Massively parallel sequencing using unlabeled nucleotides

Abstract

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

Description

Technical Field

[0001] The present invention relates to nucleic acid sequencing and its use in medicine and biological sciences.

[0002] Sequence Listing

[0003] This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to U.S. Provisional Patent Application No. 62 / 758,317 filed on November 9, 2018, U.S. Provisional Patent Application No. 62 / 914,877 filed on October 14, 2019, U.S. Provisional Patent Application No. 62 / 914,940 filed on October 14, 2019, and U.S. Provisional Patent Application No. 62 / 914,915 filed on October 14, 2019, each of which is incorporated herein by reference.

[0006] This application is related to U.S. Patent Publication No. US 2018 / 0223358 and International Patent Application No. PCT / US2018 / 012425 published as WO 2018 / 129214, both of which are incorporated herein by reference. Background Art

[0007] 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. Patents 6,210,891, 6,828,100, 6,833,246, and 6,911,345 and U.S. Patent Publication No. 2016 / 0130647.

[0008] SBS requires 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, thereby preventing a series of uncontrolled incorporations from occurring. In one approach, reversible terminator nucleotides (RTs) are used to determine the sequence of the DNA template. In the most commonly used SBS method, each RT contains a modified nucleotide that includes: (1) a capping group that ensures that only a single base is added to the 3' end of the growing DNA copy strand by a DNA polymerase, and (2) a fluorescent label that can be detected by a camera. In the most common SBS method, the template and sequencing primer are fixed to 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, 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.

[0009] Although SBS is widely used, improvements are still needed. 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

[0010] The present invention relates to methods and compositions for nucleic acid analysis and sequencing. A SBS sequencing method is disclosed herein, wherein the last incorporated nucleotide base is identified by binding an affinity reagent (e.g., antibody, aptamer, affimer, knottin, etc.) that recognizes the base, sugar, cleavable capping group, or a combination of these components in the last incorporated nucleotide. The binding is directly or indirectly associated with the generation of a detectable signal.

[0011] According to one embodiment, the invention provides a method for sequencing using non-labeled reversible terminator (NLRT) nucleotides. Reversible terminator (RT) nucleotides are modified deoxynucleotide triphosphates (dNTPs) or dNTP analogs containing removable blocking groups, which ensure that only a single base can be added to the 3' end of the growing DNA copy chain by DNA polymerase. As is well known, the 3' end of the dNTP (2'-deoxynucleoside triphosphate) incorporated into the growing chain during DNA synthesis involves the release of pyrophosphate, and when dNTP is incorporated into the DNA chain, the incorporated part is a monophosphate nucleotide (or more precisely, a nucleotide monomer connected to one or two adjacent nucleotide monomers by a phosphodiester bond). Reversible terminator (RT) nucleotides are modified deoxynucleotide triphosphates (dNTPs) or dNTP analogs containing removable blocking groups, which ensure that only a single base can be added to the 3' end of the growing DNA copy chain by DNA polymerase. Non-labeled RT nucleotides do not contain detectable labels. 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 nucleotide or nucleotide analog. After the image is taken, the blocking group and the labeled affinity reagent are removed from the DNA, thereby allowing the next sequencing cycle to begin. In some embodiments, the epitope identified by the affinity reagent is formed by the incorporated nucleoside itself (i.e., base sugar) or nucleotide and 3' blocking group. 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 a deoxyribose combination.

[0012] 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 where 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:

[0013]

[0014] Where: R 1 is a 3'-O reversible blocking group; R 2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R 3(a) containing or consisting of one or more phosphates; (b) contacting the unlabeled extension product with an affinity reagent under conditions where 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 nucleotides 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.

[0015] 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, e.g., U.S. Patent Publication No. 2002 / 0227131. In contrast, in the dNTP analogs of the present invention, typically R 2 A nucleobase that is not conjugated to a dye or other detectable label via a linker.

[0016] According to another embodiment, the method further comprises (d) removing the reversible blocking group from the RT to produce 3'-OH; and (e) removing the affinity agent from the RT.

[0017] 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 said nucleic acid template is determined.

[0018] According to another embodiment, the method comprises removing the reversible blocking group and the affinity reagent in the same reaction.

[0019] 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.

[0020] In this method, the affinity reagent may include an antibody (including binding fragments of antibodies, single-chain antibodies, bispecific antibodies, etc.), an aptamer, an affimer, a desmin, or any other known reagent that binds to the incorporated NLRT with suitable specificity and affinity. In one embodiment, the affinity reagent is an antibody. In another embodiment, the affinity reagent is an antibody comprising a detectable label, and the detectable label is a fluorescent label.

[0021] According to one embodiment, R 1is selected from the group consisting of 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, propadienyl, cis-cyanovinyl, trans-cyanovinyl, cis-cyanofluorovinyl, trans-cyanofluorovinyl, cis-trifluoromethylvinyl, trans-trifluoromethylvinyl, biscyanovinyl, bisfluoroethyl The invention also includes but is not limited to alkenyl, 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 nitronapthyl.

[0022] According to another embodiment, R 2 is a nucleobase selected from the group consisting of adenine (A), cytosine (C), guanine (G) and thymine (T).

[0023] According to another embodiment, R 3 Consists of or comprises one or more phosphate esters.

[0024] The term non-labeled reversible terminator (NLRT) may refer to the triphosphate form of the nucleotide analog, or may refer to the incorporated NLRT.

[0025] 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 where primers are extended to incorporate an unlabeled RT into a sequence complementary to at least some of the plurality of template DNA molecules, thereby generating an unlabeled extension product comprising the RT, the nucleic acid primer being complementary to a portion of each of the template DNA molecules:

[0026]

[0027] Where: R 1 is a 3'-O reversible blocking group; R 2is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and analogs thereof; and R 3 (c) contacting the unlabeled extension product with the affinity reagent comprising a detectable label under conditions where 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.

[0028] According to one embodiment of the present invention, the method comprises: (b) contacting the DNA array with a nucleic acid primer, a polymerase and an unlabeled RT set of formula I under conditions where primer extension is performed to incorporate unlabeled RT into sequences complementary to at least some of the plurality of template DNA molecules, thereby generating unlabeled extension products comprising the RT, wherein the nucleic acid primer is complementary to a portion of each of the template DNA molecules, and the unlabeled RT set of formula I comprises wherein R 2 is the first RT of A, where R 2 is the second RT of T, where R 2 is the third RT of C, and where R 2The method comprises the steps of: (i) contacting the unlabeled extension product with the affinity reagent set under conditions in which the affinity reagent set specifically binds to the incorporated RT 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; and (d) identifying the RT in the labeled extension product 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 different amounts 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.

[0029] According to another embodiment of the present invention, a DNA array is provided. This array comprises: a plurality of template DNA molecules, each DNA molecule attached at one position of the array; a complementary DNA sequence, which is base-paired 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, which is specifically attached 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.

[0030] According to another embodiment of the present invention, a kit is provided, comprising: (a) an unlabeled RT of Formula I:

[0031]

[0032] Where: R 1 is a 3'-O reversible blocking group; R 2(a) a nucleic acid base 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 reagents. According to another embodiment, such a kit comprises: a plurality of RTs, each of which comprises a different nucleic acid base; and a plurality of affinity reagents, each of which specifically binds to one of the RTs.

[0033] In any of the foregoing embodiments, the affinity agent can be a monoclonal antibody selected from the group consisting of 2C5, 3B12, 17H7, 18B7, 1B8, 2B9, 4C8, 1A10, 3B7, 3G6, 5F6, 4B8, 7C8, 2D4, 2D10, 1F9, 3B7, and 4G8, and variants and derivatives thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A -H shows the alignment of the heavy chain amino acid sequence and the light chain amino acid sequence of the following monoclonal antibodies with specificity: 3'-azidomethyl-dA (N3A): mAbs 2C5, 3B12, 17H7 and 18B7; 3'-azidomethyl-dC (N3C): mAbs 1B8, 2B9, 4C8, 1A10, and 3B7; 3'-azidomethyl-dG (N3G): mAbs 3G6, 5F6, 4B8 and 7C8; and 3'-azidomethyl-dT (N3T): mAbs 2D4, 2D10, 1F9 and 3B7. Specifically, Figure 1A The N3A light chain sequence is shown; Figure 1B N3A heavy chain sequence is shown; Figure 1C The N3C light chain sequence is shown; Figure 1D N3C heavy chain sequence is shown; Figure 1E The N3G light chain sequence is shown; Figure 1F N3G heavy chain sequence is shown; Figure 1G The N3T light chain sequence is shown; and Figure 1H The N3T heavy chain sequence is shown.

[0035] Figure 2A is a scatter plot showing the fluorescence intensity of the DNB population in two channels within a single imaging area after binding to the labeled antibody.

[0036] Figure 2B is a graph of detected fluorescence showing that antibody binding is dependent on both the base and the sugar with a 3' azidomethyl block.

[0037] Figure 2Cis a data graph showing the rapid kinetics of antibody binding to detect primer extension in DNA nanoball (DNB) sequencing. Binding of labeled antibodies to unlabeled RT nucleotides is shown within 30 seconds, 60 seconds, and 90 seconds.

[0038] Figure 2D Intensity data are compared showing the effect of removing the fluorescent antibody after binding to RT under several reaction conditions.

[0039] Figure 2E The relative intensities of the base-labeled nucleotides were compared within the first 10 cycle positions before returning to base-labeled RT, followed by an additional 80 cycle positions for antibody-labeled detection.

[0040] Figure 2F is a scatter plot comparing the signals of a set of DNBs in two consecutive cycles, showing independent labeling of different bases.

[0041] Figure 3A The average called base intensity of DNBs in selected regions of the array is shown, illustrating the change in label intensity over 200 cycles of single-end reading.

[0042] Figure 3B Figure 2 shows the positional discordance of 200 sequencing cycles. The data showed high accuracy and 94% sequencing yield.

[0043] Figure 4A Graph showing PE150 intensity of a human DNA library with background subtracted and spectral crosstalk corrected.

[0044] Figure 4B and 4C Shown are PE150 Lag of the same DNA library and PE100 Lag of an E. coli library with optimized Ph29 removal.

[0045] Figure 5 An example of an NLRT structure is shown: Figure 5 A: 3'-O-azidomethyl-2'-deoxyguanine; Figure 5 B: 3'-O-amino-2'-deoxyguanine; Figure 5 C: 3'-O-cyanoethylene-2'-deoxyguanine; Figure 5 D: 3'-O-phosphate; Figure 5 E: 3'-ethyldisulfide-methylene-2'-deoxythymidine.

[0046] Figure 6 Various protecting groups that can be used in the practice of the present invention are shown. "~" indicates the point of attachment of a molecule to the rest of the structure. DETAILED DESCRIPTION

[0047] 1. Overview

[0048] In some aspects, the present invention provides methods and compositions for carrying out sequencing by synthesis (SBS) of nucleic acid using unlabeled reversible terminator nucleotides. In one method, SBS is carried out by generating fixed single-stranded template DNA at the position on the array. In most methods, each fixed single-stranded template DNA is located at a position with a large number of copies (such as amplicons) of similar sequences. For example, bridge PCR (for example, as described in WO1998044151) can be used to generate template sequence clusters at positions (Illumina) on the array, or rolling circle replication can be used to generate single-stranded concatemers, or DNA nanoballs (DNBs) (see, for example, U.S. Patent No. 8,445,194), which have many copies of template sequences (Complete Genomics, Inc., San Jose, CA). In one method, SBS is carried out by hybridizing one or more primers with template DNA and extending the primers to produce "extension primers" or "growing DNA chains" (GDS). Extension primers refer to adding ("incorporating") nucleotides at the 3' end of the primer DNA chain while allowing it to hybridize with the template. The nucleotide incorporated at the 3' end is complementary to the corresponding nucleotide of the primer, so that the nucleotide sequence of the template can be determined by determining the identity of the incorporated nucleotide at each sequencing cycle. As used herein, "extension primer" and "growing DNA strand" (GDS) and "growing DNA replication strand" have the same meaning and are used interchangeably.

[0049] In a prior art approach, labeled nucleotide analogs are incorporated into GDS. Typically, labeled nucleotide analogs include capping groups that ensure that only single nucleotides can be incorporated into each step, and dyes (typically fluorescent dyes) are attached to nucleotides via cleavable joints. Each sequencing cycle encompasses incorporating labeled nucleotide analogs at one end of the GDS, detecting the nucleotide analog labels of the incorporated labels, removing the labels from the incorporated nucleotide analogs, and removing the capping groups from the incorporated nucleotide analogs to allow incorporation of newly labeled nucleotide analogs. In contrast, the present invention does not require labeled nucleotide analogs that include dyes attached to bases or sugars via cleavable joints.

[0050] In the alternative approach described in US Patent Publication 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 method, the affinity tag is biotin. After incorporation, the incorporated nucleotide is exposed to an affinity reagent comprising a second member of the SBP (e.g., streptavidin) and a detectably labeled. 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 method, the affinity tag is an antigen and the affinity reagent is a fluorescently labeled antibody that specifically binds to the antigen. In contrast, the present invention does not require an affinity tag, and in some aspects, an affinity reagent is used, rather than an affinity tag, and the affinity reagent binds to a nucleobase, a sugar unit (moiety), a cleavable capping group, or a combination or subcombination thereof.

[0051] According to one aspect of the methods disclosed herein, a non-labeled reversible terminator, i.e., a nucleotide analog containing a reversible terminator or a blocking group (Non-Labeled Reversible Terminator or NLRT) is incorporated at the 3' end of the GDS, and then exposed to an affinity reagent (e.g., an antibody) that specifically binds to the incorporated NLRT ("binding event"). After the binding event is detected, the affinity reagent is removed. In one method, 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, optionally in the same step. In this method, each sequencing cycle includes: (i) incorporation of a NLRT containing a capping group by a DNA polymerase, followed by washing away 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 capping 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), 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. The affinity reagent (e.g., an antibody) may be directly labeled (e.g., a fluorescently labeled antibody) or may be indirectly detected (e.g., by binding to a labeled anti-affinity reagent secondary affinity reagent). Thus, it should be understood that a "labeled affinity reagent" may be directly labeled, for example, by conjugation to a fluorophore, or may be indirectly labeled.

[0052] In another method, 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 method, each sequencing cycle includes: (i) incorporating a NLRT containing a blocking group by a DNA polymerase, optionally followed by removal (washing off) of unincorporated NLRT; (ii) contacting the incorporated nucleotide analog with a labeled affinity reagent that recognizes and specifically binds to the incorporated NLRT; (iii) detecting the binding of the affinity reagent; (iv) removing the blocking group in a manner that regenerates the hydroxyl (OH) at the 3' position of the deoxyribonucleotide; (iv) removing the blocking group in a manner that regenerates the hydroxyl (OH) at the 3' position of the deoxyribose unit, which allows the incorporation of additional nucleotide analogs (e.g., generating a hydroxyl at the 3' position of the deoxyribose moiety), and (v) removing the affinity reagent. 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.

[0053] SBS involves two or more primer extension cycles, wherein nucleotides are incorporated at the 3' end of the extension primer. The present invention utilizes affinity reagents, such as antibodies, to (i) detect the nucleotides incorporated at the 3' end of the extension primer ("3' terminal nucleotide") and (ii) identify the nucleobase of the 3' terminal nucleotide and distinguish one from another nucleobase (e.g., distinguish A from G). It is not desirable to be bound by a specific mechanism, which is possible because each affinity reagent is designed to distinguish the 3' terminal nucleotide from other "inner" nucleotides of the extension primer, even when the 3' terminal nucleotide and the inner nucleotide contain the same nucleobase. Each affinity reagent (or in some cases, a combination of affinity reagents) is also designed to detect the properties of the 3' terminal nucleotide of the nucleobase associated with the 3' terminal nucleotide. 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.

[0054] In some methods, the SBS reaction of the present invention is carried out using nucleotides with 3' reversible terminator parts. In these methods, based on the position and existence of the reversible terminator parts, the 3' terminal nucleotides incorporated are different from the internal nucleotides. Therefore, the affinity reagents combined with the reversible terminator parts in the extension primers bind (and therefore detect) the 3' terminal nucleotides, thereby distinguishing them from the internal nucleotides. In different methods, 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 reagents combined with the free 3'-OH groups in the extension primers bind to the 3' terminal nucleotides, bind (and therefore detect) the 3' terminal nucleotides, thereby distinguishing them from the internal nucleotides. In some methods, the free 3'-OH group is generated by the reversible terminator in the nucleotide analogs incorporated by cleavage. In another method, the free 3'-OH group is produced by the incorporation of nucleotides (such as naturally occurring nucleotides) that do not contain reversible terminator units. In additional methods that can be combined with either of the above two methods, the incorporated 3' terminal nucleotide is different from the internal nucleotides based on other structural difference characteristics of the 3' terminal nucleotide, including but not limited to: the affinity agent is more accessible to the deoxyribose of the 3' terminal nucleotide relative to the deoxyribose of the internal nucleotides; the affinity agent is more accessible to the nucleobase of the 3' terminal nucleotide relative to the accessibility of the affinity agent to the deoxyribose of the internal nucleotides, and other molecular and conformational differences between the 3' terminal nucleotide and the internal nucleotides.

[0055] Thus, in one aspect of the invention, affinity reagents are used to detect these structural differences between the 3' terminal nucleotide and the other nucleotides of the extended primer.

[0056] Also provided are many strategies, methods and materials for detecting the properties of the 3' terminal nucleotide of the nucleobase of the 3' terminal nucleotide. In one method, naturally occurring nucleotides or nucleotide analogs comprising naturally occurring nucleobases (e.g., A, T, C and G) are used for sequencing reactions and incorporated into primer extension products. Affinity reagents that specifically bind to a nucleobase (e.g., A) and distinguish the nucleobase from other nucleobases (e.g., T, C and G) that are not combined with it are used to identify the nucleobase of the 3' terminal nucleotide. In another method, nucleotide analogs comprising modified (i.e., non-naturally occurring) nucleobases are used for sequencing reactions and incorporated into primer extension products. Affinity reagents specifically bind to a modified nucleobase (e.g., modified A) and distinguish the modified nucleobase from other modified or natural nucleobases. Affinity reagents that specifically bind to modified nucleobases generally recognize modifications, so that the combination with the modified nucleobase is different from the combination with the naturally occurring nucleobase in the absence of modification. For example, affinity reagents that bind to adenosine analogs, wherein position 7 (N 7) is replaced by a methylated carbon and 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 portion (in this case, the modified nucleobase) achieves the above 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.

[0057] In another approach, nucleotides (reversible terminator nucleotides) with 3' reversible blocking groups are incorporated into primer extension products. The blocking groups are removed in each sequencing cycle so that only the last nucleotides incorporated of primer extension products include blocking groups. In the method, an affinity reagent in conjunction with blocking groups is used. In one aspect of the method, at least two nucleotide analogs (that is, they have different core bases) used in the sequencing reaction include different blocking groups. To illustrate, by using the first blocking group (for example, 3'-O-azidomethyl) for the nucleotides comprising adenine or adenine analogs, using different second blocking groups (for example, 3'-O-cyanoethylene) for the nucleotides comprising guanine or guanine analogs, etc., the specificity of the affinity reagent will identify the associated core bases. For example, when extending as described above, if the 3' terminal nucleotides are identified by the 3'-O-cyanoethylene specific affinity reagent, this indicates that the associated core bases are guanine or guanine analogs, and the template base at this position is cytosine. In a variation of this approach, capping 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.

[0058] As described below, in one aspect of the invention, an affinity reagent (e.g., an affinity reagent that identifies and specifically binds to a nucleotide or nucleotide analog based on a combination of structural features) is used (e.g., an affinity reagent that identifies a specific end-capping group and a specific core base with a specific modification). In this regard, nucleotides or nucleotide analogs are designed and / or selected for properties identified by specific affinity reagents. In some cases, affinity reagents in combination with a variety of structural features have the advantage of being more strongly and more specifically combined with affinity reagents. The following table 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 with affinity reagents to provide enough binding efficiencies in the last incorporated nucleotides; and / or based on those features distinguishing between the last incorporated nucleotides and internal nucleotide regions (column 3).

[0059] Table 1

[0060]

[0061] 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 capping group, or a combination of a nucleobase and / or a capping group with a sugar portion of the nucleotide analog. See Table 1. The 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 capping group at the 3' end of the GDS and a similar nucleotide analog (lacking a capping group) located in or within the GDS. The binding of the labeled affinity reagent also depends on the nucleobase itself, such that the affinity reagent binds to a target NLRT incorporated at the end of the GDS (e.g., NLRT-A) at one position on the array but does not bind to other NLRTs incorporated at the end of the GDS (e.g., NLRT-C, -T, or -G) at a different position on the array.

[0062] 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 the method, the affinity reagent can include multiple fluorescent units, and provide a stronger signal than a single fluorescent dye attached to dNTP according to a common method. The method can also cause less light damage because a lower excitation power or a shorter exposure time can be used. The method disclosed herein allows longer high-accuracy readings (e.g., longer than 500 bases or longer than 1000 bases) and / or longer than 50, 100 or 200 bases More accurate readings (e.g., with less than one error in 2000 bases or less than one error in 5000 bases). The composition and method of the present invention can also be more economical than the labeled reversible terminator (RT) method commonly used for SBS. The unlabeled RT cost is lower than the labeled RT. In standard SBS using labeled RT, a high concentration of labeled RT is used to drive the incorporation of complete RT, and most of the labeled RT (70-99% or more) is not incorporated by the polymerase and is washed away. The use of lower cost unlabeled RT thus reduces this cost. In addition, in the labeling step of the present invention (wherein a labeled affinity reagent is used), it is not necessary that each copy of the target sequence at the array site is bound by the affinity reagent, especially when the affinity reagent is labeled with multiple dye molecules (e.g., per molecule of affinity reagent, an average of 2, 3, 4, 5, at least 2, at least 3, at least 4, at least 5, 2-5 or 3-5 dye molecules). For example, 50 copies of the template sequence may be present at one site of the array (e.g., a concatemer at one site on the array may contain 50 copies of the template sequence). In one method, one molecule of the affinity reagent is labeled with multiple dye molecules and less than about 50% of the copies of the template sequence are bound by the affinity reagent. In some embodiments, less than about 30%, less than about 25%, less than about 20%, or less than about 15% of the copies of the target sequence are bound by the affinity reagent. A higher level of binding may be preferred if the affinity reagent carries only a single labeled molecule (e.g., 50% or more or 70%).

[0063] 2. Definitions and Terminology

[0064] As used herein, in the context of nucleotide analogs, the terms "unlabeled" and "non-labeled" are used interchangeably.

[0065] 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 or analog, and a cleavable capping group. The non-labeled reversible terminator nucleotide may 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 incorporated into the "inner" portion of the primer extension product after additional incorporation cycles (if any).

[0066] As used herein, "dNTPs" include naturally occurring deoxyribonucleotide triphosphates and analogs thereof, including analogs having 3'-O cleavable capping groups.

[0067] As used herein, in the context of cleavable capping groups of nucleotide analogs, the designation 3'-O-" is sometimes implied rather than explicit. For example, the terms "azidomethyl", "3'-O-azidomethyl" are interchangeable, as will be apparent from the context.

[0068] "Amplicon" means the product of a polynucleotide amplification reaction, i.e., a polynucleotide population 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. Patents 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. Patent Publication 2006 / 0024711).

[0069] As used herein, "antigen" means 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 capping groups, dNTPs, polypeptides, small molecules, lipids, or nucleic acids.

[0070] "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 a non-planar interrogable structure having a surface such as a bead or a well. 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 is well known, the array is typically contained within a flow cell.

[0071] As used herein, "random array" or "random microarray" refers to a microarray in which the identity of oligonucleotides or polynucleotides cannot be identified, at least not initially, by their position, but can be determined by specific biochemical detection techniques performed on the array.

[0072] The terms "reversible," "removable," and "cleavable" when referring to a capping group have the same meaning.

[0073] The term "reversible blocking group" of a reversible terminator nucleotide may also be referred to as a "removable blocking group," "cleavable linker," "blocking moiety," "blocking group," "reversible terminator blocking group," and the like. A reversible blocking group is a chemical unit attached to a nucleotide sugar (e.g., deoxyribose) typically at the 3'-O position of the sugar moiety that prevents the addition of nucleotides by a polymerase at that position. A reversible blocking group can be cleaved by an enzyme (e.g., a phosphatase or esterase), a chemical reaction, heat, light, and 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.

[0074] "Derivative" or "analog" means such a compound or molecule, whose core structure is identical or very similar to the core structure of the parent compound, but it has chemical or physical modifications, such as different or additional side groups, or 2' and / or 3' blocking groups, which allow the derivative nucleotide or nucleoside 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 "analog" 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 may also include modified phosphodiester linkages, 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 adenosine, 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 iodoxuridine and trifluridine. As used herein, "derivatives", "analogs" and "modified" are used interchangeably and are covered by the terms "nucleotide" and "nucleoside" as defined herein.

[0075] "Incorporation" means becoming part of a nucleic acid molecule. In SBS, incorporation of RT occurs, i.e., RT is added to a DNA chain, 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 of one nucleotide (i.e., the 3' nucleotide on the DNA chain) and the 5' position of the pentose on the adjacent nucleotide.

[0076] In the context of the affinity reagent of labeling, "labeling" means any atom or molecule that can be used to provide detectable and / or quantifiable signals. 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. Fluorescent labels can be any label that can emit light when 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 fluorescent dyes commercially available under the Alexa[image] 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 alkyls, fluorescent proteins, non-fluorescent dyes, and photoproteins.

[0077] "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.

[0078] As used herein, "naturally occurring nucleobase" means adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). In some cases, the naturally occurring nucleobase refers to A, C, G, and T (the naturally occurring bases found in DNA).

[0079] "Nucleotides" consist of a nucleobase, a sugar, and one or more phosphate groups. They are the monomeric unit of a nucleic acid sequence. In RNA, the sugar is ribose, while in DNA, the sugar is deoxyribose, i.e., a sugar lacking 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 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 C-5 of the sugar. Nucleotides are typically monophosphates, diphosphates, or triphosphates. "Nucleosides" are similar in structure to nucleotides, but do not include the phosphate unit. Common abbreviations include "dNTP" for deoxynucleotide triphosphates.

[0080] "Nucleic acid" means a polymer of nucleotide monomers. As used herein, the term may 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., base pairing, base stacking, Hoogsteen or reverse Hoogsteen base pairing, etc.) to form a duplex or triplex form. This monomer and its internucleoside bonding may be naturally occurring or may be its analog, such as a naturally occurring or non-naturally occurring analog. Non-naturally occurring analogs may include peptide nucleic acids, locked nucleic acids, thiophosphate internucleoside bonding, bases containing linking groups that allow labeling to attach, such as fluorophores or haptens, etc. The size of nucleic acids is typically several monomer units (e.g., 5-40, when they are typically referred to as "oligonucleotides") or even hundreds of thousands or more monomer units. Whenever a nucleic acid or oligonucleotide is represented by a sequence of letters (uppercase or lowercase), such as "AGCT", 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 terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Typically, nucleic acids contain natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester bonds; however, they may also contain non-natural nucleotide analogs, such as modified bases, sugars, or internucleoside linkages. For those skilled in the art, when the enzyme has a specific oligonucleotide or nucleic acid substrate requirement for activity (e.g., single-stranded DNA, RNA / DNA duplex, etc.), the selection of a composition suitable for the oligonucleotide or nucleic acid substrate is within the knowledge of the ordinary technician, 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.

[0081] "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.

[0082] "Polynucleotide" is used interchangeably with the term "nucleic acid" to refer to DNA, RNA, and hybrid and synthetic nucleic acids, and may 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.

[0083] "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. A solid support may include an ordered or disordered array with fixed sites or wells.

[0084] The "identity" percentage between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps to obtain the maximum sequence identity percentage, if necessary. For short (e.g., less than 150 amino acids) sequences, a pair of opf ​​sequences can be manually aligned and visually inspected to determine the amino acid sequence identity percentage. Alternatively, publicly available computer software, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA software. In some embodiments, CLUSTAL OMEGA software is used to perform the alignment. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0085] "Conservative substitution" or "conservative amino acid substitution" refers to the substitution of one or more amino acids by one or more chemically or functionally similar amino acids. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences with such substitutions are referred to as "conservatively modified variants". In addition to, but not excluding, polypeptide variants, such conservatively modified variants are also interspecies homologs and alleles. In certain embodiments, selected amino acid groups are considered to be conservative substitutions of each other. For example, substitutions within the following residue groups are conservative substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); 8) cysteine ​​(C), methionine (M). Additional conservative substitutions can be found in, for example, Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) WH Freeman & Co., New York, NY. Proteins with conservative substitutions relative to a reference protein can be referred to as conservatively substituted variants.

[0086] 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 reagent or a mixture of reagents, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art.

[0087] Unless otherwise defined, 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 described in the publications and which may be used in conjunction with the present invention.

[0088] Where a range of values ​​is provided, it is understood that, unless the context clearly dictates otherwise, each intermediate value to one tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intermediate values ​​within the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits within the stated ranges. Where the stated range includes one or two limits, ranges excluding one or both of these included limits are also encompassed within the present invention.

[0089] In the following description, many 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 cases, well-known features and procedures well known to those skilled in the art are not described in order to avoid obscuring the present invention.

[0090] While the invention has been described primarily with reference to particular 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.

[0091] Unless otherwise indicated, the practice of the present invention may employ conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and immunology, which are within the technical scope of the art. These conventional techniques include polymer array synthesis, hybridization, connection (ligation) and hybridization detection using labels. Specific descriptions of suitable techniques may be obtained by reference to the examples below. However, other equivalent conventional procedures may certainly be used. 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.

[0092] Nucleotides and nucleotide analogs

[0093] In various embodiments, SBS according to the present invention can use unlabeled reversible terminators ("NLRTs") (e.g., nucleotide analogs with blocking groups), unlabeled naturally occurring nucleotides (e.g., dATP, dTTP, dCTP, and dGTP), or unlabeled nucleotide analogs that do not include blocking groups.

[0094] Non-labeled reversible terminator (NLRT)

[0095] The unlabeled reversible terminator ("NLRT") of the present invention is a nucleotide analog that contains a removable blocking group at the 3'-OH position of the deoxyribose. Although many reversible terminators have been described, and reversible terminators are widely used in SBS, the unlabeled reversible terminators used according to the present invention are different from those used commercially because they are unlabeled and because they are used in combination with the affinity reagents described below. In one aspect, the NLRT of the present invention is 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.

[0096] In some embodiments, prior to incorporation of the NLRT into a DNA strand, an exemplary NLRT has the following structure I.

[0097]

[0098] Where R 1 is a 3'-O reversible blocking group, R 2 is or includes a nucleobase; and R 3 Contains at least one phosphate group or its analog.

[0099] After incorporation of NLRT into the DNA strand, the reversible capping group R can be removed 1 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 may be used. Exemplary reversible blocking groups are described below.

[0100] Nucleobase R 2It 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 according to the nucleobase; for example, an NLRT with an A nucleobase is referred to as NLRT-A. Therefore, 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.

[0101] Nucleobase R 2 Any nucleobase or nucleobase analog (e.g., an analog of adenine, cytosine, guanine, thymine, uracil, or inosine) may be used. For example, naturally occurring nucleobases may be modified to increase the immune response to the analog when raising antibodies, or to increase the specificity of antibodies to a particular nucleobase.

[0102] R 3 There may be 1-10 phosphate or phosphate analog groups. Phosphate analogs include phosphorothioate (PS), in which 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, R 3 There may be 1 to 10 phosphate groups. 3 There may be 3 to 12 phosphate groups. In some cases, the nucleotide analog is a nucleoside triphosphate.

[0103] In certain embodiments, R of Formula I 1 The MW of the peptide is less than 184, usually less than 174, usually less than 164, usually less than 154, usually less than 144, usually less than 134, usually less than 124, usually less than 114, usually less than 104, usually less than 94, and sometimes less than 84. When conjugated to a larger carrier molecule such as KLH, R 1 Can act as a hapten and elicit an immune response.

[0104] It should 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 size and structure of the reversible blocking group should make the NLRT a substrate for at least some DNA polymerases. The incorporation of the NLRT can be completed by terminal transferase, polymerase or reverse transcriptase. Any DNA polymerase for sequencing can be used, including, for example, DNA polymerases from Thermococcus, such as 9 ° N or its mutants, including A485L, including double mutants Y409V and A485L. As known in the art, polymerases have a high degree of discrimination in the properties of the 3' blocking group. Therefore, it is usually necessary to mutate the polymerase protein to promote effective incorporation. Exemplary DNA polymerases and methods that can be used in the present invention include those described in the following documents: 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 Publications WO2005 / 024010 and WO2006 / 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 double mutants Y409V and A485L. Other examples include KOD polymerase (Kitabayashi et al., 2002. Biosci. Biotechnol. Biochem. 66:10, 2194; Fujii et al., 1999. J. Mol. Biol. 289:835), Taq polymerase, Escherichia 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.

[0105] It will be appreciated that modifications to the capping groups should not interfere with the reversible terminator function. That is, they should be cleavable to generate 3'-OH deoxyribonucleotides.

[0106] In one embodiment, prior to incorporation of RT into a DNA strand, RT has the following structure II.

[0107] Structure II

[0108]

[0109] Where R 1 is a 3'-O reversible blocking group, R 4 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U); and R 3 Contains at least one (e.g., 1-10) phosphate ester. In some cases, R 3 It is a triphosphate.

[0110] In one embodiment, after incorporation of RT into a DNA strand, RT has the following structure III.

[0111] Structure III

[0112]

[0113] Where R 1 is a 3'-O-reversible capping group, R 2 is a nucleobase, 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).

[0114] In another embodiment, after incorporation and removal of the reversible blocking group, RT has structure IV.

[0115] Structure IV

[0116]

[0117] R 6 is H and R 7 is a polynucleotide as defined above comprising 10-1000 nucleosides linked by phosphate-sugar bonds (e.g. GDS), or is R as defined above 3 .

[0118] In certain embodiments of structures I, III and IV, R 2is a nucleobase analog (e.g., an analog of A, T, G, C, U) whose modification does not alter the binding specificity of the base (i.e., an A analog binds to T, a T analog binds to A, etc.), (ii) but which renders 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 groups are not removed from the nucleotide when they are incorporated into the GDS such that the GDS comprises a plurality of nucleotides comprising the modification. In such embodiments, the affinity reagent binds to the terminal nucleotide analog comprising the modification, but binds to the internal nucleotides with much lower affinity with the modification.

[0119] 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 non-target ends, such as by different capping groups or by attaching a "contaminated" end to a 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 with RT to prevent binding to the molecular binders described herein.

[0120] Reversible terminator capping group

[0121] NLRTs for use in the present invention may include any suitable capping group. In some embodiments, a suitable capping group is a group that can be removed by chemical or enzymatic treatment to produce a 3'-OH group. The chemical treatment should not significantly degrade the template or primer extension strand. Various molecular units of the 3'-capping group of the reversible terminator 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 capping groups include -O-azidomethyl and -O-cyanovinyl. Other exemplary RT capping groups are Figure 5 and 6 The diagrams are shown for purposes of illustration and not limitation.

[0122] In other embodiments, R of Formula I (above) 1is 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. 1 It can be selected from propadienyl, 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.

[0123] Various 3'-O reversible blocking groups (R in Formula I 1 ) can be used in the practice of the present invention. According to one embodiment of the method of the present invention, R 1 is selected from the group consisting of 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-trifluoromethylvinyl, trans-trifluoromethylvinyl The invention also includes but is not limited to: 1-hydroxy-2-nitro-1, 2-hydroxy-2-nitro-1, 2-hydroxy-2-nitro-2, 2-hydroxy-2-nitro-3, 2-hydroxy-2-nitro-3, 2-hydroxy-2-nitro-4, 2-hydroxy-2-nitro-5, 2-hydroxy-2-nitro-6, 2-hydroxy-2-nitro-7, 2-hydroxy-2-nitro-8, 2-hydroxy-2-nitro-9, 2-hydroxy-2-nitro-1, 2-hydroxy-2-nitro-2, 2-hydroxy-2-nitro-3, 2-hydroxy-2-nitro-1, 2-hydroxy-2-nitro-2, 2-hydroxy-2-nitro-3, 2-hydroxy-2-nitro-1, 2-hydroxy-2-nitro-2

[0124] 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 C 1 -C 10 Alkyl, C 2 -C 10 Alkenyl and C 2 -C 10 Additional ranges of these groups that can be used in the compounds and methods described herein include C 1 -C 8 Alkyl, C2 -C 8 Alkenyl, C 2 -C 8 Alkynyl, C 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Alkynyl, C 1 -C 4 Alkyl, C 2 -C 4 Alkenyl and C 2 -C 4 Alkynyl.

[0125] "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 C 1 -C 10 Heteroalkyl, C 2 -C 10 Heteroalkenyl and C 2 -C 10 Additional ranges of these groups that can be used in the compounds and methods described herein include C 1 -C 8 Heteroalkyl, C 2 -C 8 Heteroalkenyl, C 2 -C 8 Heteroalkynyl, C 1 -C 6 Heteroalkyl, C 2 -C 6 Heteroalkenyl, C 2 -C 6 Heteroalkynyl, C 1 -C 4 Heteroalkyl, C 2 -C 4 Heteroalkenyl and C 2 -C 4 Heteroalkynyl.

[0126] 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 alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl groups at the position attached to the main chain of alkoxy, aryloxy, amino, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloalkyl or heterocycloalkyl, 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 alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl has complete hydrogen, i.e., comparable to its saturation level, without substitution, for example, linear butane (-(CH 2 ) 3 -CH 3 ).

[0127] In other embodiments, the reversible capping group is an amino-containing capping group (e.g., NH 2 -). See, Hutter et al., 2010, Nucleosides Nucleotides Nucleic Acids 29 (11) (incorporated herein by reference), which describes exemplary amino-containing reversible end-capping groups. In some embodiments, the reversible end-capping group is an allyl-containing end-capping group (e.g., CH2=CHCH2-). In some embodiments, the reversible end-capping group comprises a cyano group (e.g., a cyanovinyl or cyanoethyl group). In some embodiments, the reversible end-capping group is an azide-containing end-capping group (e.g., N3-). In some embodiments, the reversible capping group is an azidomethyl group ((N3CH2-). In some embodiments, the reversible capping group is an alkoxy-containing capping group (e.g., CH3CH2O-). In some embodiments, the reversible capping group contains a polyethylene glycol (PEG) unit having one or more ethylene glycol moieties. In some embodiments, the reversible capping group is a substituted or unsubstituted alkyl group (i.e., a substituted or unsubstituted hydrocarbon). In some embodiments, the reversible capping group is an acyl group. See, U.S. Patent 6,232,465, which is incorporated herein by reference. In some embodiments, the reversible capping group is or contains a methoxymethyl group. In some embodiments, the reversible capping group is or contains an aminooxy group (H2NO-). In some embodiments, the reversible capping group is or contains a carbonyl group (O=CH-). In some embodiments, the reversible capping group comprises an ester or phosphate group.

[0128] In some embodiments, the reversible capping group is a nitrobenzyl group (C6H4(NO2)-CH2-). In some embodiments, the reversible capping 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 group reversible capping group is covalently attached to the 3'-O of the nucleotide analog.

[0129] In some embodiments, the reversible capping group is nitronapthyl (ie, contains a nitronaphthalene unit or a derivative thereof), wherein, for example, either of the CH carbons of the nitronapthyl reversible capping group is covalently attached to the 3'-O of the nucleoside analog.

[0130] In some embodiments, the reversible capping group is selected from:

[0131]

[0132] Where R 3 and R 4 is H or alkyl, and R 5 It is alkyl, cycloalkyl, alkenyl, cycloalkenyl and benzyl.

[0133] Other reversible capping groups suitable for use in the present invention are those described in the literature as capping groups for labeled reversible terminators.In general, any suitable reversible capping group for sequencing by synthesis may be used in the practice of the present invention.

[0134] Properties of reversible terminator capping groups and nucleotides containing them

[0135] Preferably, for sequencing applications, the capping group of the RT is removable under reaction conditions that do not interfere with the integrity of the DNA being sequenced. An ideal capping 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 cause damage to the polynucleotide structure.

[0136] In certain embodiments of the invention, the capping 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, R of Formula I 3 Having 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.

[0137] The molecular weight of deoxyribose monophosphate nucleotide esters 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 the GDS (i.e., excluding the pyrophosphate of the dNTP), 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.

[0138] Phosphate-containing units

[0139] In some embodiments, R 3 The unit comprises one or more phosphate and / or phosphate analog units. 3 The 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 may be an alkyl group or any of the various linkers described in the art. See, for example, U.S. Pat. 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 the alpha phosphate) is removed from the nucleotide so that X is not present in the incorporated reversible terminator deoxyribonucleotide. In certain embodiments, X may 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 that X is not present in the incorporated reversible terminator deoxyribonucleotide.

[0140]

[0141] NLRT group

[0142] In some methods, SBS sequencing according to the present invention includes contacting a sequencing array with a plurality of NLRTs (e.g., NLRT-A, NLRT-T, NLRT-C, and NLRT-G). The contact can be performed sequentially, one NLRT at a time. Alternatively, four NLRTs can be contacted with the sequencing array at the same time, most commonly as a mixture of four NLRTs. The four NLRTs together constitute a "NLRT group". The NLRTs of the NRRT group 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 include unequal amounts in a mixture of the four NLRTs. One embodiment of the NLRT group members (NLRT-A, NLRT-T, NLRT-C, and NLRT-G) include naturally occurring nucleobases and 3' azidomethyl capping groups.

[0143] In one embodiment, each NLRT in the NLRT set comprises the same capping group (e.g., azidomethyl). In one embodiment, the NLRTs in the NLRT set comprise different capping 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 capping groups are used, such capping groups are optionally selected so that different capping groups can be removed by the same treatment. Alternatively, the capping groups can be selected to 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.

[0144] 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 different NLRTs. The structurally different NLRTs can differ in their respective nucleobases. For example, the mixture can contain four structurally different NLRTs, each NLRT comprising one of the four natural DNA nucleobases (i.e., adenine, cytosine, guanine, and thymine) or a derivative thereof.

[0145] 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., premixed). A kit comprising a NLRT set (wherein the different NLRTs are packaged in separate containers or as a mixture in the same container) can be provided.

[0146] Nucleobase analogs with groups that improve affinity reagent binding

[0147] In one embodiment, the nucleobase includes a non-removable chemical group that increases the specificity or affinity of the affinity reagent for the nucleobase when present at the 3' end of the growing DNA strand (i.e., as the last base incorporated), 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 less affinity or less efficiency relative to the same modification in the 3' terminal nucleotide.

[0148] By way of illustration and not limitation, examples of such modified nucleobases include:

[0149]

[0150] R 6 , R 7 , R 8 and R 9 : can be the same or different, each selected from H, I, Br, F, structures XIX-XXVIII, or any group that does not interfere with base pairing. Note R 9 This is the case for the methyl group structure XVIII in thymidine. In some cases, the modification has the additional beneficial effect of increasing the antigenicity of the nucleotide.

[0151]

[0152] 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 no greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 Da of the molecular weight of the natural bases.

[0153] Unblocked dNTPs

[0154] In one embodiment, a natural dNTP (e.g., dATP, dGTP, dCTP, or dTTP) or a dNTP analog without a 3'-O-capping group is used for sequencing. In some embodiments, nucleotides are incorporated one at a time during the sequencing process (such as in pyrophosphate sequencing), or incorporated by a 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 use in the present invention by removing the label and / or the joint connecting the label to the RT. In some methods, 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.

[0155] Deoxyribose analogs

[0156] In some embodiments of the invention, the sugar (deoxyribose) unit is modified. For example, using an affinity reagent that recognizes the capping group and the sugar unit, an NLRT having a nucleobase adenine, a capping group azidomethyl, and a sugar deoxyribose can be distinguished from an NLRT having a nucleobase cytosine, a capping group azidomethyl, and a sugar-modified deoxyribose.

[0157] Nucleotides without 3'-O reversible terminators

[0158] In various aspects useful for 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).

[0159] The examples given above include reversible capping groups attached to nucleotides via the 3'-O of the deoxyribose sugar unit. The present invention also includes NLRTs with reversible and irreversible capping groups attached to the 2'-O- of the deoxyribose sugar. These embodiments can be used for single base detection (single or several base primer extensions), monitoring gaps and gaps 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' instead of 3' capping groups.

[0160] Affinity reagents

[0161] The present invention uses an affinity reagent that specifically binds to the NLRT at the 3' end of the GDS, for example, after incorporation into the end of the growing DNA strand by a polymerase 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.

[0162] Common affinity reagents

[0163] 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. The affinity reagent is a molecule or macromolecule that specifically binds to the 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.

[0164] For illustration, one example of an affinity agent is a monoclonal antibody (mAb) that binds with high affinity to an NLRT incorporated at the 3' end of a DNA strand when the NLRT comprises a nucleobase adenosine and an azidomethyl reversible capping group. Exemplary affinity agents include antibodies (including binding fragments of antibodies, single-chain antibodies, etc.), nucleic acid aptamers, affimers, and desmins, as described in U.S. Patent Publication No. 2018 / 0223358. To illustrate, one example of an affinity agent is a monoclonal antibody (mAb) that binds with high affinity to a NLRT incorporated at the 3' end of a DNA strand when the NLRT comprises the nucleobase adenosine and an azidomethyl reversible blocking group, but does not bind with high affinity to a NLRT incorporated at the 3' end of a DNA strand when the NLRT comprises the nucleobase adenosine but has a 3' hydroxyl group instead of an azidomethyl reversible blocking agent, and does not bind with high affinity to a NLRT incorporated at the 3' end of a DNA strand that comprises the nucleobase cytosine, guanine, or thymine (each with or without an azidomethyl reversible blocking group). The affinity agent may be directly or indirectly labeled.

[0165] "Specificity" is the degree to which an affinity reagent discriminates between different molecules (e.g., NLRTs), as measured, for example, by the relative binding affinity of the affinity reagent to the molecule. With respect to the affinity reagents of the present invention, the affinity reagent should have a significantly 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). In addition, when incorporated by a polymerase at the 3' end of a growing DNA chain, 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 chain. If there are multiple template polynucleotides (e.g., a template polynucleotide array), wherein the 3'-end of the GDS includes NLRT-A, NLRT-T, NLRT-C, NLRT-G (e.g., in an array), the affinity reagent is specific 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 not to the second structure or binds to the second structure more weakly (ie, with lower affinity) or less efficiently.

[0166] 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) as compared to an NLRT with a different nucleobase (NLRT-T, NLRT-C, or NLRT-G), a different capping group, or no capping 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 The measured value is less than 10 -6 M -1 Affinity of the value). Greater than 10 -8 M -1 Affinity of 100% 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 that bind to the target NLRT in a sequencing reaction. As discussed below, the specificity of binding can be determined by positive and negative binding assays.

[0167] The specific binding interaction between an affinity agent (e.g., an antibody) and an incorporated reversible terminator deoxyribonucleotide can be described in a variety of ways, including by reference to a portion or portion of the incorporated reversible terminator deoxyribonucleotide that is responsible for specificity. An analogy is useful here: Imagine a protein with two domains (domain 1 and domain 2). Two different antibodies can specifically bind to the 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 hypothesis, if a modification is made in domain 1, this may affect the binding of the first antibody to the protein without changing the binding of the second antibody. In this case, the binding of the first 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 change the binding properties of antibody 1 (e.g., eliminate binding, increase binding affinity, reduce 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 structural features of one moiety (e.g., a capping group) and not be affected by other moieties (e.g., a nucleobase) or by the structure of the other moieties. Alternatively, the specificity of binding may be due to structural features of multiple moieties (e.g., a nucleobase and a capping 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, having those structural features may be "responsible for" binding by the affinity agent, or the binding of the affinity agent may be "dependent on" the presence of a moiety having those structural features.

[0168] 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 both A and A', and thus may not be considered to "specifically bind" A. However, in a reaction or sample containing A, B, C, and D, the affinity reagent only binds 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 A and A' with different affinities or efficiencies, and thus binding to A and binding to A' can be distinguished based on this.

[0169] Another related term is "discriminating" (or sometimes "differentiating"). An affinity reagent that binds to an incorporated reversible terminator deoxyribonucleotide only when a particular capping group (e.g., an azidomethyl group) is present, but binds to an incorporated reversible terminator deoxyribonucleotide with an azidomethyl capping group regardless of what nucleobase is present, can be said to "discriminate" between incorporated reversible terminator deoxyribonucleotides with and without an azidomethyl capping group, or more broadly, can be said to "discriminate based on the capping group."

[0170] The specificity of an affinity reagent is a result of the process used to prepare the affinity reagent. For example, reagents that recognize an azidomethyl blocking moiety can be tested empirically 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 moiety. In one approach, an azidomethyl conjugated to keyhole limpet hemocyanin is used to raise antibodies against the hapten O-azidomethyl. The desired antibodies can be selected for binding to 3'-O-azidomethyl-2'-deoxyguanine, but against 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 against binding to other azidomethyl NLRTs (such as 3'-O-azidomethyl-2'-deoxyadenosine; 3'-O-azidomethyl-2'-deoxycytosine; and 3'-O-azidomethyl-2'-deoxythymine).

[0171] The nature of the antibody-hapten interaction can also be determined using methods known in the art, such as those described in the following references: 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 should be understood that describing an affinity agent as binding 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 a nucleobase and a sugar unit may also bind a capping group.

[0172] Affinity reagents can specifically recognize a nucleobase, a sugar (e.g., deoxyribose), a capping group, or any other part or combination thereof in a target NLRT. In one method, an affinity reagent recognizes an epitope comprising a capping group. In another method, an affinity reagent recognizes an epitope comprising a nucleobase. In another method, an affinity reagent recognizes an epitope comprising a nucleobase and a capping group. It should be understood that even if the affinity reagent is not in contact with a part, the part can determine the position of the other parts. For example, for an affinity reagent that discriminates NLRT based on a nucleobase and a 3' capping group, a deoxyribose part is required to position the nucleobase and the 3' capping group for recognition.

[0173] 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'-azidomethyl-dC are titrated continuously for 3'-O-azidomethyl-dC (positive specificity assay) and nucleotides such as 3'-O-azidomethyl-dG or -dA or 3'-OH-dC (negative specificity assay).

[0174] In some embodiments, the base-specific binding of the 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 the 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.

[0175] 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.

[0176] Affinity agents with the desired specificity can be selected using positive selection (i.e., binding to the target molecule) and negative selection (i.e., not binding to molecules that are not the target molecule). In the case where the affinity agent is a monoclonal antibody, a selection scheme is described below in the "Screening and Selection of Monoclonal Antibodies" section.

[0177] The affinity reagent can bind both dNTPs in solution and the corresponding nucleotides 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, in general, binding of the affinity reagent to unincorporated NLRTs does not occur during sequencing because the unincorporated NLRTs are removed (washed away) before the introduction of the affinity reagent. Alternatively, the complex formed by the affinity reagent bound to the NLRT is removed (washed away) before imaging.

[0178] In one approach, an affinity reagent specifically binds to a nucleobase and distinguishes 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 an incorporated nucleotide from within the GDS (not the 3' end). In some cases, an affinity reagent that recognizes a specific nucleobase also distinguishes the presence or absence of a 3'-OH group, thereby identifying an incorporated NLRT as having a 3' terminal nucleotide of a specific nucleobase.

[0179] In one approach, an affinity reagent recognizes an epitope containing a capping group but does not discriminate between bases. For example, given four RT capping 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 between these four capping groups. To illustrate, given the deoxyguanine analogs labeled A to D below, an affinity reagent can be selected that recognizes only one NLRT and not the other three NLRTs.

[0180] A. 3'-O-azidomethyl-2'-deoxyguanine

[0181] B. 3'-O-2-(Cyanoethoxy)methyl-2'-deoxyguanine

[0182] C. 3'-O-(2-nitrobenzyl)-2'-deoxyguanine

[0183] D. 3'-O-allyl-2'-deoxyguanine

[0184] In some embodiments, the selected affinity reagent does not discriminate between nucleotides with different nucleobases, as long as the nucleotides have the same capping 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.

[0185] Although the above examples describe embodiments in which the four nucleotides have different capping groups with very different structures (e.g., azidomethyl vs. 2-(cyanoethoxy)methyl), in some embodiments of the invention, there are only small differences between the capping groups bound by different affinity reagents. For example, in the capping group, the hydrogen atom can be replaced by a fluorine atom or a methyl group to generate three related capping groups that can be distinguished by the affinity reagent group [capping group, F-substituted capping group, methyl-substituted capping group].

[0186] In some embodiments of the invention, sequencing is performed using four NLRTs, each of which has a 3'-O-capping group, wherein two or more, alternatively three or more, alternatively all four of the capping groups are structurally similar in the following sense: (1) they have the same number of atoms or the number of atoms differs by no more than a small number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); (2) the molecular formulas of the capping group units differ by 1 to 10 atoms (e.g., there are 3 differences: a single H is replaced by a CH 3In some embodiments, the capping group unit may have any of the properties described in the section entitled "Properties of Reversible Terminator Capping Groups and Nucleotides Containing Them" above.

[0187] 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).

[0188] 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.

[0189] 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.

[0190] 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).

[0191] Exemplary Affinity Reagents

[0192] In some embodiments, the affinity agent is an antibody.Any method known in the art for producing antibodies can be used.

[0193] Antibody

[0194] As used herein, "antibody" refers to immunoglobulin molecules or compositions (eg, monoclonal and polyclonal antibodies), as well as genetically engineered forms, such as chimeric antibodies and other antibodies described herein.

[0195] The immunoglobulin G molecule is a tetramer with two heavy chains and two light chains. The heavy and light chains contain constant and variable regions (VH and VL). The VH and VL regions can be further subdivided into hypervariable regions (hypervariable regions (HVRs), also called complementarity determining regions (CDRs)), interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each VH and VL typically contains three CDRs and four FRs, which are arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. See, Kabat et al., (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.) The CDR sequences on the heavy chain (VH) can be designated as CDRH1,2,3, whereas the CDR sequences on the light chain (Vv) can be designated as CDRL1,2,3.

[0196] Antibodies can be derived from recombinant sources and / or produced 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, minibodies, nanobodies, diabodies, and multimers thereof, and bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab') can be generated by treating an antibody with pepsin. 2 The resulting F(ab') 2 The 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, dimers, miniantibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant technology. The antibody can be any useful isotype, including IgM and IgG, such as IgG1, IgG2, IgG3 and IgG4.

[0197] Antibodies can be chimeric antibodies, wherein a portion of the heavy chain and / or light chain is derived from a specific source or species, whereas the remainder of the heavy chain and / or light chain is derived from a different source or species. The CDR-grafted antibody comprises a CDR sequence from one source (e.g., rabbit) and framework residues from a different source (e.g., goat). For example, the CDR from rabbit IgG can be spliced ​​into a mouse antibody framework or scaffold. For illustration, an antibody can be a "humanized" form of a non-human antibody. A humanized antibody is a chimeric antibody comprising a minimal sequence derived from a non-human antibody. A humanized antibody is typically a human immunoglobulin (receptor antibody), wherein residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody with desired specificity, affinity, or biological effect. In some cases, the selected framework region residues of the receptor antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies may also contain residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further improve antibody function. For more details, see Jones et al., Nature, 1986, 321: 522-525; Riechmann et al., Nature, 1988, 332: 323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2: 593-596, each of which is incorporated by reference in its entirety. Humanized antibodies are primarily used for therapeutic purposes and have no unique value in sequencing. They are discussed here to illustrate the types of modifications they can make to antibodies. Similar chimeric antibodies can be prepared in which both the donor antibody and the recipient antibody are non-human.

[0198] In some embodiments, the affinity agent is a miniantibody. Other antibody binding moieties include "single-chain Fv" or "sFv" or "scFv" fragments comprising a VH domain and a VL domain in a single polypeptide chain. VH and VL are usually connected by a peptide linker. See, Pluckthun A. (1994). Antibodies from Escherichia coli in Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315), Springer-Verlag, New York, which is incorporated by reference in its entirety. In some embodiments, the linker can be a single amino acid. In some embodiments, the linker can be a chemical bond.

[0199] Miniantibodies are engineered single-chain antibody constructs, which are composed of variable heavy chains (VH) and variable light chains (VL) domains of natural antibodies fused to the hinge region and CH3 domains of immunoglobulin molecules. Therefore, miniantibodies are small versions of complete antibodies encoded in a single protein chain, which retain the antigen binding region, CH3 domain to allow assembly into divalent molecules and allow antibody hinges to adapt to dimerization by disulfide bonding. Single domain antibodies (sdAb) can also be used. Single domain antibodies or NANOBODY (Ablynx) are antibody fragments with single monomer variable antibody domains. See, Holt et al., Trends in Biotechnol., 2003, 21: 484-490, which are incorporated by reference in their entirety. Single domain antibodies selectively bind specific antigens, and are smaller than conventional antibodies (MW is 12-15kDa). Other antibody binding moieties include heavy chain antibodies. "Heavy chain antibody" refers to an antibody comprising at least two heavy chains and lacking a light chain. See, Harmesen et al., Applied Microbiology and Biotechnology, 77: 13-22, 2007; and Hamers-Casterman et al., Nature, 1993, 363: 446-448; each of which is incorporated by reference in its entirety. Other antibody binding portions include antibodies that are naturally free of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds different from those derived from antibodies. Single domain antibodies can be any antibody in the art, or any future single domain antibodies. Single domain antibodies can be derived from any species, including but not limited to mice, rats, guinea pigs, pigs, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. Single domain antibodies are described, for example, in International Application Publication WO 94 / 04678. Other antibody binding portions include single light chain antibodies provided in Masat et al., Proc. Natl. Acad. Sci. USA, 1994, 91: 893-896.

[0200] Other affinity reagents include "alternative scaffolds" such as those derived from fibronectin (e.g., Adnectins TM), β-sandwich (e.g., iMab), lipocalin (e.g., Anticalins), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamer, protein A (e.g., Affibody), ankyrin repeats (e.g., DARPins), γ-B-crystallin / ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), and (LDLR-A module) (e.g., Avimers). Additional information on alternative scaffolds is provided in Binz et al., Nat. Biotechnol., 2005 23: 1257-1268; and Skerra, Current Opin. in Biotech., 2007 18: 295-304, each of which is incorporated by reference in its entirety.

[0201] Antibody Fusion Affinity Reagents

[0202] In addition, fusions that directly link recombinant antibody fragments (e.g., single-chain Fv fragments (scFv)) to reporter proteins can be used (Skerrah 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). For example, Morino et al. (J. Immunol. Methods 257:175-184, 2001) described fusions of single-chain Fv-CL fragments fused to green fluorescent protein (GFP) or red fluorescent protein (RFP), and Luria et al. (mAbs 4:3, 373-384, 2012) described that full-length IgG antibodies fused to Superfolder GFP (SFGFP) and mCherry functioned in antigen binding while maintaining fluorescence intensity, and additionally linked multiple SFGFPs in series to each IgG, where the fluorescence intensity increased accordingly.

[0203] Antibody production

[0204] Methods for culturing polyclonal antibodies are known, and can be used to produce NLRT-specific antibodies. For one method, see Example 2 of WO 2018 / 129214. According to a method for culturing polyclonal antibodies specific to a specific NLRT (e.g., NLRT-A), rabbits are injected with NLRT-A (conjugated with an immunogen) to culture antibodies, and the antibodies are selected to not bind: lacking a capping group (e.g., having 3'-OH) and the same structure of other NLRTs (NLRT-T, NLRT-G, and NLRT-C). Therefore, the generated polyclonal antibodies 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 of the array. (The polyclonal antibody can also recognize unincorporated NLRT-A, but the unincorporated NLRT has been washed away before the incorporated NLRT is detected using a labeled affinity reagent).

[0205] It should be recognized that, depending on the needs of the researcher, it is not always necessary to raise antibodies against the entire NLRT. For example, if an antibody specific for a capping group is desired, the hapten can be a deoxyribose with a 3'-O-capping group (i.e., no nucleobase) or only a 3'-O-capping group. In some embodiments, antibodies are raised against the polynucleotide of the NLRT of interest at the 3' end. In some embodiments, antibodies are raised against the polynucleotide annealed to the template molecule.

[0206] For example, to produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from animals immunized with an immunogen comprising NLRT, and fused with myeloma cells by standard somatic cell fusion methods, thereby immortalizing these cells and harvesting hybridoma cells. These techniques are well known in the art (e.g., hybridoma technology originally 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 (Cole et al., 1986, Methods Enzymol, 121: 140-67) for producing human monoclonal antibodies, and screening of combinatorial antibody libraries (Huse et al., 1989, Science 246: 1275)). Hybridoma cells can be screened immunochemically to produce antibodies that specifically react with a particular RT, and monoclonal antibodies can be isolated. In vitro production of monoclonal antibodies can be performed using methods known in the art. See, for example, Li, N. et al., MAbs. 2, 466–477 (2010); Shukla, A. & J. Trends in Biotechnology. 28,253–261(2010).

[0207] 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, which 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).

[0208] In addition, antibodies specific to the target NLRT are easily isolated by screening antibody phage display libraries. For example, antibody phage libraries are optionally screened by using to identify antibody fragments specific to the target NLRT. Methods for screening antibody phage libraries are well known in the art.

[0209] Anti-NLRT antibodies can also be produced in a cell-free system. 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.

[0210] Antibody purification:

[0211] Anti-NLRT antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices and / or column chromatography (e.g., affinity chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and ion exchange chromatography). In one approach, affinity purification using Ig binding proteins (e.g., protein A, protein G, protein A / G, and protein L) can be immobilized on a resin and used to purify the antibody of interest.

[0212] Screening and selection of monoclonal antibodies

[0213] A person of ordinary skill guided by the present disclosure will be able to generate polyclonal antisera for any desired NLRT (e.g., NLRT-A, NLRT-T, NLRT-C, NLRT-G) having a blocking moiety at the 3'-OH of the deoxyribose. In one method, as described in Example 1 and US 2018 / 0223358, a test animal (e.g., rabbit) is immunized with a KLH antigen every two weeks for 3 months. Serum is collected one week after immunization and tested by ELISA for target (e.g., NLRT-A) and non-target (e.g., NLRT-T, NLRT-C, NLRT-G) antigens to determine the antibody response. Splenocytes are obtained from animals that produce good specific responses. Splenocytes are tested for binding to the target NLRT-A. For example, biotinylated NLRT-A can be used to detect with fluorescent streptavidin and spleen cells can be sorted by FACS to create culture colony plates from single cells. Nucleotide analogs with zero or one phosphate can be used in the immunization and / or FACS sorting steps.

[0214] The supernatants from these single cell cultures are tested for these target and non-target antigens by ELISA. In one method, the supernatant is tested for the target and non-target BSA-NLRT bound to the wells of the ELISA plate (see, Figure 9 of WO 2018 / 129214). In the second method, positive and negative ELISA screening is performed, wherein the ELISA target antigen is a complex comprising a fixed template DNA, which is hybridized with an extension primer and a target (for positive screening) or non-target (for negative screening) NLRT is incorporated at the 3' end of the extension primer. The complex is a partial duplex, so the template chain extends beyond the 3' primer end, thereby simulating the DNA structure generated in sequencing. In one method, a complex for screening is formed by fixing a 3' biotinylated DNA template on a streptavidin-coated surface (e.g., a well of an ELISA plate), hybridizing primers, and incorporating NLRT. By using different primers in the incorporation step, different nucleotide specificities can be screened using the same template. In a related approach, a hairpin oligonucleotide (biotinylated in the loop for fluorescent streptavidin detection) incorporates a reversible terminator into the duplex of the hairpin at the 3' end and is used in a binding assay. In another approach, a biotinylated primer hybridized to the template can be used to add a 3'-NLRT. The template can be removed (e.g., by denaturation) and the primer captured on streptavidin. The resulting structure can be used in screening to mimic partially denatured DNA ends.

[0215] Select high-performance splenocyte clones and clone and express antibodies using IgG coding sequences. One method is to clone the sequence into a linear expression module (LEM) for transfection into a HEK cell line (HEK cell), and then clone the productive LEM into a plasmid to transfect and produce purified antibodies. The selected antibody can be further changed, for example, to improve affinity to the target, for example, by affinity maturation. See, Marks et al. (Bio / Technology, 1992, 10: 779-783), which describes affinity maturation by mixing VH and VL domains. See also, Barbas et al., (Proc. Nat. Acad. Sci. USA., 1994, 91: 3809-3813 (describing random mutagenesis of CDR and / or framework residues).

[0216] Exemplary Monoclonal Antibodies

[0217] Exemplary rabbit anti-NLRT antibodies were generated as described in the Examples. A number of monoclonal antibodies that specifically bind to the target NLRT are discussed herein, including, but not limited to, monoclonal antibodies specific for 3'-azidomethyl dA (N3A): mAb2C5, 3B12, 17H7, and 18B7; and monoclonal antibodies specific for 3'-azidomethyl dC (N3C): mAbs 1B8, 2B9, 4C8, 1A10, and 3B7; monoclonal antibodies specific for 3'-azidomethyl dG (N3G): mAbs 3G6, 5F6, 4B8, 4G8, and 7C8; and monoclonal antibodies specific for 3'-azidomethyl dT (N3T): mAbs 2D4, 2D10, 1F9, and 3B7. The amino acid sequences of the heavy and light chains (including the signal peptide) are as follows: Figure 1A -H and are provided in Table 2 below.

[0218] Table 2

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] Obviously, the antibody chain name can be read as N3X_ABC_Y, where X is a nuclear base specificity (e.g., A, T, G or C), ABC is the antibody name, and Y represents a heavy (H) or light (L) chain sequence. It should also be recognized that heavy and light chains with a common name (ABC) can be produced as heterodimers (HL) or HV dimers optionally combined with antibody constant regions. Antibody chain sequences 1-36 include signal peptides. It will be recognized that mature antibodies will not include signal peptide sequences.

[0228] Affinity reagents can be selected from the antibodies disclosed above or derivatives of such antibodies. In some cases, mAbs 18B7 (A), 4G8 (C), 7C8 (G) and 2D10 (T) are used. All other combinations or subcombinations with appropriate specificity combinations can be used. Typically, mAbs specific for A, T, G and C are used together. However, other combinations can be used; for example, in some methods, only three affinity reagents or only three labeled affinity reagents are used, and one affinity reagent is omitted (therefore, there is no signal recognizing the 3' terminal base).

[0229] Other useful affinity reagents include antibodies (or other affinity reagents) that compete with an affinity reagent selected from mAb 2C5, 3B12, 17H7, 18B7, 1B8, 2B9, 4C8, 1A10, 3B7, 3G6, 5F6, 4B8, 7C8, 2D4, 2D10, 1F9, 3B7, and 4G8 for binding to a target structure. In this context, a "target structure" refers to a 3' biotinylated DNA template on a streptavidin-coated surface (e.g., a well of an ELISA plate) that hybridizes to a primer that incorporates a NLRT nucleotide at the end, as discussed above in the context of antibody screening. Competition assays can be used to identify antibody pairs that bind to the same epitope (or to epitopes that overlap or are close together). Thus, when used herein in the context of two or more affinity reagents, the term "compete with..." means that two or more affinity reagents compete for binding to a target antigen. Competitive binding assays are well known (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). In an exemplary assay, one of the "reference mAbs" (mAb 2C5, 3B12, 17H7, 18B7, 1B8, 2B9, 4C8, 1A10, 3B7, 3G6, 5F6, 4B8, 7C8, 2D4, 2D10, 1F9, 3B7, or 4G8) is allowed to bind to the target antigen (e.g., in an ELISA format or sequencing array), and a candidate affinity agent is added to the target antigen. If the presence of the candidate reduces the binding of the reference mAb, the candidate affinity agent will compete with the reference mAb. In some embodiments, the presence of the candidate will reduce the binding of an equimolar amount of the reference mAb to no more than 50% of the binding in the absence of the candidate (i.e., the candidate reduces the reference binding by half). In some cases, the candidate inhibits binding to the reference mAb by at least 50%, sometimes by at least 75% or by at least 90%. In another competitive assay, the reference mAb is immobilized on a matrix and various concentrations of the candidate and soluble target antigen are added to detect and measure competition. In this case, the soluble antigen can be a hairpin oligonucleotide (biotinylated in the loop portion for detection of fluorescent streptavidin), with a reversible terminator incorporated into the duplex of the hairpin at the 3' end as described above. Therefore, in one aspect of the invention, sequencing is determined as described herein, wherein at least one affinity reagent is an affinity reagent (e.g., an antibody) that competes with one of mAbs 2C5, 3B12, 17H7, 18B7, 1B8, 2B9, 4C8, 1A10, 3B7, 3G6, 5F6, 4B8, 7C8, 2D4, 2D10, 1F9, 3B7 or 4G8. In some embodiments, at least three or at least four affinity agents compete with one of the mAbs.

[0230] In some embodiments, the affinity agent is an antibody or antigen-binding portion thereof comprising a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprising an amino acid sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 (optionally excluding the signal peptide, e.g., the amino-terminal about 19 amino acids), the light chain variable region comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: An amino acid sequence that is at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any of NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 (optionally excluding the signal peptide, e.g., the amino-terminal about 22 amino acids).

[0231] Exemplary CDR sequences

[0232] As mentioned above, antibody variants can be prepared using CDR sequences from donor antibodies of known specificity. For example, monoclonal antibody sequences can be used to produce chimeric or CDR-grafted antibodies, for example, by combining the variable region of an antibody with the constant region of another antibody, or by inserting the complementary determining region (CDR) fragment of the donor antibody into the acceptor antibody scaffold by recombinant DNA technology (reviewed in Almagro and Fransson, Frontiers in Bioscience 13, 1619-1633, 2008), while retaining the specificity of the original monoclonal antibody.

[0233] The amino acid sequence boundaries of the CDRs can be determined by one skilled in the art using any of a number of known numbering schemes, including those described by Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0234] Table 3 below provides the CDR sequences of the heavy and light chains of the antibodies listed in Table 2. As described above, the CDRs confer antigen specificity and binding affinity to the antibody, and these CDR sequences can be incorporated into chimeric humanized antibodies, single chain antibodies, nanobodies, and other antibodies described above.

[0235] The skilled person will recognize that Table 3 identifies three CDR sequences for each of 18 light chains and 18 heavy chains, corresponding to 18 four-chain antibodies comprising a combination of 2 light variable regions and 2 heavy variable regions (VH-VL dimers). Each combination of heavy and light chains from the same mAb can be referred to as a "homologous set". The present invention encompasses related affinity reagents (e.g., single-chain antibodies) comprising one or more CDR sequences in Table 3. In particular, the present invention encompasses affinity reagents comprising three corresponding CDRs from heavy or light chains in Table 3. Each group of three CDRs from the same IgG chain is referred to as a "corresponding set". In addition, the present invention encompasses affinity reagents comprising six CDR sequences from 18 listed antibodies. In addition, the present invention includes the use of such affinity reagents in sequencing methods described herein. In one aspect, the present invention includes the use of a combination of 3 or 4 affinity reagents, each of which includes CDR sequences that confer specificity to different nucleotide analogs (i.e., A, T, G, or C).

[0236] In one aspect, the invention includes an affinity reagent (e.g., an antibody or an antigen-binding portion thereof) comprising: a heavy chain variable region comprising a corresponding set of CDRs, including (i) VH CDR1, (ii) VH CDR2, (iii) VH CDR3. For example, a heavy chain variable region having CDRs comprising SEQ ID Nos: 37, 74, and 80.

[0237] In one aspect, the invention includes an affinity reagent (e.g., an antibody or antigen-binding portion thereof) comprising: a light chain variable region comprising a corresponding set of CDRs, including (i) VL CDL1; (ii) VL CDL2; (iii) VL CDL3. For example, a light chain variable region having CDRs comprising SEQ ID Nos: 85, 90, and 95.

[0238] In one aspect, the invention includes an affinity reagent comprising a heavy chain variable region, the heavy chain variable region including a corresponding CDR set, which includes (e.g., an antibody or an antigen-binding portion thereof), including: a light chain variable region, the light chain variable region including a corresponding CDR set, including (i) VL CDL1; (ii) VL CDL2; (iii) VL CDL3. For example, a light chain variable region having CDRs comprising SEQ ID Nos: 85, 90, and 95.

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] Labeled affinity reagent

[0247] Fluorescent detectable label

[0248] Affinity reagents for practicing 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 300nm to about 1,000nm or at least about 5,000, more preferably at least about 10,000, and most preferably at least about 50,000cm-1M-1, and have 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 about 0.1 to about 1. A labeling strategy for labeling affinity reagents adapted to multiple dye molecules is described below.

[0249] There is a wealth of practical guidance in the literature for selecting an appropriate detectable label to attach to an affinity reagent, 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 that provide lists of fluorescent molecules and their associated 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 portions thereof, as demonstrated in Ullman et al., U.S. Pat. No. 3,996,345; Khanna et al., U.S. Pat. No. 4,351,760; etc. Each of the above publications is incorporated herein by reference in its entirety for all purposes.

[0250] Exemplary fluorescent dyes include, but are not limited to, acridine dyes, cyanine dyes, fluorescent dyes, oxazine dyes, phenanthridine dyes, and rhodamine dyes. Exemplary fluorescent dyes include, but are not limited to, fluorescein, FITC, Texas Red, ROX, Cy3, AlexaFluor dyes (e.g., Alexa Fluor 647 or 488), ATTO dyes (e.g., ATTO 532 or 655), and Cy5. Exemplary fluorescent dyes may also include dyes used in or compatible with two-channel or four-channel SBS chemicals and workflows. Exemplary labeling molecules may be selected from xanthene dyes (including fluorescein) and rhodamine dyes. Many suitable forms of these compounds are generally commercially available, with substituents on their phenyl moieties, which can be used as sites connected to affinity reagents. Another group of fluorescent compounds is naphthylamine, which has an amino group in the α or β position. 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. In some embodiments, the label is selected from fluorescein and rhodamine dyes. These dyes and appropriate attachment methods 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 5,188,934; Menchen et al., European Patent Application 87310256.0; and Bergot et al., International Application 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, CAL Fluor Orange 560, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 615, CALFluor Red 635 and Texas Red (Molecular Probes).

[0251] 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, e.g., 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).

[0252] Enzyme-labeled affinity reagents

[0253] In one method, an affinity reagent (e.g., an antibody or an affinity) is enzymatically 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 method, an affinity reagent (e.g., an antibody or an affinity) is directly enzymatically labeled. In one method, 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 method, an affinity reagent is coupled to luciferase or other proteins that can be used to generate chemiluminescent signals (or are part of their fusion proteins). In another approach, the affinity reagent can be coupled / fused to an enzyme system selected to produce a non-optical signal, such as a pH change in a proton that can be detected (e.g., 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 resulting from signal amplification and the ability to customize sequencing methods for a variety of instruments. Enzyme reporter systems are reviewed in Rashidian et al., Bioconjugate Chem. 24: 1277–1294, 2013.

[0254] Indirect and direct detection methods

[0255] 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, knottin) binds to the primary affinity reagent. In some methods, the primary and / or secondary affinity reagent is an antibody. For example, in one method, 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 methods, the use of a secondary affinity reagent provides advantageous signal amplification.

[0256] In the case of indirect detection, the assay includes two distinct parts: first, an incubation period (usually one hour) of unlabeled primary antibody during which the antibody binds to the antigen (assuming, of course, that the antigen is present). Excess unbound primary antibody is then washed off and a labeled secondary reagent is added. After a period of incubation (another hour), excess secondary reagent is 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 in an increase in the amount of light emitted at a specific wavelength. In the absence of antigen, there is no binding of the primary antibody, and there is no binding of the secondary reagent, so there is no signal. With direct detection, the prior 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.

[0257] Secondary antibody specificity

[0258] 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 (usually 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) antibodies for each antigen are 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.

[0259] Pre-combined primary and secondary antibodies

[0260] The primary antibody and secondary antibody or other agents can be added to the sequencing array sequentially, and can be pre-combined under conditions where the secondary antibody binds to the primary antibody and is added to the array as a complex.

[0261] Methods for labeling antibodies and other affinity reagents

[0262] Labeled affinity reagents can be used to sequence template nucleic acids by a variety of methods. Any method of labeling antibodies and other affinity reagents of the present invention can be used. 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, 49). thed.; Elsevier: Amsterdam, the Netherlands, 2013; Kobayashi and Oyama, Analyst 136:642-651, 2011). Enzymes, biotin, fluorophores, and radioisotopes are commonly used to provide detection signals in biological assays and can be linked or conjugated to affinity reagents such as antibodies.

[0263] Most antibody labeling strategies use one of the following three targets: (1) Primary amines (-NH2): They are present on lysine residues and the N-terminus of each polypeptide chain. They are numerous and distributed throughout the antibody; (2) Sulfhydryl groups (-SH): They are present on cysteine ​​residues and in disulfide bonds that stabilize the overall molecular structure. Hinge region 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 cis-diol-containing polysaccharide units can be oxidized to produce reactive aldehydes (-CHO) for conjugation. The four main chemical methods for antibody labeling are summarized below:

[0264] 1. NHS esters. In the case of fluorescent dye labels, labels are usually purchased in activated form with built-in NHS esters (also called "succinimidyl esters"). 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.

[0265] 2. Heterobifunctional reagents. If the label is a protein molecule [e.g., 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 of the lysines on one molecule (e.g., the antibody) to create a new reactive group (X) and a lysine on the label to create another reactive group (Y). 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.

[0266] 3. Carbodiimides. These reagents (EDC is a very common example) are used to create covalent linkages between amine-containing molecules and carboxyl-containing molecules. Carbodiimides activate 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).

[0267] 4. Sodium periodate. This chemical cannot be used with the vast majority of 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 can react with the lysines on the antibody molecule. Since HRP itself has very few lysines, it is relatively easy to produce antibody-HRP conjugates without significant HRP aggregation.

[0268] For 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 causes 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.

[0269] Random Marking

[0270] In one method, antibodies are specifically labeled with a limited number of dye molecules (e.g., 1, 2, 3, 4 or 5 dye molecules per antibody) (e.g., at a specific site on the antibody). In another method, antibodies are randomly labeled, for example, by reacting the free amines available on the protein with fluorescent dyes activated by NHS esters (Mattson et al., A practical approach to crosslinking. Mol. Biol. Rep. 17, 167–183 (1993), which is incorporated herein by reference). Random labeling processes can be used to produce antibodies labeled with multiple dye molecules per antibody. Similarly, specific labeling methods can be used to produce antibodies labeled with multiple dye molecules per antibody. If each antibody has multiple dye molecules, the dyes on a given antibody protein (e.g., tetramers) can be the same or different (e.g., two different dyes). Therefore, in one method, antibodies in an antibody group (wherein the antibody group includes antibodies with the same nucleobase specificity, such as nucleobase-specific monoclonal antibodies) are labeled with 2 or more dye molecules (e.g., two fluorescein molecules) of the same dye. In one approach, antibodies in a panel are labeled with two or more non-identical dye molecules (eg, one fluorescein molecule and one rhodamine molecule).

[0271] Labeling without dye purification to remove free dye

[0272] In one method, antibodies are labeled by the reaction of available free amines on the antibody protein with NHS ester-activated fluorescent dyes. For example, the NHS ester-activated fluorophore is diluted in anhydrous DMSO and reacted with a concentration (10-100uM). The antibody at a relatively low concentration is adjusted to pH 8 in a bicarbonate buffer and reacted with an NHS ester dye. The antibody concentration at this stage can be about (1mg / ml), or in various embodiments, can be, for example, 0.1 to 0.5mg / ml, 0.5 to 5mg / ml, 0.3-1mg / ml or 0.3 to 2mg / ml. Incubate at room temperature for 45 minutes. Optionally, unreacted dye is quenched in tris-buffered saline (pH7.4). This labeling method can provide a strong signal without adversely affecting antibody binding or specificity.

[0273] In order to bind the antibody in the final sequencing reaction, the labeled antibody composition is diluted (usually 30-300 times, such as greater than 50 times, often greater than 100 times, and sometimes greater than 500 times). In the final sequencing reaction mixture incubated on the nucleic acid array, an excess of antibody can be used, such as a concentration of about 1 to about 10 ug / ml, which results in a final dye concentration of about 0.2 uM in the antibody binding reaction compared to the commonly used concentration of more than 1 uM of the labeled nucleotides of highly purified base labels.

[0274] Surprisingly, we found that these labeled antibodies can be stored at -20°C and used without purifying free unreacted dye from the antibodies, and surprisingly, we found that it is not necessary to remove free unreacted dye from the labeled antibody preparations prior to use in sequencing reactions.

[0275] In one aspect, the present invention provides a composition comprising an anti-NLRT antibody labeled with a fluorescent dye and a free (i.e., not conjugated to a protein) dye, wherein the composition comprises greater than 10 nanomoles of free dye per 1 mg of antibody, typically greater than 20 nanomoles of free dye, typically greater than 50 nanomoles of free dye per 1 mg of antibody, wherein the antibody is typically labeled with an average of more than one dye molecule. In one embodiment, the dye is an NHS ester-activated fluorophore. After optional "quenching" of unused dye molecules, the labeled antibodies can be stored at -4°C or -20°C or -80°C, even in the absence of glycerol. Four labeled antibodies can be mixed and stored, or stored in a concentration range of 1ug / ml to 10ug / ul.

[0276] Thus, one aspect of the invention includes: (1) labeling an affinity reagent (e.g., a protein, such as an antibody) with a dye (e.g., a fluorescent dye, such as an NHS ester-activated fluorescent dye) to produce a composition comprising a labeled affinity reagent and unreacted dye; (2) using the composition in affinity reagent-based sequencing as described herein without removing unreacted dye molecules (without purification). Affinity reagent (antibody)-based synthetic sequencing is performed using NLRT, wherein a base-specific labeled antibody is used in a binding reaction in the presence of a non-incorporated dye at a concentration greater than 10 nanomolar / mg labeled antibody protein, sometimes greater than 20 nanomolar, and sometimes greater than 50 nanomolar.

[0277] Sequencing system

[0278] Array-based sequencing

[0279] Various SBS methods can be used with NLRT and the antibodies of the present application, such as disclosed in PCT patent publications WO1999 / 019341; WO2005 / 082098; WO 2006 / 073504; WO 2018 / 129214; and Shendure et al., 2005, Science, 309: 1728-1739. The SBS method can employ an ordered array of DNA nanospheres, which is described, for example, in U.S. Patent Publications 2010 / 0105052, 2007 / 099208, and US 2009 / 0264299 and PCT Patent Publications WO 2007 / 120208, WO 2006 / 073504, and WO 2007 / 133831, which are incorporated by reference in their entirety for all purposes.

[0280] In some embodiments, the nucleic acid template is fixed on a solid surface (e.g., silicon, glass, gold, polymer, PDMS, beads), typically in a wall. In some embodiments, the nucleic acid template is fixed or contained in a droplet (optionally fixed on beads or other substrates in the droplet). Typically, an array (sometimes referred to as an array chi) is contained in a flow cell, which is a fluid device that transports a reagent solution to an array template. Typically, the reagent solution is transported to a reaction chamber formed between the array surface and a cover glass. Referring to U.S. Patent Publication 2013 / 0281305, which is incorporated by reference.

[0281] 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 represented by a DNA concatemer comprising multiple copies of a target sequence and an "adapter sequence", such as a DNA nanoball (DNB). In some embodiments, the DNA template is a DNA concatemer, and there is a single concatemer at each position. See, PCT Patent Publication WO 2007 / 133831, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0282] In some embodiments, the nucleic acid template at each position of the array is a clonal population of DNA fragments. In some embodiments, the clonal population of DNA fragments is produced by bridge PCR. In some embodiments, the template is a single polynucleotide molecule. In some embodiments, the template exists as a clonal population of template molecules (e.g., a clonal population produced by bridge amplification or Wildfire amplification).

[0283] Suitable template nucleic acids including DNBs, clusters, polony, and arrays or groups thereof are further described in U.S. Patents 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.

[0284] In one aspect, the present invention provides a DNA array comprising: a plurality of template DNA molecules, each template DNA molecule attached at one 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 method, the DNA array comprises a primer extension product 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 product.

[0285] The method for detecting the combination of antibodies and incorporated RT will vary with the nature of the detectable label used. Many methods are known in the art and are commercially available. For fluorescent labels, one method is to pass a laser through an array to activate the fluorescent label. Use a camera (e.g., a camera based on CCD or CMOS) to detect fluorescence and record it on a computer, for example, as a collection of tiled fluorescence or luminescent images recorded after each iterative sequencing step. (Or as described below, collect more than once in each complete cycle. Different dyes emit light with different wavelengths (or different colors) and intensities). In one method, each color generates a separate image (acquiring signals of different wavelengths), and then the images are compared. Use various techniques and algorithms that can be executed on one or more computer systems. A variety of methods known in the art can be used to distinguish dyes of different colors. A common method is to use multiple lasers to activate dyes and / or filters with different excitation wavelengths to capture light of different wavelengths. Such filters and methods generally capture light on a wavelength spectrum that can be referred to as "color" (e.g., red or green), "band" or "detection channel". In one method, each channel produces a different image so that images can be compared to determine the nature of the signal at each array position. Commercially available sequencers can be tuned to 1, 2, or 4 colors based on the presence or absence of filters, illumination sources, and software.

[0286] One color sequencing is particularly suitable for methods in which chemiluminescent (rather than fluorescent) labels are used or non-luminescent labels are used, and affinity reagents labeled with chemiluminescent dyes and surrogate labels can be used in the methods disclosed herein.

[0287] Removal of blocking groups and removal of affinity reagents

[0288] The blocking groups and affinity reagents may be removed simultaneously or may be uncoupled or occur at different times. In one approach, the array is exposed to conditions that simultaneously remove the 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 the affinity reagents (e.g., high salt, small molecule competitors, proteases, etc.) are combined with reagents that cleave the blocking groups.

[0289] In some cases, removal of the 3' blocking group results in removal of the affinity agent. Without wishing to be bound by a particular mechanism, it is believed that in these cases, removal of the blocking unit destroys the desired epitope to which the antibody or other affinity agent binds.

[0290] In a different approach, the removal of the affinity reagent and the capping group are uncoupled, thereby removing the affinity reagent but not cleaving the capping group from the nucleotide sugar. In one aspect of the invention, SBS is performed on a DNA array using NLRT, wherein the base-specific labeled antibody is removed after imaging but before removing the capping group. The antibody is typically removed at elevated temperatures (greater than 50° C., sometimes greater than 60° C.), and removal is substantially complete within 40 seconds after the removal conditions are introduced (some of which are discussed below).

[0291] It will be appreciated that removal conditions, ie, removal conditions for the affinity reagent and / or the blocking group, will be selected to preserve the integrity of the DNA being sequenced.

[0292] Removal of blocking groups

[0293] Nucleoside analogs or NLRTs include those that are 3'-O reversibly blocked. In some aspects, the capping 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).

[0294] Typically, in each sequencing cycle using a NLRT, the capping group is removed and the affinity reagent is dissociated from the NLRT. These steps can be performed simultaneously. For example, the azidomethyl capping 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 the affinity reagent (e.g., a phosphine contained in a high salt buffer). In some embodiments, if, for example, the capping group is required for affinity reagent binding, the removal of the capping group results in the dissociation of the affinity reagent.

[0295] 3'-0 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, the blocking group is removed by contacting the nucleoside of reversible blocking with a reducing agent, such as dithiothreitol (DTT) or a phosphine reagent [such as tris (2-carboxyethyl) phosphine (TCEP), tris (hydroxymethyl) phosphine (THP) or tris (hydroxypropyl) phosphine]. In some cases, the blocking group is removed by washing out the blocking group from the nucleotide analogs incorporated using a reducing agent (e.g., a phosphine reagent). In some cases, the blocking group is photolabile, and the blocking group can be removed by applying, for example, UV light. In some cases, the blocking group can be removed by contacting the nucleoside analog with a transition metal catalyzed reaction using, for example, a palladium (Pd) aqueous solution. In some cases, the blocking group can be removed by contacting the nucleoside analog with a nitrite aqueous solution. Additionally, or alternatively, the blocking group can be removed by changing the pH of a solution or mixture containing the incorporated nucleotide analog. For example, in some cases, the blocking group can be removed by contacting the nucleoside analog with an acid or low pH (e.g., less than 4) buffered aqueous solution. As another example, in some cases, the blocking group can be removed by contacting the nucleoside analog with a base or a high pH (e.g., greater than 10) buffered aqueous solution.

[0296] 3'-O reversible capping groups that can be cleaved by a reducing agent (e.g., phosphine) include, but are not limited to, azidomethyl. 3'-O reversible capping groups that can be cleaved by UV light include, but are not limited to, nitrobenzyl. 3'-O reversible capping groups that can be cleaved by contact with a Pd aqueous solution include, but are not limited to, allyl. 3'-O reversible capping groups that can be cleaved by acid include, but are not limited to, methoxymethyl. 3'-O reversible capping groups that can be cleaved by contact with a sodium nitrite buffer (pH 5.5) aqueous solution include, but are not limited to, aminoalkoxy.

[0297] Removal of affinity reagents

[0298] Antibody-based affinity reagents can be removed by low pH, high pH, ​​high 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. In addition, 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.

[0299] In one approach, high temperature (e.g., 50-60°C or 55C-65°C or 60-70°C) or a combination of high temperature (e.g., 60-65°C) and high pH (8.5-9.5) can also be used to quantitatively remove antibodies in less than 30s or less than 20s or less than 10s. Rapid complete or near complete removal of antibodies without cleavage of the 3' blocking group allows for i) optimal cleavage conditions or ii) rapid sequential binding / detection / removal of each antibody or two antibodies at a time.

[0300] As described above, the affinity agent can also be removed by destroying the ability of the agent to bind to the incorporated NLRT. This typically occurs when the 3' blocking group is cleaved from the incorporated nucleotide analog. In cases where the affinity agent binding is dependent on the presence of the blocking group (e.g., where the epitope recognized by the 1 antibody includes the blocking group or a portion thereof), removal of the blocking group also results in release of the affinity agent.

[0301] Simultaneous removal of the affinity agent and the blocking group can also be achieved by adding a solution containing a blocking group cleavage component (eg, a phosphine reagent) and an affinity agent releasing agent (eg, a high salt).

[0302] Simultaneous second incorporation and antibody removal

[0303] In one aspect of the invention, SBS is used to incorporate NLRT into a growing primer chain ("first incorporation"), and an affinity reagent (e.g., a monoclonal antibody) is used to detect the incorporation. In one method, after detection, the affinity reagent is removed, and the reversible blocking group is removed ("deblocking"). In one method, after detection and before deblocking, a second NLRT incorporation step is performed while or after the affinity reagent is removed. The second incorporation solves the problem of non-simultaneity (heterophase incorporation). SBS is typically performed using a large clonal population of templates at a certain position on an array. Exemplary large clonal populations of templates include DNBs and template clusters (which can be generated by bridge PCR or similar methods). In some cases, for certain DNA template copies at a certain position on the array, the DNA polymerase may not be able to incorporate the complementary RT into the GDS, and therefore, the sequencing reactions on a large number of DNA templates on the DNA array may be incomplete or non-simultaneous (heterophase). That is, not all primers hybridized to all templates are extended with the same efficiency, and this difference increases with the increase in the number of cycles, resulting in lower quality sequencing data. When there is complementarity between the RT and the bases on the DNA template, the second incorporation step provides a second opportunity for the DNA polymerase to incorporate the RT in each sequencing cycle, thereby increasing the proportion of templates that are extended in each sequencing cycle. Antibody removal and second incorporation can be performed simultaneously under the same conditions. This eliminates the need to take steps to change conditions to accommodate two different types of reactions and significantly reduces costs and cycle times.

[0304] In one approach, after detecting a signal from a labeled extension product and prior to deblocking, the DNA array (and the labeled extension products immobilized thereon) is subjected to dissociation conditions under which: (1) the labeled affinity reagent is dissociated from the extension product and (2) further incorporation of the NLRT is performed at any template position that did not incorporate a blocked nucleotide in the first incorporation step ("second incorporation"). The second incorporation comprises adding additional polymerase and, optionally, additional NLRT under antibody dissociation conditions. The addition of the polymerase and NLRT and the removal of the affinity reagent can occur simultaneously (i.e., both under dissociation conditions). Alternatively, the affinity reagent can be removed or partially removed under dissociation conditions and subsequently the polymerase / / NLRT added under the same or similar dissociation conditions, typically without an intermediate wash step (to remove the dissociated affinity reagent). The careful reader will recognize that the second incorporation step is performed under dissociation conditions.

[0305] After the second incorporation, the capping groups of both the NLRT incorporated by the first incorporation and the NLRT incorporated by the second incorporation are removed, allowing the next extension cycle of the growing DNA copy strand and identification of the subsequently incorporated NLRT.

[0306] The sequencing methods disclosed herein use reagents that allow the second incorporation to be performed under the same conditions as the antibody dissociation step. These conditions result in the separation of the labeled antibodies from their target RTs on the array, but are still suitable for the polymerase to correctly perform the polymerization reaction (eg, the second incorporation).

[0307] As used herein, "first incorporation" or "second incorporation" refers to the incorporation of RT at the 3' end of a nucleic acid primer or a growing DNA strand. RT incorporated by the first incorporation will be identified by antibody binding, whereas RT incorporated by the second incorporation will not be subject to antibody binding. The second incorporation occurs after the first incorporation, antibody binding, and detection.

[0308] The NLRT used in the first incorporation step and the second incorporation step typically have the same capping group. However, different capping groups may be used. When multiple different capping groups are used, preferably all groups can be cleaved with compatible cleaving agents under the same conditions, such as at a common temperature, pH and salt concentration.

[0309] In one method, one cycle of a sequencing reaction includes the following steps: i) forming an unlabeled extension product by incorporating RT at the 3' end of a nucleic acid primer or a growing DNA copy strand hybridized to a plurality of DNA templates on an array ("first incorporation"), ii) forming a labeled extension product by binding a labeled affinity reagent (e.g., an antibody) to the extension product, iii) detecting the labeled extension product, iv) removing the bound labeled antibody and incorporating an additional amount of RT ("second incorporation") under conditions that allow both processes to occur simultaneously (simultaneously or under the same conditions). After removing the blocking group, these steps can be repeated to perform additional cycles of the sequencing reaction.

[0310] First incorporation

[0311] As described above, the first incorporation step involves the extension of a nucleic acid primer hybridized to a template nucleic acid on a DNA array or the extension of a primer extension product produced in an earlier sequencing cycle. The reaction includes a DNA polymerase, NLRT, and a buffer suitable for primer extension. In one method, the NLRT used in the method disclosed herein is a mixture of A, G, C, and T (i.e., NLRT-A, NLRT-G, NLRT-C, and NLRT-T). Alternatively, each NLRT or a combination of NLRTs can be individually incorporated into a separate step (e.g., in certain two-color schemes described herein). In each cycle, the NLRT is incorporated into the growing DNA chain of one of the template DNA molecules to form an unlabeled extension product. In some cases, after an incorporation, the unincorporated NLRT is washed off and removed from the sequencing reaction.

[0312] Antibody Binding and Detection

[0313] The unlabeled extension product formed by the first incorporation is then combined with a labeled antibody. Each antibody used in the method can specifically bind to a nucleobase (e.g., A) and distinguish the nucleobase from other nucleobases (e.g., T, C, and G) that are not combined at all or ineffectively combined. For example, if the 3' terminal nucleotide is recognized by a specific antibody of a guanosine nucleobase (e.g., a guanosine nucleotide blocked by 3'-OH is incorporated into the growing chain of the template primer duplex), it indicates that the related nucleobase is guanine and the template base at this position is cytosine. The labeled antibody is combined with the unlabeled extension product to form a labeled extension product, and then the labeled extension product is detected using methods known in the art. Optionally, the unbound labeled antibody is washed off before the detection step.

[0314] The binding of the antibody to the RT incorporated into the growing DNA chain is generally carried out under conditions suitable for antibody-antigen interactions ("binding conditions"). For example, in some embodiments, the binding occurs at a temperature in the range of 30 to 45°C or 35-50°C. In some embodiments, the binding occurs in an environment with a pH value of 7 to 8.5, typically 7 to 7.5. In some embodiments, the binding is carried out and the binding conditions include a temperature in the range of 30-45°C and / or an environment with a pH range of 7 to 7.5. Under certain conditions on the DNB array, a low salt (e.g., 30-70 mM) Tris buffer ( 1-20 mM) promoted binding, suggesting that the composition of a good binding reaction achieves more efficient terminal breathing of the extended primer.

[0315] After binding, excess (unbound) antibody can be removed under removal conditions, typically at higher salt concentrations of 150 mM to 1000 mM (e.g., 150 mM to 400 mM, 150 mM to 350 mM) and near neutral pH conditions (e.g., pH ranges from 6 to 8, from 6.5 to 7.5, such as about 7). Washing can be performed at a temperature of 20 to 50° C., such as 25 to 40° C. or about 30° C. In another approach, another possibility is low salt (30-100 mM or 50-150 mM) in a near neutral buffer (e.g., pH 6.8 to 7.2).

[0316] Antibody dissociation and secondary incorporation

[0317] After detection, the DNA array is subjected to dissociation conditions (e.g., by increasing temperature and / or pH) under which the bound labeled antibody dissociates from the DNA template. The DNA polymerase used in the second incorporation step should retain its polymerase activity under these dissociation conditions so that incorporation of the additional RT can occur under conditions of antibody dissociation. Additional NLRTs and additional polymerase are typically added to the sequencing reaction after detection.

[0318] The same spike-in reaction components (typically pH 9, with enzyme and NLRT) used for the first spike-in can be used at an appropriate temperature and for 10-60 seconds to simultaneously perform the second spike-in and antibody removal. Multiple spike-in reaction aliquots can be pushed into the flow cell, for example, 2-3 aliquots are incubated for 10-20 seconds each. The presence of NLRT in the solution facilitates complete or near-complete removal of the labeled antibody at lower temperatures (e.g., less than 60°C) or shorter times (e.g., 20-50% shorter), which may be due to competition for antibody binding.

[0319] In some embodiments, depending on the choice of a wash step prior to the second incorporation, only an additional NLRT (depending on residual polymerase from the first incorporation step) or an additional polymerase (depending on residual NLRTS from the first incorporation) may be added. The additional polymerase may be the same or different from the polymerase used in the first incorporation, and the additional NLRT may be the same or different (e.g., a different blocking moiety) from that used in the first incorporation.

[0320] Exemplary dissociation conditions include high temperatures, such as 50°C to 75°C, and sometimes 55°C to 75°C, such as 60°C to 70°C. In some embodiments, the high pH is greater than 7, greater than 8, such as about 9. Exemplary dissociation conditions include high pH environments (pH in the range of pH 8 to 10). In preferred embodiments, dissociation conditions include high temperatures and high pH. In addition, in some embodiments, the removal and second incorporation of antibodies are carried out in a reaction mixture containing a salt, the concentration of which is less than 100mM, such as less than 90mM or less than 80mM. Under preferred dissociation conditions, the removal or dissociation of antibodies can usually be carried out in less than 60 seconds, such as less than 40 seconds or less than 30 seconds. In some embodiments, the dissociation conditions are such conditions that at least about 90%, sometimes at least about 95%, and sometimes at least about 99% of the bound labeled antibodies are dissociated from the template DNA molecule in less than 5 minutes, less than 60 seconds, such as less than 40 seconds, or less than 30 seconds, less than 20s, or less than 10s.

[0321] For the second incorporation, preferably a DNA polymerase is used which retains at least 90% of its polymerase activity under dissociative conditions compared to its activity under known optimal conditions. The optimal conditions for each DNA polymerase can usually be found in the manufacturer's instructions.

[0322] Non-limiting examples of suitable DNA polymerases that can be used in the methods disclosed herein include DNA polymerases from Thermococcus sp., such as 9°N or mutants thereof, including A485L, including double mutants Y409V and A485L, as described in, for example, WO2018 / 129214. Other non-limiting examples of DNA polymerases include Taq polymerase, Bst DNA polymerase, and KOD polymerase.

[0323] The desired dissociation conditions are typically due, at least in part, to a change in the buffer in contact with the array (e.g., by replenishing a previous buffer (e.g., a binding buffer or by buffer exchange) with an added reagent (e.g., a NLRT). That is, the dissociation conditions are typically generated by the introduction of a dissociation buffer that is introduced into the DNA array (e.g., injected into a flow cell), optionally with direct heating or cooling of the flow cell.

[0324] Timing

[0325] In some embodiments, antibody dissociation and the second incorporation occur substantially simultaneously (e.g., under the same conditions in the same reaction buffer). In some embodiments, a dissociation buffer without polymerase and / or without reversible terminator nucleotides is introduced into the initial group and quickly supplemented by adding polymerase and / or reversible terminator nucleotides to the buffer or performing a buffer exchange in which the second buffer comprises polymerase and / or reversible terminator nucleotides. However, antibody dissociation and the second incorporation may occur in any order, e.g., antibody dissociation may occur before, after, or substantially simultaneously with the second incorporation.

[0326] The duration of time that the DNA array is subjected to the high temperature and high pH conditions is very short (usually less than 60 seconds or less than 30 seconds). These relatively mild dissociation conditions advantageously minimize the adverse effects on the incorporated RT and subsequent extension reactions. According to the methods disclosed herein, antibody removal and second incorporation can occur under the same conditions, which means that the conditions can be changed to accommodate two different reactions without any action. Therefore, these methods significantly improve efficiency and reduce sequencing costs and cycle time.

[0327] washing

[0328] After removal of the antibody following the second incorporation step, the array can be washed to remove the antibody and unincorporated RT. The removable capping group of the RT is then removed to allow the next cycle of primer extension, antibody binding, and detection.

[0329] cycle

[0330] In some methods, each cycle of a sequencing reaction on a DNA array includes: (i) incorporating a RT comprising a removable blocking group into at least some of a plurality of template DNA molecules on the array to form an unlabeled extension product; (ii) contacting the incorporated RT on the unlabeled extension product with a labeled antibody that specifically binds to the incorporated RT, and the binding event forms a labeled extension product; (iii) detecting the binding of the antibody, optionally followed by washing away the unbound antibody; (iv) subjecting the labeled extension product hybridized to the DNA array to conditions that both dissociate the bound antibody and incorporate additional RT, and (v) 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 moiety). This step may be followed by one or more new cycles in which new RT is incorporated and detected. The antibody may be directly labeled (e.g., a fluorescently labeled antibody) or may be detected indirectly (e.g., by binding to a labeled secondary antibody).

[0331] In the case of simultaneous removal of the blocking group and the affinity reagent, the DNA polymerase used to incorporate the NLRT is able to mediate polymerization under conditions that are also suitable for dissociating the labeled antibody from its target, i.e., the incorporated RT is at the 3' end of the primer extension product. As disclosed above, these conditions, referred to as dissociation conditions, typically involve higher temperatures (e.g., between 50-75°C, between 55-75°C or between 60-70°C), high pH (pH 8 to 10, such as pH 9) and low salt conditions (salt is present in the reaction at a concentration of less than 100 mM). In some embodiments, the polymerase used in the present invention is able to retain at least 80%, at least 85%, or at least 90% of its polymerase activity. The use of a DNA polymerase having these properties allows antibody removal and the second incorporation of RT to be performed under the same conditions, thereby improving sequencing efficiency and reducing costs.

[0332] Table 4

[0333]

[0334] Sequencing with fewer than four channels or images

[0335] General approach

[0336] Imagers with two or one detection channels (detecting one or two wavelength bands) are more efficient (e.g., more light is detected, no dye crosstalk) and cheaper than 4-channel imagers. Some of these imagers can provide electronic detection or other detection equivalent to one channel detection. It is advantageous to use these imagers for SBS on DNA arrays by generating 1, 2, 3, 4 or more images per cycle (per DNA position). However, sequencing that requires less than 4 images per position is faster and results in processing less data. The use of NLRT and labeled base-specific antibodies provides many benefits in these types of sequencing methods, especially i) more accurate sequencing using less than 4 images, or ii) using two or more separate antibody combinations and imaging to effectively generate 4 or more images (including re-detection) steps to achieve excellent accuracy. In some methods, these methods can use only three labeled antibodies, one of which is unlabeled or absent) or all four labeled antibodies, and only use one or two or more different dyes or other markers that can be detected in one or two channels available for each imager. For example, by attaching different numbers of dye molecules per antibody, especially in combination with dyes of different brightness, four antibodies can generate four different intensities (e.g., relative numbers of 0.5, 1, 2, 4) to distinguish four incorporated nucleotides in one image. An alternative to this single-channel imager is to generate two images, each of which uses two sequential antibody binding, imaging, and removal steps to detect two antibodies with two different intensities (e.g., 1 and 4).

[0337] Compared with labeled dNTPs, it is economically and chemically feasible to attach multiple dye molecules to the antibodies of the present invention. Each antibody attaches multiple dye molecules to provide a stronger signal than one dye molecule attached to the base, thereby achieving more efficient high-quality imaging with less illumination light. In addition, we have realized that this can develop new detection strategies. For example, each antibody attaches multiple dye molecules to allow us to balance the signal intensity in the aforementioned 2-color MPS sequencing, in which one nucleotide must be detected in two different wavelength channels. See U.S. Patent 8,617,81. More dye molecules can be attached to antibodies, where 50% of the antibody molecules must be labeled with one dye and 50% of the antibodies are labeled with another dye.

[0338] Antibody / NLRT sequencing and detection methods are provided that use directly or indirectly labeled antibodies or other affinity reagents for single-color, two-color, three-color, or four-color detection. In some embodiments, one affinity reagent is unlabeled.

[0339] As used herein, dyes with similar emission wavelengths are considered to be "the same color" if they are detected in the same channel of an automatic sequencing system, and the emission in a 200 nm band (preferably a 100 nm band, sometimes 50 nm or narrower) is detected in the same channel of an automatic sequencing system. It will be understood by those skilled in the art that dyes of different colors can be selected to avoid or minimize crosstalk or overlapping emission spectra. Sequencing using the methods of the present invention can be two-color, three-color or four-color sequencing. In one method (four-color sequencing), each affinity reagent is directly or indirectly labeled with a combination of different detectable labels (e.g., fluorescent dyes) or labels that produce unique signals. It should be understood that when a single antigen is recognized with two or more dyes (or other labels), it is possible (but not necessary) to label a single affinity reagent molecule with two (or all) dyes or other labels. On the contrary, 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.

[0340] According to one such method, an array is provided that includes a single-stranded nucleic acid template disposed at a location on a surface. Sequencing is performed by extension or SBS so that the identity of a nucleotide at a detection location in the nucleic acid template is determined in multiple sequencing cycles by: (i) binding (or incorporating) an unlabeled complementary nucleotide (NLRT) to (or incorporating) a nucleotide at the detection location, (ii) labeling the NLRT by binding it to a directly or indirectly labeled affinity reagent that specifically binds to such NLRT; (iii) detecting the presence or absence of a signal associated with the complementary NLRT at the detection location, 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 location 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 a first signal at the detection location 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) both the first signal and the second signal are detected at the detection position, identifying 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) neither the first signal nor the second signal is detected at the said position, identifying 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.

[0341] 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 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 using a NLRT set and a corresponding affinity reagent set in one or more synthesis sequencing cycles, wherein the NLRT set and the corresponding affinity reagent set are, 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 in each cycle of synthesis sequencing, 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 to determine the target nucleic acid sequence. An alternative to the above method 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).

[0342] 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 cycles of sequencing by synthesis using a set of NLRTs and a corresponding set of 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 comprises the following steps: (i) a first affinity reagent that binds to a second NLRT and comprises a second affinity reagent that binds to a second NLRT and comprises a second intensity label; (ii) a third NLRT and a third affinity reagent that specifically binds to the third NLRT and comprises a third intensity label; 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 a 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.

[0343] In another approach, affinity reagents are used that are labeled with one or the same number of molecules of a single dye, but the four NLRTs are discriminated due to different binding efficiencies (i.e., the average number of affinity reagents that bind to a single spot on the array, such as 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 capping group, and affinity reagents with different affinities for their targets are selected. In another approach, the capping 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 capping group can produce the highest signal (100% signal), a capping group with modification 1 can produce a lower level of signal (e.g., 50%), a capping group with modification 2 can produce a lower or even less signal (25%), and so on.

[0344] In another approach, only one affinity reagent is used. A mixture of nucleotides with different ratios of blocking groups recognized by the affinity reagent is used to generate distinguishable signal levels. The remaining nucleotides in the mixture have one blocking group but no corresponding affinity reagent. The following is used for illustration:

[0345] dA 0% Capping Group 1, 100% Capping Group 2

[0346] dG 25% Capping Group 1, 75% Capping Group 2

[0347] dC 50% Capping Group 1, 50% Capping Group 2

[0348] dT 100% Capping Group 1, 0% Capping Group 2

[0349] In another embodiment, the antibody can recognize two bases (nucleotide dimer) where the downstream base is modified by the addition of a cleavable or non-cleavable group.

[0350] In another embodiment, the last base incorporated 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 capping group. The identity of the terminal base can only be determined when the two affinity reagents are bound and / or in close spatial proximity, such as when the two affinity reagents include a FRET donor-acceptor pair as their respective "tags". Alternatively, the binding of one of the affinity reagents may result in a conformational change that allows or enhances the binding of the second affinity reagent.

[0351] Nucleoside analogs described herein can be used for a variety of sequencing methods. For example, analogs can be used for 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.

[0352] Exemplary single-label sequencing methods include, but are not limited to, methods in which nucleoside analogs with different core bases (e.g., A, C, G, T) are continuously delivered and incorporation is detected by detecting the presence or absence of the same signal or label for each different core base. Therefore, single-label methods are sometimes referred to as monochrome methods, because for all core bases, detection signals and / or labels are the same, even if it may be different in terms of intensity (or presence) for each nucleoside analog. For example, by detecting the pyrophosphate cleaved from nucleoside pyrophosphate, it is possible to detect the incorporation of nucleosides into primers by template-directed polymerization mediated by DNA polymerase. Pyrophosphate can be detected using a coupled assay, wherein ATP sulfurylase converts pyrophosphate into ATP in the presence of 5' adenosine 5' phosphosulfate, which in turn serves as a substrate for the conversion of luciferin mediated by luciferase into oxidized luciferin, thereby generating visible light in an amount proportional to ATP generation.

[0353] According to another embodiment, RT and affinity reagents described herein can be used for double labeling or dual-color sequencing, using two distinguishable signals in a combined manner 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 No. 8,617,811, the contents of which, particularly the disclosures related to double labeling sequencing, are incorporated herein by reference as a whole for all purposes. In brief, in double labeling sequencing, the incorporation of the first RT (e.g., RT-A) is detected by marking the newly incorporated RT, and the labeling of the newly incorporated RT is performed by specifically binding to a first affinity reagent comprising a first label, and then detecting the presence of the first label. The incorporation of the second RT (e.g., RT-C) is detected by labeling the second RT, and the labeling of the second RT is performed by specifically binding to a second affinity reagent comprising a second label, and then detecting the presence of the second label. The incorporation of the 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 to 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 the fourth RT (e.g., RT-G) is detected by detecting the absence of both the first label and the second label due to the binding of an unlabeled fourth affinity reagent or due to the fact that the fourth affinity reagent is not included 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.

[0354] According to another embodiment, a first RT labeled by specifically binding to a first affinity reagent comprising a first label, a second RT labeled by specifically binding to a second affinity reagent comprising a second label, and a third RT labeled by specifically binding to a third affinity reagent comprising a third label can be used for three-label sequencing. 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 in the label and specifically binding to the fourth RT). The first label, the second label, and the third label can be distinguished from each other.

[0355] Similarly, four-label sequencing can be performed using a first NLRT labeled by specific binding to a first affinity reagent comprising a first label, a second NLRT labeled by specific binding to a second affinity reagent comprising a second label, a third NLRT labeled by specific binding to a third affinity reagent comprising a third label, and a fourth NLRT labeled by specific binding to a fourth affinity reagent comprising a fourth label. Likewise, the first label, the second label, the third label, and the fourth label can be distinguished from each other.

[0356] Two-color sequencing in which three types of affinity reagents are labeled

[0357] In one approach, the extension primers on the array are labeled by contacting the array with a set of at least three different affinity reagents (hereinafter referred to as antibodies for the sake of clarity and not for the purpose of limitation), including the following:

[0358] (a) a composition comprising a first antibody specific for one of the four nucleotide analogs, the first antibody carrying a first label that fluoresces at a first wavelength or produces a product that fluoresces at a first wavelength;

[0359] (b) a composition comprising a second antibody specific for another of the four nucleotide analogs, the second antibody carrying a second label that fluoresces at a second wavelength or produces a product that fluoresces at a second wavelength;

[0360] (c) a composition comprising a third antibody specific for one of the two remaining nucleotide analogs, which carries a first and a second label. The fourth antibody may be absent or unlabeled.

[0361] In one method, the composition in (c) comprises a mixture of antibodies, some of which (e.g., 50%) comprise a first marker, and some of which (e.g., 50%) comprise a second marker. In this embodiment, the density of the first marker in composition (c) (dye molecules per antibody labeled with the first marker) is greater than the density of the first marker in composition (a), and the density of the second marker in composition (c) (dye molecules per antibody labeled with the second marker) is greater than the density of the second marker in composition (b). For example, the antibodies in composition (a) may comprise 2 molecules of the first marker or an average of 2 molecules of the first marker, and the antibodies labeled with the first marker in composition (c) may comprise 3 or 4 or more molecules of the first marker or an average of 3 or 4 or more molecules of the first marker, and similarly, the antibodies in composition (b) may comprise 2 molecules of the second marker or an average of 2 molecules of the second marker, and the molecules of the antibodies labeled with the second marker in composition (c) may comprise 3 or 4 or more molecules of the second marker or an average of 3 or 4 or more molecules of the second marker. Antibodies specific for nucleotides identified based on emission at two wavelengths can be more densely labeled.More dye molecules can be attached to the antibodies, where 50% of the antibody molecules must be labeled with one dye and 50% must be labeled with a different dye.

[0362] Table 5

[0363]

[0364] Table 4 above shows the equilibrium when the affinity reagent labeled with two different dyes is divided into two equal parts and each of the four nucleotides has equal incorporation. It will be apparent to the reader that the general principles shown can be applied to situations where the affinity reagent is divided into unequal proportions.

[0365] In another method, the composition in (c) comprises antibodies, wherein a single antibody (e.g., a tetramer) has a first label and a second label attached thereto, wherein the density of the first label in the composition (c) is greater than the density of the first label in the antibody of the composition (a), and the density of the first label in the antibody of the composition (c) is greater than the density of the first label in the antibody of the composition (b). For example, the antibody in the composition (a) may comprise 1 first label molecule, or comprise 1 first label molecule on average, and the antibody in the composition (c) may comprise 2 first label molecules, and / or comprise 2 first label molecules and 2 second label molecules on average, or comprise 2 second label molecules on average.

[0366] The compositions in (a), (b) and (c) can be combined into a single composition, for example, to allow for the simultaneous addition of affinity reagents. Alternatively, the compositions can be different and can be combined on the array at about the same time (simultaneously). Alternatively, the compositions can be added to the array one at a time, sequentially.

[0367] Optionally, the affinity reagent set further comprises a fourth affinity reagent that specifically binds to the fourth nucleotide analog, but does not emit detectable fluorescence or produce a fluorescent product at either the first or second wavelength. In this context, the term "detectable" refers to fluorescence that is scored negative, below a threshold value for scoring positive, when the detection device is adjusted to accurately distinguish between positive and negative signals from the first and second affinity reagents.

[0368] After the affinity reagent binds to the extension primer, the unreacted affinity reagent is washed away, and the nucleotide analogs that have been incorporated into the extension primer are determined by detecting or measuring the labels at each site on the array. Fluorescence only at the first wavelength indicates that the first nucleotide analog has been incorporated, fluorescence only at the second wavelength indicates that the second nucleotide analog has been added, fluorescence at both the first wavelength and the second wavelength indicates that the third nucleotide analog has been incorporated; no fluorescence at both wavelengths indicates that the fourth nucleotide analog has been incorporated. This is shown in the following table:

[0369] Table 6

[0370]

[0371] Table 6 is provided by way of example only. Any combination of affinity agent specificities in column 3 and labels in columns 4 and 5 may be used so that the interpretation of the nucleotides in the target nucleic acid in column 1 can occur in any order.

[0372] As described above, to accomplish the two-color approach, the third affinity reagent is labeled so as to be detected or imaged simultaneously with the first and second affinity reagents. As described above, the signal intensity of the third affinity reagent at each wavelength can be matched in intensity to the first and second affinity reagents. Possible techniques for matching intensities include the following:

[0373] (1) The third affinity reagent comprises a specific antibody or its equivalent with a combination of two different labels, wherein the two different labels fluoresce or produce a product that fluoresces at a first and a second wavelength, respectively. The two different labels on the antibody in the third affinity reagent can be the same as the labels used for the antibodies in the first and second affinity reagents, respectively. In order to match the same intensity, the antibody in the third affinity reagent carries the same density of each label as each antibody in the first and second reagents, and the density is twice as high in total.

[0374] (2) Alternatively, the third affinity reagent can be labeled with one or more labels that are different from the labels on the first and second affinity reagents. By selecting a label for the third affinity reagent, the fluorescence intensity of the third affinity reagent at each of the two wavelengths can be matched to that of the first and second affinity reagents, and when excited at the same wavelength, the third affinity reagent fluoresces at a higher intensity (perhaps twice the intensity) at the wavelength used to detect each label on the first and second affinity reagents.

[0375] (3) In another alternative, the third affinity reagent comprises a mixture of at least two specific antibodies or their equivalents, wherein the first antibody carries a label that fluoresces or produces a fluorescent product at a first wavelength, and wherein the second antibody carries a label that fluoresces or produces a fluorescent product at a second wavelength. For example, the first antibody carries the same label as the antibody in the first affinity reagent at about twice the density, and the second antibody carries the same label as the antibody in the second affinity reagent at about twice the density.

[0376] For ease of detection, it is helpful to match the intensity of the two labels on the third affinity reagent with the respective labels of the first and second reagents when measurements are performed separately. When two different antibodies that fluoresce at the first wavelength and the second wavelength are present in the labels carried by the third reagent, the intensity can be matched by doubling the density of the labels on each antibody and doubling the total amount of antibodies in the reagent. In this way, the extension primer labeled with the third reagent fluoresces at a first wavelength with an intensity comparable to the intensity of the first analog labeled by the first reagent; and fluoresces at a second wavelength with an intensity that matches or is comparable to the intensity of the second analog labeled by the second reagent. In this article, the intensity of "matching" or "comparable" means that the intensity of each label in the double-labeled reagent is at least at least about 75% of the label intensity in any one of the single-labeled reagents, and is usually no more than about 135% or 150%.

[0377] Two-color sequencing with two or more binding reactions

[0378] In some methods for detecting which nucleotide has been incorporated into an extended primer on a single or dual channel instrument, multiple separate labeling reactions are performed.

[0379] In the method, a total of four images (one for each base) were acquired as follows:

[0380] In the first reaction, an extended primer containing four incorporated nucleotides (e.g., A, T, G, and C) at the 3' end is contacted with an affinity reagent to form a first reaction product under the following conditions, wherein the first affinity reagent specifically binds to the first nucleotide analog, the second affinity reagent specifically binds to the second nucleotide analog, the first affinity reagent carries a label that fluoresces or produces a fluorescent product at a first wavelength, and the second affinity reagent carries a label that fluoresces or produces a fluorescent product at a second wavelength. After optionally removing unbound reagents, the newly added incorporated nucleotides in each of the two first reaction products are determined by detecting and / or measuring the fluorescence at the first wavelength and the second wavelength. The first affinity reagent and the second affinity reagent (or the label thereon) are then removed (or modified so that it no longer emits a signal), and then a second labeling reaction can be performed and interpreted. In one method, the label is attached to the affinity reagent through a cleavable linker, and the affinity reagent is modified so that they no longer emit a related signal due to the cleavage of the label.

[0381] In the second reaction, the extended primer is contacted with an affinity reagent under the following conditions to form a second reaction product, wherein a third affinity reagent specifically binds to a third nucleotide analog, a fourth affinity reagent specifically binds to a fourth nucleotide analog, the third affinity reagent comprises a label that fluoresces or produces a fluorescent product at a first wavelength, and the fourth affinity reagent comprises a label that fluoresces or produces a fluorescent product at a second wavelength. After optionally removing unreacted reagents, the nucleotide analog that has been added to each of the second reaction products is determined by measuring the fluorescence at the first wavelength and the second wavelength.

[0382] Therefore, the four affinity reagents are as follows:

[0383] (a) a first affinity reagent specific for one of the nucleotide analogs, which is labeled with a fluorescent product that fluoresces at a first wavelength or produces a fluorescent product that fluoresces;

[0384] (b) a second affinity reagent specific for another of the nucleotide analogs and labeled to fluoresce at a second wavelength or to produce a fluorescent product that fluoresces;

[0385] (c) a third affinity reagent specific for one of the two remaining nucleotide analogs and having a label that fluoresces or produces a product that fluoresces at the same wavelength as the first affinity reagent, and

[0386] (d) a fourth affinity reagent specific for a fourth nucleotide analogue and labeled to fluoresce or produce a product that fluoresces at the same wavelength as the second affinity reagent.

[0387] In certain embodiments, the first affinity reagent and the third affinity reagent comprise the same label (e.g., the same dye), and the second affinity reagent and the fourth affinity reagent comprise the same label (e.g., the same dye), which is different from the label on the first affinity reagent and the third affinity reagent. In other embodiments, the dyes / labels detected in the same channel are different, having similar or different brightness.

[0388] The results of labeling and detection are explained as follows: the fluorescence of the first reaction product at the first wavelength indicates that the first nucleotide analog has been incorporated, the fluorescence of the first reaction product at the second wavelength indicates that the second nucleotide analog has been incorporated, the fluorescence of the second reaction product at the first wavelength indicates that the third nucleotide analog has been incorporated, and the fluorescence of the second reaction product at the second wavelength indicates that the fourth nucleotide analog has been incorporated. As shown in the following table:

[0389] Table 7

[0390]

[0391] Table 7 is provided by way of example only. As previously described, any combination of affinity agent specificities in column 3 and labels in columns 4 and 5 may be used so that the interpretation of the nucleotides in the target nucleic acid in column 1 may be performed in any order.

[0392] As described above, when applying the labeling and detection schemes proposed above, an exemplary affinity agent is a monoclonal antibody (or an antigen-binding fragment derived therefrom) having the desired specificity. As discussed elsewhere herein, the affinity agent can be labeled directly or indirectly. For example, when the affinity agent is an unlabeled antibody, it can be directly bound to the corresponding nucleotide analog and then labeled using a secondary antibody that specifically binds to the primary antibody.

[0393] Exemplary labels are fluorescent moieties that can be distinguished under different conditions (emission wavelengths), which are directly attached to the corresponding antibodies or affinity reagents. The present disclosure also includes dual-color detection using labels that do not fluoresce per se but produce fluorescent products. Such labels include enzymes that convert small molecule substrates that are substantially non-fluorescent at the detection wavelength into products that fluoresce at the detection wavelength. Such substrates include L-alanine 4-methoxy-β-naphthamide hydrochloride, 3-amino-9-ethylcarbazole, dansulphonamide, dihydrorhodamine, fluorescein di(β-D-pyranogalactoside), L-methionine 7-amido-4-methylcoumarin trifluoroacetate, 4-methylumbelliferone α-D-pyranogalactoside, resorcinol ethyl ether, tyramine, available from Sigma Aldrich and Thermofisher Scientific. The reader can refer to the latest version of the "The Molecular Probes" manual (invitrogen).

[0394] In order to implement the two-color method, the first, second and third affinity reagents in the two enzyme-labeled detection systems can be used. The two enzymes convert the substrate into two different products, respectively, which fluoresce at two different wavelengths. Under some reaction conditions, each enzyme part will produce a plurality of fluorescent molecules. This can enhance the signal, so the user will usually time the reaction to obtain the required intensity. The binary detection scheme of the present invention can also be realized by labeling antibodies or affinity reagents with a label, which can be detected by other means, through necessary modifications, as its conjugate, bioluminescence measurement or other suitable technology.

[0395] The discussion of labels in the above description does not necessarily require that each antibody or other affinity reagent is labeled with a single labeling moiety (e.g., a fluorescent dye or enzyme). More typically, the affinity reagent is labeled so that multiple labeling moieties are placed on each affinity reagent molecule (e.g., in a Poisson distribution), whereby the labeling intensity is determined by the average number of entities of each affinity reagent (i.e., the total number of moieties in the aliquot divided by the total number of affinity reagents in the aliquot). The affinity reagents of the aliquots may have some molecules that are unlabeled. Typically, this does not interfere with the efficiency of the detection, since the nucleic acid molecules to be sequenced on the array are typically amplicons of DNA fragments, presenting multiple binding sites.

[0396] Unless explicitly stated or required, labels that fluoresce at the same wavelength are not necessarily the same label. The emission intensity of a fluorescent label at a particular wavelength can be adjusted by adjusting the amount of label per affinity reagent, and / or by selecting different labels that fluoresce at the same detection wavelength with different intensities per label moiety.

[0397] In the labeling and detection methods proposed above, the reactions can be carried out in any effective order. For example, the target nucleic acid is usually contacted with all nucleotide analogs at the same time, and then contacted with the affinity reagent at the same time. However, the target nucleic acid is allowed to contact with the analogs and then contact with the affinity reagent in a sequential manner. The different steps in the protocol can also be determined in an effective manner: for example, reacting the hybrid with some but not all analogs, detecting the incorporated analogs, then reacting the hybrid with other analogs and then detecting the analogs.

[0398] The above method can be adapted to a two-color method for sequencing a DNA molecule as follows: A sequencing primer is hybridized to a DNA molecule. Subsequently, the user performs the following multiple cycles:

[0399] A. contacting the sequencing primer with a nucleotide analog to form an extension primer, and

[0400] B. determine the nucleotide analogs that are incorporated into the extension primer; then

[0401] C. Removal of labeled affinity reagents and fluorescent labels, and

[0402] D. Convert the terminator nucleotide on each extension primer to a non-terminator nucleotide, thereby allowing further primer extension in subsequent sequencing cycles.

[0403] Optionally, steps B and C are repeated (two half cycles) using two pairs of antibodies with different specificities as described above.

[0404] Any desired number of cycles may be performed, such as 5 or 10 cycles, and more typically in excess of 25, 50, 100 or 200 cycles.

[0405] The present disclosure also provides a kit or reagent set for sequencing a DNA molecule. For example, in order to provide reagents for sequencing using the first detection scheme described above, the set of reagents may include: (1) four different nucleotide analogs that extend a sequencing primer hybridized to a DNA molecule according to whether the complementary nucleotide on the DNA is adenine, thymine, cytosine, or guanine; (2) at least three affinity reagents, wherein

[0406] (a) a first affinity reagent specific for one of the four nucleotide analogs and having a label that fluoresces or produces a product that fluoresces at a first wavelength;

[0407] (b) a second affinity reagent specific for another of the four nucleotide analogs and labeled to fluoresce or produce a product that fluoresces at a second wavelength;

[0408] (c) a third affinity reagent specific for one of the two remaining nucleotide analogs, which carries one or more labels that fluoresce at both the first and second wavelengths; and optionally

[0409] (d) a fourth reagent specific for a fourth nucleotide analogue which is not labeled with a label that fluoresces or produces a product that fluoresces at the first or second wavelength.

[0410] To provide reagents for sequencing using the second detection scheme described above, the reagent set may include: (1) four different nucleotide analogs that extend a sequencing primer hybridized to a DNA molecule depending on whether the complementary nucleotide on the DNA is adenine, thymine, cytosine, or guanine; and (2) four affinity reagents, wherein:

[0411] (a) a first affinity reagent specific for one of the nucleotide analogs, which is labeled to fluoresce at a first wavelength or to produce a fluorescent product that fluoresces;

[0412] (b) a second affinity reagent specific for another of the nucleotide analogs and labeled to fluoresce at a second wavelength or to produce a fluorescent product that fluoresces;

[0413] (c) a third affinity reagent specific for one of the two remaining nucleotide analogs and having a label (optionally the same label as used for the first affinity reagent) that fluoresces or produces a product that fluoresces at the same wavelength as the first affinity reagent; and

[0414] (d) a fourth reagent specific for a fourth nucleotide analogue and having a label (optionally the same label as used for the second affinity reagent) that fluoresces or produces a product that fluoresces at the same wavelength as the second affinity reagent.

[0415] In another approach, two binding reactions are used. The first bound antibody is not removed. The second pair of images detects the two nucleotides separately.

[0416] In another approach, four binding reactions are performed to obtain four images on a single channel imager. In the first reaction, one affinity agent is bound. In the second reaction, a second affinity agent is bound but the first affinity agent is not removed. The third and fourth affinity agents are similarly bound and the results are interpreted as shown in the table below. This method can be used on a single channel (monochrome) sequencer.

[0417] Image 1 Image 2 Image 3 Image 4 A + + + + T + + + G + + C +

[0418] A similar protocol using three labeled antibodies can be used on a single channel imager by acquiring two sequential images. For example: Image 1: Antibodies A, C labeled with dye 1. Image 2: Antibodies T, C labeled with dye 2:

[0419] Image 1 Image 2 A + none C + + T none + G None or does not exist None or does not exist

[0420] In another method, by using substrate to change the signal, four binding reactions are carried out to obtain four images on a single channel imager. If after combining these two antibodies, first a detectable signal is produced from an antibody, and then a detectable signal is produced from the second combined antibody, two binding reactions can be used on a single channel imager to obtain four images (one for each base). For example, if every kind of antibody is bound to different luciferases, and wherein every kind of luciferase acts on different substrates to emit bioluminescence, then by adding the first substrate, the first antibody will be detected. Then the substrate can be removed and replaced with the second substrate to detect the second antibody.

[0421] Re-detection

[0422] As described in Section 8 above, according to the present invention, it is possible to decouple the removal of affinity reagents (e.g., antibodies) and 3' protecting groups. 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. Any given base position can be detected once and re-probed 0 times, 1 time, 2 times, or more than 2 times. Generally, a single round of re-probing is considered to be sufficient. Just for convenience, in the case of twice probing base positions, the first round of probing can be referred to as the first half cycle, and the second round of probing can be referred to as the second half cycle.

[0423] When reprobing, 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 may be probed with anti-A, anti-T, anti-C, and anti-G, and in the second half cycle, the array may be probed with anti-purine and anti-pyrimidine.

[0424] In one approach, four NLRTs are blocked using two capping groups, e.g., 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 show signals characteristic of 3'-O-azidomethyl-2'-deoxyribose and purines will be identified as having guanine bases, and so on.

[0425] Affinity reagent set

[0426] "Affinity reagent set" is used to label the RT used in SBS. For example, in one embodiment, for an RT group 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 affinity reagent specifically recognizing and binding 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 form, as a mixture, or in a separate container and / or (ii) contacted with a sequencing array or combined on a sequencing array (e.g., in a sequencing flow cell). It is envisioned that the affinity reagents of the present invention include at least one of the above-mentioned affinity reagents, which include one or more (e.g., 3 of 6) CDRs shown in Table 3. It should be understood that the envisioned group will include affinity reagents that include at least one (e.g., 2) antibody chains described in Table 2.

[0427] According to one embodiment, each member of the affinity reagent set has a different, distinguishable detectable label, such as in four-color SBS. According to another embodiment, one member of the affinity reagent set is unlabeled, while the other members are labeled. Alternatively, the affinity reagent set can simply exclude unlabeled affinity reagents and include only labeled affinity reagents.

[0428] 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). Such an affinity reagent set can be used for three-color sequencing.

[0429] 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 not labeled (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.

[0430] According to another embodiment, only a single detectable label (or a single combination of two or more labels) is used, but the intensity varies between the members of the set, such as when the affinity reagents contain different amounts of labels (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 not labeled (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.

[0431] Reaction Mixtures and Kits

[0432] Reaction mixture

[0433] 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) a NLRT or a mixture of a plurality of NLRTs. An exemplary sequencing reaction mixture of the present invention includes, but is 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).

[0434] 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 together with a second affinity reagent that recognizes a capping 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 the first affinity reagent is removed in the case of re-probing (as discussed below).

[0435] The components described in the present application can be used in a reaction mixture for nucleic acid sequencing. Exemplary reaction mixtures include, but are not limited to, those: (a) a nucleic acid array comprising a clonal population of multiple nucleic acid template molecules at positions on an array substrate; and (b) a polymerase; (c) a primer extension product; d) a mixture of 3'-O reversibly blocked nucleoside analogs (e.g., 3'-O-reversible terminator deoxyribonucleotides) with structurally different nucleobases; and (e) one or more labeled antibodies that can specifically bind to one or more of the 3'-O reversibly blocked nucleoside analogs with structurally different nucleobases, wherein at least 95% of the antibody molecules are free in solution (i.e., dissociated from the nucleic acid template)), and wherein the reaction mixture is at an elevated temperature and pH (i.e., dissociation conditions as described above), and the salt concentration is typically less than 100 mM.

[0436] In some embodiments, the reaction mixture comprises (a) a DNA polymerase, wherein the polymerase is capable of mediating polymerization at a temperature of 60° C., pH 9, and 50 mM salt; (c) an oligonucleotide primer; (d) a 3′-O-reversible terminator deoxyribonucleotide, or a mixture of 3′-O-reversible blocking nucleoside analogs having structurally different nucleobases; and (e) one or more labeled antibodies that specifically bind to one or more of the 3′-O-reversible blocking nucleoside analogs having structurally different nucleobases, and at least 95% of the labeled antibody molecules in the reaction mixture do not dissociate from their target 3′-O-reversible blocking nucleoside analogs.

[0437] An exemplary sequencing reaction mixture of the present invention may further include a wash buffer and / or an array comprising a plurality of template nucleic acids immobilized at different locations on the array. The template nucleic acids on the array may have different sequences.

[0438] Reagent test kit

[0439] Kits can be provided for practicing the present invention. As described above, NLRTs and NLRT groups can be provided in kit form. Also as described above, affinity reagents and affinity reagent groups can be provided in kit form. Kits comprising NLRTs and NLRT groups and affinity reagents or affinity reagent groups are also contemplated. For example, the present invention provides a kit including, but not limited to, (a) a reversible terminator nucleotide (RT) or a RT group comprising one, two, three, four or more different individual RTs; (b) a corresponding affinity reagent or an affinity reagent group 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. It is contemplated that the kit of the present invention includes at least one of the above-mentioned affinity reagents, which includes one or more (e.g., 3 of 6) CDRs described in Table 3. It should be understood that the envisioned kit will include an affinity reagent comprising at least one (e.g., 2) antibody chain as described in Table 2.

[0440] 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. It will be appreciated that the kit of the invention comprises at least one of the above affinity reagents, which comprises one or more (e.g., 3 of 6) CDRs described in Table 3. It should be understood that the envisioned kit will comprise an affinity reagent comprising at least one (e.g., 2) antibody chain as described in Table 2.

[0441] 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, which specifically binds to the reversible terminator nucleotide when incorporated at the 3' end of the primer extension product; and (c) (a) and (b) packaging. In one method, the kit contains a plurality of reversible terminator deoxyribonucleotides, each of which comprises a different nucleobase; and a plurality of first affinity reagents, each of which specifically binds to a different one of the reversible terminator deoxyribonucleotides. In some embodiments, the first affinity reagent is detectably labeled and distinguishable from one another. 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.

[0442] The kit may include one or more NLRTs, DNA polymerases, and antibodies as described above. For example, the present invention provides a kit including, but not limited to (a) a NLRT or a group of NLRTs including one, two, three, four, or more NLRTs having different structural nucleobases; (b) a corresponding affinity agent, each affinity agent being able to bind to one NLRT in a nucleobase-specific manner; (c) a DNA polymerase capable of mediating polymerization at 50-75° C. (e.g., 60° C.) and pH 8-10 (e.g., pH 9); (c) packaging materials and / or instructions for use of (a)-(c). In some embodiments, the affinity agent or affinity agent group is detectably labeled and distinguishable from each other. In some embodiments, the kit comprises a second affinity agent. In some embodiments, the first and / or second affinity agent is an antibody. In some embodiments, the kit further comprises a first wash buffer, wherein the pH of the first wash buffer is in the range of 6-8 (e.g., pH 6.5-7.5) and can be used to wash away unbound NLRTs. In some embodiments, the kit further comprises a second wash buffer, wherein the second buffer comprises 150 mM-1000 mM or 150 mM-400 mM salt.

[0443] Unlabeled reversible terminator nucleotide (RT)

[0444] In various embodiments, the sequencing method according to the present invention includes contacting the DNA array with multiple unlabeled RTs (e.g., RT-A, RT-T, RT-C, and RT-G). The contact can be performed sequentially, one RT at a time. Alternatively, four RTs can be contacted with the sequencing array at the same time, most commonly as a mixture of four RTs. In some embodiments, the four RTs are provided together as a "RT group". In one embodiment, the RT group includes RT-A, RT-T, RT-C, and RT-G. In one embodiment, the RT group includes RT-A, RT-U, RT-C, and RT-G. In one embodiment, one or more RTs in one group include modified (non-naturally occurring) nucleobases conjugated to removable capping groups.

[0445] The RT of the RT group can be packaged as a mixture or as a kit, wherein the kit containing each different RT is a separate container. In the mixture of the four RTs, each base can be contained in an equal proportion or can contain unequal amounts.

[0446] In some embodiments, the 3'-O removable blocking group of RT used in the present invention can be cleaved by a reducing agent, such as a phosphine, including but not limited to azidomethyl and tris(hydroxypropyl)phosphine (THPP). In some embodiments, the 3'-O reversible blocking group of RT used in the present invention can be cleaved by UV light, including but not limited to nitrobenzyl. In some embodiments, the 3'-O reversible blocking group of RT used in the present invention can be cleaved by contact with a Pd aqueous solution. The Pd aqueous solution includes but is not limited to allyl. In some embodiments, the 3'-O reversible blocking group can be cleaved by acid. Suitable acids include but are not limited to methoxymethyl. The 3'-O reversible blocking group that can be cleaved by contact with a buffered aqueous solution of sodium nitrite (pH 5.5) includes but is not limited to aminoalkoxy.

[0447] In one embodiment, each RT in the RT group comprises the same end-capping group (e.g., azidomethyl). In one embodiment, the RTs in the RT group comprise different end-capping groups (e.g., RT-A comprises azidomethyl, and RT-T comprises cyanovinyl; or RT-A and RT-G comprise azidomethyl, and RT-C and RT-T comprise cyanovinyl). If different protecting groups are used, such end-capping groups are optionally selected so that different end-capping groups can be removed by the same treatment. Alternatively, the end-capping groups can be selected to be removed by different treatments, optionally at different times.

[0448] Example

[0449] WO 2018 / 129214 provides useful embodiments for understanding the present invention and is a prerequisite for the following embodiments. The preparation of conjugated 3′-O-azidomethyl-2′-dG, -dC, -dA and -dT antigens is described in Example 1 of WO 2018 / 129214. Polyclonal antibodies against non-labeled reversible terminator (NLRT) antigens are prepared as described in Example 2 of WO 2018 / 129214. DNA nanoball (DNB) arrays of Escherichia coli genomic DNA libraries are used for sequencing experiments. These arrays are described in Example 3 of WO 2018 / 129214. In short, circular library constructs are prepared from fragments of Escherichia coli genomic DNA, and the library constructs are amplified by rolling circle amplification (RCA) to produce DNBs containing genomic DNA inserts with adjacent primer binding sites. DNBs are arranged in a DNA sequencing flow cell (e.g., a BGISEQ-500 flow cell or a BGISEQ-1000 flow cell). See, Drmanac et al., 2010, Science 327:78–81 and Huang et al., 2017, Gigascience 6:1-9. Example 4 of WO 2018 / 129214 describes the use of dN-azidomethyl-specific rabbit polyclonal antibodies and labeled goat anti-rabbit secondary antibodies to detect NLRT incorporated into DNB arrays. Example 5 of WO 2018 / 129214 describes DNA sequencing using fluorescently labeled RT-A, -C and -T and unlabeled RT-G. Example 6 of WO 2018 / 129214 describes DNA sequencing using four unlabeled RTs and unlabeled anti-NLRT polyclonal antibodies. Example 7 of WO 2018 / 129214 describes 50 sequencing cycles, wherein unlabeled rt-g is detected using anti-RT-G rabbit primary antibodies and labeled goat anti-rabbit secondary antibodies. Example 8 of WO 2018 / 129214 describes antibodies that bind to NLRT with sufficient specificity to produce signal-to-noise ratio (SNR) values ​​suitable for base detection analysis. Example 9 of WO 2018 / 129214 describes 25 cycles of sequencing using labeled anti-NLRT polyclonal antibodies. Example 11 of WO 2018 / 129214 describes the removal of anti-NLRT antibodies without removing the 3' blocking group. As described elsewhere herein, the removal of the antibody (dissociation from the primer extension product) can be decoupled from the cleavage and removal of the 3' blocking group. In one method, the antibody is removed by specific competition. Primer extension is performed on a DNB array containing an E. coli library using nucleotides containing four unlabeled 3'-azidomethyl bases. Staining is the simultaneous incubation of all four anti-3'-azidomethyl base antibodies directly labeled with a Color Set 1 fluorophore.Specific competition was used to remove the detection affinity reagent by incubation in 50% WB1, 50% Ab buffer at 57°C for 2 minutes in the presence of 20 μM free antigen (3'-O-azidomethyl-2'-deoxyguanine, deoxyadenine, deoxycytosine, deoxythymine, each in the form of triphosphates). The program for removing Ab was (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 pH9 50mM, Tween 20 0.05%, EDTA 1mM. pH 9.0; SRE NaCl 400mM, Tris HCl pH7 1000mM, Sodium Ascorbate L 100mM, Tween 20 0.05%, pH 7.0.

[0450] Example 1: Rabbit anti-NLRT monoclonal antibodies (mAbs) and sequences

[0451] Rabbit monoclonal antibodies (Yurogen Biosystems, Worcester, MA) were generated against KLH-conjugated 3'-azidomethyl-dA (N3A), 3'-azidomethyl-dC (N3C), 3'-azidomethyl-dG (N3G), or 3'-azidomethyl-dT (N3T). Briefly, eight rabbits were immunized with four different KLH-conjugated NLRTs, two rabbits for each of the four molecules. Bleeding analysis by ELISA was performed using each NLRT. On day 63 after immunization, rabbits were sacrificed and peripheral blood mononuclear cells (PBMCs) or splenocytes were isolated. Rabbits were selected for cell sorting and culture of antibody-secreting B cells. Co-culture supernatants were screened using NLRTs. Five or ten different anti-NLRT antibody clones (depending on the target) were prepared for each of the four NLRTs, resulting in >30 mAb preparations.

[0452] Rabbit IgG genes were cloned from specific B cells identified by antigen screening. Heavy and light chain IgG antibody sequences of selected monoclonal antibodies binding to each target antigen were obtained. Figure 1A -H shows the aligned heavy and light chain sequences of monoclonal antibodies specific for each of the four NLRTs.

[0453] A linear expression module was constructed. Recombinant rabbit mAbs were expressed in human embryonic kidney (HEK) 293T cells by microtransient expression. The supernatants of transfected 293T cells were screened by ELISA.

[0454] The heavy and light chain sequences were subcloned into separate expression vectors and expressed in HEK293 cells. The expressed recombinant rabbit mAbs were verified by binding to the antigen by indirect ELISA.

[0455] Example 2: Detection of dN-azidomethyl-specific rabbit monoclonal antibody and labeled goat anti-rabbit secondary antibody Detection of NLRT incorporated into DNB array

[0456] Rabbit monoclonal antibodies N3A, N3T, N3G and N3C were used in this experiment. The DNB array containing the E. coli genomic DNA insert was sensitized and primers were extended using BG9 DNA polymerase (BGI, Shenzhen, China). Thirty antibody preparations were applied to separate lanes on the DNB array at 3 μg / mL or 25% culture supernatant as indicated and incubated for 5 minutes at 35°C (30 independent incubations). At the end of the incubation, the array was washed with antibody buffer (AbB) (Tris buffered saline pH 7.4 + 0.1% BSA and 0.05% Tween-20) at 35°C to remove unbound primary antibodies. The array was then incubated for 5 minutes at 35°C with Cy3-labeled goat anti-rabbit secondary antibodies (Fab fragments) from Jackson Immune Research (West Grove, PA, USA). The array was washed with AbB to remove unbound secondary antibodies and imaged using the BGISEQ-1000 sequencing system. As described above, each of the 30 antibody preparations stained with a single primary antibody is expected to bind incorporated NLRT at approximately 25% of the DNA sites.

[0457] Preliminary screening was performed to determine whether the ELISA positive clones were also positive in functional assays, i.e., sequencing. The control lanes (lanes 1, 8) in the sequencing array were generated by sensitizing DNBs and using all four 3'-azidomethyl dNTPs (primer extension by fluorophore labels connected to bases via cleavable joints). The ACG control value displayed is Cy3. The T antibody data uses the second antibody labeled with ROXtra, and the control value is for ROX. The results are shown in Table 7.

[0458] Table 8

[0459]

[0460]

[0461] Even with a given antibody concentration, the signal can vary between clones. Some antibodies that are positive by ELISA perform poorly on the DNB array.

[0462] Example 3: Sequencing by Synthesis Using Labeled Anti-NLRT Monoclonal Antibodies

[0463] E. coli genomic DNA libraries were prepared as described previously and arrayed on a BGISEQ-500 flow cell. Primers were added and sequencing by synthesis was performed by primer extension using a target unlabeled nucleotide 3'-azidomethyl reversible terminator (dATP, dCTP, dGTP, dTTP) and three conventionally labeled reversible terminators in the following ratios: A-AF532 25% labeled, C-IF700 40% labeled, G-Cy5 35% labeled, and T-ROX 35% labeled (in one experiment, two of the RTs (RT-A and RT-C) were conventionally labeled, and two of the RTs (RT-G and RT-T) were detected by labeled monoclonal antibodies). For each nucleotide, the total concentration of 3' blocked dNTPs was 1 μM and BG9 DNA incorporation was used at 55°C and each cycle lasted 1 minute. After incorporation and washing to remove unincorporated nucleotides, the target 3'-azidomethyl base nucleotides are detected by incubating the array with a mixture of four directly labeled anti-3'-azidomethyl base antibodies (ranging from 1-3 μg / mL). The antibodies are incubated on the array at 35°C for 2X2 minutes per cycle, where "2x2" means incubating with the antibodies for 2 minutes, and then incubating for another 2 minutes after adding the additional antibodies. The array is washed twice to remove any unbound antibodies, and then incubated at 35°C with antibodies labeled with appropriate fluorescent dyes, 2X2min in each cycle. The array is washed twice to remove any unbound antibodies. Table 9 shows the identity of the fluorophores directly conjugated to each secondary antibody.

[0464] Table 9

[0465]

[0466] The fluorescence signal at each position on the DNB array is determined by scanning during laser excitation of the fluorophore for 40 ms. After the identity of the DNB base is determined, the 3' blocking group is removed by reduction with THPP (26 mM) at 57°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 the dissociation of the antibody from the primer extension product.

[0467] This series of steps (extension, antibody incubation, detection and deblocking) was repeated for a total of 25 cycles of DNA sequencing.

[0468] Table 10 shows the results of 25-30 sequencing cycles using labeled anti-NLRT monoclonal antibodies (using the E. coli genome as the reference genome).

[0469] Table 10

[0470]

[0471]

[0472] These data demonstrate that multiple cycles of DNA sequencing can be performed using unlabeled reversible terminators and monoclonal antibodies that bind to both the blocking group and the base.

[0473] Example 4: Obtaining and labeling monoclonal antibody - MPS antibody

[0474] As described above, to demonstrate antibody-MPS, native unlabeled adenosine, cytosine, guanosine and thymidine, azidomethyl-3'-modified monophosphate nucleotides were used.

[0475] The nucleotides were linked to NHS via monophosphates, which were then linked to the KLH protein, and rabbits were immunized every two weeks. Sera were collected from immunized rabbits over a three-month period and screened by ELISA to determine the immune response. The antigen used for ELISA screening was an azidomethyl-3'-blocking nucleotide linked to BSA coated in microtiter plate wells.

[0476] Splenocyte screening: Splenocytes collected from seropositive rabbits were FACS sorted for positive antibody expression using antigens conjugated to fluorescently labeled streptavidin via biotin. Single cells with positive expression of immunogen-reactive surface-bound IgG were selected by FACS for further growth in 384-well plates. This allowed for confirmatory screening of expressed antibodies.

[0477] Antibody Screening: Following spleen cell expansion, the supernatant of each single cell-derived clonal culture was screened for all 4 nucleotide variants (A, C, G, and T) to identify clones with high reactivity to specific nucleobase antigens and low or non-detectable reactivity to 3 non-targeted bases. For this ELISA screen, antigens that mimic the DNA structure generated in sequencing were used. Four biotinylated DNA templates with hybridization primers were used to incorporate unlabeled azidomethyl RT and bound to streptavidin plates for positive and negative ELISA screening. Antibodies with high non-specific binding (>20%), as indicated by high ELISA positive signals for non-targeted bases, were not considered.

[0478] Cloning and expression of antibodies: Selected spleen cell cultures have the coding regions for antibody heavy and light chains cloned into a plasmid expression system. These plasmids are used to transiently transfect 293 cell lines to produce monoclonal antibodies. The expressed antibodies are purified by protein A capture columns and eluted in low pH buffer before the buffer is exchanged to phosphate-buffered saline.

[0479] The antibodies are labeled by reaction of available free amines on the protein with NHS ester-activated fluorescent dyes (14). The NHS ester-activated fluorophore is diluted in anhydrous DMSO and reacted at a concentration (10-100 uM) that provides a strong signal without adversely affecting antibody binding or specificity. A relatively low and readily available antibody concentration (1 mg / ml) is adjusted to pH 8 in bicarbonate buffer and reacted with the NHS ester dye. Incubation is continued at room temperature for 45 minutes, and unreacted dye is then quenched in tris-buffered saline (pH 7.4). Without any purification, these labeled antibodies are aliquoted and stored at -20°C. The random labeling process under these conditions balances the number of fluorophores per antibody and antibody inactivation. Antibody-MPS antibodies can be labeled with multiple dye molecules per antibody molecule, thereby providing a stronger sequencing signal.

[0480] Example 5: Characterization of Antibodies-MPS Antibodies in Sequencing Assays

[0481] Sequencing Platform

[0482] DNBSEQ-G400 was used to test and implement the antibody-MPS process. The DNBSEQ platform utilizes a PCR-free nanoarray of DNA nanoballs (DNBs); linear concatemers of DNA copies generated by rolling circle replication that are bound to defined locations on a patterned nanoarray (4). For popular double-end (PE) or second-end sequencing, a controlled multiple displacement amplification (MDA) process is used on the DNB array described in US20160237488, which is incorporated by reference for all purposes. After the first read is generated on the DNB, the extension product can be further extended (optionally using additional primers) using natural unblocked nucleotides by a strand displacement polymerase (e.g., Phi29) in a controlled and fully synchronized manner. This process produces single-stranded (ss) DNA branches that are complementary to the original DNB and are still bound to the DNB through the undisplaced region (Figure 3). The resulting "branched DNBs" typically contain 1-3 template copies per branch, providing more priming sites and stronger signals in the second end read than in the first end read.

[0483] Complementary strand preparation and paired-end sequencing were performed on the DNBSEQ MPS platform.

[0484] Referring to U.S. Patent 10,227,647, it describes the method for double-end sequencing. In the method for the embodiment, a copy DNA nanoball (DNB) containing an adapter sequence and the inserted genomic DNA is hybridized with a primer as a concatemer to perform first end sequencing. After the first end reading is generated, a controlled continuous extension is performed by a chain displacement DNA polymerase to produce multiple complementary chains. When the 3' end of the newly synthesized chain reaches the 5' end of the downstream chain, the 5' end is replaced by a DNA polymerase, producing a ssDNA overhang, forming a "branched DNB". The second end sequencing primer is hybridized with the adapter copy in the newly formed branched chain to produce a second end reading.

[0485] For sequencing, a modified standard MPS kit is used to implement the antibody-MPS process. The labeled RT is replaced with an unlabeled RT with natural nucleobases, and a mixture of four labeled antibodies (specific for each natural nucleobase) in binding buffer is added to the cartridge. The antibodies are labeled with fluorescent dyes to enable imaging on current sequencers, and the fluorescent dyes have similar excitation and emission spectra as those used in the labeled RT. Each sequencing cycle includes the incorporation of a reversible terminator using a modified polymerase, followed by antibody binding. After washing excess unbound antibody, standard imaging is performed, followed by removal of bound antibody, and standard 3' deblocking is performed as one step or in two separate steps.

[0486] Example 6: Antibody Evaluation in Sequencing Assays on the DNBSEQ Platform

[0487] Specificity

[0488] In the initial DNBSEQ screening of several ELISA-positive antibodies for each of the four nucleotides, up to 50% were found to have relatively weak positive signals. Possible explanations are clonal expansion failure or ELISA false positives. A group of four antibodies with good signals and low background were selected. Then, the key properties of these antibodies required for sequencing were evaluated. The same was true for primary splenocyte supernatants from promising clones.

[0489] Accurate sequence determination requires that the antibody be specific for the base associated with the 3' reversible terminator ribose. To demonstrate that each antibody species is specific for each individual base, arrays of DNA nanoballs were formed and hybridized with primers, which were then extended by one nucleotide with a reversible terminator.

[0490] Figure 2AThe fluorescence intensity of the DNB clusters in two channels in a single imaging field of view after binding to fluorescent antibodies is shown. Channel pairs without spectral dye crosstalk (e.g., AG, AC, TG, TC) do not show any antibody cross-binding. DNBs are negative in both channels, or positive in one channel, but not positive in the other (DNB clusters on the x and y axes). Using oligonucleotide constructs that simulate RT incorporated during sequencing to select positive and negative antibodies helps to achieve high antibody specificity.

[0491] The DNB intensity generated by the antibody-MPS from one cycle is plotted in paired imaging channels. A random selection of 100,000 DNBs in the FOV is represented. The intensity of the background subtracted without dye crosstalk correction is proposed. Only paired channels without dye crosstalk are shown. For each pair, if there is no antibody cross-binding on the XY coordinate representation, 3 DNB clusters are expected to exist: - / -: low X and Y intensity, + / -: high X intensity and low Y intensity, - / +: low X intensity and high Y intensity. If there is cross-binding, the + / - or - / + cluster will be offset from X or Y at a certain angle. In all four pairs, only strong binding of one antibody was observed (relative signal is within the range of 1000 counts), but there was no detectable cross-binding.

[0492] Example 7: Antibodies used in these examples recognize 3' blocking groups

[0493] The immunogen used a nucleotide with a 3' azidomethyl blocking group. After confirming the base specificity, it was next determined whether the azidomethyl group was required for strong binding.

[0494] Figure 2B Is a graph of detected fluorescence, which shows that antibody binding depends on bases and sugars with 3' azidomethyl blocks. In three conventional sequencing cycles, the 3' blocking group is removed after antibody binding and imaging, followed by three cycles in which the 3' azidomethyl is cleaved before antibody binding and imaging. The intensity of background subtraction, phase correction and spectral crosstalk correction is shown, and / or each imaging channel (corresponding to each base) is depicted, with the average intensity of the DNB with the highest intensity in the channel.

[0495] During the first 3 cycles, when a single antibody was incubated with surface-associated DNBs, fluorescence intensity was achieved. Here, the intensity is reported as a background-subtracted and spectral crosstalk-corrected measurement of the average population intensity of DNBs assigned to the fluorophore channel within the imaging field (with the strongest intensity in that channel).

[0496] All four antibodies produced strong signals (400-600 counts) when the azidomethyl group was present during antibody binding. Starting from the 4th cycle, each cycle had a cleavage step before binding the antibody. After removing the 3' azidomethyl blocking group, there was no obvious detection signal, indicating that in addition to the base, this chemical part is also important for strong antibody binding, which may prevent the antibody from binding to other target bases in the DNA. The bases on the non-terminal nucleotides can also be distinguished by other spatial or chemical features because they have stacked bases and phosphates on the 5' and 3' sides.

[0497] Example 8: Fast Binding Kinetics

[0498] When optimizing antibody binding conditions, it was found that low salt (50 mM) Tris buffer (pH 7.6) provided efficient binding at 35-40°C.

[0499] See also Figure 2C , showing the effect of 30, 60 or 90 seconds of labeled antibody binding to unlabeled RT nucleotides and incorporation by DNBSEQ sequencing. Minimal increases in fluorescence intensity were observed with increasing incubation time. While this suggests that incubation times may be shorter than 30 seconds, it must be remembered that this represents a population average and the behavior of a specific sequence environment may behave differently.

[0500] During the three incubation times at 35°C, the same concentration of antibody ( 4ug / ml, providing excess antibody) binds to DNBs. 30 seconds of incubation has produced greater than 90% of the maximum signal, thus demonstrating the fast binding kinetics of all 4 selected antibodies. The intensity of background subtraction, phase correction and spectral crosstalk correction is shown. For each imaging channel (corresponding to each base), the average intensity of the DNB with the highest intensity in that channel is depicted.

[0501] Example 9: Efficient Removal of Bound Antibodies

[0502] After imaging and before the next cycle of nucleotide incorporation, sufficient removal (e.g., at least 95% or complete removal) of bound antibodies is critical for high-quality sequencing. In some cases, antibody removal and 3' blocking cleavage are performed simultaneously. In some cases, antibody removal and second incorporation are performed simultaneously, see above.

[0503] Figure 2Dis a graph of intensity data showing the effect of removing the fluorescent antibody after binding to RT. In cycles 1-10, the flow cell was briefly washed with pH 7 SSC buffer at 40°C and then imaged at 20°C. In cycles 11-20, the flow cell was incubated at 57°C for 1 minute in 50mM Tris pH 9 buffer (including RT) for 60 seconds and then imaged. Cycles 21-30 show the intensity before imaging after incubation for 60 seconds in the same buffer without nucleotides. Using the intensity subtracted from the background and corrected for spectral crosstalk, and / or depicting the average intensity of the DNB with the highest intensity in each imaging channel (corresponding to each base) in that channel.

[0504] High pH (> pH 8) and temperatures above 55°C were found to be effective in quantitative antibody removal. It was also found that the inclusion of unlabeled RT in the removal buffer can accelerate the rate of dissociation. Buffer conditions that do not include RT are compatible with the azidomethyl cleavage reaction.

[0505] Example 10: Labeled Antibodies Produce Stronger Signals Than Labeled RT

[0506] Due to proximity quenching, labeled RTs can only have one dye attached to the base. To minimize the adverse effects of base scarring, typically only 60-70% is labeled. Antibody-MPS antibodies can be labeled with multiple dye molecules per antibody molecule, potentially providing a stronger sequencing signal.

[0507] The signal intensity provided by the current random labeling process was tested, which balances the number of fluorophores and antibody inactivation of each antibody. It is found that in the composition comprising the labeled antibody, a single antibody molecule is usually labeled or unlabeled by 1-5 fluorophores. In some embodiments, at least 50% (mol %) of the antibody is labeled by more than one fluorophore molecule (e.g., 2-5 fluorophore molecules). In some embodiments, at least 75% of the antibody is labeled by more than one fluorophore molecule (e.g., 2-5 fluorophore molecules). For illustration and not limitation, exemplary dyes are described in Drmanac et al.

[0059] ,

[0171] -

[0174] .

[0508] Figure 2Eis a plot comparing the relative intensities of base-labeled nucleotides at the first 10 cycle positions, followed by another 80 cycle positions, using antibody-labeled detection before returning to base-labeled RT. Antibody detection produces a stronger signal relative to base-labeled nucleotides, with some fluorophores showing more than 200% increase in intensity relative to their base-labeled counterparts. The range of responses for different fluorophores may reflect labeling efficiency of the dye for a specific antibody, antibody binding affinity, or fluorophore quenching. Beneficial effects of the increased intensity include retention of adequate signal in low-copy DNBs throughout long sequencing runs, low exposure times, or more rapid imaging.

[0509] Ten base-labeled sequencing cycles were performed, followed by switching to antibody-MPS sequencing (cycles 10-90), and then back to standard direct base-labeled sequencing. Background-subtracted, phase-corrected, and spectral crosstalk-corrected intensities are shown or for each imaging channel (corresponding to each base), and for each cycle, the average intensity of the DNB with the highest intensity in that channel is depicted.

[0510] Example 11: No signal suppression

[0511] Labeled RT was observed to produce some signal suppression (e.g., quenching) in subsequent cycles, most likely due to modified ("scarred") bases. Since antibody-MPS uses unlabeled RT, this effect is not expected.

[0512] Figure 2F Data are provided that compare the signals in the DNB groups in two consecutive cycles (from one field of view) and demonstrate that when using labeled RT, DNBs with G in the previous cycle and T in the current cycle suppress the T signal. Lower than expected T signals cause the GT clusters to shift from the diagonal to the Y axis, representing the G signal. No inhibition was observed in antibody-MPS using unlabeled RT with natural bases and without any scars. In addition, the dye on the T antibody is far from the G base, thus avoiding quenching.

[0513] These data show that the antibody-MPS technology eliminates signal suppression. The DNB signal in the DNB group is compared in the channel G (Y-axis) of the previous cycle and the channel T (X-axis) of the current cycle. The labeled RT chemistry and antibody-MPS chemistry (natural unlabeled bases, labeled base-specific antibodies) are shown. Each point on the figure is a DNB, which forms 4 clusters: no G / no T, G / no T, T / no G and G / T. In the case of labeled RT, it is observed that the T signal is lower than expected (the cluster of GT DNB is offset to the Y-axis). Inhibition is not observed in antibody-MPS.

[0514] Example 12: Full Sequencing Test of Antibody-MPS Chemistry

[0515] Generate 200 base reads: SE200 sequencing

[0516] MPS reads longer than 100 bases are very useful. As an initial validation test of the antibody-MPS potential, 200 base reads were obtained. 200 sequencing cycles were performed on DNBs in the lanes loaded into the DNBSEQ-G400 sequencer flow cell. DNBs were prepared from a standard 300 base library of E. coli DNA using the MGI protocol.

[0517] Figure 3A The average called base intensity of DNBs in selected areas of the array with optimal fluidics and optics is shown to highlight the potential of this new chemistry. The change in label intensity over 200 cycles of single-end reading is shown. The intensity is shown with background subtracted, phase corrected, and spectral crosstalk corrected, and for each imaging channel (corresponding to each base), the average intensity of the DNB with the highest intensity in that channel is depicted.

[0518] As previously observed with directly labeled nucleotides, a decrease in intensity was observed as the cycle proceeded. Multiple factors contributing to this include: i) out-of-phase signals, ii) partly due to irreversible termination of impure RT and DNA damage during imaging; or ii) DNA loss. It was found that even without antibody binding and imaging, only incorporation cycles with unlabeled RT (data not shown), similar signals were lost. This does not include the effects of antibody binding, imaging or removal. It is speculated that the difference in the rate of decline between bases is due to changes in background or the effects of illumination efficiency of light collection during the cycle. Although a decrease in dye intensity may occur in the cartridge during the run, this is a small reason because a minimal increase in intensity was observed when fresh reagents were added during the run (data not shown). However, the remaining signal after 200 cycles was still high, which supports the possibility of longer antibody-MPS readings.

[0519] As with standard MPS with reversible terminators, positional discordance increases with cycling. This is due to: i) accumulation of out-of-phase signal, which becomes confounded with dye crosstalk; and ii) loss of signal relative to background, particularly affecting DNBs with low template copy number. Hysteresis (-1 signal) and continuity (+1 signal) are relatively low (<0.1%) per cycle, but still accumulate to 90% within 200 cycles. 30% of the combination is out of phase.

[0520] Figure 3BPositional inconsistencies for 200 cycles of SE sequencing are shown. Note: After cycle 185, the high inconsistency rate increases due to shorter insert fragments and reads in the adapter region that do not match the human reference. After filtering out 5% of empty spots and mixed DNBs from all binding spots in the array, the mapping rate of the remaining 95% of DNBs was 99%, with an overall inconsistency of 0.11%, which further decreased to 0.06% in base calls with quality scores >Q10. This is a very promising result for 200 base reads, showing high accuracy and 94% sequencing yield (0.95 filtered reads x 0.99 mapping rate).

[0521] The sequence inconsistency in 100 base reads from the PCR-free E. coli library was further evaluated. An overall inconsistency of 0.029% was obtained (1 difference from the reference in 3500 called bases). Then, the inconsistency under different base call quality filtering conditions was calculated. Base calls with quality scores >20 (close to 99.8% of all base calls) had five to six times fewer errors (inconsistency close to 0.0005% or one mismatch in 20,000 bases). The remaining high-quality inconsistencies may be caused by DNA replication errors, DNA damage, or actual sequencing errors. This shows the great potential of antibody-MPS for high-quality sequencing, with extremely low overall error rates and very few high-quality errors.

[0522] Table 11

[0523] Reference Escherichia coli Number of cycles 200 ESR% 95.09 >Q30％ 95.23 Mapping rate % 97.83 Average inconsistency rate % 0.11 <＝Q10％ 0.21 >Q10_Inconsistency rate % 0.06 Hysteresis % per cycle 0.07 Continue % per loop 0.07

[0524] Example 13: High-quality 150-base paired-end reads: PE150 sequencing

[0525] Paired-end (PE) sequencing provides very useful MPS reads that bridge repetitive sequences that are longer than the read and minimize the need for long continuous reads. The most commonly used is PE150 (150 bases on both ends of a 300-600b insert).

[0526] Antibody-MPS PE150 was tested to demonstrate that the use of the antibody would not interfere with the DNBSEQ PE process of the controlled MDA. Figure 4A The intensity variation in 150 cycles of the first strand is shown, followed by a good recovery of the intensity on the second strand as a complementary template, and the corresponding sequencing primer is used for extension. In this test, the antibody concentration used for the second strand is twice that of the first strand. Overall, the intensity values ​​within 150 positions of the first strand drop by about 30-50%, and the intensity values ​​of the second strand drop by 40-50%, which is partly due to the higher incorporation incompleteness (hysteresis) in the second strand.

[0527] After filtering out approximately 11-13% of empty arrays and low-quality array spots, the mapping rates were >99% and the discordance rates were 0.08% and 0.26% for the first strand of the E. coli (300b insert) and human (400b insert) DNA libraries, respectively ( Figure 4B ). For the second strand, the mapping rate was approximately 99% and the discordance rates were 0.22% and 0.62%, respectively. After filtering 0.4% and 0.8% of the base calls with quality scores <10, the combined discordance in the E. coli and human DNA libraries dropped to 0.06% and 0.24%, respectively. Part of the discordance is due to PCR errors introduced during library preparation. Higher discordance is expected for the human library due to the polymorphism of the samples relative to the human reference.

[0528] The higher discordance in the second read despite the higher signal is due to the longer lag time and lower quality threshold used for DNB filtering. The higher lag in the second read (out-of-phase -1) is likely due to incomplete removal of the Phi 29 polymerase used at high concentrations for complementary strand preparation. This was confirmed in a PE100 run with optimized Phi29 removal, which reduced the accumulated lag from about 15% to about 11% ( Figure 4B and 4C ). In addition, the hysteresis accumulation is more linear, indicating less -2 phase. This result illustrates the complexity of the MPS process (with many biochemical steps that are multiple and interdependent), which requires careful balanced optimization.

[0529] See also Figure 4A , PE150 intensity of a human DNA library is shown, with background subtracted and spectral crosstalk corrected, or for each imaging channel (corresponding to each base), the average intensity of the DNB with the highest intensity in that channel is depicted.

[0530] Table 12

[0531]

[0532]

[0533] Figure 4B Shows the Figure 4A PE150 hysteresis during the same process (out-of-phase incorporation of -1). The hysteresis represents the intensity contribution of the previous (-1) base to the current cycle.

[0534] Figure 4CShown is the PE100 hysteresis in the PE100 run (E. coli library) utilizing optimized Ph29 removal.There are many tools developed to further optimize the antibody-MPS process, including replacing complete antibodies with smaller versions (such as nano antibodies expressed in ScFv or bacterial hosts), and effectively marking at the target site.Compared with many common programs (such as Western blot, ELISA) utilizing antibodies to detect, the binding time of antibodies is proven to be relatively fast, only with 30 seconds, thus proving that effective generation of enough intensity is provided to detect bases with low errors.The increase of antibody binding time has the least effect on the increase of intensity, thus showing that most available target sites are occupied within 30 seconds.In addition, the antibody of about 4ug / ml is enough to bind most of the RTs incorporated.

[0535] This is surprising since the target nucleotide is present in dsDNA and the immunogen used is a single monophosphate reversibly terminated nucleotide. Most likely there is some transient dsDNA end melting, thus enabling antibody binding. The preferred low salt, Mg++-free binding buffer (which aids breathing of DNA ends) supports this explanation.

[0536] A particular benefit of antibody-MPS is the possibility of step-by-step base detection after a single reaction incorporation of all unlabeled RTs. This is achieved by rapid binding and removal of the labeled antibodies but without removal of the 3' blocking group. Each base can be detected in a separate image using a more efficient and cost-effective 2-color or 1-color imager, without dye crosstalk on a 4-color imager. For a 2-color imager, two antibodies labeled with different dyes will first be combined and two images will be generated. After rapid removal of the bound antibodies, two additional antibodies labeled with the same pair of dyes will be combined to produce more than two images, one for each base. For a fast imager, the entire process will take a little longer, but the expected sequence quality is much higher because a 2-color imager can collect more than 2-3 types of light (wider filter bands) without dye crosstalk.

[0537] In addition to the PCR-free DNBSEQ MPS platform, Antibody-MPS can be used in any MPS platform, including PCR-based clonal arrays (PCR clusters on supports or beads) or single-molecule arrays. The combination of high-quality, low-cost Antibody-MPS chemistry and PCR-free, cost-effective DNB nanoarrays creates a new advanced MPS platform to advance the implementation of genomics-based health monitoring that requires screening tests based on comprehensive, accurate, and evaluative sequencing.

[0538] Although the foregoing invention has been described in detail by way of illustration and example for the purpose of clear 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 each reference is incorporated by reference individually. If there is a conflict between the present application and the references provided herein, the present application shall prevail. Sequence Listing <110> Shenzhen MGI Technology Co., Ltd. Shenzhen BGI Life Sciences Institute <120> Massively parallel sequencing using unlabeled nucleotides <130> 092171-1164247 <150> 62 / 914,915 <151> 2019-10-14 <150> 62 / 914,940 <151> 2019-10-14 <150> 62 / 914,877 <151> 2019-10-14 <150> 62 / 758,317 <151> 2018-11-09 <160> 165 <170> PatentIn Version 3.5 <210> 1 <211> 464 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 1 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Glu Glu Ser Gly Gly Asp Leu Val Lys 20 25 30 Pro Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Phe 35 40 45 Ser Ser Tyr Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Ile Ala Cys Ile Tyr Gly Gly Ser Ser Gly Thr Thr Tyr 65 70 75 80 Tyr Ala Ser Trp Pro Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser 85 90 95 Thr Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Met Arg Gly Ala Asn Gly Ala Gly Phe Gly Asp Ala 115 120 125 Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro 130 135 140 Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro 145 150 155 160 Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu 165 170 175 Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg 180 185 190 Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala 210 215 220 His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr 225 230 235 240 Cys Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser 245 250 255 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro 275 280 285 Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala 290 295 300 Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val 305 310 315 320 Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe 325 330 335 Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met 355 360 365 Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys 370 375 380 Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys 385 390 395 400 Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp 405 410 415 Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser 420 425 430 Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455 460 <210> 2 <211> 238 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 2 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser 35 40 45 Pro Ser Val Tyr Ser Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Arg Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 85 90 95 Thr Ile Ser Asp Val Gln Cys Asp Asp Ala Ala Asn Tyr Tyr Cys Ala 100 105 110 Gly Gly Tyr Thr Tyr Thr Ser Asp Ser Ile Trp Ala Phe Gly Gly Gly 115 120 125 Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 130 135 140 Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val 145 150 155 160 Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val 165 170 175 Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln 180 185 190 Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr 195 200 205 Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln 210 215 220 Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 3 <211> 464 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 3 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Glu Glu Ala Gly Gly Asp Leu Val Lys 20 25 30 Pro Glu Gly Ser Leu Arg Leu Thr Cys Lys Ala Ser Gly Phe Asp Phe 35 40 45 Ser Ser Tyr Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Ile Ala Cys Ile Tyr Gly Gly Ala Ser Gly Thr Thr Tyr 65 70 75 80 Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser 85 90 95 Thr Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Met Arg Gly Ala Asn Gly Ala Gly Phe Gly Asp Ala 115 120 125 Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro 130 135 140 Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro 145 150 155 160 Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu 165 170 175 Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg 180 185 190 Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala 210 215 220 His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr 225 230 235 240 Cys Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser 245 250 255 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro 275 280 285 Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala 290 295 300 Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val 305 310 315 320 Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe 325 330 335 Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 340 345 350 Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met 355 360 365 Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys 370 375 380 Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys 385 390 395 400 Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp 405 410 415 Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser 420 425 430 Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala 435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455 460 <210> 4 <211> 238 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 4 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser 35 40 45 Pro Ser Val Tyr Ser Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Arg Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 85 90 95 Thr Ile Ser Asp Val Gln Cys Asp Asp Ala Ala Asn Tyr Tyr Cys Ala 100 105 110 Gly Gly Tyr Thr Tyr Thr Ser Asp Ser Ile Trp Ala Phe Gly Gly Gly 115 120 125 Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 130 135 140 Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val 145 150 155 160 Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val 165 170 175 Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln 180 185 190 Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr 195 200 205 Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln 210 215 220 Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 5 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 5 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Gln Gln Met Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Ile Asp Phe 35 40 45 Ser Ser Tyr Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Leu Ile Ala Cys Ile Tyr Leu Ser Ser Gly Ser Thr Trp Tyr 65 70 75 80 Ala Ser Trp Val Asn Gly Arg Phe Thr Ile Ser Arg Ser Thr Ser Leu 85 90 95 Asn Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Gly Gly Phe Cys Thr Ala Tyr Ser Gly Asp 115 120 125 Gly Cys Tyr Phe Thr Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser 130 135 140 Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys 145 150 155 160 Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly 165 170 175 Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr 180 185 190 Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr 195 200 205 Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr 210 215 220 Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val 225 230 235 240 Ala Pro Ser Thr Cys Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro 245 250 255 Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln 290 295 300 Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr 305 310 315 320 Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg 325 330 335 Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys 355 360 365 Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val 370 375 380 Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val 385 390 395 400 Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro 405 410 415 Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser 420 425 430 Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg 450 455 460 Ser Pro Gly Lys 465 <210> 6 <211> 236 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 6 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Ile Lys Met Thr Gln Pro Pro Ala Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Arg Ala Ser 35 40 45 Glu Asp Ile Asp Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Gln Leu Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr 85 90 95 Ile Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Ser 100 105 110 Thr Tyr Tyr Ser Ser Asn Pro Glu Gly Val Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro 130 135 140 Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val 145 150 155 160 Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly 165 170 175 Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser 180 185 190 Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr 195 200 205 Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr 210 215 220 Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 7 <211> 462 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 7 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Glu Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe 35 40 45 Ser Ser Tyr Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Leu Ser Ala Cys Ile Asp Thr Gly Ser Gly Ser Thr Trp Tyr 65 70 75 80 Pro Ser Trp Val Asn Gly Arg Phe Thr Ile Ser Arg Ser Thr Ser Leu 85 90 95 Asn Thr Val Asp Leu Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Glu Tyr Ser Thr Ala Trp Tyr Phe Asn Leu 115 120 125 Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser 145 150 155 160 Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val 165 170 175 Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe 180 185 190 Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro 210 215 220 Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser 225 230 235 240 Lys Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe 245 250 255 Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val 275 280 285 Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro 290 295 300 Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr 305 310 315 320 Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys 325 330 335 Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro 355 360 365 Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile 370 375 380 Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly 385 390 395 400 Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp 405 410 415 Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp 420 425 430 Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455 460 <210> 8 <211> 236 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 8 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Ile Lys Met Thr Gln Thr Pro Gly Ser 20 25 30 Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40 45 Gln Ser Ile Ser Thr Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Arg Pro Lys Leu Leu Ile Tyr Asp Ala Ser Arg Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys His Gln 100 105 110 Gly Phe Gly Ala Ser Asn Val Asp Asn Pro Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Glu Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro 130 135 140 Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val 145 150 155 160 Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly 165 170 175 Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser 180 185 190 Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr 195 200 205 Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr 210 215 220 Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 9 <211> 461 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 9 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Glu Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe 35 40 45 Ser Asp Asn Ala Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Ile Gly Cys Ile Tyr Ile Gly Ser Ser Ser Thr Tyr Tyr 65 70 75 80 Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Thr Ser Ser Thr 85 90 95 Thr Val Asn Leu Gln Met Thr Ser Leu Thr Asp Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Gly Arg Asp Pro Thr Ala Ala Trp Gly Gly Gly Leu Trp 115 120 125 Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr 165 170 175 Val Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro 180 185 190 Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser 195 200 205 Val Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala 210 215 220 Thr Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys 225 230 235 240 Pro Met Cys Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln 275 280 285 Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro 290 295 300 Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu 305 310 315 320 Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys 325 330 335 Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Ala Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro 355 360 365 Arg Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys 385 390 395 400 Ala Glu Asp Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly 405 410 415 Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln 420 425 430 Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455 460 <210> 10 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequence description: Synthesis of peptide <400> 10 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Ile Cys Asp Pro Val Met Thr Gln Thr Pro Ser Ser 20 25 30 Thr Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser 35 40 45 Gln Ser Val Tyr Asn Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Arg Leu Ile Tyr Glu Ser Ser Lys Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Arg Gly Ser Gly Ser Gly Ala Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Ala Tyr Tyr Thr Thr Leu Asp Phe Gly Gly Gly Thr Glu Val 115 120 125 Val Val Arg Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro 130 135 140 Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala 145 150 155 160 Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr 165 170 175 Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala 180 185 190 Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Leu Thr Ser Thr Gln 195 200 205 Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr 210 215 220 Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 11 <211> 458 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 11 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30 Gly Ala Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Ile Asp Phe Ser 35 40 45 Ser Ser Tyr Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Ala Cys Ile Asp Thr Gly Ser Ser Gly Ser Thr Tyr Tyr 65 70 75 80 Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Pro Ser Ser Thr 85 90 95 Thr Val Ser Leu Gln Met Thr Ser Leu Gln Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Lys Gly Asp Gly Thr Asp Leu Trp Gly Pro Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp 165 170 175 Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser 195 200 205 Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr 210 215 220 Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Met Cys 225 230 235 240 Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp 275 280 285 Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu 290 295 300 Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala 305 310 315 320 His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly 340 345 350 Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu 355 360 365 Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp 385 390 395 400 Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe 405 410 415 Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp 420 425 430 Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455 <210> 12 <211> 236 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 12 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Leu Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Gln 50 55 60 Pro Pro Lys Asn Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Asn Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser 100 105 110 Tyr Gly Gly Tyr Ser Ile Tyr Gly Leu Val Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro 130 135 140 Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val 145 150 155 160 Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly 165 170 175 Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser 180 185 190 Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr 195 200 205 Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr 210 215 220 Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 13 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 13 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Glu Glu Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe 35 40 45 Ile Ser Ser Asp Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Ile Ala Cys Ile Tyr Ile Gly Gly His Thr Pro Tyr Tyr 65 70 75 80 Ala Ser Trp Ala Arg Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr 85 90 95 Ala Val Thr Leu Gln Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Gly Ile Ala Gly Pro Ala Leu Trp Gly Pro Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp 165 170 175 Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser 195 200 205 Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr 210 215 220 Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Met Cys 225 230 235 240 Pro Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro 245 250 255 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 260 265 270 Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp 275 280 285 Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu 290 295 300 Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala 305 310 315 320 His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn 325 330 335 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly 340 345 350 Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu 355 360 365 Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr 370 375 380 Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp 385 390 395 400 Asn Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe 405 410 415 Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp 420 425 430 Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 435 440 445 Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys 450 455 <210> 14 <211> 235 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptide <400> 14 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40 45 Gln Ser Val Phe Arg Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Thr Gln Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Ala Thr Ser Ser Ile Ile Ile Phe Gly Gly Gly Thr Glu Val 115 120 125 Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile...

Claims

1. A method for determining the nucleotides of each of a plurality of DNA molecules to be sequenced, wherein each of the DNA molecules hybridizes with a sequencing primer to form a hybrid, the method comprising: (I) contacting the hybrid with a first set of four different nucleotide analogs and a first DNA polymerase, whereby the sequencing primer is extended by incorporating one of the four nucleotide analogs to form an extended primer, wherein the nucleotide analog is a non-labeled reversible terminating nucleotide NLRT comprising a 3' blocking group, wherein a different one of the four nucleotide analogs is added to the primer depending on whether the complementary nucleotide on the DNA molecule is adenine, thymine, cytosine or guanine; (II) performing a first labeling reaction by contacting the extended primer with an affinity reagent to form a first reaction product, wherein the affinity reagent comprises: (a) a first affinity reagent specific for a first of the four nucleotide analogs, wherein the first affinity reagent: With a label that fluoresces or luminesces at a first wavelength, or A label is provided for producing a product that fluoresces or luminesces at a first wavelength, or is recognized by a secondary affinity reagent that is labeled to fluoresce or luminesce at a first wavelength, or is recognized by a secondary affinity reagent that produces a product that fluoresces or luminesces at a first wavelength, and (b) a second affinity reagent specific for a second of the four nucleotide analogs, wherein the second affinity reagent: carries a label that fluoresces or luminesces at a second wavelength, or A label that produces a product that fluoresces or luminesces at a second wavelength, or is recognized by a secondary affinity reagent that is labeled to fluoresce or luminesce at a second wavelength, or is recognized by a secondary affinity reagent that carries a label that produces a product that fluoresces or luminesces at a second wavelength; wherein no affinity reagent specific for the third and fourth of the four nucleotide analogs is present; determining the nucleotide analog that has been added to the first reaction product by measuring fluorescence or luminescence at first and second wavelengths, wherein detection of the fluorescence or luminescence at the first wavelength indicates incorporation of the first nucleotide analog and detection of the fluorescence or luminescence at the second wavelength indicates incorporation of the second nucleotide analog; (III) removing the first and second affinity reagents from the extended primer; (IV) performing a second labeling reaction by contacting the extended primer with an affinity reagent to form a second reaction product, wherein the affinity reagent comprises: (c) a third affinity reagent specific for a third of the four nucleotide analogs, wherein the third affinity reagent: With a label that fluoresces or luminesces at a first wavelength, or A label is provided for producing a product that fluoresces or luminesces at a first wavelength, or is recognized by a secondary affinity reagent that is labeled to fluoresce or luminesce at a first wavelength, or is recognized by a secondary affinity reagent that produces a product that fluoresces or luminesces at a first wavelength, and (d) a fourth affinity reagent specific for a fourth of the four nucleotide analogs, wherein the fourth affinity reagent carries a label that fluoresces or luminesces at a second wavelength, or A label that produces a product that fluoresces or luminesces at a second wavelength, or is recognized by a secondary affinity reagent that is labeled to fluoresce or luminesce at a second wavelength, or is recognized by a secondary affinity reagent having a label that produces a product that fluoresces or emits light at a second wavelength; wherein no affinity reagents specific for the first and second of the four nucleotide analogs are present; and The nucleotide analogs added to the second reaction product are determined by measuring fluorescence or luminescence emitted at first and second wavelengths, wherein detection of fluorescence or luminescence emitted at the first wavelength indicates incorporation of the third nucleotide analog and detection of fluorescence or luminescence emitted at the second wavelength indicates incorporation of the fourth nucleotide analog.

2. The method of claim 1, wherein the first, second, third and fourth affinity reagents are antibodies.

3. The method of claim 2, wherein at least one of the antibodies is a monoclonal antibody, a single chain antibody, or a single chain Fv-reporter fusion protein.

4. The method of any one of claims 1 to 3, wherein the labels in parts (a), (b), (c) and (d) emit fluorescence.

5. The method of any one of claims 1-3, wherein the third affinity reagent is labeled with the same fluorescent label as the first affinity reagent, and the fourth affinity reagent is labeled with the same fluorescent label as the second affinity reagent.

6. The method according to any one of claims 1 to 3, further comprising the following steps: (V) removing the third and fourth affinity reagents from the extended primer.

7. The method of claim 6, wherein the third and fourth affinity reagents expose the hybrid to dissociation conditions, wherein the dissociation conditions comprise one or more of: i) adding unincorporated non-labeled reversible terminator nucleotide NLRT; ii) pH from 8 to 10; and iii) Temperature range is 50 to 75°C.

8. The method according to any one of claims 1 to 3, further comprising the steps of: (VI) Removing the 3' blocking group.

9. The method of claim 8, wherein the third and fourth affinity reagents and the 3' blocking group are removed simultaneously.

10. A massively parallel sequencing method comprising performing multiple cycles of steps (I) to (VI) according to claim 8 or 9, except that in step (I), the extended primer generated in the previous round is extended by incorporating the nucleotide analogue.

11. The method of claim 10, wherein the DNA molecules being sequenced are distributed among locations on an array, wherein a plurality of locations on the array comprise clonal populations of template DNA sequences.

12. The method of claim 8, wherein after step (V) and before step (VI), the hybrid is allowed to contact with a second DNA polymerase that is the same as or different from the first DNA polymerase and a second group of four different nucleotide analogs, wherein the second group of nucleotide analogs is an unlabeled reversible terminating nucleotide NLRT comprising a 3' blocking group that is the same as or different from the 3' blocking group of the first group.

13. The method of any one of claims 1-3, wherein the hybrid is contacted with the first, second, third and fourth nucleotide analogs simultaneously, the extended primer is contacted with the first and second affinity reagents simultaneously in the first reaction, and the extended primer is contacted with the third and fourth affinity reagents simultaneously in the second reaction.

14. The method according to any one of claims 1 to 3, wherein the four nucleotide analogs are 3'-O-azidomethyl-2'deoxyguanine; 3'-O-azidomethyl-2'deoxyadenylic acid; 3'-O-azidomethyl-2'-deoxycytosine; and 3'-O-azidomethyl-2'-deoxythymine.

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