulfurylase converts pyrophosphate into ATP in the presence of 5' adenosine 5' phosphosulfate, which serves as the substrate for converting the luciferin mediated by luciferase into oxidized luciferin, thereby generating visible light with an amount directly proportional to ATP generation.

[0181] According to another embodiment, RT as described herein and affinity reagent can be used to carry out double labeling or two-color sequencing, using two distinguishable signals of combination mode to detect the incorporation of four different RTs.Exemplary double labeling systems, methods and compositions include but are not limited to those described in U.S. Patent number 8,617,811, the content of this patent, particularly the disclosure relevant to double labeling sequencing, is incorporated herein by reference in its entirety for all purposes.In brief, in double labeling sequencing, the incorporation of the first RT (for example, RT-A) is detected by marking the newly incorporated RT, and the labeling of the newly incorporated RT is carried out by specifically binding to the first affinity reagent comprising the first label, and then detecting the presence of the first label.The incorporation of the second RT (for example, RT-C) is detected by labeling the second RT, and the labeling of the second RT is carried out by specifically binding to the second affinity reagent comprising the second label, and then detecting the presence of the second label. The incorporation of a third RT (e.g., RT-T) is detected by labeling the third RT, and the labeling of the third RT is performed by specifically binding a third affinity reagent containing a first label and a second label, and then detecting the presence of the first label and the second label; and the incorporation of a fourth RT (e.g., RT-G) is detected by detecting the absence of both the first label and the second label due to binding of an unlabeled fourth affinity reagent or due to the absence of the fourth affinity reagent in the affinity reagent set used. In dual-color sequencing, the first label can be distinguished from the second label, and the combination of the first label and the second label can be distinguished from the first label and the second label used alone.

[0182] According to another embodiment, triple-label sequencing can be performed using a first RT labeled by a first affinity reagent containing a first label, a second RT labeled by a second affinity reagent containing a second label, and a third RT labeled by a third affinity reagent containing a third label. For the fourth RT, the corresponding affinity reagent is omitted from the affinity reagent set, or is unlabeled, or includes a combination of two or more of the first label, the second label, and the third label (or a mixture of affinity reagents labeled with a different label among the labels and specifically binding to the fourth RT). The first label, the second label, and the third label can be distinguished from each other.

[0183] Similarly, four-labeled sequencing can be performed using a first NLRT labeled by specific binding to a first affinity reagent containing a first label, a second NLRT labeled by specific binding to a second affinity reagent containing a second label, a third NLRT labeled by specific binding to a third affinity reagent containing a third label, and a fourth NLRT labeled by specific binding to a fourth affinity reagent containing a fourth label. Again, the first label, the second label, the third label, and the fourth label are distinguishable from each other. 4.6 Affinity reagents used in combination

[0184] Affinity reagents that recognize different epitopes of a single NLRT can be used in combination. For example, a first affinity reagent that recognizes the nucleobase portion of an incorporated NLRT can be used with a second affinity reagent that recognizes a blocking group. Staining can be performed simultaneously or sequentially. In sequential staining, the second affinity reagent can be applied while the first affinity reagent remains bound to the NLRT or after removal of the first affinity reagent in the case of re-probing (as discussed below). 4.7 Affinity reagent set

[0185] An "affinity reagent set" is used to label RTs used in SBS. For example, in one embodiment, for an RT set comprising four RTs (RT-A, RT-T, RT-C, and RT-G), there may be a corresponding affinity reagent set of four affinity reagents, each of which specifically recognizes and binds to one of the RTs (anti-A, anti-T, anti-C, and anti-G). An affinity reagent set describes a combination of affinity reagents that can be (i) provided in a kit format, as a mixture, or in separate containers and / or (ii) combined in contact with or on a sequencing array (e.g., within a sequencing flow cell).

[0186] According to one embodiment, each member of the affinity reagent set has a different, distinguishable detectable label, such as in four-color SBS.

[0187] According to another embodiment, one member of the affinity reagent set is unlabeled, while the other members are labeled. Alternatively, the affinity reagent set may simply exclude unlabeled affinity reagents and include only labeled affinity reagents.

[0188] For example, according to one embodiment, one affinity reagent is labeled with a first label (e.g., anti-A); a second affinity reagent is labeled with a second label (e.g., anti-T); a third affinity reagent is labeled with a third label (e.g., anti-C); and a fourth affinity reagent is unlabeled or simply excluded from the affinity reagent set (e.g., anti-G). This affinity reagent set can be used for three-color sequencing.

[0189] According to another embodiment, one affinity reagent (e.g., anti-A) is labeled with a first label; a second affinity reagent (e.g., anti-T) is labeled with a second label; a third affinity reagent (e.g., anti-C) is labeled with a first label and a second label; and a fourth affinity reagent (e.g., anti-G) is unlabeled (or excluded from the affinity reagent set). Alternatively, the third affinity reagent may include a mixture of affinity reagent molecules, all of which specifically bind to a specific base (e.g., all anti-C), but some include a first label and others include a second label. This affinity reagent set can be used for dual-color sequencing.

[0190] According to another embodiment, only a single detectable label is used (or a single combination of two or more labels), but the intensity varies between the members of the set, such as when the affinity reagents comprise different amounts of the label (or different amounts of at least one label in a combination of two or more labels). For example, in one embodiment, a first affinity reagent (e.g., anti-A) is labeled with a label of a first intensity; a second affinity reagent (e.g., anti-T) is labeled with the same label but of a second intensity; a third affinity reagent (e.g., anti-C) is labeled with the same label but of a third intensity; and a fourth affinity reagent (e.g., anti-G) is unlabeled (or the fourth affinity reagent is excluded from the set of affinity reagents). In another embodiment, a first affinity reagent (e.g., anti-A) is labeled with a first label of a first intensity, and a second label; a second affinity reagent (e.g., anti-T) is labeled with the same first label but of a second intensity, and the same second label; a third affinity reagent (e.g., anti-C) is labeled with the same first label but of a third intensity, and the same second label; and a fourth affinity reagent (e.g., anti-G) is unlabeled, labeled only with the second label, or is excluded from the set of affinity reagents. 4.8 Reaction mixture

[0191] Nucleoside analogs (e.g., NLRTs) and oligonucleotides or polynucleotides containing such nucleoside analogs or their reaction products can be used as components of a reaction mixture. For example, such components can be used in a reaction mixture to perform nucleic acid sequencing (e.g., SBS). Exemplary reaction mixtures include, but are not limited to, those containing: (a) a template nucleic acid; (b) a polymerase; (c) an oligonucleotide primer; (d) a 3'-O reversibly blocked nucleoside analog, or a mixture of 3'-O reversibly blocked nucleoside analogs having structurally different nucleobases; and (e) a labeled affinity reagent. Exemplary sequencing reaction mixtures of the present invention include, but are not limited to, an array comprising a plurality of different template nucleic acids, the plurality of different template nucleic acids being fixed at different positions on the array; (b) a polymerase; (c) an oligonucleotide primer; and (d) one NLRT or a mixture of multiple NLRTs. Exemplary sequencing reaction mixtures of the present invention include, but are not limited to, an array comprising a plurality of different template nucleic acids immobilized at different positions on the array; (b) a growing DNA strand (GDS) (which may comprise a 3' NLRT); and (c) one or more affinity reagents (e.g., an affinity reagent set as described above). 5. Template nucleic acid and nucleic acid array

[0192] In various embodiments, the template polynucleotide is DNA (e.g., cDNA, genomic DNA, transcriptome or microbiome DNA, amplification products, etc.) or RNA. In various embodiments, the polynucleotide is double-stranded or single-stranded.

[0193] In some embodiments, the template nucleic acid is fixed on a solid surface. In some embodiments, the template nucleic acid is fixed on a substrate (e.g., a channel in a bead, a flow cell, a pad, a microfluidic device, etc.). The substrate may include silicon, glass, gold, polymer, PDMS, etc.

[0194] In some embodiments, the template nucleic acid is immobilized or contained within the droplet (optionally immobilized on a bead or other substrate within the droplet).

[0195] In some embodiments, template nucleic acid is an immobilized DNA concatemer comprising multiple copies of a target sequence. In some embodiments, template nucleic acid is expressed as a DNA concatemer comprising multiple copies of a target sequence and an "adapter sequence", such as a DNA nanoball (DNB). See PCT Patent Publication No. WO 2007 / 133831, the contents of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, template is a single polynucleotide molecule. In some embodiments, template exists as a clonal group (e.g., a clonal group produced by bridge amplification or Wildfire amplification) of a template molecule.

[0196] It should be understood that the method is not limited to a particular form of template, and that the template can be any template, such as, for example, a DNA concatemer, a dendrimer, a clonal population of templates (e.g., as produced by Bridge amplification or Wildfire amplification), or a single polynucleotide molecule. Thus, when reading the specification, each reference to a template should be read as if it were an alternative reference to a concatemer template, a dendrimer, a clonal population (e.g., a clonal population of short linear templates), a single molecule template (e.g., in a zero-mode waveguide), and other forms of template.

[0197] Suitable template nucleic acids including DNBs, clusters, polymerase polony and arrays or groups thereof are further described in U.S. Patent Nos. 8,440,397; 8,445,194; 8,133,719; 8,445,196; 8,445,197; 7,709,197; 12 / 335,168; 7,901,891; 7,960,104; 7,910,354; 7,910,302; 8,105,771; 7,910 ,304; 7,906,285; 8,278,039; 7,901,890; 7,897,344; 8,298,768; 8,415,099; 8,671,811; 7,115,400; 8,236,499, and U.S. Patent Publication Nos. 2015 / 0353926; 2010 / 0311602; 2014 / 0228223; and 2013 / 0338008, all of which are incorporated herein by reference in their entirety.

[0198] In one aspect, the present invention provides a DNA array comprising: a plurality of template DNA molecules, each template DNA molecule attached at a position of the array; a complementary DNA sequence that base pairs with a portion of the template DNA molecules at the plurality of positions, wherein the complementary DNA sequence comprises a first reversible terminator deoxyribonucleotide incorporated at its 3' end; and a first affinity reagent that specifically binds to at least some of the first reversible terminator deoxyribonucleotides. In one approach, the DNA array comprises primer extension products having a 3' terminal nucleotide comprising an A, T, G, or C nucleobase or an analog thereof; and an affinity reagent that binds to the primer extension products. 6. Reagent test kit

[0199] Kits can be provided for practicing the present invention. As described above, NLRTs and NLRT sets can be provided in kit form. Also as described above, affinity reagents and affinity reagent sets can be provided in kit form. Kits comprising NLRTs and NLRT sets and affinity reagents or affinity reagent sets are also contemplated. For example, the present invention provides kits comprising, but not limited to, (a) a reversible terminator nucleotide (RT) or a set of RTs comprising one, two, three, four, or more different individual RTs; (b) a corresponding affinity reagent or an affinity reagent set comprising one, two, three, four, or more affinity reagents, each affinity reagent being specific for one of the RTs; and (c) packaging materials and / or instructions for use.

[0200] According to another embodiment, such a kit comprises a plurality of RTs, wherein each RT comprises a different nucleobase; and a plurality of affinity reagents, wherein each affinity reagent specifically binds to one of the RTs.

[0201] In one example, the present invention provides a kit comprising (a) a reversible terminator nucleotide as described herein, which can be incorporated into a primer extension product; (b) a first affinity reagent that specifically binds to the reversible terminator nucleotide when incorporated at the 3' end of the primer extension product; and (c) packaging (a) and (b). In one approach, the kit contains a plurality of reversible terminator deoxyribonucleotides, wherein each reversible terminator deoxyribonucleotide comprises a different nucleobase; and a plurality of first affinity reagents, wherein each first affinity reagent specifically binds to a different one of the reversible terminator deoxyribonucleotides. In some embodiments, the first affinity reagent is detectably labeled and can be distinguished from each other. In some embodiments, the kit comprises a second affinity reagent. In some embodiments, the first affinity reagent and / or the second affinity reagent are antibodies.

[0202] In one example, the reversible terminator deoxyribonucleotide has the structure of Formula I: wherein R1 is a 3'-O reversible blocking group; R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R3 comprises one or more phosphates. 7. application

[0203] In addition to the SBS applications described above, the novel affinity reagents, NLRTs, kits, and methods described herein can be used in many other applications, such as detecting species at the ends of naturally or experimentally fragmented DNA (or within DNA gaps); capturing oligonucleotides or polynucleotides with specific end-bases (at the ends of the molecule or within the gaps of the strands) with or without specific modifications. 5' or 3' end / gap bases can be detected. Affinity reagents can be used for ligation, hybridization, and other detection.

[0204] It will be appreciated that the methods of the present invention may also be used for direct RNA sequencing. 8. method 8.1 Removal of blocking groups, removal of affinity reagents, and detection

[0205] Blocking groups and affinity reagents can be removed simultaneously. In one approach, the array is exposed to conditions that simultaneously remove blocking groups and affinity reagents. In one approach, the array is contacted with a solution having a combination of reagents, wherein some reagents that result in removal of affinity reagents (e.g., high salt, small molecule competitors, proteases, etc.) are combined with reagents that cleave blocking groups.

[0206] In some cases, removal of the 3' blocking group results in removal of the affinity reagent. Without wishing to be bound by a particular mechanism, it is believed that in these cases, removal of the blocking unit destroys the epitope to which the antibody or other affinity reagent binds.

[0207] In a different approach, the removal of the affinity reagent and the blocking group are decoupled, so that the affinity reagent is removed but the blocking group is not cleaved from the nucleotide sugar. This is useful when reprobing is required. See Figure 2 , Section 9 below and Example 11.

[0208] It will be understood that removal conditions, ie, removal conditions for the affinity reagent and / or blocking group, will be selected to maintain the integrity of the DNA being sequenced. 8.1.1 Removal of blocking groups

[0209] Nucleoside analogs or NLRTs include those that are 3'-O reversibly blocked. In some aspects, the blocking group provides for controlled incorporation of a single 3'-O reversibly blocked NLRT at the 3' end of a primer (eg, a GDS extended in a previous sequencing cycle).

[0210] Typically, in each sequencing cycle using an NLRT, the blocking group is removed and the affinity reagent is dissociated from the NLRT. These steps can be performed simultaneously. For example, the azidomethyl blocking group can be removed by treatment with a phosphine (a widely used process), and the antibody affinity reagent can be removed by treatment with a low pH (e.g., 100 mM glycine pH 2.8) or high pH (e.g., 100 mM glycine pH 10), high salt, or chaotropic stripping buffer. In one embodiment, a single treatment or condition can be used to remove the NLRT and affinity reagent (e.g., a phosphine contained in a high salt buffer). In some embodiments, if, for example, the blocking group is required for affinity reagent binding, removal of the blocking group results in dissociation of the affinity reagent.

[0211] 3'-O reversible blocking groups can be removed by enzymatic cleavage or chemical cleavage (e.g., hydrolysis). Removal conditions can be selected by those of ordinary skill in the art based on the description provided herein, the chemical identity of the blocking group to be cleaved, and nucleic acid chemistry principles known in the art. In some embodiments, blocking groups are removed by contacting the nucleoside of reversible blocking with a reducing agent, such as dithiothreitol (DTT) or a phosphine reagent [e.g., tris (2-carboxyethyl) phosphine (TCEP), tris (hydroxymethyl) phosphine (THP), or tris (hydroxypropyl) phosphine]. In some cases, blocking groups are removed by washing out the blocking groups from the nucleotide analogs incorporated using a reducing agent (e.g., a phosphine reagent). In some cases, blocking groups are photolabile, and can be removed by applying, for example, UV light. In some cases, blocking groups can be removed by contacting nucleoside analogs with transition metal catalyzed reactions using, for example, palladium (Pd) aqueous solutions. In some cases, blocking groups can be removed by contacting nucleoside analogs with nitrite aqueous solutions. Additionally, or alternatively, blocking groups can be removed by changing the pH of the solution or mixture containing the incorporated nucleotide analog. For example, in some cases, blocking groups can be removed by contacting the nucleoside analog with an acid or a low pH (e.g., less than 4) buffered aqueous solution. As another example, in some cases, blocking groups can be removed by contacting the nucleoside analog with a base or a high pH (e.g., greater than 10) buffered aqueous solution.

[0212] 3'-O reversible blocking groups that can be cleaved by reducing agents (e.g., phosphines) include, but are not limited to, azidomethyl. 3'-O reversible blocking groups that can be cleaved by UV light include, but are not limited to, nitrobenzyl. 3'-O reversible blocking groups that can be cleaved by contact with a Pd aqueous solution include, but are not limited to, allyl. 3'-O reversible blocking groups that can be cleaved by acid include, but are not limited to, methoxymethyl. 3'-O reversible blocking groups that can be cleaved by contact with a sodium nitrite buffered (pH 5.5) aqueous solution include, but are not limited to, aminoalkoxy. 8.1.2 Removal of affinity reagents

[0213] Antibody-based affinity reagents can be removed by low pH, high pH, high salt or low salt or denaturants (e.g., chaotropic stripping buffers). Other types of affinity reagents (e.g., aptamers) can be removed by any method known in the art. Additionally, affinity reagents, such as antibodies, can be removed by introducing an agent that competes with the bound epitope for binding to the affinity reagent, for example, as shown in Example 10 below.

[0214] In addition, the affinity reagent can be removed by destroying the ability of the reagent to bind to the incorporated NLRT. This typically occurs when the 3' blocking group is cleaved from the incorporated nucleotide analog. In cases where affinity reagent binding depends on the presence of a blocking group (e.g., in cases where binding of the affinity reagent is dependent on the presence of a blocking group), 0 In cases where the epitope recognized by the antibody includes a blocking group or portion thereof, removal of the blocking group also results in the release of the affinity reagent.

[0215] The affinity agent and the blocking group can also be removed simultaneously by adding a solution containing a blocking group cleavage component (eg, a phosphine reagent) and an affinity agent releasing agent (eg, high salt).

[0216] Alternatively, the affinity reagent can be removed without removing the 3' blocking group. This approach is useful when reprobing is required (as described in Section 9 below). 8.1.3 Detection

[0217] The method for detecting the binding event will vary with the nature of the detectable label used and is well known in the art. Detection (e.g., detection of a fluorescent signal) is typically performed before removing the blocking group. However, detection can be performed before or after removing the blocking group, as long as the labeled affinity reagent remains bound. 8.2 Antibody production

[0218] For example, the complexity of small compounds (drugs or peptides) themselves is not enough to induce an immune response or to be processed in a manner that triggers the production of specific antibodies. In order to successfully produce antibodies with small antigens, the small antigens must be chemically conjugated to immunogenic carrier proteins, such as keyhole limpet hemocyanin (KLH). Adjuvants can be mixed with immunogens and injected to increase the intensity of the immune response. In this section about antibody production, carrier protein conjugation, the use of adjuvants and other issues related to the preparation of injection samples are described. Standard procedures for generating, purifying and modifying antibodies used as antigen-specific probes can be used. See, for example, Harlow and Lane, "Antibodies: A Laboratory Manual" (1988) and Harlow and Lane, "Using Antibodies: A Laboratory Manual" (1999).

[0219] Hapten Small molecules that act as antigens are called haptens. They serve as recognition sites for specific antibodies but cannot stimulate the necessary immune response on their own. Haptens can be made immunogenic by coupling them to a suitable carrier molecule. Epitope An epitope is a specific site on an antigen to which an antibody binds. For very small antigens, virtually the entire chemical structure can serve as a single epitope. Depending on its complexity and size, an antigen can generate antibodies targeting numerous epitopes. Polyclonal antibodies are mixtures of serum immunoglobulins that may collectively bind to multiple epitopes on an antigen.

[0220] Keyhole limpet hemocyanin (KLH) Keyhole limpet hemocyanin (KLH) is the most widely used carrier protein.

[0221] Bovine serum albumin Bovine serum albumin (BSA; 67 kDa) belongs to the class of serum proteins known as albumins. 8.3 Immunization regimen

[0222] Immunization protocols are well known and are described here in general terms only. For further details, see Example 2 below. The concentration of the immunogen prior to mixing with the adjuvant will ultimately determine the amount of conjugate that will be administered per injection. Immunization schedule for mice: Day 0: Collect pre-immune serum from mice to serve as a blank for ELISA screening after immunization. Store frozen. Each mouse is injected with 50 to 100 μg of immunogen (equal to 100 to 200 μL of antigen-adjuvant mixture). Typical injection routes include intraperitoneal (ip) or subcutaneous (sc). Each animal can receive one or two such injections. Day 14: Boost with an equal amount of immunogen in adjuvant. Day 21: Test bleeding and determine antibody response by ELISA. [Typically, mice are bled under anesthesia via the tail vein or retro-orbital plexis]. Day 28: Boost again if necessary. Continue with a similar schedule of alternating boosts and test bleeding until a satisfactory response is observed. For the production of monoclonal antibodies, the cells were injected ip or intravenously (iv) 4 to 5 days prior to fusion with the immunogen dissolved in saline (without adjuvant).

[0223] Rabbit immunization schedule: Day 0: Collect pre-immune serum from the rabbit to use as a blank when performing ELISA after immunization. Store frozen. Inject 100 μg of immunogen (equivalent to approximately 200 μL of antigen-adjuvant mixture) into each of 8 to 10 subcutaneous sites on the rabbit's back. Other injection routes can also be used, but this is by far the easiest route for rabbit injection. Day 14: Push with an equal amount of adjuvant. Day 21: Test bleeding and determine antibody response by ELISA. (Usually, the rabbit is bled through the ear vein without anesthesia). It is not difficult to collect 5 to 10 mL of blood (which is sufficient to measure the antibody response). Day 28: Push again if necessary. Continue a similar schedule of alternating pushes and test bleeding until a satisfactory response is observed.

[0224] General purification of immunoglobulins Because antibodies have predictable structures, including relatively invariant domains, it is possible to identify certain protein ligands that are generally capable of binding to antibodies, regardless of the antibody's specificity for the antigen. Protein A, protein G, and protein L are three bacterial proteins whose antibody-binding properties have been well characterized. These proteins are recombinantly produced and routinely used for affinity purification of key antibody types from various species. Genetically engineered recombinant forms of proteins A and G, termed protein A / G, are also available. These antibody-binding proteins can be immobilized on beaded agarose resin. 8.4 Affinity purification of antibodies:

[0225] A variety of methods are used to enrich or purify a protein of interest from other proteins and components in crude cell lysates or other samples. The most powerful of these methods is affinity chromatography, also known as affinity purification, whereby a protein of interest is purified by virtue of its specific binding properties to an immobilized ligand.

[0226] Proteins and other macromolecules of interest can be purified from crude extracts or other complex mixtures by various methods. Selective precipitation is probably the simplest method for separating one macromolecule from another.

[0227] However, most purification methods involve some form of chromatography, whereby molecules in a solution (mobile phase) are separated based on differences in their chemical or physical interactions with a stationary material (solid phase). Gel filtration (also known as size exclusion chromatography or SEC) uses a porous resin material to separate molecules based on size (i.e., physical exclusion). In ion exchange chromatography, molecules are separated based on the strength of their total ionic interactions (i.e., nonspecific interactions) with the solid phase material.

[0228] In contrast, affinity chromatography (also known as affinity purification) exploits specific binding interactions between molecules. A specific ligand is chemically immobilized or "coupled" to a solid support so that when a complex mixture passes through the column, only those molecules with a specific binding affinity for the ligand are bound. After washing away other sample components, the bound molecules are stripped from the support, thereby purifying them from the original sample.

[0229] Each specific affinity system requires its own set of conditions and presents its own unique challenges for a given research goal. Other protein methods articles describe the factors and conditions associated with specific purification systems. 8.5 Antibody labeling

[0230] Antibody structure and modification sitesLike other proteins, antibodies can be covalently modified in a variety of ways to suit the purpose of a specific assay. Many immunological methods involve the use of labeled antibodies, and a variety of reagents have been created to allow for the labeling of antibodies. Enzymes, biotin, fluorophores, and radioisotopes are all commonly used to provide detection signals in biological assays. Understanding the functional groups available on antibodies is key to choosing the best method for modification, whether for labeling, crosslinking, or covalent immobilization. Most antibody labeling strategies use one of three targets: (1) primary amines (-NH2): These are present on lysine residues and the N-terminus of each polypeptide chain, are numerous, and are distributed throughout the antibody; (2) sulfhydryl groups (-SH): These are present on cysteine residues and in disulfide bonds that stabilize the overall molecular structure. Hinge disulfides can be selectively reduced to make free sulfhydryl groups available for targeted labeling; (3) carbohydrates (sugars): Glycosylation occurs primarily in the Fc region of antibodies (IgG). The component sugars in these polysaccharide units containing cis-diols can be oxidized to produce reactive aldehydes (-CHO) for conjugation.

[0231] Antibody labeling method Any known method for labeling antibodies can be used in the practice of the present invention. Antibodies, like all proteins, are composed of amino acids, and the side chain of lysine terminated in a primary amine (-NH2) is commonly used to covalently attach a label to the antibody molecule.

[0232] The four main chemistries for antibody labeling are summarized below:

[0233] 1. NHS esters. In the case of fluorescent dye labels, labels are typically purchased in an activated form with a built-in NHS ester (also known as a "succinimidyl ester"). The activated dye can react with antibodies (all of which have multiple lysine groups) under appropriate conditions. Excess reactive dye is removed by one of several possible methods (usually column chromatography), after which the labeled antibody can be used in an immunoassay.

[0234] 2. Heterobifunctional Reagents. If the label is a protein molecule [such as horseradish peroxidase (HRP), alkaline phosphatase, or phycoerythrin], the antibody labeling procedure is complicated by the fact that both the antibody and the label have multiple amines. In this case, it is common to modify some lysines on one molecule (e.g., the antibody) to create a new reactive group (X) and a lysine to create another reactive group (Y) on the label. A "heterobifunctional reagent" is used to introduce the Y group, which then reacts with the X group when the antibody and label are mixed, thereby creating a heterodimeric conjugate. There are many variations on this theme, and one will find hundreds of examples in the literature of the use of heterobifunctional reagents to produce labeled antibodies and other labeled biomolecules.

[0235] 3. Carbodiimides. These reagents (EDC is a very common example) are used to create covalent linkages between molecules containing amines and molecules containing carboxyl groups. The carbodiimide activates the carboxyl group, and the activated intermediate is then attacked by an amine (e.g., provided by a lysine residue on an antibody). Carbodiimides are commonly used to conjugate antibodies to carboxylated particles (e.g., latex particles, magnetic beads) and other carboxylated surfaces (e.g., microplate or chip surfaces). Carbodiimides are rarely used to attach dyes or protein labels to antibodies, although they are important in the production of NHS-activated dyes (see above).

[0236] 4. Sodium periodate. This chemical cannot be used with most labels, but it is a very important reagent because it works with the most commonly used diagnostic enzyme, HRP. Periodate activates the carbohydrate chains on the HRP molecule to produce aldehyde groups that react with lysines on the antibody molecule. Because HRP itself has very few lysines, it is relatively easy to produce antibody-HRP conjugates without significant HRP aggregation.

[0237] In any particular antibody clone, lysine (primary amine) may be prominently present in the antigen binding site. Therefore, the only disadvantage of this labeling strategy is that it occasionally leads to a significant reduction in the antigen binding activity of the antibody. The reduction may be particularly evident when working with monoclonal antibodies or when attempting to add high-density labels to each antibody molecule. 9. Re-detection

[0238] As described above in Section 8, according to the present invention, it is possible to decouple the removal of the affinity reagent (e.g., antibody) and the 3' protecting group. Because the affinity reagent can be removed without removing the blocking unit, it is possible to advantageously re-probe some or all base positions to improve the accuracy of base calls, test the integrity of the chip, or for other purposes. See Examples 11 and 12 below. Figure 2 Any given base position can be probed once and reprobed 0, 1, 2, or more than 2 times. Generally, a single round of reprobing is considered sufficient. For convenience only, in the case of a base position probed twice, the first round of reprobing may be referred to as the first half-cycle, and the second round of reprobing may be referred to as the second half-cycle.

[0239] When re-probing, it is possible to probe each position twice with the same affinity reagent (e.g., the same primary antibody). More commonly, different affinity reagents are used, such as different antibody preparations (e.g., different monoclonal antibodies), different classes of affinity reagents (e.g., using antibody probing in the first half-cycle and aptamer probing in the second half-cycle), or affinity reagents with different specificities. For example, in the first half-cycle, the array can be probed with anti-A, anti-T, anti-C, and anti-G, and in the second half-cycle, the array can be probed with anti-purine and anti-pyrimidine.

[0240] In one approach, four NLRTs are blocked using two blocking groups, such as azidomethyl-T, azidomethyl-G, cyanovinyl-C, and cyanovinyl-A, and the array is probed once with two affinity reagents (one specific for 3'-O-azidomethyl-2'-deoxyribose and the other specific for 3'-O-cyanovinyl-2'-deoxyribose), and a second time with a different pair of affinity reagents (one specific for purines and the other specific for pyrimidines). Addresses on the array that exhibit signals characteristic of 3'-O-azidomethyl-2'-deoxyribose and purines will be identified as having guanine bases, and so on. 10. Sequencing process

[0241] Figure 1 and Figure 2 Additional guidance is provided for the reader but should not be construed as limiting. For example, when using an affinity reagent to detect the terminal 3'-OH of the extension product (see Section 3.8 above), the blocking group would be removed (Step 8b) prior to antibody staining (Step 5).

[0242] As discussed above, in one aspect, the present invention relates to a synthetic sequencing (SBS) method using unlabeled reversible terminator nucleotides. The SBS method is well known, including but not limited to the methods described in the references cited herein, each of which is incorporated by reference for all purposes. Typically, SBS determines the sequence of a single-stranded nucleic acid template fixed on a surface. As known to those of ordinary skill in the art, there are typically many copies of a template at a position on the surface. For illustration and not limitation, a DNA nanoball (DNB) method or a bridge PCR method is typically used to produce template copies. The DNB method produces single-stranded concatemers having many copies of a template (e.g., a genomic DNA sequence and adjacent primer binding sites). The bridge PCR method produces clonal clusters of template molecules (e.g., genomic DNA sequences flanked by adapters that can be used as primer binding sites). In bridge PCR, both chains of the template nucleic acid can exist as separate single chains. It should be understood that a "template" nucleic acid (i.e., singular grammatical form) or equivalent terms mentioned herein also refers to multiple copies of the template at a given position on a substrate. It should also be recognized that although reference may be made herein to determining the sequence of a template nucleic acid or template nucleic acid sequence (i.e., in the singular grammatical form), it is contemplated that the methods of the invention are performed using arrays comprising a plurality (typically hundreds of millions) of positions containing one or more template nucleic acid molecules.

[0243] As used in this context, "array" is used in the broadest sense and, unless otherwise specified, includes both ordered arrays (meaning that the template binding regions are arranged in an ordered, usually rectilinear pattern (e.g., a grid, spiral, or other pattern) and disordered arrays (meaning that the template binding regions are at random positions). In one approach, the identity of the template at any particular position (or "address") on the array may be known before the template is sequenced. More commonly, the array is a "random array," in which the identity of the template at a given address is not known before sequencing. Unless otherwise specified, in this disclosure, "array" is not limited to positions on a plane, but may include bead arrays, droplet arrays, and the like.

[0244] A variety of SBS methods can be used with the nucleoside analogs and affinity reagents of the present invention. In some aspects, the SBS method can be selected from U.S. Patent Nos. 6,210,891; 6,828,100, 6,833,246; 6,911,345; 6,969,488; 6,897,023; 6,833,246; and 6,787,308; U.S. Patent Publication Nos. 2003 / 0064398; 2003 / 0022207; 2016 / 0130647; and PCT Patent Publication No. WO 2016 / 133764; Margulies et al., 2005, Nature 437:376-380; Ronaghi et al., 1996, Anal. Biochem. 242:84-89; Constans, A, 2003, The Scientist 17(13):36; and Bentley et al., 2008, Nature 456(7218):53-59. DNA sequencers that perform sequencing by synthesis are commercially available from, for example, Illumina Inc. (San Diego, CA), including the MiniSeq, MiSeq, NextSeq, HiSeq, HiSeq X, and NovaSeq sequencing systems. Other DNA sequencing systems that can be used with the compositions and methods of the present invention include BGISEQ-50, BGISEQ-500, BGISEQ-1000, MGI-200, and MGISEQ-2000 (BGI, Shenzhen, People's Republic of China); and the GeneReader sequencing platform (QIAGEN, Manchester, UK).

[0245] Some SBS embodiments include detecting protons released when nucleotides are incorporated into extension products. For example, sequencing based on detection of released protons can use electronic detectors and associated technologies commercially available from IonTorrent (Guilford, Conn., a subsidiary of Life Technologies); or can use the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082A1; 2009 / 0127589A1; 2010 / 0137143A1; or 2010 / 0282617A1.

[0246] Another sequencing procedure using a circular reaction can be used together with the compositions and methods of the present invention, for example, such as pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Anal Biochem 242:84-89, 1996; Ronaghi, Genome Res. 11:3-11, 2001; Ronaghi et al., Science 281:363, 1998; and U.S. Patent No. 6,210,891; 6,258,568 and 6,274,320). In pyrophosphate sequencing, the released PPi can be detected by converting it into adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected via photons generated by luciferase. Therefore, the sequencing reaction can be monitored via a luminescent detection system. The excitation radiation source for a fluorescence-based detection system is not necessary for the pyrophosphate sequencing procedure. Useful fluidics systems, detectors, and procedures for applying pyrosequencing to the arrays of the present disclosure are described in, for example, WIPO Patent Application Serial No. PCT / US11 / 57111, US Patent Application Publication No. 2005 / 0191698A1, US Patent No. 7,595,883, and US Patent No. 7,244,559.

[0247] In some aspects, sequencing by ligation can be selected from PCT Patent Publication No. WO 1999 / 019341; WO 2005 / 082098; WO 2006 / 073504; and those described in Shendure et al., 2005, Science, 309:1728-1739. The SBS method can employ an ordered DNA nanosphere array, as described in, for example, U.S. Patent Publication Nos. 2010 / 0105052, 2007 / 099208, and US2009 / 0264299, and PCT Patent Publication Nos. WO 2007 / 120208, WO 2006 / 073504, and WO 2007 / 133831. The patents and non-patent publications listed above in this paragraph are incorporated herein by reference in their entirety for all purposes.

[0248] According to one embodiment, an array of ordered DNA nanoballs (DNBs) is sequenced. By rolling circle replication of circular library constructs (each containing a fragment of a genome or other target nucleic acid), linear single-stranded DNA concatemers containing multiple copies of the circular construct are generated, which disintegrate in an aqueous solution to form a compact spherical structure, thereby producing DNBs. DNBs are arranged on the surface of a two-dimensional planar substrate to form a random array of single molecules. DNBs can be directly or indirectly fixed on the surface by a variety of techniques (including covalent attachment and non-covalent attachment). In some embodiments, a patterned substrate with a two-dimensional spot array is used to produce a DNB array. The spots are activated to capture and hold the DNBs, and the DNBs are not retained in the region between the spots. Typically, the DNBs on the spots will repel other DNBs, resulting in one DNB per spot. Because DNBs are three-dimensional (i.e., not linear short DNA pieces), the array of the present invention results in more DNA copies per square nanometer binding surface compared to traditional DNA arrays. This three-dimensional quality further reduces the number of sequencing reagents required, thereby producing brighter spots and more effective imaging. The occupancy of the DNB array is typically over 90%, but the occupancy can range from 50% to 100%. In these embodiments, the high-density DNB array is essentially "self-assembled" from DNBs in solution because the DNBs are disposed on the surface and then adhere to the activated spots.

[0249] When such a DNB array is used for sequencing by synthesis, the DNB array is contacted with a primer, and in each sequencing cycle the primer is extended by one complementary base by a polymerase. Because a specific affinity reagent binds to its corresponding RT, the identity of the RT incorporated by the polymerase at each position in the array is revealed. For example, in four-color sequencing, the result is a DNB array, each DNB labeled with an affinity reagent, such that the identity of the RT incorporated at a specific position on the array can be identified by a fluorescent marker (or other detectable marker) that is part of the affinity reagent that binds to the RT.

[0250] In the SBS method using a reversible terminator, the template nucleic acid is fixed on the surface, and an oligonucleotide primer is hybridized to a predetermined position (i.e., primer binding site) on the template. In the primer extension reaction, a nucleotide analog is incorporated into the 3' end of the primer, in which deoxyribose 3'-OH is replaced by a removable blocking unit (e.g., 3'-O-azidomethyl). The nucleotide analog incorporated is complementary to the nucleotide at the corresponding position on the template and forms a base pair with it. Routinely, the nucleotide analog comprises a detectable label, and the detectable label identifies the core base of the nucleotide analog incorporated, and therefore also identifies the base of the complementary nucleotide in the template. In the SBS method commonly used, the nucleotide analog comprises a fluorescent label that is attached to the core base by a cleavable linker.

[0251] In the SBS method using reversible terminators, after the incorporation of nucleotide analogs is detected, the blocking group is removed (usually chemically or enzymatically removed) to produce an incorporated nucleotide with a 3'-OH group. Additional rounds of incorporation, detection, and deblocking of 3'-blocked nucleotide analogs can be performed in additional primer extension reactions. Although nucleotides are added in each round of primer extension, the process can be referred to as an extension of the primer, but it may be more accurate to say that the extension product of the previous round (rather than the original oligonucleotide primer) is extended. The primer extension chain can be referred to in various ways, including "growing DNA chain (GDS)", "primer extension product" or "extension primer".

[0252] It should be understood that when a dNTP (i.e., nucleoside triphosphate) is added to the 3' end of a primer, the pyrophosphate is removed, thereby incorporating a nucleoside monophosphate (or nucleotide). Unless otherwise indicated, an unlabeled or non-labeled reversible terminator nucleotide may refer to either form (free nucleoside triphosphate or incorporated monophosphate nucleotide), as is apparent from the context. An unlabeled or non-labeled reversible terminator nucleotide may be referred to as an NLRT.

[0253] In one aspect of the invention, the incorporated dNTP analog is not detectably labeled. In this context, "not detectably labeled" means that the incorporated dNTP is not conjugated to a dye that produces a detectable (e.g., fluorescent) signal or an enzyme that produces a detectable (e.g., chemiluminescent) signal in the presence of a substrate. As used herein, a "reversible terminator nucleotide" refers to a naturally occurring nucleotide, or a nucleotide analog, in which the deoxyribose 3'-OH is replaced by a removable blocking unit (e.g., 3'-O-azidomethyl).

[0254] In one aspect of the invention, an incorporated NLRT is detected in a sequencing reaction by an affinity reagent (e.g., an antibody) that distinguishes the 3' terminal nucleotide of a primer extension product, thereby identifying the nucleobase of the 3' terminal nucleotide of the template. In one approach, the affinity reagent specifically binds to an incorporated NLRT containing a particular base (e.g., A, T, G, or C) or an analog of a particular base with an affinity that is much greater than its affinity for binding to incorporated NLRTs having other bases or other base analogs present in the sequencing reaction. In another approach, the affinity reagent binds to an incorporated NLRT containing a particular base (e.g., A, T, G, or C) or an analog of a particular base with a characteristic affinity or efficiency that is different from its affinity or efficiency for binding to other bases or other base analogs present in the sequencing reaction.

[0255] According to the present invention, the affinity reagent can also distinguish between an NLRT incorporated at the 3' terminus of a primer extension product and a previously incorporated "internal" nucleotide that is not at the 3' terminus. Typically, the NLRT at the 3' terminus of a primer extension product differs from the previously incorporated nucleotide by the presence of a free 3'-OH (which is replaced by a phosphodiester linkage in internal nucleotides) or the presence of a 3' blocking unit, as well as differential accessibility of the sugar and nucleobase.

[0256] According to the present invention, an SBS reaction is performed using four NLRTs having different nitrogenous bases, such as A, T, G, and C. In the SBS reaction, different affinity reagents are used (e.g., 2, 3, or 4 different affinity reagents), each of which binds to the NLRT having a specific nitrogenous base but does not bind to the NLRT having a different nitrogenous base, or in some embodiments, binds to the NLRT having a different nitrogenous base or a different blocking group, but with different efficiency levels.

[0257] Affinity reagents can distinguish one incorporated NLRT from a different NLRT based on structural differences in the nitrogenous base, sugar, cleavable blocking group, or a combination of these elements. In some cases, different NLRTs are distinguished due to, for example, significant structural differences in the nitrogenous base (e.g., adenosine vs. guanine) and / or significant structural differences in the blocking group (e.g., azidomethyl vs. cyanoethylene).

[0258] In addition, affinity reagents can distinguish an incorporated NLRT from a different NLRT based on small structural differences (e.g., in some cases, additions or substitutions of fewer than 5 atoms), preferably in combination with natural differences. These small structural changes can be made in nitrogenous bases, sugars, and / or blocking groups. Affinity reagents, such as antibodies, can be prepared that distinguish such small differences between different NLRTs.

[0259] According to one aspect of the invention, each of the affinity reagents can be distinguished from the other affinity reagents present in the sequencing reaction (eg, because each is differently labeled) or bound by a different secondary binding agent.

[0260] According to the present invention, there are constraints on the structure of each of the nitrogenous base, sugar, and cleavable blocking group.

[0261] For example, suitable modified bases will retain normal Watson-Crick binding specificity and should be compatible with incorporation by DNA polymerase. In some embodiments, the base analogs do not have fluorescent properties (Renatus et al., 2010, Chem Rev. 110(5):2579-2619).

[0262] Similarly, the sugar portion of the NLRT can be modified. Nucleic acids with such modified NLRTs should retain the ability to anneal to the template strand and should be compatible with incorporation by DNA polymerases.

[0263] Similarly, NLRTs with only slightly different blocking groups can be used. For example, 2, 3 or 4 different such NLRTs can be used.

[0264] In certain embodiments of the invention, the blocking group (excluding the 3' oxygen atom of the deoxyribose sugar) has a molecular weight (MW) of less than 184, typically less than 174, typically less than 164, typically less than 154, and often less than 144. Typically less than 134, typically less than 124, typically less than 114, typically less than 104, typically less than 94, and sometimes less than 84.

[0265] In certain embodiments, the molecular weight of the deoxyribose monophosphate nucleotide esters is in the range of about 300 to 325 (dAMP 331.2, dCMP 307.2, dGMP 347.2, and dTMP 322.2). In certain embodiments, the NLRT unit, when incorporated into a primer extension product (i.e., including a reversible terminator blocking group but not including the pyrophosphate of a dNTP), has a molecular weight of less than 700, less than 600, less than 550, typically less than 540, typically less than 530, typically less than 520, typically less than 510, typically less than 500, typically less than 490, typically less than 480, typically less than 470, and sometimes less than 460.

[0266] In certain embodiments, the methods of the invention are used to generate sequencing reads that are longer than 1000 nucleotides, sometimes 0-500 nucleotides, sometimes 10-250 nucleotides, sometimes more than 25, and sometimes more than 50 nucleotides. In some cases, sequencing is performed with less than one error per 2000 bases and one error per 5000 bases. 11. Example 11.1 Example 1. Preparation of conjugated 3'-O-azidomethyl-2'-dG, -dC, -dA and -dT antigens .

[0267] Synthesis of Active Esters of 3'-O-Azidomethyl-2'-deoxyguanine . Figure 5The synthesis of the amino-reactive N-hydroxysuccinimide (NHS) ester of 3'-O-azidomethyl-2'-deoxyguanine is shown in Figure 2. Compound G1 (416 mg, 0.708 mmol), anhydrous DMF (3 mL) and 1,1'-carbonyldiimidazole (CDI) (171 mg, 1.054 mmol) were added to a 50 mL flask. The reaction mixture was stirred at room temperature for 20 h. Ethyl 4-aminobutyrate hydrochloride (384 mg, 2.29 mmol) and triethylamine (300 μL, 2.155 mmol) were added. The mixture was stirred at 40 °C for 10 h. Most of the DMF was removed on a rotary evaporator (or rotary evaporator) under vacuum to give crude compound G2.

[0268] To the crude compound G2 was added EtOH (5 mL) and 1N NaOH / H2O (7 mL). The mixture was stirred at room temperature for three days. 1N HCl / H2O was added to adjust the pH to 7.4. Most of the EtOH was removed on a rotary evaporator and then filtered. The filtrate was purified by preparative HPLC using 25 mM TEAB buffer (triethylamine bicarbonate, pH 8.0 at room temperature) and CH3CN to give compound G3 (341 mg) as a white solid. LCMS: 452.1 (MS + ).

[0269] In a 5 mL vial, compound G3 (42 mg, 0.076 mmol), anhydrous dimethylformamide (DMF) (0.6 mL) and O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) (19 mg, 0.063 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour to obtain the desired activated NHS ester G4 for use in the preparation of bioconjugates. LCMS: 548 (MS+).

[0270] Conjugation of 3'-O-azidomethyl-dG to bovine serum albumin (BSA) 20 mg of BSA (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH = 9.0) with 150 mM NaCl was reacted with 5 mg of compound G4. The reaction was carried out at room temperature for 1 hour and the mixture was purified by desalting column [ The reaction mixture was purified using 5-mercaptoethanol (5-mercaptoethanol) polyacrylamide beads (P6DG beads, Bio-Rad Laboratories, Hercules, CA). The conjugate was lyophilized to obtain a white powder.

[0271] Conjugation of 3'-O-azidomethyl-dG to keyhole limpet hemocyanin (KLH)20 mg of KLH (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 7 mg of compound G4. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P-6DG beads) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0272] Figure 9 The use of Figure 5 A slightly different synthetic approach using different linkers was used as shown in to conjugate 3'-O-azidomethyl-dC-NHS ester to BSA, KLH, and agarose resin.

[0273] Conjugation of 3'-O-azidomethyl-dG to amine-activated agarose resin 20 ml of wet amine-activated agarose resin (5 μmol activated groups / ml) was washed with 30 mL of 50 mM sodium bicarbonate buffer (pH 9.0) and 150 mM NaCl. 70 mg of Compound G4 was added to the 20 ml wet beads, and the reaction was incubated with rotation at room temperature (RT) for 2 hours. Following the reaction, the resin was washed with 50 mL of phosphate-buffered saline until the absorbance at 260 nm was less than 0.02, yielding the desired purified resin.

[0274] Synthesis of Amino-Reactive NHS Ester of 3'-O-Azidomethyl-2'-deoxycytidine (C8) . Figure 6 The synthesis of amino-reactive NHS ester (C8) of 3'-O-azidomethyl-2'-deoxycytidine is shown. Compound C5 (410 mg, 1.061 mmol), anhydrous DMF (3 mL) and 1,1'-carbonyldiimidazole (CDI) (213 mg, 1.314 mmol) were added to a 50 mL flask. The reaction mixture was stirred at room temperature for 20 hours. Ethyl 4-aminobutyrate hydrochloride (223 mg, 1.330 mmol) and triethylamine (200 μL, 1.437 mmol) were added. The mixture was stirred at 40 ° C for 6 hours. Most of the DMF was removed on a rotary evaporator under vacuum to give crude compound C6.

[0275] To the crude compound C6 was added EtOH (5 mL) and 1N NaOH / H2O (5 mL). The mixture was stirred at room temperature for 24 hours. 1N HCl / H2O was added to adjust the pH to 7.4, most of the EtOH was removed on a rotary evaporator, and the mixture was filtered. The filtrate was purified by preparative HPLC using 25 mM TEAB buffer and CH3CN to give compound C7 (518 mg) as a white solid. LCMS: 412.1 (MS + ).

[0276] In a 5 mL vial, compound C7 (49 mg, 0.096 mmol), anhydrous DMF (0.8 mL) and TSTU (27 mg, 0.090 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour to obtain the desired activated NHS ester C8 for the preparation of bioconjugates. LCMS: 509.2 (MS + ).

[0277] Conjugation of 3'-O-azidomethyl-dC to bovine serum albumin (BSA) 20 mg of BSA (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 5 mg of compound C8. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P6DG beads) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0278] Conjugation of 3'-O-azidomethyl-dC to keyhole limpet hemocyanin (KLH) 20 mg of KLH (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 7 mg of compound C8. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P-6DG beads, Bio-Rad Laboratories, Inc.) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0279] Conjugation of 3'-O-azidomethyl-dC to amine-activated agarose resin 20 ml of wet amine-activated agarose resin (5 μmol activated groups / ml) was washed with 30 mL of 50 mM sodium bicarbonate buffer (pH 9.0) and 150 mM NaCl. 70 mg of compound C8 was added to the 20 ml wet beads, and the reaction was incubated with rotation at room temperature for 2 hours. Following the reaction, the resin was washed with 50 mL of phosphate-buffered saline until the absorbance at 260 nm was less than 0.02, yielding the desired purified resin.

[0280] Synthesis of Amino-Reactive NHS Ester of 3'-O-Azidomethyl-2'-deoxyadenine (A12) . Figure 7 The synthesis of the amino-reactive NHS ester (A12) of 3'-O-azidomethyl-2'-deoxyadenine is shown. Compound A9 (111 mg, 0.270 mmol), anhydrous DMF (1 mL) and 1,1'-carbonyldiimidazole (CDI) (70 mg, 0.431 mmol) were added to a 25 mL flask. The reaction mixture was stirred at room temperature for 24 hours. Ethyl 4-aminobutyrate hydrochloride (78 mg, 0.465 mmol) and triethylamine (75 μL, 0.539 mmol) were added. The mixture was stirred at 40 ° C for 16 hours. Most of the DMF was removed on a rotary evaporator under vacuum to give crude compound A10.

[0281] To the crude compound A10 was added EtOH (2 mL) and 1N NaOH / H2O (4 mL). The mixture was stirred at 40°C for 24 hours. 1N HCl / H2O was added to adjust the pH to 8.5, most of the EtOH was removed on a rotary evaporator, and the mixture was filtered. The filtrate was purified by preparative HPLC using 25 mM TEAB buffer and CH3CN to give compound A11 (107 mg) as a white solid. LCMS: 435.9 (MS + ).

[0282] In a 5 mL vial, compound 11 (61 mg, 0.114 mmol), anhydrous DMF (1 mL) and TSTU (20 mg, 0.066 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour to obtain the desired activated NHS ester A12 for the preparation of bioconjugates. LCMS: 555.2 (MS + ).

[0283] Conjugation of 3'-O-azidomethyl-dA to bovine serum albumin (BSA) 20 mg of BSA (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 5 mg of compound A12. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P-6DG beads) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0284] Conjugation of 3'-O-azidomethyl-dA to keyhole limpet hemocyanin (KLH) 20 mg of KLH (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 7 mg of compound A12. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P-6DG beads) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0285] Conjugation of 3'-O-azidomethyl-dC to amine-activated agarose resin 20 ml of wet amine-activated agarose resin (5 μmol activated groups / ml) was washed with 30 mL of 50 mM sodium bicarbonate buffer (pH 9.0) and 150 mM NaCl. 70 mg of Compound A12 was added to the 20 ml of wet beads, and the reaction was incubated at room temperature with rotation for 2 hours. Following the reaction, the resin was washed with 50 mL of phosphate-buffered saline until the absorbance at 260 nm was less than 0.02, yielding the desired purified resin.

[0286] Synthesis of Amino-Reactive NHS Ester of 3'-O-Azidomethyl-2'-deoxythymine (T16) . Figure 8The synthesis of an amino-reactive NHS ester of 3'-O-azidomethyl-2'-deoxythymine (T16) is shown in Figure 2. Compound T13 (108 mg, 0.363 mmol), anhydrous DMF (1 mL), and 1,1'-carbonyldiimidazole (CDI) (74 mg, 0.456 mmol) were added to a 25 mL flask. The reaction mixture was stirred at room temperature for 24 hours. Ethyl 4-aminobutyrate hydrochloride (80 mg, 0.477 mmol) and triethylamine (75 μL, 0.539 mmol) were added. The mixture was stirred at 40°C for 6 hours. Most of the DMF was removed on a rotary evaporator under vacuum to give crude compound T14.

[0287] To the crude compound T14 was added EtOH (2 mL) and 1N NaOH / H2O (2 mL). The mixture was stirred at room temperature for 1 hour. 1N HCl / H2O was added to adjust the pH to 7.5, and then most of the EtOH was removed on a rotary evaporator and the mixture was filtered. The filtrate was purified by preparative HPLC using 25 mM TEAB buffer and CH3CN to give compound T15 (286 mg) as a white solid. LCMS: 426.5 (MS + ).

[0288] In a 5 mL vial, compound 15 (121 mg, 0.225 mmol), anhydrous DMF (1 mL) and TSTU (40 mg, 0.132 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour to obtain the desired activated NHS ester T16 for the preparation of bioconjugates. LCMS: 546.1 (MS+Na + ).

[0289] Conjugation of 3'-O-azidomethyl-dT to bovine serum albumin (BSA) 20 mg of BSA (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 5 mg of compound T16. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P-6DG beads) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0290] Conjugation of 3'-O-azidomethyl-dT to keyhole limpet hemocyanin (KLH) 20 mg of KLH (10 mg / ml) in 50 mM sodium bicarbonate buffer (pH 9.0) containing 150 mM NaCl was reacted with 7 mg of compound T16. The reaction was carried out at room temperature for 1 hour, and the reaction mixture was purified using a desalting column (P-6DG beads) in phosphate-buffered saline. The conjugate was lyophilized to obtain a white powder.

[0291] 3 Conjugation of '-O-azidomethyl-dT to amine-activated agarose resinWash 20 ml of wet amine-activated agarose resin (5 μmol activated groups / ml) with 30 mL of 50 mM sodium bicarbonate buffer (pH 9.0) and 150 mM NaCl. Add 70 mg of Compound T16 to the 20 ml wet beads, and incubate the reaction at room temperature with rotation for 2 hours. After the reaction, wash the resin with 50 mL of phosphate-buffered saline until the absorbance at 260 nm is less than 0.02, yielding the desired purified resin. 11.2 Example 2. Preparation of polyclonal antibodies against non-labeled reversible terminator (NLRT)

[0292] This example describes a protocol for immunization and antibody purification to generate reagents for sequencing. This protocol has been used to prepare polyclonal antisera with antibodies specific for NLRT-A, NLRT-T, NLRT-G, and NLRT-C, with an azidomethyl group as the 3' blocking group.

[0293] Material The following materials were used to immunize rabbits: 3 mg KLH-antigen (for rabbit injection), 3 mg BSA-antigen (for titration) and 2-3 ml agarose-antigen (for purification).

[0294] Immunization of two rabbits : Rabbits were immunized with the KLH antigen described in Example 1.

[0295] In one approach, a 70-day immunization schedule was followed: the first immunization was on day 1; the second immunization was on day 20; the third immunization was on day 40; and the fourth immunization was on day 60. 5 ml of pre-immune serum was collected before the first immunization, and 5 ml of test blood was collected after the third immunization for quality control. Finally, a total of 100 ml of antiserum was collected from two rabbits 10 days after the fourth immunization.

[0296] Polyclonal antibody titer : Monitor titers using the following protocol: a) Coat each well of a plate with 3'-O-azidomethyl-2'-deoxyguanine-BSA at a concentration of 1 μg / well (100 μl) at 4°C overnight or at 37°C for 2-3 hours. b) 100 μl of serially diluted antiserum from immunized rabbits was added to each well and incubated at 37°C for 30 min. c) Wash three times with excess 1X PBS. d) Add 100 μl of HRP-conjugated goat anti-rabbit IgG (1:4000) to each well and incubate at 37°C for 30 min. e) Wash three times with excess 1X PBS. f) Add 100 μl of ABTS substrate solution to each well and incubate at room temperature for 20 min. g) In A 405nm Read the board. The same protocol was used to generate antisera against 3'-O-azidomethyl-2'-deoxy-cytosine, -adenine, and -thymine.

[0297] purification Antibodies were purified from serum using the following protocol. Affi-Gel (Bio-Rad) was prepared by conjugating 3'-azidomethyl-2'-deoxynucleobases to Sepharose 6B via an aminohexanoic acid linker and filled with Affi-Gel purification columns. Antisera recovered from one or two rabbits (up to 100 ml) were applied to an affinity column of Sepharose 6G immobilized with azido-dG, azido-dC, azido-dA, or azido-dT. High-titer polyclonal antibodies specific for each 3'-azidomethyl NLRT were obtained.

[0298] We also use 50-day and 90-day immunization schedules to generate polyclonal antibodies. For example, four rabbits were immunized with the antigen KLH-3'-azido-2'-deoxyguanine conjugate. The immunization schedule was as follows: first immunization, day 1; second immunization, day 14; third immunization, day 28; and fourth immunization, day 42. Before the first immunization, 5 ml of preimmune serum was collected, and after the third immunization, 5 ml of test blood was collected for quality control. Finally, a total of 100 ml of antiserum was collected from two rabbits 10 days after the fourth immunization. 11.3 Example 3. Preparation of Escherichia coli DNA library

[0299] DNA nanoball (DNB) arrays of E. coli genomic DNA libraries were used in the sequencing experiments described in the Examples. DNBs and DNB arrays are described, for example, in Drmanac et al., 2010, "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays," Science 327:7881, which is incorporated herein by reference. During sample preparation, circular library constructs were prepared from fragments of E. coli genomic DNA, and the library constructs were amplified by rolling circle amplification (RCA) to produce DNBs containing genomic DNA inserts with adjacent primer binding sites. DNBs were arrayed in a DNA sequencing flow cell (e.g., a BGISEQ-500 flow cell or a BGISEQ-1000 flow cell) and sequenced using a BGISEQ-500 (BGI, Shenzhen, China; see Huang et al., 2017, “A reference human genome dataset of the BGISEQ-500 sequencer” Gigascience 6:1-9) or a BGISEQ-1000 (BGI, Shenzhen, China). 11.4 Example 4. Use of dN-azidomethyl-specific rabbit polyclonal antibody and labeled goat anti-rabbit secondary Antibodies to detect NLRT incorporated into DNB arrays

[0300] Serum-derived antibodies raised against KLH conjugates of 3'-azidomethyl-dA, 3'-azidomethyl-dC, 3'-azidomethyl-dG, or 3'-azidomethyl-dT were used in this experiment as described in Example 2. Four (4) different purified preparations of anti-NLRT antibodies were prepared for each of the four bases (i.e., RT-A, RT-C, RT-G, and RT-T), resulting in sixteen (16) antibody preparations designated A1-A4, C1-C4, G1-G4, and T1-T4. The DNB array containing the E. coli genomic DNA insert as described in Example 4 was primed and the primers were extended using BG9 DNA polymerase (BGI, Shenzhen, China), which was engineered to incorporate 3'-modified dNTPs and four non-labeled reversible terminators with 3'-azidomethyl blocking groups (e.g., 3'-azidomethyl-dATP, -dCTP, -dGTP, and -dTTP). Sixteen (16) antibody preparations were applied to separate lanes on the DNB array at 10 μg / mL and incubated at 35°C for 5 min (16 separate incubations). At the end of the incubation, unbound primary antibodies on the array were removed by washing with antibody buffer (AbB) (Tris-buffered saline pH 7.4 + 0.1% BSA and 0.05% Tween-20) at 35°C. The array was then incubated with AF488-labeled goat anti-rabbit secondary antibody (Fab fragment) obtained from Jackson Immune Research (West Grove, PA, USA) for 5 min at 35°C. The array was washed with AbB to remove unbound secondary antibody and imaged using the BGISEQ-1000 sequencing system. It should be understood that each of the 16 antibody preparations stained with a single primary antibody is expected to bind to NLRTs incorporated at approximately 25% of DNA sites.

[0301] Four control lanes were generated in the sequencing array by priming with DNBs and extending the primers using all four 3'-azidomethyl dNTPs labeled with fluorophores attached to the bases via cleavable linkers. The control signal values shown here are for C-AF488.

[0302] Table 1 shows the signals obtained using the control array and the antibody array. The highest levels of antibody-mediated signal are shown in bold. Although variations in signal intensity were observed between arrays (depending on the specific preparation of the rabbit polyclonal antibody used), the results show that it is possible to match or exceed control signal intensities at relatively low antibody concentrations using this indirect detection technique. Table 1 11.5 Example 5. DNA Sequencing Using Fluorescently Labeled RT-A, RT-C, and RT-T and Unlabeled RT-G

[0303] The DNA nanosphere E. coli genomic DNA library was sequenced using fluorescently labeled RT-A, RT-C, and RT-T, and unlabeled RT-G (all with a 3'-azidomethyl blocking group). Sequencing was performed using a BGISEQ-500 sequencer (BGI, Shenzhen, China), and the data were analyzed using the base call analysis report provided with the sequencer. Sequencing was performed for 5 cycles ( Figure 10A and Figure 10C ) or 10 cycles ( Figure 10B and Figure 10D ). Figure 10A and Figure 10C Displays Rho values. (Rho values are calculated by subtracting the background from the signal intensity obtained after image analysis. Signal normalization, including crosstalk correction, is also applied.) Figure 10B and Figure 10D The signal-to-noise ratio (SNR) is shown.

[0304] Data from successful base call reports, including intensity of Rho and SNS (signal-to-noise ratio), indicate that sequencing can be successfully performed using unlabeled RT and labeled affinity reagents that specifically bind to RT. 11.6 Example 6. DNA Sequencing Using Four Unlabeled RTs and Unlabeled Anti-NLRT Polyclonal Antibodies

[0305] Table 2 illustrates the sequencing data generated using a BGISEQ-1000 sequencer with an 8-lane chip array (BGI, Shenzhen, China) (see Fehlmann et al., Clin. Epigenetics 8:123, 2016). Column 5 shows the sequencing data using non-labeled reversible terminators (NLRT-A, NLRT-T, NLRT-C, and NLRT-G) with a 3'-O-azidomethyl group as a cleavable blocking unit and polyclonal antibodies against each of the four NLRTs ("1 0 Antibody”) results. Using 2 0 Antibody binding was detected using an AF488-labeled goat anti-rabbit Fab fragment obtained from Jackson ImmunoResearch. Signals were measured in the FIT channel. Each primary antibody was applied individually (i.e., in a separate channel) and detected in a non-control lane (e.g., Table 2, columns 3-8). Raw signal values are shown.

[0306] The rows of Table 2 correspond to an NLRT with a 3' cleavable azidomethyl group as a reversible blocking group. Each row of Table 2 is associated with a target dNTP, and each column shows the test for that target. The columns are as follows:

[0307] Column 1: 1 used in column 5 0Specificity and concentration (in μg / mL) of the antibody (positive control 2).

[0308] Columns 2 and 9: Extension using four fluorescently labeled ("hot") reversible terminator dNTPs (labeled with a fluorescent dye attached to the base via a cleavable linker). (Positive control for DNA array).

[0309] Column 3: Extension using BG9 DNA polymerase. 0 The concentration of the antibodies was 100 μg / ml in all cases (positive control 1).

[0310] Column 4: primary antibody omitted (negative control; secondary antibody only background).

[0311] Column 5: Extension using four (4) unlabeled azidomethyl NLRTs. Results show staining for each primary antibody used at the concentrations in column 1.

[0312] Column 6: extension performed omitting the target NLRT but including 3 non-target NLRTs (antibody specificity control 1);

[0313] Column 7: Negative control, where the target base at the 3' end of the GDS has a 3'-OH instead of an azidomethyl blocking group (Antibody Specificity Control 2).

[0314] Column 8: Negative control, where no sequencing primer was used (specificity control 3). Table 2 Sequencing data of BGISeq-1000 with 8 chip arrays 11.7 Example 7. Detection of non-labeled RT-G using anti-RT-G rabbit primary antibody and labeled goat anti-rabbit secondary antibody 50 sequencing cycles of RT-G

[0315] This example shows the results of fifty (50) cycles of synthesis sequencing (SBS) using a BGISEQ-1000 DNA sequencer and an E. coli genomic DNB library. DNA primers complementary to sequences flanking the genomic DNA insert were hybridized to the DNB array and primer extension was performed using 3'-azidomethyl reversible terminators (RT-A, RT-C, RT-G, RT-T) at a concentration of 2 μM each. Three reversible terminators (azidomethyl-A, azidomethyl-C, azidomethyl-T) were fluorescently labeled by attachment to the bases via a cleavable linker (used in a 50% labeled / 50% non-labeled ratio), while one reversible terminator (3'-azidomethyl-dGTP) was unlabeled. Primer extension was performed at 35°C for 2 min using BG9 DNA.

[0316] After one primer extension cycle, the array was washed to remove unincorporated nucleotides. Incorporated 3'-azidomethyl-dG was detected by incubation with an anti-3'-azidomethyl-dG rabbit primary antibody pre-combined with an AF647-labeled goat anti-rabbit fluorescently labeled Fab fragment. The primary antibody and secondary antibody (Fab fragment) were pre-combined by incubating together at 35°C for 15 minutes. This pre-combined complex was incubated on the array at 35°C for 10 minutes at a concentration of 25 μg / mL primary and 50 μg / mL secondary, and the array was washed three times to remove any unbound antibody.

[0317] After antibody incubation, the three labeled RTs (RT-A, RT-C, RT-T) were detected using their unique fluorescent labels, and the non-labeled base (RT-G) was detected using a fluorescent label conjugated to a goat anti-rabbit fragment secondary antibody. After the identity of the DNB base was determined via fluorescence wavelength detection, the linkers (RT-A, RT-C, RT-T) and 3' blocking groups (RT-G, RT-A, RT-C, RT-T) used for labeling were removed by reduction with 13mM THPP at 35°C for 2min, thereby achieving the regeneration of the 3'-OH group and the ability to further extend the nascent DNA chain. This series of steps (extension, antibody incubation, detection, and unblocking) was repeated for a total of 50 sequence identification cycles.

[0318] Figure 11A The base call information content (BIC) percentages are shown. This figure demonstrates that the identity of the unlabeled bases provides sufficient information for base call analysis and identification of unknown DNA residues when detected indirectly via an anti-3'-azidomethyl-dG rabbit primary antibody pre-combined with an anti-rabbit AF647 fluorescently labeled fragment secondary antibody.

[0319] Figure 11B The signal intensity and trend of each unique fluorescent label are shown. Three nucleotides (dATP, dCTP, dTTP) containing fluorescently labeled cleavable linkers attached to the bases were used in a 50% labeled / 50% non-labeled ratio and corresponded to Cy3, FITC and TxR, respectively. Unlabeled 3'-azidomethyl-dGTP was detected by an anti-3'-azidomethyl-dG rabbit primary antibody pre-combined with an anti-rabbit AF647 fluorescently labeled fragment secondary antibody. These data show that the rate of signal intensity degradation of the non-labeled 3'-azidomethyl-dG base is less than the rate of degradation of the nucleotide labeled with the cleavable linker. This degradation of signal intensity indicates that using this technology it is possible to obtain longer readings than the conventional method of using a cleavable linker to label dNTPs. 11.8 Example 8. Antibodies bind to NLRTs that are specific enough to generate sequences suitable for base calling. Analyzed signal-to-noise ratio (SNR) values

[0320] The data in Tables 3, 4, 5, and 6 show Top signal, Rho (background and crosstalk subtracted signal), background signal ("back"), and signal-to-noise ratio (SNR) values obtained by staining non-labeled reversible terminators on E. coli genomic DNA using directly labeled anti-azidomethyl-base antibodies and using labeled azidomethyl-base for control arrays. The experiment was performed on a BGISEQ-500 flow cell array with an E. coli genomic DNA library and scanned on a BGISEQ-500 DNA sequencer.

[0321] The control values are the results of sequencing using four labeled 3'-azidomethyl RTs (labels attached to the bases via cleavable linkers). 3'-azidomethyl RTs were used in a ratio of 60% labeled ("hot") and 40% unlabeled ("cold") in Tables 4, 5, and 6, and in a ratio of 25% labeled and 75% unlabeled in Table 3.

[0322] Pre-stained values were scanned after one round of primer extension but before addition of antibodies.

[0323] Staining values were obtained by scanning after incubation with the appropriate anti-azidomethyl-base antibodies at the indicated concentrations (2 x 2 min at 35°C). Anti-azidomethyl-base antibodies were directly labeled with the indicated fluorophores. Values corresponding to anti-azidomethyl-base binding are shown in bold.

[0324] Table 3 shows the results using a polyclonal antibody against 3'-O-azidomethyl-2'-deoxyadenine. Table 4 shows the results using a polyclonal antibody against 3'-O-azidomethyl-2'-deoxycytosine. Table 5 shows the results using a polyclonal antibody against 3'-O-azidomethyl-2'-deoxyguanine. Table 6 shows the results using a polyclonal antibody against 3'-O-azidomethyl-2'-deoxythymidine. Table 3 Table 4 Table 5 Table 6 11.9 Example 9. 25-cycle sequencing using labeled anti-NLRT polyclonal antibodies

[0325] As described in Example 2, an E. coli genomic DNA library was prepared and arrayed on a BGISEQ-500 flow cell. Primers were added and sequencing by synthesis was performed using primer extension using unlabeled nucleotide 3'-azidomethyl reversible terminators (dATP, dCTP, dGTP, dTTP). Unlabeled 3'-blocked dNTPs were each present at a concentration of 1 μM and incorporated using BG9 DNA at 55°C for 1 min per cycle. After incorporation and washing to remove unincorporated nucleotides, four 3'-azidomethyl-base nucleotides were detected by contacting the array with a mixture of four directly labeled anti-3'-azidomethyl-base antibodies having a concentration as shown in Table 11 (ranging from 10 to 100 μg / mL), with each cycle incubated at 35°C for 2x2 min on the array. "2x 2" refers to incubation with antibodies for 2 minutes, followed by incubation for another 2 minutes after adding additional antibodies. The array was washed three times to remove any unbound antibodies. Table 7 shows the identity of the fluorophore directly conjugated to each detection antibody. Table 7

[0326] The fluorescence signal at each position on the DNB array is determined by scanning for 80 milliseconds during laser excitation of the fluorophore. After the DNB base identity is determined, the 3' blocking group is removed by reduction with 13 mM THPP at 35°C for 2 minutes, thereby regenerating the 3'-OH group and allowing further extension of the nascent DNA chain. Removal of the 3' blocking group also results in dissociation of the antibody from the primer extension product.

[0327] This series of steps (extension, antibody incubation, detection and unblocking) was repeated for a total of 25 cycles of DNA sequence identity.

[0328] Figure 12A and 12B The Rho and signal-to-noise ratio (SNR) for each base in each sequencing cycle are shown. Table 8 shows the number of DNB reads, as well as the mapping rate and error rate compared to the reference E. coli genome.

[0329] These data, including the intensity of Rho and SNR reported from successful base calls, confirm that multiple cycles of DNA sequencing can be performed using unlabeled reversible terminators and antibodies that bind blocking groups and bases. Table 8 Total reading (M) 0.57 >Q30％ 73.84 Mapping rate % 88.3 Average error rate % 1.58 11.10 Example 10: Comparative results of different labeled antibody groups

[0330] Tables 9 and 10 show Top signal, Rho (background and crosstalk subtraction signal), background signal, and signal-to-noise ratio (SNR) values obtained by staining non-labeled reversible terminators on E. coli genomic DNA using directly labeled anti-azidomethyl-base antibodies. The experiment was performed on a BGISEQ-500 flow cell array with an E. coli genomic DNA library and scanned on a BGISEQ-500 DNA sequencer. Primers were hybridized to fixed DNBs and extended for 2 minutes at 35°C using BG9 polymerase in the presence of all four unlabeled nucleotides 3'-azidomethyl RT (dATP, dCTP, dGTP, dTTP, each at a concentration of 1 μM). After incorporation and washing to remove unincorporated nucleotides, incorporated 3'-azidomethyl-base nucleotides were detected simultaneously by incubating (two consecutive incubations at 35°C for 2 minutes) with all four labeled anti-3'-azidomethyl-base antibodies (Antibody Set 1) at the indicated concentrations (e.g., "@30" means 30 μg / mL).

[0331] The second array was probed using Antibody Set 2. Antibody Set 2 contained the same antibody preparation, but with different antibody labels. Table 10 shows the signal after application of Antibody Set 2. The data demonstrate that the signal and SNR values are suitable for base calling analysis and are not affected by the identity of the directly labeled fluorophore. Table 9 *Antibody specificity and concentration (mg / mL) Table 10 11.11 Example 11: Removal of anti-NLRT antibodies without removal of the 3' blocking group

[0332] As discussed elsewhere herein, antibody removal (dissociation from primer extension products) can be decoupled from the cleavage and removal of the 3' blocking group, and Table 11 shows the results of experiments in which antibodies were removed by specific competition. Primer extension was performed on a DNB array containing an E. coli library using four unlabeled 3'-azidomethyl-base nucleotides. Simultaneous staining was performed by incubating all four anti-3'-azidomethyl-base antibodies directly labeled with color set 1 fluorophores (see Example 10). Specific competition was used to remove the affinity reagent for detection by incubating for 2 min in 50% WB1, 50% Ab buffer at 57°C in the presence of 20 μM free antigen (3'-O-azidomethyl-2'-deoxyguanine, deoxyadenine, deoxycytosine, deoxythymine, each in triphosphate form). The Ab removal procedure was as follows: (1) WB1, 55°C; (2) removal solution; (3) WB1, 20°C; (4) WB2; (5) SRE. WB1: NaCl 0.75M, sodium citrate 0.075M, Tween 20 0.05%, pH 7.0; WB2: NaCl 50mM, Tris-HCl pH 9 50mM, Tween 20 0.05%, EDTA 1mM, pH 9.0; SRE: NaCl 400mM, Tris-HCl pH 7 1000mM, sodium ascorbate 100mM, Tween 20 0.05%, pH 7.0.

[0333] The data showed a significant decrease in signal and SNR, indicating that most of the affinity detection reagents were removed from the DNB array. Table 11 11.12 Example 12: Removal of anti-NLRT antibodies and reprobing in multiple sequencing cycles

[0334] This example describes a method in which (1) the identity of a base at a first position is determined by detecting the binding of a first primary antibody specific for the base and a 3' blocking group; (2) the first primary antibody is removed without removing the 3' blocking group; and (3) the same position is reprobed using a second primary antibody specific for the base and the 3' blocking group. The results of these experiments are summarized in Table 12.

[0335] Table 12 illustrates that when for each position of nascent synthetic sequencing chain (read 20 positions in total), two independent readings from different fluorescent color combinations (as described in Example 10) are combined, the DNA sequence consistency mapping rate is improved. "Odd indep" represents the initial reading of the "conventional color" for each sequencing position. "Even indep" represents the subsequent reading in the "alternative color" after specific competitive removal using the program outlined in Table 12. "Combo" represents comparing each of the two independent readings and weighting the result to a higher intensity (therefore the confidence value of the two readings is higher). The results show that when using this technology to combine two independent readings, the mapping rate is significantly higher and the mismatch rate is significantly lower. Table 12

[0336] Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, it will be appreciated by those skilled in the art that certain changes and modifications may be implemented within the scope of the appended claims. In addition, each reference provided herein is incorporated herein by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between the present application and a reference provided herein, the present application shall prevail.

Claims

1. A method for detecting the incorporation of a 3'-O-reversible terminator deoxyribonucleotide at the 3' end of a primer extension product, wherein the reversible terminator deoxyribonucleotide comprises a nucleobase, a sugar unit and a cleavable blocking group, the method comprising: (a) combining (i) the primer extension product comprising the incorporated reversible terminator deoxyribonucleotide and (ii) a first affinity reagent that binds to the incorporated reversible terminator deoxyribonucleotide, wherein the first affinity reagent binds to the nucleobase, the cleavable blocking group, or both, and (b) detecting binding of the first affinity reagent to the incorporated reversible terminator deoxyribonucleotide.

2. The method of claim 1, wherein the first affinity reagent binds to the sugar unit.

3. The method of claim 1, wherein the first affinity reagent binds to the nucleobase and the cleavable blocking group.

4. A method for detecting the incorporation of a 3'-O-reversible terminator deoxyribonucleotide at the 3' end of a primer extension product, wherein the reversible terminator deoxyribonucleotide comprises a nucleobase, a sugar unit and a cleavable blocking group, the method comprising: (a) generating a primer extension product comprising a 3'-O-reversible terminator deoxyribonucleotide at the 3' end; (b) removing the cleavable blocking group of the incorporated reversible terminator deoxyribonucleotide to produce a 3'-OH deoxyribonucleotide extension product comprising a 3'-OH unit; (c) combining the extension product from (b) with a first affinity reagent that binds to the 3'-OH deoxyribonucleotide extension product, wherein the first affinity reagent binds to the nucleobase and the 3'-OH unit, and (d) detecting the binding of the first affinity reagent to the extension product.

5. A method for performing a sequencing-by-synthesis reaction, comprising the steps of: (a) providing a plurality of immobilized template nucleic acids comprising a plurality of different sequences; (b) annealing an oligonucleotide primer to the template nucleic acid, wherein the oligonucleotide primer hybridizes to a predetermined position on the template nucleic acid; (c) combining the template nucleic acid and the primers annealed to the template nucleic acid with a polymerase and four reversible terminator deoxyribonucleotide triphosphates (dNTPs), each deoxyribonucleotide comprising a nucleobase (N), a sugar unit, and a cleavable blocking group, under conditions whereby a plurality of the oligonucleotide primers are extended by incorporation of a single reversible terminator deoxyribonucleotide to produce a plurality of primer extension products, wherein N is adenine (A) or an analog thereof (A'), guanine (G) or an analog thereof (G'), thymine (T) or an analog thereof (T'), and cytosine (C) or an analog thereof (C'), wherein at least one of the four dNTPs is unlabeled, some of the primer extension products include an A or A' incorporated at the 3' terminus, some of the primer extension products include a T or T' incorporated at the 3' terminus, some of the primer extension products include a G or G' incorporated at the 3' terminus, and some of the primer extension products include a C or C' incorporated at the 3' terminus; (d) contacting the plurality of primer extension products with one or more first affinity reagents under conditions such that each of the one or more first affinity reagents binds only to one of the four incorporated reversible terminator deoxyribonucleotides, wherein the first affinity reagents bind to the nucleobase, the sugar unit, the cleavable blocking group, or a combination thereof, of the one of the four incorporated reversible terminator deoxyribonucleotides; (e) detecting said binding of said one or more first affinity reagents, wherein binding of a first affinity reagent to a primer extension product comprising an incorporated reversible terminator deoxyribonucleotide identifies a nucleobase of the incorporated reversibly terminated deoxyribonucleotide that is complementary to a nucleobase of the template nucleotide, or a nucleobase of the template nucleotide that is complementary to a nucleobase of the incorporated reversible terminator deoxyribonucleotide.

6. A method for performing sequencing-by-synthesis of a deoxyribonucleotide at the 3' end of a primer extension product, wherein the reversible terminator deoxyribonucleotide comprises a nucleobase, a sugar unit, and a cleavable blocking group, the method comprising: (a) generating a primer extension product comprising a 3'-O-reversible terminator deoxyribonucleotide at the 3' end; (b) removing the cleavable blocking group of the incorporated reversible terminator deoxyribonucleotide to produce a 3'-OH deoxyribonucleotide extension product; (c) combining the extension product from (b) with a first affinity reagent that specifically binds to the 3'-OH deoxyribonucleotide extension product, wherein the first affinity reagent specifically binds to the nucleobase and the 3'OH unit, and (d) detecting the binding of the first affinity reagent to the extension product.

7. A method for nucleic acid sequencing, comprising: (a) contacting a nucleic acid template comprising the nucleic acid, a nucleic acid primer complementary to a portion of the template, a polymerase, and an unlabeled RT of Formula I under conditions where the primer is extended to incorporate the unlabeled RT into a sequence complementary to the nucleic acid template, thereby generating an unlabeled extension product comprising the RT: in: R1 is a 3'-O reversible blocking group; R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R3 consists of one or more phosphates; (b) contacting the unlabeled extension product with an affinity reagent comprising a detectable label under conditions in which the affinity reagent specifically binds to the RT to produce a labeled extension product comprising the RT; and (c) identifying the RT in the labeled extension product to identify at least a portion of the sequence of the nucleic acid.

8. A DNA array comprising: A plurality of template DNA molecules, each DNA molecule is attached to a position of the array, a complementary DNA sequence that base pairs with a portion of the template DNA molecule at a plurality of said positions, wherein the complementary DNA sequence includes a first reversible terminator deoxyribonucleotide incorporated at its 3' end; and A first affinity reagent that specifically binds to at least some of the first reversible terminator deoxyribonucleotides.

9. A kit comprising: (a) an unlabeled reversible terminator deoxyribonucleotide of formula I: in: R1 is a 3'-O reversible blocking group; R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R3 consists of one or more phosphates; (b) a first affinity reagent that specifically binds to the reversible terminator nucleotide when incorporated at the 3' end of the primer extension product; and (c) Packaging (a) and (b).

10. A kit comprising (i) a first affinity reagent that binds a first reversible terminator deoxyribonucleotide comprising a first naturally occurring nucleobase or an analog thereof, and (ii) a first affinity reagent that binds a second reversible terminator deoxyribonucleotide comprising a second naturally occurring nucleobase or an analog thereof, wherein the first nucleobase and the second nucleobase are different.

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