Novel nucleotide analogs and methods of use

Abstract

The invention provides a plurality of orthogonal nucleotide analogues and methods for sequencing by synthesis using combinations of the plurality of orthogonal nucleotide analogues.

Description

Technical Field

[0001] This application is a divisional application of Chinese invention patent application filed on July 9, 2020, with application number 202080063617.1 and entitled "Novel Nucleotide Analog and Method of Use".

[0002] This application claims priority to U.S. Provisional Application No. 62 / 872,164, filed July 9, 2019, the contents of which are hereby incorporated by reference.

[0003] Throughout this application, various publications and patents have been cited. Full citations of these references can be found at the end of the description immediately preceding the claims. The disclosures of these publications and patents, in their entirety, are incorporated herein by reference to provide a more comprehensive description of the current state of the art to which this invention pertains.

[0004] This application incorporates by reference nucleotide and / or amino acid sequences present in a file named “200824_90884-A-PCT_Sequence_Listing_BI.txt”, which is 2.81 kilobytes in size and was created on August 19, 2020, in an IBM-PC machine format compatible with the MS-Windows operating system. This file is included as part of this application in a text file filed on August 24, 2020. Background Technology

[0005] DNA sequencing is a fundamental tool in biological and medical research and is particularly important for the personalized medicine paradigm. To ultimately achieve the goal of a $1,000 genome, various new DNA sequencing methods have been investigated; the primary method is sequencing-by-synthesis (SBS), a method that determines the DNA sequence during a polymerase reaction (Hyman 1988; Ronaghi et al. 1998; Ju et al. 2003; Li 2003; Braslavsky et al. 2003; Ruparel et al. 2005; Margulies et al. 2005; Ju et al. 2006; Wu et al. 2007; Guo et al. 2008; Bentley et al. 2008; Harris et al. 2008; Eid et al. 2009; Rothberg et al. 2011). Summary of the Invention

[0006] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; The cleavable connector includes DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivatives and is linked to the base via the 5-position of a pyrimidine (C, U) or the 7-position of a desaturated purine (A, G, I); and The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for dye bonding, clusters of anchors for dye bonding, or anchors and dyes.

[0007] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; R includes methyl, ethyl, propyl, tert-butyl, aryl, and alkylaryl; Cuttable connectors include DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivatives; and The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for linking fluorescent dyes, clusters of anchors for linking fluorescent dyes, or anchors and dyes.

[0008] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; Cleavable connectors include DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivatives, or more than one of these cleavable connectors, including the special case where one cleavable connector is present between the base and the blocker and a second different cleavable connector is present between the blocker and the marker; The blocking agent is a nucleotide or oligonucleotide comprising 2-50 monomeric units of a basic sugar or modified nucleoside or a combination thereof; and the blocking agent is linked to the 5-position of pyrimidine (C, U) and the 7-position of desaminoglycan (A, G, I) via a cleavable linker. The blocking agent is the part that prevents further incorporation of other nucleotides or nucleotide analogs into the primer chain after incorporation; and The markers include fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for linking fluorescent dyes, clusters of anchors for linking fluorescent dyes, or anchors and dyes, wherein the markers are linked to the blocking agent.

[0009] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, uracil, thymine, hypoxanthine, or analogues thereof; and R is a cleavable chemical group, including alkyl DTM, azo, 2-nitrobenzyl, allyl, and azidomethyl derivatives.

[0010] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; and The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for dye bonding, clusters of anchors for dye bonding, or anchors and dyes.

[0011] This invention provides a nucleotide analog having the following structure: , The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof.

[0012] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for dye bonding, clusters of anchors for dye bonding, or anchors and dyes; and R includes methyl, ethyl, propyl, tert-butyl, aryl, and alkylaryl.

[0013] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; and R includes methyl, ethyl, propyl, tert-butyl, aryl, and alkylaryl.

[0014] This invention provides a nucleotide analog having the following structure: , The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof.

[0015] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; and (B) an anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) an anchor-labeled nucleotide analog comprising a base and an anchor attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable connector and the 3'-OH group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) an anchor-labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and an anchor connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable joint described herein can be cut with the same cutting agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog provided in step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signal generated due to the binding of the anchor binding group to any anchor-tagged nucleotide analog incorporated in step (b); g) Repeat steps (b)-(f) with two different labeled nucleotide analogs, the two different labeled nucleotide analogs being different from the two different labeled nucleotide analogs from the previous iterations of step (b); h) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any markers, anchors or blocking groups from the incorporated nucleotide analogue of step (b); i) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0016] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each of which hybridizes with primers, wherein each template has the same sequence as the nucleic acid to be sequenced, and provide a nucleic acid polymerase; b) The nucleic acid template is contacted with four different labeled nucleotide analogs (A, C, T, G), and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; (B) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label linked to the base via a cleavable linker; (C) Two different anchor-labeled dideoxynucleotide analogs, wherein each analog comprises a different anchor linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (C) Two different anchor-labeled nucleotide analogs comprising a base and an anchor attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein each analog comprises a different anchor attached to the base via a cleavable linker. The cuttable connector and the 3'-O blocking group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a fluorescent label attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A pH-responsive fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a pH label attached to the base distal to the blocking group. The responsive fluorescent label, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, and (C) two different anchor-labeled nucleotide analogs, the two different anchor-labeled nucleotide analogs comprising a base, a blocking group attached to the base via a cleavable linker, and an anchor attached to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, wherein each analog comprises a different anchor attached to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); e) Contact the incorporated nucleotide analog from step (b) with: (A) an anchor-binding group that binds to the anchor of only one of the anchor-labeled nucleotide analogs of step (b), wherein the anchor-binding group comprises the same fluorescent label as the fluorescently labeled nucleotide analog of step (b); and (B) an anchor-binding group that binds to the anchor of only the remaining anchor-labeled nucleotide analogs, wherein the anchor-binding group comprises the same pH-responsive fluorescent label as the pH-responsive fluorescently labeled nucleotide analog of step (b). f) Wash away any unincorporated nucleotide analogs at pH at which the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the anchor-labeled nucleotide analog from step (b). g) Wash the incorporated nucleotide analog from step (b) at a pH where the pH-responsive fluorescent label no longer has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b). h) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any markers, anchors or blocking groups from the incorporated nucleotide analogue of step (b); i) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0017] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; and (B) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a pH-responsive fluorescently labeled nucleotide analog comprising a base and a pH-responsive fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable connector and the 3'-O blocking group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a pH-responsive fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a pH-responsive fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the cleavable linker is cleavable by the same cleaving agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); e) Repeat steps (b)-(d) with two different labeled nucleotide analogs, which are different from the two different labeled nucleotide analogs from the previous iterations of step (b); f) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); Steps (e) and (f) can be performed in reverse order; g) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any labeling or blocking groups from the incorporated nucleotide analogue of step (b); h) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0018] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a first cleavable linker; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a carbamoyl TCO linker; (C) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via the first cleavable linker; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via a carbamoyl TCO linker. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label linked to the base via a first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label linked to the base via a carbamoyl TCO linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) Anchor-labeled nucleotide analog, wherein... Anchor-tagged nucleotide analogs include a base and an anchor linked to the base via a first cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) anchor-tagged nucleotide analogs comprising a base and an anchor linked to the base via a carbamoyl TCO linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the 3'-O blocking group and the first cleavable linker can be cleaved by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a carbamoyl TCO linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. (C) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a first cleavable linker, and an anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain; and (D) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a carbamoyl TCO linker, and an anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain. The fluorescent label on each analogue is identical. The anchors on each analogue are identical; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog provided in step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signal generated due to the binding of the anchor binding group to any anchor-tagged nucleotide analog incorporated in step (b); g) Contact the incorporated nucleotide analog with the tetrazine derivative to click the TCO portion of the carbamoyl TCO linker to release any tags or anchors attached through the carbamoyl TCO linker, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b). h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the first cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0019] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a first anchor; (C) Anchor-labeled dideoxynucleotide analog comprising a base and a first anchor and a second anchor attached to the base via a cleavable linker; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and a second anchor attached to the base via a cleavable linker, wherein the cleavable linker is cleavable by the same cleaving agent. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a first anchor and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) Anchor-labeled nucleotide analog, wherein the anchor... The labeled nucleotide analogue comprises a base and a first anchor and a second anchor connected to the base via the cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analogue into the extended primer chain, and (D) an anchor-labeled nucleotide analogue comprising a base and a second anchor connected to the base via the cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analogue into the extended primer chain, wherein the cleavable linker and the 3'-O blocking group can be cleaved by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group and a first anchor, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (C) Anchor-labeled nucleotides. (A) Anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a cleavable linker, and a first anchor and a second anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, and (D) Anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a cleavable linker, and a second anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, wherein the cleavable linker can be cleaved by the same cleaving agent. The fluorescent labeling on each analogue is identical; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the second anchor of the nucleotide analog from step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signals generated due to the binding of the anchoring group to the anchoring group of any incorporated nucleotide analogue in step (b); g) Contact the incorporated nucleotide analog with a second anchoring group, the second anchoring group being bound to the first anchor of the nucleotide analog in step (b) and including a portion that quenches the fluorescence signal of any fluorescently labeled nucleotide analog linked to the anchoring group, and identifies any fluorescence signal generated due to the incorporation of the fluorescently labeled nucleotide analog. h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0020] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable adapter; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via the first cleavable adapter and a carbamoyl TCO adapter attached to the distal end of the first cleavable adapter; (C) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor attached to the base via the first cleavable adapter and a carbamoyl TCO adapter attached to the distal end of the first cleavable adapter; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor attached to the base via the first cleavable adapter. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a carbamoyl TCO linker attached to the distal end of the first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) Anchor-labeled nucleotide analog, wherein... Anchor-tagged nucleotide analogs include a base and an anchor linked to the base via a first cleavable adapter and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) anchor-tagged nucleotide analogs comprising a base and an anchor linked to the base via the first cleavable adapter and a carbamoyl TCO adapter connected to the distal end of the first cleavable adapter, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the 3'-OH blocking group and the first cleavable adapter can be cleaved by the same reagent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via the first cleavable linker, and a fluorescent label connected to the base via a carbamoyl TCO linker distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. (C) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a first cleavable adapter, and an anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain; and (D) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via the first cleavable adapter, and an anchor connected to the base via a carbamoyl TCO adapter at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain. The fluorescent label on each analogue is identical. The anchors on each analogue are identical; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals generated due to the incorporation of the fluorescently labeled nucleotide analog in step (b); e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog provided in step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signal generated due to the binding of the anchor binding group to any anchor-tagged nucleotide analog incorporated in step (b); g) Contact the incorporated nucleotide analog with the tetrazine derivative to click the TCO portion of the carbamoyl TCO linker to release any tags or anchors attached through the carbamoyl TCO linker, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b). h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the first cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0021] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable adapter; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via the first cleavable adapter and a carbamoyl TCO adapter attached to the distal end of the first cleavable adapter; (C) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label attached to the base via the first cleavable adapter and a carbamoyl TCO adapter attached to the distal end of the first cleavable adapter; and (D) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label attached to the base via the first cleavable adapter. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a carbamoyl TCO linker attached to the distal end of the first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a carbamoyl TCO linker attached to the distal end of the first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; and (D) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a carbamoyl TCO linker attached to the distal end of the first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; and (E) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a carbamoyl TCO linker attached to the distal end of the first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; and (F) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a fluorescent label attached to the base via a first cleavable linker and a blocking group at the distal end of the first cleavable linker and a blocking group at the distal end Nucleotide analogs include a base and a pH-responsive fluorescent label linked to the base via a first cleavable adapter and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) pH-responsive fluorescently labeled nucleotide analogs, wherein the pH-responsive fluorescently labeled nucleotide analogs include a base and a pH-responsive fluorescent label linked to the base via the first cleavable adapter and a carbamoyl TCO adapter linked to the distal end of the first cleavable adapter, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the 3'-OH blocking group and the first cleavable adapter can be cleaved by the same reagent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via the first cleavable linker, and a fluorescent label connected to the base via a carbamoyl TCO linker distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (C) A pH-responsive fluorescently labeled nucleotide analog. Nucleotide analogs, the pH-responsive fluorescently labeled nucleotide analogs comprising a base, a blocking group connected to the base via a first cleavable linker, and a pH-responsive fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) pH-responsive fluorescently labeled nucleotide analogs comprising a base, a blocking group connected to the base via the first cleavable linker, and a pH-responsive fluorescent label connected to the base via a carbamoyl TCO linker at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analogs at a pH where the pH-responsive fluorescent label does not have the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b); e) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b), wherein steps (d) and (e) may be performed in reverse order. f) Contact the incorporated nucleotide analogue with a tetrazine to click the TCO portion of the carbamoyl TCO linker to release any tags attached through the carbamoyl TCO linker; g) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the first cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0022] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label and an anchor attached to the base via a cleavable linker; (C) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label attached to the base via a cleavable linker; and (D) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label and an anchor attached to the base via a cleavable linker, wherein the cleavable linker can be cleaved by the same cleaving agent. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label and anchor attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) A pH-responsive fluorescently labeled nucleotide analog, wherein the pH-responsive fluorescent label... The nucleotide analogue comprises a base and a pH-responsive fluorescent label linked to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analogue into the extended primer chain; and (D) a pH-responsive fluorescently labeled nucleotide analogue comprising a base and a pH-responsive fluorescent label and anchor linked to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analogue into the extended primer chain, wherein the cleavable linker and the 3'-O blocking group can be cleaved by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a fluorescent label attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a fluorescent label and anchor attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into an extended primer chain; (C) A pH-responsive fluorescently labeled nucleotide analog, wherein the pH The responsive fluorescently labeled nucleotide analogue comprises a base, a blocking group attached to the base via a cleavable linker, and a pH-responsive fluorescent label attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analogue into the extended primer chain, and (D) a pH-responsive fluorescently labeled nucleotide analogue comprising a base, a blocking group attached to the base via a cleavable linker, and a pH-responsive fluorescent label and anchor attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analogue into the extended primer chain, wherein the cleavable linker can be cleaved by the same cleaving agent. c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analogs at a pH where the pH-responsive fluorescent label does not have the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b); e) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b), wherein steps (d) and (e) may be performed in reverse order. f) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the nucleotide analog from step (b), wherein the anchor binding group includes a portion that quenches the fluorescent label of the fluorescently labeled nucleotide analog from step (b). g) Wash away any unbound anchoring groups, including quenching moieties, at pH at which the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b). h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0023] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker; and (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label and an anchor attached to the base via a cleavable linker, wherein the cleavable linker can be cleaved by the same cleaving agent. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; and (B) a fluorescently labeled nucleotide analog comprising a base and a fluorescent label and anchor attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain, wherein the cleavable linker and the 3'-OH blocking group are cleavable by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a fluorescent label attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a fluorescent label and anchor attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the cleavable linker is cleavable by the same cleaving agent. c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Repeat steps (b)-(d) with two different labeled nucleotide analogs, which are different from the two different labeled nucleotide analogs from the previous iterations of step (b); f) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the nucleotide analog from step (b), wherein the anchor binding group includes a portion that quenches the fluorescent label of the fluorescently labeled nucleotide analog from step (b). g) Identify any fluorescent signals generated due to the incorporation of fluorescently labeled nucleotide analogs in step (b); h) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any markers, anchors or blocking groups from the incorporated nucleotide analogue of step (b); i) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0024] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each of which hybridizes with primers, wherein each template has the same sequence as the nucleic acid to be sequenced, and provide a nucleic acid polymerase; b) The nucleic acid template is contacted with four different labeled nucleotide analogs (A, C, T, G), and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via an uncleavable linker; (C) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor attached to the base via a cleavable linker; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor attached to the base via an uncleavable linker. c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals generated due to the incorporation of the fluorescently labeled nucleotide analog in step (b); e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog from step (b); f) Identify any fluorescent signals generated due to the incorporation of the fluorescently labeled nucleotide analog in step (b); g) Contact the incorporated nucleotide analog in step (b) with a reagent that cleaves the cleavable linker of the nucleotide analog in step (b) and cleaves the 3'-O blocking group of the nucleotide analog in step (c). h) Identify any fluorescent signals generated due to the incorporation of fluorescently labeled nucleotide analogs in step (b); i) The nucleotide analog incorporated in step (b) of photobleaching step (b) is used to photobleach any remaining fluorescent labeling; and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0025] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) a fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and (B) a fluorescently labeled dideoxynucleotide analog comprising a base and a different fluorescent label attached to the base via a cleavable linker, wherein the cleavable linker is cleavable by the same cleaving agent. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a fluorescently labeled nucleotide analog comprising a base and a different fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the cleavable linker and the 3'-OH blocking group are cleavable by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a different fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the cleavable linker is cleavable by the same cleaving agent; c) Contact the nucleic acid template with an unlabeled nucleotide analog (A, C, T, G) that does not have any base modifications and includes a 3'-O blocking group, wherein the 3'-O blocking group can be cleaved by the same cleavable linker and / or the blocking group of the two labeled nucleotide analogs of step (b), and extend any unextended primers with the unlabeled nucleotide analogs, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Repeat steps (b)-(d) with two different labeled nucleotide analogs different from the two different labeled nucleotide analogs from the previous iterations of step (b), but only with two unlabeled nucleotides including a 3'-O blocking group that are different from the two labeled nucleotide analogs added in this step. f) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any labeling or blocking groups from the incorporated nucleotide analogue of steps (b) and (c); g) Identify any fluorescent signal generated due to the incorporation of the fluorescently labeled nucleotide analog in step (b); and h) Repeat steps (b) to (g) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0026] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each of which hybridizes with primers, wherein each template has the same sequence as the nucleic acid to be sequenced, and provide a nucleic acid polymerase; b) The nucleic acid template is contacted with four different labeled nucleotide analogs (A, C, T, G), and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; (B) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label linked to the base via a cleavable linker; (C) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label and an anchor linked to the base via a cleavable linker; and (D) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label and an anchor linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; (C) Fluorescent... (A) Optically labeled nucleotide analogs, the fluorescently labeled nucleotide analogs comprising a base and a fluorescent label and anchor linked to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (D) pH-responsive fluorescently labeled nucleotide analogs, the pH-responsive fluorescently labeled nucleotide analogs comprising a base and a fluorescent label and the same anchor linked to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable connector and the 3'-O blocking group can be cut by the same cutting agent; or (iii) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A pH-responsive fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a pH-responsive fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (C) A fluorescently labeled... The fluorescently labeled nucleotide analogue comprises a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label and anchor connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analogue into the extended primer chain, and (D) a pH-responsive fluorescently labeled nucleotide analogue comprising a base, a blocking group connected to the base via a cleavable linker, and a pH-responsive fluorescent label and the same anchor connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analogue into the extended primer chain. The cuttable joint described herein can be cut with the same cutting agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analogs at a pH where the pH-responsive fluorescent label does not have the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b); e) Wash away any unincorporated nucleotide analogs at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the anchor-labeled nucleotide analog from step (b); f) Contact the incorporated nucleotide analog with an anchor binding group, the anchor binding group being bound to the anchor of the nucleotide analog in step (b) and including a portion that quenches the fluorescence signal of any fluorescently labeled nucleotide analog linked to the anchor binding group, and identify any fluorescence signal generated due to the incorporation of the fluorescently labeled nucleotide analog. g) Contacting the incorporated nucleotide analog with a cleaving agent that cleaves the cleavable linker and any 3'-O blocking group; and h) Repeat steps (b) to (g) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0027] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each of which hybridizes with the primers; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) Two fluorescently labeled nucleotide analogs, said two fluorescently labeled nucleotide analogs comprising a base and a fluorescent label serving as an energy transfer donor, connected to said base via a cleavable linker, an anchor for connecting an energy transfer acceptor label, and a blocking group at the 3'-OH position, said blocking group preventing subsequent incorporation of the nucleotide analog into the extended primer chain, The cuttable connector and the 3'-O blocking group can be cut by the same cutting agent; Each of the nucleotide analogues described herein has a different anchor; or (ii) Two fluorescently labeled nucleotide analogs, said two fluorescently labeled nucleotide analogs comprising a base, a blocking group connected to said base via a cleavable linker, and a fluorescent energy transfer donor label connected to a base linker distal to said blocking group and an anchor for connecting an energy transfer acceptor label, wherein said blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, The cutable joint described herein can be cut with the same cutting agent, and Each of the nucleotide analogues has a different anchor. c) Wash away any unincorporated nucleotide analogs and contact the incorporated nucleotide analogs with two anchoring groups that specifically bind to each of the anchors of the nucleotide analogs from step (b) and include a portion that functions as an energy transfer acceptor. The energy transfer acceptor on one of the anchoring groups is a pH-insensitive marker, and the energy transfer acceptor on the other anchoring group is a pH-responsive marker. d) Wash away any free label at a pH where the pH-responsive fluorescent energy transfer receptor dye label has the same or similar absorption and emission curves as the pH-non-responsive fluorescent energy transfer receptor label; e) Expose the incorporated nucleotide to a wavelength capable of exciting the energy transfer donor dye, and identify any fluorescence signals resulting from energy transfer and emission of the energy transfer acceptor dye linked to the nucleotide analogue due to the labeling reaction performed in step (c); f) Repeat steps (b) to (e) with two different labeled nucleotide analogs, which are different from the two different labeled nucleotide analogs in (b), but otherwise have all the other properties described in (b); g) Change the buffer solution to a pH when the pH-responsive fluorescent label does not have the same or similar absorption and emission curves as the pH-non-responsive fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescence signal generated due to the incorporation of the anchor-labeled nucleotide analog from step (b) or (f), wherein the order of steps (e) and (g) may be reversed. h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the cleavable linker and the 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0028] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each of which hybridizes with primers, wherein each template has the same sequence as the nucleic acid to be sequenced, and provide a nucleic acid polymerase; b) The nucleic acid template is contacted with four different labeled nucleotide analogs (A, C, G, T), and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) A fluorescently labeled nucleotide analog comprising a base, and a fluorescent label serving as an energy transfer donor and an anchor (anchor 1) for linking a pH-insensitive energy transfer acceptor label connected to the base via a first cleavable adapter (cleavable adapter 1), and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; (b) A fluorescently labeled nucleotide analog comprising a base, and both a fluorescent label serving as an energy transfer donor and a second anchor (anchor 2) for linking a pH-responsive energy transfer acceptor label connected to the base via the same cleavable adapter (cleavable adapter 1), and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; (c) A fluorescent... A fluorescently labeled nucleotide analog comprising a base, and a fluorescent label serving as an energy transfer donor connected to the base via a second cleavable adapter (cleavable adapter 2), and a first anchor (anchor 1) for connecting a pH-insensitive energy transfer acceptor label, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) a fluorescently labeled nucleotide analog comprising a base, and a fluorescent label serving as an energy transfer donor connected to the base via a second cleavable adapter (cleavable adapter 2), and a second anchor (anchor 2) for connecting a pH-responsive energy transfer acceptor label, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, The first cuttable connector and the 3'-O blocking group can be cut by the same cutting agent, and the second cuttable connector can be cut by a different cutting agent; or (ii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable adapter (cleavable adapter 1), a fluorescent energy transfer donor tag connected to a base adapter distal to the blocking group, and an anchor (anchor 1) for connecting a pH-insensitive energy transfer acceptor tag, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable adapter (cleavable adapter 1), a fluorescent energy transfer donor tag connected to a base adapter distal to the blocking group, and a second anchor (anchor 2) for connecting a pH-responsive energy transfer acceptor tag, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, (C) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a second cleavable adapter (cleavable adapter 2), a fluorescent energy transfer donor tag connected to a base adapter distal to the blocking group, and a first anchor (anchor 1) for attaching a pH-insensitive energy transfer acceptor tag, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) a fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a second cleavable adapter (cleavable adapter 2), a fluorescent energy transfer donor tag connected to a base adapter distal to the blocking group, and a second anchor (anchor 2) for attaching a pH-responsive energy transfer acceptor tag, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, Each cuttable joint can be cut with a different cutting agent; c) Wash away any unincorporated nucleotide analogs and contact the incorporated nucleotide analogs with two anchoring groups that specifically bind to each of the anchors of the nucleotide analogs from step (b) and include a portion that functions as an energy transfer acceptor. The energy transfer acceptor on one of the anchoring groups is a pH-insensitive marker, and the energy transfer acceptor on the other anchoring group is a pH-responsive marker. d) Wash away any free label at a pH where the pH-responsive fluorescent energy transfer receptor dye label has the same or similar absorption and emission curves as the pH-non-responsive fluorescent energy transfer receptor label; e) Expose the incorporated nucleotide to a wavelength capable of exciting the energy transfer donor dye and identify any fluorescence signals generated due to energy transfer and emission of the energy transfer acceptor dye linked to the nucleotide analogue incorporated in step (b) as a result of the labeling reaction performed in step (c); f) Change the buffer solution to a pH when the pH-responsive fluorescent label does not have the same or similar absorption and emission curves as the pH-non-responsive fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescence signal generated due to the incorporation of the anchor-labeled nucleotide analog from step (b) as a result of the labeling reaction performed in step (c), wherein steps (d) and (f) may be reversed. g) Contact the incorporated nucleotide analogue with the cutting agent that cuts the second cuttable connector; h) Wash away the cleavage agent and the released label at a pH where the pH-responsive fluorescent energy transfer receptor dye label has the same or similar absorption and emission curves as the pH-non-responsive fluorescent energy transfer receptor label; i) Repeat step (e); j) Contacting the incorporated nucleotide analog with a cleaving agent that cleaves the first cleavable linker and the 3'-O blocking group; and k) Repeat steps (b) to (j) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained. Attached Figure Description

[0029] Figure 1 A general set of dyes and anchors for single-color SBS labeling consists of cleavable ddNTP analogs and labeling reagents: two dideoxynucleotide analogs with anchors (e.g., biotin) and two with dyes (e.g., Cy5). The labeled molecule consists of a molecule (streptavidin) capable of specifically binding to the anchor and the same dye. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0030] Figure 2 : Used for the use of cleavable nucleotide analogs (such as in Figure 3A simplified representation of a monochromatic SBS scheme for the cleavable nucleotide analogs presented in the diagram. Two of the ddNTPs have Cy5 linked via an SS linker, and the other two have biotin linked via an SS linker. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is C, G, T, or A from left to right. When using Therminator IX polymerase, four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-) are used to extend most primers. O After incubation with two ddNTP analogs (i.e., ddT with Cy5 and ddA with biotin) and streptavidin-Cy5, imaging reveals a positive signal in the right rectangular region (indicating primer extension with T) and a background signal in the remaining region. After labeling with streptavidin-Cy5, imaging reveals a new positive signal in the third region, indicating A incorporation. Next, incubation with the remaining ddNTP analogs (i.e., ddC with biotin and ddG with Cy5) (along with excess A and T NRT) is performed, and imaging reveals a new positive signal in the left region, indicating G incorporation. Relabeling with streptavidin-Cy5 results in a signal in the remaining rectangular region, indicating C incorporation. Finally, THP treatment is used to cleave the SS adapter and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, 3, and 4 on the left represent the cumulative signal at each of the four indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all four imaging steps, 0111 represents A, 0001 represents C, 0011 represents G, and 1111 represents T; considering only the first three of these imaging steps, 011 represents A, 000 represents C, 001 represents G, and 111 represents T).

[0031] Figure 3 Example ddNTP analogues for Figure 4.

[0032] Figures 4A-4B Monochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs, two of which contained biotin and two of which contained Cy5, with only SS adapters. ddNTP-cleavable adapter-dyes (ddGTP-7-SS-Cy5, ddTTP-5-SS-Cy5), ddNTP-cleavable adapter-anchors (ddCTP-5-SS-Biotin, ddATP-7-SS-Biotin), and 3'-O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) are used to perform monochromatic DNA SBS. Step 1 involves using Therminator IX DNA polymerase, two of the ddNTP analogs (ddTTP-5-SS-Cy5, ddATP-7-SS-Biotin), and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding ddTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template allows for the incorporation of complementary nucleotide reversible terminator analogs into most (>90%) of the growing DNA strand to terminate DNA synthesis, and allows for the extension of a small subset of primers with ddA or ddT analogs (complementary to T or A in the template strand). Step 2: After washing away unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal primers extended with ddTTP-5-SS-Cy5. Step 3: Adding streptavidin-Cy5 to label any incorporated ddATP-7-SS-biotin analogs. Step 4: After washing away unused labeling reagents, a second imaging step will reveal ddA incorporation. Step 5: Subsequently, using Therminator IX DNA polymerase and the remaining ddNTP analogs (ddGTP-SS-Cy5, ddCTP-5-SS-biotin) and 3'- to ensure incorporation fidelity... O -azidomethyl-dATP and 3'- O -Azide-methyl dTTP extension will extend most of the remaining fixed primer-bound DNA template, especially those strands opposite G and C. Following this step, the growing DNA strand terminates with one of four dye-labeled dideoxynucleotide analogs (A, C, G, T) or the same nucleotide analog among four 3'-blocking reversible terminator nucleotide analogs (A, C, G, T) without dye. Step 6, after washing away unincorporated nucleotides, performs a third imaging step. A positive signal will indicate the incorporation of ddG. At this point, four 3'- O- Azidoxymethyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either ddNTPs or NRT analogs. Step 7: Streptavidin-Cy5 is used again to ligate Cy5 to any incorporated ddC-5-SS-biotin analogs. Step 8: After washing away unused labeling reagents, a fourth round of imaging is performed. The gain of the Cy5 signal indicates the incorporation of ddC. Step 9: The SS adapter is cleaved by adding THP to the extended DNA strand, thus removing all dye from the ddNTP analogs and also restoring the 3'- O -Azide-methyl-dNTP extended 3'-OH groups on any growing strand. After washing away the cleaved dye, an optional final round of imaging is performed. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 3 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0033] Figure 5 A general set of cleavable dNTP-blocking (virtual terminator) analogs and labeling reagents for single-color SBS dye and anchor labeling: two of the virtual terminator analogs have an anchor (e.g., biotin) and two have a dye (e.g., Cy5). The labeled molecule consists of a molecule (streptavidin) capable of specifically binding to the anchor and the same dye. Four unlabeled reversible terminators (e.g., 3'- O (-azidomethyl dNTP) is further extended.

[0034] Figure 6 : Used for the use of cleavable nucleotide analogs (such as in Figure 5 A simplified representation of a monochromatic SBS scheme for the cleavable nucleotide analogs presented in the diagram. Two of the dNTP-blocking virtual terminators have Cy5 linked via an SS linker, and the other two have biotin linked via an SS linker. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template is C, G, T, or A from left to right. When using Therminator IX polymerase, four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-) are used to extend most primers. OAfter incubation with two of the dNTPs (-azidomethyl dNTPs) and two of the virtual terminator analogs (i.e., dT with Cy5 and dA with biotin), imaging will reveal a positive signal in the right rectangular region (indicating primer extension with T) and a background signal in the remaining region. After labeling with streptavidin-Cy5, imaging will reveal a new positive signal in the third region, indicating A incorporation. Next, incubation with the remaining virtual terminator analogs (i.e., dC with biotin and dG with Cy5) (as well as excess A and T NRTs) is performed, and imaging will reveal a new positive signal in the left region, indicating G incorporation. Relabeling with streptavidin-Cy5 will result in a signal in the remaining rectangular region, indicating C incorporation. Finally, THP treatment is used to cleave the SS adapter and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, 3, and 4 on the left represent the cumulative signal at each of the four indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all four imaging steps, 0111 represents A, 0001 represents C, 0011 represents G, and 1111 represents T; considering only the first three of these imaging steps, 011 represents A, 000 represents C, 001 represents G, and 111 represents T).

[0035] Figure 7 Examples of virtual terminator analogs and labeled molecules used in Figure 8.

[0036] Figures 8A-8B Monochrome sequencing-by-synthesis was performed using a set of dNTP-blocker (virtual terminator) analogues, two of which contained biotin and two contained Cy5, with only SS adapters. The following methods were used: dNTP-cleavable adapter-blocker-dye (dGTP-7-SS-blocker-Cy5, dTTP-5-SS-blocker-Cy5), dNTP-cleavable adapter-blocker-anchor (dCTP-5-SS-blocker-Biotin, dATP-7-SS-blocker-Biotin), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O-Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) are used to perform monochromatic DNA SBS. Step 1 involves using Therminator IX DNA polymerase, two of the dNTP-blocker (virtual terminator) analogues (dTTP-5-SS-blocker-Cy5, dATP-7-SS-blocker-biotin), and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding dTTP (-azidomethyl-dTTP) to a fixed, primer-bound DNA template allows for the incorporation of a complementary nucleotide reversible terminator analog into most (>90%) of the growing DNA strand to terminate DNA synthesis, and allows for the extension of a small subset of primers with dA or dT analogs (complementary to T or A in the template strand). Step 2, after washing away unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal primers extended with dTTP-5-SS-blocker-Cy5. Step 3, adding streptavidin-Cy5 to label any incorporated dATP-7-SS-blocker-biotin analogs. Step 4, after washing away unused labeling reagents, a second imaging step will reveal dA incorporation. Step 5, followed by Therminator IX DNA polymerase and the remaining dNTP-blocking (virtual terminator) analogs (dGTP-SS-blocker-Cy5, dCTP-5-SS-blocker-Biotin) and 3'- to ensure incorporation fidelity. O -azidomethyl-dATP and 3'- O -Azide-methyl dTTP extension will extend most of the remaining immobilized primer-bound DNA template, especially those strands opposite G and C. Following this step, the growing DNA strand terminates with one of four labeled dummy terminator nucleotide analogs (A, C, G, T) or the same nucleotide analog of one of four 3'-blocking reversible terminator nucleotide analogs (A, C, G, T) without dye. Step 6: After washing away unincorporated nucleotides, a third imaging step is performed. A positive signal will indicate the incorporation of dG. At this point, four 3'- O- Azidoxymethyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either a dNTP dummy terminator or an NRT analog. Step 7: The primer is re-labeled with streptavidin-Cy5 to ligate any incorporated dC-5-SS-blocker-biotin analog. Step 8: After washing away unused labeling reagents, a fourth round of imaging is performed. The gain of the Cy5 signal indicates the incorporation of dC. Step 9: The SS adapter is cleaved by adding THP to the extended DNA strand, such that all dye on the dNTP dummy terminator nucleotide analog is removed, and the 3'- O -Azide-methyl-dNTP extended 3'-OH groups on any growing strand. After washing away the cleaved dye, an optional final round of imaging is performed. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 7 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0037] Figure 9 A general set of dye and anchor labeling agents for monochromatic SBS using cleavable 3'-blocking reversible terminator analogs and labeling reagents: two of the reversible terminator analogs have an anchor (e.g., biotin) and two have a dye (e.g., Cy5). The labeled molecule consists of a molecule (streptavidin) capable of specifically binding to the anchor and the same dye. Four unlabeled reversible terminators (e.g., 3'- O (-azidomethyl dNTP) is further extended.

[0038] Figure 10 : Used for the use of cleavable nucleotide analogs (such as in Figure 9 A simplified representation of a monochromatic SBS scheme for the cleavable nucleotide analogues presented in the diagram. Two of the 3'-blocking reversible nucleotide terminators have Cy5 linked via an SS linker, and the other two have biotin linked via an SS linker. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template is C, G, T, or A from left to right. When using Therminator IX polymerase, with four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-) used to extend most primers. OAfter incubation with two of the labeled reversible terminator analogs (i.e., dT with Cy5 and dA with biotin), imaging will reveal a positive signal in the right rectangular region (indicating primer extension with T) and a background signal in the remaining region. After labeling with streptavidin-Cy5, imaging will reveal a new positive signal in the third region, indicating A incorporation. Next, incubation with the remaining reversible terminator analogs (i.e., dC with biotin and dG with Cy5) (along with excess unlabeled A and T NRT) is performed, and imaging will reveal a new positive signal in the left region, indicating G incorporation. Relabeling with streptavidin-Cy5 will result in a signal in the remaining rectangular region, indicating C incorporation. Finally, THP treatment is used to cleave the SS adapter and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, 3, and 4 on the left represent the cumulative signal at each of the four indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all four imaging steps, 0111 represents A, 0001 represents C, 0011 represents G, and 1111 represents T; considering only the first three of these imaging steps, 011 represents A, 000 represents C, 001 represents G, and 111 represents T).

[0039] Figure 11 Examples of labeled reversible terminator analogs and labeled molecules used in Figure 12.

[0040] Figure 12A-12B Use a set of 3'- O - Blocked nucleotide reversible terminator analogs were synthesized and sequenced in monochromatic mode, the set of 3'- O - Two of the blocked nucleotide reversible terminator analogs contain biotin and two contain Cy5, with only one having an SS linker. 。 Use 3'- O -SS-dNTP-cuttable connector-dye (3'- O -SS-dGTP-7-SS-Cy5、3'- O -SS-dTTP-5-SS-Cy5), 3'- O -SS-dNTP-Cutable Connector-Anchor (3'- O -SS-dCTP-5-SS-Biotin, 3'- O -SS-dATP-7-SS-Biotin), 3'- O -Azide-methyl-dNTP(3'- O-azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase, 3'- O -SS-dNTP-dyne analogues or 3'- O Two types of -SS-dNTP-anchor analogues (3'- O -SS-dTTP-5-SS-Cy5、3'- O -SS-dATP-7-SS-Biotin) and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding dTTP (-azidomethyl-dTTP) to a fixed, primer-bound DNA template allows for the incorporation of complementary, unlabeled nucleotide reversible terminator analogs into most (>90%) of the growing DNA strand to terminate DNA synthesis, and allows for the extension of a small subset of primers with labeled dA or dT analogs (complementary to T or A in the template strand). Step 2, after washing away the unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal those primers extended with 3'- O -SS-dTTP-5-SS-Cy5 extended primers. Step 3, add streptavidin-Cy5 to label any incorporated 3'- O -SS-dATP-7-SS-Biotin analogue. Step 4, after washing away unused labeling reagents, a second imaging step will reveal the incorporation of dA. Step 5, subsequently using Therminator IX DNA polymerase and the remaining 3'- O -SS-dNTP-dyne analogues or 3'- O -SS-dNTP-anchor analogue (3'- O -SS-dGTP-SS-Cy5、3'- O -SS-dCTP-5-SS-Biotin) and 3'- O -azidomethyl-dATP and 3'- O-Azide-methyl dTTP extension will extend most of the remaining immobilized primer-bound DNA template, especially those strands opposite G and C. Following this step, the growing DNA strand terminates with one of four labeled reversible terminator nucleotide analogs (A, C, G, T) or the same nucleotide analog of one of the four unlabeled 3'-blocking reversible terminator nucleotide analogs (A, C, G, T). Step 6, after washing away unincorporated nucleotides, performs a third imaging step. A positive signal will indicate the incorporation of dG. Four 3'- O - Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either a labeled dNTP reversible terminator or an unlabeled NRT analog. Step 7, again labeled with streptavidin-Cy5 to link Cy5 to any incorporated 3'- O -SS-dC-5-SS-Biotin analogue. Step 8: After washing away unused labeling reagents, perform a fourth round of imaging. The gain of the Cy5 signal indicates the incorporation of dC. Step 9: Cleave the SS adapter by adding THP to the elongated DNA strand, thereby removing all dye from the labeled reversible terminator nucleotide analogue and also restoring any 3'-OH groups on the elongated strand. After washing away the cleaved dye, perform an optional final round of imaging. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 11 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0041] Figure 13 A general set of dye and anchor labeling reagents for monochromatic SBS with two spectrally equivalent dyes (i.e., Cy5 and HCyC-646, where the latter dye is pH-responsive): one dideoxynucleotide analog with a biotin anchor, one with a tetrazine anchor, one with a Cy5 dye, and one with HCyC-646. The labeling molecule consists of a molecule capable of specifically binding to one of the anchors (for biotin, the molecule is streptavidin; or for tetrazine, the molecule is TCO) and the same two dyes. This hybrid SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0042] Figure 14 : Used for the use of cleavable nucleotide analogs (such as in Figure 15A simplified representation of a monochromatic SBS scheme for cleavable nucleotide analogs (presented in the text). One of the ddNTPs has Cy5 linked to a base via an SS linker, one has HCyC-646 linked to a base via an SS linker, one has biotin linked to a base via an SS linker, and one has tetrazine linked to a base via an SS linker. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is C, G, T, or A from left to right. When using Therminator IX polymerase, four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-) are used to extend most primers. O After incubation with 3'-azidomethyl dNTPs, further extension was performed using Thermo Sequenase and four ddNTP analogs: ddA linked to biotin, ddT linked to Cy5, ddC linked to tetrazine, and ddG linked to HCyC-646. Finally, washing at pH 5 ensured that HCyC-646 would emit a signal (at the same or similar emission wavelength as Cy5). Imaging revealed positive signals in the left and right rectangular regions (indicating primer chain extension with G or T) and background signals in the remaining regions. After simultaneous labeling with streptavidin-Cy5 and TCO-HCyC-646, which would bind to biotin and tetrazine respectively, followed by another wash at pH 5, imaging revealed new positive signals in the two central rectangular regions, indicating the incorporation of A or C. Next, washing at pH 8.5–9 eliminated the signal from the HCyC-646 dye, which is now present on the C and G ddNTP analogs. Using four 3'- O - Optional additional extension of the azidomethyl dNTPs is performed to ensure that substantially all primers have been extended. Finally, THP treatment is used to cleave the SS adapters, thereby removing the dye on the incorporated ddNTPs and removing any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (011 for A, 010 for C, 110 for G, and 111 for T when considering all three imaging steps; 01 for A, 00 for C, 10 for G, and 11 for T when considering only the first and third of these imaging steps).

[0043] Figure 15 Examples of ddNTP analogs and labeled binding molecules used in Figure 16.

[0044] Figures 16A-16B Monochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs, one containing Cy5, one containing HCyC-646, one containing biotin, and one containing tetrazine, all with SS adapters. ddNTP-cleavable adapter-dyes (ddGTP-7-SS-HCyC-646, ddTTP-5-SS-Cy5), ddNTP-cleavable adapter-anchors (ddCTP-5-SS-tetrazine, ddATP-7-SS-biotin), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecules-dyes (streptavidin-Cy5, TCO-HCyC-646) are used to perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- OAdding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template allows complementary nucleotide analogs to be incorporated into most of the growing DNA strand (>90%) to terminate DNA synthesis. Step 2 involves adding Thermo Sequenase and four ddNTP analogs (ddGTP-7-SS-HCyC-646, ddTTP-5-SS-Cy5, ddCTP-5-SS-tetraazine, ddATP-7-SS-biotin) to the immobilized primer-bound DNA template, enabling ddNTP incorporation into most of the remaining template-loop-primer. Step 3 involves washing away unincorporated nucleotide analogs at pH 5, followed by fluorescence imaging to reveal primers extended with ddTTP-5-SS-Cy5 or ddGTP-SS-HCyC-646. Step 4: Add streptavidin-Cy5 and TCO-HCyC-646 to label any incorporated ddATP-7-SS-biotin or ddCTP-5-SS-tetraazine analogs. Step 5: After washing away unused labeling reagents at pH 5, a second imaging step will reveal the incorporation of ddA or ddC. Step 6: After this additional washing step at pH 9, perform a third imaging step. Since the fluorescence of HCyC-646 is pH-responsive, it only fluoresces below pH 6, therefore no fluorescence will be displayed in this step. Thus, the fluorescence loss first observed in Step 3 will indicate the extension of the ddG analog, while the remaining fluorescence will indicate the incorporation of the ddT analog. Similarly, the fluorescence loss first visualized in Step 5 will indicate the extension of the ddC analog, and the remaining fluorescence will indicate the incorporation of the ddA analog. At this point, four 3'- O - Azide-methyl dNTPs perform an optional additional extension step to ensure that virtually every primer has been extended with either ddNTPs or NRT analogs. Step 8, cleaving the SS adapter by adding THP to the extended DNA strand, removes all dye from the ddNTP analogs and also restores the 3'- O -Azide-methyl-dNTP extended 3'-OH groups on any growing strand. After washing away the cleaved dye, an optional final round of imaging is performed. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 15 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0045] Figure 17A general set of dye and anchor labeling reagents for monochromatic SBS with two spectrally equivalent dyes (i.e., Cy5 and HCyC-646, where the latter dye is pH-responsive) for cleavable dNTP-blocking agent (virtual terminator) analogs and labeling agents: one of the virtual terminator analogs has a biotin anchor, one has a tetrazine anchor, one has a Cy5 dye, and one has HCyC-646. The labeled molecule consists of a molecule capable of specifically binding to one of the anchors (for biotin, said molecule is streptavidin; or for tetrazine, said molecule is TCO) and the same two dyes. Additional extension molecules are a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0046] Figure 18 : Used for the use of cleavable nucleotide analogs (such as in Figure 17 A simplified representation of the monochromatic SBS scheme using cleavable nucleotide analogs (as presented in the diagram). One of the dNTP-blocking (virtual terminator) nucleotides is Cy5, linked to a base via an SS linker; one is HCyC-646, linked to a base via an SS linker; one is biotin, linked to a base via an SS linker; and one is tetrazine, linked to a base via an SS linker. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is C, G, T, or A from left to right. Extension is performed using Thermo Sequenase and the following four virtual terminator analogs (containing a blocking group between the base and the label): dA linked to biotin, dT linked to Cy5, dC linked to tetrazine, and dG linked to HCyC-646. Then, using Therminator IX polymerase, four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-) are used. O Additional extension is performed using a 3'-azidomethyl dNTP to extend the remaining primers. Finally, washing at pH 5 ensures that HCyC-646 will emit a signal (at the same or similar emission wavelength as Cy5). Imaging will reveal positive signals in the left and right rectangular regions (indicating primer chain extension with G or T) and background signal in the remaining regions. After simultaneous labeling with streptavidin-Cy5 and TCO-HCyC-646, which will bind to biotin and tetrazine respectively, followed by another wash at pH 5, imaging will reveal new positive signals in the two central rectangular regions, indicating the incorporation of A or C. Next, washing at pH 8.5–9 will eliminate the fluorescent signal from the HCyC-646 dye, now present on the C and G ddNTP analogs. Using four 3'- O- Optional additional extension of the azidomethyl dNTPs is performed to ensure that substantially all primers have been extended. Finally, THP treatment is used to cleave the SS adapter, thereby removing the dye on the incorporated dummy terminator and removing any azidomethyl groups on the primers extended with NRT in preparation for the next sequencing cycle. The 1, 2, and 3 numeric codes on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numeric code (011 for A, 010 for C, 110 for G, and 111 for T when considering all three imaging steps; 01 for A, 00 for C, 10 for G, and 11 for T when considering only the first and third of these imaging steps).

[0047] Figure 19 Example 3'-blocker (virtual terminator) analogues and labeled molecules used in Figure 20.

[0048] Figures 20A-20B Monochrome sequencing-by-synthesis was performed using a set of dNTP-blocker (virtual terminator) analogues, one of which contained Cy5, one contained HCyC-646, one contained biotin, and one contained tetrazine, all of which had SS adapters. dNTP-cleavable adapter-blocker-dyes (dGTP-7-SS-blocker-HCyC-646, dTTP-5-SS-blocker-Cy5), dNTP-cleavable adapter-blocker-anchors (dCTP-5-SS-blocker-tetrazine, dATP-7-SS-blocker-biotin), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecules-dyes (streptavidin-Cy5, TCO-HCyC-646) are used to perform monochromatic DNA SBS. Step 1: ThermoSequenase and four dNTP-blocking virtual terminator analogs (dGTP-7-SS-blocker-HCyC-646, dTTP-5-SS-blocker-Cy5, dCTP-5-SS-blocker-tetraazine, dATP-7-SS-blocker-biotin) are added to the immobilized primer-bound DNA template to enable the incorporation of these virtual terminators. Step 2: Therminator IX DNA polymerase and four reversible terminators (3'-O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template allows for the incorporation of complementary nucleotide analogs into any primer strands not extended with a dummy terminator. Step 3: After washing away unincorporated nucleotide analogs at pH 5, fluorescence imaging will reveal primers extended with dTTP-5-SS-blocker-Cy5 or dGTP-SS-blocker-HCyC-646. Step 4: Adding streptavidin-Cy5 and TCO-HCyC-646 to label any incorporated dATP-7-SS-blocker-biotin or dCTP-5-SS-blocker-tetraazine analogs. Step 5: After washing away unused labeling reagents at pH 5, a second imaging step will reveal the incorporation of dA or dC. Step 6: Following this additional washing step at pH 9, a third imaging step is performed. Since HCyC-646's fluorescence is pH-responsive, fluorescing only below pH 6, no fluorescence will be observed in this step. Therefore, the fluorescence loss observed in step 3 will indicate the extension of the dG analog, while the remaining fluorescence will indicate the incorporation of the dT analog. Similarly, the fluorescence loss initially visualized in step 5 will indicate the extension of the dC analog, and the remaining fluorescence will indicate the incorporation of the dA analog. At this point, four 3'- O - Azide-methyl dNTPs perform an optional additional extension step to ensure that virtually every primer has been extended with either a dummy terminator or an NRT analog. Step 7, cleaving the SS adapter by adding THP to the extended DNA strand, removes all dye from the dummy terminator analog and also restores the 3'- O -Azide-methyl-dNTP extended 3'-OH groups on any growing strand. After washing away the cleaved dye, an optional final round of imaging is performed. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 19 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0049] Figure 21A general set of dye and anchor labeling reagents for monochromatic SBS with two spectrally equivalent dyes (i.e., Cy5 and HCyC-646, where the latter dye is pH-responsive) using 3'-blocking reversible terminator analogs and labeling agents: one of the 3'-blocking reversible terminator analogs has a biotin anchor, one has a tetrazine anchor, one has a Cy5 dye, and one has HCyC-646. The labeled molecule consists of a molecule capable of specifically binding to one of the anchors (for biotin, the molecule is streptavidin; or for tetrazine, the molecule is TCO) and the same two dyes. Additional extension molecules are a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0050] Figure 22 One of the 3'-blocking nucleotide reversible terminators has Cy5 linked to a base via an SS linker, one has HCyC-646 linked to a base via an SS linker, one has biotin linked to a base via an SS linker, and one has tetrazine linked to a base via an SS linker. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is C, G, T, or A from left to right. Extension is performed using Therminator IX and the following four 3'-blocking labeled nucleotide reversible terminator analogs: dA linked to biotin, dT linked to Cy5, dC linked to tetrazine, and dG linked to HCyC-646. Then, using Therminator IX polymerase, extension is performed with four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'- O Additional extension was performed using a 3'-azidomethyl dNTP to extend the remaining primers. Finally, washing at pH 5 ensured that HCyC-646 would emit a signal (at the same or similar emission wavelength as Cy5). Imaging revealed positive signals in the left and right rectangular regions (indicating primer chain extension with G or T) and background signal in the remaining regions. After simultaneous labeling with streptavidin-Cy5 and TCO-HCyC-646, which will bind to biotin and tetrazine respectively, followed by another wash at pH 5, imaging revealed new positive signals in the two central rectangular regions, indicating the incorporation of A or C. Next, washing at pH 8.5–9 eliminated the fluorescent signal from the HCyC-646 dye, now present on the C and G reversible terminator analogs. Using four 3'- O- Optional additional extensions of the azidomethyl dNTPs are performed to ensure that substantially all primers have been extended. Finally, THP treatment is used to cleave the SS adapters, thereby removing the dye on the reversible terminator of the incorporated nucleotide and removing the blocking groups on the primers extended with labeled or unlabeled NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (011 for A, 010 for C, 110 for G, and 111 for T when considering all three imaging steps; 01 for A, 00 for C, 10 for G, and 11 for T when considering only the first and third of these imaging steps).

[0051] Figure 23 Used for Figure 24-25 Examples include labeled reversible terminator analogs and labeled molecules.

[0052] Figure 24-25 Monochrome sequencing-by-synthesis was performed using a set of reversibly 3'-blocking nucleotide terminator analogs, one of which has Cy5, one has HCyC-646, one has biotin, and one has tetrazine, all with SS adapters. Using 3'- O -SS-dNTP-cuttable connector-dye (3'- O -SS-dGTP-7-SS-HCyC-646、3'- O -SS-dTTP-5-SS-Cy5), 3'- O -SS-dNTP-Cutable Connector-Anchor (3'- O -SS-dCTP-5-SS-tetraazine, 3'- O -SS-dATP-7-SS-Biotin), 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecules-dyes (streptavidin-Cy5, TCO-HCyC-646) perform monochromatic DNA SBS. Step 1, Therminator IX and four 3'- O -SS-dNTP reversible terminator analogue (3'- O-SS-dGTP-7-SS-HCyC-646、3'- O -SS-dTTP-5-SS-Cy5、3'- O -SS-dCTP-5-SS-tetraazine, 3'- O Adding 3'-SS-dATP-7'-SS-biotin to a fixed primer-bound DNA template enables the incorporation of these nucleotide reversible terminators. Step 2 involves adding Therminator IX DNA polymerase and four unlabeled reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding 3'-azidomethyl-dTTP to a fixed primer-bound DNA template allows for the incorporation of complementary nucleotide analogs into any primer strand not extended with a labeled reversible terminator. Step 3, after washing away unincorporated nucleotide analogs at pH 5, fluorescence imaging will reveal those primers extended with a 3'- O -SS-dTTP-5-SS-Cy5 or 3'- O -SS-dGTP-7-SS-HCyC-646 extended primers. Step 4, add streptavidin-Cy5 and TCO-HCyC-646 to label any incorporated 3'- O -SS-dATP-7-SS-Biotin or 3'- O -SS-dCTP-5-SS-tetraazine analogs. Step 5: After washing away unused labeling reagents at pH 5, a second imaging step will reveal the incorporation of dA or dC. Step 6: After this additional washing step at pH 9, a third imaging step is performed. Since the fluorescence ability of HCyC-646 is pH-responsive, it only fluoresces below pH 6, so no fluorescence will be shown in this step. Therefore, the fluorescence loss shown in step 3 will indicate the extension of the dG analog, while the remaining fluorescence will indicate the incorporation of the dT analog. Similarly, the fluorescence loss initially visualized in step 5 will indicate the extension of the dC analog, and the remaining fluorescence will indicate the incorporation of the dA analog. At this point, four 3'- O - Azide-methyl dNTPs perform an optional additional extension step to ensure that virtually every primer has been extended with one of an unlabeled or labeled NRT analog. Step 7, the SS adapter is cleaved by adding THP to the extended DNA strand, such that the 3'- O All dyes on the -SS-dNTP terminator analog were removed, and the 3'-OH group of 3'-O-SS-dNTP or 3'- O-Azide-methyl-dNTPs. After washing away the cut dye, an optional final round of imaging is performed. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 23 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0053] Figure 26 This invention relates to cleavable and non-cleavable ddNTP analogs and labeling reagents for colorimetric SBS with photobleachable anchors: two dideoxynucleotide analogs with linked Cy5, one via a cleavable linker and one via a non-cleavable linker. Two other dideoxynucleotide analogs have linked biotin anchors, one via a cleavable linker and one via a non-cleavable linker. The labeled molecule can specifically bind to one of the biotin anchors and has the same dye. A photobleachable dye, such as Cy5, is required. This hybrid SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0054] Figure 27 : Used with cleavable and non-cleavable nucleotide analogs (such as in Figure 26 A simplified representation of a scheme for monochromatic SBS of the cleavable and non-cleavable nucleotide analogues presented in the diagram. Two of the ddNTPs have Cy5 linked via an SS or non-cleavable linker, and the other two have biotin linked via an SS or non-cleavable linker. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is C, G, T, or A from left to right. The representation is based on four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'- OAfter incubation with Therminator IX to extend most of the primers, extension was performed using Thermo Sequenase and four ddNTP analogs: ddATP linked to biotin via an SS adapter, ddTTP linked to Cy5 via an SS adapter, ddGTP linked to Cy5 via a non-cleavable adapter, and ddCTP linked to biotin via a non-cleavable adapter. Imaging revealed positive signals (indicating primer extension with G or T) in the left and right rectangular regions and background signals in the remaining regions. After labeling with streptavidin-Cy5, imaging revealed new positive signals in the remaining regions, indicating the incorporation of A or C. Treatment with THP cleaved the SS adapters on the A and T ddNTP analogs and removed any azidomethyl groups from primers extended with NRT in preparation for the next sequencing cycle. Finally, photobleaching was performed to destroy any remaining dyes linked to C and G, which do not have cleavable adapters. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (010 for A, 011 for C, 111 for G, and 110 for T if all three imaging steps are considered; 00 for A, 01 for C, 11 for G, and 10 for T if only the first and last of these imaging steps are considered).

[0055] Figure 28 Example ddNTP analogues used in Figure 29.

[0056] Figures 29A-29B Using a photobleaching step, monochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs. One of these ddNTP analogs had an SS adapter and Cy5, another had an SS adapter and biotin, one had an uncuttable adapter and Cy5, and one had an uncuttable adapter and biotin. The following analogs were used: ddNTP-cleavable adapter-dye (ddTTP-5-SS-Cy5), ddNTP-cleavable adapter-anchor (ddATP-7-SS-Biotin), ddNTP-uncuttable adapter-dye (ddGTP-7-Cy5), ddNTP-uncuttable adapter-dye (ddCTP-5-Biotin), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O-Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) are used to perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Step 2 involves adding ddNTP (-azidomethyl-dTTP) to a fixed primer-bound DNA template, enabling the incorporation of complementary nucleotide analogs into most (>90%) of the growing DNA strand to terminate DNA synthesis. Thermo Sequenase and four ddNTP analogs (ddATP-7-SS-Biotin, ddTTP-5-SS-Cy5, ddCTP-5-Biotin, ddGTP-7-Cy5) to the fixed primer-bound DNA template, allowing the incorporation of ddNTPs into most of the remaining primers. Step 3 involves imaging against Cy5 fluorescence after washing away any unincorporated nucleotide analogs, revealing primers extended with ddTTP-5-SS-Cy5 or ddGTP-7-Cy5. Step 4 involves adding streptavidin-Cy5 to label any incorporated ddATP-7-SS-Biotin or ddCTP-5-Biotin analogs. Step 5, after washing away unused labeling reagents, the second imaging step will reveal the incorporation of ddA or ddC. At this point, four 3'- O - Azide-methyl dNTPs perform an optional additional extension step to ensure that virtually every primer has been extended with either ddNTP or an NRT analog. Step 6, cleaving the SS adapter by adding THP to the extended DNA strand, removes the dyes on the ddATP and ddTTP analogs and also restores the 3'- O -Azide-methyl-dNTP extension on any growing chain with a 3'-OH group. Step 7: After washing to remove THP, perform an imaging step. In the case of previously identified ddGTP or ddTTP analog incorporation, loss of Cy5 signal indicates ddT, and remaining signal indicates ddG incorporation. Similarly, in the case of previously identified ddATP or ddCTP analog incorporation, loss of Cy5 signal indicates ddA, and remaining signal indicates ddC incorporation. Step 8: Perform a photobleaching step to eliminate any fluorescence generated due to the incorporation of ddCTP or ddGTP analogs. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 28The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0057] Figure 30 Used for Figure 31 Examples of ddNTP analogues.

[0058] Figure 31 Two-color sequencing-by-synthesis was performed using a set of ddNTP analogs, two of which had Alexa488 and two had Cy5, with only SS adapters. ddNTP-cleavable adapter-dyes (ddTTP-5-SS-Cy5, ddATP-7-SS-Alexa488, ddGTP-7-SS-Cy5, ddCTP-5-SS-Alexa488) and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) performs two-color DNA SBS. Step 1, Therminator IX DNA polymerase, two of the four ddNTP analogs (ddTTP-5-SS-Cy5, ddATP-7-SS-Alexa488) and an excess of the four 3'-blocking dNTPs (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template enables the incorporation of complementary nucleotide reversible terminators into most of the growing DNA strand (>90%) and the incorporation of ddA or ddT analogs into some of the remaining primers (opposite to the template T or A portion) to terminate DNA synthesis. Step 2, after washing away the unincorporated nucleotide analogs, imaging against Cy5 and Alexa488 fluorescence will reveal those primers specifically extended with ddTTP-5-SS-Cy5 and ddATP-7-Alexa488. Step 3, Therminator IX DNA polymerase, two of the four ddNTP analogs (ddGTP-7-SS-Cy5, ddCTP-5-SS-Alexa488), and two of the four 3'-blocking dNTPs (3'-...) to ensure incorporation fidelity are added.O -azidomethyl-dATP, 3'- O Adding dTTP to the immobilized primer-bound DNA template allows for the incorporation of ddC or ddG analogs into some of the remaining primers (opposite to the G or C portion of the template) to terminate DNA synthesis. Step 4, after washing away unused labeling reagents, a second imaging step will reveal the incorporation of ddC or ddG. At this point, four 3'- O - Azide-methyl dNTPs perform an optional additional extension step to ensure that virtually every primer has been extended with either ddNTPs or NRT analogs. Step 5, cleaving the SS adapter by adding THP to the extended DNA strand, removes all dye from the ddNTP analogs and also restores the 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 30 The structure of the nucleotides used in this scheme is shown in the figure. Fluorescence induced by Cy5 is shown as black squares, and fluorescence induced by Alexa488 is shown as black circles.

[0059] Figure 32 A general set of dyes and anchors for labeling monochromatic SBS using click-release chemistry includes cleavable ddNTP analogs and labeling reagents: two dideoxynucleotide analogs with linked Cy5, one via an SS linker and one via a carbamoyl TCO linker. Two other dideoxynucleotide analogs have linked biotin anchors, one via an SS linker and one via a carbamoyl TCO linker. The labeled molecule can specifically bind to one of the biotin anchors and has the same dye. Clicking the tetrazine onto the TCO triggers an elimination reaction that initiates dye or anchor cleavage. This hybrid SBS approach requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0060] Figure 33 : Used for the use of cleavable nucleotide analogs (such as in Figure 32 A simplified representation of a scheme for monochromatic SBS of the cleavable nucleotide analogs presented in the diagram. Two of the ddNTPs have Cy5 linked via an SS linker or a carbamoyl TCO linker, and the other two have biotin linked via an SS linker or a carbamoyl TCO linker. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. The diagram also includes four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-O After incubation with Therminator IX to extend most of the primers, extension was performed using ThermoSequenase and four ddNTP analogs: ddATP linked to Cy5 via the SS adapter, ddTTP linked to Cy5 via the carbamoyl TCO adapter, ddGTP linked to biotin via the SS adapter, and ddCTP linked to biotin via the carbamoyl TCO adapter. Imaging revealed positive signals (indicating primer extension with A or T) in the left and right rectangular regions and background signals in the remaining regions. After labeling with streptavidin-Cy5, imaging revealed new positive signals in the remaining regions, indicating C or G incorporation. Tetraazine treatment was used to cleave the TCO adapters on the ddC and ddT analogs. Finally, THP treatment was used to cleave away any remaining dye and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all three imaging steps, 111 represents A, 010 represents C, 011 represents G, and 110 represents T; considering only the first and last of these imaging steps, 11 represents A, 00 represents C, 01 represents G, and 10 represents T).

[0061] Figure 34 Example ddNTP analogues for Figure 35.

[0062] Figures 35A-35B Monochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs, one of which had an SS adapter and Cy5, one had an SS adapter and biotin, one had a TCO-carbamate adapter and Cy5, and one had a TCO-carbamate adapter and biotin. ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddTTP-5-TCO-Cy5), ddNTP-cleavable adapter-anchors (ddGTP-7-SS-Biotin, ddCTP-5-TCO-Biotin), and 3'- O -Azide-methyl dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O-Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) perform monochromatic DNA SBS. This is essentially the same as the dual-linker scheme proposed in a recently filed patent application (Ju et al. PCT / US2019 / 022326), but instead of an azo linker that can be cleaved by sodium dithionite, a TCO carbamate linker that undergoes a click-release reaction with tetrazine is used here. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Step 2 involves adding ddNTPs (-azidomethyl-dTTP) to a fixed, primer-bound DNA template, enabling the incorporation of complementary nucleotide analogs into most (>90%) of the growing DNA strand to terminate DNA synthesis. Thermo Sequenase and four ddNTP analogs (ddATP-7-SS-Cy5, ddTTP-5-TCO-Cy5, ddGTP-7-SS-Biotin, ddCTP-5-TCO-Biotin) to the fixed, primer-bound DNA template, allowing the incorporation of ddNTPs into most of the remaining template-loop-primer. Step 3 involves washing away unincorporated nucleotide analogs, followed by imaging against Cy5 fluorescence to reveal primers extended with ddATP-7-SS-Cy5 or ddTTP-5-TCO-Cy5. Step 4 involves adding streptavidin-Cy5 to label any incorporated ddGTP-7-SS-Biotin or ddCTP-5-TCO-Biotin analogs. Step 5, after washing away unused labeling reagents, the second imaging step will reveal the incorporation of ddC or ddG. At this point, or just before, four 3'- O - Azide-methyl dNTPs perform an optional additional extension step to ensure that virtually every primer has been extended with either ddNTP or NRT analogs. Step 6, cleaving the SS adapter by adding tetrazine to the extended DNA strand, removes the dyes on the ddCTP and ddTTP analogs and also restores the 3'- O-Azide-methyl-dNTP extends any growing chain with a 3'-OH group. Step 7, after washing away excess tetrazine, imaging is performed. In the case of previously determined ddATP or ddTTP analog incorporation, loss of Cy5 signal indicates ddT, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously determined ddCTP or ddGTP analog incorporation, loss of Cy5 signal indicates ddC, and the remaining signal indicates ddG incorporation. Step 8, SS adapters are cleaved by adding THP to the extended DNA strand, such that the dye on ddATP and ddGTP analogs is removed, and the 3'-OH group is also restored. O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 34 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0063] Figure 36 A general set of dyes and anchors for labeling cleavable ddNTP analogs, labeling, and quenching reagents using monochromatic SBS with quenching: one dideoxynucleotide analog with Cy5 linked to a base, one with biotin linked, one with both branched biotin and TCO linked, and a last with both branched Cy5 and TCO linked. All four dideoxynucleotide analogs are linked via SS linkers. The labeled molecule can specifically bind to one of the biotin anchors and has the same dye. The quenching molecule (e.g., BHQ3) binds via a TCO anchor. This hybrid SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0064] Figure 37 : Used for the use of cleavable nucleotide analogs (such as in Figure 36 A simplified representation of a scheme for monochromatic SBS of the cleavable nucleotide analogs presented in the diagram. Each type of ddNTP has one of the following linked via an SS adapter: Cy5, biotin, biotin-TCO, or Cy5-TCO. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. This is in contrast to four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- OAfter incubation with Therminator IX to extend most of the primers, extension was performed using Thermo Sequenase and four ddNTP analogs: ddATP linked to Cy5, ddTTP linked to Cy5-TCO, ddGTP linked to biotin, and ddCTP linked to biotin-TCO. Imaging revealed positive signals (indicating primer extension with A or T) in the left and right rectangular regions and background signals in the remaining regions. After labeling with streptavidin-Cy5, imaging revealed new positive signals in the remaining regions, indicating C or G incorporation. Treatment with tetrazine-BHQ3 quenched Cy5 fluorescence on the C and T ddNTP analogs. Finally, treatment with THP cleaved the remaining dye and removed any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all three imaging steps, 111 represents A, 010 represents C, 011 represents G, and 110 represents T; considering only the first and last of these imaging steps, 11 represents A, 00 represents C, 01 represents G, and 10 represents T).

[0065] Figure 38 Examples of ddNTP analogs and quencher-anchored molecules used in Figure 39.

[0066] Figures 39A-39B Using dye quenchers, monochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs, one of which contained Cy5, one contained biotin, one contained both Cy5 and biotin, and one contained both biotin and a TCO anchor, all of which had SS adapters. The following analogs were used: ddNTP-cleavable adapter-dye (ddATP-7-SS-Cy5), ddNTP-cleavable adapter-anchor 1 (ddGTP-7-SS-Biotin), ddNTP-cleavable adapter-branched anchors 1 and 2 (ddCTP-5-SS-Biotin / TCO), ddNTP-cleavable adapter-dye-anchor (ddTTP-5-SS-Cy5-TCO), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O-Azide-methyl-dTTP), anchor-binding molecule-dye (streptavidin-Cy5), and anchor-binding molecule-quencher (tetraazine-BHQ) are used to perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Step 2 involves adding ddNTPs (-azidomethyl-dTTP) to a fixed primer-bound DNA template, enabling the incorporation of complementary nucleotide analogs into most (>90%) of the growing DNA strand to terminate DNA synthesis. Thermo Sequenase and four ddNTP analogs (ddATP-7-SS-Cy5, ddGTP-7-SS-Biotin, ddCTP-5-SS-Biotin / TCO, ddTTP-5-SS-Cy5-TCO) to the fixed primer-bound DNA template, allowing the incorporation of ddNTPs into most of the remaining primers. Step 3 involves washing away any unincorporated nucleotide analogs, followed by imaging against Cy5 fluorescence to reveal primers extended with ddATP-7-SS-Cy5 or ddTTP-5-SS-Cy5-TCO. Step 4 involves adding streptavidin-Cy5 to label any incorporated ddGTP-7-SS-Biotin or ddCTP-5-SS-Biotin / TCO analogs. Step 5: After washing away unused labeling reagents, perform the second imaging step, and the new fluorescence signal will confirm the incorporation of ddC or ddG. At this point, or just before, four 3'- O -Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of the ddNTP or NRT analogs. Step 6: Incubation with tetrazine-BHQ is performed to quench the fluorescence of the dye on the ddC or ddT analogs. Step 7: After washing to remove any free tetrazine-BHQ, a third imaging step is performed. In the case of previously identified ddATP or ddTTP analog incorporation, a significant loss of Cy5 signal indicates ddT, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously identified ddCTP or ddGTP analog incorporation, a significant loss of Cy5 signal indicates ddC, and the remaining signal indicates ddG incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, thereby removing the dye and quencher on the nucleotide analogs and also restoring the fluorescence with 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 38 The structure of the nucleotides used in this protocol is shown in the image. In the imaging animation at each step, black indicates a positive Cy5 signal, and white or light gray indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0067] Figure 40 A general set of cleavable virtual terminator nucleotide analogs, labels, and quenchers for labeling with single-color SBS dyes and anchors using quenching: one virtual terminator analog has Cy5 linked to a base, one has biotin linked, one has both branched biotin and TCO linked, and the last has both branched Cy5 and TCO linked. All four virtual terminator analogs achieve this linkage via an SS linker. The labeled molecule can specifically bind to one of the biotin anchors and has the same dye. The quencher molecule binds via a TCO anchor. Additional extension reactions are performed using four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP) is used.

[0068] Figure 41 : Used for the use of cleavable nucleotide analogs (such as in Figure 40 A simplified representation of the scheme for monochrome SBS using the cleavable nucleotide analogs presented in the diagram. Each type of virtual terminator has one of the following linked via an SS linker: Cy5, biotin, biotin-TCO, or Cy5-TCO. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extensions were performed using Thermo Sequenase and four virtual terminator analogs (i.e., dATP linked to Cy5, dTTP linked to Cy5-TCO, dGTP linked to biotin, and dCTP linked to biotin-TCO). Extensions were performed using four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- O(-Azide-methyl dNTP) and Therminator IX perform additional extensions to extend all remaining primers. Imaging will reveal positive signals (indicating primer chains extended with A or T) in the left and right rectangular regions and background signals in the remaining regions. After labeling with streptavidin-Cy5, imaging will reveal new positive signals in the remaining regions, indicating the incorporation of C or G. Treatment with tetrazine-BHQ quenches Cy5 fluorescence on C and T dummy terminator analogs. Finally, treatment with THP cleaves the remaining dye and removes any azidomethyl groups from primers extended with NRT in preparation for the next sequencing cycle. The 1, 2, and 3 numeric codes on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 010 for C, 011 for G and 110 for T, taking all three imaging steps into account; 11 for A, 00 for C, 01 for G and 10 for T, taking only the first and last of these imaging steps into account).

[0069] Figure 42 Examples of dNTP analogues (virtual terminators) and quencher-anchored molecules used in Figure 43.

[0070] Figures 43A-43B Monochromatic sequencing-by-synthesis was performed using a set of virtual terminator nucleotide analogs, one of which contained Cy5, one contained biotin, one contained both Cy5 and biotin, and one contained both biotin and a TCO anchor, all of which had SS adapters. The following were used: dNTP-cleavable adapter-blocker-dye (dATP-7-SS-blocker-Cy5), dNTP-cleavable adapter-blocker anchor (ddGTP-7-SS-blocker-Biotin), dNTP-cleavable adapter-blocker-branched anchors 1 and 2 (dCTP-5-SS-blocker-Biotin / TCO), ddNTP-cleavable adapter-blocker-dye-anchor (ddTTP-5-SS-blocker-Cy5-TCO), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- OMonochromatic DNA SBS is performed using dNTP-azide-methyl-dTTP, anchor-binding molecule-dye (streptavidin-Cy5), and anchor-binding molecule-quencher (tetraazine-BHQ). Step 1: Thermo Sequenase and four dNTP-blocker virtual terminator analogs (dATP-7-SS-blocker-Cy5, ddGTP-7-SS-blocker-biotin, dCTP-5-SS-blocker-biotin / TCO, ddTTP-5-SS-blocker-Cy5-TCO) are added to a primer-bound DNA template to enable incorporation of these virtual terminators. Step 2: Therminator IX DNA polymerase and four unlabeled reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O The additional extension of the primer-bound DNA template (-azidomethyl-dTTP) allows for the incorporation of complementary nucleotide analogs into any primer strand not terminated with a dummy terminator. Step 3, after washing away the unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal primers extended with ddATP-7-SS-blocker-Cy5 or ddTTP-5-SS-blocker-Cy5-TCO. Step 4, add streptavidin-Cy5 to label any incorporated ddGTP-7-SS-blocker-biotin or ddCTP-5-SS-blocker-biotin / TCO analogs. Step 5, after washing away unused labeling reagents, perform a second imaging step, and the new fluorescence signal will confirm the incorporation of the C or G dummy terminator. If the additional extension step was not performed earlier, at this time, or just before this, four 3'- O -Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either a dummy terminator or an NRT analog. Step 6: Incubate with tetrazine-BHQ to quench the fluorescence of the dye on the C or T dummy terminator. Step 7: After washing to remove any free tetrazine-BHQ, a third imaging step is performed. In the case of previously identified dATP or dTTP dummy terminator analog incorporation, a significant loss of Cy5 signal indicates dT, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dGTP dummy terminator analog incorporation, a significant loss of Cy5 signal indicates dC, and the remaining signal indicates dG incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, thereby removing the dye and quencher on the nucleotide analog and also restoring the fluorescence of the 3'- O-Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 42 The structure of the nucleotides used in this protocol is shown in the image. In the imaging animation at each step, black indicates a positive Cy5 signal, and white or light gray indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0071] Figure 44 A general set of dyes and anchors for labeling reversibly cleavable 3'-blocking nucleotide terminator analogs, labels, and quenchers using monochromatic SBS dyes: one of the reversible terminator analogs has Cy5 linked to a base, one has biotin linked, one has both branched biotin and TCO linked, and the last has both branched Cy5 and TCO linked. All four reversible terminator analogs achieve this linkage via an SS linker. The labeled molecule can specifically bind to one of the biotin anchors and has the same dye. The quencher molecule binds via a TCO anchor. Further extension reactions are performed using four unlabeled reversible terminators (e.g., 3'-...). O -Azide-methyl dNTP) is used.

[0072] Figure 45 : Used for the use of cleavable nucleotide analogs (such as in Figure 44 A simplified representation of the monochromatic SBS scheme for the cleavable nucleotide analogues presented in the diagram. Each type of nucleotide reversible terminator (A, C, G, and T) has one of the following linked via an SS linker: Cy5, biotin, biotin, and TCO or Cy5-TCO. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand from left to right is T, G, C, or A. Therminator IX and four nucleotide reversible terminator analogues (i.e., 3'- linked with Cy5) are used. O- tert-butyl-SS-ATP, 3'- linked to Cy5-TCO O- tert-butyl-SS-dTTP, biotin-linked 3'- O- tert-butyl-SS-dGTP and the 3'-linked biotin and TCO O- Extension was performed using tert-butyl-SS-dCTP (all ligations were achieved via SS linkers). Four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'-) were used. O(-Azide-methyl dNTP) and Therminator IX perform additional extensions to extend all remaining primers. Imaging will reveal positive signals (indicating primer chains extended with A or T) in the left and right rectangular regions and background signals in the remaining regions. After labeling with streptavidin-Cy5, imaging will reveal new positive signals in the remaining regions, indicating C or G incorporation. Treatment with tetrazine-BHQ quenches Cy5 fluorescence on C and T reversible terminator analogs. Finally, treatment with THP cleaves the remaining dye and removes any azidomethyl groups from primers extended with NRT in preparation for the next sequencing cycle. The 1, 2, and 3 numeric codes on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 010 for C, 011 for G and 110 for T, taking all three imaging steps into account; 11 for A, 00 for C, 01 for G and 10 for T, taking only the first and last of these imaging steps into account).

[0073] Figure 46 Examples of 3'-SS-dNTP analogues (reversible terminators) and quencher-anchored molecules used in Figure 47.

[0074] Figures 47A-47B Monochromatic sequencing-while-synthesizing was performed using a set of nucleotide reversible terminator analogs, one containing Cy5, one containing biotin, one containing both Cy5 and biotin, and one containing both biotin and a TCO anchor, all with SS adapters. A 3'-SS-dNTP-cleavable adapter-dye (3'- O -SS-dATP-7-SS-Cy5), 3'- O -SS-dNTP-Cutable Connector-Anchor (3'- O -SS-ddGTP-7-SS-Biotin), 3'- O -SS-dNTP-cuttable connector-blocking agent-branched anchors 1 and 2 (3'- O -SS-dCTP-5-SS-Biotin / TCO), 3'- O -SS-ddNTP-cuttable connector-dye-anchor (3'- O -SS-dTTP-5-SS-Cy5-TCO), 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O-Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP), anchor-binding molecule-dye (streptavidin-Cy5), and anchor-binding molecule-quencher (tetraazine-BHQ) are used to perform monochromatic DNA SBS. Step 1, Therminator IX and four 3'-blocking reversible terminator analogs (3'- O -SS-dATP-7-SS-Cy5, 3'- O -SS-dGTP-7-SS-Biotin, 3'- O -SS-dCTP-5-SS-Biotin / TCO, 3'- O Adding SS-dTTP-5-SS-Cy5-TCO to the immobilized primer-bound DNA template allows for the incorporation of these dummy terminators. Step 2 involves using Therminator IX DNA polymerase and four unlabeled reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O The addition of α-azidomethyl-dTTP to the primer-bound DNA template allows for the incorporation of complementary nucleotide analogs into any primer strand not terminated with a labeled reversible terminator. Step 3, after washing away the unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal those primers terminated with 3'- O -SS-dATP-7-SS-Cy5 or 3'- O -SS-dTTP-5-SS-Cy5-TCO extended primers. Step 4, add streptavidin-Cy5 to label any incorporated 3'- O -SS-ddGTP-7-SS-Biotin or 3'- O -SS-dCTP-5-SS-Biotin / TCO analogues. Step 5: After washing away unused labeling reagents, perform a second imaging step, and the new fluorescence signal will confirm the incorporation of the C or G reversible terminator. If an additional extension step was not performed earlier, at this time, or just before this, four 3'- O-Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of the unlabeled or labeled NRT analogs. Step 6: Incubate with tetrazine-BHQ to quench the fluorescence of the dye on the 3'-blocked reversible terminator C or T. Step 7: After washing to remove any free tetrazine-BHQ, a third imaging step is performed. In the case of previously identified ATP or TTP reversible terminator analog incorporation, a significant loss of Cy5 signal indicates T, and the remaining signal indicates A incorporation. Similarly, in the case of previously identified CTP or GTP reversible terminator analog incorporation, a significant loss of Cy5 signal indicates C, and the remaining signal indicates G incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, such that the dye on the reversible terminator analog is removed, and the fluorescence of the 3'-blocked NRT analog is also restored. O -SS-dNTP or 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 46 The structure of the nucleotides used in this protocol is shown in the image. In the imaging animation at each step, black indicates a positive Cy5 signal, and white or light gray indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0075] Figure 48 A general set of dye-labeled, labeling, and quenching reagents for single-color SBS using pH-responsive dyes and quenching: one dideoxynucleotide analog with Cy5 linked to a base, one with HCyC-646 linked, one with a branched configuration of Cy5 and TCO linked, and the last with a branched configuration of HCyC-646 and TCO linked, all dideoxynucleotide analogs achieving the linkage via SS linkers. HCyC-646 exhibits pH-responsive fluorescence. The quenching molecule binds via a TCO anchor. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0076] Figure 49 : Used for the use of cleavable nucleotide analogs (such as in Figure 48A simplified representation of a monochromatic SBS scheme for the cleavable nucleotide analogs presented in the diagram. Each type of ddNTP has one of the following linked via an SS adapter: Cy5, HCyC-646, Cy5-TCO, or HCyC-646-TCO. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. This is in contrast to four unlabeled nucleotide reversible terminators (NRTs, e.g., 3'- O After incubation with Therminator IX to extend most of the primers, extension was performed using Thermo Sequenase and four ddNTP analogs: ddATP linked to Cy5, ddTTP linked to HCyC-646, ddGTP linked to Cy5-TCO, and ddCTP linked to HCyC-646-TCO. Following washing at pH 9, imaging revealed positive signals in the first and third rectangular regions (indicating primer extension with A or G) and background signals in the remaining regions. After washing at pH 5, imaging revealed new positive signals in the remaining regions due to protonation of HCyC-646, indicating C or T incorporation. Treatment with tetrazine-BHQ quenched the fluorescence of HCyC-646 and Cy5 on the C and G ddNTP analogs. Finally, treatment with THP cleaved the remaining dye and removed any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all three imaging steps, 111 represents A, 010 represents C, 110 represents G, and 011 represents T; considering only the first and last of these imaging steps, 11 represents A, 00 represents C, 10 represents G, and 01 represents T).

[0077] Figure 50 Examples of ddNTP analogs and quencher-anchored molecules used in Figure 51.

[0078] Figures 51A-51BMonochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs, including one containing Cy5, one containing the pH-responsive fluorescent dye HCyC-646, one containing Cy5 and a TCO anchor, and one containing HCyC-646 and a TCO anchor, all linked to bases via SS adapters. ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddTTP-5-SS-HCyC-646), ddNTP-cleavable adapter-dye-anchors (ddGTP-7-SS-Cy5-TCO, ddCTP-5-SS-HCyC-646-TCO), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecule-quencher (tetraazine-BHQ) are used to perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template allows complementary nucleotide analogs to be incorporated into most (>90%) of the growing DNA strand to terminate DNA synthesis. Step 2 involves adding Thermo Sequenase and four ddNTP analogs (ddATP-7-SS-Cy5, ddTTP-5-SS-HCyC-646, ddGTP-7-SS-Cy5-TCO, ddCTP-5-SS-HCyC-646) to the immobilized primer-bound DNA template, enabling ddNTP incorporation into most of the remaining template-loop-primer. Step 3 involves washing away unincorporated nucleotide analogs at pH 9, followed by imaging against Cy5 fluorescence to reveal primers extended with either ddATP-7-SS-Cy5 or ddGTP-7-SS-Cy5-TCO. Step 4, a second wash at pH 5, will allow HCyC-646 on ddTTP-5-SS-HCyC-646 and ddCTP-5-SS-HCyC-646-TCO to fluoresce. A second imaging step is performed at pH 5, and the new fluorescence signal will confirm the incorporation of ddC or ddT. At this point, or just before, four 3'-O -Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of the ddNTP or NRT analogs. Step 5: Incubate with tetrazine-BHQ to quench the dye on the ddG or ddC analogs. Step 6: After washing to remove any free tetrazine-BHQ, perform a third imaging step at pH 5. In the case of previously identified ddATP or ddGTP analog incorporation, loss of Cy5 signal indicates ddG, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously identified ddCTP or ddTTP analog incorporation, loss of fluorescence signal indicates ddC, and the remaining signal indicates ddT incorporation. Step 7: Cleave the SS adapter by adding THP to the extended DNA strand, thereby removing the dye on the nucleotide analogs and also restoring the 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 50 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white or light gray indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0079] Figure 52 A general set of cleavable ddNTP analogs and labeling reagents for single-color SBS labeled with dyes or anchors using click-cutting linkers and quenching: one of the dideoxynucleotide analogs has Cy5 linked to a base via an SS linker, one has biotin linked to a base via an SS linker, one has Cy5 linked to a base via a linker containing SS (shown as cleavable linker 1) and TCO (shown as cleavable linker 2), and the last has biotin linked to a base via a linker containing both SS and TCO. The binding molecule is dye-labeled streptavidin, and the dye can be released by a click-cutting reaction at cleavable linker 2 or by standard cleavage at cleavable linker 1. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0080] Figure 53 : Used for the use of cleavable nucleotide analogs (such as in Figure 52A simplified representation of a scheme for monochromatic SBS of the cleavable nucleotide analogs presented in the diagram. Each type of ddNTP has one of the following linked via an SS linker or a linker having both SS and TCO groups: Cy5 or biotin. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. This is in contrast to four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- O After incubation with Therminator IX to extend most of the primers (-azidomethyl dNTP), extension was performed using Thermo Sequenase and four ddNTP analogs (i.e., ddATP linked to Cy5 via an SS linker, ddTTP linked to biotin via an SS linker, ddGTP linked to Cy5 via a linker containing both SS and TCO, and ddCTP linked to biotin via a linker containing both SS and TCO). After washing, imaging revealed positive signals in the first and third rectangular regions (indicating primer extension with A or G) and background signals in the remaining regions. Treatment with streptavidin-Cy5 labeled ddCTP and ddTTP. After washing, imaging revealed new positive signals in the second and fourth rectangular regions (indicating primer extension with C or T). The reaction of tetrazine with TCO released Cy5 from the ddCTP and ddGTP nucleotide analogs. After washing, fluorescence loss specifically revealed the incorporation of C and G, while the remaining fluorescence revealed the incorporation of A and T, respectively. Finally, THP treatment is used to cleave away any remaining dye and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The 1, 2, and 3 numeric codes on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numeric code (considering all three imaging steps, 111 represents A, 010 represents C, 110 represents G, and 011 represents T; considering only the first and last of these imaging steps, 11 represents A, 00 represents C, 10 represents G, and 01 represents T).

[0081] Figure 54 Example ddNTP analogues for Figure 55.

[0082] Figures 55A-55BThe process involved a streptavidin-Cy5 labeling step followed by monochromatic sequencing-by-synthesis using a set of orthogonal ddNTP analogs containing Cy5 or biotin and either an SS adapter only or an SS plus TCO adapter. ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddGTP-7-SS-TCO-Cy5), ddNTP-cleavable adapter-dye-anchors (ddTTP-5-SS-Biotin, ddCTP-5-SS-TCO-Biotin), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) are used to perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Step 2 involves adding ddNTPs (-azidomethyl-dTTP) to a fixed primer-bound DNA template, enabling the incorporation of complementary nucleotide analogs into most (>90%) of the growing DNA strand to terminate DNA synthesis. Thermo Sequenase and four ddNTP analogs (ddATP-7-SS-Cy5, ddGTP-7-SS-TCO-Cy5, ddTTP-5-SS-Biotin, and ddCTP-5-SS-TCO-Biotin) to the fixed primer-bound DNA template, allowing the incorporation of ddNTPs into most of the remaining template-loop-primer. Step 3 involves washing away any unincorporated nucleotide analogs, followed by imaging against Cy5 fluorescence to reveal primers extended with either ddATP-7-SS-Cy5 or ddGTP-7-SS-TCO-Cy5. Step 4, labeling with streptavidin-Cy5 will link Cy5 to ddTTP-5-SS-Biotin and ddCTP-5-SS-TCO-Biotin via biotin anchors. Step 5, perform the second imaging step, and the new fluorescence signal will confirm the incorporation of ddC or ddT. At this point, or just before, four 3'- O-Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of the ddNTP or NRT analogs. Step 6: Incubate with tetrazine to cleave the dye on the ddC or ddG analogs. Step 7: After washing to remove any free tetrazine, a third imaging step is performed. In the case of previously determined ddATP or ddGTP analog incorporation, loss of Cy5 signal indicates ddG, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously determined ddCTP or ddTTP analog incorporation, loss of Cy5 signal indicates ddC, and the remaining signal indicates ddT incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, thereby removing the dye on the nucleotide analogs and also restoring the dye with 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 54 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0083] Figure 56 A general set of cleavable virtual terminator nucleotide analogs and labeling reagents for labeling with single-color SBS dyes or anchors using click-cutting linkers and quenching: one of the virtual terminator analogs has Cy5 linked to a base via an SS linker, one has biotin linked to a base via an SS linker, one has Cy5 linked to a base via a linker containing SS (shown as cleavable linker 1) and TCO (shown as cleavable linker 2), and the last has biotin linked to a base via a linker containing SS and TCO. The binding molecule is dye-labeled streptavidin, and the dye can be released by click-cutting at cleavable linker 2 or by standard cleavage at cleavable linker 1. Additional extension reactions are performed using four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP) is used.

[0084] Figure 57 : Used for the use of cleavable nucleotide analogs (such as in Figure 56A simplified representation of the scheme for monochrome SBS using the cleavable nucleotide analogs presented in the diagram. Each type of virtual terminator nucleotide analog has one of the following linked via an SS linker or a linker having both SS and TCO groups: Cy5 or biotin. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extensions were performed using Thermo Sequenase and four virtual terminator analogs (i.e., dATP linked to Cy5 via an SS linker, dTTP linked to biotin via an SS linker, dGTP linked to Cy5 via a linker containing both SS and TCO, and dCTP linked to biotin via a linker containing both SS and TCO). Extensions were performed using four unlabeled reversible nucleotide terminators (NRT, e.g., 3'- O -Azide-methyl dNTP) and Therminator IX perform additional extensions to extend all remaining primers. After washing, imaging reveals positive signals in the first and third rectangular regions (indicating primer extension with A or G) and background signals in the remaining regions. Treatment with streptavidin-Cy5 tags the dCTP and dTTP dummy terminator analogs. After washing, imaging reveals new positive signals in the second and fourth rectangular regions (indicating primer extension with C or T). The reaction between tetrazine and TCO releases Cy5 from the dCTP and dGTP dummy terminator analogs. After washing, fluorescence loss specifically reveals the incorporation of C and G, while the remaining fluorescence reveals the incorporation of A and T, respectively. Finally, treatment with THP cleaves the remaining dye and removes any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The 1, 2, and 3 numeric codes on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 010 for C, 110 for G and 011 for T, taking all three imaging steps into account; 11 for A, 00 for C, 10 for G and 01 for T, taking only the first and last of these imaging steps into account).

[0085] Figure 58 Example virtual terminator nucleotide analogues used in Figure 59.

[0086] Figures 59A-59BThe process involved monochromatic sequencing-by-synthesis using a streptavidin-Cy5 labeling step and a set of orthogonal virtual terminator nucleotide analogs containing Cy5 or biotin and either an SS adapter only or an SS plus TCO adapter. dNTP-blocker-cleavable adapter-dye (dATP-7-SS-blocker-Cy5, dGTP-7-SS-blocker-TCO-Cy5), dNTP-cleavable adapter-blocker-dye-anchor (dTTP-5-SS-blocker-Biotin, dCTP-5-SS-blocker-TCO-Biotin), and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Monochromatic DNA SBS is performed using a combination of dTP (-azidomethyl-dTTP) and an anchoring molecule-dye (streptavidin-Cy5). Step 1: Thermo Sequenase and four virtual terminator analogs (dATP-7-SS-blocker-Cy5, dGTP-7-SS-blocker-TCO-Cy5, dTTP-5-SS-blocker-biotin, dCTP-5-SS-blocker-TCO-biotin) are added to a fixed primer-bound DNA template to enable the incorporation of virtual terminators onto the template-loop-primer (or other template-binding primer arrangement). Step 2: TherminatorIX DNA polymerase and four unlabeled reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Additional extension of the dTTP (-azidomethyl-dTTP) allows for the incorporation of complementary nucleotide analogs into the remaining growing DNA strand. Step 3, after washing away unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal primers extended with either dATP-7-SS-blocker-Cy5 or dGTP-7-SS-blocker-TCO-Cy5. Step 4, labeling with streptavidin-Cy5 will link Cy5 to dTTP-5-SS-blocker-biotin and dCTP-5-SS-blocker-TCO-biotin via biotin anchors. Step 5, performing a second imaging step, and the new fluorescence signal will confirm the incorporation of either dC or dT dummy terminators. At this point, or just before, four 3'- O-Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either a virtual terminator or an NRT analog. Step 6: Incubate with tetrazine to cleave the dye on the dC or dG virtual terminator analog. Step 7: After washing to remove any free tetrazine, a third imaging step is performed. In the case of previously identified dATP or dGTP virtual terminator analog incorporation, loss of Cy5 signal indicates dG, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dTTP virtual terminator analog incorporation, loss of Cy5 signal indicates dC, and the remaining signal indicates dT incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, thereby removing the dye on the virtual terminator analog and also restoring the dye with 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 58 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0087] Figure 60 A general set of dyes or anchors for single-color SBS using click-cutting connectors for cleavable nucleotide reversible terminator analogs and labeling reagents: one of the nucleotide reversible terminator analogs has Cy5 linked to a base via an SS connector, one has biotin linked to a base via an SS connector, one has Cy5 linked to a base via a connector containing SS (shown as cleavable connector 1) and TCO (shown as cleavable connector 2), and the last has biotin linked to a base via a connector containing SS and TCO. The binding molecule is dye-labeled streptavidin, and the dye can be released by a click-cutting reaction at cleavable connector 2 or by standard cleavage at cleavable connector 1. Additional extension reactions are performed using four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP) is used.

[0088] Figure 61 : Used for the use of cleavable nucleotide analogs (such as in Figure 60A simplified representation of the monochromatic SBS scheme for the cleavable nucleotide analogs presented in the diagram. Each type of reversible nucleotide terminator analog has one of the following linked via an SS linker or a linker having both SS and TCO groups: Cy5 or biotin. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Therminator IX and four reversible terminator analogs (i.e., 3'- linked via an SS linker to Cy5) are used. O- tert-butyl-SS-dATP, 3'-linked to biotin via the SS linker O- tert-butyl-SS-dTTP, 3'- connected to Cy5 via a connector containing both SS and TCO. O- tert-butyl-SS-dGTP and 3'-linked to biotin via a linker containing both SS and TCO. O- Extension was performed using tert-butyl-SS-dCTP. Four unlabeled nucleotide reversible terminators (NRT, e.g., 3'-) were used. O -Azide-methyl dNTP) and Therminator IX perform additional extensions to extend all remaining primers. After washing, imaging reveals positive signals in the first and third rectangular regions (indicating primer extension with A or G) and background signals in the remaining regions. Treatment with streptavidin-Cy5 tags the dCTP and dTTP reversible terminator analogs. After washing, imaging reveals new positive signals in the second and fourth rectangular regions (indicating primer extension with C or T). The reaction of tetrazine with TCO releases Cy5 from the dCTP and dGTP reversible terminator analogs. After washing, fluorescence loss specifically reveals the incorporation of C and G, while the remaining fluorescence reveals the incorporation of A and T, respectively. Finally, treatment with THP cleaves the remaining dye and removes any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., positive signal indicated by 1 and background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 010 for C, 110 for G and 011 for T, taking all three imaging steps into account; 11 for A, 00 for C, 10 for G and 01 for T, taking only the first and last of these imaging steps into account).

[0089] Figure 62 Example 3'-SS-dNTP analogue (reversible terminator) for Figure 63.

[0090] Figures 63A-63B The process involved monochromatic sequencing-by-synthesis using a streptavidin-Cy5 labeling step and a set of orthogonal nucleotide reversible terminator analogs containing Cy5 or biotin and either an SS adapter only or an SS plus TCO adapter. A 3'- O -SS-dNTP-cuttable connector-dye (3'- O -SS-ATP-7-SS-Cy5、3'- O -SS-dGTP-7-SS-TCO-Cy5), 3'- O -SS-dNTP-cuttable connector-dye-anchor (3'- O -SS-dTTP-5-SS-Biotin, 3'- O -SS-dCTP-5-SS-TCO-Biotin), 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecule-dye (streptavidin-Cy5) are used to perform monochromatic DNA SBS. Step 1, Therminator IX and four virtual terminator analogs (3'- O -SS-ATP-7-SS-Cy5、3'- O -SS-dGTP-7-SS-TCO-Cy5、3'- O -SS-dTTP-5-SS-Biotin, 3'- O Adding SS-dCTP-5-SS-TCO-Biotin to a fixed primer-bound DNA template allows for the incorporation of a 3'-blocking reversible terminator onto the template-loop-primer. Step 2 involves using Therminator IX DNA polymerase and four unlabeled reversible terminators (3'-...). O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O The additional extension (-azidomethyl-dTTP) allows for the incorporation of complementary nucleotide analogs into the remaining growing DNA strand. Step 3, after washing away unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal those incorporated using 3'- O -SS-ATP-7-SS-Cy5 or 3'- O-SS-dGTP-7-SS-TCO-Cy5 extended primers. Step 4, labeling with streptavidin-Cy5 will link Cy5 to the 3'- 3'- β ... O -SS-dTTP-5-SS-Biotin and 3'- O -SS-dCTP-5-SS-TCO-Biotin. Step 5, perform the second imaging step, and the new fluorescence signal will confirm the incorporation of the dC or dT reversible terminator. At this time or just before, four 3'- O -Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of the labeled or unlabeled NRT analogs. Step 6: Incubate with tetrazine to cleave the dye on the dC or dG reversible terminator analogs. Step 7: After washing to remove any free tetrazine, a third imaging step is performed. In the case of previously identified dATP or dGTP reversible terminator analog incorporation, loss of Cy5 signal indicates dG, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dTTP reversible terminator analog incorporation, loss of Cy5 signal indicates dC, and the remaining signal indicates dT incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, such that the dye on the dATP and dGTP reversible terminator analogs is removed, and the 3'- O -SS-dNTP or 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 62 The structure of the nucleotides used in this protocol is shown. In the imaging animation at each step, black indicates a positive Cy5 signal, and white indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0091] Figure 64A general set of dye-labeled cleavable ddNTP analogs for monochromatic SBS using click-cutting adapters and pH-responsive dyes: one of the dideoxynucleotide analogs has Cy5 linked to a base via an SS adapter, one has HCyC-646 linked to a base via an SS adapter, one has Cy5 linked to a base via an adapter containing SS (shown as cleavable adapter 1) and TCO (shown as cleavable adapter 2), and the last has HCyC-646 linked to a base via an adapter containing both SS and TCO. HCyC-646 is a pH-responsive dye that fluoresces below pH 6. The dye can be released by click-cutting at cleavable adapter 2 or by standard cleavage at cleavable adapter 1. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0092] Figure 65 : Used for the use of cleavable nucleotide analogs (such as in Figure 64 A simplified representation of a scheme for monochromatic SBS of the cleavable nucleotide analogs presented in the diagram. Each type of ddNTP has one of the following linked via an SS linker or a linker having both SS and TCO groups: Cy5 or HCyC-646. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. This is in contrast to four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- OAfter incubation with Therminator IX to extend most of the primers (-azidomethyl dNTP), extension was performed using Thermo Sequenase and four ddNTP analogs (i.e., ddATP linked to Cy5 via an SS linker, ddTTP linked to HCyC-646 via an SS linker, ddGTP linked to Cy5 via a linker containing both SS and TCO, and ddCTP linked to HCyC-646 via a linker containing both SS and TCO). Following washing at pH 9, imaging revealed positive signals in the first and third rectangular regions due to Cy5 fluorescence (indicating primer extension with A or G) and background signals in the remaining regions. Following washing at pH 5, imaging revealed new positive signals in the second and fourth rectangular regions (indicating primer extension with C or T) due to HCyC-646's ability to fluoresce below pH 6. The reaction of the tetrazine with TCO released Cy5 from the ddCTP and ddGTP nucleotide analogs. After washing, fluorescence loss will specifically reveal the incorporation of C and G, while the remaining fluorescence will reveal the incorporation of A and T, respectively. Finally, THP treatment is used to cleave away any remaining dye and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all three imaging steps, 111 represents A, 010 represents C, 110 represents G, and 011 represents T; considering only the first and last of these imaging steps, 11 represents A, 00 represents C, 10 represents G, and 01 represents T).

[0093] Figure 66 Example ddNTP analogues for Figure 67.

[0094] Figures 67A-67B Monochromatic sequencing-by-synthesis was performed using a set of orthogonal ddNTP analogs containing Cy5 or HCyC-646 and either an SS adapter only or an SS plus TCO adapter. The ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddGTP-7-SS-TCO-Cy5, ddTTP-5-SS-HCyC-646, ddCTP-5-SS-TCO-HCyC-646) and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'-O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) performs monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Step 2 involves adding ddNTP analogs (ddATP-7-SS-Cy5, ddGTP-7-SS-TCO-Cy5, ddTTP-SS-HCyC-646, and ddCTP-SS-TCO-HCyC-646) to the immobilized primer-bound DNA template, enabling the incorporation of ddNTPs into most of the remaining primers. Step 3 involves washing away any unincorporated nucleotide analogs at pH 9, followed by imaging against Cy5 fluorescence to reveal primers extended with either ddATP-7-SS-Cy5 or ddGTP-7-SS-TCO-Cy5. Step 4, washing at pH 5 will allow the HCyC-646 dye on ddTTP-5-SS-HCyC-646 and ddCTP-5-SS-TCO-HCyC-646 to fluoresce. A second imaging step is performed at pH 5, and the new fluorescence signal will confirm the incorporation of ddC or ddT. At this point, or just before, four 3'- O -Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of the ddNTP or NRT analogs. Step 5: Incubate with tetrazine to cleave the dye on the ddC or ddG analog. Step 6: After washing at pH 5 to remove any free tetrazine, a third imaging step is performed at pH 5. In the case of previously determined ddATP or ddGTP analog incorporation, loss of HCyC-646 fluorescence signal indicates ddG, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously determined ddCTP or ddTTP analog incorporation, loss of Cy5 signal indicates ddC, and the remaining signal indicates ddT incorporation. Step 7: The SS adapter is cleaved by adding THP to the extended DNA strand, such that the remaining blocking agent and dye on the dummy terminator nucleotide analog are removed, and the 3'- O-Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 66 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0095] Figure 68 A general set of dye-labeled cleavable dNTP-blocker (virtual terminator) analogs for monochromatic SBS using click-cutting connectors and pH-responsive dyes: one of the virtual terminator analogs has Cy5 linked to a base via an SS connector, one has HCyC-646 linked to a base via an SS connector, one has Cy5 linked to a base via a connector containing SS (shown as cleavable connector 1) and TCO (shown as cleavable connector 2), and the last has HCyC-646 linked to a base via a connector containing both SS and TCO. HCyC-646 is a pH-responsive dye that fluoresces below pH 6. The dye can be released by click-cutting at cleavable connector 2 or by standard cleavage at cleavable connector 1. Four unlabeled reversible terminators (e.g., 3'-) are also required. O (-azidomethyl dNTP) undergoes an additional extension step.

[0096] Figure 69 : Used for the use of cleavable nucleotide analogs (such as in Figure 68 A simplified representation of the scheme for monochrome SBS using the cleavable nucleotide analogs presented in the diagram. Each type of virtual terminator has one of the following connected via an SS linker or a linker containing both SS and TCO groups: Cy5 or HCyC-646. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extension was performed using Thermo Sequenase and four virtual terminator analogs (i.e., dATP connected to Cy5 via an SS linker, dTTP connected to HCyC-646 via an SS linker, dGTP connected to Cy5 via a linker containing both SS and TCO, and dCTP connected to HCyC-646 via a linker containing both SS and TCO). Extension was performed using four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- O-Azide-methyl dNTP) and Therminator IX perform additional extensions to extend all remaining primers. After washing at pH 9, imaging will reveal positive signals (indicating primer extension with A or G) due to Cy5 fluorescence in the first and third rectangular regions and background signals in the remaining regions. After washing at pH 5, imaging will reveal new positive signals (indicating primer extension with C or T) in the second and fourth rectangular regions due to the ability of HCyC-646 to fluoresce below pH 6. The reaction of tetrazine with TCO will release Cy5 on dCTP and dGTP nucleotide analogs. After washing, fluorescence loss will specifically reveal the incorporation of C and G, while the remaining fluorescence will reveal the incorporation of A and T, respectively. Finally, treatment with THP is used to cleave away the remaining dye and remove any azidomethyl groups on the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., positive signal indicated by 1 and background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 010 for C, 110 for G and 011 for T, taking all three imaging steps into account; 11 for A, 00 for C, 10 for G and 01 for T, taking only the first and last of these imaging steps into account).

[0097] Figure 70 Example dNTP analogues for Figure 71.

[0098] Figures 71A-71B Monochromatic sequencing-by-synthesis was performed using a set of orthogonal dNTP-blocker (virtual terminator) analogs containing Cy5 or HCyC-646 and either an SS adapter only or an SS plus TCO adapter. The dNTP-cleavable adapter-blocker-dye (dATP-7-SS-blocker-Cy5, dGTP-7-SS-blocker-TCO-Cy5, dTTP-5-SS-blocker-HCyC-646, dCTP-5-SS-blocker-TCO-HCyC-646) and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O-Azide-methyl-dTTP) performs monochromatic DNA SBS. Step 1, Thermo Sequenase and four dNTP-blocking virtual terminator analogs (dATP-7-SS-blocker-Cy5, dGTP-7-SS-blocker-TCO-Cy5, dTTP-5-SS-blocker-HCyC-646, dCTP-5-SS-blocker-TCO-HCyC-646) are added to a fixed primer-bound DNA template to enable these dNTPs to be incorporated into the 3' end of the template-loop-primer (or other template-binding primer arrangement) so as to be opposite to the complementary bases on the template strand. Step 2, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O The addition of primer-bound DNA templates to the dTTP (-azidomethyl-dTTP) extension immobilization allows for the incorporation of complementary nucleotide analogs into most of the remaining growing DNA strands to terminate DNA synthesis. Step 3, after washing away unincorporated nucleotide analogs at pH 9, imaging against Cy5 fluorescence will reveal primers extended with dATP-7-SS-blocker-Cy5 or dGTP-7-SS-blocker-TCO-Cy5. Step 4, washing at pH 5 will allow HCyC-646 dyes on dTTP-5-SS-blocker-HCyC-646 and dCTP-5-SS-blocker-TCO-HCyC-646 to fluoresce. A second imaging step is performed at pH 5, and the new fluorescence signal will confirm the incorporation of dC or dT. At this point, or just before, four 3'- O -Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either a dummy terminator or an NRT analog. Step 5: Incubate with tetrazine to cleave the dye on the dC or dG analog. Step 6: After washing at pH 5 to remove any free tetrazine, perform a third imaging step at pH 5. In the case of previously determined dATP or dGTP analog incorporation, loss of HCyC-646 fluorescence signal indicates dG, and the remaining signal indicates dA incorporation. Similarly, in the case of previously determined dCTP or dTTP analog incorporation, loss of Cy5 signal indicates dC, and the remaining signal indicates dT incorporation. Step 7: Cleave the SS adapter by adding THP to the extended DNA strand, thereby removing any remaining blocking agent and dye from the incorporated dummy terminator, and also restoring the 3'- O-Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 70 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0099] Figure 72 A general set of dye-labeled, cleavable 3'-blocking reversible terminator analogs for monochromatic SBS using click-cutting connectors and pH-responsive dyes: one of the reversible terminator analogs has Cy5 linked to a base via an SS connector, one has HCyC-646 linked to a base via an SS connector, one has Cy5 linked to a base via a connector containing SS (shown as cleavable connector 1) and TCO (shown as cleavable connector 2), and the last has HCyC-646 linked to a base via a connector containing both SS and TCO. HCyC-646 is a pH-responsive dye that fluoresces below pH 6. The dye can be released by a click-cutting reaction at cleavable connector 2 or by standard cleavage at cleavable connector 1. Four unlabeled reversible terminators (e.g., 3'-) are also required. O (-azidomethyl dNTP) undergoes an additional extension step.

[0100] Figure 73 : Used for the use of cleavable nucleotide analogs (such as in Figure 72 A simplified representation of the scheme for a single-color SBS using the cleavable nucleotide analogs presented in the diagram. Each type of 3'-blocking nucleotide reversible terminator has one of the following linked via an SS linker or a linker containing both SS and TCO groups: Cy5 or HCyC-646. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extensions are performed using Therminator IX and four reversible terminator analogs (i.e., dATP linked to Cy5 via an SS linker, dTTP linked to HCyC-646 via an SS linker, dGTP linked to Cy5 via a linker containing both SS and TCO, and dCTP linked to HCyC-646 via a linker containing both SS and TCO). Extensions are performed using four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- O-Azide-methyl dNTP) and Therminator IX perform additional extensions to extend all remaining primers. After washing at pH 9, imaging will reveal positive signals due to Cy5 fluorescence in the first and third rectangular regions (indicating primer extension with A or G) and background signals in the remaining regions. After washing at pH 5, imaging will reveal new positive signals in the second and fourth rectangular regions (indicating primer extension with C or T) due to the ability of HCyC-646 to fluoresce below pH 6. The reaction of tetrazine with TCO will release Cy5 on the dCTP and dGTP nucleotide reversible terminator analogs. After washing, fluorescence loss will specifically reveal the incorporation of C and G, while the remaining fluorescence will reveal the incorporation of A and T, respectively. Finally, treatment with THP is used to cleave the remaining dye and remove any azidomethyl groups on the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (considering all three imaging steps, 111 represents A, 010 represents C, 110 represents G, and 011 represents T; considering only the first and last of these imaging steps, 11 represents A, 00 represents C, 10 represents G, and 01 represents T).

[0101] Figure 74 Example 3'-SS-dNTP analogue used in Figure 75.

[0102] Figures 75A-75B : Using a set of orthogonal 3'- O - Monochromatic sequencing-while-synthesizing was performed using a blocking nucleotide reversible terminator analog containing Cy5 or HCyC-646 and either an SS adapter only or an SS plus TCO adapter. Using 3'- O -SS-dNTP-cuttable connector-dye (3'- O -SS-dATP-7-SS-Cy5, 3'- O -SS-dGTP-7-SS-TCO-Cy5、3'- O -SS-dTTP-5-SS-HCyC-646、3'- O -SS-dCTP-5-SS-TCO-HCyC-646) and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'-O -Azide-methyl-dTTP) performs monochromatic DNA SBS. Step 1, Therminator IX and four 3'- O -SS-dNTP analogues (3' -O -SS-dATP-7-SS-Cy5,3' -O -SS-dGTP-7-SS-TCO-Cy5、3' -O -SS-dTTP-5-SS-HCyC-646、3' -O Adding SS-dCTP-5-SS-TCO-HCyC-646 to a fixed primer-bound DNA template allows these dye-labeled reversible terminators to be incorporated into the 3' end of the template-loop-primer (or other template-binding primer arrangements) to align with complementary bases on the template strand. Step 2 involves using Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O The addition of primer-bound DNA templates with 3'-azidomethyl-dTTP (-azidomethyl-dTTP) to extend and fix the DNA allows for the incorporation of complementary nucleotide analogs into most of the remaining growing DNA strands to terminate DNA synthesis. Step 3, after washing away unincorporated nucleotide analogs at pH 9, imaging against Cy5 fluorescence will reveal those containing 3'- O -SS-dATP-7-SS-Cy5 or 3'- O -SS-dGTP-7-SS-TCO-Cy5 extended primers. Step 4, washing at pH 5 will allow 3'- O -SS-dTTP-5-SS-HCyC-646 and 3'- O The HCyC-646 dye on -SS-dCTP-5-SS-TCO-HCyC-646 emits fluorescence. A second imaging step is performed at pH 5, and the new fluorescence signal will confirm the incorporation of dC or dT. At this point, or just before, four 3'- O-Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either a dye-labeled nucleotide reversible terminator or an unlabeled NRT analog. Step 5: Incubate with tetrazine to cleave the dye on the dC or dG analog. Step 6: After washing at pH 5 to remove any free tetrazine, a third imaging step is performed. In the case of previously determined dATP or dGTP analog incorporation, loss of HCyC-646 fluorescence signal indicates dG, and the remaining signal indicates dA incorporation. Similarly, in the case of previously determined dCTP or dTTP analog incorporation, loss of Cy5 signal indicates dC, and the remaining signal indicates dT incorporation. Step 7: The SS adapter is cleaved by adding THP to the extended DNA strand, thereby removing the remaining dye on the nucleotide analog and also restoring the 3'- O -SS-dNTP or 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 74 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0103] Figure 76 A general set of dye-labeled cleavable ddNTP analogs for monochromatic SBS using pH-responsive dyes: two of the dideoxynucleotide analogs have Cy5 linked to the base via an SS linker, and the other two have HCyC-646 linked to the base via an SS linker. HCyC-646 is a pH-responsive dye that fluoresces below pH 6. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0104] Figure 77 : Used for the use of cleavable nucleotide analogs (such as in Figure 76A simplified representation of the monochromatic SBS scheme for the cleavable nucleotide analogs presented in the image. Each type of ddNTP has one of the following linked via an SS linker: Cy5 or HCyC-646. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Therminator IX and two of the ddNTP analogs (i.e., ddATP linked to Cy5 via an SS linker and ddTTP linked to HCyC-646 via an SS linker) and excess 3'- O -Azide-methyl dNTPs are extended. After washing at pH 5, imaging will reveal positive signals in the first and fourth rectangular regions due to Cy5 or HCyC-646 fluorescence, indicating the incorporation of A or T. Next, ddGTP linked to Cy5 via the SS linker and ddCTP linked to HCyC-646 via the SS linker, along with an excess of 3'- , are used to ensure fidelity. O -azidomethyl-dATP and 3'- O Incubation with -azidomethyl-dTTP and washing at pH 5 will result in new positive signals in the second and third rectangular regions, indicating C or G incorporation. After washing at pH 9, imaging will reveal a loss of positive signals in the second and fourth rectangular regions, as HCyC-646 fluoresces below pH 6 but not at pH 9. Therefore, if A or T incorporation was previously identified, the fluorescence loss will indicate T incorporation, and if C or G incorporation was previously identified, the fluorescence loss will indicate C incorporation. Remaining fluorescence indicates A and G incorporation, respectively. Finally, THP treatment is used to cleave remaining dye and remove any azidomethyl groups from primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the four indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 010 for C, 011 for G and 110 for T, taking into account all four imaging steps).

[0105] Figure 78 Example ddNTP analogues used in Figure 79.

[0106] Figures 79A-79BMonochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs containing either Cy5 or HCyC-646. The ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddGTP-7-SS-Cy5, ddTTP-5-SS-HCyC-646, ddCTP-5-SS-HCyC-646) and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) performs monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase, two of the ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddTTP-5-SS-HCyC-646) and excess of four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding 3'-azidomethyl-dTTP to a fixed primer-bound DNA template enables the complementary 3'- O -Azide-methyl-dNTPs are incorporated into most of the growing DNA strands (>95%), and ddATP-7-SS-Cy5 and ddTTP-5-SS-HCyC-646 are incorporated into most of the remaining primers to terminate DNA synthesis. Step 2, after washing away unincorporated nucleotide analogs at pH 5, imaging against Cy5 or HCyC-646 fluorescence (the two dyes absorb and emit light at substantially the same wavelength) will reveal those primers extended with ddATP-7-SS-Cy5 or ddT-5-SS-HCyC-646. Step 3, Therminator IX DNA polymerase, the remaining two ddNTP-cleavable adapter-dyes (ddGTP-7-SS-Cy5 and ddCTP-SS-HCyC-646), and two other reversible terminators (3'- O -azidomethyl-dATP, 3'- OAdding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template enables high-fidelity incorporation of ddCTP-5-SS-HCyC-646 and ddGTP-7-SS-Cy5. Step 4, after washing away unincorporated nucleotides at pH 5, performs a second imaging step to reveal Cy5 or HCyC-646 fluorescence, and the new fluorescence signal will confirm the incorporation of ddC or ddG. Step 5, after washing at pH 9 to eliminate the fluorescence of the HCyC-646 dye on ddCTP-SS-HCyC-646 and ddTTP-SS-HCyC-646, a third imaging step will reveal which nucleotide has been incorporated. Therefore, if ddA or ddT is determined to have been added in imaging step 2, the loss of fluorescence signal indicates the incorporation of T, and the remaining signal indicates the incorporation of A. If ddC or ddG is determined to have been added in imaging step 4, the loss of fluorescence signal indicates the incorporation of C, and the remaining signal indicates the incorporation of G. At this point, or just before, an optional additional extension step can be performed using four 3'-O-azidomethyl dNTPs to ensure that virtually every primer has been extended with one of the ddNTPs or NRT analogs. Step 6, cleaving the SS adapter by adding THP to the extended DNA strand, removes the dye from the nucleotide analogs and also restores the 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 78 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0107] Figure 80 A general set of dye-labeled cleavable ddNTP analogs for monochromatic SBS using pH-responsive dyes and anchors for linking dye quencher molecules: two of the dideoxynucleotide analogs have Cy5 linked to the base via an SS linker, and the other two have HCyC-646 linked to the base via an SS linker. The anchor for linking the quencher is present on one of the ddNTPs containing Cy5 and one of the ddNTPs containing HCyC-646. HCyC-646 is a pH-responsive dye that fluoresces below pH 6. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0108] Figure 81 : Used for the use of cleavable nucleotide analogs (such as in Figure 80A simplified representation of a scheme for monochromatic SBS of the cleavable nucleotide analogs presented in the diagram. Each type of ddNTP has one of the following linked via an SS adapter: Cy5, Cy5-tetraazine, HCyC-646, or HCyC-646-tetraazine. Rectangles represent regions on a substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. This is in contrast to four unlabeled nucleotide reversible terminators (NRT, e.g., 3'- O After incubation with Therminator IX to extend most of the primers (-azidomethyl dNTP), extension was performed using Thermo Sequenase and four ddNTP analogs (i.e., ddATP linked to Cy5, ddTTP linked to HCyC-646, ddGTP linked to both tetrazine and Cy5, and ddCTP linked to both tetrazine and HCyC-646). Following washing at pH 9, imaging revealed positive signals in the first and third rectangular regions due to Cy5 fluorescence, indicating A or G incorporation. Switching to pH 5, imaging revealed novel fluorescence in the second and fourth rectangular regions due to the low pH dependence of HCyC-646 fluorescence, indicating C or T incorporation. Incubation with TCO-BHQ3 will quench the tetrazine anchors on ddCTP and ddGTP, and after washing at pH 5, imaging will reveal fluorescence quenching that significantly reduces the fluorescence of both nucleotide analogs, specifically indicating the incorporation of C or G, while the absence of fluorescence loss will specifically indicate the incorporation of A or T. Finally, treatment with THP is used to cleave away any remaining dye and remove any azidomethyl groups from the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogs will be revealed by a unique numerical code (111 for A, 010 for C, 110 for G, and 011 for T considering all three imaging steps; or 11 for A, 00 for C, 10 for G, and 01 for T considering only the first and third imaging steps).

[0109] Figure 82 Examples of ddNTP analogs and quencher-anchored molecules used in Figure 83.

[0110] Figures 83A-83BMonochromatic sequencing-by-synthesis was performed using a set of orthogonal ddNTP analogs containing Cy5, HCyC-646, tetrazine-Cy5, or tetrazine-HCyC-646, and quenched with TCO-BHQ3. ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddGTP-7-SS-tetrazine / Cy5, ddTTP-5-SS-HCyC-646, ddCTP-5-SS-tetrazine / HCyC-646) and 3'- O -Azide-methyl dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O -Azide-methyl-dTTP) and anchor-binding molecule-quencher (TCO-BHQ3) are used to perform monochromatic DNA SBS. Step 1, Therminator IX DNA polymerase and four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template allows complementary nucleotide analogs to be incorporated into most (>90%) of the growing DNA strand to terminate DNA synthesis. Step 2 involves adding Thermo Sequenase and four ddNTP analogs (ddATP-7-SS-Cy5, ddGTP-7-SS-tetraazine / Cy5, ddTTP-5-SS-HCyC-646, ddCTP-5-SS-tetraazine / HCyC-646) to the immobilized primer-bound DNA template, enabling ddNTP incorporation into most of the remaining template-loop-primer. Step 3 involves washing away unincorporated nucleotide analogs at pH 9, followed by imaging against Cy5 fluorescence to reveal primers extended with either ddATP-7-SS-Cy5 or ddGTP-7-SS-tetraazine / Cy5. Step 4, washing at pH 5 will allow the HCyC-646 dye on ddTTP-5-SS-HCyC-646 and ddCTP-5-SS-tetraazine / HCyC-646 to fluoresce. A second imaging step is performed at pH 5, and the new fluorescence signal will confirm the incorporation of ddC or ddT. At this point, or just before, four 3'- O-Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with either ddNTP or NRT analogs. Step 5, incubation with TCO-BHQ3 will link the BHQ quencher to the tetrazine anchors on ddC and ddG. Step 6, after washing at pH 5 to remove any free TCO-BHQ, a third imaging step is performed. In the case of previously identified ddATP or ddGTP analog incorporation, a significant decrease in fluorescence signal indicates ddG, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously identified ddCTP or ddTTP analog incorporation, a significant loss of fluorescence signal indicates ddC, and the remaining signal indicates ddT incorporation. Step 7, cleaving the SS adapter by adding THP to the extended DNA strand removes the dye on the ddNTP analogs and also restores the 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 82 The structure of the nucleotides used in this protocol is shown in the image. In the imaging animation at each step, black indicates a positive Cy5 signal, and white or light gray indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0111] Figure 84 A general set of cleavable ddNTP analogs for dye labeling in monochromatic SBS and anchors for linking dye quencher molecules: all dideoxynucleotide analogs have a Cy5 base linked to the base via an SS linker, and two of the dideoxynucleotide analogs have anchors for linking the dye quencher. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'- O -Azide-methyl dNTP).

[0112] Figure 85 : Used for the use of cleavable nucleotide analogs (such as in Figure 84 A simplified representation of the monochromatic SBS scheme for the cleavable nucleotide analogs presented in the diagram. Each type of ddNTP has one of the following linked via an SS linker: Cy5 or Cy5 and tetrazine. Rectangles represent regions containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Two of the TherminatorIX and ddNTP analogs (i.e., ddATP linked to Cy5 via an SS linker and ddTTP linked to both tetrazine and Cy5 via an SS linker) and excess 3'- O-Azide-methyl dNTPs are extended. After washing, imaging will reveal positive signals in the first and fourth rectangular regions due to Cy5 fluorescence, indicating the incorporation of A or T. Next, ddGTP linked to Cy5 via the SS linker and ddCTP linked to both tetrazine and Cy5 via the SS linker, along with excess 3'- O -azidomethyl-dATP and 3'- O Incubation with -azidomethyl-dTTP followed by washing and imaging will produce a neutral positive signal in the second and third rectangular regions, indicating C or G incorporation. Incubation with TCO-BHQ3 will attach the quencher to the tetrazine anchor on ddCTP and ddTTP to ensure fidelity, and after washing, imaging will reveal fluorescence quenching that significantly reduces the fluorescence of both nucleotide analogs (in the second and fourth rectangular regions), specifically indicating C or T incorporation, while no fluorescence loss (in the first and third rectangular regions) will specifically indicate A or G incorporation. Finally, treatment with THP is used to cleave away any remaining dye and remove any azidomethyl groups on the primers extended with NRT in preparation for the next sequencing cycle. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogs will be revealed by a unique digital code (111 for A, 010 for C, 011 for G and 110 for T, taking all three imaging steps into account; or 11 for A, 00 for C, 01 for G and 10 for T, taking only the first and third imaging steps into account).

[0113] Figure 86 Examples of ddNTP analogs and quencher-anchored molecules used in Figure 87.

[0114] Figures 87A-87B Monochromatic sequencing-by-synthesis was performed using a set of ddNTP analogs containing Cy5 or Cy5-anchored components, along with a quenching step. The ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddGTP-7-SS-Cy5, ddTTP-5-SS-tetraazine / Cy5, ddCTP-5-SS-tetraazine / Cy5) and 3'- O -Azide-methyl dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- OMonochromatic DNA SBS is performed using a combination of dTTP (-azidomethyl-dTTP) and an anchor-binding molecule-quencher (TCO-BHQ3). Step 1 involves adding TherminatorIX DNA polymerase, two of the ddNTP-cleavable adapter-dyes (ddATP-7-SS-Cy5, ddTTP-5-SS-tetraazine / Cy5), and an excess of four reversible terminators (3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- O Adding 3'-O-azidomethyl-dTTP to the immobilized primer-bound DNA template allows complementary 3'-O-azidomethyl-dNTPs to be incorporated into most of the growing DNA strands (>95%) and ddATP-7-SS-Cy5 and ddTTP-5-SS-tetraazine / Cy5 to be incorporated into most of the remaining primers to terminate DNA synthesis. Step 2, after washing away unincorporated nucleotide analogs, imaging against Cy5 fluorescence will reveal primers extended with ddATP-7-SS-Cy5 or ddTTP-5-SS-tetraazine / Cy5. Step 3, Therminator IX DNA polymerase, the remaining two ddNTP-cleavable adapter-dyes (ddGTP-7-SS-Cy5 and ddCTP-5-SS-tetraazine / Cy5), and two other reversible terminators (3'- O -azidomethyl-dATP, 3'- O Adding dTTP (-azidomethyl-dTTP) to the immobilized primer-bound DNA template allows for the precise incorporation of ddGTP-7-SS-Cy5 and ddCTP-5-SS-tetraazine / Cy5. In step 4, after washing away unincorporated nucleotides, a third imaging step is performed to reveal Cy5 fluorescence, and the new fluorescence signal will confirm the incorporation of ddC or ddG. At this point, or just before, four 3'- O-Azide-methyl dNTPs are used to perform an optional additional extension step to ensure that virtually every primer has been extended with one of ddNTPs or NRT analogs. Step 5, incubation with TCO-BHQ3 will link the BHQ quencher to the tetrazine anchors on ddC and ddT. Step 6, after washing to remove any free tetrazine-BHQ, a third imaging step is performed. In the case of previously identified ddATP or ddTTP analog incorporation, a significant reduction in Cy5 signal indicates ddT, and the remaining signal indicates ddA incorporation. Similarly, in the case of previously identified ddCTP or ddGTP analog incorporation, a significant loss of Cy5 signal indicates ddC, and the remaining signal indicates ddG incorporation. Step 7, cleaving the SS adapter by adding THP to the extended DNA strand removes the dye on the ddATP and ddGTP analogs and also restores the 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 86 The structure of the nucleotides used in this protocol is shown in the image. In the imaging animation at each step, black indicates a positive Cy5 signal, and white or light gray indicates a background signal. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0115] Figure 88 Synthesis of ddNTP-SS-dye-TCO (using ddGTP-7-SS-Cy5-TCO as an example). This type of compound was used in Examples 6 and 7.

[0116] Figure 89 Synthesis of ddNTP-SS-dye-TCO (short connector type) (using ddGTP-7-SS-Cy5-TCO as an example). This type of compound is used in Example 6.

[0117] Figure 90 Synthesis of ddNTP-SS-anchored-TCO (using ddCTP-5-SS-biotin-TCO as an example). This type of compound is used in Example 6.

[0118] Figure 91 Synthesis of combined molecular-quenchers (e.g., tetrazine-BHQ3). This type of compound is used in Examples 6 and 7.

[0119] Figure 92 Synthesis of a combination molecule-dye (using TCO-HCyC-646 as an example). This type of compound is used in Example 2.

[0120] Figure 93Synthesis of combined molecular-quencher (TCO-BHQ3 as an example). This type of compound is used in Examples 11 and 12.

[0121] Figure 94 Synthesis of HCyC-646 NHS ester. Compounds of this type were used in Examples 2, 7, and 10.

[0122] Figure 95 Synthesis of ddNTP-SS-dyes (using ddTTP-5-SS-HCyc-646 as an example). Compounds of this type were used in Examples 2, 7, and 10.

[0123] Figure 96 Synthesis of dye-TCO connector-NHS ester (using Cy5-TCO-NHS ester as an example). Compounds of this type were used in Examples 5, 8, and 9.

[0124] Figure 97 Synthesis of ddNTP-TCO connector-anchor (using ddCTP-5-TCO-biotin as an example). This type of compound was used in Example 5.

[0125] Figure 98 Synthesis of ddNTP-SS-dyed-tetraazine (using ddGTP-7-SS-Cy5-tetraazine as an example). This type of compound is used in Examples 11 and 12.

[0126] Figure 99 Synthesis of ddNTP-SS-dyed-tetraazine (using ddGTP-7-SS-Cy5-tetraazine as an example). This type of compound is used in Examples 11 and 12.

[0127] Figure 100 Synthesis of dNTP-SS-blocker-TCO-anchor (using dCTP-SS-blocker-TCO-biotin as an example, the bases can be A, C, T, or G). This type of compound is used in Example 8.

[0128] Figure 101 Synthesis of dNTP-SS-blocker-TCO-dye (using dCTP-SS-blocker-TCO-HCyC-646 as an example, where the bases can be A, C, T, or G, and the dye can be Cy5). This type of compound is used in Examples 8 and 9.

[0129] Figure 102 Synthesis of 3'-SS-dNTP-SS--TCO- dyes (taking 3'-SS-dGTP-SS--TCO-HCyC-646 as an example, where the bases can be A, C, T, or G, and the dye can also be Cy5). This type of compound is used in Example 8.

[0130] Figure 103 Synthesis of 3'-SS-dNTP-SS--TCO-anchors (using 3'-SS-dCTP-SS--TCO-biotin as an example, where the bases can be A, C, T, or G). This type of compound is used in Examples 8 and 9.

[0131] Figure 104 For two different fixed DNA templates, ddCTP-5-SS-Cy5, ddGTP-7-SS-Cy5, ddATP-7-SS-Biotin, ddTTP-5-SS-Biotin, streptavidin-Cy5, and four 3'- O The results of thirteen monochromatic sequencing-by-synthesis cycles using azidomethyl dNTPs. Each cycle consists of the following steps (with an intermediate washing step): (1) using Therminator IX DNA polymerase with four 3'- O - Azide-methyl dNTPs were extended to extend ~95% of the primer-loop-template molecule at each spot on the slide; (2) ddCTP-SS-Cy5, ddATP-SS-biotin, 3'- O -Azide-methyl-dGTP and 3'- O(3) Extension with 3'-O-azidomethyl-dTTP (“E-ddAddC”); (4) Incubation with streptavidin-Cy5 (first “labeling” step); (5) Extension with ddGTP-SS-Cy5, ddTTP-SS-biotin, 3'-O-azidomethyl-dATP and 3'-O-azidomethyl-dCTP (“E-ddGddT”); (6) Additional extension with four 3'-O-azidomethyl-dNTPs; (7) Treatment with THP to remove the dye and restore the 3'-OH group on the incorporated reversible terminator (3'-O-azidomethyl-dNTP) (“cleavage”). Signals below 700 are considered background and encoded as “0”; signals above 850 are considered positive and encoded as “1”. In the bar chart, each group of four bars represents one cycle, and the bars from left to right represent the fluorescence images of any unit of the first extension, first labeling, second extension, and second labeling, respectively. Examining the top bar chart, the imaging result of cycle 1 (0011) indicates the incorporation of G, cycle 2 (0111) indicates A, cycle 3 (0011) indicates G, cycle 4 (1111) indicates C, cycle 5 (0111) indicates A, cycle 6 (0001) indicates T, and so on, thus revealing that the first 13 bases of the template sequence are 3'-CTCGTAGTTCAAA-5' (SEQ ID NO: 1), which perfectly matches the expected template sequence attached to the surface of the slide region. Similarly, examining the bottom bar chart, the obtained sequence is 3'-GTAGTTCAAACCC-5' (SEQ ID NO: 2), which is also perfectly consistent with the expected sequence of the template attached to the surface of the slide region. These results demonstrate that the SBS method described in Example 1 of this application can be used for successful and accurate DNA sequencing.

[0132] Figure 105 : A group of nucleotide analogs (ddCTP-SS-Cy5, ddGTP-SS-Cy5, ddTTP-SS-HCyC-646, ddATP-SS-HCyC-646, 3'-O-CH2-N3-dATP, 3'-O-CH2-N3-dCTP, 3'-O-CH2-N3-dGTP and 3'-O-CH2-N3-dTTP) for enabling monochromatic sequencing-by-synthesis without the labeling step in Example 2.

[0133] Figure 106 : Used for Figure 105 The diagram shows a scheme for single-color sequencing of a set of nucleotides.

[0134] Figure 107Synthesis of the pH-responsive dye HCyC-646 and the conjugation of HCyC-646 NHS with 5-amino-SS-dTTP. Detailed procedures are described in Example 10 in the main text.

[0135] Figure 108 The connection of HCyC-646 NHS with 7-amino-SS-dATP. Detailed protocol is described in Example 10 of the text.

[0136] Figure 109 MALDI-TOF-MS spectra of ddTTP-5-SS-HCyC-646 synthesized and purified as described in Example 10. Expected MW (1298 Da); Obtained value (1302 Da).

[0137] Figure 110 MALDI-TOF-MS spectra of ddA-7-SS-HCyC-646 synthesized and purified as described in Example 10. Expected MW (1321 Da); Obtained value (1326 Da).

[0138] Figure 111 Examples of protonated and deprotonated forms of HCyC-646 linked to ddNTPs (shown as ddATP).

[0139] Figure 112 MALDI-TOF-MS spectra of primers extending ddTTP-5-SS-HCyC-646. The protocol is described in Example 10 in the text. Expected product size (6286 Da); obtained value (6287 Da). This indicates that the ddTTP-5-SS-HCyC-646 nucleotide was recognized by DNA polymerase (Therminator IX in this case).

[0140] Figure 113Sequencing was performed using dTTP-5-SS-CyC-646 as described in Example 10. Three cycles of extension, pH washing, and cleavage were performed. Example 10 details the protocol, and the individual steps are indicated in the figures. Four images are shown for cycle 1, and two images are shown for cycles 2 and 3 each. Note the fluorescence loss when the slide, previously washed with a low pH buffer (below 7), is then washed with a high pH buffer (above 9). The expected sequences for the first three positions of the template are TAG in the left rectangular region of the slide, GAG in the second region, CAT in the third region, and ATT in the rightmost region of the slide. Therefore, using dTTP-5-SS-CyC-646, it was expected to be incorporated into the left region in cycle 1, the right region in cycle 2, and the two rightmost regions of the slide in cycle 3, which is exactly what was observed. Similar successful characterization was also performed on dATP-SS-HCyC-646.

[0141] Figure 114 :use Figure 106 The protocol shown here is an example of sequencing cycles using ddCTP-5-SS-Cy5, ddATP-7-SS-HCyC-646, ddGTP-7-SS-Cy5, ddTTP-5-SS-HCyC-646, and four 3'-O-azidomethyl dNTPs. A simplified protocol is shown in the box on the left of the figure, and a detailed protocol is provided in the accompanying text for Example 10. As expected, after extension with ddCTP-5-SS-Cy5, ddATP-7-SS-HCyC-646, and 3'-O-azidomethyl dGTP and 3'-O-azidomethyl dTTP, followed by washing at pH 5, a positive fluorescent signal for Cy5 or HCyC-646 was obtained, indicating the incorporation of C or A. A second extension was performed using ddGTP-7-SS-Cy5, ddTTP-5-SS-HCyC-646, and 3'-O-azidomethyl dCTP and 3'-O-azidomethyl dATP, followed by a pH 5 wash to produce a positive fluorescence signal due to the incorporation of G or T. Washing with a pH 9 buffer eliminated the signal generated by the fluorescence of HCyC-646. Therefore, if A or C incorporation was previously determined, fluorescence loss indicates A incorporation, while remaining fluorescence indicates C incorporation. Similarly, if G or T incorporation was previously determined, fluorescence loss indicates T incorporation, while remaining fluorescence indicates G incorporation. Finally, THP treatment cleaves the disulfide bonds, thereby removing the dye from the ddNTP analogues (indicated by background fluorescence only) and restoring the 3'-OH group on any incorporated 3'-O-azidomethyl dNTP.

[0142] Figure 115Sequencing-by-synthesis (SBS) was performed for four cycles using ddCTP-5-SS-Cy5, ddATP-7-SS-HCyC-646, ddGTP-7-SS-Cy5, ddTTP-5-SS-HCyC-646, and four 3'-O-azidomethyl dNTPs against four different templates. Two of the four different templates were replicated in different portions of the slide. The four consecutive SBS cycles were executed in... Figure 106 Described in Figure 114 The procedure is illustrated for one cycle. The bar graph in the upper left corner shows the results of the first cycle. Six groups of three bars each represent different templates. The first bar in each group (E-ddAC5) represents the fluorescence result obtained after the first extension with ddATP-7-SS-HCyC-646 and ddCTP-5-SS-Cy5, followed by washing at pH 5. The second bar in each group (E-ddGT5) represents the fluorescence result obtained after the second extension with ddTTP-7-SS-HCyC-646 and ddGTP-5-SS-Cy5, followed by washing at pH 5. The last bar in each group (pH 9) represents the fluorescence result obtained after switching to pH 9 buffer to significantly reduce fluorescence caused by the pH-sensitive dye HCyC-646. As an example, the background fluorescence after the first extension (below 700 arbitrary units), the positive fluorescence after the second extension, and the background fluorescence after switching to pH 9 are digitally recorded as 010 and indicate the incorporation of T. The digital readings 011, 111, and 110 indicate G, C, and A incorporation, respectively. The lower left bar indicates the result of the second SBS cycle, the lower right bar indicates the result of the third SBS cycle, and the upper right bar indicates the result of the fourth SBS cycle. Correct results were obtained in each of the four cycles using the template DNA.

[0143] Figure 116 This is a general set of nucleotide analogs (virtual terminators) with base-blocking anchors and dye-labeled molecules for single-molecule energy transfer (SBS), which utilize donor dyes and anchors for linking pH-responsive or pH-unresponsive dye acceptor molecules. All nucleotide analogs have Cy3 and biotin or tetrazine anchors linked to the bases via SS linkers. The anchoring molecules streptavidin and TCO are linked to Cy5 and HCyC-646, respectively. The latter is a pH-responsive dye that fluoresces below pH 6.

[0144] Figure 117 : Used with nucleotide analogs (virtual terminators) that have blocking agents at the bases (such as in Figure 116This is a simplified representation of a scheme for single-molecule energy transfer (SBS) using nucleotide analogs presented in the diagram. Each type of nucleotide analog has a Cy3 and a biotin or tetrazine anchor linked to a base via an SS linker. Rectangles represent regions on the substrate containing a single-template DNA molecule consisting of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extension is performed using both Thermo Sequenase and dNTP analogs (i.e., dATP linked to Cy3 and biotin, and dTTP linked to Cy3 and tetrazine). Labeling is then performed using both streptavidin-Cy5 and TCO-HCyC-646. After washing at pH 5, excitation and imaging of Cy3 due to energy transfer from Cy3 to Cy5 or HCyC-646 dye will reveal positive signals in the first and fourth rectangular regions, indicating the incorporation of A or T. A second extension was performed using Thermo Sequenase and the remaining two nucleotide analogs (i.e., dCTP linked to Cy3 and biotin, and dGTP linked to Cy3 and tetrazine). Labeling was again performed using both streptavidin-Cy5 and TCO-HCyC-646. After washing at pH 5, excitation and imaging of Cy3 revealed new fluorescence in the second and third rectangular regions, indicating C or G incorporation, due to energy transfer to Cy5 or HCyC-646. After washing at pH 9, imaging revealed a significant decrease in HCyC-646 fluorescence on the T and G nucleotide analogs (third and fourth rectangular regions on the slide), but no loss of Cy5 fluorescence on the A and C nucleotide analogs (first and second rectangular regions). Finally, treatment with THP was used to cleave the remaining dye and restore the 3'-OH groups on these nucleotides. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 011 for C, 010 for G and 110 for T, taking all three imaging steps into account; or 11 for A, 01 for C, 00 for G and 10 for T, taking only the first and third imaging steps into account).

[0145] Figure 118 Example dNTP virtual terminator analogues for connecting donor dye (Cy3) and anchor molecule (tetraazine or biotin) and corresponding binding molecule (TCO or streptavidin)-acceptor dye (Cy5 or HCyc-646) conjugates for Figure 119.

[0146] Figure 119A-D: Single-molecule energy transfer sequencing-by-synthesis (SBS) is performed using a set of virtual terminator analogs containing Cy3 and biotin or tetrazine for linking Cy5 or the pH-responsive dye HCyC-646. Single-molecule energy transfer DNA SBS is performed using dNTP-blocker-cleavable adapter-anchor / dye (dATP-7-SS-blocker-biotin / Cy3, dTTP-5-SS-blocker-tetrazine / Cy3, dCTP-5-SS-blocker-biotin / Cy3, and dGTP-7-SS-blocker-tetrazine / Cy3) and anchor-binding molecule-dye molecules (streptavidin-Cy5 and TCO-HCyC-646). Step 1: Thermo Sequenase DNA polymerase and two of the four virtual terminator analogs (dATP-7-SS-blocker-biotin / Cy3 and dTTP-5-SS-blocker-tetraazine / Cy3) are added to the immobilized primer-bound DNA template. Step 2: After washing away any unincorporated nucleotides, streptavidin-Cy5 and TCO-HCyC-646 are added together to label the dATP and dTTP nucleotide analogs with biotin and tetraazine anchors. Step 3: After washing at pH 5 and exciting Cy3, the fluorescence of Cy5 and HCyC-646 will reveal primers extended with either dATP-7-SS-blocker-biotin / Cy3 or dTTP-5-SS-blocker-tetraazine / Cy3 due to energy transfer from Cy3. Step 4: Thermo Sequenase DNA polymerase and the remaining virtual terminator analogs (dCTP-5-SS-blocker-biotin / Cy3 and dGTP-7-SS-blocker-tetraazine / Cy3) are added to the immobilized primer-bound DNA template. Step 5: After washing away any unincorporated nucleotides, streptavidin-Cy5 and TCO-HCyC-646 are added again to label the dCTP and dGTP nucleotide analogs with biotin and tetraazine anchors. Step 6: After washing at pH 5 and exciting Cy3, the emergence of new Cy5 and HCyC-646 fluorescent signals due to energy transfer from Cy3 will reveal primers extended with either dCTP-5-SS-blocker-biotin / Cy3 or dGTP-7-SS-blocker-tetraazine / Cy3. Step 7: After washing at pH 9, a third imaging step is performed to obtain a positive Cy5 fluorescence signal but only background HCyC-646 fluorescence. In the case of previously identified dATP or dTTP analog incorporation, a significant reduction in fluorescence signal indicates dT, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dGTP analog incorporation, a significant loss of fluorescence signal indicates dG, and the remaining signal indicates dC incorporation.Step 8 involves cleaving the SS adapter by adding THP to the elongated DNA strand, thereby removing the dye from the reversible terminator analog and restoring its 3'-OH group. The DNA product is now ready for the next cycle of the DNA sequencing reaction. Figure 118 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive Cy5 or HCyC-646 signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0147] Figure 120 This is a general set of anchors and dye-labeled cleavable 3'-blocking dNTP analogs (reversible terminators) for single-molecule energy transfer (SBS) using donor dyes and anchors for linking pH-responsive or pH-unresponsive dye acceptor molecules. All nucleotide analogs have Cy3 and biotin or tetrazine anchors linked to bases via SS linkers. The anchor-binding molecules streptavidin and TCO are linked to Cy5 and HCyC-646, respectively. The latter is a pH-responsive dye that fluoresces below pH 6.

[0148] Figure 121 : Used with nucleotide analogs that can cleave 3'-block (such as in Figure 120This is a simplified representation of a scheme for single-molecule energy transfer (SBS) using cleavable 3'-blocking nucleotide analogs. Each type of dNTP has a Cy3 and biotin or tetrazine anchor linked to a base via an SS adapter. Rectangles represent regions on the substrate containing a single-template DNA molecule consisting of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extension is performed using Therminator IX and two of the dNTP analogs (i.e., 3'-SS-dATP linked to Cy3 and biotin, and 3'-SS-dTTP linked to Cy3 and tetrazine). Labeling is then performed using both streptavidin-Cy5 and TCO-HCyC-646. After washing at pH 5, excitation and imaging of Cy3 due to energy transfer from Cy3 to Cy5 or HCyC-646 dye will reveal a positive signal in the first and fourth rectangular regions, indicating the incorporation of A or T. A second extension was performed using Therminator IX and the remaining two dNTP analogs (i.e., 3'-SS-dCTP linked to Cy3 and biotin, and 3'-SS-dGTP linked to Cy3 and tetrazine). Labeling was again performed using both streptavidin-Cy5 and TCO-HCyC-646. After washing at pH 5, excitation and imaging of Cy3 revealed novel fluorescence in the second and third rectangular regions, indicating C or G incorporation, due to energy transfer to Cy5 or HCyC-646. After washing at pH 9, imaging revealed a significant decrease in fluorescence of HCyC-646 on the T and G nucleotide analogs (third and fourth rectangular regions on the slide), but no loss of fluorescence of Cy5 on the A and C nucleotide analogs (first and second rectangular regions). Finally, treatment with THP was used to cleave the remaining dye and restore the 3'-OH groups on these nucleotides. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps: a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique numerical code (111 for A, 011 for C, 010 for G, and 110 for T if all three imaging steps are considered; or 11 for A, 01 for C, 00 for G, and 10 for T if only the first and third imaging steps are considered).

[0149] Figure 122 Example 3'-SS-dNTP reversible terminator analogue used in Figure 123, which connects both the donor dye (Cy3) and the anchor molecule (tetraazine or biotin), and the corresponding binding molecule (TCO or streptavidin)-acceptor dye (Cy5 or HCyc-646) conjugate.

[0150] Figure 123A -D: Single-molecule energy transfer sequencing-by-synthesis is performed using a set of nucleotide reversible terminator analogs containing Cy3 and biotin or tetrazine for linking Cy5 or the pH-responsive dye HCyC-646. 3'-blocking reversible terminator analogs are used (3'- O -SS-dATP-7-SS-Biotin / Cy3,3'- O -SS-dGTP-7-SS-tetraazine / Cy3,3'- O -SS-dTTP-5-SS-tetraazine / Cy3,3'- O -SS-dCTP-5-SS-Biotin / Cy3) and anchor-binding molecules-dye molecules (streptavidin-Cy5 and TCO-HCyC-646) perform single-molecule energy transfer DNA SBS. Step 1, Therminator IX DNA polymerase and two of the four 3'-blocking dNTP analogs (3'- O -SS-dATP-7-SS-Biotin / Cy3 and 3'- O (-SS-dTTP-5-SS-tetraazine / Cy3) is added to the immobilized primer-bound DNA template. Step 2, after washing away any unincorporated nucleotides, streptavidin-Cy5 and TCO-HCyC-646 are added together to label dATP and dTTP nucleotide analogs via biotin and tetraazine anchors. Step 3, after washing at pH 5 and exciting Cy3, the fluorescence of Cy5 and HCyC-646 will reveal those labeled with 3'- O -SS-dATP-7-SS-Biotin / Cy3 or 3'- O -SS-dTTP-5-SS-tetraazine / Cy3 extension primers. Step 4, add Therminator IX DNA polymerase and the remaining 3'-blocking dNTP analog (3'- O -SS-dCTP-5-SS-Biotin / Cy3 and 3'- O (-SS-dGTP-7-SS-tetraazine / Cy3) is added to the immobilized primer-bound DNA template. Step 5, after washing away any unincorporated nucleotides, streptavidin-Cy5 and TCO-HCyC-646 are added again to label dCTP and dGTP nucleotide analogs via biotin and tetraazine anchors. Step 6, after washing at pH 5 and exciting Cy3, the emergence of new Cy5 and HCyC-646 fluorescent signals due to energy transfer from Cy3 will reveal those labeled with 3'- O -SS-dCTP-5-SS-Biotin / Cy3 or 3'-O -SS-dGTP-7-SS-tetraazine / Cy3 extended primers. Step 7: After washing at pH 9, a third imaging step is performed to obtain a positive Cy5 fluorescence signal but only background HCyC-646 fluorescence. In the case of previously identified dATP or dTTP analog incorporation, a significant decrease in fluorescence signal indicates dT, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dGTP analog incorporation, a significant loss of fluorescence signal indicates dG, and the remaining signal indicates dC incorporation. Step 8: The SS adapter is cleaved by adding THP to the extended DNA strand, such that the dye on the reversible terminator analog is removed, and its 3'-OH group is also restored. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 122 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive Cy5 or HCyC-646 signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0151] Figure 124 This is a general set of nucleotide analogs (virtual terminators) for single-molecule energy transfer (SBS) using base-blocking anchors and dye-labeled nucleotide analogs, employing donor dyes and anchors for linking pH-responsive or pH-unresponsive dye acceptor molecules. Two of the nucleotide analogs have Cy3 and biotin or tetrazine anchors linked to the bases via SS linkers. Two other nucleotide analogs have Cy3 and biotin or tetrazine anchors linked to the bases via azo (N=N) linkers. The anchoring molecules streptavidin and TCO are linked to Cy5 and HCyC-646, respectively. The latter is a pH-responsive dye that fluoresces below pH 6.

[0152] Figure 125 : Used with nucleotide analogs (virtual terminators) that have blocking agents at the bases (such as in Figure 124A simplified representation of a scheme for single-molecule energy transfer (SBS) using nucleotide analogs presented in the diagram. Each type of nucleotide analog has both a Cy3 and a biotin or tetrazine anchor linked to a base via an SS or azo linker, with all combinations of linkers and anchors: SS and biotin at A, SS and tetrazine at C, azo and tetrazine at G, and azo and biotin at T. Rectangles represent regions on the substrate containing a single-template DNA molecule consisting of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extension was performed using Thermo Sequenase and four dNTP analogs. Labeling was then performed using both streptavidin-Cy5 and TCO-HCyC-646. Following washing at pH 5, excitation and imaging of Cy3 will reveal positive signals in all four rectangular regions due to energy transfer from Cy3 to Cy5 or HCyC-646 dye, indicating incorporation of A, C, G, or T. Following washing at pH 9, excitation and imaging of Cy3 will reveal fluorescence loss in the second and third rectangular regions due to the low pH dependence of HCyC-646 fluorescence, indicating incorporation of C or G. Residual fluorescence due to the lack of pH responsiveness of Cy5 indicates incorporation of A or T. Treatment with sodium dithionite will cleave the azo linkers on G and T, thereby removing the dye attached to these nucleotides. Therefore, fluorescent signals will be present only in the first and second rectangular regions. Finally, treatment with THP will cleave away the remaining dye and restore the 3'-OH groups on these nucleotides. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 101 for C, 100 for G and 110 for T, taking all three imaging steps into account; or 11 for A, 01 for C, 00 for G and 10 for T, taking only the second and third imaging steps into account).

[0153] Figure 126 Example 3'-dNTP virtual terminator analogues of Figure 127, which connect both the donor dye (Cy3) and the anchor molecule (tetraazine or biotin) via orthogonal cuttable joints (SS and azo joints) and the corresponding binding molecule (TCO or streptavidin)-acceptor dye (Cy5 or HCyc-646) conjugates.

[0154] Figure 127A-D: Single-molecule energy transfer sequencing-by-synthesis (SBS) is performed using a set of orthogonal virtual terminator analogs containing Cy3 and a biotin or tetrazine linked to a base via an SS or azo linker for conjugating Cy5 or the pH-responsive dye HCyC-646. Single-molecule energy transfer DNA SBS is performed using dNTP-blocker-cleavable linker-anchor / dye (dATP-7-SS-blocker-biotin / Cy3, dTTP-5-SS-blocker-azo-biotin / Cy3, dCTP-5-SS-blocker-tetrazine / Cy3, and dGTP-7-SS-blocker-azo-tetrazine / Cy3) and anchor-binding molecule-dye molecules (streptavidin-Cy5 and TCO-HCyC-646). Step 1: ThermoSequenase DNA polymerase and four virtual terminator analogs (dATP-7-SS-blocker-biotin / Cy3, dTTP-5-SS-blocker-azo-biotin / Cy3, dCTP-5-SS-blocker-tetraazine / Cy3, and dGTP-7-SS-blocker-azo-tetraazine / Cy3) are added to a primer-bound DNA template. Step 2: After washing away any unincorporated nucleotides, streptavidin-Cy5 and TCO-HCyC-646 are added together to label the nucleotide analogs with biotin and tetraazine anchors. Step 3: After washing at pH 5 and exciting Cy3, the fluorescence of Cy5 and HCyC-646 will reveal primers extended with any of the four virtual terminator nucleotide analogs due to energy transfer from Cy3. Step 4: After washing and exciting Cy3 at pH 9, a significant loss of fluorescence signal due to energy transfer from Cy3 will reveal primers extended with dC or dG nucleotide analogs labeled HCyC-646, while the remaining fluorescence due to Cy5 will indicate the incorporation of dA and dT nucleotide analogs. Step 5: Cleavage of the azo groups in the linkers connecting the dye and anchor to the bases of the dG and dT nucleotide analogs will remove the dye from these nucleotides. Step 6: Excite Cy3 and image the fluorescence of Cy5 or HCyC-646. In the case of previously identified dATP or dTTP analog incorporation, a significant decrease in fluorescence signal indicates dT, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dGTP analog incorporation, a significant loss of fluorescence signal indicates dG, and the remaining signal indicates dC incorporation. Step 7 involves cleaving the SS adapter by adding THP to the elongated DNA strand, thereby removing the dye from the reversible terminator analog and restoring its 3'-OH group. The DNA product is now ready for the next cycle of the DNA sequencing reaction. Figure 126The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive Cy5 or HCyC-646 signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0155] Figure 128 This is a general set of anchors and dye-labeled cleavable 3'-blocking dNTP analogs (reversible terminators) for single-molecule energy transfer (SBS) using donor dyes and anchors for linking pH-responsive or pH-unresponsive dye acceptor molecules. Two of the nucleotide analogs have Cy3 and biotin or tetrazine anchors linked to the bases via SS linkers. The other two nucleotide analogs have Cy3 and biotin or tetrazine anchors linked to the bases via azo (N=N) linkers. The anchor binding molecules streptavidin and TCO are linked to Cy5 and HCyC-646, respectively. The latter is a pH-responsive dye that fluoresces below pH 6.

[0156] Figure 129 : Used with nucleotide analogs that can cleave 3'-block (such as in Figure 128This is a simplified representation of a scheme for single-molecule energy transfer (SBS) using cleavable 3'-blocking nucleotide analogs. Each type of dNTP has both a Cy3 and a biotin or tetrazine anchor linked to a base via an SS or azo linker, with all combinations of linkers and anchors: SS and biotin at A, SS and tetrazine at C, azo and tetrazine at G, and azo and biotin at T. Rectangles represent regions on the substrate containing a single-template DNA molecule consisting of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Extension was performed using Therminator IX and four dNTP analogs. Labeling was then performed using both streptavidin-Cy5 and TCO-HCyC-646. Following washing at pH 5, excitation and imaging of Cy3 will reveal positive signals in all four rectangular regions due to energy transfer from Cy3 to Cy5 or HCyC-646 dye, indicating incorporation of A, C, G, or T. Following washing at pH 9, excitation and imaging of Cy3 will reveal fluorescence loss in the second and third rectangular regions due to the low pH dependence of HCyC-646 fluorescence, indicating incorporation of C or G. Residual fluorescence due to the lack of pH responsiveness of Cy5 indicates incorporation of A or T. Treatment with sodium dithionite will cleave the azo linkers on G and T, thereby removing the dye attached to these nucleotides. Therefore, fluorescent signals will be present only in the first and second rectangular regions. Finally, treatment with THP will cleave away the remaining dye and restore the 3'-OH groups on these nucleotides. The numerical codes 1, 2, and 3 on the left represent the cumulative signal at each of the three indicated imaging steps, i.e., a positive signal indicated by 1 and a background signal indicated by 0. The incorporation of each of the four possible nucleotide analogues will be revealed by a unique digital code (111 for A, 101 for C, 100 for G and 110 for T, taking all three imaging steps into account; or 11 for A, 01 for C, 00 for G and 10 for T, taking only the second and third imaging steps into account).

[0157] Figure 130 Example 3'-SS-dNTP reversible terminator analogues of Figure 131, which connect both the donor dye (Cy3) and the anchoring molecule (tetraazine and biotin) via orthogonal cleavable connectors (SS and azo connectors) and the corresponding binding molecule (TCO or streptavidin)-acceptor dye (Cy5 or HCyc-646) conjugates.

[0158] Figure 131A-D: Sequencing-by-synthesis based on single-molecule energy transfer using a set of orthogonal nucleotide reversible terminator analogs containing Cy3 and a biotin or tetrazine linked to Cy5 or the pH-responsive dye HCyC-646 via an SS or azo linker. 3'-blocking reversible terminator analogs (3'- O -SS-dATP-7-SS-Biotin / Cy3,3'- O -SS-dGTP-7-azo-tetraazine / Cy3,3'- O -SS-dTTP-5-azo-tetraazine / Cy3,3'- O -SS-dCTP-5-SS-tetraazine / Cy3) and anchoring molecules-dye molecules (streptavidin-Cy5 and TCO-HCyC-646) perform single-molecule energy transfer DNA SBS. Step 1, Therminator IX DNA polymerase and four 3'-blocking dNTP analogs (3'- O -SS-dATP-7-SS-Biotin / Cy3,3'- O -SS-dGTP-7-azo-tetraazine / Cy3,3'- O -SS-dTTP-5-azo-biotin / Cy3 and 3'- OStep 2: After washing away any unincorporated nucleotides, streptavidin-Cy5 and TCO-HCyC-646 are added together to label the nucleotide reversible terminator analogs with biotin and tetrazine anchors. Step 3: After washing and exciting Cy3 at pH 5, the fluorescence of Cy5 and HCyC-646 will reveal primers extended with any of the four nucleotide reversible terminator nucleotide analogs due to energy transfer from Cy3. Step 4: After washing and exciting Cy3 at pH 9, a significant loss of fluorescence signal due to energy transfer from Cy3 will reveal primers extended with dC or dG nucleotide analogs labeled with HCyC-646, while the remaining fluorescence due to Cy5 will indicate the incorporation of dA and dT nucleotide analogs. Step 5: Cleavage of the azo group in the linker between the dye and anchor to the bases of the dG and dT nucleotide analogs removes the dye from these nucleotides. Step 6: Excite Cy3 and image the fluorescence of Cy5 or HCyC-646. In the case of previously identified dATP or dTTP analog incorporation, a significant decrease in fluorescence signal indicates dT, and the remaining signal indicates dA incorporation. Similarly, in the case of previously identified dCTP or dGTP analog incorporation, a significant loss of fluorescence signal indicates dG, and the remaining signal indicates dC incorporation. Step 7: Cleavage of the SS linker by adding THP to the elongated DNA strand removes the dye from the reversible terminator analog and also restores its 3'-OH group. The DNA product is now ready for the next cycle of the DNA sequencing reaction. Figure 130 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive Cy5 or HCyC-646 signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0159] Figure 132 A common set of dye-labeled, reversible terminators for monochromatic SBS using pH-responsive dyes: two of the nucleotide analogs have Cy5 linked to the base via an SS linker, and two others have HCyC-646 linked to the base via an SS linker. HCyC-646 is a pH-responsive dye that fluoresces below pH 6. This hybridization SBS method requires a separate set of four unlabeled reversible terminators (e.g., 3'-O-azidomethyl dNTPs).

[0160] Figure 133 : Used for the use of cleavable nucleotide analogs (such as in Figure 132A simplified representation of the monochromatic SBS scheme for the cleavable nucleotide analogs presented in the diagram. Each type of reversible nucleotide has one of the following linked via an SS linker: Cy5 or HCyC-646. Rectangles represent regions on the substrate containing multiple copies of the linked primer-loop-template molecule (or other template-binding primer arrangement), where the next base in the template strand is T, G, C, or A from left to right. Two of the Therminator IX and dNTP analogs (i.e., 3'-tBu-dATP linked to HCyC-646 via an SS linker and 3'-tBu-dCTP linked to Cy5 via an SS linker) and a small amount of 3'-... are used to improve fidelity. O -Azide-methyl-dGTP and 3'- O -Azidemethyl-dTTP is extended. After washing at pH 5, imaging will reveal positive signals in the first and second rectangular regions due to Cy5 or HCyC-646 fluorescence, indicating A or C incorporation. Next, incubation with 3'-tBu-dGTP linked to Cy5 via an SS linker, 3'-tBu-dTTP linked to HCyC-646 via an SS linker, 3'-O-azidomethyl-dATP and 3'-O-azidomethyl-dCTP for fidelity enhancement, and washing at pH 5 will produce new positive signals in the third and fourth rectangular regions, indicating G or T incorporation. After washing at pH 9, imaging will reveal the loss of positive signals in the first and fourth rectangular regions because HCyC-646 is able to fluoresce below pH 6 but not at pH 9. Therefore, if A or C incorporation was previously determined, fluorescence loss will indicate A incorporation, and if G or T incorporation was previously determined, fluorescence loss will indicate T incorporation. Remaining fluorescence indicates C and G incorporation, respectively. Finally, THP treatment is used to cleave away any remaining dye and remove any azidomethyl groups from primers extended with NRT in preparation for the next sequencing cycle. The 1, 2, and 3 numeric codes on the left represent the cumulative signal at each imaging step indicated, i.e., a positive signal indicated by 1 and a background signal indicated by 0. Incorporation of each of the four possible nucleotide analogues will be revealed by a unique numeric code that takes into account all three imaging steps (110 for A, 111 for C, 011 for G, and 010 for T).

[0161] Figure 134 Used for Figure 133 Example structure of a reversible terminator.

[0162] Figure 135A-D: Monochromatic sequencing-while-synthesis was performed using a set of fluorescent 3'-tert-butyl-SS nucleotide analogs containing Cy5 or HCyC-646. 3'-tBu-SS-dNTP-cleavable adapter-dyes (3'-tBu-SS-dATP-7-SS-HCyC-646, 3'-tBu-SS-dCTP-5-SS-Cy5, 3'-tBu-SS-dGTP-7-SS-Cy5, 3'-tBu-SS-dTTP-5-SS-HCyC-646) and 3'- O -Azide-methyl-dNTP(3'- O -azidomethyl-dATP, 3'- O -Azide-methyl-dCTP, 3'- O -azidomethyl-dGTP, 3'- OPerform a monochromatic DNA SBS using 3'-Azidemethyl-dTTP. Step 1 involves adding Therminator IX DNA polymerase, two of the 3'-tert-butyl-SS-dNTP-cleavable adapter-dyes (3'-tBu-SS-dATP-7-SS-HCyC-646 and 3'-tBu-SS-dCTP-5-SS-Cy5), and small amounts of 3'-O-azidomethyl-dGTP and 3'-O-azidomethyl-dTTP to the immobilized primer-bound DNA template to allow incorporation of 3'-tBu-SS-dATP-7-SS-HCyC-646 and 3'-tBu-SS-dCTP-5-SS-Cy5 or 3'-O-azidomethyl-dNTP to terminate DNA synthesis. Step 2, after washing away the unincorporated nucleotide analogs at pH 5, imaging against Cy5 or dCTP-646 fluorescence (the two dyes absorb and emit light at substantially the same wavelengths) will reveal those primers extended with 3'-tBu-SS-dATP-7-SS-HCyC-646 and 3'-tBu-SS-dCTP-5-SS-Cy5. Step 3: Therminator IX DNA polymerase, the remaining two 3'-tert-butyl-SS-cleavable adapter dyes (3'-tBu-SS-dGTP-7-SS-Cy5 and 3'-tBu-SS-dTTP-5-SS-HCyC-646), and 3'-O-azidomethyl-dATP and 3'-O-azidomethyl-dCTP for fidelity enhancement are added to the immobilized primer-bound DNA template to enable incorporation of 3'-tBu-SS-dGTP-7-SS-Cy5 and 3'-tBu-SS-dTTP-5-SS-HCyC-646 or 3'-O-azidomethyl-dNTP. Step 4: After washing away unincorporated nucleotides at pH 5, a second imaging step is performed to reveal Cy5 or HCyC-646 fluorescence, and the new fluorescence signal will confirm the incorporation of G or T. Step 5, after washing at pH 9 to reduce the fluorescence of HCyC-646 dye on 3'-tBu-SS-dATP-7-SS-HCyC-646 or 3'-tBu-SS-dTTP-5-SS-HCyC-646, the third imaging step will reveal which nucleotide has been incorporated. Therefore, if A or C is determined to have been added in imaging step 1, the loss of fluorescence signal indicates the incorporation of A, and the remaining signal indicates the incorporation of C. If G or T is determined to have been added in imaging step 2, the loss of fluorescence signal indicates the incorporation of T, and the remaining signal indicates the incorporation of G. At this point, or just before, with four 3'- O-Azide-methyl dNTPs are used to perform additional extension steps to ensure that almost every primer has been extended with either a dye-labeled 3'-SS-dNTP or an NRT analog, especially since 3'- dNTPs were not added in the first and second extension reactions. O In the case of -azidomethyl dNTP. Step 6, by adding THP to the elongated DNA strand to cleave the SS adapter, the dye on the nucleotide analog is removed, and the 3'- O -Azide-methyl-dNTP extension of any growing chain with a 3'-OH group. The DNA product is ready for the next cycle of the DNA sequencing reaction. Figure 134 The structure of the nucleotides used in this protocol is shown in the imaging animation. In the imaging animation at each step, black indicates positive fluorescent signals, and white indicates background signals. The coding in the final summary animation indicates the template sequence, not the incorporated nucleotide.

[0163] Figure 136 Twenty consecutive sequencing-by-synthesis cycles were performed using 3'-tBu-SS-dATP-7-SS-HCyC-646, 3'-tBu-SS-dCTP-5-SS-Cy5, 3'-tBu-SS-dGTP-7-SS-Cy5, 3'-tBu-SS-dTTP-5-SS-HCyC-646, and four 3'-O-azidomethyl dNTPs. The sequencing-by-synthesis cycle was performed for each of the 20 consecutive cycles. Figure 133The procedure described in 135 is followed, with the template and primers shown at the top of the figure. However, 3'-O-azidomethyl-dNTPs were not added in steps 1 and 3, but only during the additional extension in step 6. White bars in each cycle represent fluorescence results after extension with the first two nucleotide analogs (3'-tBu-SS-dATP-7-SS-HCyC-646 and 3'-tBu-SS-dCTP-5-SS-Cy5) and washing at pH 5, black bars represent fluorescence results after extension with the second two nucleotide analogs (3'-tBu-SS-dGTP-7-SS-Cy5 and 3'-tBu-SS-dTTP-5-SS-HCyC-646) and washing at pH 5, and shaded bars represent fluorescence results after washing at pH 9. The expected codes were obtained (010 for T, 011 for G, 111 for C, and 110 for A), indicating successful sequencing at each cycle. At the end of each cycle, treatment with THP brings the fluorescence to background levels (not shown). Note that in cycles 4 and 5, the first extension is performed with 3'-tBu-SS-dGTP-7-SS-Cy5 and 3'-tBu-SS-dTTP-5-SS-HCyC-646, and the second extension is performed with 3'-tBu-SS-dATP-7-SS-HCyC-646 and 3'-tBu-SS-dCTP-5-SS-Cy5. This is the reverse order of addition in the other 18 cycles and makes the coding of cycles 4 and 5 different (010 represents A, 011 represents C, 111 represents G and 110 represents T). Detailed Implementation

[0164] The widely used high-throughput SBS technology (Bentley et al. 2008) utilizes previously developed cleavable fluorescent nucleotide reversible terminator (NRT) sequencing chemistry (Ju et al. 2003; Ju et al. 2006). These cleavable fluorescent NRTs are designed based on the principle of modifying each of the four nucleotides (A, C, G, T) by attaching a unique cleavable fluorophore to a specific position on the base and capping the 3'-OH group with a small, reversible portion, so that it remains recognized as a substrate by DNA polymerase. Therefore, cleavable fluorescent NRTs involve two site modifications (Ju et al. 2003; Ju et al. 2006): a fluorescent dye acting as a reporter group on the base and a small chemical portion that caps the 3'-OH group to temporarily terminate the polymerase reaction for sequencing after nucleotide incorporation. After incorporation and signal detection, the fluorophore is cleaved and the 3'-OH capping portion is removed to resume the polymerase reaction in the next cycle. These cleavable fluorescent NRTs have proven to be excellent substrates for reengineered polymerases and have been widely used in next-generation DNA sequencing systems (Ju et al. 2006; Bentley et al. 2008). Furthermore, the cleavable fluorescent NRTs enable accurate determination of homopolymer sequences because only one base is identified in each cycle.

[0165] The SBS method, which uses cleavable fluorescent nucleotide analogs as reversible terminators for sequencing surface-fixed DNA, has been used (Ju et al. 2003; Li et al. 2003; Ruparel et al. 2005; Ju et al. 2006; Wu et al. 2007; Guo et al. 2008). In this method, the nucleotides are modified at two specific positions so that they are still recognized as substrates by DNA polymerase: ( i Different fluorophores with different fluorescence emissions are connected to specific sites of each of the four bases via cleavable linkers, and ( ii The 3'-OH group is capped with a small, chemically reversible portion. DNA polymerase incorporates only mononucleotide analogs complementary to the bases covalently linked to the DNA template on the surface. After incorporation, a unique fluorescent emission is detected to identify the incorporated nucleotide. The fluorophore is then removed and the 3'-OH group is chemically regenerated, allowing for the next cycle of the polymerase reaction. Because the large surface area on the DNA chip can accommodate a high density of different dotted DNA templates, many bases can be identified in parallel per cycle, enabling simultaneous sequencing of large numbers of DNA molecules. Previous research has firmly established SBS's molecular-level strategy of rationally modifying nucleotides by linking cleavable fluorescent dyes to bases and reversibly capping the 3'-OH group with a small portion.

[0166] A class of nucleotide analogs with unprotected 3'-OH and cleavable disulfide linkers between the bases and fluorescent dyes has been reported (Turcatti et al. 2008; Mitra et al. 2003). However, after DNA polymerase-catalyzed extension on the primer / template and imaging of the incorporated bases, the cleavage of the disulfide bond produces a free reactive -SH group, which must be capped with the alkylating agent iodoacetamide before a second extension can be performed. This capping step not only adds an extra step to the process but also limits the sequential addition of multiple nucleotides due to the long residual tail region on the nucleotide base moiety. In this method, the sequencing read length is limited to 10 bases (Turcatti et al. 2008). Other disulfide-based methods require similar capping reactions to render the free SH group non-reactive (Mitra et al. 2003).

[0167] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; The cleavable connector includes DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivatives and is linked to the base via the 5-position of a pyrimidine (C, U) or the 7-position of a desaturated purine (A, G, I); and The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for dye bonding, clusters of anchors for dye bonding, or anchors and dyes.

[0168] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; R includes methyl, ethyl, propyl, tert-butyl, aryl, and alkylaryl; Cuttable connectors include DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivatives; and The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for linking fluorescent dyes, clusters of anchors for linking fluorescent dyes, or anchors and dyes.

[0169] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; Cleavable connectors include DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivatives, or more than one of these cleavable connectors, including the special case where one cleavable connector is present between the base and the blocker and a second different cleavable connector is present between the blocker and the marker; The blocking agent is a nucleotide or oligonucleotide comprising 2-50 monomeric units of a basic sugar or modified nucleoside or a combination thereof; and the blocking agent is linked to the 5-position of pyrimidine (C, U) and the 7-position of desaminoglycan (A, G, I) via a cleavable linker. The blocking agent is the part that prevents further incorporation of other nucleotides or nucleotide analogs into the primer chain after incorporation; and The markers include fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for linking fluorescent dyes, clusters of anchors for linking fluorescent dyes, or anchors and dyes, wherein the markers are linked to the blocking agent.

[0170] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, uracil, thymine, hypoxanthine, or analogues thereof; and R is a cleavable chemical group, including alkyl DTM, azo, 2-nitrobenzyl, allyl, and azidomethyl derivatives.

[0171] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; and The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for dye bonding, clusters of anchors for dye bonding, or anchors and dyes.

[0172] This invention provides a nucleotide analog having the following structure: , The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof.

[0173] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; The labeling includes fluorescent dyes, pH-responsive fluorescent dyes, clusters of fluorescent dyes, pH-responsive fluorescent dye clusters, anchors for dye bonding, clusters of anchors for dye bonding, or anchors and dyes; and R includes methyl, ethyl, propyl, tert-butyl, aryl, and alkylaryl.

[0174] This invention provides a nucleotide analog having the following structure: , in: The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; and R includes methyl, ethyl, propyl, tert-butyl, aryl, and alkylaryl.

[0175] This invention provides a nucleotide analog having the following structure: , The bases include adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof.

[0176] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; and (B) an anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) an anchor-labeled nucleotide analog comprising a base and an anchor attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable connector and the 3'-OH group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) an anchor-labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and an anchor connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable joint described herein can be cut with the same cutting agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog provided in step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signal generated due to the binding of the anchor binding group to any anchor-tagged nucleotide analog incorporated in step (b); g) Repeat steps (b)-(f) with two different labeled nucleotide analogs, the two different labeled nucleotide analogs being different from the two different labeled nucleotide analogs from the previous iterations of step (b); h) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any markers, anchors or blocking groups from the incorporated nucleotide analogue of step (b); i) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0177] In one embodiment, the nucleotide analogue in step (b) is selected from... Figure 3 , Figure 7 or Figure 11 A group composed of nucleotide analogs.

[0178] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each of which hybridizes with primers, wherein each template has the same sequence as the nucleic acid to be sequenced, and provide a nucleic acid polymerase; b) The nucleic acid template is contacted with four different labeled nucleotide analogs (A, C, T, G), and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; (B) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label linked to the base via a cleavable linker; (C) Two different anchor-labeled dideoxynucleotide analogs, wherein each analog comprises a different anchor linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) A pH-responsive fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (C) Two different anchor-labeled nucleotide analogs comprising a base and an anchor attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein each analog comprises a different anchor attached to the base via a cleavable linker. The cuttable connector and the 3'-O blocking group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a fluorescent label attached to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A pH-responsive fluorescently labeled nucleotide analog comprising a base, a blocking group attached to the base via a cleavable linker, and a pH label attached to the base distal to the blocking group. The responsive fluorescent label, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, and (C) two different anchor-labeled nucleotide analogs, the two different anchor-labeled nucleotide analogs comprising a base, a blocking group attached to the base via a cleavable linker, and an anchor attached to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, wherein each analog comprises a different anchor attached to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); e) Contact the incorporated nucleotide analog from step (b) with: (A) an anchor-binding group that binds to the anchor of only one of the anchor-labeled nucleotide analogs of step (b), wherein the anchor-binding group comprises the same fluorescent label as the fluorescently labeled nucleotide analog of step (b); and (B) an anchor-binding group that binds to the anchor of only the remaining anchor-labeled nucleotide analogs, wherein the anchor-binding group comprises the same pH-responsive fluorescent label as the pH-responsive fluorescently labeled nucleotide analog of step (b). f) Wash away any unincorporated nucleotide analogs at pH at which the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the anchor-labeled nucleotide analog from step (b). g) Wash the incorporated nucleotide analog from step (b) at a pH where the pH-responsive fluorescent label no longer has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b). h) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any markers, anchors or blocking groups from the incorporated nucleotide analogue of step (b); i) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0179] In one embodiment, the nucleotide analogue in step (b) is selected from... Figure 15 , Figure 18 or Figure 19 The group consists of nucleotide analogs. In one embodiment, the label with pH-responsive fluorescence is HCyC-646, and the label with pH-non-responsive fluorescence is Cy5.

[0180] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; and (B) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label linked to the base via a cleavable linker. The cuttable joint described herein can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a pH-responsive fluorescently labeled nucleotide analog comprising a base and a pH-responsive fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable connector and the 3'-O blocking group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a pH-responsive fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a pH-responsive fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the cleavable linker is cleavable by the same cleaving agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); e) Repeat steps (b)-(d) with two different labeled nucleotide analogs, which are different from the two different labeled nucleotide analogs from the previous iterations of step (b); f) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); Steps (e) and (f) can be performed in reverse order; g) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any labeling or blocking groups from the incorporated nucleotide analogue of step (b); h) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0181] In one embodiment, the nucleotide analogue in step (b) is selected from... Figure 78 The group consists of nucleotide analogs. In one embodiment, the label with pH-responsive fluorescence is HCyC-646, and the label with pH-non-responsive fluorescence is Cy5.

[0182] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a first cleavable linker; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a carbamoyl TCO linker; (C) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via the first cleavable linker; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via a carbamoyl TCO linker. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label linked to the base via a first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label linked to the base via a carbamoyl TCO linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) Anchor-labeled nucleotide analog, wherein... Anchor-tagged nucleotide analogs include a base and an anchor linked to the base via a first cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) anchor-tagged nucleotide analogs comprising a base and an anchor linked to the base via a carbamoyl TCO linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the 3'-O blocking group and the first cleavable linker can be cleaved by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a carbamoyl TCO linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. (C) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a first cleavable linker, and an anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain; and (D) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a carbamoyl TCO linker, and an anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain. The fluorescent label on each analogue is identical. The anchors on each analogue are identical; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog provided in step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signal generated due to the binding of the anchor binding group to any anchor-tagged nucleotide analog incorporated in step (b); g) Contact the incorporated nucleotide analog with the tetrazine derivative to click the TCO portion of the carbamoyl TCO linker to release any tags or anchors attached through the carbamoyl TCO linker, and identify any fluorescent signals generated due to the incorporation of the labeled nucleotide analog from step (b). h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the first cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0183] In one embodiment, the nucleotide analogue in step (b) is selected from... Figure 34 A group composed of nucleotide analogs.

[0184] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a first anchor; (C) Anchor-labeled dideoxynucleotide analog comprising a base and a first anchor and a second anchor attached to the base via a cleavable linker; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and a second anchor attached to the base via a cleavable linker, wherein the cleavable linker is cleavable by the same cleaving agent. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker and a first anchor and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) Anchor-labeled nucleotide analog, wherein the anchor... The labeled nucleotide analogue comprises a base and a first anchor and a second anchor connected to the base via the cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analogue into the extended primer chain, and (D) an anchor-labeled nucleotide analogue comprising a base and a second anchor connected to the base via the cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analogue into the extended primer chain, wherein the cleavable linker and the 3'-O blocking group can be cleaved by the same cleaving agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group and a first anchor, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (C) Anchor-labeled nucleotides. (A) Anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a cleavable linker, and a first anchor and a second anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, and (D) Anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a cleavable linker, and a second anchor connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of nucleotide analogs into the extended primer chain, wherein the cleavable linker can be cleaved by the same cleaving agent. The fluorescent labeling on each analogue is identical; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the second anchor of the nucleotide analog from step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signals generated due to the binding of the anchoring group to the anchoring group of any incorporated nucleotide analogue in step (b); g) Contact the incorporated nucleotide analog with a second anchoring group, the second anchoring group being bound to the first anchor of the nucleotide analog in step (b) and including a portion that quenches the fluorescence signal of any fluorescently labeled nucleotide analog linked to the anchoring group, and identifies any fluorescence signal generated due to the incorporation of the fluorescently labeled nucleotide analog. h) Contact the incorporated nucleotide analogue with a cleaving agent that cleaves the cleavable linker and any 3'-O blocking group; and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

[0185] In one embodiment, the nucleotide analogue in step (b) is selected from... Figure 38 , Figure 42 or Figure 46 A group composed of nucleotide analogs.

[0186] This invention provides a method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with four different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with the nucleotide analog if one of the labeled nucleotide analogs is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the four different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable adapter; (B) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label attached to the base via the first cleavable adapter and a carbamoyl TCO adapter attached to the distal end of the first cleavable adapter; (C) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor attached to the base via the first cleavable adapter and a carbamoyl TCO adapter attached to the distal end of the first cleavable adapter; and (D) Anchor-labeled dideoxynucleotide analog comprising a base and an anchor attached to the base via the first cleavable adapter. (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a first cleavable linker and a carbamoyl TCO linker attached to the distal end of the first cleavable linker and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into an extended primer chain; (C) Anchor-labeled nucleotide analog, wherein... Anchor-tagged nucleotide analogs include a base and an anchor linked to the base via a first cleavable adapter and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, and (D) anchor-tagged nucleotide analogs comprising a base and an anchor linked to the base via the first cleavable adapter and a carbamoyl TCO adapter connected to the distal end of the first cleavable adapter, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the 3'-OH blocking group and the first cleavable adapter can be cleaved by the same reagent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a first cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; (B) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via the first cleavable linker, and a fluorescent label connected to the base via a carbamoyl TCO linker distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. (C) Anchor-labeled nucleotide analogs, the anchor-labeled nucleotide analogs comprising a base, a blocking group connected to the base via a first cleavable adapter, and an anchor connected to the base at the distal end of the blocking group, wherein the bloc...

Claims

1. A nucleotide analog having the following structure: ， in: The bases are adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; R is methyl, ethyl, propyl, tert-butyl, aryl, or alkylaryl; The cuttable connector is a DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivative, and The markers consist of anchors and dyes.

2. The nucleotide analogue according to claim 1, wherein (a) The base is thymine; (b) R is tert-butyl; and / or (c) The dye is a fluorescent dye or a cluster of fluorescent dyes, and the anchor is an anchor for connecting the fluorescent dye, or an anchor cluster for connecting the fluorescent dye; preferably, the fluorescent dye or cluster of fluorescent dyes is Rhodamine 110, R6G, TAMRA, ROX, Cy5, HCyC-646, or Alexa 647, and the anchor for connecting the fluorescent dye, or the anchor cluster for connecting the fluorescent dye, is biotin, DBCO, TCO, or tetrazine; more preferably, the fluorescent dye or cluster of fluorescent dyes is Cy5, and the anchor for connecting the fluorescent dye, or the anchor cluster for connecting the fluorescent dye, is TCO; Preferably, the nucleotide analog has the following structure: 。 3. A composition comprising the nucleotide analogue according to claim 2; preferably, the composition further comprising one or more nucleotide analogues having the following structure: ， in: The bases are adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; R is methyl, ethyl, propyl, tert-butyl, aryl, or alkylaryl; The cuttable connector is a DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivative; and The marker is a fluorescent dye, a cluster of fluorescent dyes, an anchor for connecting the fluorescent dye, or a cluster of anchors for connecting the fluorescent dye; preferably, wherein... (a) The bases of one or more nucleotide analogs are adenine or guanine; (b) In one or more of the said nucleotide analogues, R is tert-butyl; and / or (c) In the one or more nucleotide analogs, the label is a fluorescent dye, a fluorescent dye cluster, or an anchor for linking a fluorescent dye, or an anchor cluster for linking a fluorescent dye; preferably, in the one or more nucleotide analogs, the fluorescent dye or fluorescent dye cluster is Rhodamine 110, R6G, TAMRA, ROX, Cy5, HCyC-646, or Alexa 647, and the anchor for linking a fluorescent dye or the anchor cluster for linking a fluorescent dye is biotin, DBCO, TCO, or tetrazine; more preferably, in the one or more nucleotide analogs, the fluorescent dye or fluorescent dye cluster is Cy5, and the anchor for linking a fluorescent dye or the anchor cluster for linking a fluorescent dye is biotin; Preferably, the one or more nucleotide analogs have the following structure: , and / or ； More preferably, the one or more nucleotide analogs have the following structure: , and / or 。 4. The composition according to claim 3, further comprising one or more anchoring molecules; preferably, wherein the one or more anchoring molecules are streptavidin, azide, tetrazine, or TCO and further comprising a single dye, a single dye cluster, or an energy transfer dye; more preferably, wherein the one or more anchoring molecules are and / or .

5. The composition according to claim 4, further comprising a nucleotide analog having the following structure: ， in: The bases are adenine, guanine, cytosine, uracil, thymine, hypoxanthine, or analogues thereof; and R is a cleavable chemical group, including alkyl DTM, azo, 2-nitrobenzyl, allyl, and azidomethyl derivatives.

6. The composition according to claim 5, wherein... (a) In the nucleotide analogue, the base is cytosine and R is an alkyl DTM; and / or (b) The nucleotide analog binds to an anchoring molecule, wherein the anchoring molecule is streptavidin, azide, tetrazine, or TCO and further comprises a single dye, a single dye cluster, or an energy transfer dye; preferably, wherein the anchoring molecule is an anchoring molecule having a quencher; more preferably, wherein the anchoring molecule having a quencher is tetrazine-BHQ.

7. A composition comprising the nucleotide analogue according to claim 6; preferably, the composition further comprising one or more nucleotide analogues having the following structures: ， in: The bases are adenine, guanine, cytosine, thymine, uracil, hypoxanthine, or analogues thereof; R is methyl, ethyl, propyl, tert-butyl, aryl, or alkylaryl; The cuttable connector is a DTM, azo, 2-nitrobenzyl, allyl, azidomethyl, or TCO derivative; and The marker is a fluorescent dye, a cluster of fluorescent dyes, an anchor used to connect fluorescent dyes, or a cluster of anchors used to connect fluorescent dyes; Preferably, wherein (a) The bases of one or more nucleotide analogs are adenine or guanine; (b) In one or more of the nucleotide analogues, R is tert-butyl; (c) In one or more nucleotide analogs, the marker is a fluorescent dye, a cluster of fluorescent dyes, or an anchor for linking a fluorescent dye, or a cluster of anchors for linking a fluorescent dye; preferably, in one or more nucleotide analogs, the marker is a fluorescent dye or a cluster of fluorescent dyes; (d) The cuttable connector is a DTM or TCO derivative; (e) In one or more nucleotide analogs, the label is a fluorescent dye or a cluster of fluorescent dyes; (f) In one or more of the nucleotide analogues, the fluorescent dye or fluorescent dye cluster is Rhodamine 110, R6G, TAMRA, ROX, Cy5, HCyC-646, or Alexa 647; and / or (g) In one or more nucleotide analogs, the fluorescent dye or fluorescent dye cluster is Cy5; Preferably, the one or more nucleotide analogs have the following structure: , and / or ； Preferably, the one or more nucleotide analogs have the following structure: , and / or 。 8. The composition according to claim 7, further comprising one or more anchoring molecules; preferably, said one or more anchoring molecules are streptavidin, azide, tetrazine or TCO and further comprising a single dye, a single dye cluster or an energy transfer dye; More preferably, one or more anchoring molecules are ； More preferably, the composition further comprises a nucleotide analog having the following structure: ， in: The bases are adenine, guanine, cytosine, uracil, thymine, hypoxanthine, or analogues thereof; and R is a cleavable chemical group, which includes alkyl DTM, azo, 2-nitrobenzyl, allyl, and azidomethyl derivatives; More preferably, in the nucleotide analogue, the base is cytosine and R is an alkyl DTM.

9. A method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with one of the labeled nucleotide analogs if the nucleotide analog is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; and (B) an anchor-labeled dideoxynucleotide analog comprising a base and an anchor linked to the base via a cleavable linker. The cuttable joint described above can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) an anchor-labeled nucleotide analog comprising a base and an anchor attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable connector and the 3'-OH group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) an anchor-labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and an anchor connected to the base distal to the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable joint described above can be cut with the same cutting agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Identify any fluorescent signals produced due to the incorporation of fluorescently labeled nucleotide analogs; e) Contact the incorporated nucleotide analog from step (b) with an anchor binding group, the anchor binding group binding to the anchor of the anchor-tagged nucleotide analog provided in step (b), wherein the anchor binding group includes the same fluorescent label as the fluorescent label of the fluorescently labeled nucleotide analog from step (b). f) Identify any fluorescent signal generated due to the binding of the anchor binding group to any anchor-tagged nucleotide analog incorporated in step (b); g) Repeat steps (b)-(f) with two different labeled nucleotide analogs, the two different labeled nucleotide analogs being different from the two different labeled nucleotide analogs from the previous iterations of step (b); h) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any markers, anchors or blocking groups from the incorporated nucleotide analogue of step (b); i) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and j) Repeat steps (b) to (i) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

10. A method for sequencing nucleic acids, the method comprising: a) Provide multiple nucleic acid templates, each hybridizing with a primer, and a nucleic acid polymerase, wherein each template has the same sequence as the nucleic acid to be sequenced; b) The nucleic acid template is contacted with two different labeled nucleotide analogs, and the contact is performed under the condition that the nucleic acid polymerase is allowed to extend the primer with one of the labeled nucleotide analogs if the nucleotide analog is complementary to the 5' nucleotide residue of the nucleic acid template that hybridizes to the 3' nucleotide residue of the primer, wherein the two different labeled nucleotide analogs are: (i) (A) A fluorescently labeled dideoxynucleotide analog comprising a base and a fluorescent label linked to the base via a cleavable linker; and (B) A pH-responsive fluorescently labeled dideoxynucleotide analog comprising a base and a pH-responsive fluorescent label linked to the base via a cleavable linker. The cuttable joint described above can be cut with the same cutting agent; (ii) (A) A fluorescently labeled nucleotide analog comprising a base and a fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a pH-responsive fluorescently labeled nucleotide analog comprising a base and a pH-responsive fluorescent label attached to the base via a cleavable linker, and a blocking group at the 3'-OH position, wherein the blocking group prevents subsequent incorporation of the nucleotide analog into the extended primer chain. The cuttable joint and the 3'-O blocking group can be cut by the same cutting agent; or (iii) (A) A fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain; and (B) a pH-responsive fluorescently labeled nucleotide analog comprising a base, a blocking group connected to the base via a cleavable linker, and a pH-responsive fluorescent label connected to the base at the distal end of the blocking group, wherein the blocking group prevents or greatly reduces subsequent incorporation of the nucleotide analog into the extended primer chain, wherein the cleavable linker can be cleaved by the same cleaving agent; c) Extending the unextended primer with a nucleotide analog that does not have any base modifications and includes a 3'-O blocking group, wherein step (c) occurs before, simultaneously with or after step (b); d) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); e) Repeat steps (b)-(d) with two different labeled nucleotide analogs, which are different from the two different labeled nucleotide analogs from the previous iterations of step (b); f) Wash away any unincorporated nucleotide analog at pH where the pH-responsive fluorescent label has the same or similar absorption and emission curves as the fluorescent label on the fluorescently labeled nucleotide analog, and identify any fluorescent signal generated due to the incorporation of the labeled nucleotide analog from step (b); Steps (e) and (f) can be performed in reverse order; g) Cleavage the cleavable linker from the incorporated nucleotide analogue, thereby removing any labeling or blocking groups from the incorporated nucleotide analogue of step (b); h) cleaving the 3'-O blocking group from any incorporated nucleotide analogue from step (c); and i) Repeat steps (b) to (h) iteratively for each residue of the nucleic acid to be sequenced. The sequence of the nucleic acid is thus obtained.

Images