Compositions and methods for sequencing multiple regions of a template molecule using read segment capping nucleotide analogs

Abstract

This disclosure provides compositions comprising read-capped nucleotide analogs and methods for sequencing using said read-capped nucleotide analogs. When sequencing of a first region of a template molecule is complete, the read-capped nucleotide analog can be incorporated at the 3' end of a sequencing read to produce a first-capped sequencing read. The first-capped sequencing read is not removed from the template molecule before sequencing a second region of the template molecule. Removal of the first-capped sequencing read is avoided because the incorporated read-capped nucleotide analog blocks the binding and incorporation of nucleotide reagents used for sequencing the second region of the nucleic acid template molecule. Using said read-capped nucleotide analogs without removing the sequencing read reduces damage to the template molecule and preserves an intact template molecule after multiple sequencing cycles.

Description

[0001] Cross-reference of priority claims

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 607,234, filed on December 7, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of high-throughput sequencing and provides compositions comprising read-capped nucleotide analogs and methods of using read-capped nucleotide analogs. Background Technology

[0004] Traditional multiplex sequencing workflows require sequencing a first region (e.g., the target sequence region) using a first sequencing primer and a second region (e.g., the sample index region) using a second sequencing primer. Before sequencing the second region, the sequencing reads synthesized by sequencing the first region must be efficiently removed without damaging the template molecule. Traditional sequencing workflows that use chemical-based reagents (e.g., NaOH and / or formamide) to denature the sequencing reads can be harsh, resulting in template molecule damage. Milder chemical-based denaturation conditions may cause less damage to the template molecule but are not efficient at removing sequencing reads that contribute to residual signals. The accumulation of residual signals in subsequent sequencing cycles can reduce sequencing accuracy. Therefore, additional methods are needed to efficiently remove sequencing reads from previous sequencing cycles or prevent previous sequencing reads from affecting subsequent sequencing cycles. This disclosure provides compositions and methods for sequentially sequencing different regions of the same nucleic acid template molecule and reducing residual signals that lead to poor sequencing quality. Summary of the Invention

[0005] This disclosure provides a method for sequencing two or more regions of a nucleic acid template molecule, the method comprising: (a) providing a plurality of nucleic acid template molecules, wherein each template molecule includes (i) a first region and a first universal sequencing primer binding site, and (ii) a second region and a second universal sequencing primer binding site; (b) providing a plurality of first nucleic acid sequencing primers and a plurality of second nucleic acid sequencing primers; (c) hybridizing each first nucleic acid sequencing primer to the first universal sequencing primer binding site on the template molecule; (d) sequencing the first region of the plurality of nucleic acid template molecules, thereby generating a plurality of first sequencing reads; and (e) performing a plurality of first capping reactions, the plurality of first capping reactions comprising incorporating a read-capping nucleotide analog into the terminal 3' end of each first sequencing read, thereby generating a plurality of capped first sequencing reads, wherein the reads The capped nucleotide analog comprises (i) a heterocyclic base, (ii) a sugar, and (iii) a polyphosphate chain, wherein the heterocyclic base is linked to a termination portion that blocks the binding of a multivalent molecule to the 3' end of the capped first sequencing read, wherein each multivalent molecule comprises a core attached to multiple nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; and wherein the termination portion blocks the polymerase-catalyzed incorporation of subsequent nucleotides into the capped first sequencing read, (f) retaining the multiple capped first sequencing reads hybridized to a nucleic acid template molecule, wherein the termination portion is not cleaved or removed; (g) hybridizing each second nucleic acid sequencing primer to the second universal sequencing primer binding site on each template molecule; and (h) sequencing the second region on the multiple template molecules, thereby generating multiple second sequencing reads.

[0006] In some embodiments of the method disclosed herein, the method further includes: (i) performing a plurality of second capping reactions, the plurality of second capping reactions including incorporating a read-capping nucleotide analog into the terminal 3' end of a respective second sequencing read product, thereby generating a plurality of capped second sequencing read products, wherein the read-capping nucleotide analog comprises (i) a heterocyclic base, (ii) a sugar, and (iii) a polyphosphate chain, wherein the heterocyclic base is linked to a termination portion that blocks the binding of a multivalent molecule to the terminal 3' end of the capped second sequencing read product, wherein each multivalent molecule comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit; wherein the termination portion blocks the subsequent polymerase-catalyzed incorporation of nucleotides into the capped second sequencing read product; and (j) retaining the plurality of capped second sequencing read products hybridized with a nucleic acid template molecule, wherein the termination portion is not cleaved or removed.

[0007] In some embodiments of the methods disclosed herein, each sequencing template molecule comprises at least one capped first sequencing read product hybridized thereon and at least one capped second sequencing read product hybridized thereon.

[0008] In some embodiments of the methods disclosed herein, the capped nucleotide analog incorporated at the 3' end of each capped first sequencing read product is not modified to convert the sugar 3' position into an extendable 3'OH group. In some embodiments, the capped nucleotide analog incorporated at the 3' end of each capped second sequencing read product is not modified to convert the sugar 3' position into an extendable 3'OH group.

[0009] In some embodiments of the method disclosed herein, the heterocyclic base is connected to the termination portion via an alkyne bond, an olefin bond, or an alkane bond.

[0010] In some embodiments of the methods disclosed herein, the termination portion comprises a polymer portion. In some embodiments, the polymer portion is selected from the group consisting of: a polyether portion, a polyethylene glycol portion, a polypropylene glycol portion, a polyvinyl acetate portion, a polylactic acid portion, a polyglycolic acid portion, a polyamide portion, and a polyester portion. In some embodiments, the polymer portion comprises a poly(glycerol) portion, a poly(oxazoline) portion, a poly(hydroxypropyl methacrylate) portion (PHPMA), a poly(2-hydroxyethyl methacrylate) portion (PHEMA), a poly(N-(2-hydroxypropyl)methacrylamide) portion (HPMA), a poly(vinylpyrrolidone) portion (PVP), a poly(N,N-dimethylacrylamide) portion (PDMA), or a poly(N-acryloylmorpholine) portion (PAcM).

[0011] In some embodiments of the methods disclosed herein, the termination portion comprises a polyethylene glycol (PEG) portion. In some embodiments, the PEG portion has a molecular weight of 1K to 20K (kilodaltons or kDa). In some embodiments, the termination portion comprises a propargylamino portion, an allylamino portion, a propylamino portion, an ethyl mercapto portion, a hydroxymethyl portion, an aryl mercapto portion, a 1-X-1H-1,2,3-triazol-4-yl portion, a 5-X-1H-1,2,3-triazol-1-methyl portion, a hydrazone portion, or an O-alkyl oxime portion, wherein "X" comprises a polymer.

[0012] In some embodiments of the method disclosed herein, the heterocyclic base includes a chain-terminating portion at the 3' sugar group. In some embodiments, the chain-terminating portion includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azidoyl group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate ester group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0013] In some embodiments of the methods disclosed herein, each of the plurality of nucleic acid template molecules is a single-stranded or double-stranded nucleic acid molecule.

[0014] In some embodiments of the methods disclosed herein, the plurality of nucleic acid template molecules are immobilized to a support or to a coating on the support. In some embodiments, each of the plurality of nucleic acid template molecules is covalently linked to an immobilized surface capture primer, or wherein each of the plurality of nucleic acid template molecules hybridizes to an immobilized surface capture primer, wherein the immobilized surface capture primer is immobilized on the support.

[0015] In some embodiments of the methods disclosed herein, at least one of the plurality of nucleic acid template molecules contains uridine, or at least one of the plurality of nucleic acid template molecules lacks uridine.

[0016] In some embodiments of the methods disclosed herein, each of the plurality of nucleic acid template molecules comprises a cloned and amplified nucleic acid molecule. In some embodiments, each of the plurality of nucleic acid template molecules comprises at least one copy of a target sequence and at least one universal adaptor sequence. In some embodiments, each of the plurality of nucleic acid template molecules comprises a multiplicative template molecule having two or more tandem repeat units, wherein each repeat unit comprises the target sequence and at least one universal adaptor sequence.

[0017] In some embodiments of the methods disclosed herein, the support comprises glass or plastic. In some embodiments, the support is disposed on a flow cell or inside a capillary lumen. In some embodiments, the support comprises at least one hydrophilic polymer coating and a plurality of surface-capturing primers immobilized to the at least one hydrophilic polymer coating, wherein the at least one hydrophilic polymer coating has a water contact angle of no more than 45 degrees. In some embodiments, the at least one hydrophilic polymer coating comprises polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran. In some embodiments, the at least one hydrophilic polymer coating comprises polymer molecules having a molecular weight of at least 1000 Daltons. In some embodiments, the at least one hydrophilic polymer coating comprises branched polymer molecules having 4 to 8 branches. In some embodiments, the support comprises: a) a first coating comprising a first monolayer of hydrophilic polymer molecules tethered to the support; b) a second coating comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and c) a third coating comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first, second, or third layer comprise a branched polymer layer.

[0018] In some embodiments of the method disclosed herein, the plurality of surface-capturing primers are immobilized to the hydrophilic polymer molecules of the second or third monolayer, and the surface-capturing primers are distributed at multiple depths throughout the second or third layer. In some embodiments, one or more of the at least one hydrophilic polymer coating has a surface density of at least 1000 / µm. 2 Multiple surface-capturing primers.

[0019] In some embodiments of the method disclosed herein, the method includes contacting the plurality of nucleic acid template molecules with conditions suitable for hybridizing soluble sequencing primers with the respective nucleic acid template molecules to generate a plurality of nucleic acid duplexes on the respective nucleic acid template molecules: (i) the first plurality of sequencing primers, wherein the respective nucleic acid template molecules are immobilized and the plurality of sequencing primers are soluble, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents, wherein the conditions are suitable for binding the nucleic acid duplexes with the sequencing polymerases and the nucleotide reagents.

[0020] In some embodiments of the methods disclosed herein, the various nucleotide reagents in the plurality of nucleotide reagents comprise an aromatic base, a pentose sugar, and 1 to 10 phosphate ester groups. In some embodiments, the plurality of nucleotide reagents comprises one or more types of nucleotide reagents selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the plurality of nucleotide reagents comprises a combination of two or more types of nucleotide reagents selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, at least one nucleotide reagent in the plurality of nucleotide reagents lacks a detectable reporter portion. In some embodiments, at least one nucleotide reagent in the plurality of nucleotide reagents is labeled with a detectable reporter portion.

[0021] In some embodiments of the methods disclosed herein, the various nucleotide reagents among the multiple nucleotide reagents comprise at least one chain-terminating nucleotide, the at least one chain-terminating nucleotide comprising (i) an aromatic base, (ii) a sugar having a 3' chain-terminating portion that inhibits polymerase-catalyzed nucleotide incorporation, and (iii) 1 to 10 phosphate groups. In some embodiments, the at least one chain-terminating nucleotide comprises a removable chain-terminating portion at the 3' sugar group. In some embodiments, the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the at least one chain-terminating nucleotide comprises a removable chain-terminating portion at the 3' sugar group. In some embodiments, the removable chain-terminating portion comprises a 3'-O-amino group, a 3'-O-aminomethyl group, a 3'-O-methylamino group, or a derivative thereof. In some embodiments, the at least one chain-terminating portion may be cleaved by a chemical compound or may be removed therefrom to produce an extendable 3'OH portion on the sugar group.

[0022] In some embodiments of the method disclosed herein, sequencing the first region on the plurality of template molecules comprises: (a) contacting the plurality of nucleic acid template molecules with (i) a first plurality of sequencing polymerases and (ii) a plurality of first nucleic acid sequencing primers, wherein the nucleic acid template molecules are immobilized and the plurality of first nucleic acid sequencing primers are soluble, and wherein the contact is performed under conditions suitable for forming a plurality of complex polymerases comprising a sequencing polymerase that binds to a nucleic acid duplex, wherein the nucleic acid duplex comprises a portion of each nucleic acid template molecule hybridized to each of the respective first nucleic acid sequencing primers; (b) contacting the plurality of complex sequencing polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the complex sequencing polymerases, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorescent group and having a removable chain termination portion at the 3' position of the sugar; (c) incorporating at least one nucleotide into the 3' end of the first nucleic acid sequencing primer, thereby generating a plurality of first sequencing reads by extending the first nucleic acid sequencing primer; and (d) detecting the at least one nucleotide and identifying the nucleobases of the at least one nucleotide.

[0023] In some embodiments of the methods disclosed herein, the multiple nucleotide reagent comprises at least one multivalent molecule. In some embodiments, the at least one multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms comprising (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit. In some embodiments, the nucleotide unit comprises a base, a sugar, and 1 to 10 phosphate groups, and the linker is attached to the nucleotide unit via the base. In some embodiments, each nucleotide arm of the at least one multivalent molecule comprises a linker having an aliphatic chain or an oligoethylene glycol chain, and wherein the linker chain comprises 2 to 6 subunits. In some embodiments, the core of the at least one multivalent molecule comprises streptavidin, and the core attachment portion comprises biotin. In some embodiments, the plurality of nucleotide arms comprise the same type of nucleotide unit, wherein the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0024] In some embodiments of the methods disclosed herein, the multiple nucleotide reagent comprises a plurality of multivalent molecules, wherein each of the plurality of multivalent molecules comprises the same type of nucleotide unit. In some embodiments, the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. In some embodiments, the multiple nucleotide reagent comprises a plurality of multivalent molecules comprising a mixture of two or more types of multivalent molecules, each type of multivalent molecule comprising one or more nucleotide unit types selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0025] In some embodiments of the methods disclosed herein, the multiple nucleotide reagent comprises at least one fluorophore-tagged multivalent molecule.

[0026] In some embodiments of the methods disclosed herein, sequencing the first region includes performing a two-step sequencing method. In some embodiments, the first step includes: (a) contacting the plurality of nucleic acid template molecules with (i) a first plurality of sequencing polymerases and (ii) a plurality of first sequencing primers, wherein the nucleic acid template molecules are immobilized and the plurality of first sequencing primers are soluble, and wherein the contact is performed under conditions suitable for forming a plurality of first complex polymerases, each complex polymerase comprising a sequencing polymerase that binds to a nucleic acid duplex, wherein the nucleic acid duplex comprises a portion of the nucleic acid template molecule that has hybridized with the first soluble sequencing primer; (b) contacting the plurality of complex sequencing polymerases with a plurality of nucleotide reagents comprising at least one multivalent molecule, wherein the at least one multivalent molecule is a separably labeled multivalent molecule, wherein complementary nucleotide units of the multivalent molecule bind to at least two of the plurality of first complex polymerases, thereby forming a plurality of multivalent complex polymerases, and wherein incorporation of the complementary nucleotide units into the first sequencing primers is inhibited; (c) detecting the plurality of multivalent complex polymerases; and (d) identifying the nucleobases of the complementary nucleotide units bound to the plurality of first complex polymerases in the plurality of multivalent complex polymerases, thereby determining the sequence of the nucleic acid template. In some embodiments, the complementary nucleotide unit is complementary to the nucleotide at the 5' end of the nucleic acid double strand of the nucleic acid template molecule.

[0027] In some embodiments of the methods disclosed herein, sequencing the first region on the plurality of template molecules includes forming an affinity complex, wherein the method includes: (i) binding a first sequencing primer, a first sequencing polymerase, and a first detectably labeled multivalent molecule to the first region of the multivalent template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first detectably labeled multivalent molecule binds to the first sequencing polymerase; and (ii) binding a second sequencing primer, a second sequencing polymerase, and the first detectably labeled multivalent molecule to the second region of the multivalent template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, wherein the first binding complex and the second binding complex form an affinity complex. The multiply template molecule comprises two or more tandem repeat units, each tandem repeat unit comprising a target sequence and a universal primer binding site for binding a first universal sequencing primer and a second universal sequencing primer, and wherein the contact is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of the first nucleotide unit and the second nucleotide unit into the first binding complex and the second binding complex, respectively; (iii) detecting the first binding complex and the second binding complex on the same multiply template molecule, and (iv) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first region of the multiply template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second region of the multiply template molecule.

[0028] In some embodiments of the method disclosed herein, the second step includes: (e) dissociating the plurality of multivalent complex polymerases and removing the plurality of first sequencing polymerases and the multivalent molecule, while retaining the nucleic acid duplex; (f) contacting the nucleic acid duplex from step (e) with a plurality of second sequencing polymerases, wherein the contact is performed under conditions suitable for binding the plurality of second sequencing polymerases to the nucleic acid duplex to form a plurality of second complex polymerases; and (g) contacting the plurality of second complex polymerases with a plurality of nucleotides, the plurality of nucleotides comprising at least one nucleotide analog having a removable chain termination portion, wherein the contact is performed under conditions suitable for binding complementary nucleotides present in the plurality of nucleotides to at least two of the complex polymerases in step (f) to form a plurality of nucleotide complex polymerases, and wherein the conditions are suitable for promoting the incorporation of the bound complementary nucleotide into the first sequencing primer of the nucleotide complex polymerase, thereby producing the plurality of first sequencing reads. In some embodiments, the removable chain termination portion is located at the 3' position of the sugar. In some embodiments, the complementary nucleotide is complementary to the nucleotide immediately adjacent to the 5' of the nucleic acid duplex of the nucleic acid template molecule. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating an exemplary single-stranded template molecule comprising: a second surface primer binding site (e.g., SP2; surface pinning primer binding site); a second index sequence (second index); a first sequencing primer binding site (e.g., forward sequencing primer binding site or FWD sequencing); a target sequence (e.g., an insert); a second sequencing primer binding site (e.g., reverse sequencing primer binding site or REV sequencing); a first index sequence (first index); and a first surface primer binding site (e.g., SP1; surface capture primer binding site). Figure 1 The template molecule shown can be a copy template molecule containing one copy of the target sequence and one copy of various universal adaptor sequences. Alternatively, Figure 1 The template molecule shown is a unit of a multiplex having two or more tandem copies of the depicted unit, and each unit contains a copy of the target sequence and various universal connector sequences. In some embodiments, the first index sequence includes a first sample index sequence. In some embodiments, the first sample index sequence includes a short random sequence (e.g., NNN).

[0030] Figure 2 This is a schematic diagram illustrating an exemplary single-stranded template molecule comprising: a second surface primer binding site (e.g., SP2; surface pinning primer binding site); a first sequencing primer binding site (e.g., forward sequencing primer binding site or FWD sequencing); a first index sequence; a target sequence (e.g., an insert); a second index sequence; a second sequencing primer binding site (e.g., reverse sequencing primer binding site or REV sequencing); and a first surface primer binding site (e.g., SP1; capture primer binding site). Figure 2 The template molecule shown can be a copy template molecule containing one copy of the target sequence and one copy of various universal adaptor sequences. Alternatively, Figure 2 The template molecule shown is a unit of a multiplex having two or more tandem copies of the depicted unit, and each unit contains a copy of the target sequence and various universal connector sequences. In some embodiments, the first index sequence includes a first sample index sequence. In some embodiments, the first sample index sequence includes a short random sequence (e.g., NNN).

[0031] Figure 3A This is a schematic diagram illustrating a fixed multiply template molecule containing at least two tandem copies of a unit, wherein each unit contains an insert sequence (e.g., a target sequence) and various universal sequences containing universal primer binding sites. The multiply template molecule may lack a cleavable portion (e.g., uracil). Alternatively, the multiply template molecule may contain at least one cleavable portion (e.g., uracil). Figure 3A The schematic diagram also shows a first plurality of soluble sequencing primers and a second plurality of soluble sequencing primers, which can hybridize with their corresponding universal sequencing primer binding sites on the template molecule. Figure 3A , Figure 4A , Figure 5A , Figure 6A and Figure 7A The diagram in the image shows the... Figure 3A The workflow described herein is for sequencing fixed multiply template molecules, where the sequencing workflow uses read-capped nucleotide analogs. This can be achieved through... Figure 16A Find legends of various general sequences.

[0032] Figure 3B This is a schematic diagram illustrating a linear template molecule having an insert sequence (e.g., a target sequence) and a copy of a universal sequence including universal primer binding sites (tagged FWD sequencing and REV sequencing). The linear template molecule may be immobilized to a support. The template molecule may lack a cleavable portion (e.g., uracil). Alternatively, the template molecule may contain at least one cleavable portion (e.g., uracil). Figure 3B The schematic diagram also shows a first plurality of soluble sequencing primers and a second plurality of soluble sequencing primers, which can hybridize with their corresponding universal sequencing primer binding sites on the template molecule. Figure 3B , Figure 4B , Figure 5B , Figure 6B and Figure 7B The diagram in the image shows the... Figure 3B The workflow for sequencing linear template molecules described in the diagram uses read-capped nucleotide analogs. Figure 16A Find legends of various general sequences.

[0033] Figure 4A This is a schematic diagram showing how each first sequencing primer hybridizes with its corresponding first universal sequencing primer binding site on the nucleic acid multiply template molecule and sequences the first region of the template molecule, thereby producing the first multiple sequencing reads.

[0034] Figure 4B This is a schematic diagram showing the hybridization of the first sequencing primer with the first universal sequencing primer binding site on the template molecule and the sequencing of the first region of the nucleic acid multiply template molecule, thereby producing the first sequencing read product.

[0035] Figure 5AThis is a schematic diagram illustrating the first capping reaction, which involves incorporating a capping nucleotide analog into the 3' end of each first sequencing read product, thereby producing a first plurality of capped sequencing read products. The capping nucleotide analog is depicted as a solid "X".

[0036] Figure 5B This is a schematic diagram illustrating the first capping reaction, which involves incorporating a capping nucleotide analog into the 3' end of the first sequencing read product, thereby producing a first-capped sequencing read product. The capping nucleotide analog is depicted as a solid "X".

[0037] Figure 6A This is a schematic diagram showing the retention of multiple capped first sequencing reads (which are retained to hybridize with the nucleic acid polymorph template molecule), hybridization of each second sequencing primer with its corresponding second universal sequencing primer binding site on the polymorph template molecule, and sequencing of the second region of the template molecule to generate multiple second sequencing reads.

[0038] Figure 6B This is a schematic diagram showing the process of retaining the first capped sequencing read (which retains hybridization with the nucleic acid polymorph template molecule), hybridizing the second sequencing primer with the second universal sequencing primer binding site on the template molecule, and sequencing the second region of the nucleic acid polymorph template molecule to produce the second sequencing read.

[0039] Figure 7A This is a schematic diagram illustrating the second capping reaction, which is carried out by incorporating a capped nucleotide analog into the 3' end of each second sequencing read product, thereby generating multiple capped second sequencing read products and retaining multiple capped second sequencing read products that are retained to hybridize with the nucleic acid multiply template molecule.

[0040] Figure 7B This is a schematic diagram illustrating the second capping reaction, which is carried out by incorporating a capped nucleotide analog into the 3' end of the second sequencing read product, thereby producing a capped second sequencing read product, and retaining the capped second sequencing read product for hybridization with the nucleic acid multiply template molecule.

[0041] Figures 8A to 8J Several examples of read-capped nucleotide analogs are shown, each containing a heterocyclic base linked to a termination portion. Figure 8A The terminating portion contains a propargylamino moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8B The terminating portion contains an allylamino moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8C The terminating portion contains a propylamino moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8D The terminating portion contains an ethyl mercapto moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8E The terminating portion contains a hydroxymethyl portion, where X can be a polymer (e.g., a PEG portion). Figure 8F The terminating portion contains an aryl thiol moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8G The terminating portion contains a 1-X-1H-1,2,3-triazol-4-yl moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8H The terminating portion contains a 5-X-1H-1,2,3-triazol-1-methyl moiety, where X can be a polymer (e.g., a PEG moiety). Figure 8I The terminating portion contains an hydrazone portion, where X can be a polymer (e.g., a PEG portion). Figure 8J The terminating portion contains an O-alkyl oxime moiety, where X can be a polymer (e.g., a PEG moiety).

[0042] Figure 9 Several examples of capped nucleotide analogs are shown, each comprising a heterocyclic base linked to a termination moiety. The termination moiety comprises a polyethylene glycol (PEG) moiety or a derivative thereof. In some embodiments, the PEG moiety comprises a molecular weight of about 1 kDa to 20 kDa (KDa: kilodaltons).

[0043] Figure 10 This is a schematic diagram illustrating an exemplary sequencing sequence: read 1 (e.g., full-length sequencing of the target sequence, "first sequencing read product"); first sample index ("second sequencing read product"); and second sample index ("third sequencing read product").

[0044] Figure 11 This is a schematic diagram illustrating an exemplary sequencing sequence: read 1 (e.g., sequencing only a portion of the target sequence, "first sequencing read product"); first sample index ("second sequencing read product"); second sample index ("third sequencing read product") and read 1 (e.g., sequencing the full length of the target sequence, "fourth sequencing read product").

[0045] Figure 12 This is a schematic diagram illustrating an exemplary sequencing sequence: First sample index (“First sequencing read product”); Second sample index (“Second sequencing read product”); Read 1 (e.g., for full-length sequencing of the target sequence, “Third sequencing read product”).

[0046] Figure 13This is a schematic diagram illustrating an exemplary sequencing sequence: a first sample index (“first sequencing read product”); read 1 (e.g., sequencing only a portion of the target sequence, “second sequencing read product”); a second sample index (“third sequencing read product”); and read 1 (e.g., sequencing the full-length target sequence, “fourth sequencing read product”).

[0047] Figure 14 This is a schematic diagram illustrating an exemplary sequencing sequence for a paired workflow: forward first sample index (“first FWD sequencing read product”); forward second sample index (“second FWD sequencing read product”); forward read 1 (e.g., full-length sequencing of the target sequence, “third FWD sequencing read product”); paired turn; reverse read 2 (e.g., full-length sequencing of the target sequence, “first REV sequencing read product”).

[0048] Figure 15 This is a schematic diagram illustrating an exemplary sequencing sequence for a paired workflow: forward read 1 (e.g., full-length sequencing of the target sequence, “first FWD sequencing read product”); paired turn; reverse first sample index (“first REV sequencing read product”); reverse second sample index (“second REV sequencing read product”); and reverse read 2 (e.g., full-length sequencing of the target sequence, “third REV sequencing read product”).

[0049] Figure 16A This is a schematic diagram illustrating an exemplary single-stranded nucleic acid multiplicative template molecule immobilized to an immobilized first surface primer. The multiplicative template molecule is covalently linked to the immobilized first surface primer. The immobilized multiplicative template molecule contains at least one nucleotide having a cleavable portion that can be cleaved to generate a base-free site in the immobilized multiplicative template molecule. The immobilized multiplicative template molecule can be generated by a support-based rolling circle amplification reaction using a covalently closed circular library molecule carrying a universal adaptor sequence that can hybridize with the immobilized first surface primer. The arrangement of the sequence of interest and various universal adaptor sequences is for illustrative purposes. Those skilled in the art will understand that many other arrangements are possible. Figures 16B to 16H It shows the Figure 16A The workflow for pair sequencing of fixed multiply template molecules described in the paper.

[0050] Figure 16B It is shown in Figure 16A This diagram illustrates an exemplary forward sequencing reaction performed on a fixed multiply template molecule. The forward sequencing reaction can be performed using multiple soluble forward sequencing primers, producing multiple first forward sequencing reads. The fixed multiply template molecule can contain two or more first forward sequencing reads hybridized thereon.

[0051] Figure 16C This is a schematic diagram illustrating an exemplary method for replacing the first forward sequencing read product with a forward extension strand produced by using a strand substitution polymerase to perform a primer extension reaction without adding soluble primers. The strand substitution polymerase can use the upstream first forward sequencing read product to initiate the primer extension reaction.

[0052] Figure 16D This is a schematic diagram illustrating an exemplary method for replacing the first forward sequencing read product with a forward extension strand generated by removing the first forward sequencing read product and performing a primer extension reaction with new soluble forward sequencing primers.

[0053] Figure 16E This is a schematic diagram illustrating an exemplary method for generating base-free sites in an immobilized single-stranded multiplex template molecule at nucleotides with easily cleavable portions, and creating gaps at these base-free sites to generate multiple multiplex template molecules containing gaps, while retaining the forward extension strand and the immobilized first surface primer. For example, this can be achieved through... Figure 16C or Figure 16D The method described in the paper generates a positive extension chain.

[0054] Figure 16F It shows that in the removal such as Figure 16E The diagram shows an exemplary retained forward extension chain after a multi-linked template molecule containing gaps.

[0055] Figure 16G It is shown in Figure 16F The diagram illustrates an exemplary reverse sequencing reaction performed on the retained forward extension strand. The reverse sequencing reaction can be performed using multiple soluble reverse sequencing primers. The retained forward extension strand may contain two or more first reverse sequencing read products hybridized thereon. The first reverse sequencing read products do not hybridize with the first surface primers, or are not covalently linked to the first surface primers. Therefore, the first reverse sequencing read products are not immobilized to the support. For simplicity, Figures 16A to 16G An exemplary fixed multiply molecule is shown, having two copies of the target sequence and various universal primer binding sites. Those skilled in the art will understand that a fixed multiply molecule can contain more than two tandem copies of a single unit, wherein each unit includes the target sequence and various universal primer binding sites.

[0056] Figure 16H This is a schematic diagram showing an exemplary support on which a first surface primer and a second surface primer are fixed. Figure 16AA portion of the immobilized multiply template molecule shown hybridizes with an immobilized second surface primer. The immobilized second surface primer is used to pin a portion of the immobilized multiply template molecule downwards to a support. The immobilized multiply template molecule may have two or more copies of a universal binding sequence for the immobilized second surface primer. A portion of the immobilized multiply template molecule containing the universal binding sequence for the immobilized second surface primer can hybridize with the immobilized second surface primer.

[0057] Figure 17 This is a schematic diagram illustrating an exemplary single-stranded nucleic acid multiply template molecule immobilized to an immobilized first surface primer. The multiply template molecule hybridizes with the immobilized first surface primer. The immobilized multiply template molecule contains at least one nucleotide having a cleavable portion that can be cleaved to generate a base-free site in the immobilized multiply template molecule. In some embodiments, the immobilized multiply template molecule can be generated by performing a rolling circle amplification reaction in solution and distributing the rolling circle amplification reaction onto a support. The arrangement of the sequence of interest and various universal adaptor sequences is for illustrative purposes. Those skilled in the art will understand that many other arrangements are possible. The immobilized multiply template molecule can be subjected to… Figures 16A to 16G The described pairwise sequencing workflow.

[0058] Figure 18 This is a schematic diagram of an exemplary low-binding support, comprising a glass substrate and alternating hydrophilic coatings covalently or non-covalently adhered to the glass, and further comprising chemically reactive functional groups that act as attachment sites for oligonucleotide primers (e.g., capturing oligonucleotides). In an alternative embodiment, the support may be made of any material, such as glass, plastic, or polymeric materials.

[0059] Figure 19 These are schematic diagrams of various exemplary configurations of multivalent molecules. Left (Class I): Schematic diagram of multivalent molecules with a "starburst" or "helter-skelter" configuration. Middle (Class II): Schematic diagram of multivalent molecules with a dendritic macromolecular configuration. Right (Class III): Schematic diagram of multiple multivalent molecules formed by the reaction of streptavidin with a 4-arm or 8-arm PEG-NHS containing biotin and dNTPs. Nucleotide units are designated as 'N', biotin is designated as 'B', and streptavidin is designated as 'SA'.

[0060] Figure 20 This is a schematic diagram of an exemplary multivalent molecule comprising a universal nucleus attached to multiple nucleotide arms.

[0061] Figure 21 This is a schematic diagram of an exemplary multivalent molecule comprising a dendritic nucleus attached to multiple nucleotide arms.

[0062] Figure 22 A schematic diagram of an exemplary multivalent molecule is shown, comprising a nucleus attached to multiple nucleotide arms, wherein the nucleotide arms include biotin, spacers, linkers, and nucleotide units.

[0063] Figure 23 It is a schematic diagram of an exemplary nucleotide arm that includes a core attachment portion, spacers, linkers, and nucleotide units.

[0064] Figure 24 The chemical structures of exemplary spacers (top) and various exemplary connectors, including 11-atom connectors, 16-atom connectors, 23-atom connectors and N3 connectors (bottom), are shown.

[0065] Figure 25 The chemical structures of various exemplary connectors (including connectors 1 to 9) are shown.

[0066] Figure 26A The chemical structures of various exemplary connectors that bind / attach to nucleotide units are shown.

[0067] Figure 26B The chemical structures of various exemplary connectors that bind / attach to nucleotide units are shown.

[0068] Figure 26C The chemical structures of various exemplary connectors that bind / attach to nucleotide units are shown.

[0069] Figure 27 The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide unit is attached to the linker via propargylamine attachment at the 5-position of the pyrimidine base or the 7-position of the purine base. Detailed Implementation

[0070] definition

[0071] The headings provided herein are not intended to limit any aspect of this disclosure, which is to be understood by referring to the entire specification.

[0072] Unless otherwise defined, the technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. Generally, terms related to the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with the laboratory procedures and techniques described herein is that which is well-known and commonly used in the art.

[0073] Unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. Unless explicitly and definitively limited to a single referent, the singular forms “a / an” and “the,” as well as the singular use of any word, shall include multiple referents.

[0074] It should be understood that the use of alternative terms (e.g., "or") is considered to refer to one or both of the alternatives or any combination thereof.

[0075] As used herein, the term “and / or” will be considered to mean that each of the specified features or components is explicitly disclosed with or without the other. For example, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). Similarly, the term “and / or” as used herein, such as “A, B and / or C”, is intended to cover each of the following aspects: “A, B and C”; “A, B or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0076] As used herein and in the appended claims, the terms “comprising,” “including,” “having,” and “containing,” and their grammatical variations, are intended to be non-limiting, such that one or more items in the list do not exclude other items that may be substituted for or added to the listed items. It should be understood that whenever an aspect is described herein with the language “comprising,” other similar aspects described as “consisting of” and / or “substantially consisting of” are also provided.

[0077] As used herein, the terms “about” and “approximately” mean a value or composition within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, “about” or “approximately” may mean within one or more standard deviations. Alternatively, “about” or “approximately” may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. Furthermore, particularly for biological systems or methods, the term may mean a value of up to one order of magnitude or up to five times. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Furthermore, in the case of providing ranges and / or subranges of values, the range and / or subranges may include the endpoints of the range and / or subranges.

[0078] The term "cellular biological sample" refers to a section of a single cell, multiple cells, tissue, organ, organism, or any of these cellular biological samples. Cellular biological samples can be extracted from an organism (e.g., via biopsy) or obtained from cell cultures grown in liquid or in petri dishes. Cellular biological samples can be fresh, frozen, fresh-frozen, or archived (e.g., formalin-fixed paraffin-embedded; FFPE). Cellular biological samples can be embedded in wax, resin, epoxy resin, or agar. Cellular biological samples can be fixed in any one or more of, such as acetone, ethanol, methanol, formaldehyde, paraformaldehyde-triton, or glutaraldehyde. Cellular biological samples can be sectioned or unsectioned. Cellular biological samples can be stained, destained, or unstained.

[0079] As used herein, the term "polymerase" and its variants refer to an enzyme containing a domain that binds a nucleotide (or nucleoside), wherein the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. A polymerase may have one or more activities, including but not limited to: base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand substitution activity, and nucleotide binding and recognition. A polymerase can be any enzyme capable of catalyzing the polymerization of nucleotides (including their analogs) into a nucleic acid chain. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase contains other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand substitution activity. Polymerases may include, but are not limited to: naturally occurring polymerases and any subunits and truncated thereof, mutant polymerases, variant polymerases, recombinant, fusion, or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases may be catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes containing nucleotide-binding domains. In some embodiments, polymerases may be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases may be expressed in prokaryotes, eukaryotes, viruses, or bacteriophages. In some embodiments, polymerases may be post-translational modified proteins or fragments thereof. Polymerases may be derived from prokaryotes, eukaryotes, viruses, or bacteriophages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases. Exemplary polymerases are described, for example, in U.S. Patent No. 11,859,241, the contents of which are incorporated herein by reference in their entirety.

[0080] The term "strand displacement" refers to the ability of a polymerase to locally separate double-stranded nucleic acid strands and synthesize a new strand in a template-based manner. A strand displacement polymerase displaces the complementary strand from the template strand and catalyzes the synthesis of the new strand. During synthesis, a nucleotide complementary to the template strand can be incorporated into the 3' end of the new strand. Strand displacement polymerases include mesophilic and thermophilic polymerases. Strand displacement polymerases include wild-type enzymes and variants (including exonuclease depletion mutants, mutant versions, chimeric enzymes, and truncated enzymes). Examples of strand displacement polymerases include phi29 DNA polymerase, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, Deep Vent® DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon). TM ), or variant EquiPhi29 TM DNA polymerase (e.g., from ThermoFisher Scientific®), or chimeric QualiPhi TM DNA polymerase (e.g., from 4basebio).

[0081] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,” as used herein, and other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded (ss) or double-stranded (ds). Nucleic acids comprise polymers of nucleotides, wherein the nucleotides comprise natural or non-natural bases and / or sugars. Nucleic acids comprise naturally occurring nucleoside bonds, such as phosphodiester bonds. Nucleic acids comprise non-natural nucleoside bonds, including thiophosphate, sulfur-containing phosphate, or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids comprise one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0082] Desired nucleic acids can be extracted from cells or cellular biological samples using any of a variety of techniques known to those skilled in the art. For example, a typical DNA extraction procedure includes: (i) collecting a cell or tissue sample from which DNA is to be extracted; (ii) disrupting the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components; (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA; and (iv) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell membrane lysis. A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to: the QIAamp® kit (for isolating genomic DNA from human samples) and DNAeasy® kit (for isolating genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep™ series kits from Promega (Madison, WI).

[0083] The term "nucleotide" and related terms refer to a molecule comprising a heterocyclic base, a pentose sugar (e.g., ribose or deoxyribose), and at least one phosphate ester group. Canonical or non-canonical nucleotides are used in accordance with this terminology. In some embodiments, a nucleotide comprises a monophosphate, diphosphate, or triphosphate, or a corresponding phosphate analog. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides may be unlabeled or partially labeled with a detectable reporter.

[0084] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, which are commonly found in nucleic acids, including naturally occurring, substituted, modified, or engineered variants. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Husstein hydrogen bonds with suitable complementary bases. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine; 2,6-diaminopurine; adenine (A); ethylene adenine, N... 6 -Δ 2 -Isopentenyladenine (6iA); N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA); N 6 -Methyladenine; Guanine (G), isoguanine; N 2 -Dimethylguanine (dmG); 7-methylguanine (7mG); 2-thiopyrimidine; 6-thioguanine (6sG); hypoxanthine and O 6-Methylguanine; 7-denitro-purine, such as 7-denitro-adenine (7-denitro-A) and 7-denitro-guanine (7-denitro-G); pyrimidine, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole, such as nitroindole and 4-methylindole; pyrrole, such as nitropyrrole; muscarin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated, and acylated base moieties; etc. Further exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385–394, CRC Press, Boca Raton, Fla.

[0085] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), an acyclic moiety (Martinez et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7: 3013-3016), and other sugar moieties (Joeng et al., 1993 J. Med. Chem. 36: 2627-2638; Kim et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284: 2118-2124; and US Patent No. 5,558,991). The sugar moiety includes: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0086] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analogue having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S, or BH3). In some embodiments, the chain comprises a phosphate ester group substituted with an analogue, including phosphoramide, thiophosphate, dithiophosphate, and O-methylphosphoramide groups.

[0087] As used herein, the terms “operably linked” and “operably conjoined,” or related terms, refer to the juxtaposition of components (i.e., adjacent components). The juxtaposed components can be covalently or non-covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond conjoining the two components comprises a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can be linked together, wherein the first nucleic acid component can confer function to the second nucleic acid component. For example, a bond between a primer-binding sequence and a target sequence forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a target nucleic acid sequence) can be linked to a support, wherein the bond allows the transgene sequence contained in the support to be expressed or function. Those skilled in the art will understand that components can be operably linked, but direct physical linkage is not required.

[0088] The terms “connected,” “joined,” “attached,” “attached,” and variations thereof encompass any type of fusion, binding, attachment, or association between any combination of compounds or molecules that possesses sufficient stability to withstand use in a particular procedure. This procedure may include, but is not limited to: nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of chain terminations); washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds, or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and so on. In some embodiments, such bonds occur intramolecularly, for example, linking the ends of single-stranded or double-stranded linear nucleic acid molecules together to form a cyclic molecule. In some embodiments, such bonds may occur between combinations of different molecules or between molecules and non-molecules, including, but not limited to: bonds between nucleic acid molecules and solid surfaces; bonds between proteins and detectable reporter moieties; bonds between nucleotides and detectable reporter moieties; and so on. Some examples of the key can be found in Hermanson, G., “Bioconjugate Techniques”, 2nd edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0089] As used herein, the term "primer" and related terms refer to oligonucleotides capable of hybridizing with DNA and / or RNA polynucleotide templates to form double-stranded molecules. Primers may be single-stranded along their entire length or contain both single-stranded and double-stranded portions. Primers may include natural nucleotides and / or nucleotide analogs. Primers may be recombinant nucleic acid molecules or synthetic nucleic acid molecules. Primers can have any length, but typically range from 4 to 50 nucleotides. Typical primers include a 5' end and a 3' end. The 3' end of a primer may contain a 3' OH portion that acts as the initiation site for nucleotide polymerization in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer may lack a 3' OH portion, or may contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any nucleotide or more along the length of the primer may be labeled with a detectable reporter portion. Primers may be in solution (e.g., soluble primers) or may be immobilized to a support (e.g., capture primers).

[0090] The terms “template nucleic acid,” “template polynucleotide,” “target nucleic acid,” “target polynucleotide,” “template strand,” and other variations refer to the nucleic acid strand used as the base nucleic acid molecule in any amplification and / or sequencing method described herein. The template nucleic acid can be single-stranded or double-stranded, or it can contain single-stranded or double-stranded portions. The template nucleic acid can be obtained from naturally occurring sources, in recombinant forms, or chemically synthesized to include any type of nucleic acid analogue. The template nucleic acid can be linear, multiplicative, circular, or other forms.

[0091] The term "adaptor" and related terms refer to an oligonucleotide that can be operatively linked (attached) to a target polynucleotide, wherein the adaptor confers function on the co-conjugated adaptor-target molecule. The adaptor may comprise DNA, RNA, chimeric DNA / RNA, or the like. The adaptor may contain at least one ribonucleoside residue. The adaptor may be single-stranded, double-stranded, or contain single-stranded and / or double-stranded portions. The adaptor may be configured in linear, stem-loop, hairpin, or Y-shaped form. The adaptor may be of any length, comprising 4 to 100 nucleotides or longer. The adaptor may have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' and 3' overhanging ends. The 5' end of a single-stranded adaptor or one strand of a double-stranded adaptor may contain a 5' phosphate group or lack a 5' phosphate group. The adaptor may contain a 5' tail that has not hybridized to the target polynucleotide (e.g., a tailed adaptor), or the adaptor may be tailless. At least a portion of the adaptor may contain a known and predetermined sequence. The adaptor may contain a sequence complementary to at least a portion of a primer (such as an amplification primer, sequencing primer, or capture primer (e.g., a soluble or immobilized capture primer)). The adaptor may contain a random or degenerate sequence. The adaptor may contain at least one inosine residue. The adaptor may contain at least one thiophosphate, thiophosphate, and / or phosphoramide bond. The adaptor may contain at least one barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex assays. The adaptor may contain at least one unique identification sequence (e.g., a molecular identifier) ​​that can be used to uniquely identify the nucleic acid molecule to which the adaptor is attached. In some embodiments, the unique identification sequence comprises 2 to 12 or more nucleotides having a known sequence. For example, the unique identification sequence comprises a known random sequence, wherein the nucleotides at each position are randomly selected from nucleotides having bases A, G, C, T, or U. The adaptor may contain at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from the group consisting of: type I, type II, type III, type IV, Hs, or type IIB.

[0092] The term "universal sequence" and related terms refer to a sequence common to two or more polynucleotide molecules in a nucleic acid molecule. For example, an adaptor with a universal sequence can operatively bind to multiple polynucleotides such that a population of co-conjugated molecules carries the same universal adaptor sequence. Examples of universal adaptor sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0093] When used to refer to nucleic acid molecules, the terms "hybridize," "hybridizing," "hybridization," or other related terms refer to the hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes the hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridized molecule with double-stranded regions. Hybridization can involve Watson-Crick or Hoogstein binding to form double-stranded nucleic acids or double-stranded regions within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be completely complementary or partially complementary. Complementary nucleic acid strands do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing, or it can be other forms of base pairing interactions. Double-stranded nucleic acids can contain mismatched base-paired nucleotides.

[0094] When used to refer to nucleic acids, the terms "extend," "extending," "extension," and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3' OH end of the nucleic acid chain, resulting in the extension of the nucleic acid chain. Nucleotide incorporation can be performed using native nucleotides and / or nucleotide analogs. Typically, but not always, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0095] The term "rolling circle amplification" or "RCA" generally refers to an amplification method that employs a circular nucleic acid template molecule containing a target sequence, an amplification primer binding sequence, and optionally one or more adaptor sequences, such as sequencing primer binding sites and / or sample index sequences. Rolling circle amplification reactions can be performed under isothermal amplification conditions and include a circular nucleic acid template molecule, amplification primers, a strand displacement polymerase, and multiple nucleotides to produce a multiplicative molecule containing a tandem repeat sequence of the circular template molecule (including any adaptor sequences present in the original circular nucleic acid template molecule). The multiplicative template molecule can self-collapse to form nucleic acid nanospheres. The shape and size of the nanospheres can be further compressed by including a pair of inverted repeat sequences in the circular template molecule, or by performing the rolling circle amplification reaction with one or more compressed oligonucleotides. One advantage of using rolling circle amplification to generate clonal amplicones for sequencing workflows is that repetitive copies of the target sequence in the nanospheres can be sequenced simultaneously to increase signal strength. In some embodiments, the rolling circle amplification reaction can be performed in the presence of multiple compressed oligonucleotides having at least four consecutive guanines. Rolling circle amplification (RoBA) produces a polymorphic template molecule containing repetitive copies of a universal binding sequence for a compressed oligonucleotide. At least one compressed oligonucleotide can form a guanine tetramer and hybridize with the universal binding sequence for the compressed oligonucleotide, and the resulting polymorphic template molecule can fold to form an intramolecular G-quadruplex structure. The polymorphic template molecule can self-collapse to form compressed nanospheres. The formation of the guanine tetramer and G-quadruplex within the nanospheres increases their stability, maintaining their compact size and shape, thus allowing them to withstand repeated flows of reagents used in any sequencing workflow described herein.

[0096] When used to refer to nucleic acids, the terms "amplify," "amplifying," and other related terms include generating multiple copies of the original polynucleotide template molecule, wherein the copies contain sequences complementary to the template sequence, and / or the copies contain sequences identical to the template sequence. In some embodiments, the copies contain sequences substantially identical to the template sequence, and / or sequences substantially identical to sequences complementary to the template sequence.

[0097] The terms "reporter moiety," "reporter moieties," or related terms refer to compounds that produce or cause a detectable signal. A reporter moiety is sometimes referred to as a "tag." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemical, mass spectrometry, Raman spectroscopy, haptens, affinity tags, atoms, or enzymes. A reporter moiety produces a detectable signal caused by a chemical or physical change, such as heat, light, electricity, pH, salt concentration, enzyme activity, or proximity events. Proximity events include two reporter moieties coming close to each other, associating with each other, or binding together. Those skilled in the art are well aware of selecting reporter moieties such that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing for the monitoring of the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties with spectrally distinct emission profiles or with minimal overlap spectral emission profiles can be selected. The reporter portion may be linked to a nucleotide, nucleoside, nucleic acid, enzyme (e.g., polymerase or reverse transcriptase) or support (e.g., surface) (e.g., operatively linked).

[0098] The report portion (or label) may include fluorescent labels or fluorophores. Exemplary fluorescent portions that can be used as fluorescent markers or fluorophores include, but are not limited to, fluoresceins and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthol fluorescein, isothiocyanate fluorescein, NHS-fluorescein, iodoacetamide-fluorescein, maleimide fluorescein, SAMSA-fluorescein, aminothiourea fluorescein, hydrazine methylthioacetamide fluorescein, rhodamine and rhodamine derivatives, such as TRITC, TMR, lissamine rhodamine, Texas red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazine, Texas red sulfonyl chloride, Texas red hydrazine, coumarin and coumarin derivatives, such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazine, BODIPY® and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY530 / 550 C3-SE, BODIPY 530 / 550 C3 acylhydrazine, BODIPY 493 / 503 C3 acylhydrazine, BODIPY FL C3 acylhydrazine, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue® and its derivatives, such as Cascade Blue acetylazide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue acylhydrazine, Lucifer Yellow and its derivatives, such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, anthocyanins and their derivatives, such as indolonyl anthocyanin dyes, benzoindolonyl anthocyanin dyes, pyridiniumyl anthocyanin dyes, thiazolonyl anthocyanin dyes, quinolinelonyl anthocyanin dyes, imidazolonyl anthocyanin dyes, Cy 3. Cy5, lanthanide chelates and derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor® dyes, DyLight® dyes, Atto TM Dyes, LightCycler® Red dyes, CALFluor dyes, JOE and its derivatives, Oregon Green TMDyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and other dyes known in the art, such as the fluorescent moieties described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition; Lakowicz, Principles of Fluorescence Spectroscopy, 2nd edition, Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd edition, or derivatives thereof; or any combination thereof. Anthocyanin dyes may be present in sulfonated or non-sulfonated forms and may consist of two pseudoindole, benzoindoline, pyridinium, thiazoline, and / or quinolinium groups separated by a polymethystylene bridge between two nitrogen atoms.Commercially available anthocyanin fluorophores include, for example, Cy3 (which may contain 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{ ...3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidone-1-yloxy)-6-oxohexyl]-2- [[1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl]-3,3-dimethyl-5-sulfonyl-1,3-dihydro-2H-indol-2-ylide]prop-1-en-1-yl)-3,3-dimethyl-3H-indolon-5-sulfonate], Cy5 (which may contain 1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidone-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indololin-2-ylide) )pent-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-onium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfonoindoline-2-ylidene)pent-1,3-dien-1-yl)-3,3-dimethyl-3H-indole-1-onium-5-sulfonate) and Cy7 (which may contain 1-(5-carboxypentyl) Cy2 is an oxazole derivative, not a pseudoindole, and the benzo[Cy]-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indole-2-ylidene)hept-1,3,5-trien-1-yl]-3H-indole-5-sulfonate, where “Cy” stands for 'cyanine' and the first number indicates the number of carbon atoms between the two pseudoindole groups. Cy2 is an oxazole derivative, not a pseudoindole, and the benzo[Cy]-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. Other dyes are described, for example, in U.S. Patent Application Publication No. 2024 / 0240249, the contents of which are incorporated herein by reference in their entirety.

[0099] In some embodiments, the reporter portion may be a fluorescence resonance energy transfer (FRET) pair, allowing for multiple classifications in a single excitation and imaging step. As used herein, FRET may include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0100] The term "persistence time" and related terms refer to the length of time a binding complex remains stable without dissociation of any component. An exemplary binding complex comprises a nucleic acid template and primer, a polymerase, a nucleotide unit of a multivalent molecule, or a free (e.g., unconjugated) nucleotide. The nucleotide unit or free nucleotide may be complementary or non-complementary to nucleotide residues in the template molecule. The nucleotide unit or free nucleotide may bind to the 3' end of the nucleic acid primer at a position opposite to the complementary nucleotide residue in the nucleic acid template molecule. Persistence time indicates the stability of the binding complex and the strength of the binding interaction. Persistence time can be measured by observing the onset and / or duration of the binding complex (e.g., by observing a signal from a labeled component of the binding complex). For example, a labeled nucleotide or a labeled reagent containing one or more nucleotides may be present in the binding complex, thus allowing a signal from the label to be detected during the persistence time of the binding complex. An exemplary label is a fluorescent label. The binding complex (e.g., a ternary complex) remains stable before being subjected to conditions that cause dissociation between the polymerase, template molecule, primer, and / or nucleotide unit or nucleotide. For example, dissociation conditions include contacting the bound complex with any or any combination of detergent, EDTA, and / or water.

[0101] As used herein, the term "support" refers to a substrate designed for the deposition of biological molecules or biological samples for assays and / or analyses. Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), peptides, carbohydrates, lipids, single cells, or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs. Exemplary supports may include the inner surface of capillaries or flow cells.

[0102] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is any combination of porous, semi-porous, non-porous, or porous. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0103] In some embodiments, the surface of the support may be substantially smooth. In some embodiments, the support may have a regular or irregular texture, including bumps, etching, holes, three-dimensional supports, or any combination thereof.

[0104] In some embodiments, the support comprises beads of any shape, including spherical, hemispherical, cylindrical, barrel-shaped, ring-shaped, disc-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or linear.

[0105] The support can be made of any material, including but not limited to: glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)) or any combination thereof. Various combinations of both glass and plastic substrates are envisioned.

[0106] This disclosure provides a plurality of (e.g., two or more) nucleic acid template molecules immobilized (i.e., attached to) a support. In some embodiments, the immobilized plurality of nucleic acid template molecules have the same sequence or different sequences. In some embodiments, each of the plurality of nucleic acid template molecules is immobilized to a different site on the support. In some embodiments, two or more of the plurality of nucleic acid template molecules are immobilized to sites on the support.

[0107] The term "array" refers to a support containing multiple sites located at predetermined positions on a support to form a site array. The sites can be discrete and separated by interstitial space. In some embodiments, the predetermined sites on the support can be arranged in one-dimensional rows or columns, or in two-dimensional rows and columns. In some embodiments, the multiple predetermined sites are arranged in an organized manner on the support. In some embodiments, the multiple predetermined sites are arranged in any organized pattern, including straight lines, hexagonal patterns, grid patterns, patterns with reflective symmetry, patterns with rotational symmetry, etc. The distances between different pairs of sites can be the same or different. In some embodiments, the support includes at least 10 2 At least 10 sites 3 At least 10 sites 4 At least 10 sites 5 At least 10 sites 6 At least 10 sites 7 At least 10 sites 8 At least 10 sites 9 At least 10 sites 10 At least 10 sites11 At least 10 sites 12 At least 10 sites 13 At least 10 sites 14 At least 10 sites 15 One or more sites, wherein the site is located at a predetermined position on the support. In some embodiments, a plurality of predetermined sites on the support (e.g., 10 sites) 2 Up to 10 15 (One or more sites) contain immobilized nucleic acid template molecules to form a nucleic acid template array. In some embodiments, nucleic acid template molecules are immobilized at multiple predetermined sites by hybridization with immobilized surface capture primers. In some embodiments, nucleic acid template molecules are immobilized at multiple predetermined sites by covalent attachment with surface capture primers. In some embodiments, nucleic acid template molecules are immobilized at multiple predetermined sites, for example, at 10 sites. 2 Up to 10 15 Multiple sites or more. In some embodiments, the immobilized nucleic acid template molecule is amplified clonally to generate immobilized nucleic acid polymerase clones at the multiple predetermined sites. In some embodiments, each immobilized nucleic acid polymerase clone contains a linear single-copy molecule or contains a single-stranded or double-stranded nucleic acid multiplicand template molecule.

[0108] A support comprising multiple sites located at random positions on a support may herein be referred to as a support having randomly positioned sites thereon. The positions of the randomly positioned sites on the support are not predetermined. These multiple randomly positioned sites are arranged on the support in a disordered and / or unpredictable manner. In some embodiments, the support comprises at least 10 2 At least 10 sites 3 At least 10 sites 4 At least 10 sites 5 At least 10 sites 6 At least 10 sites 7 At least 10 sites 8 At least 10 sites 9 At least 10 sites 10 At least 10 sites 11 At least 10 sites 12 At least 10 sites 13 At least 10 sites 14 At least 10 sites 15 One or more sites, wherein the sites are randomly located on the support. In some embodiments, multiple randomly located sites on the support (e.g., 10 sites) 2 Up to 10 15(One or more sites) contain a nucleic acid template molecule immobilized. In some embodiments, the nucleic acid template molecule is immobilized at a plurality of randomly located sites by hybridization with immobilized surface capture primers. In some embodiments, the nucleic acid template molecule is immobilized at a plurality of randomly located sites by covalent attachment with surface capture primers. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites, for example, at 10 sites. 2 Up to 10 15 At one or more sites. In some embodiments, a fixed nucleic acid template is amplified clonally to generate fixed nucleic acid polymerase clones at the plurality of randomly located sites. In some embodiments, each fixed nucleic acid polymerase clone comprises a linear molecule containing a single copy of the target sequence and optionally an adaptor or primer-binding sequence. In alternative embodiments, each fixed nucleic acid polymerase clone comprises a linear molecule containing a single-stranded or double-stranded multiplicative template molecule.

[0109] In some embodiments, a plurality of fixed surface capture primers on a support (e.g., located at predetermined or random positions on the support) are fluidly connected to each other to allow solutions of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, etc.) to flow onto the support, such that the plurality of fixed surface capture primers on the support can react with the reagents substantially simultaneously in a large-scale parallel manner. In some embodiments, the fluid connectivity of the plurality of fixed surface capture primers can be used to perform nucleic acid amplification reactions (e.g., rolling circle amplification [RCA], multiplex substitution amplification [MDA], polymerase chain reaction [PCR], bridging amplification, etc.) substantially simultaneously on the plurality of fixed surface capture primers. An exemplary support that allows reagent solutions to flow and is fluidly connected between various positions on the support is a flow cell.

[0110] In some embodiments, multiple immobilized nucleic acid polymerase clones on a support are fluidly connected to each other to allow reagent solutions (e.g., enzymes, nucleotides, divalent cations, etc.) to flow onto the support, such that the multiple immobilized nucleic acid polymerase clones on the support can react with the reagents substantially simultaneously in a large-scale parallel manner. In some embodiments, the fluid connectivity of the multiple immobilized nucleic acid polymerase clones can be used for substantially simultaneous nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​the multiple immobilized nucleic acid polymerase clones, and optionally for detection and imaging for large-scale parallel sequencing.

[0111] The terms "fixed" or "fixed to" and related terms refer to nucleic acid molecules attached to a support, or to a coating on a support, or embedded within a matrix formed by a coating on a support via covalent or non-covalent interactions, wherein the nucleic acid molecules include surface capture primers, nucleic acid template molecules, and extension products of the capture primers. Extension products of the capture primers include nucleic acid multimers (e.g., nucleic acid polymerase clones). Nucleic acid molecules can be fixed at predetermined or random sites on the support. Nucleic acid molecules can be fixed at predetermined or random sites on or within a passivated coating on the support. In some cases, nucleic acid molecules can be fixed to the support by hybridization with complementary sequences of other nucleic acid molecules, which in turn are fixed to the support.

[0112] The term "fixed" and related terms can also refer to an enzyme (e.g., a polymerase) attached to a support, or to a coating on a support, or embedded within a matrix formed by a coating on a support via covalent bonds or non-covalent interactions. The enzyme can be fixed at predetermined or random locations on the support. The enzyme can be fixed at predetermined or random locations on or within a passivated coating on the support.

[0113] In some embodiments, one or more nucleic acid template molecules are immobilized on a support, for example, at sites on the support. In some embodiments, one or more nucleic acid template molecules are cloned and amplified. In some embodiments, one or more nucleic acid template molecules are cloned and amplified from a support (e.g., in solution), then deposited onto the support and immobilized. In some embodiments, the cloning and amplification reaction of one or more nucleic acid template molecules is performed on the support, resulting in immobilization on the support. In some embodiments, the cloning and amplification of one or more nucleic acid template molecules using nucleic acid amplification reactions (e.g., in solution or on a support) includes any one or any combination of the following: polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridging amplification, isothermal bridging amplification, rolling circle amplification (RCA), loop-to-loop amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-strand-binding (SSB) protein-dependent amplification.

[0114] The term "surface primer" and related terms refer to a single-stranded oligonucleotide immobilized to a support and containing a sequence capable of hybridizing with at least a portion of a nucleic acid template molecule. "Surface capture primers" can be used to immobilize a template molecule to a support via hybridization. Surface capture primers can be immobilized to the support in a manner resistant to primer removal during flow, washing, aspiration, and changes in temperature, pH, salt, chemical, and / or enzymatic conditions. Typically, but not necessarily, the 5' end of a surface capture primer can be immobilized to the support or a coating on the support (or embedded in a coating on the support). Alternatively, the interior or 3' end of a surface capture primer can be immobilized to the support. "Surface pinning primers" can be used to hybridize with one or more additional portions of a nucleic acid multiply template molecule captured by a surface capture primer and pin a second (or additional) portion of the nucleic acid template molecule downwards to the support (see [link to relevant documentation]). Figure 16H At least a portion of the surface-pinning primer can hybridize with a portion of the nucleic acid multiplicon template molecule to pin a portion of the multiplicon molecule downwards to the support.

[0115] The sequence of the surface capture primer may be completely or partially complementary to at least a portion of the nucleic acid template molecule along its length. The support may contain multiple fixed surface capture primers having the same sequence or two or more different sequences. The surface capture primers may be of any length, such as 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer.

[0116] Surface capture primers may have a terminal 3' nucleotide with a sugar 3' OH moiety that can be extended for nucleotide polymerization (e.g., polymerase-catalyzed polymerization). Surface capture primers may have a terminal 3' nucleotide with a 3' sugar position connected to a chain-terminating moiety that inhibits nucleotide polymerization. The 3' chain-terminating moiety can be removed (e.g., deblocked) using a deblocking agent to convert the 3' end into an extendable 3' OH end. Examples of chain-terminating moieties include alkyl groups, alkenyl groups, alkynyl groups, allyl groups, aryl groups, benzyl groups, azido groups, amine groups, amide groups, ketol groups, isocyanate groups, phosphate groups, thio groups, disulfide groups, carbonate groups, urea groups, or silyl groups, or any of the 3' chain-terminating moieties described herein. Azide-type chain-terminating moieties include azido, azido, and azidomethyl groups. Examples of deblocking agents include phosphine compounds, such as tris(2-carboxyethyl)phosphine (TCEP) and bissulfotriphenylphosphine (BS-TPP), for azide, azido, and azidomethyl chain-terminating groups. Examples of deblocking agents include tetra(triphenylphosphine)palladium(O) (Pd(PPh3)4) with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), for alkyl, alkenyl, alkynyl, and allyl chain-terminating groups. Examples of deblocking agents include H2Pd / C, for the benzyl chain-terminating group. Examples of deblocking agents include phosphine, β-mercaptoethanol, or dithiothreitol (DTT), for the amine, amide, ketone, isocyanate, phosphate, thio, and disulfide chain-terminating groups. Examples of deblocking agents include potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, and Zn in acetic acid (AcOH), for the carbonate chain-terminating group. Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, and ammonium fluoride and triethylamine trihydrofluoride, used for chain-terminating groups urea and silyl groups.

[0117] The term "sequencing" and related terms refer to methods for obtaining nucleotide sequence information from nucleic acid molecules, typically by determining the identity of at least some nucleotides (including their nucleobase components) within the nucleic acid molecule and their order within the molecule. In some embodiments, sequence information for a given region of the nucleic acid molecule includes identifying each nucleotide within the sequenced region. In some embodiments, sequencing information identifies only some nucleotides in the region, while the identity of some nucleotides remains undetermined or incorrectly determined. Any suitable sequencing method can be used. In exemplary embodiments, sequencing may include label-free or ion-based sequencing methods. In some embodiments, sequencing may include labeled or dye-based or fluorescence-based nucleotide sequencing methods. In some embodiments, sequencing may include polymerase cloning-based sequencing or bridge sequencing methods. In some embodiments, sequencing employs polymerases and multivalent molecules to generate at least one affinity complex, wherein each multivalent molecule contains multiple nucleotide units tethered to a core. In some embodiments, sequencing employs polymerases and free nucleotides for sequencing-by-synthesis. In some embodiments, sequencing employs ligases and multiple sequence-specific oligonucleotides for sequencing-by-ligation.

[0118] introduction

[0119] This disclosure provides compositions and methods for sequentially sequencing different regions of the same nucleic acid template molecule and reducing residual signals that lead to poor sequencing quality. The provided compositions and methods can be used in pairwise sequencing workflows. The provided compositions and methods can be used in singleton and multiplex sequencing workflows.

[0120] Traditional multiplex sequencing workflows require sequencing a first region (e.g., the target sequence region) using a first sequencing primer and a second region (e.g., the sample index region) using a second sequencing primer. The first and second primers are designed to hybridize with different regions of the same template molecule to perform separate sequencing reactions, producing separate reads for the target sequence and the sample index region. Sequencing reads are typically generated through a polymerase-catalyzed primer extension reaction. Before sequencing the second region, the sequencing reads synthesized from sequencing the first region must be efficiently removed without damaging the template molecule. Traditional sequencing workflows that use chemical-based reagents (e.g., NaOH and / or formamide) to denature the sequencing reads can be harsh, resulting in template molecule damage. Switching to milder chemical-based denaturation conditions may cause less damage to the template molecule but cannot efficiently remove sequencing reads that contribute to residual signals. After multiple sequencing cycles, residual signals can accumulate, making it difficult to image and detect the true sequencing signal. Residual signals lead to poor sequencing quality and reduced sequencing accuracy.

[0121] The sequencing order of the sequence of interest and the sample index region also presents challenges. When sequencing the sequence of interest region before the sample index, longer sequencing reads of the sequence of interest region must be removed before sequencing the shorter sample index region. Inefficient removal of sequencing reads from the sequence of interest region in previous sequencing cycles will produce residual signals in subsequent sequencing cycles of the sample index region. The accumulation of residual signals in subsequent sequencing cycles can reduce sequencing accuracy, which may lead to misalignment of the sample index with its proper target sequence region. This phenomenon can cause index skipping when data from a sample associated with one sample index “jumps” to different sample indices during sequencing.

[0122] Therefore, efficiently removing sequencing reads from previous sequencing cycles will improve the overall accuracy of sequencing data. High-accuracy sequencing of different regions of the template molecule depends on many parameters, including high signal intensity, reduced residual signal from previous sequencing reads, and maintaining an intact template molecule after multiple sequencing cycles.

[0123] This disclosure provides compositions comprising read-capped nucleotide analogs and methods for using read-capped nucleotide analogs. In some embodiments, read-capped nucleotide analogs can be used for sequential sequencing of different regions of the same nucleic acid template molecule. Read-capped nucleotide analogs offer several advantages over conventional chemical-based reagents that use formamide, alkaline conditions, and / or high temperatures to denature / remove sequencing read products during the sequencing workflow. Read-capped nucleotide analogs do not require formamide or alkaline conditions. The read-capping workflow can be performed at low temperatures, which reduces the risk of damaging the template molecule.

[0124] It is well known that formamide lowers the melting temperature of double-stranded DNA in a linear manner of approximately 2.4°C to 2.9°C per mole of formamide or approximately 0.6°C per percentage of formamide. The length of the double-stranded region and the G+C composition also affect the melting temperature of double-stranded DNA. Common chemical-based methods for removing sequencing reads involve denaturation at 50°C to 75°C in the presence of 20% to 40% formamide. Denaturation of double-stranded DNA at high temperatures can lead to strand breaks and depurination, which can result in the loss of intact template molecules after multiple sequencing cycles and reduced signal strength. Furthermore, formamide is classified as a reproductive toxicant that can be inhaled, ingested, or absorbed through the skin. Laboratory safety regulations stipulate that any reagent containing formamide must be disposed of as chemical waste.

[0125] In contrast, when sequencing the first region of the template molecule is complete, a read-capping nucleotide analog can be incorporated into the 3' end of the sequencing read to produce a first-capped sequencing read. This first-capped sequencing read is not removed from the template molecule before sequencing the second region. Removal of the first-capped sequencing read is avoided because once incorporated into the read (“incorporated read-capping nucleotide analog”), it blocks the binding and incorporation of the nucleotide reagents used to sequence the second region of the nucleic acid template molecule. Therefore, the template molecule is not subjected to chemically based dehybridization reagents and the high temperatures typically used to remove previously generated sequencing reads. Using read-capping nucleotide analogs without removing the sequencing read reduces damage to the template molecule and preserves the intact template molecule after multiple sequencing cycles. When the fixed template molecule is a multiplexed structure that collapses to form nucleic acid nanospheres, sequencing methods using read-capped nucleotide analogs can reduce nanosphere dispersion and preserve the shape and size of the nanospheres after multiple sequencing cycles. Therefore, the read-capped nucleotide analog method can improve the full width at half maximum (FWHM) of the nanosphere dot pattern after multiple sequencing cycles.

[0126] Capped nucleotide analogues

[0127] In one aspect, this disclosure provides read-capped nucleotide analogs comprising: (i) a heterocyclic base, (ii) a ribose, and (iii) a polyphosphate chain, wherein the heterocyclic base is linked to a termination portion (e.g., a base-linked chain termination portion). In some embodiments, this disclosure provides read-capped nucleotide analogs that can be incorporated into the 3' end of a primer in a polymerase-catalyzed primer extension reaction to produce a read-capped primer product. In some embodiments, this disclosure provides read-capped nucleotide analogs that can be incorporated into the 3' end of a nascent primer in a polymerase-catalyzed primer extension reaction to produce a capped primer product.

[0128] In some embodiments, a read-capped nucleotide analog is incorporated into the 3' end of the primer product. Once incorporated, the read-capped nucleotide analog can block polymerase-mediated binding of the nucleotide reagent to the 3' end of the read-capped primer product, wherein the nucleotide reagent comprises a multivalent molecule. In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. In some embodiments, the multivalent molecule comprises (1) a core; and (2) a plurality of nucleotide arms, each arm comprising (i) a core attachment portion, (ii) a spacer comprising, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (e.g., see [link to relevant documentation]). Figures 19 to 23 ).

[0129] In some embodiments, after incorporation into the 3' end of the primer product, the read-capped nucleotide analog can block polymerase-catalyzed incorporation of the nucleotide reagent into the 3' end of the read-capped primer product. The nucleotide reagent may comprise a typical nucleotide or a chain-terminating nucleotide. In some embodiments, the chain-terminating nucleotide comprises (i) a heterocyclic base, (ii) a ribose (e.g., ribose or deoxyribose), and (iii) a polyphosphate chain, wherein the 2' or 3' sugar position is connected to the chain-terminating portion via a cleavable or non-cleavable linker. Many chain-terminating portions are described below.

[0130] In another aspect, this disclosure provides read-capped nucleotide analogs comprising: (i) a heterocyclic base, (ii) a ribose, and (iii) at least one phosphate ester group, wherein the heterocyclic base is linked to a termination portion. In some embodiments, the termination portion is linked to the heterocyclic base via a non-cleavable or cleavable linker. In some embodiments, the read-capped nucleotide analog may be unlabeled or partially labeled with a detectable reporter component. Figures 8A to 8J and Figure 9 Illustrative readings of capped nucleotide analogs are shown.

[0131] In some embodiments, the capped nucleotide analog comprises a heterocyclic base comprising a substituted nitrogen-containing parent heteroaromatic ring, including naturally occurring, substituted, modified, or engineered variants. The bases of the capped nucleotide analog are capable of forming Watson-Crick and / or Husstein hydrogen bonds with suitable complementary bases. Illustrative bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine; 2,6-diaminopurine; adenine (A); ethylene adenine; N... 6 -Δ 2 -Isopentenyladenine (6iA); N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA); N 6 -Methyladenine; Guanine (G); Isoguanine; N 2 -Dimethylguanine (dmG); 7-methylguanine (7mG); 2-thiopyrimidine; 6-thioguanine (6sG); hypoxanthine and O 6-Methylguanine; 7-denitro-purine, such as 7-denitro-adenine (7-denitro-A) and 7-denitro-guanine (7-denitro-G); pyrimidine, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole, such as nitroindole and 4-methylindole; pyrrole, such as nitropyrrole; muscarin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated, and acylated base moieties; etc. Further exemplary bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385–394, CRC Press, Boca Raton, Fla.

[0132] In some embodiments, the capped nucleotide analogue includes a base-linked termination portion that is linked to a nucleobase via an alkyl bond. In some embodiments, the bond comprises an alkyl group. In some embodiments, the bond comprises an alkenyl group. In some embodiments, the bond comprises an alkynyl group.

[0133] In some embodiments, the terminating portion of the base link comprises a polymer portion. In some embodiments, the terminating portion of the base link comprises a polyether portion. In some embodiments, the terminating portion of the base link comprises polyethylene glycol; in some embodiments, the terminating portion of the base link comprises polypropylene glycol; in some embodiments, the terminating portion of the base link comprises polyvinyl acetate. In some embodiments, the terminating portion of the base link comprises polylactic acid. In some embodiments, the terminating portion of the base link comprises polyglycolic acid. In some embodiments, the terminating portion of the base link comprises a polyamide portion. In some embodiments, the terminating portion of the base link comprises a polyester portion. In some embodiments, the terminating portion of the base link comprises a poly(glycerol) portion. In some embodiments, the terminating portion of the base link comprises a poly(oxazoline) portion. In some embodiments, the terminating portion of the base link comprises a poly(hydroxypropyl methacrylate) portion (PHPMA). In some embodiments, the terminating portion of the base link comprises a poly(2-hydroxyethyl methacrylate) portion (PHEMA). In some embodiments, the terminating portion of the base link comprises a poly(N-(2-hydroxypropyl)methacrylamide) portion (HPMA). In some embodiments, the terminating portion of the base link comprises a poly(vinylpyrrolidone) portion (PVP). In some embodiments, the terminating portion of the base link comprises a poly(N,N-dimethylacrylamide) portion (PDMA). In some embodiments, the terminating portion of the base link comprises a poly(N-acryloylmorpholine) portion (PAcM). In some embodiments, the terminating portion of the base link comprises a synthetic zwitterionic portion, such as a poly(carboxybetaine acrylamide) portion, a poly(carboxybetaine methacrylate) portion, a poly(sulfobetaine methacrylate) portion, or a poly(methacryloyloxyethyl phosphorylcholine) portion. In some embodiments, the terminating portion of the base link comprises a polyglutamic acid portion. In some embodiments, the terminating portion of the base link comprises a polyaspartic acid portion. In some embodiments, the terminating portion of the base link comprises a polylysine portion. In some embodiments, the terminating portion of the base link comprises a polyethyleneimine portion. In some embodiments, the terminating portion of the base link comprises a polysialic acid portion.

[0134] In some embodiments, the termination portion of the base linkage includes a connector, for example... Figure 24 The 11-atom connector, 16-atom connector, 23-atom connector, or N3 connector shown. In some embodiments, the termination portion of the base linkage comprises a polymer connector, such as... Figure 25 Any of the connectors 1 to 9 shown.

[0135] In some embodiments, the terminating portion of the base linkage comprises an acetylated amino moiety, an allylamino moiety, a propylamino moiety, an ethyl mercapto moiety, a hydroxymethyl moiety, or an aryl mercapto moiety (e.g., see [link to relevant documentation]). Figures 8A to 8F In some embodiments, the terminating portion of the base linkage comprises a 1-X-1H-1,2,3-triazol-4-yl moiety, a 5-X-1H-1,2,3-triazol-1-methyl moiety, a hydrazone moiety, or an O-alkyl oxime moiety (e.g., see [link to relevant documentation]). Figures 8G to 8J In some embodiments, Figures 8A to 8J The “X” in the text contains polymers, such as any of the polymers mentioned above.

[0136] In some embodiments, the terminating portion of the base linkage comprises a hydrophilic polymer of ethylene oxide. In some embodiments, the terminating portion of the base linkage comprises a polyethylene glycol (PEG) portion (e.g., H-(OCH2CH2)). n -OH). In some embodiments, the PEG portion comprises PEG with a molecular weight of about 100-200 Da, 200-300 Da, 300-400 Da, 400-500 Da, 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15 K Da, 20K Da, 30K Da, 40K Da, 50K Da, or greater. In some embodiments, the value of n can be 1, or at least 2, or at least 5, or at least 10, at least 20, at least 25, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1000, or 2000 or greater (see, for example...). Figure 9 In some embodiments, the PEG portion comprises a linear PEG molecule. In some embodiments, the PEG portion comprises a branched PEG molecule. In some embodiments, the branched PEG portion molecule comprises 4 to 8 branches. In some embodiments, the terminating portion comprises an aromatic group having a six-carbon ring structure. In some embodiments, the PEG portion may be functionalized with a methoxy group (OCH3), an amino group (NH2), a carboxyl group (COOH), and / or a hydroxyl group (OH). For example, the base-linked terminating portion comprises polyethylene glycol methyl ether (mPEG) (e.g., CH3(OCH2CH2)). n -OH). In some embodiments, Figures 8A to 8J The “X” in the text contains the PEG portion or its derivatives, such as any of the PEG portions mentioned above.

[0137] In some embodiments, a set of capped nucleotide analogues comprises four types of capped nucleotide analogues, including dATP, dGTP, dCTP, and dTTP / dUTP, wherein the nucleobases of each type of capped nucleotide analogue are linked to the same type of termination portion. For example, the same type of PEG portion is linked to the nucleobases of dATP, dGTP, dCTP, and dTTP / dUTP.

[0138] In some embodiments, a set of capped nucleotide analogs comprises four types of capped nucleotide analogs, including dATP, dGTP, dCTP, and dTTP / dUTP, wherein the nuclei of at least two types of capped nucleotide analogs are linked to different termination portions. For example, at least one type of capped nucleotide analog (e.g., dATP) contains nuclei linked to a PEG portion that is different from the PEG portions of other capped nucleotide analogs (e.g., dGTP, dCTP, or dTTP / dUTP). In some embodiments, the length of the PEG portion may vary. In some embodiments, the molecular weight of the PEG portion may vary. In some embodiments, the nuclei of one, two, three, or four types of capped nucleotide analogs may be linked to the PEG portion via different bonds (e.g., different alkyl, alkenyl, or alkynyl bonds). In some embodiments, the nuclei of one, two, three, or four types of capped nucleotide analogs may be linked to PEG portions with different functional groups, including methoxy groups (OCH3), amino groups (NH2), carboxyl groups (COOH), and / or hydroxy groups (OH).

[0139] In some embodiments, the capped nucleotide analogue comprises a sugar moiety, such as a cyclic moiety (see, for example, Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48, which is incorporated herein by reference to illustrate examples of cyclic moieties that may be present in the capped nucleotide analogue described herein), an acyclic moiety (see, for example, Martinez et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7: 3013-3016, each incorporated herein by reference to illustrate examples of acyclic moieties that may be present in the capped nucleotide analogue described herein), or other sugar moieties (see, for example, Joeng et al., 1993 J. Med. Chem. 36: 2627-2638; Kim et al., 1993 J. Med. Chem. 36: 2627-2638). 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Patent No. 5,558,991, each incorporated herein by reference for examples of sugar moieties that may be present in the capped nucleotide analogues described herein. In some embodiments, the capped nucleotide analogue comprises a sugar moiety comprising: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or otherwise modified sugars.

[0140] In some embodiments, the capped nucleotide analogue includes a hydroxyl group at the 3' sugar position. In some embodiments, the capped nucleotide analogue includes a chain termination portion at the 3' sugar position. The chain termination portion may include an H, F, NH2, or an amino group.

[0141] In some embodiments, the capped nucleotide analog comprises a sugar moiety having a 3'OH group. In some embodiments, the capped nucleotide analog comprises a chain termination portion at the 2' or 3' sugar position. In some embodiments, the chain termination portion may be removed from or cleaved from the 3' sugar position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain termination portion may be cleaved from the nucleotide and / or removed, for example, by reacting the chain termination portion with a chemical agent, by a pH change, by light, or by heat. For example, in some embodiments, chain-terminating portions comprising alkyl, alkenyl, alkynyl, and allyl groups can be cleaved with tetra(triphenylphosphine)palladium(O) (Pd(PPh3)4) using piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, chain-terminating portions comprising benzyl groups can be cleaved with H2 Pd / C. In some embodiments, chain-terminating portions comprising amines, amides, ketones, isocyanates, phosphates, thioides, or disulfides can be cleaved with phosphine or with thiol groups, including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, chain-terminating portions comprising carbonates can be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, chain-terminating portions comprising urea and silyl groups can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, chain-terminating portions can be cleaved or removed with nitrous acid. In some embodiments, the chain termination portion can be cleaved or removed using a solution containing nitrite (such as, for example, a combination of nitrite and an acid, such as an organic acid (e.g., acetic acid), sulfuric acid, or nitric acid).

[0142] In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog comprises an azide group, an azide group, or an azidemethyl group. In some embodiments, the chain termination portion comprises a 3'-O-azido or a 3'-O-azidomethyl group. In some embodiments, the chain termination portion comprising an azide, an azide, and an azidemethyl group can be cleaved or removed by a cleavage agent, such as a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine moiety. In some embodiments, the phosphine compound comprises a derived triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the phosphine compound comprises bis(sulfotriphenylphosphine) (BS-TPP). In some embodiments, the phosphine compound comprises tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent comprises 4-dimethylaminopyridine (4-DMAP).

[0143] In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog contains a 3'-O-amino group or a derivative thereof, which can be cleaved with nitrous acid by utilizing a nitrous acid mechanism or using a solution containing nitrous acid. In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog contains a 3'-O-aminomethyl group or a derivative thereof, which can be cleaved with nitrous acid by utilizing a nitrous acid mechanism or using a solution containing nitrous acid. In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog contains a 3'-O-methylamino group or a derivative thereof, which can be cleaved with nitrous acid by utilizing a nitrous acid mechanism or using a solution containing nitrous acid.

[0144] In some embodiments, the chain-terminating portion comprises a 3'-O-amino group or a derivative thereof, which can be cleaved using a solution containing nitrite. In some embodiments, the chain-terminating portion comprises a 3'-O-aminomethyl group or a derivative thereof, which can be cleaved using a solution containing nitrite. In some embodiments, the chain-terminating portion comprises a 3'-O-methylamino group or a derivative thereof, which can be cleaved using a solution containing nitrite. In some embodiments, for example, the nitrite may be combined with or contacted with an acid such as acetic acid, sulfuric acid, or nitric acid. In some other embodiments, for example, the nitrite may be combined with or contacted with an organic acid such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, etc.

[0145] In some embodiments, the capped nucleotide analog comprises a 3'-deoxynucleotide, a 2',3'-dideoxynucleotide, or a nucleotide comprising one of the following 3' modifications: 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl or derivatives thereof.

[0146] In some embodiments, the capped nucleotide analog comprises a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene, or ethylene group. In some embodiments, the phosphorus atoms in the chain comprise substituted side groups, such as O, S, or BH3. In some embodiments, the chain comprises phosphate ester analogs, such as aminophosphate, thiophosphate, dithiophosphate, and O-methylphosphoramide groups. In some embodiments, the chain is a polyphosphate chain. In some embodiments, the chain is a mixture of phosphate and aminophosphate groups. In some embodiments, the chain is a mixture of phosphate and thiophosphate groups. In some embodiments, the chain is a mixture of phosphate, aminophosphate, and thiophosphate groups.

[0147] Complex polymerases containing capped sequencing reads

[0148] In another aspect, this disclosure provides a composition comprising a plurality of complex polymerases, wherein each complex polymerase comprises at least one polymerase that is complexed with a single-stranded nucleic acid template molecule having two or more sites along a single-stranded nucleic acid template molecule, the single-stranded nucleic acid template molecule hybridizing with capped sequencing read products (e.g., a plurality of capped sequencing read products hybridized thereon). In some embodiments, each capped sequencing read product along the single-stranded nucleic acid template molecule is bound to a sequencing polymerase. In some embodiments, each capped sequencing read product comprises a sequencing primer (e.g., an oligonucleotide) linked to a polynucleotide having a sequence complementary to at least a portion of the single-stranded nucleic acid template molecule, and the 3' end of each capped sequencing read product comprises an incorporated read-capped nucleotide analog. In some embodiments, the polynucleotide may be synthesized in a template-dependent manner via a polymerase-catalyzed primer extension reaction. In some embodiments, the plurality of single-stranded nucleic acid template molecules may be immobilized to a support or may be immobilized to a coating on a support.

[0149] In some embodiments, each single-stranded nucleic acid template molecule has been hybridized along its length with multiple capped sequencing reads. In some embodiments, each capped sequencing read contains a read-capped nucleotide analog incorporated at its 3' end, wherein the read-capped nucleotide analog can inhibit the binding of the multivalent molecule to the complex polymerase, or can significantly reduce the stability of the binding between the multivalent molecule and the complex polymerase to significantly reduce the duration. Thus, the binding between the complex polymerase and the detectably labeled multivalent molecule is undetectable or exhibits significantly reduced detectability. In some embodiments, the multivalent molecule comprises a core attached to multiple nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. In some embodiments, the multivalent molecule comprises (1) a core; and (2) multiple nucleotide arms, each arm comprising (i) a core attachment portion, (ii) a spacer containing a linker, and (iii) a nucleotide unit, wherein the core is attached to the multiple nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (e.g., see...). Figures 19 to 23 ).

[0150] In some embodiments, each template molecule has been hybridized along its length to multiple capped sequencing reads. In some embodiments, each capped sequencing read contains a read-capping nucleotide analog incorporated at its 3' end, wherein the read-capping nucleotide analog can inhibit or significantly reduce polymerase-catalyzed incorporation of nucleotides or chain-termining nucleotides. Therefore, the read-capping nucleotide analog prevents primer extension by polymerases that complex with the template molecule.

[0151] In some embodiments, the capped sequencing read product comprises a sequencing primer (e.g., a nucleic acid oligonucleotide) linked to a polynucleotide having a sequence complementary to at least a portion of a single-stranded nucleic acid template molecule, and the 3' end of the capped sequencing read product comprises an incorporated read-capping nucleotide analog. The capped sequencing read product can hybridize to a portion of the single-stranded template molecule. The capped sequencing read product can be synthesized in a template-dependent manner via a polymerase-catalyzed primer extension reaction. At least a portion of the complementary polynucleotide can be generated by any type of sequencing workflow including a polymerase-catalyzed primer extension reaction in which the sequence of the single-stranded template molecule is determined. At least a portion of the complementary polynucleotide can be generated by any type of polymerase-catalyzed primer extension reaction in which the sequence of the single-stranded template molecule is not determined.

[0152] In some embodiments, any portion of the single-stranded template molecule is anchored to a support or a coating on the support. For example, the 5' or 3' end of the template molecule, or an internal portion of the template molecule, or a combination thereof, may be anchored to the support. In some embodiments, the 5' and / or 3' ends of the single-stranded nucleic acid template molecule are covalently attached to a surface primer, which is anchored to the support or a coating on the support. In some embodiments, the 5' and / or 3' regions of the single-stranded nucleic acid template molecule hybridize with a surface primer, which is anchored to the support or a coating on the support.

[0153] In some embodiments, single-stranded nucleic acid template molecules can be generated through a cloning amplification workflow. In some embodiments, single-stranded nucleic acid template molecules are not generated through a cloning amplification workflow.

[0154] In some embodiments, the single-stranded nucleic acid template molecule contains at least one uridine nucleotide. In some embodiments, the single-stranded nucleic acid template molecule does not contain uridine nucleotides.

[0155] In some embodiments, a single-stranded nucleic acid template molecule contains one copy of the sequence of interest. A single-copy template molecule can be generated by bridge amplification. In some embodiments, a single-stranded nucleic acid template molecule contains two or more tandem copies of the sequence of interest. For example, a tandem copy template molecule contains multiples that can be generated by rolling circle amplification (RCA).

[0156] In some embodiments, the single-stranded nucleic acid template molecule contains at least one target sequence (e.g., an insertion region). In some embodiments, the single-stranded nucleic acid template molecule further contains at least one universal adaptor sequence. The universal adaptor sequence may contain a first surface primer binding site (e.g., a capture primer binding site); a second surface primer binding site (e.g., a surface pinning primer binding site); a first sequencing primer binding site (e.g., a forward sequencing primer binding site, sometimes also referred to as a first universal sequencing primer binding site); a second sequencing primer binding site (e.g., a reverse sequencing primer binding site, sometimes also referred to as a second universal sequencing primer binding site); a first amplification primer binding site (e.g., a forward amplification primer binding site); a second amplification primer binding site (e.g., a reverse amplification primer binding site); a first sample index sequence; a second sample index sequence; a first unique molecular tag sequence; a second unique molecular tag sequence; a first compressed oligonucleotide binding site; and / or a second compressed oligonucleotide binding site.

[0157] In some embodiments, the single-stranded nucleic acid template molecule includes a first surface primer binding site. In some embodiments, the first surface primer binding site (or its complementary sequence) can hybridize with at least a portion of an immobilized first surface primer. In some embodiments, the single-stranded nucleic acid template molecule includes a second surface primer binding site. In some embodiments, the second surface primer binding site (or its complementary sequence) can hybridize with at least a portion of an immobilized second surface primer. In some embodiments, the single-stranded nucleic acid template molecule includes a first sequencing primer binding site. In some embodiments, the first sequencing primer site (or its complementary sequence) can hybridize with at least a portion of a forward sequencing primer (sometimes referred to as a first universal sequencing primer). In some embodiments, the single-stranded nucleic acid template molecule includes a second sequencing primer binding site. In some embodiments, the second sequencing primer site (or its complementary sequence) can hybridize with at least a portion of a reverse sequencing primer (sometimes referred to as a second universal sequencing primer). In some embodiments, the single-stranded nucleic acid template molecule includes a first amplification primer binding site. In some embodiments, the first amplification primer binding site (or its complementary sequence) can hybridize with at least a portion of a forward amplification primer. In some embodiments, the single-stranded nucleic acid template molecule includes a second amplification primer binding site. In some embodiments, the second amplification primer binding site (or its complementary sequence) may hybridize with at least a portion of the reverse amplification primer. In some embodiments, the single-stranded nucleic acid template molecule includes a first compressed oligonucleotide binding site. In some embodiments, the first compressed oligonucleotide binding site (or its complementary sequence) may hybridize with at least a portion of the first compressed oligonucleotide. In some embodiments, the single-stranded nucleic acid template molecule includes a second compressed oligonucleotide binding site. In some embodiments, the second compressed oligonucleotide binding site (or its complementary sequence) may hybridize with at least a portion of the second compressed oligonucleotide.

[0158] In some embodiments, the support is a planar support (e.g., a glass slide). In some embodiments, the support is a non-planar support (e.g., beads). In some embodiments, the support comprises or is composed of a solid material. In some embodiments, the support comprises or is composed of a semi-solid material. In some embodiments, the support comprises or is composed of a porous material. In some embodiments, the support comprises or is composed of a semi-porous material. In some embodiments, the support comprises or is composed of a non-porous material. The support may be made of any suitable material known in the art or described herein, such as glass, plastic, or polymeric materials.

[0159] In some embodiments, the surface of the support may be coated with one or more compounds to create a passivation layer on the support. In some embodiments, the passivation layer forms a porous layer. In some embodiments, the passivation layer forms a semi-porous layer. In some embodiments, surface primers or single-stranded nucleic acid template molecules may be attached to the passivation layer to immobilize the primers or template molecules to the support. In some embodiments, the support includes a low-nonspecific binding surface that enables improved nucleic acid hybridization, amplification, and sequencing performance on the support. Generally, the support may include one or more layers of covalently or non-covalently attached low-binding chemically modified layers (e.g., silane layers, polymer membranes), and one or more oligonucleotides that can be used to immobilize multiple nucleic acid template molecules to the support via covalent or non-covalent attachment. In some embodiments, the support may include a functionalized polymer coating at least covalently bound to a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of hydrophilic polymer coating and at least one layer of multiple oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise branched PEG having at least four branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity, which can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees.

[0160] In some embodiments, in a composition comprising a complex polymerase, the support comprises a plurality of single-stranded nucleic acid template molecules immobilized to the support or to a coating thereof. In some embodiments, about 10 2 Up to 10 15Multiple template molecules are immobilized to a support, with each template molecule immobilized to a different site on the support. In some embodiments, multiple template molecules are immobilized to predetermined sites (e.g., locations) on the support. In some embodiments, multiple template molecules are immobilized to random sites (e.g., locations) on the support. In some embodiments, multiple immobilized template molecules are fluidly connected to each other to allow reagent solutions (e.g., enzymes, including polymerases, multivalent molecules, nucleotides, and / or divalent cations, etc.) to flow onto the support, such that multiple immobilized template molecules on the support can react with the reagent solution in a large-scale parallel manner.

[0161] Using read-capped nucleotide analogs—sequencing template strands

[0162] In one aspect, this disclosure provides a method for sequencing two or more regions of a nucleic acid template molecule, the method comprising the step (a): providing a plurality of nucleic acid template molecules, wherein each template molecule comprises (i) a first region and a first universal sequencing primer binding site, and (ii) a second region and a second universal sequencing primer binding site (e.g., ... Figure 3A or Figure 3B (As shown in the figure). In some embodiments, each template molecule in a plurality of nucleic acid template molecules comprises a single-stranded nucleic acid template molecule. In some embodiments, each template molecule in a plurality of nucleic acid template molecules comprises a double-stranded nucleic acid template molecule. In some embodiments, the template molecule comprises at least one target sequence (e.g., an insert sequence) and at least one universal adaptor sequence. In some embodiments, the first region in the template molecule of step (a) comprises the target sequence and / or the universal adaptor sequence. In some embodiments, the second region in the template molecule of step (a) comprises the target sequence and / or the universal adaptor sequence. In some embodiments, each template molecule comprises one copy of the target sequence and at least one universal adaptor sequence. In some embodiments, each template molecule comprises a nucleic acid polymer comprising two or more tandem copies of a unit, wherein each unit comprises the target sequence and one or more universal adaptor sequences. In some embodiments, the first region and the second region comprise the same target sequence. In some embodiments, the first universal sequencing primer binding site and the second universal sequencing primer binding site have the same sequence. In some embodiments, the first region and the second region comprise the same target sequence and / or the same universal adaptor sequence.

[0163] In some embodiments, in step (a), each template molecule contains any one or any combination of two or more universal adaptor sequences. The universal adaptor sequence may contain (i) a universal binding sequence for forward sequencing primers, (ii) a universal binding sequence for reverse sequencing primers, (iii) a universal binding sequence for a fixed first surface primer, (iv) a universal binding sequence for a fixed second surface primer, (v) a universal binding sequence for a first amplification primer, (vi) a universal binding sequence for a second amplification primer, (vii) a universal binding sequence for a compressed oligonucleotide, and / or (viii) a sample barcode sequence. In some embodiments, each template molecule contains one or more unique molecular index (UMI) sequences.

[0164] In some embodiments, in step (a), the universal adaptor sequence includes a universal binding sequence for a forward sequencing primer. In some embodiments, the universal binding sequence for a forward sequencing primer (or its complementary sequence) may hybridize with at least a portion of the forward sequencing primer. In some embodiments, the universal adaptor sequence includes a universal binding sequence for a reverse sequencing primer. In some embodiments, the universal binding sequence for a reverse sequencing primer (or its complementary sequence) may hybridize with at least a portion of the reverse sequencing primer. In some embodiments, the universal adaptor sequence includes a universal binding sequence for a fixed first surface primer. In some embodiments, the universal binding sequence for a fixed first surface primer (or its complementary sequence) may hybridize with at least a portion of the fixed first surface primer. In some embodiments, the universal adaptor sequence includes a universal binding sequence for a fixed second surface primer. In some embodiments, the universal binding sequence for a fixed second surface primer (or its complementary sequence) may hybridize with at least a portion of the fixed second surface primer. In some embodiments, the universal adaptor sequence includes a universal binding sequence for a first amplification primer. In some embodiments, the universal binding sequence for a first amplification primer (or its complementary sequence) may hybridize with at least a portion of the first amplification primer. In some embodiments, the universal adaptor sequence includes a universal binding sequence for a second amplification primer. In some embodiments, a universal binding sequence (or its complementary sequence) for the second amplification primer may hybridize with at least a portion of the second amplification primer. In some embodiments, the universal adaptor sequence comprises a universal binding sequence for a compressed oligonucleotide. In some embodiments, a universal binding sequence (or its complementary sequence) for a compressed oligonucleotide may hybridize with at least a portion of the compressed oligonucleotide.

[0165] In some embodiments, the method includes step (b): providing a plurality of first nucleic acid sequencing primers and a plurality of second nucleic acid sequencing primers (e.g., such as...). Figure 3A or Figure 3B(As shown in the figure). In some embodiments, a plurality of first nucleic acid sequencing primers comprise soluble oligonucleotides that hybridize to at least a portion of a first region of a template molecule near or adjacent to a universal binding sequence. In some embodiments, the first sequencing primers comprise a 3' OH extension end. In some embodiments, the first sequencing primers comprise a 3' blocking portion that can be removed to produce a 3' OH extension end. In some embodiments, a plurality of second nucleic acid sequencing primers comprise soluble oligonucleotides that hybridize to at least a portion of a second region of a template molecule near or adjacent to a universal binding sequence. In some embodiments, the second sequencing primers comprise a 3' OH extension end. In some embodiments, the second sequencing primers comprise a 3' blocking portion that can be removed to produce a 3' OH extension end.

[0166] In some embodiments, the method includes step (c): hybridizing each first nucleic acid sequencing primer with a first universal sequencing primer binding site on each template molecule (e.g., Figure 4A or Figure 4B (as shown in the figure). In some embodiments, one or more first sequencing primers are hybridized to the first universal sequencing primer binding site on each template molecule to produce one or more nucleic acid duplexes.

[0167] In some embodiments, the method includes step (d): sequencing a first region on a plurality of nucleic acid template molecules, thereby generating a plurality of first sequencing reads (see example...). Figure 4A or Figure 4B In some embodiments, step (d) can be performed using any type of sequencing workflow. In some embodiments, step (d) includes contacting the nucleic acid duplex from step (c) with a plurality of sequencing polymerases under conditions suitable for binding the sequencing polymerases to the nucleic acid duplex and suitable for generating a plurality of complex polymerases. In some embodiments, step (d) includes contacting the plurality of complex polymerases with a plurality of nucleotide reagents. In some embodiments, the various nucleotide reagents in the plurality of nucleotide reagents comprise typical nucleotides, chain-termining nucleotides, or multivalent molecules.

[0168] In some embodiments, step (d) includes contacting a plurality of complex polymerases with a plurality of detectably labeled chain-termining nucleotides under conditions suitable for incorporating the labeled chain-termining nucleotides into the 3' end of a first sequencing primer for hybridization, and detecting and identifying the incorporated chain-termining nucleotides. In some embodiments, step (d) includes performing a nucleotide incorporation reaction catalyzed by at least one polymerase on a plurality of template molecules to produce a plurality of first sequencing reads comprising a first sequencing primer linked to a polynucleotide having a sequence complementary to at least a portion of a nucleic acid template molecule. In some embodiments, step (d) includes removing the chain-termining portion and / or removing the detectable label from the incorporated chain-termining nucleotide to generate an extendable 3'OH sugar group on the chain-termining nucleotide. In some embodiments, step (d) includes repeating the following steps at least once: (i) incorporating the detectably labeled chain-termining nucleotide into the 3' end of a first sequencing primer for hybridization; (ii) detecting and identifying the chain-termining nucleotide; and (iii) removing the chain-termining portion and / or removing the detectable label from the chain-termining nucleotide to generate an extendable 3'OH sugar group on the chain-termining nucleotide.

[0169] In some embodiments, step (d) includes performing a two-stage sequencing workflow. A first sequencing stage of the two-stage sequencing workflow includes contacting a plurality of complex polymerases with a plurality of detectably labeled multivalent molecules. In some embodiments, each multivalent molecule includes a core attached to a plurality of nucleotide arms, and the nucleotide arms are attached to nucleotide units. In some embodiments, the plurality of complex polymerases and the plurality of multivalent molecules are contacted under conditions suitable for binding nucleotide units to the complex polymerases and for inhibiting incorporation of nucleotide units into the 3' end of a first sequencing primer for hybridization. The first sequencing stage of the two-stage sequencing workflow may further include detecting and identifying the nucleotide units bound to the detectably labeled multivalent molecules. In some embodiments, the plurality of complex polymerases and multivalent molecules are removed. In some embodiments, a second sequencing stage of the two-stage sequencing workflow includes contacting a nucleic acid duplex with a second plurality of sequencing polymerases under conditions suitable for binding the second sequencing polymerase to the nucleic acid duplex to generate a second plurality of complex polymerases. In some embodiments, the second sequencing stage of the two-stage sequencing workflow includes contacting the second plurality of complex polymerases with a plurality of unlabeled nucleotide analogs under conditions suitable for incorporating the nucleotide analogs into the 3' end of a first sequencing primer for hybridization. In some embodiments, step (d) includes one or more cycles of a two-stage sequencing workflow on a plurality of template molecules to produce a first plurality of sequencing reads containing a first sequencing primer linked to a polynucleotide having a sequence complementary to at least a portion of the nucleic acid template molecule.

[0170] In some embodiments, a method for sequencing two or more regions of a nucleic acid template molecule includes step (e): performing a plurality of first capping reactions, the capping reactions comprising incorporating a read-capping nucleotide analog into the terminal 3' end of each first sequencing read product, thereby generating a plurality of capped first sequencing read products (e.g. Figure 5A or Figure 5B (as shown in the diagram). In some embodiments, the plurality of first capping reactions include a nucleotide incorporation reaction catalyzed by one or more polymerases. In some embodiments, the plurality of first capping reactions include contacting a plurality of first sequencing read products with a plurality of polymerases and a plurality of read-capping nucleotide analogs under conditions suitable for incorporating the read-capping nucleotide analogs into the 3' end of each of the first sequencing read products, thereby producing a plurality of capped first sequencing read products. In some embodiments, the polymerase comprises a sequencing polymerase capable of catalyzing the incorporation of a nucleotide analog having a base-linked terminator portion. In some embodiments, one or more read-capping nucleotide analogs are incorporated into the 3' end of each of the first sequencing read products. In some embodiments, the incorporation of the read-capping nucleotide analogs may be performed at a temperature of about 25°C to 50°C or about 50°C to 55°C. In some embodiments, the incorporation of the read-capping nucleotide analogs may be performed for about 2 to 20 minutes.

[0171] In some embodiments, in step (e), the read-capped nucleotide analog comprises (i) a heterocyclic base, (ii) a sugar, and (iii) a polyphosphate chain, wherein the heterocyclic base is attached to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the first-capped sequencing read product. In some embodiments, the read-capped nucleotide analog further comprises a sugar moiety having an extendable 3'OH group. In some embodiments, the read-capped nucleotide analog further comprises a sugar moiety having a non-extendable chain-terminating group at the 3' position. Exemplary read-capped nucleotide analogs are shown in Figures 8A to 8J and Figure 9 And described in more detail below. In some embodiments, the sugar contains 5 carbons, namely a pentose. In some embodiments, the sugar contains ribose or deoxyribose.

[0172] In some embodiments, in step (e), the read-capped nucleotide analog includes a heterocyclic base linked to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the first-capped sequencing read product. In some embodiments, each multivalent molecule includes a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit (see, for example...). Figures 19 to 23 In some embodiments, the termination portion of the base linkage also blocks the incorporation of subsequent nucleotides (e.g., typical nucleotides or chain-terminating nucleotides) into the capped sequencing read product via polymerase catalysis.

[0173] In some embodiments, the method includes step (f): retaining multiple capped first sequencing reads (e.g., hybridized with their corresponding nucleic acid template molecules) Figure 5A , Figure 5B , Figure 6A , Figure 6B , Figure 7A and Figure 7B (As shown in the figure). In some embodiments, the terminating portion at the 3' end of each of the first-capped sequencing read products is not cleaved or removed. In some embodiments, the retained multiple capped first sequencing read products are denatured from their respective nucleic acid template molecules without heat and / or chemical denaturation reagents. In some embodiments, the retained multiple capped first sequencing read products are not degraded by enzymes having 5' to 3' exonuclease activity (e.g., T7 exonuclease or λ exonuclease). In some embodiments, the retained multiple capped first sequencing read products are not degraded by enzymes having 3' to 5' exonuclease activity (e.g., E. coli exonuclease I or E. coli exonuclease III).

[0174] In some embodiments, the method includes step (g): hybridizing a plurality of second sequencing primers to a plurality of second universal sequencing primer binding sites, wherein the second universal primer binding sites are present on each nucleic acid template molecule (see, for example...). Figure 6A or Figure 6B In some embodiments, multiple second sequencing primers are hybridized to multiple second universal sequencing primer binding sites to generate one or more nucleic acid duplexes. In some embodiments, first sequencing primers and second sequencing primers hybridize to different and non-overlapping regions of the same nucleic acid template molecule. In some embodiments, first sequencing primers and second sequencing primers hybridize to overlapping regions of the same nucleic acid template molecule. In some embodiments, first sequencing primers and second sequencing primers hybridize to the same region of the same nucleic acid template molecule. In some embodiments, step (g) includes hybridizing each second nucleic acid sequencing primer to a second universal sequencing primer binding site on a respective template molecule.

[0175] In some embodiments, the method includes step (h): sequencing a second region on a plurality of nucleic acid template molecules, thereby generating a plurality of second sequencing read products (e.g. Figure 6A or Figure 6B (as shown in the diagram). In some embodiments, sequencing in step (h) includes contacting the individual nucleic acid duplexes from step (g) with a plurality of sequencing polymerases under conditions suitable for binding the sequencing polymerases with the nucleic acid duplexes to produce a plurality of complex polymerases. In some embodiments, sequencing includes contacting the plurality of complex polymerases with a plurality of nucleotide reagents. In some embodiments, the nucleotide reagents comprise typical nucleotides, chain-terminating nucleotides, and / or multivalent molecules.

[0176] In some embodiments, step (h) includes contacting a plurality of complex polymerases with a plurality of detectably labeled chain-termining nucleotides under conditions suitable for incorporating the labeled chain-termining nucleotides into the 3' end of a hybridization second sequencing primer, and detecting and identifying the chain-termining nucleotides. In some embodiments, the sequencing in step (h) includes performing a nucleotide incorporation reaction catalyzed by at least one polymerase on a plurality of template molecules to produce a plurality of second sequencing reads, wherein each of the plurality of second sequencing reads comprises a second sequencing primer linked to a polynucleotide having a sequence complementary to at least a portion of the nucleic acid template molecule.

[0177] In some embodiments, step (h) includes performing a two-stage sequencing workflow. The first sequencing stage of the two-stage sequencing workflow may include contacting a complex polymerase with a plurality of detectably labeled multivalent molecules. In some embodiments, each multivalent molecule includes a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. In some embodiments, the complex polymerase and multivalent molecules are contacted under conditions suitable for binding the nucleotide unit to the complex polymerase and for inhibiting incorporation of the nucleotide unit into the 3' end of a hybridization second sequencing primer. The first sequencing stage of the two-stage sequencing workflow may further include detecting and identifying the nucleotide unit bound to the detectably labeled multivalent molecule. In some embodiments, the complex polymerase and multivalent molecules are removed. In some embodiments, the second sequencing stage of the two-stage sequencing workflow includes contacting a plurality of nucleic acid duplexes with a second plurality of sequencing polymerases under conditions suitable for binding the second sequencing polymerase to the nucleic acid duplexes to generate a second plurality of complex polymerases. In some embodiments, the second sequencing stage of the two-stage sequencing workflow further includes contacting the second plurality of complex polymerases with a plurality of unlabeled nucleotide analogs under conditions suitable for incorporating the nucleotide analogs into the 3' end of a hybridization second sequencing primer. In some embodiments, the sequencing in step (h) includes one or more cycles of a two-stage sequencing workflow on a plurality of template molecules to produce a plurality of second sequencing reads, wherein each second sequencing read contains a second sequencing primer linked to a polynucleotide having a sequence complementary to at least a portion of the nucleic acid template molecule.

[0178] In some embodiments, the method includes step (i): performing a plurality of second capping reactions, the plurality of second capping reactions including incorporating a read-capping nucleotide analog into the terminal 3' end of each of the plurality of second sequencing read products, thereby producing a second plurality of capped sequencing read products (e.g. Figure 7A or Figure 7B(as shown in the diagram). In some embodiments, the plurality of second capping reactions include a nucleotide incorporation reaction catalyzed by one or more polymerases. In some embodiments, the plurality of second capping reactions include contacting a plurality of second sequencing read products with a plurality of polymerases and a plurality of read-capping nucleotide analogs under conditions suitable for incorporating the read-capping nucleotide analogs into the 3' end of each of the second sequencing read products, thereby producing a second plurality of capped sequencing read products. In some embodiments, the polymerase comprises a sequencing polymerase capable of catalyzing the incorporation of a nucleotide analog having a base-linked terminator portion. In some embodiments, one or more read-capping nucleotide analogs are incorporated into the 3' end of each of the plurality of second sequencing read products. In some embodiments, the incorporation of the read-capping nucleotide analogs may be performed at a temperature of about 25°C to 50°C or about 50°C to 55°C. In some embodiments, the incorporation of the read-capping nucleotide analogs may be performed for about 2 to 20 minutes.

[0179] In some embodiments, in step (i), each read-capped nucleotide analog comprises (i) a heterocyclic base, (ii) a sugar, and (iii) a polyphosphate chain, wherein the heterocyclic base is attached to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the second-capped sequencing read product. In some embodiments, each read-capped nucleotide analog comprises a sugar moiety having an extendable 3'OH group. In some embodiments, each read-capped nucleotide analog comprises a sugar moiety having a non-extendable chain termination group at the 3' position. In some embodiments, the sugar comprises 5 carbons, i.e., a pentose sugar. In some embodiments, the sugar comprises ribose or deoxyribose. Illustrative read-capped nucleotide analogs are shown in Figures 8A to 8J and Figure 9 And described in more detail in this article.

[0180] In some embodiments, in step (i), each capped nucleotide analog contains a heterocyclic base linked to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the sequencing read in a plurality of capped second sequencing reads. In some embodiments, each multivalent molecule contains a core attached to a plurality of nucleotide arms, wherein each nucleotide arm is attached to a nucleotide unit. In some embodiments, the base-linked termination portion also blocks the polymerase-catalyzed incorporation of subsequent nucleotides (e.g., typical nucleotides or chain-terminating nucleotides) into the capped sequencing read.

[0181] In some embodiments, in step (i), each sequencing template molecule comprises at least one capped first sequencing read product hybridized thereon and at least one capped second sequencing read product hybridized thereon.

[0182] In some embodiments, a method for sequencing two or more regions of a nucleic acid template molecule includes step (j1): removing a plurality of capped second sequencing reads that have hybridized with their respective template molecules. In some embodiments, the plurality of capped second sequencing reads may be denatured from their respective template molecules by heat and / or by a chemical denaturing agent. In some embodiments, the plurality of capped second sequencing reads may be degraded by an enzyme having 5' to 3' exonuclease activity (e.g., T7 exonuclease or λ exonuclease). In some embodiments, the plurality of capped second sequencing reads may be degraded by an enzyme having 3' to 5' exonuclease activity (e.g., E. coli exonuclease I or E. coli exonuclease III).

[0183] In some embodiments, a method for sequencing two or more regions of a nucleic acid template molecule includes step (j2): retaining a plurality of capped second sequencing reads hybridized with their respective template molecules, thereby producing a plurality of retained capped second sequencing reads. In some embodiments, the termination portion at the 3' end of the read-capping nucleotide analog is not cleaved or removed from the terminal portion of each capped second sequencing read. In some embodiments, the retained plurality of capped second sequencing reads are denatured from their respective template molecules without heat and / or with chemical denaturing agents. In some embodiments, the retained plurality of capped second sequencing reads are not degraded by enzymes having 5' to 3' exonuclease activity (e.g., T7 exonuclease or λ exonuclease). In some embodiments, the retained plurality of capped second sequencing reads are not degraded by enzymes having 3' to 5' exonuclease activity (e.g., E. coli exonuclease I or E. coli exonuclease III).

[0184] In some embodiments, the method includes repeating steps (b) to (j1) or (b) to (j2) using a third sequencing primer and optionally a fourth sequencing primer, wherein the third and fourth sequencing primers hybridize with the third and fourth regions of the same template molecule, respectively. Those skilled in the art will recognize that steps (b) to (j1) or (b) to (j2) can be repeated multiple times using a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more sequencing primers that hybridize with their respective regions of the same template molecule.

[0185] In some embodiments, the third and fourth sequencing primers hybridize with different and non-overlapping regions of the same nucleic acid template molecule. In some embodiments, compared to the first and second sequencing primers, the third and fourth sequencing primers hybridize with different and non-overlapping regions of the same nucleic acid template molecule. In some embodiments, the third and fourth sequencing primers hybridize with overlapping regions of the same nucleic acid template molecule. In some embodiments, the third and fourth sequencing primers hybridize with regions on the same nucleic acid template molecule where the binding sites of the first and second sequencing primers overlap. In some embodiments, the third and fourth sequencing primers hybridize with the same region of the same nucleic acid template molecule. In some embodiments, the same region is the same region of the same nucleic acid template molecule that hybridizes with the first and second sequencing primers.

[0186] In some embodiments, any suitable portion of the single-stranded template molecule may be fixed to a support or a coating on the support. For example, the 5' or 3' end of the template molecule or an internal portion of the template molecule may be fixed to a support. In some embodiments, the 5' or 3' end of the single-stranded nucleic acid template molecule is covalently attached to a surface primer, which is fixed to a support or a coating on the support. In some embodiments, the 5' or 3' region of the single-stranded nucleic acid template molecule hybridizes with a surface primer, which is fixed to a support or a coating on the support.

[0187] In some embodiments, single-stranded nucleic acid template molecules can be generated through a cloning amplification workflow (e.g., rolling circle amplification or bridge amplification). In some embodiments, single-stranded nucleic acid template molecules are not generated through a cloning amplification workflow.

[0188] In some embodiments, the single-stranded nucleic acid template molecule contains at least one uridine nucleotide. In some embodiments, the single-stranded nucleic acid template molecule does not contain uridine nucleotides.

[0189] In some embodiments, a single-stranded nucleic acid template molecule contains one copy of the sequence of interest. For example, a single-copy template molecule can be generated by bridge amplification. In some embodiments, a single-stranded nucleic acid template molecule contains two or more tandem copies of the sequence of interest. For example, a tandem copy template molecule can contain multiples that can be generated by rolling circle amplification (RCA).

[0190] In some embodiments, the support is a support (e.g., a glass slide). In some embodiments, the support is a non-planar support (e.g., beads). In some embodiments, the support comprises or is composed of a solid material. In some embodiments, the support comprises or is composed of a semi-solid material. In some embodiments, the support comprises or is composed of a porous material. In some embodiments, the support comprises or is composed of a semi-porous material. In some embodiments, the support comprises or is composed of a non-porous material. The support may be made of any suitable material known in the art or described herein, such as glass, plastic, or polymeric materials.

[0191] In some embodiments, the surface of the support may be coated with one or more compounds to create a passivation layer on the support. In some embodiments, the passivation layer forms a porous layer. In some embodiments, the passivation layer forms a semi-porous layer. In some embodiments, surface primers or single-stranded nucleic acid template molecules may be attached to the passivation layer to immobilize the primers or template molecules to the support. In some embodiments, the support includes a low-nonspecific binding surface that enables improved nucleic acid hybridization, amplification, and sequencing performance on the support. Generally, the support may include one or more layers of covalently or non-covalently attached low-binding chemically modified layers (e.g., silane layers, polymer membranes), and one or more oligonucleotides that can be used to immobilize multiple nucleic acid template molecules to the support via covalent or non-covalent attachment. In some embodiments, the support may include a functionalized polymer coating at least covalently bound to a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of hydrophilic polymer coating and at least one layer of multiple oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise branched PEG having at least four branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity, which can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees.

[0192] In some embodiments, the support comprises a plurality of single-stranded nucleic acid template molecules immobilized to or attached to a coating on the support. In some embodiments, about 10 2 Up to 10 15Multiple template molecules are immobilized to a support, with each template molecule immobilized to a different site on the support. In some embodiments, multiple template molecules are immobilized to predetermined sites (e.g., locations) on the support. In some embodiments, multiple template molecules are immobilized to random sites (e.g., locations) on the support. In some embodiments, multiple immobilized template molecules are fluidly connected to each other to allow reagent solutions (e.g., enzymes, including polymerases, multivalent molecules, nucleotides, and / or divalent cations, etc.) to flow onto the support, such that multiple immobilized template molecules on the support can react with the reagent solution in a large-scale parallel manner.

[0193] Methods for pairwise sequencing

[0194] On the other hand, this disclosure provides a method for pairwise sequencing, comprising obtaining a first sequencing read of a first region of a first nucleic acid strand (e.g., a sense strand or a forward strand), and a second sequencing read of a second region of a second nucleic acid strand complementary to the first strand (e.g., an antisense strand or a reverse strand), wherein the first and second strands correspond to the two complementary strands of a double-stranded template molecule. The first sequencing read of the first sequencing region and the second sequencing read of the second sequencing region may contain overlapping sequences corresponding to complementary sequences present on the first and second strands of the double-stranded template molecule, respectively. The first and second sequencing reads (sometimes referred to as pairwise sequencing reads) can be aligned such that the overlapping regions of the pairwise sequencing reads can produce sequence information of the paired regions in the original double-stranded nucleic acid source (e.g., paired regions in the genome), and the accuracy of the sequence information can be determined from the first and second sequencing reads at a high confidence level. However, those skilled in the art will understand that the first sequencing read of the first sequencing region and the second sequencing read of the second sequencing region do not necessarily need to have overlapping sequences. The first and second sequencing reads may be initiated at one end of their respective template molecules or at internal locations.

[0195] This disclosure provides a pairwise sequencing method comprising the step (a): providing a plurality of single-stranded nucleic acid template molecules, wherein each nucleic acid template molecule contains at least one nucleotide having a cleavable portion. In some embodiments, each of the plurality of nucleic acid template molecules is immobilized to a first surface primer, which is immobilized to a support. In some embodiments, the first surface primer lacks the nucleotide having the cleavable portion. In some embodiments, a plurality of second surface primers are immobilized to a support. In some embodiments, no second surface primers are immobilized to a support. In some embodiments, a plurality of first surface primers and a plurality of second surface primers are immobilized to a support. In some embodiments, the template molecule is immobilized.

[0196] In some embodiments, each nucleic acid template molecule is immobilized to a support. In some embodiments, each nucleic acid template molecule comprises a multiply molecule containing multiple tandem copies of the target sequence and at least one universal adaptor sequence (e.g., a sequencing primer binding site). In some embodiments, each nucleic acid template molecule is a non-multiply molecule containing only one copy of the target sequence and at least one universal adaptor sequence (e.g., a sequencing primer binding site).

[0197] In some embodiments, each nucleic acid template molecule is covalently ligated to a fixed surface primer (e.g., a fixed first surface primer) (e.g. Figure 16A (as shown in the illustration). In some embodiments, each nucleic acid template molecule hybridizes with a fixed surface primer (e.g., a fixed first surface primer) (see, for example...). Figure 17 ).

[0198] In some embodiments, each of the plurality of nucleic acid template molecules contains at least one target sequence and at least one universal adapter sequence or any combination of two or more universal adapter sequences. A universal adaptor sequence or a combination of two or more universal adaptor sequences may include: a first surface primer binding site (or its complementary sequence) that can hybridize with any suitable portion of a fixed first surface primer; a second surface primer binding site (or its complementary sequence) that can hybridize with any suitable portion of a fixed second surface primer; a first sequencing primer site (or its complementary sequence) that can hybridize with any suitable portion of a forward sequencing primer; a second sequencing primer site (or its complementary sequence) that can hybridize with any suitable portion of a reverse sequencing primer; a first amplification primer binding site (or its complementary sequence) that can hybridize with any suitable portion of a forward amplification primer; a second amplification primer binding site (or its complementary sequence) that can hybridize with any suitable portion of a reverse amplification primer; a first compressed oligonucleotide binding site (or its complementary sequence) that can hybridize with any suitable portion of a first compressed oligonucleotide; a second compressed oligonucleotide binding site (or its complementary sequence) that can hybridize with any suitable portion of a second compressed oligonucleotide; a first sample index sequence; a second sample index sequence; a first unique molecular tag sequence; and / or a second unique molecular tag sequence.

[0199] In some embodiments, the cleavable portion of the nucleic acid template molecule in step (a) may be converted into a base-free site in the nucleic acid template molecule. In some embodiments, the cleavable portion includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Uraidine can be converted into a base-free site using uracil DNA glycosylase (UDG), 8oxoG can be converted into a base-free site using FPG glycosylase (also known as DNA-formamidopyrimidine glycosylase), and deoxyinosine can be converted into a base-free site using AlkA glycosylase (also known as 3-methyladenine-DNA glycosylase II). In some embodiments, each nucleic acid template molecule includes 1 to 20, 20 to 40, 40 to 60, 60 to 80, 80 to 100, or more nucleotides having cleavable portions. In some embodiments, about 0.1% to 1%, or about 1% to 5%, or about 5% to 10%, or about 10% to 20%, or about 20% to 30% or higher percentage of dTTP in each nucleic acid template molecule is replaced with nucleotides having cleavable portions. In some embodiments, nucleotides having cleavable portions are distributed at random locations along each nucleic acid template molecule. In some embodiments, nucleotides having cleavable portions are distributed at different locations in different nucleic acid template molecules.

[0200] In some embodiments, a first surface primer is immobilized to a support (“immobilized first surface primer”, also referred to as a “immobilized first surface capture primer”). In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising DNA. In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising RNA. In some embodiments, the immobilized first surface primer comprises a single-stranded oligonucleotide comprising a combination of RNA and DNA. The immobilized first surface primer may be immobilized to a support or to a coating on a support. The immobilized first surface primer may be embedded in and attached (coupled) to a coating on a support. In some embodiments, the 5' end of the immobilized first surface primer is immobilized to a support or to a coating on a support. In some embodiments, the internal portion of the immobilized first surface primer is immobilized to a support or to a coating on a support. In some embodiments, the 3' end of the immobilized first surface primer is immobilized to a support or to a coating on a support. In some embodiments, the support comprises a plurality of immobilized first surface primers having the same sequence. The fixed first surface primer in a plurality of first surface primers can be of any length, such as 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides or longer. In some embodiments, the 3' end of the fixed first surface primer includes an extendable 3' OH portion. In some embodiments, the 3' end of the fixed first surface primer includes a 3' non-extendable portion. In some embodiments, the fixed first surface primer lacks nucleotides with cleavable portions.

[0201] In some embodiments, each of the plurality of fixed first surface primers contains at least one phosphate-thioester diester bond at its 5' end, which makes the first surface primer resistant to exonuclease degradation. In some embodiments, each of the plurality of fixed first surface primers contains 2, 3, 4, 5 or more consecutive phosphate-thioester diester bonds at its 5' end. In some embodiments, each of the plurality of fixed first surface primers contains at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which makes the first surface primer resistant to exonuclease degradation.

[0202] In some embodiments, each of the plurality of fixed first surface primers contains at least one locked nucleic acid (LNA) containing a methylene bridge between the 2' oxygen and the 4' carbon of the pentose ring. Fixed first surface primers containing at least one LNA are resistant to nuclease digestion and can exhibit increased melting temperatures when hybridized with the forward extension strand.

[0203] In some embodiments, the nucleic acid template molecule comprises two or more copies of a universal binding sequence (or its complementary sequence) for a second surface primer (“fixed second surface primer”) immobilized to a support, the second surface primer having a sequence different from that of the first fixed surface primer. The fixed second surface primer of step (a) may comprise a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The fixed second surface primer may be immobilized to a support or to a coating on a support. The fixed second surface primer may be embedded in and attached (coupled) to a coating on a support. In some embodiments, the 5' end of the fixed second surface primer is immobilized to a support or to a coating on a support. In some embodiments, the internal portion of the fixed second surface primer is immobilized to a support or to a coating on a support. In some embodiments, the 3' end of the fixed second surface primer is immobilized to a support or to a coating on a support. In some embodiments, the support comprises a plurality of fixed second surface primers having the same sequence. The fixed second surface primer can be of any length, such as 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides or longer.

[0204] In some embodiments, the 3' end of each of the plurality of fixed second surface primers includes an extendable 3' OH portion. In some embodiments, the 3' end of the fixed second surface primer includes a 3' non-extendable portion. In some embodiments, the 3' end of the fixed second surface primer includes a portion that blocks primer extension (e.g., a non-extendable 3' end), such as, for example, a phosphate group, a dideoxycytidine group, a reverse dT, or an amino group. In some embodiments, the fixed second surface primer is non-extendable in the primer extension reaction. In some embodiments, the fixed second surface primer lacks a nucleotide with a cleavable portion.

[0205] In some embodiments, in methods for pairwise sequencing, each of the plurality of fixed second surface primers contains at least one phosphate-thioester diester bond at its 5' end, which makes the second surface primer resistant to exonuclease degradation. In some embodiments, each of the plurality of fixed second surface primers contains 2, 3, 4, 5 or more consecutive phosphate-thioester diester bonds at its 5' end. In some embodiments, each of the plurality of fixed second surface primers contains at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which makes the second surface primer resistant to exonuclease degradation.

[0206] In some embodiments, individual single-stranded nucleic acid template molecules are ligated or immobilized to immobilized first surface primers, and at least a portion of each template molecule hybridizes with an immobilized second surface primer. In such embodiments, the immobilized second surface primer is used to pin a portion of the immobilized template molecule downwards to a support (see, for example, see...). Figure 16H ).

[0207] In some embodiments, the support includes per mm 2 Approximately 10 2 Up to 10 15 A fixed first surface primer. In some embodiments, the support comprises per mm 2 Approximately 10 2 Up to 10 15 A fixed second surface primer. In some embodiments, the support comprises per mm 2 Approximately 10 2 Up to 10 15 One fixed first surface primer and approximately 10 2 Up to 10 15 A fixed second surface primer.

[0208] In some embodiments, fixed surface primers (e.g., a first surface primer and a second surface primer) are fluidly connected to each other to allow various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc., to flow onto the support, such that the plurality of fixed surface primers (and primer extension products generated from the fixed surface primers) react with the solutions in a large-scale parallel manner.

[0209] In some embodiments, the pairwise sequencing method includes step (b): sequencing a first region of a plurality of nucleic acid template molecules, thereby generating a first plurality of forward extended duplexes. Sequencing the first region in step (b) may include contacting the plurality of nucleic acid template molecules with the first plurality of forward sequencing primers under conditions suitable for hybridizing at least one of the first forward sequencing primers with at least one of the forward sequencing primer binding sites located on the nucleic acid template molecules, and performing a first set of forward sequencing reactions using one or more types of sequencing polymerases and a plurality of nucleotide reagents (e.g., nucleotides and / or multivalent molecules). In some embodiments, the nucleic acid template molecules are immobilized. In some embodiments, the first plurality of forward sequencing primers are soluble (i.e., in solution). In some embodiments, the first plurality of forward sequencing primers are soluble and may hybridize with at least one region of a universal binding sequence of a first portion of the nucleic acid template molecule to be sequenced, which is adjacent to or near the first portion of the nucleic acid template molecule. The first forward sequencing reaction may generate a plurality of first forward extended duplexes containing first forward sequencing reads hybridized with the nucleic acid template molecules. Each first forward sequencing read may contain a first forward sequencing primer linked to a polynucleotide having a sequence complementary to a first region of the nucleic acid template molecule to be sequenced. The forward sequencing primer binding site can hybridize with the forward sequencing primer and undergo a sequencing reaction. In some embodiments, the nucleic acid template molecules among the plurality of nucleic acid template molecules are multi-template template molecules. In some embodiments, each multi-template template molecule contains multiple copies of the forward sequencing primer binding site, wherein each forward sequencing primer binding site is capable of hybridizing with a first forward sequencing primer. Each multi-template template molecule may undergo two or more sequencing reactions simultaneously, wherein each sequencing reaction is initiated by a different first forward sequencing primer, and each forward sequencing primer binds to a different forward sequencing primer binding site on each multi-template template molecule (see, for example, [link to documentation]). Figure 16B Hybridization. In some embodiments, the first forward sequencing primer includes a 3' OH extendable end. In some embodiments, the first forward sequencing primer includes a 3' blocking portion that can be removed to produce a 3' OH extendable end. In some embodiments, the first forward sequencing primer lacks a nucleotide with a cleavable portion. In some embodiments, the multiple nucleotide reagent used in the sequencing reaction includes multiple nucleotides (or the like) labeled with a detectable reporter portion. In some embodiments, the multiple nucleotide reagent includes multiple multivalent molecules, each multivalent molecule including a core attached to multiple nucleotide arms, and each nucleotide arm attached to a nucleotide unit. In some embodiments, the multivalent molecule is labeled with a detectable reporter portion. In some embodiments, the core is labeled with a detectable reporter portion. In some embodiments, at least one nucleotide arm and / or at least one nucleotide unit is labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore. Illustrative nucleotide arms are shown in Figure 23 In, and illustrative multivalent molecules are shown in Figures 19 to 22 middle.

[0210] In some embodiments, step (b) includes performing a plurality of first capping reactions, the plurality of first capping reactions including incorporating a read-capping nucleotide analog into the terminal 3' end of a respective first sequencing read product, thereby producing a plurality of capped first sequencing read products. In some embodiments, the first capping reaction includes a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the plurality of first capping reactions includes contacting a plurality of first sequencing read products with a plurality of polymerases and a plurality of read-capping nucleotide analogs under conditions suitable for incorporating the read-capping nucleotide analogs into the terminal 3' end of each first sequencing read product, thereby producing a first plurality of capped sequencing read products. In some embodiments, the plurality of polymerases comprise sequencing polymerases capable of catalyzing the incorporation of nucleotide analogs having a base-linked terminator portion. In some embodiments, one or more read-capping nucleotide analogs are incorporated into the terminal 3' end of each first sequencing read product.

[0211] In some embodiments, in step (b), the read-capped nucleotide analog comprises (i) a heterocyclic base, (ii) a sugar, and (iii) a polyphosphate chain, wherein the heterocyclic base is attached to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the first-capped sequencing read product. In some embodiments, the read-capped nucleotide analog further comprises a sugar moiety having an extendable 3'OH group. In some embodiments, the read-capped nucleotide analog further comprises a sugar moiety having a non-extendable chain-terminating group at the 3' position. In some embodiments, the sugar comprises 5 carbons, i.e., a pentose sugar. In some embodiments, the sugar comprises ribose or deoxyribose. An illustrative read-capped nucleotide analog is shown in Figures 8A to 8J and Figure 9 And described in more detail below.

[0212] In some embodiments, in step (b), the read-capped nucleotide analog contains a heterocyclic base linked to a termination moiety that blocks multivalent molecules (see, for example...). Figures 19 to 23 The binding of the multivalent molecule to the 3' end of the capped first sequencing read product. In some embodiments, each multivalent molecule includes a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. In some embodiments, the termination portion of the base linkage also blocks the polymerase-catalyzed incorporation of subsequent nucleotides (e.g., typical nucleotides or chain-terminating nucleotides) into the capped sequencing read product.

[0213] In some embodiments, step (b) includes: retaining a plurality of capped first sequencing reads that have hybridized with the template molecule. In some embodiments, the termination portion at the 3' end of each capped first sequencing read, incorporating a capped nucleotide analog, is not cleaved or removed. In some embodiments, the retained plurality of capped first sequencing reads are not denatured from the template molecule by heat and / or chemical denaturing agents. In some embodiments, the retained plurality of capped first sequencing reads are not degraded by enzymes having 5' to 3' exonuclease activity (e.g., T7 exonuclease or λ exonuclease). In some embodiments, the retained plurality of capped first sequencing reads are not degraded by enzymes having 3' to 5' exonuclease activity (e.g., E. coli exonuclease I or E. coli exonuclease III).

[0214] In some embodiments, step (b) includes: hybridizing each second sequencing primer with a second universal sequencing primer binding site located on each of a plurality of nucleic acid template molecules, and performing one or more second sequencing reactions. In some embodiments, hybridizing each second sequencing primer with a second universal sequencing primer binding site produces one or more nucleic acid duplexes. In some embodiments, the second forward sequencing primer contains a soluble oligonucleotide that hybridizes to at least a portion of a second region of the nucleic acid template molecule adjacent to or adjacent to the universal binding sequence. In some embodiments, the first sequencing primer and the second sequencing primer hybridize to different and non-overlapping regions of the same nucleic acid template molecule.

[0215] In some embodiments, sequencing of the second region in step (b) includes contacting a plurality of nucleic acid template molecules with a plurality of second forward sequencing primers under conditions suitable for hybridizing at least one of the second forward sequencing primers with at least one of the second forward sequencing primer binding sites present on each of the nucleic acid template molecules, and performing one or more second forward sequencing reactions using one or more types of sequencing polymerases and a plurality of nucleotide reagents (e.g., nucleotides and / or multivalent molecules). In some embodiments, the template molecules are immobilized. In some embodiments, the plurality of second forward sequencing primers are soluble (i.e., in solution). In some embodiments, the second forward sequencing primers comprise soluble oligonucleotides that can hybridize with at least one region of a second portion of the nucleic acid template molecule adjacent to or juxtaposed with a universal binding sequence. Each of the second forward sequencing reactions can produce a plurality of second forward extended duplexes, each duplex comprising a second forward sequencing read product hybridized with the nucleic acid template molecule. In some embodiments, each of the second forward sequence read products comprises a second forward sequencing primer linked to a polynucleotide containing a sequence complementary to the second region of the nucleic acid template molecule to be sequenced. The second forward sequencing primer binding site in the nucleic acid template molecule can hybridize with a second forward sequencing primer and can undergo a sequencing reaction. In some embodiments, the nucleic acid template molecules in the plurality of template molecules are multiplicative template molecules. In some embodiments, the nucleic acid template molecules in the plurality of template molecules are immobilized to a support. In some embodiments, the multiplicative template molecule contains multiple copies of the second forward sequencing primer binding site, wherein each second forward sequencing primer binding site is capable of hybridizing with a second forward sequencing primer. The multiplicative template molecule can undergo two or more sequencing reactions, wherein each sequencing reaction is initiated by a different second forward sequencing primer, and each second forward sequencing primer binds to a different second forward sequencing primer binding site (see, for example, see...). Figure 16B Hybridization. In some embodiments, the second forward sequencing primer includes a 3' OH extendable end. In some embodiments, the second forward sequencing primer includes a 3' blocking portion that can be removed to produce a 3' OH extendable end. In some embodiments, the second forward sequencing primer lacks a nucleotide with a cleavable portion. In some embodiments, the multinucleotide reagent includes a plurality of nucleotides (or the like) labeled with a detectable reporter portion. In some embodiments, the multinucleotide reagent includes a plurality of multivalent molecules, each multivalent molecule including a core attached to a plurality of nucleotide arms, and each nucleotide arm attached to a nucleotide unit. In some embodiments, the multivalent molecule is labeled with a detectable reporter portion. In some embodiments, the core is labeled with a detectable reporter portion. In some embodiments, at least one nucleotide arm and / or at least one nucleotide unit is labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion includes a fluorophore. Illustrative nucleotide arms are shown in Figure 23In, and illustrative multivalent molecules are shown in Figures 19 to 22 middle.

[0216] In some embodiments, step (b) includes optionally performing a plurality of second capping reactions, wherein each capping reaction includes incorporating a read-capping nucleotide analog into the terminal 3' end of a respective second sequencing read product, thereby producing a plurality of capped second sequencing read products. In some embodiments, the plurality of second capping reactions include polymerase-catalyzed nucleotide incorporation reactions. In some embodiments, the plurality of second capping reactions include contacting a plurality of second sequencing read products with a plurality of polymerases and a plurality of read-capping nucleotide analogs under conditions suitable for incorporating the read-capping nucleotide analogs into the terminal 3' end of each second sequencing read product, thereby producing a plurality of capped second sequencing read products. In some embodiments, the plurality of polymerases comprise sequencing polymerases capable of catalyzing the incorporation of nucleotide analogs comprising a base-linked terminator portion. In some embodiments, one or more read-capping nucleotide analogs are incorporated into the terminal 3' end of each second sequencing read product.

[0217] In some embodiments, in step (b), the read-capped nucleotide analog comprises (i) a heterocyclic base, (ii) a sugar, and (iii) a polyphosphate chain, wherein the heterocyclic base is attached to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the capped second sequencing read product. In some embodiments, the read-capped nucleotide analog further comprises a sugar moiety having an extendable 3'OH group. In some embodiments, the read-capped nucleotide analog further comprises a sugar moiety having a non-extendable chain termination group at the 3' position. In some embodiments, the sugar comprises 5 carbons, i.e., a pentose sugar. In some embodiments, the sugar comprises ribose or deoxyribose. An illustrative read-capped nucleotide analog is shown in Figures 8A to 8J and Figure 9 And described in more detail below.

[0218] In some embodiments, in step (b), the capped nucleotide analog contains a heterocyclic base linked to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the capped second sequencing read. In some embodiments, each multivalent molecule contains a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. In some embodiments, the base-linked termination portion also blocks the polymerase-catalyzed incorporation of subsequent nucleotides (e.g., typical nucleotides or chain-terminating nucleotides) into the capped second sequencing read.

[0219] In some embodiments, step (b) includes: retaining a plurality of capped second sequencing reads that have hybridized with the nucleic acid template molecule. In some embodiments, the termination portion at the 3' end of each capped second sequencing read, incorporating a capped nucleotide analog, is not cleaved or removed. In some embodiments, the retained plurality of capped second sequencing reads are not denatured from the template molecule without heat and / or chemical denaturation. In some embodiments, the retained plurality of capped second sequencing reads are not degraded by enzymes having 5' to 3' exonuclease activity (e.g., T7 exonuclease or λ exonuclease). In some embodiments, the retained plurality of capped second sequencing reads are not degraded by enzymes having 3' to 5' exonuclease activity (e.g., E. coli exonuclease I or E. coli exonuclease III).

[0220] In some embodiments, the pairwise sequencing method includes step (c): retaining multiple single-stranded nucleic acid template molecules and replacing multiple first forward sequencing reads and multiple second forward sequencing reads with multiple forward extension strands that hybridize with the nucleic acid template molecules. In some embodiments, the pairwise sequencing method includes step (c): retaining multiple single-stranded nucleic acid template molecules and replacing multiple capped first sequencing reads and multiple capped second sequencing reads with multiple forward extension strands that hybridize with the nucleic acid template molecules. In some embodiments, the retained nucleic acid template molecules are immobilized to a support.

[0221] In some embodiments, the plurality of first forward sequencing read products and optionally the second forward sequencing read products have extendable 3' ends, which can be removed by performing a primer extension reaction and replaced with a plurality of forward extension strands (see [link to documentation]). Figure 16C and Figure 16D The chain substitution step is sometimes referred to as "second-strand synthesis" or "paired switching." Different embodiments for substituting the first forward sequencing read product and optionally the second forward sequencing read product by performing a primer extension reaction are described below.

[0222] In some embodiments, step (c) includes contacting at least one first forward sequencing read product with a plurality of strand substitution polymerases and a plurality of nucleotides in the absence of soluble amplification primers and under conditions suitable for initiating a strand substitution primer extension reaction from at least one first forward sequencing read product using at least one first forward sequencing read product, thereby generating a nascent forward extension strand covalently linked to the first forward sequencing read product, wherein the forward extension strand hybridizes with a nucleic acid template molecule. For example, the 3' end of the first forward sequencing read product can be used as a primer for the strand substitution polymerase. The strand substitution polymerase can extend the first forward sequencing read product and, while synthesizing a nascent forward extension strand that replaces the downstream first forward sequencing read product from the template molecule, displace the downstream first forward sequencing read product (see, for example...). Figure 16C The newly extended nascent strand can be covalently linked to the product of the first forward sequencing read. The fixed template molecule can be preserved.

[0223] In some embodiments, step (c) may optionally include multiple compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) for generating forward extension chains. Individual forward extension chains may collapse into nanospheres having a more compact size and / or shape compared to nanospheres generated from primer extension reactions performed without compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII). Including compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) in the primer extension reaction improves the FWHM (full width at half maximum) of the nanosphere's dot pattern. The dot pattern may be represented as Gaussian points, and the size may be measured in FWHM. Smaller dot sizes, as indicated by a smaller FWHM, are generally associated with an improved dot pattern. In some embodiments, the FWHM of the nanosphere dots may be about 10 μm or smaller.

[0224] Examples of strand substitution polymerases include phi29 DNA polymerase, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV reverse transcriptase, Deep Vent® DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon). TM ), or variant EquiPhi29 TM DNA polymerase (e.g., from ThermoFisher® Scientific), or chimeric QualiPhi TM DNA polymerase (e.g., from 4basebio).

[0225] In some embodiments, step (c) includes: (i) removing a plurality of first sequencing read products and / or a plurality of second sequencing read products, and / or removing a plurality of first-capped sequencing read products and a plurality of second-capped sequencing read products, while retaining the template molecule; and (ii) contacting the plurality of retained template molecules with a plurality of additional forward sequencing primers (e.g., a third plurality of forward sequencing primers), a plurality of nucleotides, and a plurality of primer extension polymerases under conditions suitable for hybridizing the plurality of forward sequencing primers with the plurality of retained template molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, wherein the sequencing primers hybridize with forward sequencing primer binding sites present in the retained template molecules ( Figure 16D The primer extension reaction may optionally include multiple compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) to produce a forward extension chain. The forward extension chain may collapse into nanospheres having a more compact size and / or shape compared to nanospheres generated from a primer extension reaction performed without compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII). Including compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) in the primer extension reaction improves the FWHM (full width at half maximum) of the nanosphere's dot pattern. The dot pattern may be represented as Gaussian points, and the size may be measured in FWHM. Smaller dot sizes, as indicated by a smaller FWHM, are generally associated with an improved dot pattern. In some embodiments, the FWHM of the nanosphere dots may be about 10 μm or smaller.

[0226] In some embodiments, the nucleic acid template molecule is fixed. In some embodiments, the sequencing primers (i.e., any one of the first plurality of forward sequencing primers, the second plurality of forward sequencing primers, and / or the third plurality of forward sequencing primers) are soluble (i.e., in solution).

[0227] In some embodiments, step (c) includes hybridizing a plurality of preserved nucleic acid template molecules with a plurality of sequencing primers in the presence of primer extension polymerase, a plurality of nucleotides, and a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant not greater than 115 and present in the hybridization buffer formulation in an amount capable of effectively denaturing double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25% to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5% to 10% by volume of the hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a congestant comprising polyethylene glycol (PEG) at 5% to 35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0228] In some embodiments, step (c) includes: (i) removing a plurality of first forward sequencing reads and / or a plurality of second forward sequencing reads while retaining the nucleic acid template molecule, and / or removing a plurality of capped first sequencing reads and a plurality of capped second sequencing reads while retaining the nucleic acid template molecule; and (ii) contacting the plurality of nucleic acid template molecules with a plurality of amplification primers, a plurality of nucleotides, and a plurality of primer extension polymerases under conditions suitable for hybridizing the plurality of amplification primers with the plurality of nucleic acid template molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, wherein the amplification primers hybridize with an amplification primer-binding sequence located in the nucleic acid template molecule. In some embodiments, the retained nucleic acid template molecule is immobilized to a support. In some embodiments, the amplification primers are soluble (i.e., in solution). The primer extension reaction may optionally include a plurality of compressed oligonucleotides and / or hexamines (e.g., hexaminecobalaminIII) ​​to generate forward extension strands. Individual forward-extended strands can collapse into nanospheres with a more compact size and / or shape compared to nanospheres generated from primer extension reactions performed without compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII). Including compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) in the primer extension reaction improves the FWHM (full width at half maximum) of the nanosphere's dot pattern. The dot pattern can be represented as Gaussian points, and the size can be measured in terms of FWHM. Smaller dot sizes, as indicated by a smaller FWHM, are generally associated with an improved dot pattern. In some embodiments, the FWHM of the nanosphere dots can be about 10 μm or smaller.

[0229] In some embodiments, step (c) includes hybridizing a plurality of preserved nucleic acid template molecules with a plurality of amplification primers in the presence of primer extension polymerase, multiple nucleotides, and a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant not greater than 115 and present in the hybridization buffer formulation in an amount capable of effectively denaturing double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25% to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5% to 10% by volume of the hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a congestant comprising polyethylene glycol (PEG) at 5% to 35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0230] In some embodiments, step (c) includes: (i) contacting multiple nucleic acid template molecules (e.g., preserved by immobilization as described above) with multiple soluble primers, multiple nucleotides, and multiple strand displacement polymerases under conditions suitable for hybridizing multiple soluble upstream primers with multiple nucleic acid template molecules, the multiple soluble primers hybridizing with a region upstream of at least one of the capped sequencing reads hybridizing with the nucleic acid template molecules; and (ii) performing a strand displacement reaction catalyzed by one or more polymerases, thereby generating multiple forward extension chains. The strand displacement polymerase may displace the downstream capped sequencing reads. The nucleic acid template molecules may be preserved. The nucleic acid template molecules may be immobilized. The strand displacement reaction may optionally include multiple compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) to generate forward extension chains. The forward extension chains may collapse into nanospheres having a more compact size and / or shape compared to nanospheres generated from a strand displacement reaction performed without compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII). Including compressed oligonucleotides and / or hexamines (e.g., hexaminecobaltIII) in the chain substitution reaction can improve the FWHM (full width at half maximum) of the spot image of the nanospheres. The spot image can be represented as Gaussian points, and the size can be measured in terms of FWHM. Smaller spot sizes, as indicated by a smaller FWHM, are generally associated with an improved image of the points. In some embodiments, the FWHM of the nanosphere points can be about 10 μm or smaller.

[0231] In some embodiments, step (c) includes contacting a plurality of retained nucleic acid template molecules with a plurality of soluble upstream primers in the presence of a strand displacement polymerase, a plurality of nucleotides, and a high-efficiency hybridization buffer. The nucleic acid template molecules may be retained. In some embodiments, the nucleic acid template molecules are immobilized. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant not greater than 115 and present in the hybridization buffer formulation in an amount sufficient to effectively denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent, in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25% to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5% to 10% by volume of the hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a congestant comprising polyethylene glycol (PEG) at 5% to 35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0232] In some embodiments, the primer extension polymerase in step (c) comprises a high-fidelity polymerase. In some embodiments, the primer extension polymerase in step (c) comprises a DNA polymerase (e.g., a uracil-resistant polymerase) capable of catalyzing a primer extension reaction using a template molecule containing uracil. The polymerases listed include, but are not limited to: Q5U® Hot Start high-fidelity DNA polymerase (e.g., catalog number M0515S from New England Biolabs®), Taq DNA polymerase, One Taq DNA polymerase (e.g., a mixture of Taq and Deep Vent® DNA polymerase, catalog number M0480S from New England Biolabs), LongAmp® Taq DNA polymerase (e.g., catalog number M0323S from New England Biolabs), Epimark® Hot Start Taq DNA polymerase (e.g., catalog number M0490S from New England Biolabs), Bst DNA polymerase (e.g., large fragment, catalog number M0275S from New England Biolabs), Bsu DNA polymerase (e.g., large fragment, catalog number M0330S from New England Biolabs), Phi29 DNA polymerase (e.g., catalog number M0269S from New England Biolabs), and E. coli DNA polymerase (e.g., from New England Biolabs®). Biolabs catalog number M0209S), Therminator TM DNA polymerases (e.g., catalog number M0261S from New England Biolabs), Vent® DNA polymerase, and Deep Vent® DNA polymerase.

[0233] In some embodiments, the pairwise sequencing method includes step (d): removing the plurality of nucleic acid template molecules retained in step (c) by generating a base-free site in the nucleic acid template molecule at the nucleotide with a cleavable portion and creating a gap at the base-free site, to generate a plurality of nucleic acid template molecules with gaps, while retaining a plurality of forward extension strands and a plurality of fixed surface primers (see, for example...). Figure 16E In some embodiments, the nucleic acid template molecule is fixed. In some embodiments, the template molecule is single-stranded.

[0234] Abase-free sites are generated on a nucleic acid template molecule containing a nucleotide with a cleavable moiety, which may be single-stranded. In some embodiments, the cleavable moiety present in the nucleic acid template molecule comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. Abase-free sites can be removed to generate multiple single-stranded nucleic acid template molecules with gaps, while retaining multiple forward-extending strands. Abase-free sites can be generated by contacting multiple nucleic acid template molecules with an enzyme that removes nucleosides from the nucleotide with the cleavable moiety. Uracil DNA glycosylase (UDG) can be used to convert uracil in the nucleic acid template strand to abase-free sites. FPG glycosylase can be used to convert 8oxoG in the nucleic acid template strand to abase-free sites. AlkA glycosylase can be used to convert deoxyinosine in the nucleic acid template strand to abase-free sites.

[0235] In some embodiments of step (d), the gap can be created by contacting the baseless site in the nucleic acid template molecule with an enzyme or mixture of enzymes having lysin activity that breaks the phosphodiester backbone at the 5' and 3' sides of the baseless site, thereby releasing the baseless deoxyribose and creating the gap (see, for example...). Figure 16E The removal of abase-free sites can be achieved using, for example, AP lyase, Endo IV endonuclease, FPG glycosylation enzyme / AP lyase, or Endo VIII glycosylation enzyme / AP lyase. In some embodiments, a mixture of uracil DNA glycosylation enzyme and DNA glycosylation enzyme-lyase endonuclease VIII (e.g., USER (uracil-specific excision reagent enzyme from New England Biotech Ltd.) or thermosensitive USER (also from New England Biotech Ltd.)) can be used to generate and remove abase-free sites to create gaps.

[0236] In some embodiments of step (d), multiple spaced nucleic acid template molecules can be removed from immobilized surface primers using enzymes, chemicals, and / or heat. Following the space removal procedure, the forward extension strand is retained, thereby producing multiple retained forward extension strands (e.g., see...). Figure 16F And it hybridizes with fixed surface primers.

[0237] For example, a 5' to 3' double-stranded DNA exonuclease (such as, for example, T7 exonuclease (e.g., from New England Biolabs, catalog number M0263S) or λ exonuclease (e.g., from New England Biolabs, catalog number M0262S)) can be used to enzymatically degrade multiple spaced nucleic acid template molecules. When the 5' to 3' double-stranded DNA exonuclease is used to remove spaced nucleic acid template molecules from immobilized surface primers, each of the multiple soluble amplification primers in step (c) may contain at least one phosphate thioester diester bond at its 5' end, which makes the soluble amplification primer resistant to exonuclease-induced degradation. In some embodiments, the soluble amplification primers in step (c) contain 2 to 5 or more consecutive phosphate thioester diester bonds at their 5' ends. In some embodiments, the soluble amplification primers in step (c) contain at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which makes the forward sequencing primers resistant to exonuclease degradation.

[0238] In some embodiments, chemical reagents that facilitate nucleic acid denaturation can be used to remove multiple spaced template molecules from surface primers immobilized to a support. Denaturing reagents may include formamide, acetonitrile, guanidine chloride, and / or buffers (e.g., Tris-HCl, MES, HEPES, etc.) or combinations thereof.

[0239] In some embodiments, the plurality of spaced template molecules may be removed using elevated temperatures (e.g., heat) with or without nucleic acid denaturing reagents. The spaced template molecules may be subjected to temperatures of about 45°C to 50°C, or about 50°C to 60°C, or about 60°C to 70°C, or about 70°C to 80°C, or about 80°C to 90°C, or about 90°C to 95°C or higher.

[0240] In some embodiments, formamide (e.g., 40 to 100% formamide) can be used at a temperature of about 65°C for about 3 minutes, and the mixture can be washed with a reagent containing about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5 to remove multiple interstitial template molecules.

[0241] In some embodiments, the pairwise sequencing method includes step (e): sequencing multiple retained forward extension strands, thereby generating multiple first reverse sequencing reads. In some embodiments, the sequencing in step (e) includes contacting the multiple retained forward extension strands with multiple soluble reverse sequencing primers under conditions suitable for hybridizing the reverse sequencing primers with reverse sequencing primer binding sites located on the retained forward extension strands, and subsequently performing a sequencing reaction, wherein the reverse sequencing reaction generates multiple first reverse sequencing reads (see, for example...). Figure 16GThe first reverse sequencing read product can hybridize with the retained forward extension strand. The retained forward extension strand can hybridize with the first surface primer. In some embodiments, the first reverse sequencing read product does not hybridize with the first surface primer, nor is it covalently linked to the first surface primer. Therefore, in such embodiments, the first reverse sequencing read product is not fixed to a support.

[0242] For the sake of simplicity, Figures 16F to 16G The diagram illustrates illustrative retained forward extension strands, each containing two copies of the target sequence and various universal primer binding sites. Those skilled in the art will understand that retained forward extension strands may comprise two or more tandem copies containing the target sequence and various universal primer binding sites. Therefore, reverse sequencing reactions can produce multiple first reverse sequencing reads that hybridize with the same retained forward extension strand.

[0243] In some embodiments, step (e) includes contacting a plurality of soluble reverse sequencing primers and a plurality of retained forward extension strands with a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant not greater than 40 and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant not greater than 115 and present in the hybridization buffer formulation in an amount capable of effectively denaturing double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25% to 50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5% to 10% by volume of the hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) a congestant comprising polyethylene glycol (PEG) at 5% to 35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0244] In an alternative embodiment of step (e), surface primers fixed to a support serve as sequencing primers, and step (e) includes performing a sequencing reaction to produce a plurality of first reverse sequencing reads.

[0245] In some embodiments, the reverse sequencing reaction in step (e) includes contacting a plurality of soluble reverse sequencing primers with a reverse sequencing primer binding site present on each of the retained forward extension strands, one or more types of sequencing polymerases, and a plurality of nucleotides or a plurality of multivalent molecules. In some embodiments, the soluble reverse sequencing primers contain a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primers contain a 3' blocking portion that can be removed to produce a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primers lack nucleotides with easily cleavable portions. Sequencing reactions employing nucleotides and / or multivalent molecules are described in more detail below. Reverse sequencing reactions can produce a plurality of first reverse sequencing reads. In some embodiments, the retained forward extension strand contains a plurality of copies of the reverse sequencing primer binding site, wherein each reverse sequencing primer binding site is capable of hybridizing with a reverse sequencing primer. The reverse sequencing primer binding site can hybridize with a reverse sequencing primer and can undergo a sequencing reaction. Thus, each retained forward extension strand may undergo two or more sequencing reactions, wherein each sequencing reaction begins with a reverse sequencing primer hybridizing with the reverse sequencing primer binding site (e.g., see Figure 16G In some embodiments, the sequencing reaction includes a plurality of nucleotides (or the like) labeled with a detectable reporter portion. In some embodiments, the sequencing reaction includes a plurality of multivalent molecules having nucleotide units, wherein the multivalent molecules are labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion comprises a fluorophore.

[0246] In some embodiments, at least one washing step may be performed after any of steps (a) to (e). The washing step may be performed using a washing buffer comprising a pH buffer, a metal chelating agent, a salt, and / or a detergent.

[0247] In some embodiments, the pH buffering compound in the wash buffer is Tris, Tris-HCl, Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (also known as 2-aminomethanol; MEA), citrate compounds, citrate mixtures, NaOH, KOH, or any combination thereof. In some embodiments, the pH buffer may be present in the wash buffer at a concentration of about 1 mM to 100 mM, or about 10 mM to 50 mM, or about 10 mM to 25 mM. In some embodiments, the pH of the pH buffer present in any of the reagents described herein may be adjusted to a pH of about 4 to 9, or about 5 to 9, or about 5 to 8.

[0248] In some embodiments, the metal chelating agent in the washing buffer includes: EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylenetriaminepentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, diammonium citrate, citric acid, potassium citrate, magnesium citrate, or any combination thereof. In some embodiments, the washing buffer contains a chelating agent at a concentration of about 0.01 mM to 50 mM, or about 0.1 mM to 20 mM, or about 0.2 mM to 10 mM.

[0249] In some embodiments, the salt in the wash buffer includes NaCl, KCl, NH₂SO₄, potassium glutamate, or any combination thereof. In some embodiments, the detergent includes an ionic detergent, such as SDS (sodium dodecyl sulfate). The wash buffer may contain a monovalent salt at a concentration of about 25 mM to 500 mM, or about 50 mM to 250 mM, or about 100 mM to 200 mM.

[0250] In some embodiments, the detergent in the wash buffer comprises a nonionic detergent, such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent comprises a zwitterionic detergent, such as CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate) or N-dodecyl-N,N-dimethyl-3-ammonium-1-propane sulfate (DetX). In some embodiments, the detergent comprises: LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is present in the wash buffer at a concentration of about 0.01% to 0.05%, or about 0.05% to 0.1%, or about 0.1% to 0.15%, or about 0.15% to 0.2%, or about 0.2% to 0.25%.

[0251] This disclosure provides read-capped nucleotide analogs that can be used in any of the methods described herein, including any of the methods for sequencing two or more regions of a nucleic acid template molecule and any of the pairwise sequencing methods.

[0252] In some embodiments, the capped nucleotide analogue comprises: (i) a heterocyclic base, (ii) a ribose, and (iii) a polyphosphate chain, wherein the heterocyclic base is linked to a termination portion (e.g., a base-linked chain termination portion). In some embodiments, in a polymerase-catalyzed primer extension reaction, the capped nucleotide analogue may be incorporated into the 3' end of a primer or into the 3' end of a nascent primer to produce a capped primer product.

[0253] In some embodiments of the methods described herein, once incorporated, the read-capped nucleotide analog can block polymerase-mediated binding of the nucleotide reagent to the 3' end of the read-capped primer product, wherein the nucleotide reagent comprises a multivalent molecule. In some embodiments, the multivalent molecule comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. In some embodiments, the multivalent molecule comprises (1) a core; and (2) a plurality of nucleotide arms, each arm comprising (i) a core attachment portion and (ii) a spacer comprising (a) a linker and (b) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (e.g., see [reference]). Figures 19 to 23 ).

[0254] In some embodiments, the incorporated read-capped nucleotide analog can block the polymerase-catalyzed incorporation of a nucleotide reagent into the 3' end of the read-capped primer product, wherein the nucleotide reagent is a typical nucleotide or a chain-terminating nucleotide.

[0255] In some embodiments of the methods described herein, the capped nucleotide analog comprises: (i) a heterocyclic base, (ii) a ribose, and (iii) at least one phosphate ester group, wherein the heterocyclic base is linked to a termination portion. In some embodiments, the termination portion is linked to the heterocyclic base via a non-cleavable linker. In some embodiments, the termination portion is linked to the heterocyclic base via a cleavable linker. In some embodiments, the capped nucleotide analog may be unlabeled. In some embodiments, the capped nucleotide analog may be labeled with a detectable reporter portion. Figures 8A to 8J and Figure 9 Illustrative readings of capped nucleotide analogs are shown.

[0256] In some embodiments of the methods described herein, the read-capped nucleotide analog comprises a heterocyclic base comprising a substituted nitrogen-containing parent heteroaromatic ring. In some embodiments, the substituted nitrogen-containing parent heteroaromatic ring is a naturally occurring, substituted, modified, or engineered variant. In some embodiments, the base of the read-capped nucleotide analog is capable of forming Watson-Crick and / or Husstan hydrogen bonds with suitable complementary bases. Illustrative bases that can be used in read-capped nucleotide analogs include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine; 2,6-diaminopurine; adenine (A); ethylene adenine; N 6 -Δ 2 -Isopentenyladenine (6iA); N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA); N 6 -Methyladenine; Guanine (G); Isoguanine; N 2 -Dimethylguanine (dmG); 7-methylguanine (7mG); 2-thiopyrimidine; 6-thioguanine (6sG); hypoxanthine and O 6 -Methylguanine; 7-denitro-purine, such as 7-denitro-adenine (7-denitro-A) and 7-denitro-guanine (7-denitro-G); pyrimidine, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indole, such as nitroindole and 4-methylindole; pyrrole, such as nitropyrrole; muscarin; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated, and acylated base moieties; etc. Further illustrative bases can be found in Fasman, 1989, in “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385–394, CRC Press, Boca Raton, Fla.

[0257] In some embodiments of the methods described herein, the read-capped nucleotide analog includes a base-linked termination portion connected to a nucleobase via an alkyl bond. In some embodiments, the bond comprises an alkyl group. In some embodiments, the bond comprises an alkenyl group. In some embodiments, the bond comprises an alkynyl group.

[0258] In some embodiments, the terminating portion of the base link comprises a polymer portion. In some embodiments, the terminating portion of the base link comprises a polyether portion. In some embodiments, the terminating portion of the base link comprises polyethylene glycol. In some embodiments, the terminating portion of the base link comprises polypropylene glycol. In some embodiments, the terminating portion of the base link comprises polyvinyl acetate. In some embodiments, the terminating portion of the base link comprises polylactic acid. In some embodiments, the terminating portion of the base link comprises polyglycolic acid. In some embodiments, the terminating portion of the base link comprises a polyamide portion. In some embodiments, the terminating portion of the base link comprises a polyester portion. In some embodiments, the terminating portion of the base link comprises a poly(glycerol) portion. In some embodiments, the terminating portion of the base link comprises a poly(oxazoline) portion. In some embodiments, the terminating portion of the base link comprises a poly(hydroxypropyl methacrylate) portion (PHPMA). In some embodiments, the terminating portion of the base link comprises a poly(2-hydroxyethyl methacrylate) portion (PHEMA). In some embodiments, the terminating portion of the base link comprises a poly(N-(2-hydroxypropyl)methacrylamide) portion (HPMA). In some embodiments, the terminating portion of the base link comprises a poly(vinylpyrrolidone) portion (PVP). In some embodiments, the terminating portion of the base linkage comprises a poly(N,N-dimethylacrylamide) moiety (PDMA). In some embodiments, the terminating portion of the base linkage comprises a poly(N-acryloylmorpholine) moiety (PAcM).

[0259] In some embodiments, the terminating portion of the base linkage comprises a synthetic zwitterionic portion, such as a poly(carboxybetaine acrylamide) portion, a poly(carboxybetaine methacrylate) portion, a poly(sulfobetaine methacrylate) portion, or a poly(methacryloyloxyethylphosphorylcholine) portion.

[0260] In some embodiments, the terminating portion of the base link comprises a polyglutamic acid portion. In some embodiments, the terminating portion of the base link comprises a polyaspartic acid portion. In some embodiments, the terminating portion of the base link comprises a polylysine portion. In some embodiments, the terminating portion of the base link comprises a polyethyleneimine portion. In some embodiments, the terminating portion of the base link comprises a polysialic acid portion.

[0261] In some embodiments, the terminating portion of the base link includes a connector, such as an 11-atom connector, a 16-atom connector, a 23-atom connector, or an N3 connector, examples of which are shown in Figure 24 In some embodiments, the termination portion of the base linkage comprises a polymer connector, such as... Figure 25 Any of the connectors 1 to 9 shown.

[0262] In some embodiments, the terminating portion of the base linkage comprises an acetylated amino moiety, an allylamino moiety, a propylamino moiety, an ethyl mercapto moiety, a hydroxymethyl moiety, or an aryl mercapto moiety (e.g., see [link to documentation]). Figures 8A to 8J In some embodiments, the terminating portion of the base linkage comprises a 1-X-1H-1,2,3-triazol-4-yl moiety, a 5-X-1H-1,2,3-triazol-1-methyl moiety, a hydrazone moiety, or an O-alkyl oxime moiety (e.g., see [link to relevant documentation]). Figures 8A to 8J ).

[0263] In some embodiments, the read-capped nucleotide analog comprises Figures 8A to 8J Any of the nucleotide analogs shown, where “X” includes a polymer, such as any of the polymers mentioned above, for example.

[0264] In some embodiments of the methods described herein, the terminating portion of the base linkage comprises a hydrophilic polymer of ethylene oxide. In some embodiments, the terminating portion of the base linkage comprises a polyethylene glycol (PEG) portion (e.g., H-(OCH2CH2)). n -OH, where n can be 1, or at least 2, or at least 5, or at least 10, at least 20, at least 25, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1000, or 2000 or greater. In some embodiments, the PEG portion has a molecular weight of about 100-200 Da, 200-300 Da, 300-400 Da, 400-500 Da, 1K Da, 2K Da, 3K Da, 4K Da, 5K Da, 10K Da, 15 K Da, 20K Da, 30K Da, 40K Da, 50K Da, or greater. In some embodiments, the value is [value missing]. In some embodiments, the PEG portion comprises a linear PEG molecule. In some embodiments, the PEG portion comprises a branched PEG molecule. In some embodiments, the branched PEG portion molecule comprises 4 to 8 branches. In some embodiments, the terminating portion comprises an aromatic group having a six-carbon ring structure. In some embodiments, the PEG moiety can be functionalized with methoxy (OCH3), amino (NH2), carboxyl (COOH), and / or hydroxy (OH) groups. For example, the base-linked termination portion comprises polyethylene glycol methyl ether (mPEG) (e.g., CH3(OCH2CH2)). n -OH).

[0265] In some embodiments, the read-capped nucleotide analog comprises Figures 8A to 8J Any of the nucleotide analogues shown, wherein “X” includes the PEG portion or a derivative thereof, such as any of the PEG portions described above, for example.

[0266] This document also provides a set of read-capped nucleotides that can be used in the methods described herein. In some embodiments, the set of read-capped nucleotide analogs comprises four types of read-capped nucleotide analogs, including dATP, dGTP, dCTP, and dTTP / dUTP, wherein the nucleobases of each type of read-capped nucleotide analog are linked to the same type of termination portion. For example, the same type of PEG portion is linked to the nucleobases of the four types of read-capped nucleotide analogs dATP, dGTP, dCTP, and dTTP / dUTP in the set of read-capped nucleotide analogs.

[0267] In some embodiments, a set of capped nucleotide analogs comprises four types of capped nucleotide analogs, including dATP, dGTP, dCTP, and dTTP / dUTP, wherein the nucleobase of at least one type of capped nucleotide analog is linked to a termination portion different from that of at least one of the other three capped nucleotide analogs. For example, in such embodiments, at least one type of capped nucleotide analog (e.g., dATP) comprises a nucleobase linked to a PEG portion that is different from the PEG portions of other capped nucleotide analogs (e.g., dGTP, dCTP, and cTTP / dUTP). In some embodiments, the length of the PEG portion may vary. In some embodiments, the molecular weight of the PEG portion may vary. In some embodiments, the nucleobase may be linked to the PEG portion via different bonds (e.g., different alkyl, alkenyl, or alkynyl bonds). In some embodiments, the nucleobase in each of the four types of capped nucleotide analogs may be linked to a PEG portion with different functional groups, including methoxy groups (OCH3), amino groups (NH2), carboxyl groups (COOH), and / or hydroxy groups (OH).

[0268] In some embodiments of the methods described herein, the read-capped nucleotide analogue comprises a sugar moiety, such as a cyclic moiety (see Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48, which is incorporated herein by reference to illustrate examples of cyclic moieties that may exist in the read-capped nucleotide analogues described herein), an acyclic moiety (see Martinez et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7: 3013-3016, each incorporated herein by reference to illustrate examples of acyclic moieties that may exist in the read-capped nucleotide analogues described herein), or another sugar moiety (see Joeng et al., 1993 J. Med. Chem. 36: 2627-2638; Kim et al., 1993 J. Med. Chem. 36: 2627-2638). 30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Patent No. 5,558,991, each incorporated herein by reference for examples of sugar moieties that may be present in the capped nucleotide analogues described herein. The sugar moiety can be: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0269] In some embodiments of the methods described herein, the capped nucleotide analog contains a hydroxyl group at the 3' sugar position. In some embodiments, the capped nucleotide analog contains a chain termination portion at the 3' sugar position, wherein the chain termination portion contains an H, F, NH2, or an amino group.

[0270] In some embodiments, the capped nucleotide analog comprises a sugar moiety containing a 3'OH group. In some embodiments, the capped nucleotide analog comprises a chain termination portion at the 2' or 3' sugar position. In some embodiments, the chain termination portion may be removed from or cleaved from the 3' sugar position to produce a nucleotide having a 3'OH sugar group, which may be extended with subsequent nucleotides in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain termination portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain termination portion may be cleaved or removed from the nucleotide, for example, by reacting the chain termination portion with a chemical agent, by a pH change, by light, or by heat. In some embodiments, chain-terminating portions comprising alkyl, alkenyl, alkynyl, or allyl groups may be cleaved with tetra(triphenylphosphine)-palladium(O) (Pd(PPh3)4) using piperidine or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, chain-terminating portions comprising aryl or benzyl groups may be cleaved with H2 Pd / C. In some embodiments, chain-terminating portions comprising amines, amides, ketones, isocyanates, phosphates, thioides, or disulfides may be cleaved with phosphine or with thiol groups, including β-mercaptoethanol or dithiothreitol (DTT). In some embodiments, chain-terminating portions comprising carbonates may be cleaved with potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, chain-terminating portions comprising urea or silyl groups may be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride. In some embodiments, chain-terminating portions may be cleaved or removed with nitrous acid. In some embodiments, the chain termination portion can be cleaved or removed using a solution comprising nitrite (e.g., a combination of nitrite and an acid such as acetic acid, sulfuric acid, or nitric acid). In some other embodiments, the solution may include an organic acid.

[0271] In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog comprises an azide, an azide group, or an azidemethyl group. In some embodiments, the chain termination portion comprises a 3'-O-azido or a 3'-O-azidomethyl group. In some embodiments, the chain termination portion comprising an azide, an azide group, and an azidemethyl group can be cleaved or removed using a phosphine compound. In some embodiments, the phosphine compound comprises a derived trialkylphosphine moiety or a derived triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), bis(sulfotriphenyl)phosphine (BS-TPP), or tris(hydroxypropyl)phosphine (THPP). In some embodiments, the cleavage agent comprises 4-dimethylaminopyridine (4-DMAP).

[0272] In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog contains a 3'-O-amino group or a derivative thereof, which can be cleaved with nitrous acid, by a mechanism utilizing nitrous acid, or using a solution containing nitrous acid. In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog contains a 3'-O-aminomethyl group or a derivative thereof, which can be cleaved with nitrous acid, by a mechanism utilizing nitrous acid, or using a solution containing nitrous acid. In some embodiments, the 2' or 3' chain termination portion of the capped nucleotide analog contains a 3'-O-methylamino group or a derivative thereof, which can be cleaved with nitrous acid, by a mechanism utilizing nitrous acid, or using a solution containing nitrous acid.

[0273] In some embodiments, the chain-terminating portion comprises a 3'-O-amino group or a derivative thereof, which can be cleaved using a solution containing nitrite. In some embodiments, the chain-terminating portion comprises a 3'-O-aminomethyl group or a derivative thereof, which can be cleaved using a solution containing nitrite. In some embodiments, the chain-terminating portion comprises a 3'-O-methylamino group or a derivative thereof, which can be cleaved using a solution containing nitrite. In some embodiments, for example, the nitrite may be combined with an acid (such as acetic acid, sulfuric acid, or nitric acid). In some other embodiments, for example, the nitrite may be combined with an organic acid (such as, for example, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, etc.).

[0274] In some embodiments of any of the methods described herein, the read-capped nucleotide analog comprises a 3'-deoxynucleotide, a 2',3'-dideoxynucleotide, or a nucleotide comprising one of the following 3' modifications: 3'-methyl, 3'-azido, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-mercapto, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethoxycarbonyl, 3'-tert-butoxycarbonyl, 3'-O-alkylhydroxyamino, 3'-thiophosphate, and 3-O-benzyl or derivatives thereof.

[0275] In some embodiments of the methods described herein, the read-capped nucleotide analog comprises a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, the nucleotide analog comprises a phosphorus chain, wherein the phosphorus atoms are linked together by an intermediate O, S, NH, methylene or ethylene group. In some embodiments, the phosphorus atoms in the phosphorus chain comprise substituted side groups, such as O, S or BH3. In some embodiments, the phosphorus chain comprises analog-substituted phosphate ester groups, such as phosphoramide, thiophosphate, dithiophosphate and O-methylphosphoramide groups.

[0276] In some embodiments of the methods described herein (including any of the methods for sequencing two or more regions of a nucleic acid template molecule and any of the pairwise sequencing methods), the sequencing primers comprise oligonucleotides capable of hybridizing with DNA and / or RNA template molecules to form double-stranded molecules. Sequencing primers may comprise natural nucleotides and / or nucleotide analogs. Sequencing primers may comprise recombinant nucleic acid molecules or synthetic nucleic acid molecules. Sequencing primers may have any length, but are typically in the range of 4-50 nucleotides. In some embodiments, the 3' end of the sequencing primer comprises an extendable 3' OH portion that acts as the initiation site for nucleotide polymerization in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of the sequencing primer may lack a 3' OH portion, or may comprise a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any nucleotide or more than one nucleotide of the length of the sequencing primer may be labeled with a detectable reporter portion. Sequencing primers may be in solution (e.g., soluble primers).

[0277] In some embodiments of the methods described herein, the primer hybridization step may be performed using a hybridization reagent comprising at least one aqueous solvent, a pH buffer, a monovalent cation, and a dissociating agent. In some embodiments, the hybridization reagent optionally comprises any one or any combination of two or more of the following: detergents, reducing agents, chelating agents, alcohols, zwitterions, sugar alcohols, and / or crowding agents. In some embodiments, the aqueous solvent comprises water. In some embodiments, the pH buffer comprises NaCl. In some embodiments, the dissociating agent comprises SDS (sodium dodecyl sulfate), urea, thiourea, guanidine chloride, guanidine hydrochloride, guanidine thiocyanate, guanidine isosulfate, potassium thiocyanate, lithium chloride, sodium iodide, or sodium perchlorate.

[0278] In some embodiments, the primer hybridization step may be performed at a temperature of about 20°C to 25°C, or about 25°C to 35°C, or about 35°C to 45°C, or about 45°C to 55°C, or about 55°C to 65°C, or about 65°C to 75°C or higher. In some embodiments, primer hybridization may be performed for about 10 to 60 seconds, about 1 to 3 minutes, about 3 to 6 minutes, about 6 to 9 minutes, about 9 to 12 minutes or longer.

[0279] In some embodiments, the primer hybridization step can be performed using a "step-down" temperature scheme at several different temperatures. For example, the first primer hybridization can be performed at approximately 65°C to 45°C. The second primer hybridization can be performed at approximately 25°C to 45°C. The third primer hybridization can be performed at approximately 30°C to 15°C. In some embodiments, each of the primer hybridization steps in the step-down scheme can be performed for the same or different durations. For example, any of the primer hybridization steps can be performed for approximately 10 to 60 seconds, approximately 1 to 3 minutes, approximately 3 to 6 minutes, approximately 6 to 9 minutes, approximately 9 to 12 minutes, or longer.

[0280] In some embodiments, the nucleic acid template molecule is a single-stranded nucleic acid template molecule. In some embodiments, the method includes sequencing at least one single-stranded nucleic acid template molecule.

[0281] In some embodiments of the methods described herein, at least one portion of a single-stranded nucleic acid template molecule is fixed to a support or to a coating on a support. In some embodiments, the 5' end of the template molecule is fixed to a support. In some embodiments, the 3' end of the template molecule is fixed to a support. In some embodiments, an internal portion of the template molecule is fixed to a support. In some embodiments, one or more of the 5' end, 3' end, and / or internal portion of the template molecule are fixed to a support.

[0282] In some embodiments, the 5' end of at least one single-stranded nucleic acid template molecule is covalently attached to a surface primer, which is immobilized to a support or a coating on the support. In some embodiments, the 5' end of the at least one single-stranded nucleic acid template molecule hybridizes with a surface primer, which is immobilized to a support or a coating on the support. In some embodiments, in any of the sequencing methods described herein, the 3' end of the at least one single-stranded nucleic acid template molecule is covalently attached to a surface primer, which is immobilized to a support or a coating on the support. In some embodiments, the 3' end of the at least one single-stranded nucleic acid template molecule hybridizes with a surface primer, which is immobilized to a support or a coating on the support.

[0283] In some embodiments, the at least one single-stranded nucleic acid template molecule may be generated via a cloning amplification workflow. In some embodiments, the at least one single-stranded nucleic acid template molecule may not be generated via a cloning amplification workflow.

[0284] In some embodiments, the at least one single-stranded nucleic acid template molecule comprises at least one uridine nucleotide. In some embodiments, in any of the sequencing methods described herein, the at least one single-stranded nucleic acid template molecule does not contain a uridine nucleotide.

[0285] In some embodiments, at least one single-stranded nucleic acid template molecule contains one copy of the target sequence (“single-copy template molecule”). For example, a single-copy template molecule can be generated by bridge amplification. In some embodiments, the at least one single-stranded nucleic acid template molecule contains two or more tandem copies of the target sequence. For example, the single-stranded nucleic acid template molecule contains about 2, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 700, 900, 1000, 1500, or 2000 copies of the target sequence, or any range thereof. For example, a tandem copy template molecule contains multiples that can be generated by rolling circle amplification (RCA).

[0286] In some embodiments, at least one single-stranded nucleic acid template molecule contains at least one target sequence (e.g., an insertion region). In some embodiments, at least one single-stranded nucleic acid template molecule contains at least one universal adaptor sequence, including any one or any combination of the following: a first surface primer binding site (e.g., a capture primer binding site); a second surface primer binding site (e.g., a surface pinning primer binding site); a first sequencing primer binding site (e.g., a forward sequencing primer binding site); a second sequencing primer binding site (e.g., a reverse sequencing primer binding site); a first amplification primer binding site (e.g., a forward amplification primer binding site); a second amplification primer binding site (e.g., a reverse amplification primer binding site); a first sample index sequence; a second sample index sequence; a first unique molecular tag sequence; a second unique molecular tag sequence; a first compressed oligonucleotide binding site; and / or a second compressed oligonucleotide binding site.

[0287] In some embodiments of the methods described herein, the sequencing read product comprises a sequencing primer (e.g., a nucleic acid oligonucleotide) linked to a polynucleotide having a sequence complementary to at least a portion of a single-stranded nucleic acid template molecule. At least a portion of the complementary polynucleotide can be generated by any type of sequencing workflow comprising a polymerase-catalyzed primer extension reaction in which the sequence of the single-stranded template molecule is determined. At least a portion of the complementary polynucleotide can be generated by any type of polymerase-catalyzed primer extension reaction in which the sequence of the single-stranded template molecule is not determined. In some embodiments, the sequencing primer portion of the sequencing read product has an extendable 3' end. In some embodiments, the sequencing primer portion of the sequencing read product has a non-extendable 3' end. Various methods for polymerase-catalyzed sequencing are described below.

[0288] In some embodiments of the methods described herein, the support is a planar support (e.g., a glass slide). In some embodiments, the support is a non-planar support (e.g., beads). In some embodiments, the support comprises or is composed of a solid or material. In some embodiments, the support comprises or is composed of a semi-solid material. In some embodiments, the support comprises or is composed of a porous material. In some embodiments, the support comprises or is composed of a semi-porous material. In some embodiments, the support comprises or is composed of a non-porous material. The support may be made of any suitable material known in the art or described herein, such as glass, plastic, or polymeric materials.

[0289] In some embodiments, the surface of the support may be coated with one or more compounds to create a passivation layer on the support. In some embodiments, the passivation layer forms a porous layer. In some embodiments, the passivation layer forms a semi-porous layer. In some embodiments, surface primers or single-stranded nucleic acid template molecules may be attached to the passivation layer to immobilize the primers or template molecules to the support. In some embodiments, the support includes a low-nonspecific binding surface that enables improved nucleic acid hybridization, amplification, and sequencing performance on the support. Generally, the support may include one or more layers of covalently or non-covalently attached low-binding chemically modified layers (e.g., silane layers, polymer membranes), and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize multiple nucleic acid template molecules to the support. In some embodiments, the support includes a functionalized polymer coating at least covalently bound to a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of hydrophilic polymer coating and at least one layer of multiple oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise branched PEG having at least four branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity, which can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees.

[0290] In some embodiments, the support comprises a plurality of nucleic acid template molecules fixed to the support or to a coating thereof. In some embodiments, about 10 2 Up to 10 15 Nucleic acid template molecules are immobilized to a support, wherein the nucleic acid template molecules are immobilized to different sites on the support. In some embodiments, multiple nucleic acid template molecules are immobilized to predetermined sites (e.g., positions) on the support. In some embodiments, multiple nucleic acid template molecules are immobilized to random sites (e.g., positions) on the support. In some embodiments, the multiple nucleic acid template molecules immobilized to the support are fluidly connected to each other to allow reagent solutions (e.g., enzymes, including polymerases, multivalent molecules, nucleotides, and / or divalent cations, etc.) to flow onto the support, such that the multiple nucleic acid template molecules on the support can react with the reagent solution in a large-scale parallel manner. In some embodiments, the nucleic acid template molecules are single-stranded.

[0291] In some embodiments of the methods described herein, at least one nucleic acid template molecule comprises a target sequence (e.g., an insertion region) side-attached to both sides by at least one universal adaptor sequence. An illustrative nucleic acid template molecule may comprise (ordered from 3' to 5'; see [link to documentation]). Figure 1 The template molecule may include: a first surface primer binding site (e.g., a capture primer binding site); a first sample index sequence; a second sequencing primer binding site (e.g., a reverse sequencing primer binding site); a target sequence; a first sequencing primer binding site (e.g., a forward sequencing primer binding site); a second sample index sequence; and a second surface primer binding site (e.g., a surface pinning primer binding site). Those skilled in the art will understand that many other arrangements of the target sequence and the universal adaptor sequence are possible. An illustrative template molecule may be a repeating unit of a multi-linked template molecule having two or more tandem copies of the target sequence, or an illustrative template molecule may be a single-copy template molecule having one copy of the target sequence.

[0292] In some embodiments, sequencing primers (e.g., a first sequencing primer, a second sequencing primer, a third sequencing primer, or a fourth sequencing primer) used to sequence the target sequence region hybridize with a first sequencing primer binding site (e.g., a forward sequencing primer binding site). See, for example... Figure 1 or Figure 2 .

[0293] In some embodiments, sequencing primers (e.g., a first sequencing primer, a second sequencing primer, a third sequencing primer, or a fourth sequencing primer) used to sequence the index region of a first sample hybridize with a portion of the binding site of a second sequencing primer (e.g., a reverse sequencing primer binding site). See, for example... Figure 1 .

[0294] In some embodiments, sequencing primers (e.g., a first sequencing primer, a second sequencing primer, a third sequencing primer, or a fourth sequencing primer) used to sequence the index region of a first sample hybridize with a portion of the binding site of the first sequencing primer (e.g., the binding site of the forward sequencing primer). See, for example... Figure 2 .

[0295] In some embodiments, sequencing primers (e.g., a first sequencing primer, a second sequencing primer, a third sequencing primer, or a fourth sequencing primer) used to sequence the second sample index region hybridize with a portion of a second surface primer binding site (e.g., a surface pinning primer binding site). See, for example... Figure 1 .

[0296] In some embodiments, the sequencing primers (e.g., a first sequencing primer, a second sequencing primer, a third sequencing primer, or a fourth sequencing primer) used to sequence the index region of the second sample hybridize with a portion of the second sequencing primer binding site (e.g., a reverse sequencing primer binding site). See, for example... Figure 2 .

[0297] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: read 1 (where the full length of the target sequence is sequenced); followed by the first sample index. In some embodiments, the second sample index is not sequenced. In some embodiments, the second index is sequenced.

[0298] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: read 1 (where the full length of the target sequence is sequenced); followed by a first sample index; followed by a second sample index (e.g., Figure 10 ).

[0299] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: read 1 (where only a portion of the target sequence is sequenced; for example, 5 to 25 nucleotides), followed by a first sample index, and then a second sample index. In some embodiments, sequencing of the second sample index is omitted.

[0300] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: read 1 (where only a portion of the target sequence is sequenced; for example, 5 to 25 nucleotides), followed by a first sample index, followed by a second sample index, followed by read 1 (where the full length of the target sequence is sequenced) (see example...). Figure 11 In some embodiments, sequencing of the second sample index is omitted.

[0301] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: first sample index; followed by read 1 (where the full length of the target sequence is sequenced).

[0302] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: first sample index; followed by read 1 (where only a portion of the target sequence is sequenced; for example, 5 to 25 nucleotides).

[0303] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: first sample index; followed by second sample index; followed by read 1 (where the full length of the target sequence is sequenced) (see example...). Figure 12 ).

[0304] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: first sample index; followed by second sample index; followed by read 1 (where only a portion of the target sequence is sequenced; for example, 5 to 25 nucleotides).

[0305] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: first sample index; followed by read 1 (where the full length of the target sequence is sequenced); followed by second sample index.

[0306] In some embodiments, the sequencing order of the nucleic acid template molecule is as follows: a first sample index; followed by read 1 (where only a portion of the target sequence is sequenced; for example, 5 to 25 nucleotides); followed by a second sample index; and optionally followed by read 1 (where the full length of the target sequence is sequenced) (see, for example...). Figure 13 ).

[0307] Those skilled in the art will recognize that the sequence of interest, the first sample index, and the second sample index can be sequenced in any order. They will also recognize that the full length or only a portion of the sequence of interest, the first sample index, and the second sample index can be sequenced in any order.

[0308] In some embodiments, nucleic acid template molecules can be sequenced using a pairwise sequencing workflow that includes sequencing a first template strand, performing a primer extension reaction (e.g., sometimes referred to as "pairwise turning") for a second strand synthesis step, and sequencing a template strand having a sequence complementary to the first template strand.

[0309] In some embodiments of the pairwise sequencing workflow, the sequencing order of the nucleic acid template molecule is as follows: first sample index; followed by second sample index; followed by read 1 (where the full-length target sequence is sequenced); followed by second strand synthesis; followed by read 2 (where the full-length target sequence is sequenced) (see example...). Figure 14 ).

[0310] In some embodiments of the pairwise sequencing workflow, the sequencing order of the nucleic acid template molecule is as follows: read 1 (in which the full-length target sequence is sequenced); followed by second strand synthesis (pairwise turning); followed by read 2 (in which the full-length target sequence is sequenced); followed by first sample index; followed by second sample index.

[0311] In some embodiments, in a pairwise sequencing workflow, the sequencing order of the nucleic acid template molecule is as follows: read 1 (where the full-length target sequence is sequenced); followed by second strand synthesis (pairwise turning); followed by first sample index; followed by second sample index; followed by read 2 (where the full-length target sequence is sequenced) (see example...). Figure 15 ).

[0312] Those skilled in the art will recognize that the sequence of interest, the first sample index, and the second sample index can be sequenced in any order. They will also recognize that the full length or only a portion of the sequence of interest, the first sample index, and the second sample index can be sequenced in any order.

[0313] Methods for sequencing

[0314] This disclosure provides methods for sequencing any of the nucleic acid template molecules described herein. In some embodiments, the nucleic acid template molecule is a multivalent template molecule. Any method described herein for performing rolling circle amplification can be used to generate multiple multivalent template molecules (“immobilized multivalent molecules”) fixed to a support, and sequencing reactions can be performed on the immobilized multivalent molecules. In some embodiments, the sequencing reaction employs a detectably labeled nucleotide analog. In some embodiments, the sequencing reaction employs a two-stage sequencing reaction comprising binding a detectably labeled multivalent molecule and incorporating a nucleotide analog. In some embodiments, the sequencing reaction employs an unlabeled nucleotide analog.

[0315] In some embodiments, any rolling circle amplification reaction described herein (e.g., RCA performed on a support or in solution) can be used to generate immobilized multiply molecules, each containing a target sequence and a tandem repeat unit of any linker sequence present in a covalently closed circular library molecule. Methods for generating circular libraries are described, for example, in WO2023168444 and WO2023168443, the contents of which are incorporated herein by reference in their entirety.

[0316] For example, a tandem repeat unit may include: (i) a first left universal adaptor sequence having a binding sequence for a first surface primer, (ii) a second left universal adaptor sequence having a binding sequence for a first sequencing primer, (iii) a target sequence, (iv) a second right universal adaptor sequence having a binding sequence for a second sequencing primer, (v) a first right universal adaptor sequence having a binding sequence for a second surface primer, and (vii) a first left index sequence and / or a first right index sequence (e.g., see...). Figure 1 and Figure 2In some embodiments, the tandem repeat unit further includes a first left unique identifier sequence and / or a first right unique identifier sequence.

[0317] Fixed polyp molecules can self-collapse into compact nucleic acid nanospheres. Including one or more compressed oligonucleotides during the RCA reaction can further compress the size and / or shape of the nanospheres. An increase in the number of tandem repeat units in a given polyp increases the number of sites along the polyp molecule for hybridization with multiple sequencing primers (e.g., sequencing primers with a universal sequence), which act as multiple initiation sites for the polymerase-catalyzed sequencing reaction. When the sequencing reaction employs detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., those with nucleotide units), the signals emitted by the nucleotides or nucleotide units participating in the parallel sequencing reaction along the polyp molecule produce an increased signal intensity for each polyp molecule. Multiple parts of a given polyp molecule can be sequenced simultaneously. Furthermore, multiple binding complexes can form along the polyp molecule, each containing a sequencing polymerase bound to the multivalent molecule, wherein these multiple binding complexes remain stable without dissociation, resulting in increased residence time, which increases signal intensity and reduces imaging time.

[0318] Methods for sequencing using nucleotide analogs

[0319] This disclosure provides a method for sequencing any of the nucleic acid template molecules described herein, the method comprising the step (a): contacting a sequencing polymerase with (i) the nucleic acid template molecule and (ii) nucleic acid sequencing primers, wherein the contact is performed under conditions suitable for binding the sequencing polymerase to the nucleic acid template molecule hybridizing to the nucleic acid primers. The nucleic acid template molecule hybridizing with the nucleic acid primers can form a nucleic acid duplex. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase incorporating nucleotide analogs. In some embodiments, the sequencing primers comprise a 3' extendable end. In some embodiments, the nucleic acid template molecule is fixed.

[0320] In some embodiments, the sequencing primers include a 3' extendable end. In some embodiments, the sequencing primers include a 3' non-extendable end. In some embodiments, the plurality of nucleic acid template molecules include an amplified template molecule (e.g., a cloned amplified template molecule). In some embodiments, each nucleic acid template molecule in the plurality of nucleic acid template molecules includes one copy of the target sequence. In some embodiments, each template molecule in the plurality of nucleic acid molecules includes two or more tandem copies of the target sequence (e.g., a multiplicative template molecule). In some embodiments, the nucleic acid template molecules in the plurality of nucleic acid template molecules include the same target sequence. In some embodiments, the nucleic acid template molecules in the plurality of nucleic acid template molecules include different target sequences. In some embodiments, the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are in solution. In some embodiments, the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are immobilized to a support. In some embodiments, when the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are immobilized to a support, binding with a plurality of first sequencing polymerases produces a plurality of immobilized first complex polymerases. In some embodiments, the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are immobilized to a support. 2 Up to 10 15 Multiple different sites. In some embodiments, the binding of multiple nucleic acid template molecules and multiple nucleic acid primers to multiple first sequencing polymerases produces 10 sites immobilized on a support. 2 Up to 10 15 Multiple first complex polymerases at different sites. In some embodiments, multiple immobilized first complex polymerases on the support are immobilized to predetermined sites on the support. In some embodiments, multiple immobilized first complex polymerases on the support are immobilized to random sites on the support. In some embodiments, multiple immobilized first complex polymerases are fluidly connected to each other to allow reagent solutions (e.g., enzymes including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow onto the support, such that multiple immobilized complex polymerases on the support react with the reagent solutions in a large-scale parallel manner.

[0321] In some embodiments, the method for sequencing further includes step (b): contacting a sequencing polymerase bound to a nucleic acid duplex with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the sequencing polymerase and suitable for polymerase-catalyzed incorporation of a nucleotide. In some embodiments, the sequencing polymerase is contacted with the plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain-terminating portion at a 2' or 3' position of a sugar. In some embodiments, the chain-terminating portion may be removed from the 2' or 3' position of the sugar to convert the chain-terminating portion into an OH or H group. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain-terminating portion. In some embodiments, at least one nucleotide is labeled with a detectable reporter portion (e.g., a fluorophore). In some embodiments, step (b) further comprises removing the chain-terminating portion from the incorporated chain-terminating nucleotide to generate an extendable 3'OH group. In some embodiments, step (b) includes repeating the following steps at least once: (i) incorporating a detectably labeled chain-terminating nucleotide into the 3' end of a first sequencing primer for hybridization; (ii) detecting and identifying the incorporated chain-terminating nucleotide; and (iii) removing the chain-terminating portion and / or the detectable label from the incorporated chain-terminating nucleotide to generate an extendable 3'OH sugar group on the incorporated chain-terminating nucleotide.

[0322] In some embodiments, the method for sequencing includes step (c): incorporating at least one nucleotide into the 3' end of an extendable primer under conditions suitable for incorporating at least one nucleotide. In some embodiments, the conditions suitable for nucleotide binding polymerase and for incorporating the nucleotide into the primer extension product may be the same or different. In some embodiments, the conditions suitable for incorporating the nucleotide include the addition of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, at least one nucleotide binds to a sequencing polymerase and is incorporated into the 3' end of the extendable primer. In some embodiments, incorporating the nucleotide into the 3' end of the primer in step (c) includes a primer extension reaction.

[0323] In some embodiments, the method for sequencing includes step (d): repeating at least one nucleotide incorporated into the 3' end of an extendable primer at least once in steps (b) and (c). In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter portion. The detectable reporter portion may include a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker that can be cleaved / removed from the base. In some embodiments, at least one nucleotide of the plurality of nucleotides is not labeled with a detectable reporter portion. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to a nucleotide may correspond to the identity of the nucleotide base (e.g., all dATP, dGTP, dCTP, dTTP, or dUTP are labeled with the same fluorophore) to allow detection and identification of the nucleotide base. In some embodiments, the method includes detecting at least one incorporated nucleotide at steps (c) and / or (d). In some embodiments, the method includes identifying at least one incorporated nucleotide at steps (c) and / or (d). In some embodiments, the sequence of the nucleic acid template molecule can be determined by detecting and identifying nucleotides bound to the sequencing polymerase. In some embodiments, the sequence of the nucleic acid template molecule can be determined by detecting and identifying nucleotides incorporated into the 3' end of the primer.

[0324] In some embodiments, the plurality of sequencing polymerases bound to a nucleic acid duplex comprises a plurality of complex polymerases having at least a first complex polymerase and a second complex polymerase, wherein (a) the first complex polymerase comprises a first sequencing polymerase bound to a first nucleic acid duplex, wherein the first nucleic acid duplex contains a first nucleic acid template sequence hybridized to a first nucleic acid primer; (b) the second complex polymerase comprises a second sequencing polymerase bound to a second nucleic acid duplex, wherein the second nucleic acid duplex contains a second nucleic acid template sequence hybridized to a second nucleic acid primer; (c) the first nucleic acid template sequence and the second nucleic acid template sequence contain the same or different sequences; (d) the first nucleic acid template and the second nucleic acid template are cloned and amplified; (e) the first primer and the second primer contain an extendable 3' end or a non-extendable 3' end; and (f) the plurality of complex polymerases are immobilized to a support. In some embodiments, the density of the plurality of complex polymerases immobilized to the support is about 10. 2 Up to 10 15 One complex polymerase / mm 2 .

[0325] Two-stage method for nucleic acid sequencing

[0326] This disclosure provides a two-stage method for sequencing any of the nucleic acid template molecules described herein. The first stage typically involves conjugating a multivalent molecule with a complex polymerase to form a multivalent complex polymerase, and detecting the multivalent complex polymerase. In some embodiments, the nucleic acid template molecule is immobilized (“immobilized nucleic acid template molecule”).

[0327] In some embodiments, the first stage includes step (a): contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid template molecules and (ii) a plurality of nucleic acid sequencing primers, wherein the contact is performed under conditions suitable for binding the plurality of first sequencing polymerases with the plurality of nucleic acid template molecules and the plurality of nucleic acid primers, thereby forming a plurality of first complex polymerases, each first complex polymerase comprising a first sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In some embodiments, the first sequencing polymerases comprise recombinant mutant sequencing polymerases. In some embodiments, the sequencing primers comprise a 3' extendable end.

[0328] In some embodiments, the sequencing primers include a 3' extendable end. In some embodiments, the sequencing primers include a 3' non-extendable end. In some embodiments, the plurality of nucleic acid template molecules include an amplified template molecule (e.g., a template molecule amplified in a clonal manner). In some embodiments, the plurality of nucleic acid template molecules include one copy of a target sequence. In some embodiments, the plurality of nucleic acid molecules include two or more tandem copies of a target sequence (e.g., a multiplicative template molecule). In some embodiments, the nucleic acid template molecules in the plurality of nucleic acid template molecules include the same target sequence or different target sequences. In some embodiments, the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are in solution or immobilized to a support. In some embodiments, when the plurality of nucleic acid template molecules and / or the plurality of nucleic acid primers are immobilized to a support, they bind to a first sequencing polymerase to generate a plurality of immobilized first complex polymerases. In some embodiments, the plurality of nucleic acid template molecules and / or nucleic acid primers are immobilized to a support. 2 Up to 10 15 Multiple different sites. In some embodiments, the binding of multiple nucleic acid template molecules and nucleic acid primers to multiple first sequencing polymerases produces 10 sites immobilized on a support. 2 Up to 10 15 Multiple first complex polymerases at different sites. In some embodiments, multiple immobilized first complex polymerases on the support are immobilized to predetermined or random sites on the support. In some embodiments, the multiple immobilized first complex polymerases are fluidly connected to each other to allow reagent solutions (e.g., enzymes including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow onto the support, such that the multiple immobilized complex polymerases on the support react with the reagent solutions in a large-scale parallel manner.

[0329] In some embodiments, the method for sequencing includes step (b): contacting a plurality of first complex polymerases with a plurality of multivalent molecules to form a plurality of multivalent-complex polymerases (e.g., binding complexes). In some embodiments, each of the plurality of multivalent molecules includes a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide (e.g., a nucleotide unit) (e.g., Figures 19 to 23 In some embodiments, the contact in step (b) is performed under conditions suitable for binding complementary nucleotide units of a multivalent molecule to at least two of a plurality of first complex polymerases, thereby forming a plurality of multivalent complex polymerases. In some embodiments, the conditions are adapted to inhibit the complementary nucleotide unit polymerase-catalytic incorporation into primers of the plurality of multivalent complex polymerases. In some embodiments, the plurality of multivalent molecules comprises at least one having a plurality of nucleotide arms (e.g., Figures 19 to 23 The plurality of multivalent molecules comprises at least one multivalent molecule comprising multiple nucleotide arms, each nucleotide arm having a nucleotide analog (e.g., a nucleotide analog unit) attached, wherein the nucleotide analog includes a chain termination portion located at the 2' and / or 3' positions of the sugar. In some embodiments, the plurality of multivalent molecules comprises at least one multivalent molecule comprising multiple nucleotide arms, each nucleotide arm having a nucleotide unit lacking a chain termination portion attached. In some embodiments, at least one of the plurality of multivalent molecules is labeled with a detectable reporter portion. In some embodiments, the detectable reporter portion comprises a fluorophore. In some embodiments, the contact of step (b) is performed in the presence of at least one noncatalytic cation comprising strontium, barium, and / or calcium.

[0330] In some embodiments, the method for sequencing includes step (c): detecting a plurality of multivalent complex polymerases. In some embodiments, detection includes detecting multivalent molecules bound to the complex polymerases, wherein complementary nucleotide units of the multivalent molecules are bound to primers, but incorporation of complementary nucleotide units is inhibited. In some embodiments, the multivalent molecules are partially labeled with a detectable reporter to allow detection. In some embodiments, the labeled multivalent molecules include fluorophores attached to the core, linker, and / or nucleotide units of the multivalent molecules.

[0331] In some embodiments, the method for sequencing includes step (d): identifying the nucleobases of complementary nucleotide units bound to a plurality of first complex polymerases, thereby determining the sequence of the template molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter portion corresponding to a specific nucleotide unit linked to a nucleotide arm to allow identification of complementary nucleotide units (e.g., nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to a plurality of first complex polymerases.

[0332] In some embodiments, the second stage of a two-stage sequencing method typically includes nucleotide incorporation. In some embodiments, the method for sequencing includes step (e): dissociating multiple multivalent-complex polymerases and removing multiple first sequencing polymerases and the multivalent molecules they bind, while retaining multiple nucleic acid duplexes.

[0333] In some embodiments, the method for sequencing includes step (f): contacting a plurality of retained nucleic acid duplexes from step (e) with a plurality of second sequencing polymerases, wherein the contact is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second complex polymerases, each second complex polymerase comprising a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0334] In some embodiments, the plurality of first sequencing polymerases in step (a) contain amino acid sequences that are 100% identical to the amino acid sequences of the plurality of second sequencing polymerases in step (f). In some embodiments, the plurality of first sequencing polymerases in step (a) contain amino acid sequences that are different from the amino acid sequences of the plurality of second sequencing polymerases in step (f).

[0335] In some embodiments, the method for sequencing includes step (g): contacting a plurality of second complex polymerases with a plurality of nucleotides, wherein the contact is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second complex polymerases, thereby forming a plurality of nucleotide-complex polymerases. In some embodiments, the contact in step (g) is performed under conditions suitable for promoting the polymerase-catalyzed incorporation of the complementary nucleotides into a primer of the nucleotide-complex polymerase, thereby forming a plurality of nucleotide-complex polymerases. In some embodiments, the incorporation of nucleotides into the 3' end of the primers in step (g) includes a primer extension reaction. In some embodiments, the contact in step (g) is performed in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises native nucleotides (e.g., non-analogous nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides includes removable or non-removable 2' and / or 3' chain termination portions. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter portion. The detectable reporter portion comprises a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base by a linker that may or may not be cleavable / removable from the base. In some embodiments, at least one nucleotide among the plurality of nucleotides is not labeled with a detectable reporter portion. In some embodiments, a specific detectable reporter portion (e.g., a fluorophore) attached to a nucleotide may correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow for the detection and identification of the nucleotide base. In some embodiments, the plurality of nucleotides are unlabeled nucleotides.

[0336] In some embodiments, the method for sequencing includes step (h): detecting complementary nucleotides in primers incorporated into a nucleotide-complex polymerase. In some embodiments, multiple nucleotides are partially labeled with a detectable reporter to allow detection. In some embodiments, the detection in step (h) is omitted. In some embodiments, the detection in step (h) is omitted when step (g) uses multiple unlabeled nucleotides.

[0337] In some embodiments, the method for sequencing includes step (i): identifying the bases of complementary nucleotides incorporated into the primers of a nucleotide-complex polymerase. In some embodiments, the identification of the incorporated complementary nucleotides in step (i) may be used to confirm the identity of complementary nucleotides of multivalent molecules bound to multiple first complex polymerases in step (d). In some embodiments, the identification in step (i) may be used to determine the sequence of a nucleic acid template molecule. In some embodiments, the identification in step (i) is omitted. In some embodiments, the identification in step (i) is omitted when step (g) uses multiple unlabeled nucleotides.

[0338] In some embodiments, the method for sequencing includes step (j): removing the chain termination portion from the incorporated nucleotide when step (g) is performed by contacting a plurality of second complex polymerases with a plurality of nucleotides comprising at least one nucleotide having a 2' and / or 3' chain termination portion.

[0339] In some embodiments, the method for sequencing includes step (k): repeating steps (a) to (j) at least once. In some embodiments, the sequence of the nucleic acid template molecule can be determined by detecting and identifying multivalent molecules that bind to the sequencing polymerase but are not incorporated into the 3' end of the primer at steps (c) and (d). In some embodiments, the sequence of the nucleic acid template molecule can be determined (or confirmed) by detecting and identifying nucleotides incorporated into the 3' end of the primer at steps (h) and (i).

[0340] In some embodiments, the method includes sequencing a nucleic acid multiply template molecule.

[0341] In some embodiments of the method for sequencing a multivalent nucleic acid template molecule described herein, the binding of a plurality of first complex polymerases to a plurality of multivalent molecules forms at least one affinity complex. In some embodiments, the method includes the steps of: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of the multivalent template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and (b) binding a second nucleic acid primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same multivalent template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, including the first and second binding complexes of the same multivalent molecule forming an affinity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. The multivalent template molecule comprises a target sequence and a tandem repeat sequence of at least one universal sequencing primer binding site. The first and second nucleic acid primers may bind to the sequencing primer binding site along the multivalent template molecule. Exemplary multivalent molecules are shown in Figures 19 to 23 middle.

[0342] In some embodiments of a method for sequencing a nucleic acid multivalent template molecule, the method includes binding a plurality of first complex polymerases to a plurality of multivalent molecules to form at least one affinity complex. In some embodiments, the method includes the steps of: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different portions of a nucleic acid multivalent template molecule to form at least a first complex polymerase and a second complex polymerase on the same multivalent template molecule; (b) contacting the plurality of multivalent molecules with at least the first complex polymerase and the second complex polymerase on the same nucleic acid multivalent template molecule under conditions suitable for binding a single multivalent molecule from a plurality of multiple multivalent molecules to the first complex polymerase and the second complex polymerase, wherein at least a first nucleotide unit of the single multivalent molecule binds to the first complex polymerase, the first complex polymerase comprising a first primer that hybridizes to a first portion of the nucleic acid multivalent template molecule, thereby forming a first binding complex (e.g., a first ternary complex), and wherein at least a second nucleotide unit of the single multivalent molecule binds to the second complex polymerase. The second complex polymerase binds to a second primer that hybridizes to a second portion of a nucleic acid multivalent template molecule, thereby forming a second binding complex (e.g., a second ternary complex). Contact is carried out under conditions suitable for inhibiting binding, where a first nucleotide unit and a second nucleotide unit are polymerase-catalyzed into the first and second binding complexes, and wherein the first and second binding complexes binding to the same multivalent molecule form an affinity complex; (c) detecting the first and second binding complexes on the same nucleic acid multivalent template molecule; and (d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the nucleic acid multivalent template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the nucleic acid multivalent template molecule. In some embodiments, the various sequencing polymerases comprise any wild-type or mutant sequencing polymerase described herein. The nucleic acid multivalent template molecule comprises a target sequence and a tandem repeat sequence of at least one universal sequencing primer binding site. Multiple nucleic acid primers can bind to the sequencing primer binding site along the nucleic acid multivalent template molecule. An exemplary multivalent molecule is shown in... Figures 19 to 23 middle.

[0343] Combining sequencing

[0344] This disclosure provides a method for sequencing a fixed nucleic acid template molecule described herein, wherein the sequencing method includes a sequencing by binding of unlabeled chain-terminated nucleotides (SBB) procedure. In some embodiments, the sequencing-by-binding (SBB) method includes the steps of: (a) sequentially contacting an initiated template nucleic acid with at least two separate mixtures, each of the at least two separate mixtures comprising a polymerase and a nucleotide, under conditions stabilizing the ternary complex, such sequential contact causing the initiated template nucleic acid to contact nucleotides of base types homologous to the first, second, and third base types in the template under conditions stabilizing the ternary complex; (b) examining the at least two separate mixtures to determine whether a ternary complex has formed; and (c) identifying the next correct nucleotide of the initiated template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first, second, or third base type, and wherein if a ternary complex is not present in step (b), the next correct nucleotide is extrapolated to be a homolog of a nucleotide of the fourth base type; (d) adding the next correct nucleotide to a primer of the initiated template nucleic acid after step (b), thereby producing an extended primer; and (e) repeating steps (a) through (d) at least once for the initiated template nucleic acid containing the extended primer. Exemplary binding sequencing methods are described in U.S. Patent Nos. 10,246,744 and 10,731,141 (the contents of which are incorporated herein by reference in their entirety).

[0345] Sequencing polymerase

[0346] This disclosure provides methods for sequencing nucleic acid template molecules, wherein any sequencing method described herein employs at least one type of sequencing polymerase and multiple nucleotides, or employs at least one type of sequencing polymerase and multiple nucleotides and multiple multivalent molecules. In some embodiments, the sequencing polymerase is capable of incorporating a complementary nucleotide to a nucleotide in the template molecule. In some embodiments, the sequencing polymerase is capable of binding to a complementary nucleotide unit of a multivalent molecule to a nucleotide in the template molecule. In some embodiments, the multiple sequencing polymerases comprise a recombinant mutant polymerase.

[0347] Examples of suitable polymerases for sequencing nucleotides and / or multivalent molecules include, but are not limited to: Klenow DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; candidate altiarchaeales archaea; candidate Candidatus Hadarchaeum Yellowstonense; Hadesarchaea archaea; Euryarchaeota archaea; Thermoplasmata archaea; Thermococcus polymerases, such as Thermococcus litoralis, bacteriophage T7 DNA polymerase; human α, δ, and ε DNA polymerases; bacteriophage polymerases, such as T4, RB69, and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III (subtilis); Escherichia coli DNA polymerase III α and ε; 9°N polymerase; reverse transcriptase, such as HIV M or O type reverse transcriptase; avian myeloblastoma virus reverse transcriptase; Moloney murine leukemia virus (MMLV) reverse transcriptase; or telomerase. Other non-limiting examples of DNA polymerases include those polymerases from various archaea genera (such as *Aeropyrum*, *Archaeoglobus*, *Desulfurococcus*, *Pyrobaculum*, *Pyrococcus*, *Pyrolobus*, *Pyrodictium*, *Staphylothermus*, *Stetteria*, *Sulfolobus*, *Thermococcus*, and *Vulcanisaeta*, etc., or variants thereof), including such polymerases as known in the art, such as 9°N, VENT®, DEEP VENT®, THERMINATOR. TM Polymerases such as Pfu, KOD, Pfx, Tgo, and RB69 are described. Exemplary polymerases are described, for example, in U.S. Patent No. 11,859,241, the contents of which are incorporated herein by reference in their entirety.

[0348] Nucleotides

[0349] This d...

Claims

1. A method for sequencing two or more regions of a nucleic acid template molecule, the method comprising: a) Provide multiple nucleic acid template molecules, wherein each template molecule contains (i) a first region and a first universal sequencing primer binding site, and (ii) a second region and a second universal sequencing primer binding site; b) Provide multiple first nucleic acid sequencing primers and multiple second nucleic acid sequencing primers; c) Hybridize each of the first nucleic acid sequencing primers with the first universal sequencing primer binding site on the template molecule; d) Sequencing the first region of the plurality of nucleic acid template molecules to generate a plurality of first sequencing reads; e) Performing multiple first capping reactions, said multiple first capping reactions comprising incorporating a read-capping nucleotide analog into the 3' end of each first sequencing read product, thereby generating multiple capped first sequencing read products. ●The capped nucleotide analogues described herein comprise (i) heterocyclic bases, (ii) sugars, and (iii) polyphosphate chains. ●The heterocyclic base is linked to a termination portion that blocks the binding of the multivalent molecule to the 3' end of the capped first sequencing read product. Each multivalent molecule includes a core attached to multiple nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. ●The termination portion therein blocks the incorporation of subsequent nucleotides into the capped first sequencing read product via polymerase catalysis. f) Retain the capped first sequencing reads that have hybridized with the nucleic acid template molecule, wherein the termination portion has not been cleaved or removed; g) Hybridize each of the second nucleic acid sequencing primers with the second universal sequencing primer binding site on each of the template molecules; as well as h) Sequencing the second region on the plurality of template molecules, thereby generating a plurality of second sequencing reads.

2. The method according to claim 1, further comprising: i) Performing multiple second capping reactions, said multiple second capping reactions comprising incorporating a capping nucleotide analog into the 3' end of each second sequencing read product, thereby generating multiple capped second sequencing read products. ●The capped nucleotide analogues described herein comprise (i) heterocyclic bases, (ii) sugars, and (iii) polyphosphate chains. ●The heterocyclic base is connected to the termination portion, which blocks the binding of the multivalent molecule to the 3' end of the capped second sequencing read product, wherein each multivalent molecule includes a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide unit. ●The termination portion prevents subsequent nucleotides from being incorporated into the capped second sequencing read product via polymerase catalysis; as well as j) The capped second sequencing reads that have hybridized with the nucleic acid template molecule are retained, wherein the termination portion is not cleaved or removed.

3. The method of claim 2, wherein each sequencing template molecule comprises at least one capped first sequencing read product hybridized thereon and at least one capped second sequencing read product hybridized thereon.

4. The method according to any one of claims 1 to 3, wherein the capped nucleotide analog incorporated at the 3' end of each capped first sequencing read product is not modified to convert the sugar 3' position into an extendable 3'OH group.

5. The method of claim 2, wherein the capped nucleotide analog incorporated at the 3' end of each capped second sequencing read product is not modified to convert the sugar 3' position into an extendable 3'OH group.

6. The method according to any one of claims 1 to 5, wherein the heterocyclic base is connected to the terminating portion via an alkyne bond, an olefin bond, or an alkane bond.

7. The method according to any one of claims 1 to 6, wherein the termination portion comprises a polymer portion.

8. The method of claim 7, wherein the polymer portion is selected from the group consisting of: polyether portion, polyethylene glycol portion, polypropylene glycol portion, polyvinyl acetate portion, polylactic acid portion, polyglycolic acid portion, polyamide and polyester portion.

9. The method of claim 7, wherein the polymer portion comprises a poly(glycerol) portion, a poly(oxazoline) portion, a poly(hydroxypropyl methacrylate) portion (PHPMA), a poly(2-hydroxyethyl methacrylate) portion (PHEMA), a poly(N-(2-hydroxypropyl)methacrylamide) portion (HPMA), a poly(vinylpyrrolidone) portion (PVP), a poly(N,N-dimethylacrylamide) portion (PDMA), or a poly(N-acryloylmorpholine) portion (PAcM).

10. The method according to any one of claims 1 to 9, wherein the termination portion comprises a polyethylene glycol (PEG) portion.

11. The method of claim 10, wherein the PEG portion has a molecular weight of 1K to 20K.

12. The method according to any one of claims 1 to 9, wherein the terminating portion comprises a propargylamino moiety, an allylamino moiety, a propylamino moiety, an ethyl mercapto moiety, a hydroxymethyl moiety, an aryl mercapto moiety, a 1-X-1H-1,2,3-triazol-4-yl moiety, a 5-X-1H-1,2,3-triazol-1-methyl moiety, a hydrazone moiety, or an O-alkyl oxime moiety, wherein "X" comprises a polymer.

13. The method according to any one of claims 1 to 5, wherein the heterocyclic base comprises a chain-terminating portion at the 3' sugar group, and wherein the chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azidoyl group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

14. The method according to any one of claims 1 to 13, wherein each of the plurality of nucleic acid template molecules is a single-stranded or double-stranded nucleic acid molecule.

15. The method according to any one of claims 1 to 14, wherein the plurality of nucleic acid template molecules are immobilized to a support or to a coating on the support.

16. The method of claim 15, wherein each of the plurality of nucleic acid template molecules is covalently linked to a fixed surface capture primer, or wherein each of the plurality of nucleic acid template molecules hybridizes with a fixed surface capture primer, wherein the fixed surface capture primer is fixed on the support.

17. The method according to any one of claims 1 to 16, wherein at least one of the nucleic acid template molecules of the plurality of nucleic acid template molecules contains uridine, or wherein at least one of the nucleic acid template molecules of the plurality of nucleic acid template molecules is deficient in uridine.

18. The method according to any one of claims 1 to 17, wherein each of the plurality of nucleic acid template molecules comprises a cloned and amplified nucleic acid molecule.

19. The method according to any one of claims 1 to 18, wherein each of the plurality of nucleic acid template molecules comprises at least one copy of the target sequence and at least one universal adaptor sequence.

20. The method according to any one of claims 1 to 19, wherein each of the plurality of nucleic acid template molecules comprises a multiply template molecule having two or more tandem repeat units, wherein each repeat unit comprises a target sequence and at least one universal adaptor sequence.

21. The method according to any one of claims 15 to 20, wherein the support comprises glass or plastic.

22. The method according to any one of claims 15 to 21, wherein the support is disposed on the flow cell or inside the capillary lumen.

23. The method according to any one of claims 15 to 22, wherein the support comprises at least one hydrophilic polymer coating and a plurality of surface-capturing primers fixed to the at least one hydrophilic polymer coating, and wherein the at least one hydrophilic polymer coating has a water contact angle of no more than 45 degrees.

24. The method of claim 23, wherein the at least one hydrophilic polymer coating comprises polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligomeric (ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, or dextran.

25. The method of claim 23 or 24, wherein the at least one hydrophilic polymer coating comprises polymer molecules having a molecular weight of at least 1,000 Daltons.

26. The method according to any one of claims 23 to 25, wherein the at least one hydrophilic polymer coating comprises a branched polymer molecule having 4 to 8 branches.

27. The method of any one of claims 23 to 26, wherein the support comprises: a) a first coating comprising a first monolayer of hydrophilic polymer molecules tethered to the support; b) a second coating comprising a second monolayer of hydrophilic polymer molecules tethered to the first monolayer; and c) a third coating comprising a third monolayer of hydrophilic polymer molecules tethered to the second monolayer, wherein the hydrophilic polymer molecules of the first, second, or third layer comprise a branched polymer layer.

28. The method of claim 27, wherein the plurality of surface-capturing primers are immobilized to the hydrophilic polymer molecules of the second or third monolayer, and the surface-capturing primers are distributed at a plurality of depths throughout the second or third layer.

29. The method according to any one of claims 22 to 27, wherein one or more of the at least one hydrophilic polymer coating comprises a surface density of at least 1000 / µm. 2 Multiple surface-capturing primers.

30. The method according to any one of claims 1 to 29, comprising contacting the plurality of nucleic acid template molecules with: (i) the first plurality of sequencing primers, wherein the respective nucleic acid template molecules are immobilized and the plurality of sequencing primers are soluble, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents, wherein the conditions are adapted to bind the nucleic acid duplexes to the sequencing polymerases and the nucleotide reagents.

31. The method of claim 30, wherein the various nucleotide reagents in the plurality of nucleotide reagents comprise an aromatic base, a pentose sugar, and 1 to 10 phosphate ester groups.

32. The method of claim 30 or 31, wherein the plurality of nucleotide reagents comprises one or more types of nucleotide reagents selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

33. The method according to any one of claims 30 to 32, wherein the plurality of nucleotide reagents comprises a combination of two or more types of nucleotide reagents selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

34. The method according to any one of claims 30 to 33, wherein at least one of the plurality of nucleotide reagents lacks a detectable reporter portion.

35. The method according to any one of claims 30 to 34, wherein at least one of the plurality of nucleotide reagents is labeled with a detectable reporter portion.

36. The method according to any one of claims 30 to 35, wherein the various nucleotide reagents in the plurality of nucleotide reagents comprise at least one chain-terminating nucleotide, the at least one chain-terminating nucleotide comprising (i) an aromatic base, (ii) a sugar having a 3' chain-terminating portion that inhibits polymerase-catalyzed nucleotide incorporation, and (iii) 1 to 10 phosphate ester groups.

37. The method of claim 36, wherein the at least one chain-terminating nucleotide comprises a removable chain-terminating portion at the 3' sugar group, and wherein the removable chain-terminating portion comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azidoyl group, an O-azidomethyl group, an amine group, an amide group, a ketone group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

38. The method of claim 36, wherein the at least one chain-terminating nucleotide comprises a removable chain-terminating portion at the 3' sugar group, and wherein the removable chain-terminating portion comprises a 3'-O-amino group, a 3'-O-aminomethyl group, a 3'-O-methylamino group, or a derivative thereof.

39. The method according to any one of claims 36 to 38, wherein the at least one chain termination portion may be cleaved or removed by a chemical compound to produce an extendable 3'OH moiety on the sugar group.

40. The method according to any one of claims 1 to 39, wherein sequencing the first region on the plurality of template molecules comprises: a) Contacting the plurality of nucleic acid template molecules with (i) a first plurality of sequencing polymerases and (ii) the plurality of first nucleic acid sequencing primers, wherein the nucleic acid template molecules are immobilized and the plurality of first nucleic acid sequencing primers are soluble, and wherein the contact is performed under conditions suitable for forming a plurality of complex polymerases comprising sequencing polymerases that bind to nucleic acid duplexes, wherein the nucleic acid duplexes comprise a portion of each nucleic acid template molecule that has hybridized with each of the first nucleic acid sequencing primers; b) The plurality of complex sequencing polymerases are contacted with the plurality of nucleotides under conditions suitable for binding at least one nucleotide to the complex sequencing polymerase, wherein the plurality of nucleotides comprise at least one nucleotide analog labeled with a fluorescent group and having a removable chain termination portion at the sugar 3' position. c) Incorporating at least one nucleotide into the 3' end of the first nucleic acid sequencing primer, thereby generating multiple first sequencing reads by extending the first nucleic acid sequencing primer; as well as d) Detect the at least one nucleotide and identify the nucleobases of the at least one nucleotide.

41. The method according to any one of claims 30 to 39, wherein the plurality of nucleotide reagents comprises at least one multivalent molecule.

42. The method of claim 41, wherein the at least one multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms, the plurality of nucleotide arms comprising (i) a core attachment portion, (ii) a spacer comprising a PEG portion, (iii) a linker, and (iv) nucleotide units. The core is attached to the plurality of nucleotide arms, the spacer is attached to the adapter, and the adapter is attached to the nucleotide unit.

43. The method of claim 42, wherein the nucleotide unit comprises a base, a sugar, and 1 to 10 phosphate groups, and the linker is attached to the nucleotide unit via the base.

44. The method according to claim 42 or 43, wherein each nucleotide arm of the at least one multivalent molecule comprises a linker having an aliphatic chain or an oligoethylene glycol chain, and wherein the linker chain comprises 2 to 6 subunits.

45. The method according to any one of claims 42 to 44, wherein the core of the at least one multivalent molecule comprises streptavidin, and the core attachment portion comprises biotin.

46. ​​The method according to any one of claims 42 to 45, wherein the plurality of nucleotide arms comprise nucleotide units of the same type, and wherein the nucleotide units of the same type are selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

47. The method according to any one of claims 42 to 46, wherein the plurality of nucleotide reagents comprises a plurality of multivalent molecules, wherein each of the plurality of multivalent molecules comprises the same type of nucleotide unit.

48. The method of claim 47, wherein the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

49. The method according to any one of claims 42 to 48, wherein the plurality of nucleotide reagents comprises a plurality of multivalent molecules, the plurality of multivalent molecules comprising a mixture of two or more types of multivalent molecules, each type of multivalent molecule comprising one or more nucleotide unit types selected from the group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

50. The method according to any one of claims 42 to 49, wherein the plurality of nucleotide reagents comprises at least one fluorophore-tagged multivalent molecule.

51. The method according to any one of claims 42 to 50, wherein sequencing the first region comprises performing a two-step sequencing method.

52. The method of claim 51, wherein the first step comprises: a) Contacting the plurality of nucleic acid template molecules with (i) a first plurality of sequencing polymerases and (ii) the plurality of first sequencing primers, wherein the nucleic acid template molecules are immobilized and the plurality of first sequencing primers are soluble, and wherein the contact is performed under conditions suitable for forming a plurality of first complex polymerases, each complex polymerase comprising a sequencing polymerase that binds to a nucleic acid duplex, wherein the nucleic acid duplex comprises a portion of the nucleic acid template molecule that has hybridized with the first soluble sequencing primer; b) Contact the plurality of complex sequencing polymerases with a plurality of nucleotide reagents comprising at least one multivalent molecule, wherein the at least one multivalent molecule is a separably labeled multivalent molecule, wherein the complementary nucleotide units of the multivalent molecule bind to at least two of the plurality of first complex polymerases, thereby forming a plurality of multivalent complex polymerases, and wherein the incorporation of the complementary nucleotide units into the first sequencing primers is inhibited. c) Detect the plurality of multivalent complex polymerases; as well as d) Identify the nucleobases of the complementary nucleotide units that bind to the plurality of first complex polymerases among the plurality of multivalent complex polymerases, thereby determining the sequence of the nucleic acid template.

53. The method of claim 52, wherein the complementary nucleotide unit is complementary to the nucleotide immediately adjacent to the 5' of the nucleic acid double strand of the nucleic acid template molecule.

54. The method according to any one of claims 42 to 51, wherein sequencing the first region on the plurality of template molecules comprises forming an affinity complex, wherein the method comprises: (i) A first sequencing primer, a first sequencing polymerase and a first detectably labeled multivalent molecule are bound to a first region of a multivalent template molecule to form a first binding complex, wherein a first nucleotide unit of the first detectably labeled multivalent molecule is bound to the first sequencing polymerase. (ii) A second sequencing primer, a second sequencing polymerase, and the first detectably labeled multivalent molecule are bound to a second region of the multivalent template molecule to form a second binding complex, wherein a second nucleotide unit of the first multivalent molecule is bound to the second sequencing polymerase, wherein the first binding complex and the second binding complex form an affinity complex, wherein the multivalent template molecule comprises two or more tandem repeat units, wherein each tandem repeat unit comprises a target sequence and a universal primer binding site for binding the first universal sequencing primer and the second universal sequencing primer, and wherein the contact is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of the first nucleotide unit and the second nucleotide unit into the first binding complex and the second binding complex, respectively; (iii) Detecting the first binding complex and the second binding complex on the same multiply template molecule, and (iv) Identify the first nucleotide unit in the first binding complex, thereby determining the sequence of the first region of the multiply template molecule, and identify the second nucleotide unit in the second binding complex, thereby determining the sequence of the second region of the multiply template molecule.

55. The method according to claim 51 or 52, wherein the second step comprises: e) Dissociate the plurality of multivalent complex polymerases and remove the plurality of first sequencing polymerases and multivalent molecules, while retaining the nucleic acid duplexes; f) Contact the nucleic acid duplex of step (e) with a plurality of second sequencing polymerases, wherein the contact is performed under conditions suitable for binding the plurality of second sequencing polymerases with the nucleic acid duplex to form a plurality of second complex polymerases; g) Contact the plurality of second complex polymerases with a plurality of nucleotides, the plurality of nucleotides comprising at least one nucleotide analog having a removable chain termination portion, wherein the contact is performed under conditions adapted to combine a complementary nucleotide present in the plurality of nucleotides with at least two of the complex polymerases of the plurality of second complex polymerases of step (f) thereby forming a plurality of nucleotide complex polymerases, and wherein the conditions are adapted to promote the incorporation of the combined complementary nucleotide into the first sequencing primer of the nucleotide complex polymerase, thereby producing the plurality of first sequencing reads.

56. The method of claim 55, wherein the removable chain termination portion is located at the sugar 3' position.

57. The method according to claim 55 or 56, wherein the complementary nucleotide is complementary to the nucleotide immediately adjacent to the 5' of the nucleic acid double strand of the nucleic acid template molecule.

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