Methods for increasing sequencing quality

Abstract

The present disclosure is concerned with reducing quenching that can occur during certain sequencing reactions. Provided are methods, compositions, arrays, cartridges, and kits related to producing clusters in the presence of a dATP analog, and strand resynthesis in the presence of a dATP analog.

Description

IP-2872-PCT PATENTMETHODS FOR INCREASING SEQUENCING QUALITY

[0001] FIELD

[0002] The present disclosure is concerned with reducing quenching during sequencing by synthesis to improve quality of the resulting data. In particular, the present disclosure includes methods for using dATP analogs during the production of clusters, during strand resynthesis, or the combination thereof.

[0003] BACKGROUND

[0004] Sequencing platforms rely on certain fluorescence readouts to accurately call nucleotides; however, specific DNA motifs can quench the fluorescence output of dyes. Examples of such motifs include CT rich regions and regions with high volumes of G and A nucleotides in the template. The quenching can lead to systematic errors (referred to as Sequence Specific Errors (SSEs)), in sequencing-by-synthesis (SBS) methodologies. These quenching type motifs can cause inaccurate base calling at specific locations (increased “miscall” events), which reduces sequencing quality of those regions. These motifs can occur within clinically relevant regions and thus affect the ability of clinicians to identify important mutations and make critical genome-based decisions affecting the health of patients.

[0005] SUMMARY OF THE APPLICATION

[0006] A method of the present disclosure can include providing an amplification reagent including (i) an array of amplification sites, (ii) a composition including a plurality of modified target nucleic acids, (iii) a composition including nucleotide triphosphates (NTPs), where the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog, and (iv) a composition including a polymerase. The method can include reacting the amplification reagent to produce a plurality of populated amplification sites, where the plurality of populated amplification sites each include a clonal population of amplicons from an individual modified target nucleic acid from the plurality of modified target nucleic acids.

[0007] A method of the present disclosure can include providing an amplification reagent including (i) an array of amplification sites, where each amplification site includes aIP-2872-PCT PATENT capture sequence and a single- stranded modified target nucleic acid immobilized thereto: (ii) a composition including nucleotide triphosphates (NTPs), where the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog, and (iii) a composition including a polymerase. The method can further include reacting the amplification reagent to produce a plurality of amplification sites that each include a clonal population of amplicons from the single-stranded modified target nucleic acid immobilized thereto in step (i).

[0008] A method of the present disclosure can include providing an array including a plurality of amplification sites, where the amplification sites include two populations of capture nucleic acids immobilized to the amplification sites at the 5’ end, where each population includes a capture sequence. The first population of capture nucleic acids can include at each amplification site a clonal population of a modified target nucleic acid, the 5’ end of the clonal population of the modified target nucleic acid attached to the 3’ end of the first population capture nucleic acids. The clonal population of the modified target nucleic acid at each amplification site can be a member of a sequencing library. The method can further include contacting the plurality of amplification sites to a resynthesis reagent including (i) a composition including nucleotide triphosphates (NTPs), where the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog including a nucleobase, and (ii) a composition including a polymerase. The method can further include reacting the resynthesis reagent to produce a plurality of re-populated amplification sites attached to the array, where the plurality of re-populated amplification sites each include a clonal population of a resynthesized target nucleic acids immobilized to the amplification sites at the 5’ end. The clonal population of the resynthesized target nucleic acid can include a nucleic acid sequence that is a complement of the clonal population of the modified target nucleic acid of the providing step.

[0009] A method of the present disclosure can include providing an array including a plurality of amplification sites, where the amplification sites include two populations of capture nucleic acids immobilized to the amplification sites at the 5’ end, each population including a capture sequence. A first population of capture nucleic acids can include at each amplification site a clonal population of a modified target nucleic acid, where the 5’ end of the clonal population of the modified target nucleic acid attached to the 3’ endIP-2872-PCT PATENT of the first population capture nucleic acids. A second population of capture nucleic acids can include (i) the complement of the clonal population of the modified target nucleic acid at each amplification site, where the 5’ end of the complement of the clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids, and (ii) a cleavage site. The clonal population of the modified target nucleic acid at each amplification site can be a member of a sequencing library. The method can further include contacting the amplification sites with a cleavage agent, thereby cleaving the second population of capture nucleic acids, and releasing the clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids. The method can further include removing the released clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids from the amplification sites. The method can further include contacting the plurality of amplification sites to a rcsynthcsis reagent including (i) a composition that includes nucleotide triphosphates (NTPs), where the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog, and (ii) a composition including a polymerase, and reacting the resynthesis reagent to produce a plurality of re-populated amplification sites attached to the array. The plurality of re -populated amplification sites each include a clonal population of a resynthesized target nucleic acid immobilized to the amplification sites at the 5’ end, where the clonal population of the resynthesized target nucleic acid includes a nucleic acid sequence that is a complement of the clonal population of the modified target nucleic acid of the providing step.

[0010] The present disclosure also provides arrays. In one embodiment, an array includes a plurality of populated amplification sites attached to the array, where the plurality of populated amplification sites each include a clonal population of amplicons from an individual modified target nucleic acid from a library of modified target nucleic acids. The amplicons include nucleotides dATP, dTTP, dGTP, dCTP, and a dATP analog.

[0011] The present disclosure also provides cartridges. A cartridge can be for use with a sequencing apparatus, and the cartridge can include a first chamber that has a nucleotide composition, where the nucleotide composition includes dATP, dTTP, dGTP, dCTP, and a dATP analog.IP-2872-PCT PATENT

[0012] The present disclosure also provides kits. A kit can be for use with a sequencing apparatus, and can include a cartridge. The cartridge can include a first chamber that has a nucleotide composition, where the nucleotide composition incudes dATP, dTTP, dGTP, dCTP, and a dATP analog.

[0013] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0014] As used herein, the term “amplification site” refers to a site in or on an array where one or more amplicons can be generated. An amplification site can be further configured to contain, hold or attach at least one amplicon that is generated at the site.

[0015] As used herein, the term “array” refers to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single target nucleic acid molecule having a particular sequence or a site can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof). The sites of an array can be different features located on the same substrate. Exemplary features include without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.

[0016] As used herein, the term “amplicon,” when used in reference to a nucleic acid, means the product of copying the nucleic acid, where the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, c.g., a target nucleic acid or an amplicon thereof, as aIP-2872-PCT PATENT template including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence (e.g., a polymerase extension product) or multiple copies of the nucleotide sequence (e.g., a concatemeric product of RCA). A first amplicon of a target nucleic acid is typically a complementary copy. Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target nucleic acid or from the first amplicon. A subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.

[0017] As used herein, the term “capture agent” refers to a material, chemical, molecule, or moiety thereof that is capable of attaching, retaining, or binding to a target molecule (e.g., a target nucleic acid). Exemplary capture agents include, without limitation, a capture nucleic acid that is complementary to at least a portion of a modified target nucleic acid (e.g., a universal capture binding sequence), a member of a receptor-ligand binding pair (e.g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.) capable of binding to a modified target nucleic acid (or linking moiety attached thereto), or a chemical reagent capable of forming a covalent bond with a modified target nucleic acid (or linking moiety attached thereto). In one embodiment, a capture agent is a nucleic acid. A nucleic acid capture agent can also be used as an amplification primer.

[0018] The terms “P5” and “P7” may be used when referring to a nucleic acid capture agent. The terms “P5”’ (P5 prime) and “P7”’ (P7 prime) refer to the complements of P5 and P7, respectively. It will be understood that any suitable nucleic acid capture agent can be used in the methods presented herein, and that the use of P5 and P7 are exemplary embodiments only. Uses of nucleic acid capture agents such as P5 and P7 on flow-cells is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957. One of skill in the art will recognize that a nucleic acid capture agent can also function as an amplification primer. For example, any suitable nucleic acid capture agent can act as a forward amplification primer, whether immobilized or in solution, and can be useful in the methods presented herein for hybridization to a sequence (e.g.,IP-2872-PCT PATENT a universal capture binding sequence) and amplification of a sequence. Similarly, any suitable nucleic acid capture agent can act as a reverse amplification primer, whether immobilized or in solution, and can be useful in the methods presented herein for hybridization to a sequence (e.g., a universal capture binding sequence) and amplification of a sequence. In view of the general knowledge available and the teachings of the present disclosure, one of skill in the art will understand how to design and use sequences that are suitable for capture and amplification of target nucleic acids as presented herein.

[0019] As used herein, the term “polymerase" is intended to be consistent with its use in the art and includes, for example, an enzyme that produces a complementary replicate of a nucleic acid molecule using the nucleic acid as a template strand. Typically, DNA polymerases bind to the template strand and then move down the template strand sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing strand of nucleic acid. DNA polymerases typically synthesize complementary DNA molecules from DNA templates and RNA polymerases typically synthesize RNA molecules from DNA templates (transcription). Polymerases can use a short RNA or DNA strand, called a primer, to begin strand growth. Some polymerases can displace the strand upstream of the site where they are adding bases to a chain. Such polymerases are said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase. Exemplary polymerases having strand displacing activity include, without limitation, the large fragment of Bsu (Bacillus subtilis), Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase or sequencing grade T7 exo-polymerase. Some polymerases degrade the strand in front of them, effectively replacing it with the growing chain behind (5' exonuclease activity). Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3' and / or 5' exonuclease activity. Different polymerases can be used at different times during the sequencing process, including library production (e.g., amplification or reverse transcription), production of clonal populations of amplicons at amplification sites (e.g., a polymerase for Exclusion Amplification or Bridge Amplification), or sequencing (e.g., a polymerase that can be used with 3'-blocked nucleotides).IP-2872-PCT PATENT

[0020] As used herein, the terms “nucleic acid” and “polynucleotide” are used interchangeably and are intended to be consistent with its use in the art and includes naturally occurring nucleic acids and functional analogs thereof. Particularly useful functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)). A nucleic acid can contain any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native bases. In this regard, a native deoxyribonucleic acid can have one or more bases selected from adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from uracil, adenine, cytosine or guanine. Useful non-native bases that can be included in a nucleic acid are known in the art. In some embodiments, non-native bases that can be included in a nucleic acid include a guanine modified as described herein (e.g., a guanine present in a dATP analog). The term “target,” when used in reference to a nucleic acid, is intended as a semantic identifier for the nucleic acid in the context of a method or composition set forth herein and does not necessarily limit the structure or function of the nucleic acid beyond what is otherwise explicitly indicated. A target nucleic acid having a universal sequence at each end, for instance a universal adapter at each end, can be referred to as a modified target nucleic acid.

[0021] As used herein, the symbol “” (hereinafter can be referred to as “a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example, “” indicates that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond.

[0022] The point of attachment of the organic group to the compound may be described in several ways. For example, in some embodiments, the chemical entity (or chemicalIP-2872-PCT PATENT group or moiety) may be described as the monovalent or radical of the respective functional group (e.g., alkyl for alkane, aryl for aromatic ring, aminyl for a primary or secondary amine). In some embodiments, where a general formula is shown with a covalent bond connecting a chemical moiety to a compound, the chemical moiety may be described as the common functional group. For example, if the organic group R is described relative to the formula CH3CH2CH2-R, the organic group may be described, for example, as an aromatic ring, sulfoxide, amine, or any other common functional group name.

[0023] As used herein, “alkyl” refers to a monovalent group that is a radical of an alkane and includes straight-chain, branched-chain, cyclic, and bicyclic alkyl groups, and combinations thereof, including both unsubstituted and substituted alkyl groups. Alkyl may be used to describe an alkane substituent attached to a compound. An alkyl substituent may include other functional groups, for example, including carbonyls, halogens, amines, and others.

[0024] The term "sulfonyl" means a divalent group of formula -SO2-. Sulfonyl may be used to describe a sulfone connected to a compound.

[0025] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0026] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0027] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.IP-2872-PCT PATENT

[0028] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open-ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0029] It is understood that wherever embodiments are described herein with the language "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. The term "consisting of" means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of" indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0030] Conditions that are "suitable" for an event to occur, or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and / or are conducive to the event.

[0031] As used herein, "providing" in the context of, for instance, an amplification or resynthesis reagent, an array, or a composition, means making the amplification or resynthesis reagent, an array, or composition, purchasing the amplification or resynthesis reagent, an array, or composition, or otherwise obtaining the amplification or resynthesis reagent, an array, or composition.

[0032] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.IP-2872-PCT PATENT

[0033] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0034] In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0035] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0036] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0037] BRIEF DESCRIPTION OF THE FIGURES

[0038] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0039] FIG. 1 shows a schematic representation of the direction of miscalls of bases depending on which signal, blue or green, is quenched.IP-2872-PCT PATENT

[0040] FIG. 2 shows examples of the quenching phenotype causing miscalling events. Sequencing reactions were performed as described in Example 1 and displayed as an Integrated Genome View (IGV) readout. Specific C to A and C to T miscalls are shown.

[0041] FIG. 3 shows the relative abundance of the DNA bases A, C, G, T in the regions of the human genome found to cause quenching most often on the Illumina NovaSeqX. The height of the letters in the figure indicate the relative abundance of each DNA base.

[0042] FIG. 4 shows a general block diagram of a portion of a general illustrative sequencing workflow including use of a dATP analog according to the present disclosure.

[0043] FIG. 5A - 5B show schematic drawings of embodiments that can occur during seeding of an amplification site and first strand synthesis. For simplicity, only one amplification site of an array and an associated target nucleic acid (FIG. 5A) of one amplification site of an array and an immobilized complement of a target nucleic acid (FIG. 5B) are shown. The figures use the following convention when numbering single strands of nucleic acids: the strand that is a member of a sequencing library is numbered (e.g., strand 21’ of FIG. 5A); the strand that is immobilized and is the complement of the strand that is a member of a sequencing library is numbered (e.g., strand 21 of FIG. 5B).

[0044] FIG. 6A-6D shows schematic drawings of an embodiment of producing clonal clusters. For simplicity, only one amplification site of an array and a limited number of target nucleic acids are shown.

[0045] FIG. 7A-7F shows schematic drawings of an embodiment of paired-end sequencing. For simplicity, only one amplification site of an array and a limited number of target nucleic acids are shown. The figures use the following convention when numbering capture nucleic acids: capture nucleic acids prior to cleavage are numbered (e.g., capture nucleic acid 23 of FIG. 7A); capture nucleic acids after cleavage are also numbered but the number is modified with the symbol " * " (e.g., strand 23* of FIG. 7B).

[0046] FIG. 8 shows the average percent mismatched bases (% Mismatched Bases in SSE) in the known quenching locations and Read 1 percent error (Read 1 %Error Rate) for the single read 1x151 BacPac runs. dATP analogues were spiked into the ExAmp reagentIP-2872-PCT PATENTECX1 at different concentrations relative to the natural dATP. X axis refers to the different dATP analogues. Cone Modified dATP refers to the percent ratio of dATP analogue spiked into 100% natural dATP, e.g., 0%, 2.5%, 10%, 20% or 25%. 7-deaza- dATP; 8-oxo-dATP; 7-deaza-7-bromo-dATP; 7-deaza-7-PA-dATP.

[0047] FIG. 9 shows the average percent mismatched bases (% Mismatched Bases in SSE) in the known quenching locations and Read 1 percent error (Read 1 %Error Rate) for the two read 2x151 BacPac runs. dATP analogues were spiked into the ExAmp reagent ECX1 and BridgeAmp (PET resynthesis) reagent JAM amplification mix at different concentrations relative to the natural dATP. Cone Modified dATP refers to the percent ratio of dATP analogue spiked into 100% natural dATP, e.g., 0%, 2.5%, or 20%.

[0048] FIG. 10 shows representative Integrated Genome Viewer (IGV) readouts for the quenching motif site for different modified dATP single read 1x151 BacPac runs. Boxes in the bottom of figure are locations where quenching and associated miscalling are highlighted.

[0049] FIG. 11 shows false positives plus false negatives in known quenching SSE regions in Illumina NextSeq2000 sequencing runs with human HG002 library, GATK variant caller with a range of concentrations of 2-amion-dATP added to clustering and amplification reagents.

[0050] FIG. 12 is a synthetic scheme for synthesizing 2-N(CH3)2-dATP (2-NMe2-dATP).

[0051] FIG. 13 is a synthetic scheme for synthesizing 7-SC>2Me-dATP (7-deaza-7-SC>2(CH3)- dATP).

[0052] The schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.IP-2872-PCT PATENT

[0053] DETAILED DESCRIPTION

[0054] Various fluorescence readouts are available for use in sequencing platforms, including one-, two- and four-channel chemistries. Two-channel chemistry requires dyes of two colors and two images to encode data for the four bases. For instance, sequencing platforms using two-channel approaches can use one image from a green channel and one image from a blue or a red channel. Intensities extracted from an image and compared to another image result in four distinct populations, each corresponding to a different base. Base calling determines the population to which each cluster belongs, and must be accurate or sequencing quality suffers. Certain DNA motifs have been found to quench the fluorescence output of dyes. One example of this type of motif is a CT rich quenching motif (GA rich DNA template). This motif can cause predominantly C to T miscalls (quenching of the blue signal). Other forms of miscalls, including C to A (quenching of the green signal) have also been identified (FIG. 1). FIG. 2 shows examples of C to A and C to T miscalling events due to fluorescence quenching. While this discussion is in the context of two-channel chemistries, the quenching can occur with one- and four-channel chemistries.

[0055] No single well-defined consensus motif has been identified, but it appears as if the downstream sequence (from the quench site) is more important that the upstream region, e.g., the nucleotides downstream of the quench site on the template strand influence quenching. These regions are largely characterized by being CT rich and / or having high volumes of G and A nucleotides in the template, suggesting that one type of this motif is predominantly occurring in purine rich environments. FIG. 3 shows the relative abundance of each DNA base in the most common quenching regions. The CTTC motif after incorporation is the most common, as seen in the first example in FIG. 2, but other CT rich motifs are also seen, as seen in the second example in FIG. 2. It was hypothesized that the occurrence of this specific quenching phenotype was due to the electronics of the template nucleotides surrounding these purine rich regions. One methodology for altering these environments to prevent these specific electronics from occurring is to change and / or modify the nucleotides in the template. Completely changing the template bases to different nucleotides is not feasible as it would lead to inaccurate representation of the original sample. Instead, the inventors theorized that modifications to the nucleotides, predominantly dATP, could alter the electronics of theIP-2872-PCT PATENT environment such that it prevents quenching. Any changes to dATP, however, that interfere with quenching must also be compatible with the reagents and methods used in a sequencing workflow. For instance, a modification of dATP used in the sequencing process must be compatible with one or more of cluster generation, sequencing of clusters, and paired-end turn methods.

[0056] A sequencing workflow can include sequencing library preparation (often including an amplification), cluster generation (often including seeding amplification sites, first strand extension, and amplification), sequencing (often including first read, paired end turn, and second read), and data analysis (FIG. 4). The present disclosure provides methods related to cluster generation and resynthesis between the first and second round of sequencing. Also included in the present disclosure are compositions, arrays, cartridges, and kits related to cluster generation and resynthesis. In particular, the methods, compositions, arrays, cartridges, and kits described herein include dATP analogs that reduce miscalls associated with quenching. The data obtained from subsequent sequencing includes increased representation of regions susceptible to quenching, reduced SSEs, increased output from regions susceptible to quenching, and increased quality from regions susceptible to quenching.

[0057] In one embodiment, a method of the present disclosure includes the use of dATP analogues during the production of amplification sites, e.g., during cluster formation (FIG. 4, block 32). A method can include providing an amplification reagent. An amplification reagent can include (i) an array of amplification sites, (ii) a plurality of modified target nucleic acids, (iii) nucleotide triphosphates (dNTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog, and (iv) a polymerase. In some embodiments, an amplification reagent does not include a dATP analog. The amplification reagent is reacted, for instance in an amplification reaction, to produce a plurality of populated amplification sites, where the plurality of populated amplification sites each include a clonal population of amplicons from an individual target nucleic acid from the plurality of target nucleic acids. When a dATP analog is present, the amplicons at the amplification sites will include the dATP analog incorporated in both strands. The dATP analog will be present in the amplicons at a level that is dependent on the ratio of dATP to dATP analog in the amplification reagent, and the quenching, e.g., the reduced intensity of fluorescence, that can occur during the subsequentIP-2872-PCT PATENT sequencing reaction will be decreased compared to the same amplicon that does not include a dATP analog. In embodiments where the method includes targeted sequencing (sequencing of specific regions of DNA), most or all of the amplification sites can include templates with regions that are GA rich. In other embodiments, only some of the populated amplification sites may include templates with regions that are GA rich.

[0058] In one embodiment, a method of the present disclosure includes the use of dATP analogues during the production of amplification sites, e.g., during cluster formation (FIG. 4, block 32).

[0059] In another embodiment, a method of the present disclosure includes the use of dATP analogues during paired-end turn resynthesis (FIG. 4, block 34). In one embodiment, a method includes providing a resynthesis reagent. A resynthesis reagent can include (i) an array of amplification sites, where each amplification site includes immobilized modified target nucleic acids, (ii) nucleotide triphosphates (dNTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog, and (iii) a polymerase. In some embodiments, a resynthesis reagent does not include a dATP analog. The resynthesis reagent is reacted to produce, at each amplification site, a population of strands that are complementary to the strand sequenced during the first round. The population of complementary strands are sequenced during the second round. When a dATP analog is present, the complementary strands will include the incorporated dATP analog. The dATP analog will be present in the complementary strands at a level that is dependent on the ratio of dATP to dATP analog present in the resynthesis reagent, and quenching will be reduced compared to the same amplicon that does not include a dATP analog.

[0060] dATP analogs

[0061] The methods, compositions, arrays, cartridges, and kits described herein can include 2'- deoxyadenosine triphosphate (dATP) analogs. The dATP analogs may aid in reducing quenching that can occur during sequencing. The nucleobases of the dATP analogs may include chemical functionalities that change the electronic properties of the aromatic ring system of dATP. To alter the electronics compared to dATP, the nucleobase of dATP analogs may include electron donating substituents and / or electron withdrawingIP-2872-PCT PATENT substituents on the purine ring system. Additionally or alternatively, the nucleobase of a dATP analog may have a different composition of atoms that make up the ring system. For example, the nucleobase of a dATP analog may have a C atom where an N atom is located in the ring system of dATP.

[0062] The dATP analogs can be used in certain DNA synthesis steps, for example, during cluster generation, resynthesis, or both. The terms “dATP analog” and “analog of dATP” are used interchangeably and refer to a compound having a nucleobase that differs from the nucleobase of dATP by addition of at least one component, removal of at least one component, exchange of at least one component, or any combination thereof. A component can be one or more atoms, one or more substituents, or one or more substructures. At least one component of nucleobase of dATP can be removed and replaced with at least one other component. The nucleobase of dATP has the structure shown in Formula 1. Formula 1 includes each atom in the purine ring system labelled. This numbering scheme is also used for the nucleobases of the dATP analogs described herein.

[0063] It is understood that when the nucleobases of dATP analogs are shown in a chemical formula, the point of attachment bond is covalently coupled to the T position of the deoxyribose of the dATP analog.Formula 1

[0064] In some embodiments, a dATP analog that may be used in the methods, compositions, arrays, cartridges, and kits described herein may be a substituted 2"-deoxyadenosine 5"- triphosphate (substituted dATP); 7-deaza-2"-deoxyadenosine 5 "-triphosphate (7-deaza- dATP; see Formula 2) or a substituted 7-deaza-2"-deoxy adenosine 5 "-triphosphate (substituted 7-deaza-dATP), or 8-oxo-2"-deoxyadenosine 5 '-triphosphate (8-oxo-dATP Formula 3).IP-2872-PCT PATENT

[0065] In some embodiments, the dATP analog includes a substituted dATP. A substituted dATP analog includes at least one substituent covalently bonded to an atom of the purine ring system of dATP that is different from the substituent bonded to the same atom in dATP. For example, in some embodiments, a substituted dATP analog may be a 2-substitutcd dATP where the carbon at the 2 position of the purine ring includes a substituent other than hydrogen.

[0066] In some embodiments, the dATP analog is 7-deaza-dATP (Formula 2) or a substituted 7-deaza-dATP. In 7-deaza-dATP, the nitrogen at position 7 of the purine in dATP is replaced with CH. A substituted 7-deaza-dATP includes at least one substituent covalently bonded to an atom of the ring system of 7-deaza-dATP that is different from the substituent bonded to the same atom in 7-deaza-dATP. For example, in some embodiments, a substituted 7-deaza-dATP includes a substituent other than hydrogen (H) at the 7 position of the ring system of 7-deaza-dATP.Formula 2

[0067] In some embodiments, the dATP analog is 8-oxo-dATP (Formula 3). 8-oxo-dATP includes a urea moiety spanning positions 7, 8, and 9 of the ring system.Formula 3

[0068] In some embodiments, the nucleobase of the dATP analog is of Formula 4. A nucleobase of an dATP analog that is of Formula 4 is a 2-substitued dATP.IP-2872-PCT PATENTFormula 4

[0069] In Formula 4, RAcan be halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (- OH), an amine, cyano or a group that includes a cynao moiety, or alkynyl or a group that includes an alkynyl moiety.

[0070] RAmay be halo. In some embodiments, RAis fluoro (F). In some embodiments, RAis iodo (I). In some embodiments, RAis chloro (Cl). In some embodiments, RAis bromo (Br).

[0071] RAmay be alkyl. The alkyl may be linear, branched, or cyclic. The alkyl may be a Cl to C6 alkyl. In some embodiments, RAis methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, or sec-butyl.

[0072] RAmay be acyl. RAmay be acyl of the formula -C(O)-R10where R10is alkyl. R10may be a Cl to C6 alkyl. In some embodiments, R10is methyl, ethyl, propyl, or isopropyl. In some embodiments, R1is -C(O)-CH3.

[0073] RAmay be trihaloalkyl. The trihaloalkyl includes three halos attached to the terminal carbon of an alkyl. The trihaloalkyl may be of the formula -(CH2)niC(X)3 where nl is 0, 1, 2, 3, or 4 and X is halo. X may be fluoro (F), bromo (Br), iodo (I), or chloro (Cl). In some embodiments, X is fluoro (F). In some embodiments n is 0 and X is fluoro (F). In some such embodiments, the trihaloalkyl can be referred to as trifluoromethyl.

[0074] RAmay be sulfinyl. The sulfinyl may be of the formula -S(O)-R20where R20is alkyl or trihaloalkyl. R20may be a Cl to C6 alkyl. In some embodiments, R20is methyl, ethyl, propyl, or isopropyl. When R20is trihaloalkyl, the trihaloalkyl may be of the formula - (CH2)niC(X)3 where nl is 0, 1, 2, 3, or 4 and X is halo. X may be fluoro (F), bromo (Br), iodo (I), or chloro (Cl). In some embodiments, X is fluoro (F). In some embodiments n is 1 and X is fluoro (F). In some embodiments, R1is -S(O)-CHj. In some embodiments, R1is -S(O)-CF3.IP-2872-PCT PATENT

[0075] RAmay be sulfonyl. The sulfonyl may be of the formula -S(O)2-R30where R30is alkyl or trihaloalkyl. In some embodiments, R30is methyl, ethyl, propyl, or isopropyl. In some embodiments, RAis a sulfonyl of the formula -S(O)2-CH3. When R30is trihaloalkyl, the trihaloalkyl may be of the formula -(CH2)niC(X)3 where nl is 0, 1, 2, 3, or 4 and X is halo. X may be fluoro (F), bromo (Br), iodo (I), or chloro (Cl). In some embodiments, X is fluoro (F). In some embodiments n is 1 and X is fluoro (F). In some embodiments, R1is -S(O)2-CH3. In some embodiments, R1is -S(O)2-CF3.

[0076] RAmay be an amine. The amine may be a primary, secondary, or tertiary amine. RAmay be an amine of the formula -NR1R2R3where each of R1, R2, and R3are independently H, alkyl, or a lone pair of electrons. The alkyl may be a Cl to C3 alkyl. For example, the alkyl may be methyl, ethyl, or propyl. In some embodiments, two of R1, R2, and R3are the same. For example, in some embodiments, R1and R2are H, and R3is a lone pair of electrons. In some embodiments, R1and R2are alkyl and R3is a lone pair of electrons. In some embodiments where R1and R2are both alkyl, the identity of the alkyl group for R1and R2is the same. In some embodiments where R1and R2are both alkyl, the identity of the alkyl group for R1and R2is different. In some embodiments, R1, R2, and R3are different. For example, in some embodiments, R1is H, R2alkyl, and R3is a lone pair of electrons. In some embodiments, RAis an amine of the formula -N(CH3)2.

[0077] RAmay be cyano or a group that includes a cyano moiety. RAmay be of the formula - (CH2)n2CN where n2 is 0, 1, 2, 3, or 4. In some embodiments, n2 is 1. In some embodiments, n2 is 2. In some embodiments, R1is -CN.

[0078] RAmay be alkynyl or a group that includes an alkynyl moiety. RAmay be of the formula -CC-(CH2)n3-R40where n3 is 1, 2, 3, or 4 and R40may be CH3or an amine. In some embodiments n3 is 1 or 2. The amine may be a primary amine, a secondary amine, or a tertiary amine. In some embodiments, the amine is a primary amine. In some embodiments, R1is -CC-(CH2)i-NH2.

[0079] In some embodiments, the nucleobase of the dATP analog is of Formula 5. A dATP analog of Formula 5 is a substituted 7-deaza-dATP analog.IP-2872-PCT PATENTformula 5

[0080] In Formula 5, RBcan be hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano or a group that includes a cyano moiety, or alkynyl or a group that includes an alkynyl moiety.

[0081] RBmay be halo. In some embodiments, R1is fluoro (F). In some embodiments, R1is iodo (I). In some embodiments, R1is chloro (Cl). In some embodiments, R1is bromo (Br).

[0082] RBmay be alkyl. The alkyl may be linear, branched, or cyclic. The alkyl may be a Cl to C6 alkyl. In some embodiments, RBis methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, or sec-butyl.

[0083] RBmay be acyl. RBmay be acyl of the formula -C(O)-R10where R10is alkyl. R10may be a Cl to C6 alkyl. In some embodiments, R10is methyl, ethyl, propyl, or isopropyl. In some embodiments, R1is -C(O)-CH3.

[0084] RBmay be trihaloalkyl. The trihaloalkyl includes three halos attached to the terminal carbon of an alkyl. The trihaloalkyl may be of the formula -(CH2)niC(X)3 where nl is 0, 1, 2, 3, or 4 and X is halo. X may be fluoro (F), bromo (Br), iodo (I), or chloro (Cl). In some embodiments, X is fluoro (F). In some embodiments n is 0 and X is fluoro (F). In some such embodiments, the trihaloalkyl can be referred to as trifluoromethyl.

[0085] RBmay be sulfinyl. The sulfinyl may be of the formula -S(O)-R20where R20is alkyl or trihaloalkyl. R20may be a Cl to C6 alkyl. In some embodiments, R20is methyl, ethyl, propyl, or isopropyl. When R20is trihaloalkyl, the trihaloalkyl may be of the formula - (CH2)niC(X)3 where nl is 0, 1, 2, 3, or 4 and X is halo. X may be fluoro (F), bromo (Br), iodo (I), or chloro (Cl). In some embodiments, X is fluoro (F). In some embodiments n is 1 and X is fluoro (F). In some embodiments, R1is -S(O)-CH3. In some embodiments, R1is -S(O)-CF3.IP-2872-PCT PATENT

[0086] RBmay be sulfonyl. The sulfonyl may be of the formula -S(O)2-R30where R30is alkyl or trihaloalkyl. In some embodiments, R30is methyl, ethyl, propyl, or isopropyl. In some embodiments, RAis a sulfonyl of the formula -S(O)2-CH?. When R30is trihaloalkyl, the trihaloalkyl may be of the formula -(CHzlniC X)? where nl is 0, 1, 2, 3, or 4 and X is halo. X may be fluoro (F), bromo (Br), iodo (I), or chloro (Cl). In some embodiments, X is fluoro (F). In some embodiments n is 1 and X is fluoro (F). In some embodiments, R1is -S(O)2-CH3. In some embodiments, R1is -S(O)2-CF3.RBmay be amine. The amine may be a primary, secondary, or tertiary amine. RBmay be an amine of the formula -NR1R2R3where each of R1, R2, and R3are independently H, alkyl, or a lone pair of electrons. The alkyl may be a Cl to C3 alkyl. For example, the alkyl may be methyl, ethyl, or propyl. In some embodiments, two of R1, R2, and R3are the same. For example, in some embodiments, R1and R2are H, and R3is a lone pair of electrons. In some embodiments, R1and R2are alkyl and R3is a lone pair of electrons. In some embodiments where R1and R2arc both alkyl, the identity of the alkyl group for R1and R2is the same. In some embodiments where R1and R2are both alkyl, the identity of the alkyl group for R1and R2different. In some embodiments, R1, R2, and R3are different. For example, in some embodiments, R1is H, R2alkyl, and R3is a lone pair of electrons. In some embodiments, RBis an amine of the formula -N(CH )2.

[0087] RBmay be cyano or a group that that includes a cyano moiety. RBmay be of the formula -(CH2)n2CN where n2 is 0, 1 , 2, 3, or 4. In some embodiments, n2 is 1. In some embodiments, n2 is 2. In some embodiments, RBis -CN.

[0088] RBmay be alkynyl or a group that includes an alkynyl moiety. RBmay be of the formula -CC-(CH2)n3-R40where n3 is 1 , 2, 3, or 4 and R40may be CH3 or an amine. In some embodiments n3 is 1 or 2. The amine may be a primary amine, a secondary amine, or a tertiary amine. In some embodiments, the amine is a primary amine. In some embodiments, R1is -CC-(CH2)I-NH2.

[0089] Table 1 provides the structure and the name of exemplary dATP analogs. Only the nucleobase portion of the dATP analog is shown. The point of attachment bond is covalently coupled to the 1' position of the deoxyribose of the dATP analog.IP-2872-PCT PATENTTable 1: Exemplary dATP analogsIP-2872-PCT PATENT

[0090] Arrays

[0091] Some embodiments of the methods, compositions, cartridges, and kits described herein include an array of amplification sites. An array of amplification sites can be present as one or more substrates. Exemplary types of substrate materials that can be used for an array include glass, modified glass, functionalized glass, inorganic glasses, microspheres (e.g., inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, an optical fiber or optical fiber bundles, polymers and multiwell (e.g., microtiter) plates. Exemplary plastics include acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes and Teflon™. Exemplary silica-based materials include silicon and various forms of modified silicon.

[0092] In particular embodiments, a substrate can be within or part of a vessel such as a well, tube, channel, cuvette, Petri plate, bottle or the like. A particularly useful vessel is a flow-cell, for example, as described in US Pat. No. 8,241,573 or Bentley et al., Nature 456:53-59 (2008). Exemplary flow-cells are those that are commercially available from Illumina, Inc. (San Diego, Calif.). Another particularly useful vessel is a well in a multiwell plate or microtiter plate.

[0093] In some embodiments, the sites of an array can be configured as features on a surface. The features can be present in any of a variety of desired formats. For example, the sites can be wells, pits, channels, ridges, raised regions, pegs, posts or the like. As set forth herein, the sites can contain beads. However, in particular embodiments the sites need not contain a bead or particle. Exemplary sites include wells that are present in substrates used for commercial sequencing platforms sold by 454 LifeSciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other substrates having wells include, for example, etched fiber optics and other substrates described in U.S. Pat. No. 6,266,459; U.S. Pat.IP-2872-PCT PATENTNo. 6,355,431; U.S. Pat. No. 6,770,441; U.S. Pat. No. 6,859,570; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; U.S. Pat. No. 6,274,320; U.S. Pat No. 8,262.900; U.S. Pat. No. 7,948,015; U.S. Pat. Pub. No. 2010 / 0137143; U.S. Pat. No. 8,349,167, or PCT Publication No. WO 00 / 63437. In several cases the substrates are exemplified in these references for applications that use beads in the wells. The well-containing substrates can be used with or without beads in the methods or compositions of the present disclosure. In some embodiments, wells of a substrate can include gel material (with or without beads) as set forth in U.S. Pat. No. 9,512,422.

[0094] The sites of an array can be metal features on a non-metallic surface such as glass, plastic or other materials exemplified herein. A metal layer can be deposited on a surface using methods known in the art such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering, or the like. Any of a variety of commercial instruments can be used as appropriate including, for example, the FlexAL®, OpAL®, lonfab 300Plus®, or Optofab 3000® systems (Oxford Instruments, UK). A metal layer can also be deposited by e-beam evaporation or sputtering as set forth in Thornton, Ann. Rev. Mater. Sci. 7:239-60 (1977). Metal layer deposition techniques, such as those exemplified herein, can be combined with photolithography techniques to create metal regions or patches on a surface. Exemplary methods for combining metal layer deposition techniques and photolithography techniques are provided in U.S. Pat. No. 8,778,848 and U.S. Pat. No. 8,895,249.

[0095] In particular embodiments, an array can include a collection of beads or other particles. The particles can be suspended in a solution or they can be located on the surface of a substrate. Examples of bead arrays in solution are those commercialized by Luminex (Austin, Tex.). Examples of arrays having beads located on a surface include those wherein beads are located in wells such as a BeadChip array (Illumina Inc., San Diego Calif.) or substrates used in sequencing platforms from 454 LifeSciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other arrays having beads located on a surface are described in U.S. Pat. No. 6,266,459; U.S. Pat. No. 6,355,431; U.S. Pat. No. 6,770,441; U.S. Pat. No. 6,859,570; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; U.S. Pat. No. 6,274,320; US 2009 / 0026082 Al ; US 2009 / 0127589 Al ; US 2010 / 0137143 Al ; US 2010 / 0282617IP-2872-PCT PATENTAl, or PCT Publication No. WO 00 / 63437. Several of the above references describe methods for attaching target nucleic acids to beads prior to loading the beads in or on an array substrate. It will, however, be understood that the beads can be made to include amplification primers and the beads can then be used to load an array, thereby forming amplification sites for use in a method set forth herein. As set forth previously herein, the substrates can be used without beads. For example, amplification primers can be attached directly to the wells or to gel material in wells. Thus, the references are illustrative of materials, compositions or apparatus that can be modified for use in the methods and compositions set forth herein.

[0096] In particular embodiments, a capture agent, such as a capture nucleic acid, can be attached to the amplification site. For example, the capture agent can be attached to the surface of a feature of an array. The attachment can be via an intermediate structure such as a bead, particle, or gel. An example of attachment of capture nucleic acids to an array via a gel is described in U.S. Pat. No. 8,895,249 and further exemplified by flowcells available commercially from Illumina Inc. (San Diego, Calif.) or described in WO 2008 / 093098. Exemplary gels that can be used in the methods and apparatus set forth herein include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide, SFA (see, for example, US Pat. App. Pub. No. 2011 / 0059865 Al ) or PAZAM (see, for example, U.S. Prov. Pat. App. Ser. No. 61 / 753,833 and U.S. Pat. No. 9,012,022). Attachment via a bead can be achieved as exemplified in the description and cited references set forth previously herein.

[0097] Amplification sites of an array can include a plurality of capture agents capable of binding to target nucleic acids. In one embodiment, a capture agent includes a capture nucleic acid. In typical conditions used to prepare arrays for sequencing, the nucleotide sequence of the capture nucleic acid is complementary to a sequence of one or more modified target nucleic acids, such as a universal capture binding sequence present on a target nucleic acid. In some embodiments, the capture nucleic acid can also function as a primer for amplification of the modified target nucleic acid. In some embodiments, one population of capture nucleic acid includes a P5 primer or the complement thereof, and the second population of capture nucleic acid includes a P7 primer or the complement thereof.IP-2872-PCT PATENT

[0098] A capture nucleic acid can be immobilized by single point covalent attachment to an array at or near the 5' end of the capture nucleic acid, leaving the template-specific portion of the capture nucleic acid free to anneal to its cognate universal capture binding sequence and the 3’ hydroxyl group free for extension. Any suitable covalent attachment means known in the art may be used for this puipose. The chosen attachment chemistry will depend on the nature of the solid support, and any derivatization or functionalization applied to it. The capture nucleic acid itself may include a moiety, which may be a non-nucleotide chemical modification, to facilitate attachment. In a particular embodiment, the primer may include a sulphur-containing nucleophile, such as phosphorothioate or thiophosphate, at the 5’ end.

[0099] In some embodiments, the features on the surface of an array substrate are noncontiguous, being separated by interstitial regions of the surface. Interstitial regions that have a substantially lower quantity or concentration of capture agents, compared to the features of the array, are advantageous. Interstitial regions that lack capture agents are particularly advantageous. For example, a relatively small amount or absence of capture moieties at the interstitial regions favors localization of target nucleic acids, and subsequently generated clusters, to desired features. In particular embodiments, the features can be concave features in a surface (e.g., wells) and the features can contain a gel material. The gel-containing features can be separated from each other by interstitial regions on the surface where the gel is substantially absent or, if present the gel is substantially incapable of supporting localization of nucleic acids. Methods and compositions for making and using substrates having gel containing features, such as wells, are set forth in U.S. Pat. No. 9,512,422. The size of the features and / or spacing between the regions can vary such that arrays can be high density, medium density or lower density. High density arrays are characterized as having regions separated by less than about 15 pm. Medium density arrays have regions separated by about 15 to 30 pm, while low density arrays have regions separated by greater than 30 pm. An array useful in the disclosure can have regions that are separated by less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm or 0.5 pm.

[0100] In some embodiments, the solid support comprises a patterned surface. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more of the regions can be features where one orIP-2872-PCT PATENT more amplification primers are present. In some embodiments, the pattern can be an x-y format of features that are in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and / or interstitial regions. In some embodiments, the pattern can appear as a grid of spots or patches. The features can be located in a repeating pattern or in an irregular non-repeating pattern. Particularly useful patterns are hexagonal patterns, rectilinear patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. Asymmetric patterns can also be useful. The pitch can be the same between different pairs of nearest neighbor features or the pitch can vary between different pairs of nearest neighbor features. In particular embodiments, features of an array can each have an area that is larger than about 100 nm2, 250 nm2, 500 nm2, 1 pm2, 2.5 pm2, 5 pm2, 10 pm2, 100 pm2, or 500 pm2. Alternatively, or additionally, features of an array can each have an area that is smaller than about 1 mm2, 500 prn2. 100 pm2. 25 pm2. 10 m2, 5 m2, 1 m2, 500 nm2, or 100 nm2. Indeed, a region can have a size that is in a range between an upper and lower limit selected from those exemplified above. Exemplary patterned surfaces that can be used in the methods and compositions set forth herein are described in U.S. Pat. Nos. 8,778,848, 8,778,849 and 9,079,148, and U.S. Pat. Appl. Pub. No. 2014 / 0243224.

[0101] The features in a patterned surface can be wells in an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic or other suitable solid supports with patterned, covalently-linked gel such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co- acrylamide) (PAZAM, see, for example, US Pub. No. 2013 / 184796, WO 2016 / 066586, and WO 2015 / 002813). The process can create gel pads used for sequencing that can be stable over sequencing runs with a large number of cycles. The covalent linking of the polymer to the wells is helpful for maintaining the gel in the structured features throughout the lifetime of the structured substrate during a variety of uses. However, in many embodiments the gel need not be covalently linked to the wells. For example, in some conditions silane free acrylamide (SFA, see, for example, US Pat. No. 8,563,477) which is not covalently attached to any part of the structured substrate, can be used as the gel material.IP-2872-PCT PATENT

[0102] In particular embodiments, a structured substrate can be made by patterning a solid support material with wells (e.g., microwells or nanowells), coating the patterned support with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof, such as the azidolyzed version of SFA (azido-SFA)) and polishing the gel coated support, for example via chemical or mechanical polishing, thereby retaining gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions on the surface of the structured substrate between the wells. Primer nucleic acids can be attached to gel material. A solution of modified target nucleic acids can then be contacted with the polished substrate such that individual modified target nucleic acids will seed individual wells via interactions with primers attached to the gel material; however, the target nucleic acids will not occupy the interstitial regions due to absence or inactivity of the gel material. Amplification of the modified target nucleic acids will be confined to the wells since absence or inactivity of gel in the interstitial regions prevents outward migration of the growing nucleic acid colony. The process can be conveniently manufactured, being scalable and utilizing conventional micro- or nanofabrication methods.

[0103] Target nucleic acids

[0104] Some embodiments of the methods, compositions, arrays, cartridges, and kits described herein include target nucleic acids. The terms “target nucleic acid,” “target fragment,” “target nucleic acid fragment, “target molecule,” and “target nucleic acid molecule” are used interchangeably to refer to nucleic acid molecules that are to be sequenced, such as on an array. The target nucleic acid may be essentially any nucleic acid of known or unknown sequence. It may be, for example, a fragment of genomic DNA or cDNA. Sequencing may result in determination of the sequence of the whole, or a part of the target molecule. The targets can be derived from a primary nucleic acid sample that has been randomly fragmented. In one embodiment, the targets can be processed into templates suitable for amplification by the placement of universal amplification sequences, e.g., sequences present in a universal adaptor.

[0105] The primary nucleic acid sample may originate in double-stranded DNA (dsDNA) form (e.g., genomic DNA fragments, amplification products and the like) from a sample or may have originated in single-stranded form from a sample, as DNA or RNA, and been converted to dsDNA form. By way of example, mRNA molecules may be copied intoIP-2872-PCT PATENT double- stranded cDNAs suitable for use in a method described herein using standard techniques well known in the art. The precise sequence of the polynucleotide molecules from a primary nucleic acid sample is generally not material to the disclosure, and may be known or unknown.

[0106] In one embodiment, the primary polynucleotide molecules from a primary nucleic acid sample are DNA molecules. More particularly, the primary polynucleotide molecules represent the entire genetic complement of an organism, and are genomic DNA molecules which include both intron and exon sequences, as well as non-coding regulatory sequences such as promoter and enhancer sequences. In one embodiment, particular sub-sets of polynucleotide sequences or genomic DNA can be used, such as, for example, particular chromosomes. Yet more particularly, the sequence of the primary polynucleotide molecules is not known. Still yet more particularly, the primary polynucleotide molecules are human genomic DNA molecules. The DNA target nucleic acids may be treated chemically or enzymatically either prior or subsequent to any random fragmentation processes, and prior or subsequent to the ligation of a universal sequence, such as universal adapter sequences.

[0107] The nucleic acid sample can include high molecular weight material such as genomic DNA (gDNA). The sample can include low molecular weight material such as nucleic acid molecules obtained from FFPE or archived DNA samples. In another embodiment, low molecular weight material includes enzymatically or mechanically fragmented DNA. The sample can include cell- free circulating DNA. A sample can include, but is not limited to, nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture micro-dissections, surgical resections, and other clinical or laboratory obtained samples. In some embodiments, the sample can be an epidemiological, agricultural, forensic or pathogenic sample.

[0108] The biological source of a sample is not intended to be limiting. In some embodiments, the sample can include nucleic acid molecules obtained from a eukaryote, such as an animal or a plant. Examples of an animal include, but are not limited to, a mammal including a human. In some embodiments, the sample can include nucleic acid molecules obtained from a prokaryote, such as a bacterium or archaeon. In some embodiments, the sample can include nucleic acid molecules obtained from a virus. InIP-2872-PCT PATENT some embodiments, the source of the nucleic acid molecules may be an archived or extinct sample or species.

[0109] Random fragmentation refers to the fragmentation of a polynucleotide molecule from a primary nucleic acid sample in a non-ordered fashion by enzymatic, chemical or mechanical means. Such fragmentation methods are known in the art and use standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition). In one embodiment, enzymatic fragmentation can be accomplished using a process often referred to as tagmentation. Tagmentation uses a transposome complex that can include both transposon and transposase and combines into a single step fragmentation and ligation to add universal sequences that can be used as universal adapters or for the addition of other universal sequences (Gunderson et al., WO 2016 / 130704). For the sake of clarity, generating smaller fragments of a larger piece of nucleic acid via specific PCR amplification of such smaller fragments is not equivalent to fragmenting the larger piece of nucleic acid because the larger piece of nucleic acid sequence remains in intact (i.e., is not fragmented by the PCR amplification). Moreover, random fragmentation is designed to produce fragments irrespective of the sequence identity or position of nucleotides comprising and / or surrounding the break. More particularly, the random fragmentation is by mechanical means such as nebulization or sonication to produce fragments of about 50 base pairs in length to about 1 00 base pairs in length, still more particularly 50-700 base pairs in length, yet more particularly 50-400 base pairs in length. Most particularly, the method is used to generate smaller fragments of from 50-150 base pairs in length.

[0110] Fragmentation of polynucleotide molecules by mechanical means (nebulization, sonication and Hydroshear, for example) results in fragments with a heterogeneous mix of blunt and 3'- and 5'-overhanging ends. It is therefore desirable to repair the fragment ends using methods or kits (such as the Lucigen DNA terminator End Repair Kit) known in the art to generate ends that are optimal for insertion, for example, into blunt sites of cloning vectors. In a particular embodiment, the fragment ends of the population of nucleic acids are blunt ended. More particularly, the fragment ends are blunt ended and phosphorylated. The phosphate moiety can be introduced via enzymatic treatment, for example, using polynucleotide kinase.IP-2872-PCT PATENT

[0111] A population of target nucleic acids, or amplicons thereof, can have an average strand length that is desired or appropriate for a particular application of the methods or compositions set forth herein. For example, the average strand length can be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides. Alternatively, or additionally, the average strand length can be greater than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or 100,000 nucleotides. The average strand length for population of target nucleic acids, or amplicons thereof, can be in a range between a maximum and minimum value set forth above. It will be understood that amplicons generated at an amplification site (or otherwise made or used herein) can have an average strand length that is in a range between an upper and lower limit selected from those exemplified above.

[0112] In some cases, a population of target nucleic acids can be produced under conditions or otherwise configured to have a maximum length for its members. For example, the maximum length for the members that are used in one or more steps of a method set forth herein or that are present in a particular composition can be less than 100,000 nucleotides, less than 50,000 nucleotides, less than 10,000 nucleotides, less than 5,000 nucleotides, less than 1,000 nucleotides, less than 500 nucleotides, less than 100 nucleotides, or less than 50 nucleotides. Alternatively, or additionally, a population of target nucleic acids, or amplicons thereof, can be produced under conditions or otherwise configured to have a minimum length for its members. For example, the minimum length for the members that are used in one or more steps of a method set forth herein or that are present in a particular composition can be more than 10 nucleotides, more than 50 nucleotides, more than 100 nucleotides, more than 500 nucleotides, more than 1 ,000 nucleotides, more than 5,000 nucleotides, more than 10,000 nucleotides, more than 50,000 nucleotides, or more than 100,000 nucleotides. The maximum and minimum strand length for target nucleic acids in a population can be in a range between a maximum and minimum value set forth above. It will be understood that amplicons generated at an amplification site (or otherwise made or used herein) can have maximum and / or minimum strand lengths in a range between the upper and lower limits exemplified above.IP-2872-PCT PATENT

[0113] In particular embodiments, the target nucleic acids are sized relative to the area of the amplification sites, for example, to facilitate exclusion amplification. For example, the area for each of the sites of an array can be greater than the diameter of the excluded volume of the target nucleic acids in order to achieve exclusion amplification. Taking, for example, embodiments that use an array of features on a surface, the area for each of the features can be greater than the diameter of the excluded volume of the target nucleic acids that are transported to the amplification sites. The excluded volume for a target nucleic acid and its diameter can be determined, for example, from the length of the target nucleic acid. Methods for determining the excluded volume of nucleic acids and the diameter of the excluded volume are described, for example, in U.S. Pat. No. 7,785,790; Rybenkov et al., Proc. Natl. Acad. Sci. U.S.A. 90: 5307-5311 (1993); Zimmerman et al., J. Mol. Biol. 222:599-620 (1991); or Sobel et al., Biopolymers 31: 1559-1564 (1991).

[0114] In a particular embodiment, the target fragment sequences are prepared with single overhanging nucleotides by, for example, activity of certain types of DNA polymerase such as Taq polymerase or Klenow exo minus polymerase which has a non-templatedependent terminal transferase activity that adds a single deoxynucleotide, for example, deoxyadenosine (A) to the 3' ends of a DNA molecule, for example, a PCR product. Such enzymes can be used to add a single nucleotide ‘A’ to the blunt ended 3' terminus of each strand of the double-stranded target fragments. Thus, an ‘A’ could be added to the 3' terminus of each end repaired strand of the double-stranded target fragments by reaction with Taq or Klenow exo minus polymerase, while a universal adapter polynucleotide construct could be a T-construct with a compatible ‘T’ overhang present on the 3' terminus of each region of double stranded nucleic acid of the universal adapter. This end modification also prevents self- ligation of both vector and target such that there is a bias towards formation of the combined ligated adaptor-target-adaptor molecules.

[0115] Sequencing Library Preparation

[0116] A sequencing library of the methods, compositions, arrays, cartridges, and kits described herein typically includes a target nucleic acid having a universal adapter attached one or both ends. The terms “target nucleic acid,” “target fragment,” “target nucleic acid fragment,” “target molecule,” and “target nucleic acid molecule” are usedIP-2872-PCT PATENT interchangeably to refer to nucleic acid molecules that are to be sequenced. A target nucleic acid having a universal adapter on one or both ends can be referred to as a "modified target nucleic acid." A library of target nucleic acids refers to the collection of target nucleic acids containing known common sequences at their 3' and 5' ends, and may also be referred to as a 3' and 5' modified library.

[0117] Methods for attaching a universal adapter to one of both ends of a target nucleic acid are known to the person skilled in the art. The attachment can be through standard library preparation techniques using ligation (Chesney et al. U.S. Pat. Pub. No. 2018 / 0305753 Al), through tagmentation using transposase complexes (Gunderson et al., WO 2016 / 130704), or primer extension, for instance when preparing a sample for targeted sequencing.

[0118] Target nucleic acids are often amplified during sequencing library preparation. Amplification of modified target nucleic acids during sequencing library preparation can be by linear amplification, exponential amplification, or both linear and exponential amplification steps. Amplification conditions useful during sequencing library preparation are routine and known to the person of ordinary skill in the art. For instance, amplification profiles (e.g., number of cycles and the temperature and time of each cycle), and concentrations of target nucleic acids, buffers, ions, dNTPs, and polymerase are known or can be easily determined using commercially available algorithms.

[0119] In one embodiment, double- stranded target nucleic acids from a sample, e.g., a fragmented sample, are treated by first ligating identical universal adaptor molecules to the 5' and 3' ends of the double-stranded target nucleic acids (which may be of known, partially known or unknown sequence). In some embodiments, the identical universal adaptor molecules can be ‘mismatched adaptors’, the general features of which are defined below, and further described in Gormley et al., US 7,741,463, and Bignell et al., US 8,053,192). In some embodiments, the identical universal adaptor molecules can include fully complementary polynucleotide strands. A universal adaptor typically includes the universal capture binding sequences that aid in immobilizing the target nucleic acids on an array for subsequent cluster generation. In one embodiment, library preparation of target nucleic acids having universal adaptor molecules at the 5' and 3'IP-2872-PCT PATENT ends includes one or more amplification, for instance by PCR, before immobilizing the target nucleic acids on an array for subsequent cluster generation.

[0120] In some embodiments, for instance when a universal adapter is added by tagmentation, it is desirable to modify the universal adapter present at each end of target nucleic acids before cluster generation. The modification can occur by an amplification step, such as PCR. For instance, an initial primer extension reaction is carried out using a universal primer binding site in which extension products complementary to both strands of each target nucleic acid are formed and add a universal capture binding sequence. The resulting primer extension products, and amplified copies thereof, collectively provide a library of modified target nucleic acids that can be immobilized, clonally expanded to form clusters, and then sequenced. In some embodiments, a library includes target nucleic acids originating from the same source, e.g., the same tissue, same cell, and / or same individual (for instance, a sample of cell-free DNA). The 3’ ends, and optionally the 5’ ends, of the universal adapters attached to the target nucleic acids can include a homogeneous population or a heterogeneous population of universal capture binding sequences described herein.

[0121] Generally, amplification reactions require at least two amplification primers, often denoted 'forward' and 'reverse' primers (primer oligonucleotides) that are capable of annealing specifically to a part of the nucleic acid sequence to be amplified, e.g., a universal adapter at the ends of target nucleic acids, under conditions encountered in the primer annealing step of each cycle of an amplification reaction. It will be understood by the skilled person that if the primers contain any nucleotide sequence which does not anneal to the modified target nucleic acids in the first amplification cycle then this sequence may be copied into the amplification products. For instance, the use of primers having universal capture binding sequences, i.e., sequences that do not anneal to the universal adapter at the ends of target nucleic acids, the universal capture binding sequences will be incorporated into the resulting amplicon.

[0122] Amplification primers are generally single stranded polynucleotide structures. They may also contain a mixture of natural and non-natural bases and also natural and nonnatural backbone linkages, provided that any non-natural modifications does not preclude function as a primer-that being defined as the ability to anneal to a template polynucleotide strand during conditions of the amplification reaction and to act as anIP-2872-PCT PATENT initiation point for synthesis of a new polynucleotide strand complementary to the template strand. Primers may additionally include non-nucleotide chemical modifications, for example phosphorothioates to increase exonuclease resistance, again provided such that modifications do not prevent primer function.

[0123] In some embodiments, the universal adapters used in the method of the disclosure are referred to as ‘mismatched’ adaptors because the adaptors include a region of sequence mismatch, i.e., they are not formed by annealing of fully complementary polynucleotide strands. Mismatched adaptors for use herein typically include at least one doublestranded region, also referred to as a region of double stranded nucleic acid, and at least one unmatched single-stranded region, also referred to as a region of single-stranded non-complementary nucleic acid strands. Mismatched adapters are routinely used in producing sequencing libraries, and the characteristics of useful mismatched adapters are known to the skilled person.

[0124] The ‘double- stranded region’ of the universal adapter is a short double-stranded region, typically including 5 or more consecutive base pairs, formed by annealing of the two partially complementary polynucleotide strands. As used herein, the term “double stranded,” when used in reference to a nucleic acid molecule, means that substantially all of the nucleotides in the nucleic acid molecule are hydrogen bonded to a complementary nucleotide. A partially double stranded nucleic acid can have at least 10%, 25%, 50%, 60%, 70%, 80%, 90% or 95% of its nucleotides hydrogen bonded to a complementary nucleotide.

[0125] The double-stranded region can form the ‘ligatable’ end of the adaptor, e.g., the end that is joined to a double-stranded target nucleic acid in the ligation reaction. The ligatable end of the universal adaptor may be blunt or, in other embodiments, short 5' or 3' overhangs of one or more nucleotides may be present to facilitate / promote ligation. The 5' terminal nucleotide at the ligatable end of the universal adapter is typically phosphorylated to enable phosphodiester linkage to a 3' hydroxyl group on the target polynucleotide.

[0126] The term ‘unmatched region’ refers to a region of the universal adaptor, the region of single-stranded non-complementary nucleic acid strands, wherein the sequences of the two polynucleotide strands forming the universal adaptor exhibit a degree of non-IP-2872-PCT PATENT complementarity such that the two strands are not capable of fully annealing to each other under standard annealing conditions for a primer extension or PCR reaction. The unmatched region(s) may exhibit some degree of annealing under standard reaction conditions for an enzyme-catalyzed ligation reaction, provided that the two strands revert to single stranded form under annealing conditions in an amplification reaction.

[0127] A universal adapter can include at least one universal primer binding site. A universal primer binding site is a universal sequence that can be used for amplification and / or sequencing of a target nucleic acid attached to the universal adapter. Examples of universal primer binding sites include, but are not limited to, sequences complementary to a Readl or Read2 primer.

[0128] A universal adapter can include at least one index. An index can be used as a marker characteristic of the source of particular target nucleic acid on an array. Generally, the index is a synthetic sequence of nucleotides that is part of the universal adapter which is added to the target nucleic acids as part of the library preparation step. Accordingly, an index is a nucleic acid sequence which is attached to each of the target molecules of a particular sample, the presence of which is indicative of, or is used to identify, the sample or source from which the target molecules were isolated.

[0129] In some embodiments, the index may be up to 20 nucleotides in length, more preferably 1-10 nucleotides, and most preferably 4-8 nucleotides in length. For example, a four- nucleotide index gives a possibility of multiplexing 256 (44) samples on the same array, whereas a six base index enables 4,096 (4G) samples to be processed on the same array.

[0130] In one embodiment, the universal capture binding sequence and / or universal primer binding site is part of the universal adapter when it is ligated to the double-stranded target fragments, and in another embodiment the universal capture binding sequence and / or universal primer binding site is added to the universal adapter after the universal adapter is ligated to the double- stranded target fragments. The addition can be accomplished using routine methods, including PCR-based methods.

[0131] The precise nucleotide sequence of the universal adapters is generally not material to the invention and may be selected by the user such that the desired sequence elements are ultimately included in the common sequences of the plurality of different modified target nucleic acids, for example, to provide for the universal capture binding sequencesIP-2872-PCT PATENT and universal primer binding sites for particular sets of universal primers. Additional sequence elements may be included, for example, to provide binding sites for sequencing primers, e.g., Readl and Read2 primers, which will ultimately be used in sequencing of target nucleic acids in the library, or products derived from amplification of the target nucleic acids in the library, for example on a solid support. In some embodiments, a universal adapter may include mixtures of natural and non-natural nucleotides (e.g., one or more ribonucleotides) linked by a mixture of phosphodiester and non-phosphodiester backbone linkages.

[0132] Ligation methods for adding a universal adapter to a target nucleic acid are known in the art and use standard methods. Such methods use ligase enzymes such as DNA ligase to effect or catalyze joining of the ends of the two polynucleotide strands of, in this case, the universal adapter and the double- stranded target nucleic acids, such that covalent linkages are formed. The universal adapter may contain a 5 '-phosphate moiety to facilitate ligation to the 3'-OH present on the target fragment. The double- stranded target nucleic acid contains a 5'-phosphate moiety, either residual from the shearing process, or added using an enzymatic treatment step, and has been end repaired, and optionally extended by an overhanging base or bases, to give a 3'-OH suitable for ligation.

[0133] As discussed herein, in one embodiment universal adaptors used in the ligation are complete and include a universal capture binding sequence and other universal sequences, e.g., a universal primer binding site and an index sequence. The resulting plurality of modified target nucleic acids can be amplified before immobilization for sequencing. Also, as discussed herein, in one embodiment universal adaptors used in the ligation include a universal primer binding site and an index sequence, and do not include a universal capture binding sequence. The resulting plurality of modified target nucleic acids can be further modified to include specific sequences, such as a universal capture binding sequence, and can be amplified before immobilization for sequencing.

[0134] Immobilizing Modified Target Nucleic Acids at Amplification Sites and Production of Clonal Clusters

[0135] The present disclosure includes methods, compositions, arrays, cartridges, and kits related to initial steps of cluster generation, e.g., seeding amplification sites and / or firstIP-2872-PCT PATENT strand extension. In one embodiment, a method of the present disclosure can include contacting a plurality of amplification sites of an array with a single- stranded sequencing library. Each amplification site of an array includes at least one, and in some embodiments two or more populations of capture agents immobilized to the amplification sites. The method includes using conditions suitable for attaching the universal adapter to one of the capture agents to result in a plurality of amplification sites that each include one member of the sequencing library. The conditions useful for the attaching are routinely used in sequencing workflows and are known to the skilled person.

[0136] In embodiments where the modified target nucleic acids include at least one universal capture binding sequence and a complementary capture nucleic acid is present in one of the immobilized capture agents, sequences of the universal capture binding sequence and the complementary capture nucleic acid hybridize to result in a plurality of amplification sites that each include one member of the sequencing library. The addition of a member of a sequencing library to an amplification site is referred to as “seeding” the site (FIG. 4, block 31). The seeding can be accomplished by use of a seeding reagent. A seeding reagent can include an array of amplification sites and a plurality of target nucleic acids. An example is shown in FIG. 5A, which shows an amplification site 20 containing an immobilized capture agent 24 and a member of a sequencing library 21’. The 3’ end of the member of the sequencing library 21’ is hybridized to a complementary capture nucleic acid that is present in the universal capture binding sequence 25. The skilled person will recognize that some amplification sites can include more than one member of the sequencing library at this stage and not significantly reduce the ability to obtain useful data from the subsequent sequencing reaction. The skilled person will also recognize that not all amplification sites of an array need to be occupied.

[0137] The method can further include first strand synthesis to result in immobilization of a modified target nucleic acid to an amplification site. First strand synthesis and immobilization can be accomplished by extending the 3’ end of the first capture nucleic acid associated with member of the sequencing library at the amplification sites. The extending includes the incorporation of nucleotides by a DNA polymerase using the attached member of the sequencing library as a template, and results in an extendedIP-2872-PCT PATENT nucleic acid that is immobilized to the surface of the amplification site. The immobilization can be accomplished by use of an immobilization reagent. An immobilization reagent can include an array of amplification sites, a plurality of target nucleic acids, dNTPs (e.g., dATP, dTTP, dCTP, and dGTP), and a polymerase. As shown in FIG. 5A, a polymerase extends the immobilized capture agent 24 as shown by the dashed line using the nucleotide sequence of the member of the sequencing library 21’ as template, resulting in an immobilized complement 21 of the member of the sequencing library 21’ (FIG. 5B). Under some conditions, such as when kinetic exclusion is used for cluster generation, seeding and first strand synthesis can occur essentially simultaneously.

[0138] The methods of the present disclosure can further include generating clonal clusters, e.g., producing a plurality of amplification sites that each include a clonal population of amplicons derived from the modified target nucleic acid originally present at each amplification site (FIG. 4, block 32). Quenching that can result from sequencing templates produced during cluster production, and as described herein, from sequencing templates produced during resynthesis, can result in miscalls. The present disclosure includes the use of a dATP analog during some embodiments of the step of cluster production to reduce the impact of quenching during sequencing.

[0139] In one embodiment, the method can include providing an amplification reagent and an array of amplification sites that include an immobilized nucleic acid. An amplification reagent can include (i) an array of populated amplification sites (e.g., amplification sites seeded with members of a sequencing library that are to be clonally amplified), (ii) nucleotide triphosphates (NTPs) including dATP, dTTP, dCTP, dGTP, and a dATP analog, and (iii) a polymerase. In some embodiments, the amplification reagent does not include a dATP analog. In some embodiments, the NTPs further include a dATP analog in addition to dATP, dTTP, dCTP, and dGTP. The dATP analog will be present in the amplicons at a level that is dependent on the ratio of dATP to dATP analog in the amplification reagent. Quenching results in the reduced intensity of fluorescence during sequencing, and inclusion of a dATP analog limits the reduction of fluorescence intensity. The increase of fluorescence intensity can be determined by comparing the fluorescence intensity during sequencing a template without incorporated dATP analog with the fluorescence intensity during sequencing the template with incorporated dATPIP-2872-PCT PATENT analog. The fluorescence intensity can be increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the intensity observed during sequencing a template without incorporated dATP analog.

[0140] In some embodiments, the nucleic acid at each amplification site is a modified target nucleic acid that originally seeded the site, and the clonal amplification includes first strand synthesis and the subsequent amplification. For instance, in embodiments that include clonal cluster generation by kinetic exclusion, first strand synthesis and subsequent amplification can occur essentially simultaneously. In other embodiments, the nucleic acid at each amplification site includes the complement of the modified target nucleic acid that originally seeded the site, e.g., first strand synthesis has occurred. The amplification reagent is reacted to produce a plurality of populated amplification sites, where the plurality of populated amplification sites each include a clonal population of amplicons, where each clonal population is derived from the modified target nucleic acid that originally seeded the site. FIG. 6 shows an example of generating clonal clusters. FIG. 6A shows an amplification site 20 containing immobilized strand 21. Exposure to suitable conditions results in the 3’ end of immobilized strand 21 hybridizing to complementary nucleotides of capture nucleic acid 23 (FIG. 6B), and immobilized strand 21 is used as a template for synthesis initiated from the 3’ end of capture nucleic acid 23 to result in strand 22.

[0141] The methods for cluster generation described herein can differ from typical cluster generation due to the inclusion of a dATP analog. Thus, the extension reactions that occur during cluster generation, e.g., extension from capture nucleic acid 23 of FIG. 6B), can include a dATP analog. The amount of dATP analog can be described in relation to the normal dATP present. In one embodiment, the amount of dATP analog can be expressed as a percentage of the normal dATP present in an amplification reaction. For instance, an amplification reagent can include dATP and a dATP analog, where the amount of dATP analog can be described in relation to the normal dATP present. In some embodiments, the amount of dATP analog in an amplification reaction can be at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 1 1 %, at least 12%, at least 13%, at least 14%, at least 15%, atIP-2872-PCT PATENT least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total amount of dATP. In some embodiments, the amount of dATP analog in an amplification reaction can be no greater than 99%, no greater than 98%, no greater than 97%, no greater than 96%, no greater than 95%, no greater than 94%, no greater than 93%, no greater than 92%, no greater than 91%, no greater than 90%, no greater than 89%, no greater than 88%, no greater than 87%, no greater than 86%, no greater than 85%, no greater than 84%, no greater than 83%, no greater than 82%, no greater than 81%, no greater than 80%, no greater than 79%, no greater than 78%, no greater than 77%, no greater than 76%, no greater than 75%, no greater than 74%, no greater than 73%, no greater than 72%, no greater than 71%, no greater than 70%, no greater than 69%, no greater than 68%, no greater than 67%, no greater than 66%, no greater than 65%, no greater than 64%, no greater than 63%, no greater than 62%, no greater than 61%, no greater than 60%, no greater than 59%, no greater than 58%, no greater than 57%, no greater than 56%, no greater than 55%, no greater than 54%, no greater than 53%, no greater than 52%, no greater than 51%, no greater than 50%, no greater than 49%, no greater than 48%, no greater than 47%, no greater than 46%, no greater than 45%, no greater than 44%, no greater than 43%, no greater than 42%, no greater than 41%, no greater than 40%, no greater than 39%, no greater than 38%, no greater than 37%, no greater than 36%, no greater than 35%, no greater than 34%, no greater than 33%, no greater than 32%, no greater than 31%, no greater than 30%, no greater than 29%, no greater than 28%, no greater than 27%, no greater than 26%, no greater than 25%, no greater than 24%, no greater than 23%, no greater than 22%, no greater thanIP-2872-PCT PATENT21%, no greater than 20%, no greater than 19%, no greater than 18%, no greater than 17%, no greater than 16%, no greater than 15%, no greater than 14%, no greater than 13%, no greater than 12%, no greater than 1 1%, no greater than 10%, no greater than 9%, no greater than 8%, no greater than 7%, no greater than 6%, no greater than 5%, or no greater than 4% of the total amount of dATP. In some embodiments, the amount of dATP analog in an amplification reaction is 100%, that is, there is no dATP present, only dATP analog and other dNTPs useful in an amplification, such as dGTP, dCTP, and dTTP.

[0142] Examples of ranges of the amount of dATP analog in an amplification reaction include, but are not limited to, a lower amount of the range selected from at least 3% to at least 24% and a higher amount of the range selected from no greater than 25% to no greater than 4%, for instance, at least 3% to no greater than 25%, at least 3% to no greater than 7%, at least 7% to no greater than 12%, at least 12% to no greater than 17%, or at least 17% to no greater than 22%. Other examples of ranges of the amount of dATP analog in an amplification reaction include, but are not limited to, a lower amount of the range selected from at least 3% to at least 13% and a higher amount of the range selected from no greater than 16% to no greater than 6%, for instance, at least 3% to no greater than 16%, at least 5% to no greater than 14%, or at least 7% to no greater than 12%.

[0143] dATP analogs useful in clustering include a substituted dATP, a substituted 7-deaza- dATP, 7-deaza-dATP, and 8-oxo-dATP. In some embodiments, dATP analogs useful in clustering include those having the nucleobase of Formula 3:Formula 3In some embodiments, dATP analogs useful in clustering include those having the nucleobase of Formula 4:IP-2872-PCT PATENTFormula 4 where RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-Oil), an amine, cyano or a group that includes a cynao moiety, or alkynyl or a group that includes an alkynyl moiety, as described herein. Other dATP analogs useful the amplification that occurs during resynthesis include those having the nucleobase of Formula 5:Formula 5 where RBis hydrogen, halo, sulfonyl, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), amine, cyano or a group that includes a cyano moiety, or alkynyl or a group that includes an alkynyl moiety, as described herein.

[0144] Examples of dATP analogs useful in clustering include, but are not limited to, 7-deaza- dATP, 7-deaza-7-iodo-dATP, 7-deaza-7-bromo-dATP, 7-deaza-7-SO2(CH3)-dATP, 7- SO2Me-dATP, 2-amino-dATP, 2-N(CH3)2-dATP, 8-oxo-dATP, and 7-deaza-7-PA- dATP (see Table 1 for the structures).

[0145] In some embodiments an array includes two populations of primers (e.g., capture nucleic acids) immobilized at amplification sites. In some embodiments the amplification sites of array include one population of a first primer (e.g., a first capture nucleic acids) immobilized thereto, and a second primer (e.g., a second nucleic acids) can be provided in solution during the reacting. In practice, there will be a plurality of identical first primers and / or a plurality of identical second primers immobilized at theIP-2872-PCT PATENT amplification sites, as the amplification process requires an excess of primers to sustain amplification.

[0146] As will be appreciated by the person of ordinary skill in the art, any given amplification reaction requires at least one type of forward primer and at least one type of reverse primer specific for the target nucleic acid to be amplified. However, in certain embodiments the forward and reverse primers may include target-specific portions of identical sequence and may have entirely identical nucleotide sequence and structure (including any non-nucleotide modifications). In other words, it is possible to carry out amplification at amplification sites using only one type of primer, and such singleprimer methods are encompassed within the scope of the disclosure. Other embodiments may use forward and reverse primers which contain identical targetspecific sequences but which differ in some other structural features. For example, one type of primer may contain a non-nucleotide modification which is not present in the other.

[0147] The production of a plurality of populated amplification sites on a array typically occurs by amplification at each amplification site. The term "solid-phase amplification" as used herein refers to any nucleic acid amplification reaction carried out on or in association with an array such that all or a portion of the amplified products are immobilized at amplification sites on the array as they are formed. In particular, the term encompasses solid-phase polymerase chain reaction (solid-phase PCR) and solid phase isothermal amplification which are reactions analogous to standard solution phase amplification, except that one or both of the forward and reverse capture agents include amplification primers are immobilized on the array. Solid phase PCR covers systems such as emulsions, where one primer is anchored to, for instance a bead, and the other is in free solution, and colony formation in solid phase gel matrices wherein one primer is anchored to the array and one is in free solution.

[0148] In one embodiment, a plurality of target nucleic acids is used to prepare clustered arrays of nucleic acid colonies, analogous to those described in U.S. Pub. No. 2005 / 0100900, U.S. Pat. No. 7,115,400, WO 00 / 18957 and WO 98 / 44151 by solid-phase amplification, such as solid-phase isothermal amplification. The terms "cluster" and "colony" are used interchangeably herein to refer to a discrete site on a solid support including a plurality of identical immobilized nucleic acid strands and a plurality of identical immobilizedIP-2872-PCT PATENT complementary nucleic acid strands. The term "clustered array" refers to an array formed from such clusters or colonies.

[0149] Clustered arrays can be prepared using a process of thermocycling, as described in WO 98 / 44151, a process where the temperature is maintained as a constant and the cycles of extension and denaturing are performed using changes of reagents, or a process comprising a number of enzymes that perform extension and denaturing without the need for changes in reagents or temperature. Such isothermal amplification methods include, but are not limited to, bridge amplification and exclusion amplification (ExAmp, also referred to as kinetic exclusion amplification (KEA)). Isothermal amplification methods are described in patent application numbers WO 02 / 46456, U.S. Pub. No. 2008 / 0009420, U.S. Pat. No. 8,895,249, U.S. Pub No. 2013 / 0338042, and U.S. Pat. No. 9,169,513. Isothermal amplification by exclusion amplification may be used with, for instance, the Bsu (Bacillus subtilis) DNA polymerase or large fragment of Bsu. Isothermal amplification by bridge amplification may be used with, for instance, the Bst (Bacillus stearothermophilus) DNA polymerase. Optionally, the polymerase is deficient in 5' exonuclease activity, 3' exonuclease activity, or both activities. In some embodiments, cluster generation can be accomplished using commercially available machines such as the cBot (Illumina, San Diego, CA) and certain sequencing instruments such as iSeq 100, MiniSeq, NextSeq 550 Series, NextSeq 1000 & 2000, NovaSeq 6000 Series, NovaSeq X Series, and MiSeq i 100 (Illumina, San Diego, CA).

[0150] It will be appreciated that any of the amplification methodologies described herein or generally known in the art may be used with universal or target-specific primers to amplify immobilized DNA fragments. Suitable methods for amplification include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA), as described in U.S. Pat. No. 8,003,354. The amplification methods may be employed to amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR, SDA, TMA, NASBA and the like may be utilized to amplify immobilized DNA fragments. In some embodiments, primers directed specifically to the polynucleotide of interest are included in the amplification reaction.IP-2872-PCT PATENT

[0151] Other suitable methods for amplification of target nucleic acids may include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998)) and oligonucleotide ligation assay (OLA) (See generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 Bl; EP 0 336 731 Bl; EP 0 439 182 Bl; WO 90 / 01069; WO 89 / 12696; and WO 89 / 09835) technologies. It will be appreciated that these amplification methodologies may be designed to amplify immobilized target nucleic acids. For example, in some embodiments, the amplification method may include ligation probe amplification or oligonucleotide ligation assay (OLA) reactions that contain primers directed specifically to a nucleic acid of interest. In some embodiments, the amplification method may include a primer extension-ligation reaction that contains primers directed specifically to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that may be specifically designed to amplify a nucleic acid of interest, the amplification may include primers used for the GoldcnGatc assay (Illumina, Inc., San Diego, CA) as exemplified by U.S. Pat. No. 7,582,420 and 7,611,869.

[0152] DNA nanoballs can also be used in combination with methods, systems, compositions and kits as described herein. Methods for creating and using DNA nanoballs for genomic sequencing can be found at, for example, US patents and publications U.S. Pat. No. 7,910,354, 2009 / 0264299, 2009 / 0011943, 2009 / 0005252, 2009 / 0155781 , 2009 / 0118488 and as described in, for example, Drmanac et al. (2010, Science 327(5961): 78-81). Briefly, following production of modified target nucleic acids, the modified target nucleic acids are circularized and amplified by rolling circle amplification (Lizardi et al., 1998. Nat. Genet. 19:225-232; US 2007 / 0099208 Al). The extended concatemeric structure of the amplicons promotes coiling creates compact DNA nanoballs. The DNA nanoballs can be captured on substrates, preferably to create an ordered or patterned array such that distance between each nanoball is maintained thereby allowing sequencing of the separate DNA nanoballs. In some embodiments such as those used by Complete Genomics (Mountain View, Calif.), consecutive rounds of adapter addition, amplification, and digestion are carried out prior to circularization to produce head to tail constructs having several target nucleic acids separated by adapter sequences.IP-2872-PCT PATENT

[0153] Exemplary isothermal amplification methods that may be used in a method of the present disclosure include, but are not limited to, Multiple Displacement Amplification (MDA) as exemplified by, for example Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002) or isothermal strand displacement nucleic acid amplification exemplified by, for example U.S. Pat. No. 6,214,587. Other non-PCR-based methods that may be used in the present disclosure include, for example, strand displacement amplification (SDA) which is described in, for example Walker et al.. Molecular Methods for Vims Detection, Academic Press, Inc., 1995; U.S. Pat. Nos. 5,455,166, and 5,130,238, and Walker et al., Nucl. Acids Res. 20:1691-96 (1992) or hyperbranched strand displacement amplification which is described in, for example Lage et al., Genome Res. 13:294-307 (2003). Isothermal amplification methods may be used with, for instance, the strand-displacing Phi 29 polymerase or Bst DNA polymerase large fragment, 5'->3' exo- for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high proccssivity and strand displacing activity. High processivity allows the polymerases to produce fragments that are 10-20 kb in length. As set forth herein, smaller fragments may be produced under isothermal conditions using polymerases having low processivity and strand-displacing activity such as Klenow polymerase. Additional description of amplification reactions, conditions and components are set forth in detail in the disclosure of U.S. Patent No. 7,670,810.

[0154] In some embodiments, amplification sites in an array can be, but need not be, entirely clonal. Rather, for some applications, an individual amplification site can be predominantly populated with amplicons from a first modified target nucleic acid and can also have a low level of contaminating amplicons from a second modified target nucleic acid. An array can have one or more amplification sites that have a low level of contaminating amplicons so long as the level of contamination docs not have an unacceptable impact on a subsequent use of the array. For example, when the array is to be used in a detection application, an acceptable level of contamination would be a level that does not impact signal to noise or resolution of the detection technique in an unacceptable way. Accordingly, apparent clonality will generally be relevant to a particular use or application of an array made by the methods set forth herein. Exemplary levels of contamination that can be acceptable at an individual amplification site for particular applications include, but are not limited to, at most 0.1%, 0.5%, 1%,IP-2872-PCT PATENT5%, 10% or 25% contaminating amplicons. An array can include one or more amplification sites having these exemplary levels of contaminating amplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or even 100% of the amplification sites in an array can have some contaminating amplicons. It will be understood that in an array or other collection of sites, at least 50%, 75%, 80%, 85%, 90%, 95% or 99% or more of the sites can be clonal or apparently clonal.

[0155] An amplification reagent can include further components that facilitate amplicon formation, and in some cases increase the rate of amplicon formation. An example is a recombinase in isothermal reactions including exclusion amplification. A mixture of recombinase and single-stranded binding (SSB) protein is particularly useful as SSB can further facilitate amplification. Exemplary formulations for recombinase-facilitated amplification include those sold commercially as TwistAmp kits by TwistDx (Cambridge, UK). Useful components of recombinase-facilitated amplification reagent and reaction conditions are set forth in US 5,223,414 and US 7,399,590.

[0156] Another example of a component that can be included in an amplification reagent to facilitate amplicon formation and in some cases to increase the rate of amplicon formation is a helicase. Exemplary formulations for helicase-facilitated amplification include those sold commercially as IsoAmp kits from Biohelix (Beverly, MA). Further, examples of useful formulations that include a helicase protein are described in US 7,399,590 and US 7,829,284.

[0157] Yet another example of a component that can be included in an amplification reagent to facilitate amplicon formation and in some cases increase the rate of amplicon formation is an origin binding protein.

[0158] The presence of molecular crowding reagents in the solution can be used to aid exclusion amplification. Examples of useful molecular crowding reagents include, but are not limited to, polyethylene glycol (PEG), Ficoll®, dextran, or polyvinyl alcohol. Exemplary molecular crowding reagents and formulations are set forth in U.S. Pat. No. 7,399,590.

[0159] The rate at which an amplification reaction occurs can be increased by increasing the concentration or amount of one or more of the active components of an amplification reaction. For example, the amount or concentration of polymerase, nucleotideIP-2872-PCT PATENT triphosphates, primers, recombinase, helicase or SSB can be increased to increase the amplification rate. In some cases, the one or more active components of an amplification reaction that are increased in amount or concentration (or otherwise manipulated in a method set forth herein) are non-nucleic acid components of the amplification reaction.

[0160] Amplification rate can also be increased in a method set forth herein by adjusting the temperature. For example, the rate of amplification at one or more amplification sites can be increased by increasing the temperature at the site(s) up to a maximum temperature where reaction rate declines due to denaturation or other adverse events. Optimal or desired temperatures can be determined from known properties of the amplification components in use or empirically for a given amplification reaction mixture. Such adjustments can be made based on a priori predictions of primer melting temperature (Tm) or empirically.

[0161] The rate at which an amplification reaction occurs can be increased by increasing the activity of one or more amplification reagent. For example, a cofactor that increases the extension rate of a polymerase can be added to a reaction where the polymerase is in use. In some embodiments, metal cofactors such as magnesium, zinc or manganese can be added to a polymerase reaction or betaine can be added.

[0162] In some embodiments of the methods set forth herein, it is desirable to use a population of target nucleic acids that is double- stranded. It has been observed that amplicon formation at an array of sites under exclusion amplification conditions is efficient for double- stranded target nucleic acids. For example, a plurality of amplification sites having clonal populations of amplicons can be more efficiently produced from doublestranded target nucleic acids (compared to single-stranded target nucleic acids at the same concentration) in the presence of recombinase and single- stranded binding protein. Nevertheless, it will be understood that single-stranded target nucleic acids can be used in some embodiments of the methods set forth herein.

[0163] Methods of Sequencing

[0164] An array of the present disclosure, for example, having been produced by a method set forth herein and including amplified target nucleic acids at amplification sites, can be used for any of a variety of applications. A particularly useful application is nucleicIP-2872-PCT PATENT acid sequencing. One example is sequencing-by-synthesis (SBS). In SBS, extension of a nucleic acid primer along a nucleic acid template (e.g., a target nucleic acid or amplicon thereof) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., as catalyzed by a polymerase enzyme). In a particular polymerase -based SBS embodiment, fluorescently labeled nucleotides are added to a primer (thereby extending the primer) in a template dependent fashion such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template. It is at the step of identifying the fluorescent label that the quenching described herein can occur. The use of dATP analogues reduces the quenching. A plurality of different templates at different sites of an array set forth herein can be subjected to an SBS technique under conditions where events occurring for different templates can be distinguished due to their location in the array. Examples of DNA polymerases useful for sequencing include, but arc not limited to, polymerases described in U.S. Patent No. 11,104,888, U.S. Pat. No. 11,001,816, U.S. Pat. Appl. No. 18 / 373,620; U.S. Published Patent Application No. 2023 / 0047225.

[0165] Flow cells provide a convenient format for housing an array that is produced by the methods of the present disclosure and that is subjected to an SBS or other detection technique that involves repeated delivery of reagents in cycles. For example, to initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., can be flowed into / through a flow cell that houses an array of nucleic acid templates. Those sites of an array where primer extension causes a labeled nucleotide to be incorporated can be detected. Optionally, the nucleotides can further include a reversible termination property that terminates further primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for embodiments that use reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems and detection platforms that can be readily adapted for use with an array produced by the methods of the present disclosure are described, for example, in Bentley et al., NatureIP-2872-PCT PATENT456:53-59 (2008), WO 04 / 018497; U.S. Pat. No. 7,057,026; WO 91 / 06678; WO 07 / 123,744; U.S. Pat. No. 7,329,492; U.S. Pat. No. 7,211.414; U.S. Pat. No. 7,315.019; U.S. Pat. No. 7,405,281, and U.S. Pat. No. 8,343,746. Examples nucleotides having a reversible termination property include modifications at the 3'-OH of the nucleotide sugar moiety, such as a 3’-O-azidomethyl blocking group -CH2N3, a 3’-OH acetal blocking group, or a 3'-OH thiocarbamate blocking group (U.S. Patent No. 11,293,061; U.S. Published Patent Application No. 2022 / 0396832).

[0166] Sequencing-by-ligation reactions are also useful including, for example, those described in Shendure et al. Science 309:1728-1732 (2005); U.S. Pat. No. 5,599,675; and U.S. Pat. No. 5,750,341. Some embodiments can include sequencing-by-hybridization procedures as described, for example, in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO 1989 / 10977. In both sequencing-by- ligation and sequencing-by-hybridization procedures, template nucleic acids (e.g., a target nucleic acid or amplicons thereof) that are present at sites of an array are subjected to repeated cycles of oligonucleotide delivery and detection. Fluidic systems for SBS methods as set forth herein or in references cited herein can be readily adapted for delivery of reagents for sequencing-by-ligation or sequencing-by-hybridization procedures. Typically, the oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described with regard to SBS procedures herein or in references cited herein.

[0167] Some embodiments can use methods involving the real-time monitoring of DNA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore- bearing polymerase and y-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs). Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008).

[0168] Sequencing of templates in a cluster often includes the technique of "paired-end" or "pairwise" sequencing (U.S. Pat. No. 7,754,429 and U.S. Pat. No. 8,017,335). In some embodiments, a dATP analog is used during the resynthesis that occurs during paired- end sequencing of templates at a cluster. Paired-end sequencing is a multi-step processIP-2872-PCT PATENT that allows the determination of two "reads" of sequence by sequencing both strands of a double stranded nucleic acid. The advantage of the paired-end approach is that there is significantly more information to be gained from sequencing bases from two complementary templates than from sequencing the same number of bases from each of two independent templates in a random fashion. With the use of appropriate software tools for the assembly of sequence information, it is possible to use the knowledge that the "paired-end" sequences are not completely random, but are known to occur on a single template, and are therefore linked or paired in the genome. This information greatly aids the assembly of whole genome sequences into a consensus sequence.

[0169] After production of clonal clusters, each cluster includes immobilized complementary strands. In order to provide more suitable templates for sequencing, substantially all or at least a portion of one of the immobilized strands is removed in order to generate a template which is at least partially single-stranded. The portion of the template which is single-stranded will thus be available for hybridization to a sequencing primer. The process of removing all or a portion of one immobilized strand is referred to as "linearization." There are various ways for linearization, including but not limited to enzymatic cleavage (e.g., uracil DNA glycosylase (UDG) and endonuclease VII, oxoguanine glycosylase, chemical cleavage (e.g., palladium reagents and Pd linearization, nickel reagents and Ni Pd linearization), photo-chemical cleavage. Nonlimiting examples of linearization methods are disclosed in US Serial No. 18 / 473,971 , filed Sep. 25, 2023; PCT Publication No. WO 2019 / 222264; US Published Patent Application No. 2019 / 0352327; WO 2007 / 010251; US Patent Application Publication No. 2009 / 0088327; and in US. Patent Publication No. 2009 / 0118128, which are incorporated by reference in their entireties.

[0170] Sequence data can be obtained from both immobilized complementary strands by performing a linearization to remove a strand attached by one capture nucleic acid, e.g., P5, obtaining a sequence read from the remaining first strand using a primer, copying the first strand using immobilized primers for strand resynthesis and repopulation of the cluster with the strand initially removed by the first linearization, releasing the first strand and sequencing the second, copied strand (FIG. 4, block 35). In one embodiment, resynthesis and repopulation of clusters includes use of a resynthesis reagent. A resynthesis reagent can include (i) an array of amplification sites, whereIP-2872-PCT PATENT each amplification site includes immobilized modified target nucleic acids, (ii) nucleotide triphosphates (dNTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog, and (iii) a polymerase. In some embodiments, a resynthesis reagent does not include a dATP analog. The resynthesis reagent is reacted to produce, at each amplification site, a population of strands that are complementary to the strand sequenced during the first round. The population of complementary strands are sequenced during the second round. When a dATP analog is present, the complementary strands will include the dATP analog incorporated. The dATP analog will be present in the complementary strands at a level that is dependent on the ratio of dATP to dATP analog present in the resynthesis reagent. Quenching results in the reduced intensity of fluorescence during sequencing, and inclusion of a dATP analog limits the reduction of fluorescence intensity. The increase of fluorescence intensity can be determined by comparing the fluorescence intensity during sequencing a template without incorporated dATP analog with the fluorescence intensity during sequencing the template with incorporated dATP analog. The fluorescence intensity can be increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the intensity observed during sequencing a template without incorporated dATP analog.

[0171] The present disclosure includes the use of a dATP analog during some embodiments of the step of cluster repopulation / strand resynthesis to reduce the impact of quenching during the subsequent sequencing reaction. An example of these steps is shown in FIG. 7. FIG. 7A shows an amplification site 20 containing immobilized complementary strands 21 and 22. A linearization is performed to remove strand 22 by cleaving the capture nucleic acid 23, for instance P5, at the X. Cleavage results in a cleaved capture nucleic acid 23* and one population of immobilized target nucleic acids, 21, as shown in FIG. 7B. The sequencing of strand 21 can be earned out by the sequential addition of nucleotides to the first sequencing primer using the strand 21 as the template. For instance, as shown in FIG. 7C a sequencing primer 24 is annealed to the strand 21 and is ready for extension by a DNA polymerase in a sequencing reaction. The strand extended during the sequencing reaction is not immobilized and is removed, and strand resynthesis occurs to repopulate the cluster with the strand that is the complement of theIP-2872-PCT PATENT sequenced strand. As shown in FIG. 7D, the sequenced strand 21 is used as the template to repopulate the amplification site 20 using bridge amplification. The result is shown in FIG. 7E, where the amplification site is repopulated with strand 22*, which is essentially identical to strand 22 in FIG. 7A but the cleavage site X is no longer present. Linearization is performed to remove sequenced strand 21 by cleaving the capture nucleic acid 24, for instance P7, at the Y. As shown in FIG. 7F, the repopulated amplification site 20 is ready for sequencing of the other strand, thereby resulting in pairwise sequencing.

[0172] The methods for cluster repopulation / strand resynthesis described herein can differ from typical cluster repopulation / strand resynthesis due to the inclusion of a dATP analog. Thus, the extension reactions that occur during resynsthesis, e.g., extension from capture nucleic acid 23* of FIG. 7D, can include a dATP analog. The amount of dATP analog can be described in relation to the normal dATP present. In one embodiment, the amount of dATP analog can be expressed as a percentage of the normal dATP present in an resynthesis reaction. For instance, a resynthesis reagent can include dATP and a dATP analog, where the amount of dATP analog can be described in relation to the normal dATP present. In some embodiments, the amount of dATP analog in a resynthesis reaction can be at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total amount of dATP . In some embodiments, theIP-2872-PCT PATENT amount of dATP analog in an amplification reaction can be no greater than 99%, no greater than 98%, no greater than 97%, no greater than 96%, no greater than 95%, no greater than 94%, no greater than 93%, no greater than 92%, no greater than 91%, no greater than 90%, no greater than 89%, no greater than 88%, no greater than 87%, no greater than 86%, no greater than 85%, no greater than 84%, no greater than 83%, no greater than 82%, no greater than 81%, no greater than 80%, no greater than 79%, no greater than 78%, no greater than 77%, no greater than 76%, no greater than 75%, no greater than 74%, no greater than 73%, no greater than 72%, no greater than 71 %, no greater than 70%, no greater than 69%, no greater than 68%, no greater than 67%, no greater than 66%, no greater than 65%, no greater than 64%, no greater than 63%, no greater than 62%, no greater than 61%, no greater than 60%, no greater than 59%, no greater than 58%, no greater than 57%, no greater than 56%, no greater than 55%, no greater than 54%, no greater than 53%, no greater than 52%, no greater than 51 %, no greater than 50%, no greater than 49%, no greater than 48%, no greater than 47%, no greater than 46%, no greater than 45%, no greater than 44%, no greater than 43%, no greater than 42%, no greater than 41%, no greater than 40%, no greater than 39%, no greater than 38%, no greater than 37%, no greater than 36%, no greater than 35%, no greater than 34%, no greater than 33%, no greater than 32%, no greater than 31%, no greater than 30%, no greater than 29%, no greater than 28%, no greater than 27%, no greater than 26%, no greater than 25%, no greater than 24%, no greater than 23%, no greater than 22%, no greater than 21 %, no greater than 20%, no greater than 19%, no greater than 18%, no greater than 17%, no greater than 16%, no greater than 15%, no greater than 14%, no greater than 13%, no greater than 12%, no greater than 11%, no greater than 10%, no greater than 9%, no greater than 8%, no greater than 7%, no greater than 6%, no greater than 5%, or no greater than 4% of the total amount of dATP. In some embodiments, the amount of dATP analog in an amplification reaction is 100%, that is, there is no dATP present, only dATP analog and other dNTPs useful in an amplification, such as dGTP, dCTP, and dTTP.

[0173] Examples of ranges of the amount of dATP analog in a resynthesis reaction include, but are not limited to, a lower amount of the range selected from at least 3% to at least 24% and a higher amount of the range selected from no greater than 25% to no greater than 4%, for instance, at least 3% to no greater than 25%, at least 3% to no greater than 7%, at least 7% to no greater than 12%, at least 12% to no greater than 17%, or at least 17%IP-2872-PCT PATENT to no greater than 22%. Other examples of ranges of the amount of dATP analog in a resynthesis reaction include, but are not limited to, a lower amount of the range selected from at least 3% to at least 13% and a higher amount of the range selected from no greater than 16% to no greater than 6%, for instance, at least 3% to no greater than 16%, at least 5% to no greater than 14%, or at least 7% to no greater than 12%.

[0174] dATP analogs useful in the amplification that occurs during resynthesis include a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP. In some embodiments, dATP analogs useful in the amplification that occurs during resynthesis include those having the nucleobase of Formula 3 :Formula 3In some embodiments, dATP analogs useful in the amplification that occurs during resynthesis include those having the nucleobase of Formula 4:Formula 4 where RAis alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-0H), an amine, cyano or a group that includes a cynao moiety, or alkynyl or a group that includes an alkynyl moiety, as described herein. Other dATP analogs useful the amplification that occurs during resynthesis include those having the nucleobase of Formula 5:IP-2872-PCT PATENTFormula 5 where RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano or a group that includes a cyano moiety, or alkynyl or a group that includes an alkynyl moiety, as described herein .

[0175] Examples of dATP analogs useful in resynthesis include, but are not limited to, 7- deaza-dATP, 7-deaza-7-iodo-dATP, 7-deaza-7-bromo-dATP, 7-deaza-7-SO2(CH3)- dATP (7-SO2Mc-dATP), 2-amino-dATP, 2-N(CH3)2-dATP, 8-oxo-dATP, and 7- deaza-7-PA-dATP (see Table 1 for the structures).

[0176] The present disclosure provides integrated sequencing systems capable of making an array using one or more of the methods set forth herein, e.g., producing clusters that include a dATP analog. An integrated sequencing system can be capable of detecting nucleic acids on the arrays using techniques such as those described herein, including resynthesis in the presence of a dATP analog during paired-end sequencing. Thus, an integrated sequencing system of the present disclosure can include fluidic components capable of delivering amplification reagents to an array of amplification sites such as pumps, valves, reservoirs, fluidic lines and the like. An example of useful fluidic components includes a flow cell and a cartridge. A flow cell can be configured and / or used in an integrated sequencing system to create an array of the present disclosure and to detect the array. Exemplary flow cells are described, for example, in US 2010 / 0111768 Al and U.S. Pat. No. 8,951,781. A cartridge can be configured to include the components of an amplification or resynthesis reagent in one or more chambers. As exemplified for flow cells, one or more of the fluidic components of an integrated sequencing system can be used for an amplification method and for a detection method. Taking a nucleic acid sequencing embodiment as an example, one or more of the fluidic components of an integrated sequencing system can be used for an amplification method set forth herein and for the delivery of sequencing reagents in aIP-2872-PCT PATENT sequencing method, including a resynthesis method, such as those described herein. Alternatively, an integrated sequencing system can include separate fluidic systems to cany out amplification methods and to carry out detection methods and resynthesis methods. Examples of integrated sequencing systems that are capable of creating arrays of nucleic acids and also determining the sequence of the nucleic acids include, without limitation, the MiSeq™, HiSeq™, NextSeq™, MiniSeq™, NovaSeq™ and iSeq™ platforms (Illumina, Inc., San Diego, Calif.) and devices described in U.S. Pat. No. 8,9 1 ,781.

[0177] A system capable of carrying out a method set forth herein need not be integrated with a detection device. Rather, a stand-alone system or a system integrated with other devices is also possible. Fluidic components similar to those exemplified herein in the context of an integrated sequencing system can be used in such embodiments.

[0178] A system capable of carrying out a method set forth herein, whether integrated with detection capabilities or not, can include a system controller that is capable of executing a set of instructions to perform one or more steps of a method, technique or process set forth herein. For example, the instructions can direct the performance of steps for creating an array under kinetic exclusion amplification or bridge amplification conditions. Optionally, the instructions can further direct the performance of steps for detecting nucleic acids using methods set forth previously herein. A useful system controller may include any processor-based or microprocessor-based system, including systems using microcontrollers, reduced instruction set computers (RISC), application specific integrated circuits (ASICs), field programmable gate array (FPGAs), logic circuits, and any other circuit or processor capable of executing functions described herein. A set of instructions for a system controller may be in the form of a software program. As used herein, the terms “software” and “firmware” are interchangeable, and include any computer program stored in memory for execution by a computer, including RAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatile RAM (NVRAM) memory. The software may be in various forms such as system software or application software. Further, the software may be in the form of a collection of separate programs, or a program module within a larger program or a portion of a program module. The software also may include modular programming in the form of object-oriented programming.IP-2872-PCT PATENT

[0179] Several applications for arrays of the present disclosure have been exemplified herein in the context of ensemble detection, wherein multiple amplicons present at each amplification site are detected together. In alternative embodiments, a single nucleic acid, whether a target nucleic acid or amplicon thereof, can be detected at each amplification site. For example, an amplification site can be configured to contain a single nucleic acid molecule having a target nucleotide sequence that is to be detected and a plurality of filler nucleic acids. In this example, the filler nucleic acids function to fill the capacity of the amplification site and they are not necessarily intended to be detected. The single molecule that is to be detected can be detected by a method that is capable of distinguishing the single molecule in the background of the filler nucleic acids. Any of a variety of single molecule detection techniques can be used including, for example, modifications of the ensemble detection techniques set forth herein to detect the sites at increased gain or using more sensitive labels. Other examples of single molecule detection methods that can be used arc set forth in U.S. 2011 / 0312529 Al: U.S. Pat. No. 9,279,154: and U.S. 2013 / 0085073 Al.

[0180] It will be understood that an array of the present disclosure, for example, having been produced by a method set forth herein, need not be used for a detection method. Rather, the array can be used to store a nucleic acid library. Accordingly, the array can be stored in a state that preserves the nucleic acids therein. For example, an array can be stored in a desiccated state, frozen state (e.g., in liquid nitrogen), or in a solution that is protective of nucleic acids. Alternatively, or additionally, the array can be used to replicate a nucleic acid library. For example, an array can be used to create replicate amplicons from one or more of the sites on the array.

[0181] Several embodiments of the disclosure have been exemplified herein with regard to transporting target nucleic acids to amplification sites of an array and making copies of the captured target nucleic acids at the amplification sites. Similar methods can be used for non-nucleic acid target molecules. Thus, methods set forth herein can be used with other target molecules in place of the exemplified target nucleic acids. For example, a method of the present disclosure can be carried out to transport individual target molecules from a population of different target molecules. Each target molecule can be transported to (and in some cases captured at) an individual amplification site of an array to initiate a reaction at the site of capture. The reaction at each site can, forIP-2872-PCT PATENT example, produce copies of the captured molecule or the reaction can alter the site to isolate or sequester the captured molecule. In either case, the end result can be sites of the array that are each pure with respect to the type of target molecule that is present from a population that contained different types of target molecules.

[0182] Compositions

[0183] Clusters produced by amplification, e.g., during cluster generation or during rcsynthcsis, in the presence of a dATP analog have one or more of several characteristics. In one embodiment, the analog is present in the two strands of the amplified target nucleic acids, and the percentage of normal dATP to analog present in the strands is a function of the amount of dATP analog present in an amplification reaction. In one embodiment, the analog is present in one strand of the amplified target nucleic acids. For instance, the analog is present in the population of single strands present at a cluster before the first round of sequencing, or the analog is present in the population of single strands present at a cluster before the second round of sequencing (e.g., after resynthesis in the presence of a dATP analog). The percentage of normal dATP to analog present in a strand is a function of the amount of dATP analog present in an amplification reaction.

[0184] In one embodiment, a characteristic of clusters prepared as described herein is reduced secondary structure of the amplicons compared to clusters produced in the same way but without use of a dATP analog. The present disclosure includes methods, compositions, arrays, cartridges, and kits that include clusters having one or more of these characteristics, in any combination. For instance, the present disclosure includes an array that has amplification sites populated with clusters that include a dATP analog. The array can be a flow cell, and the flow cell can be one that is configured to interact with a cartridge that can be used with a sequencing apparatus. In one embodiment, a flow cell, such as one having clusters that include a dATP analog, can be releasably attached to a cartridge.

[0185] Kits and cartridges

[0186] The present disclosure also provides kits and cartridges for carrying out the methods disclosed herein. The kits and cartridges can be configured for use with a sequencing instrument, such as an integrated sequencing system.IP-2872-PCT PATENT

[0187] In some embodiments, a cartridge for use with a sequencing system may include a chamber from which a composition (such as a composition that includes a plurality of target nucleic acids, nucleotide triphosphates (dNTPs) including dATP, dTTP, dCTP, dGTP, and an optional dATP analog, or a polymerase) may be withdrawn or expelled for use in a method disclosed herein, (e.g., cluster generation, resynthesis, or both). A cartridge may include a releasably attached flow cell.

[0188] In some embodiments, a kit includes components for use with the methods of the present disclosure. For instance, a kit may include one or more compositions configured to perform one or more of the amplification and resynthesis steps of cluster generation or amplification site repopulation. A kit may be configured for use with a cartridge. For example, a kit may include the compositions for disposing into the chambers of the cartridge.

[0189] The invention is defined in the claims. However, below there is provided a non- exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0190] Exemplary Aspects

[0191] Aspect 1. A method for reducing quenching in a subsequent sequencing reaction generating clonal amplification sites including a dATP analog, including: (a) providing an amplification reagent including (i) an array of amplification sites, (ii) a composition including a plurality of modified target nucleic acids, (iii) a composition including nucleotide triphosphates (NTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog including a nucleobase, and (iv) a composition including a polymerase; and (b) reacting the amplification reagent to produce a plurality of populated amplification sites, wherein the plurality of populated amplification sites each include a clonal population of amplicons from an individual modified target nucleic acid from the plurality of modified target nucleic acids.

[0192] Aspect 2. A method for generating clonal amplification sites including a dATP analog, including: (a) providing an amplification reagent including (i) an array of amplification sites, wherein each amplification site includes a capture sequence and aIP-2872-PCT PATENT single-stranded modified target nucleic acid immobilized thereto; (ii) a composition including nucleotide triphosphates (NTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog including a nucleobase, and (iii) a composition including a polymerase; and (b) reacting the amplification reagent to produce a plurality of amplification sites that each include a clonal population of amplicons from the single-stranded modified target nucleic acid immobilized thereto in step (a)(i).

[0193] Aspect 3. The method of any of Aspects 1 or 5-22, wherein the compositions of (ii) and (iii) are present together in a mixture, the compositions of (iii) and (iv) are present together in a mixture, or the compositions of (ii), (iii), and (iv) are present together in a mixture.

[0194] Aspect 4. The method of Aspect 2 or 5-22, wherein the compositions of (ii) and(iii) are present together in a mixture.

[0195] Aspect 5. The method of any of Aspects 1-4 or 6-22, wherein the array includes a flow cell.

[0196] Aspect 6. The method of any of Aspects 1-5 or 7-22, wherein the polymerase isBsu or Bst

[0197] Aspect 7. The method of any of Aspects 1-6 or 8-22, wherein the reacting includes kinetic exclusion amplification or bridge amplification.

[0198] Aspect 8. The method of any of Aspects 1-7 or 9-22, wherein the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP.

[0199] Aspect 9. The method of any of Aspects 1-8 or 10-22, wherein the substituted dATP includes a substituent other than hydrogen at 2 position of the purine ring system.

[0200] Aspect 10. The method of any of Aspects 1-9 or 11-22, wherein the nucleobase of the dATP analog is of Formula 4:IP-2872-PCT PATENTFormula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0201] Aspect 11. The method of any of Aspects 1-10 or 12-22, wherein RAis an amine of the formula -NR!R2R3where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

[0202] Aspect 12. The method of any of Aspects 1-11 or 13-22, wherein two of R1, R2, and R3are the same.

[0203] Aspect 13. The method of any of Aspects 1-11 or 14-22, wherein each of R1, R2, and R3are different.

[0204] Aspect 14. The method of any of Aspects 1 - 1 or 15-22, wherein at least one of R1, R2, and R3is a lone pair of electrons.

[0205] Aspect 15. The method of any of Aspects 1-14 or 16-22, wherein the nucleobase of the dATP isor

[0206] Aspect 16. The method of any of Aspects 1-15 or 17-22, wherein the dATP analog is 7-deaza-dATP.IP-2872-PCT PATENT

[0207] Aspect 17. The method of any of Aspects 1-16 or 18-22, wherein the substituted 7- deaza-dATP includes substituent other than hydrogen at the 7 position of the ring system.

[0208] Aspect 18. The method of any of Aspects 1-17 or 19-22, wherein the nucleobase of the dATP analog is of Formula 5:wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (- OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0209] Aspect 19. The method of any of Aspects 1-18 or 20-22, wherein RBis I, Br, Cl, or F.

[0210] Aspect 20. The method of any of Aspects 1-19 or 21, wherein RBa sulfonyl of the formula -S(O)2-R30where R30is a Cl to C3 alkyl.

[0211] Aspect 21. The method of any of Aspects 1-20, wherein the nucleobase of the dATP analog isIP-2872-PCT PATENT

[0212] Aspect 22. An array including a sequencing library, the array including: a plurality of populated amplification sites attached to the array, wherein the plurality of populated amplification sites each include a clonal population of amplicons from an individual modified target nucleic acid from a library of modified target nucleic acids, wherein the amplicons include nucleotides dATP, dTTP, dGTP, dCTP, and a dGTP analog including a nucleobase.

[0213] Aspect 23. The array of any of Aspects 22 or 24-37, wherein the array includes a flow cell.

[0214] Aspect 24. The array of any of Aspects 22-23 or 25-37, wherein the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP.

[0215] Aspect 25. The array of any of Aspects 22-24 or 26-37, wherein the substituted dATP includes a substituent other than hydrogen at 2 position of the purine ring system.

[0216] Aspect 26. The array of any of Aspects 22-25 or 27-37, wherein the nucleobase of the dATP analog is of Formula 4:IP-2872-PCT PATENTFormula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0217] Aspect 27. The array of any of Aspects 22-26 or 28-37, wherein RAis an amine of the formula -NR!R2R3where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

[0218] Aspect 28. The array of any of Aspects 22-27 or 29-37, wherein two of R1, R2, and R3are the same.

[0219] Aspect 29. The array of any of Aspects 22-28 or 30-37, wherein each of R1, R2, and R3are different.

[0220] Aspect 30. The array of any of Aspects 22-29 or 31-37, wherein at least one of R1, R2, and R3is a lone pair of electrons.

[0221] Aspect 31. The array of any of Aspects 22-30 or 32-37, wherein the nucleobase of the dATP isAspect 32. The array of any of Aspects 22-31 or 33-37, wherein the dATP analog is 7- deaza-dATP.IP-2872-PCT PATENT

[0222] Aspect 33. The array of any of Aspects 22-32 or 34-37, wherein the substituted 7- deaza-dATP includes substituent other than hydrogen at the 7 position of the ring system.

[0223] Aspect 34. The array of any of Aspects 22-33 or 35-37, wherein the nucleobase of the dATP analog is of Formula 5:wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0224] Aspect 35. The array of any of Aspects 22-34 or 36-37, wherein RBis 1, Br, Cl, or F.

[0225] Aspect 36. The array of any of Aspects 22-35 or 37, wherein RBa sulfonyl of the formula -S(O)2-R30where R30is a Cl to C3 alkyl.

[0226] Aspect 37. The array of any of Aspects 22-36, wherein the nucleobase of the dATP analog isIP-2872-PCT PATENT

[0227] Aspect 38. A method for reducing quenching in a subsequent sequencing reaction resynthesis of modified target nucleic acids including a dATP analog at amplification sites, including: (a) providing an array including a plurality of amplification sites, wherein the amplification sites include two populations of capture nucleic acids immobilized to the amplification sites at the 5’ end, each population including a capture sequence, wherein a first population of capture nucleic acids include at each amplification site a clonal population of a modified target nucleic acid, the 5’ end of the clonal population of the modified target nucleic acid attached to the 3’ end of the first population capture nucleic acids, wherein the clonal population of the modified target nucleic acid at each amplification site is a member of a sequencing library; (b) contacting the plurality of amplification sites to a resynthesis reagent including (i) a composition including nucleotide triphosphates (NTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog including a nucleobase, and (ii) a composition including a polymerase; and (c) reacting the resynthesis reagent to produce a plurality of re -populated amplification sites attached to the array, wherein the plurality of re-populated amplification sites each include a clonal population of a resynthesized target nucleic acids immobilized to the amplification sites at the 5’ end, wherein the clonal population of the resynthesized target nucleic acid includes a nucleic acid sequence that is a complement of the clonal population of the modified target nucleic acid of step (a).IP-2872-PCT PATENT

[0228] Aspect 39. A method for resynthesis of modified target nucleic acids including a dATP analog at amplification sites, including: (a) providing an array including a plurality of amplification sites, wherein the amplification sites include two populations of capture nucleic acids immobilized to the amplification sites at the 5’ end, each population including a capture sequence, wherein a first population of capture nucleic acids include at each amplification site a clonal population of a modified target nucleic acid, the 5’ end of the clonal population of the modified target nucleic acid attached to the 3’ end of the first population capture nucleic acids, wherein a second population of capture nucleic acids include (i) the complement of the clonal population of the modified target nucleic acid at each amplification site, the 5’ end of the complement of the clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids, and (ii) a cleavage site, wherein the clonal population of the modified target nucleic acid at each amplification site is a member of a sequencing library; (b) contacting the amplification sites with a cleavage agent, thereby cleaving the second population of capture nucleic acids, and releasing the clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids; (c) removing the released clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids from the amplification sites; (d) contacting the plurality of amplification sites to a resynthesis reagent including (i) a composition including nucleotide triphosphates (NTPs), wherein the NTPs include dATP, dTTP, dCTP, dGTP, and a dATP analog including a nucleobase, and (ii) a composition including a polymerase; and (e) reacting the resynthesis reagent to produce a plurality of re-populated amplification sites attached to the array, wherein the plurality of re-populated amplification sites each include a clonal population of a resynthesized target nucleic acid immobilized to the amplification sites at the 5’ end, wherein the clonal population of the resynthesized target nucleic acid includes a nucleic acid sequence that is a complement of the clonal population of the modified target nucleic acid of step (a).

[0229] Aspect 40. The method of Aspect 38 or 39, wherein the array includes a flow cell.

[0230] Aspect 41. The method of any of Aspects 38-40, wherein the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP.IP-2872-PCT PATENT

[0231] Aspect 42. The method of any of Aspects 38-41, wherein the substituted dATP includes a substituent other than hydrogen at 2 position of the purine ring system.

[0232] Aspect 43. The method of any of Aspects 38-42, wherein the nucleobase of the dATP analog is of Formula 4:Formula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group including a cyaao moiety, alkynyl, or a group including an alkynyl moiety.

[0233] Aspect 44. The method of any of Aspects 38-43, wherein RAis an amine of the formula -NR1R2R3where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

[0234] Aspect 45. The method of any of Aspects 38-44, wherein two of R1, R2, and R3are the same.

[0235] Aspect 46. The method of any of Aspects 38-45, wherein each of R1, R2, and R3are different.

[0236] Aspect 47. The method of any of Aspects 38-46, wherein at least one of R1, R2, andR3is a lone pair of electrons.

[0237] Aspect 48. The method of any of Aspects 38-47, wherein the nucleobase of theIP-2872-PCT PATENT

[0238] Aspect 49. The method of any of Aspects 38-48, wherein the dATP analog is 7- deaza-dATP.

[0239] Aspect 50. The method of any of Aspects 38-49, wherein the substituted 7-deaza- dATP includes substituent other than hydrogen at the 7 position of the ring system.

[0240] Aspect 51. The method of any of Aspects 38-50, wherein the nucleobase of the dATP analog is of Formula 5 :wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (- OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0241] Aspect 52. The method of any of Aspects 38-51, wherein RBis I, Br, Cl, or F.

[0242] Aspect 53. The method of any of Aspects 38-52, wherein RBa sulfonyl of the formula -S(O)2-R30where R30is a Cl to C3 alkyl.

[0243] Aspect 54. The method of any of Aspects 38-53, wherein the nucleobase of the dATP analog isIP-2872-PCT PATENT

[0244] Aspect 55. A cartridge for use with a sequencing apparatus, the cartridge including: a first chamber including a nucleotide composition, the nucleotide composition including dATP, dTTP, dGTP, dCTP, and a dATP analog including a nucleobase.

[0245] Aspect 56. The cartridge of any of Aspects 55 or 59-72, further including a flow cell, wherein the flow cell is releasably attached to the cartridge.

[0246] Aspect 57. A kit for use with a sequencing apparatus, the kit including: a cartridge, the cartridge including a first chamber including a nucleotide composition, the nucleotide composition including dATP, dTTP, dGTP, dCTP, and a dATP analog including a nucleobase.

[0247] Aspect 58. The kit of any of Aspects 57 or 59-72, further including a flow cell, wherein the flow cell is configured to releasably attach to the cartridge.

[0248] Aspect 59. The cartridge or kit of any of Aspects 55-58 or 60-72, wherein the nucleobase of the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7- deaza-dATP, or 8-oxo-dATP.

[0249] Aspect 60. The cartridge or kit of any of Aspects 55-59 or 61-72, wherein the substituted dATP includes a substituent other than hydrogen at 2 position of the purine ring system.IP-2872-PCT PATENT

[0250] Aspect 61. The cartridge or kit of any of Aspects 55-60 or 62-72, wherein the nucleobase of the dATP analog is of Formula 4:Formula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0251] Aspect 62. The cartridge or kit of any of Aspects 55-61 or 63-72, wherein RA is an amine of the formula -NR1R2R3 where each of Rl, R2, and R3 are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

[0252] Aspect 63. The cartridge or kit of any of Aspects 55-62 or 64-72, wherein two of Rl, R2, and R3 are the same.

[0253] Aspect 64. The cartridge or kit of any of Aspects 55-63 or 65-72, wherein each of Rl, R2, and R3 are different.

[0254] Aspect 65. The cartridge or kit of any of Aspects 55-64 or 66-72, wherein at least one of Rl, R2, and R3 is a lone pair of electrons.

[0255] Aspect 66. The cartridge or kit of any of Aspects 55-65 or 67-72, wherein the nucleobase of the dATP isIP-2872-PCT PATENT

[0256] Aspect 67. The cartridge or kit of any of Aspects 55-66 or 68-72, wherein the dATP analog is 7-deaza-dATP.

[0257] Aspect 68. The cartridge or kit of any of Aspects 55-67 or 69-72, wherein the substituted 7-deaza-dATP includes substituent other than hydrogen at the 7 position of the ring system.

[0258] Aspect 69. The cartridge or kit of any of Aspects 55-68 or 70-72, wherein the nucleobase of the dATP analog is of Formula 5:wherein RB is hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (- OH), an amine, cyano, group including a cyano moiety, alkynyl, or a group including an alkynyl moiety.

[0259] Aspect 70. The cartridge or kit of any of Aspects 55-69 or 71-72, wherein RB is I, Br, Cl, or F.

[0260] Aspect 71. The cartridge or kit of any of Aspects 55-70, wherein RB a sulfonyl of the formula -S(O)2-R30 where R30 is a Cl to C3 alkyl.Aspect 72. The cartridge or kit of any of Aspects 55-71, wherein the nucleobase of the dATP analog isIP-2872-PCT PATENT

[0261] EXAMPLES

[0262] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.

[0263] Example 1

[0264] Assessing dATP analogues and specific quenching

[0265] To determine if dATP analogues reduce quenching, dATP analogues were used in the steps of clustering and resynthesis, and subsequent sequencing reactions with XLEAP- SBS® chemistry were conducted and analyzed using an Illumina NextSeq2000. dATP analogues (2-amino-dATP, 7-deaza-dATP, 7-deaza-7-iodo-dATP, 7-deaza-7-bromo- dATP, 8-oxo-dATP, 7-deaza-7-MeSO2-dATP and 7-deaza-7-NMe2-dATP, and 7- Deaza-7-propargylamino-dATP; see Table 1 for the structures) were spiked into clustering reagent ECX1 and resynthesis reagent JAM amplification mix at varying concentrations (relative to the natural dATP). Different analogues were either run with a single read (clustering reagent spike-in only, no resynthesis reagent spike-in) or two read (clustering reagent spike-in and resynthesis reagent spike -in).IP-2872-PCT PATENT

[0266] To assess the ability of these analogues to prevent or reduce the severity of the SSEs resulting from quenching, a Bac library (‘BacPac’) containing a portion of the human genome including a range of regions susceptible to quenching was used. This library contains at least 32 defined locations of quenching motifs. To quantify numerically any improvement, the percent of mismatched bases within a 15 base pair region around the SSE sites was collected for each defined location and then averaged. This was repeated for all controls and treatments. The primary metrics for each of the runs was also collected to assess the impacts to the overall sequencing quality.

[0267] FIG. 8 (single read) and FIG. 9 (two read) show that all the modified dATP analogues tested except 7-deaza-7-bromo-dATP appear to reduce the percent mismatched bases within the SSE regions to at least some degree at certain concentrations. The 2-amino- dATP analogue appeared to be the most effective with concentrations of analogue to natural dATP as low as 2.5% reducing the mismatch values significantly.

[0268] FIG. 10 shows that all the analogues, including 7-deaza-7-bromo-dATP, reduced the severity of at least a portion of the overall quenching sites. The 2-amino-dATP appeared to reduce severity at all the locations even at low concentrations. The elevation of the % error rate (an undesirable result for this primary metric) of certain analogues at certain concentrations may inflate the % mismatch results and hence overestimate the average % mismatch value. This results in the appearance that the specific analogue is not reducing quenching. The severity of quenching at the specific motifs are reduced, but the overall quality is diminished such that this statistical representation is biased.

[0269] Example 2

[0270] Concentration titration of 2-Amino-dATP

[0271] Quenching reduction with 2-amino-dATP (see Table 4 for the structure of 2-amino- dATP) was investigated at a range of concentrations. Illumina NextSeq2000 with XLEAP-SBS® chemistry was used. 2-amino-dATP was spiked into clustering reagent ECX1 and resynthesis reagent JAM amplification mix at a range of concentrations relative to the natural dATP.IP-2872-PCT PATENT

[0272] FIG. 11 shows total false positives plus false negatives (FP+FN) in known quenching SSE regions, using human HG002 library and the GATK variant caller. FP+FN decreases with increasing concentration of 2-amino-dATP.

[0273] Example 3

[0274] Synthesis of various dATP analogs

[0275] 2-NMe2-dATP (2-N(CII3)2-dATP)

[0276] 2-NMc2-dATP was synthesized according to the synthetic scheme shown in FIG. 12. Dimethylamine (40 wt-% in FFO) (0.44 mL) was added to 2-chlodo-7-deaza-2'- deoxyadenosine (compound 1; 250 mg, 0.88 mmol) in methanol (2 mL). The vessel was sealed and heated to 130 °C for 1 hour then allowed to cool to room temperature. The reaction mixture was concentrated in vacuo to afford a pale yellow oil which crystallized on standing to afford an off white solid (compound 2 also called 2-NMe2- dA; 370 mg, >100%) which was carried through to the next reaction without further purification.

[0277] In a first vessel, 2-NMe2-dA (compound 2, 259 mg, 0.88 mmol) was added to triethyl phosphate (5 mL). A proton sponge (550 g, 2.55 mmol) was then added and the reaction mixture was cooled to -20 °C. In a second vessel, tributylammonium pyrophosphate (0.5 M in dimethyl formamide (DMF), 18 mL) and tributylamine (1.7 mL, 7.23 mmol) were cooled to -20 °C. Once cooled, phosphoryl chloride (POOL, 0.6 mL, 6.80 mmol) was added dropwise to the first vessel, and reaction was checked for completion after 15 minutes by HPLC. The reaction mixture from the first vessel was then transferred to the second vessel, and reaction temperature was raised to 0 °C. Upon complete consumption of monophosphate, an aqueous 2 M tetraethylammonium bromide (TEAB) solution (7.5 mL) was added dropwise to the second vessel. The reaction temperature was then set to 25 °C and the reaction was stirred overnight.

[0278] The reaction mixture was washed with ethyl acetate (2 x 15 mL) and the organic layer was back-extracted with 0.1 M TEAB (15 mL). The aqueous layer was filtered into Schott bottle through Coming filter unit, and was purified by ion-exchange and reversephase column purifications (43 pmol, 5% over 2 steps).IP-2872-PCT PATENT

[0279] 7-deaza-SO2(CH3)-dATP

[0280] 7-deaza-SO2(CH3)-dATP (7-deaza-SC>2Me-dATP) was synthesized according to the synthetic scheme shown in FIG. 13. 7-deaza-2'-deoxy-7-iodoadenosine (compound 1 ; 0.5 g, 1.3 mmol) was dissolved in DMF (5 mL). Sodium methanesulfinate (0.53 g, 5.2 mmol) and copper iodide (0.54 g, 2.9 mmol) were then added, and the reaction mixture was heated to 100 °C for 2 hours under nitrogen. The reaction mixture was diluted with ethyl acetate and extracted with water. The organic layers were combined and concentrated in vacuo to afford a white solid (7-deaza-7-SO2Me-dA, compound 2; 0.24 g, 56%).

[0281] In a first reaction vessel, 7-deaza-7-SO2Me-dA (compound 2; 0.24 g, 0.73 mmol) was added to triethyl phosphate (5.1 mL, 29.9 mmol). A proton sponge (0.47 g, 2.2 mmol) was then added and the reaction mixture was cooled to -17 °C. In a second vessel, tributylammonium pyrophosphate (0.5M in DMF, 15 mL) and tributylamine (1.5 mL, 6.2 mmol) cooled to -17 °C. Once cooled, POOL (0.1 mL, 1.1 mmol) was added dropwise to first vessel, and reaction was checked for completion after 15 minutes by HPLC. The reaction mixture from the first vessel was then transferred to the second vessel, and reaction temperature was warmed 0 °C. Upon complete consumption of monophosphate, aqueous solution of 2M TEAB (7.5 mL) was added dropwise to the second vessel. The reaction temperature was then set to 25 °C and the reaction was stirred overnight.

[0282] The reaction mixture was washed with ethyl acetate (2 x 15 mL) and the organic layer was back-extracted with 0.1 M TEAB (15 mL). The aqueous layer was filtered into Schott bottle through Coming filter unit, and was purified by ion-exchange and reversephase column purifications (0.17 mmol, 23%).

[0283] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in. e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementaryIP-2872-PCT PATENT experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[0284] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0285] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0286] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

IP-2872-PCT PATENTCLAIMS1. A method for reducing quenching in a subsequent sequencing reaction generating clonal amplification sites comprising a dATP analog, comprising:(a) providing an amplification reagent comprising:(i) an array of amplification sites,(ii) a composition comprising a plurality of modified target nucleic acids,(iii) a composition comprising nucleotide triphosphates (NTPs), wherein the NTPs comprise dATP, dTTP, dCTP, dGTP, and a dATP analog comprising a nucleobase, and(iv) a composition comprising a polymerase; and(b) reacting the amplification reagent to produce a plurality of populated amplification sites, wherein the plurality of populated amplification sites each comprise a clonal population of amplicons from an individual modified target nucleic acid from the plurality of modified target nucleic acids.

2. A method for generating clonal amplification sites comprising a dATP analog, comprising:(a) providing an amplification reagent comprising:(i) an array of amplification sites, wherein each amplification site comprises a capture sequence and a single-stranded modified target nucleic acid immobilized thereto;(ii) a composition comprising nucleotide triphosphates (NTPs), wherein the NTPs comprise dATP, dTTP, dCTP, dGTP, and a dATP analog comprising a nucleobase, and(iii) a composition comprising a polymerase; and(b) reacting the amplification reagent to produce a plurality of amplification sites that each comprise a clonal population of amplicons from the single-stranded modified target nucleic acid immobilized thereto in step (a)(i).

3. The method of claim 1, wherein the compositions of (ii) and (iii) are present together in a mixture, the compositions of (iii) and (iv) are present together in a mixture, or the compositions of (ii), (iii), and (iv) are present together in a mixture.IP-2872-PCT PATENT4. The method of claim 2, wherein the compositions of (ii) and (iii) are present together in a mixture.

5. The method of claim 1 or 2, wherein the array comprises a flow cell.

6. The method of claim 1 or 2, wherein the polymerase is Bsu or Bst.

7. The method of claim 1 or 2, wherein the reacting comprises kinetic exclusion amplification or bridge amplification.

8. The method of any one of claim 1-7, wherein the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP.

9. The method of claim 8, wherein the substituted dATP comprises a substituent other than hydrogen at 2 position of the purine ring system.

10. The method of claim 8 or 9, wherein the nucleobase of the dATP analog is of Formula 4:Formula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

11. The method of claim 10, wherein RAis an amine of the formula -NR’R^3where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.IP-2872-PCT PATENT12. The method of claim 11, wherein two of R1, R2, and R3are the same.

13. The method of claim 11, wherein each of R1, R2, and R3are different.

14. The method of any one of claims 11 to 13, wherein at least one of R1, R2, and R3is a lone pair of electrons.

15. The method of claim 8 or 10, wherein the nucleobase of the dATP is16. The method of claim 8, wherein the dATP analog is 7-deaza-dATP.

17. The method of claim 8, wherein the substituted 7-deaza-dATP comprises substituent other than hydrogen at the 7 position of the ring system.

18. The method of claim 8 or 17, wherein the nucleobase of the dATP analog is ofFormula 5 :wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

19. The method of claim 18, wherein RBis I, Br, Cl, or F.IP-2872-PCT PATENT20. The method of claim 18, wherein RBa sulfonyl of the formula -S(O)2-R30whereR30is a Cl to C3 alkyl.

21. The method of claim 8 or 18, wherein the nuclcobasc of the dATP analog is22. An array comprising a sequencing library, the array comprising: a plurality of populated amplification sites attached to the array, wherein the plurality of populated amplification sites each comprise a clonal population of amplicons from an individual modified target nucleic acid from a library of modified target nucleic acids, wherein the amplicons comprise nucleotides dATP, dTTP, dGTP, dCTP, and a dGTP analog comprising a nucleobase.

23. The array of claim 22, wherein the array comprises a flow cell.

24. The array of any one of claim 22-23, wherein the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP.

25. The array of claim 24, wherein the substituted dATP comprises a substituent other than hydrogen at 2 position of the purine ring system.IP-2872-PCT PATENT26. The array of claim 24, wherein the nucleobase of the dATP analog is of Formula4:Formula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

27. The array of claim 26, wherein RAis an amine of the formula -NR1R2R3where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

28. The array of claim 27, wherein two of R1, R2, and R3are the same.

29. The array of claim 27, wherein each of R1, R2, and R3are different.

30. The array of any one of claims 27-29, wherein at least one of R1, R2, and R3is a lone pair of electrons.

31. The array of claim 24 or 26, wherein the nucleobase of the dATP isor32. The array of claim 24, wherein the dATP analog is 7-deaza-dATP.IP-2872-PCT PATENT33. The array of claim 24, wherein the substituted 7-deaza-dATP comprises substituent other than hydrogen at the 7 position of the ring system.

34. The array of claim 24 or 33, wherein the nucleobase of the dATP analog is ofFormula 5 :wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

35. The array of claim 34, wherein RBis I, Br, Cl, or F.

36. The array of claim 34, wherein RBa sulfonyl of the formula -S(O)2-R30where R30is a Cl to C3 alkyl.

37. The array of claim 24 or 34, wherein the nucleobase of the dATP analog isIP-2872-PCT PATENT38. A method for reducing quenching in a subsequent sequencing reaction resynthesis of modified target nucleic acids comprising a dATP analog at amplification sites, comprising:(a) providing an array comprising a plurality of amplification sites, wherein the amplification sites comprise two populations of capture nucleic acids immobilized to the amplification sites at the 5’ end, each population comprising a capture sequence, wherein a first population of capture nucleic acids comprise at each amplification site a clonal population of a modified target nucleic acid, the 5’ end of the clonal population of the modified target nucleic acid attached to the 3’ end of the first population capture nucleic acids, wherein the clonal population of the modified target nucleic acid at each amplification site is a member of a sequencing library;(b) contacting the plurality of amplification sites to a resynthesis reagent comprising (i) a composition comprising nucleotide triphosphates (NTPs), wherein the NTPs comprise dATP, dTTP, dCTP, dGTP, and a dATP analog comprising a nucleobase, and (ii) a composition comprising a polymerase; and(c) reacting the resynthesis reagent to produce a plurality of re -populated amplification sites attached to the array, wherein the plurality of re-populated amplification sites each comprise a clonal population of a resynthesized target nucleic acids immobilized to the amplification sites at the 5’ end, wherein the clonal population of the resynthesized target nucleic acid comprises a nucleic acid sequence that is a complement of the clonal population of the modified target nucleic acid of step (a).

39. A method for resynthesis of modified target nucleic acids comprising a dATP analog at amplification sites, comprising:(a) providing an array comprising a plurality of amplification sites, wherein the amplification sites comprise two populations of capture nucleic acids immobilized to the amplification sites at the 5’ end, each population comprising a capture sequence, wherein a first population of capture nucleic acids comprise at each amplification site a clonal population of a modified target nucleic acid, the 5’ endIP-2872-PCT PATENT of the clonal population of the modified target nucleic acid attached to the 3’ end of the first population capture nucleic acids, wherein a second population of capture nucleic acids comprise (i) the complement of the clonal population of the modified target nucleic acid at each amplification site, the 5’ end of the complement of the clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids, and (ii) a cleavage site, wherein the clonal population of the modified target nucleic acid at each amplification site is a member of a sequencing library;(b) contacting the amplification sites with a cleavage agent, thereby cleaving the second population of capture nucleic acids, and releasing the clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids;(c) removing the released clonal population of the modified target nucleic acid attached to the 3’ end of the second population capture nucleic acids from the amplification sites;(d) contacting the plurality of amplification sites to a resynthesis reagent comprising (i) a composition comprising nucleotide triphosphates (NTPs), wherein the NTPs comprise dATP, dTTP, dCTP, dGTP, and a dATP analog comprising a nucleobase, and (ii) a composition comprising a polymerase; and(e) reacting the resynthesis reagent to produce a plurality of re-populated amplification sites attached to the array, wherein the plurality of re-populated amplification sites each comprise a clonal population of a resynthesized target nucleic acid immobilized to the amplification sites at the 5’ end, wherein the clonal population of the resynthesized target nucleic acid comprises a nucleic acid sequence that is a complement of the clonal population of the modified target nucleic acid of step (a).

40. The method of claim 38 or 39, wherein the array comprises a flow cell.

41. The method of any one of claim 38-40, wherein the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8-oxo-dATP.IP-2872-PCT PATENT42. The method of claim 41, wherein the substituted dATP comprises a substituent other than hydrogen at 2 position of the purine ring system.

43. The method of claim 41 or 42, wherein the nucleobase of the dATP analog is of Formula 4:Formula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group comprising a cyaao moiety, alkynyl, or a group comprising an alkynyl moiety.

44. The method of claim 43, wherein RAis an amine of the formula -NR1R2R3where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

45. The method of claim 44, wherein two of R1, R2, and R3are the same.

46. The method of claim 44, wherein each of R1, R2, and R3are different.

47. The method of any one of claims 44 to 46, wherein at least one of R1, R2, and R3is a lone pair of electrons.IP-2872-PCT PATENT48. The method of claim 41 or 43, wherein the nucleobase of the dATP is49. The method of claim 41, wherein the dATP analog is 7-deaza-dATP.

50. The method of claim 41, wherein the substituted 7-deaza-dATP comprises substituent other than hydrogen at the 7 position of the ring system.

51. The method of claim 41 or 50, wherein the nucleobase of the dATP analog is ofFormula 5 :wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-Oil), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

52. The method of claim 51, wherein RBis I, Br, Cl, or F.

53. The method of claim 51, wherein RBa sulfonyl of the formula -S(O)2-R30where R30is a Cl to C3 alkyl.IP-2872-PCT PATENT54. The method of claim 41 or 51, wherein the nucleobase of the dATP analog is55. A cartridge for use with a sequencing apparatus, the cartridge comprising: a first chamber comprising a nucleotide composition, the nucleotide composition comprising dATP, dTTP, dGTP, dCTP, and a dATP analog comprising a nucleobase.

56. The cartridge of claim 55, further comprising a flow cell, wherein the flow cell is releasably attached to the cartridge.

57. A kit for use with a sequencing apparatus, the kit comprising: a cartridge, the cartridge comprising a first chamber comprising a nucleotide composition, the nucleotide composition comprising dATP, dTTP, dGTP, dCTP, and a dATP analog comprising a nucleobase.IP-2872-PCT PATENT58. The kit of claim 57, further comprising a flow cell, wherein the flow cell is configured to releasably attach to the cartridge.

59. The cartridge of claim 56 or the kit of claim 57, wherein the nucleobase of the dATP analog is a substituted dATP, a substituted 7-deaza-dATP, 7-deaza-dATP, or 8- oxo-dATP.

60. The cartridge or the kit of claim 59, wherein the substituted dATP comprises a substituent other than hydrogen at 2 position of the purine ring system.

61. The cartridge or the kit of claim 59 or 60, wherein the nucleobase of the dATP analog is of Formula 4:Formula 4 wherein RAis halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-OH), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

62. The cartridge or the kit of claim 61, wherein RAis an amine of the formula - NR1R2R' where each of R1, R2, and R3are independently H, a Cl to C3 alkyl, or a lone pair of electrons.

63. The cartridge or the kit of claim 62, wherein two of R1, R2, and R3are the same.

64. The cartridge or the kit of claim 62, wherein each of R1, R2, and R3are different.

65. The cartridge or the kit of any one of claims 62 to 64, wherein at least one of R1, R2, and R3is a lone pair of electrons.IP-2872-PCT PATENT66. The cartridge or the kit of claim 59 or 61, wherein the nucleobase of the dATP67. The cartridge or the kit of claim 59, wherein the dATP analog is 7-deaza-dATP.

68. The cartridge or the kit of claim 59, wherein the substituted 7-deaza-dATP comprises substituent other than hydrogen at the 7 position of the ring system.

69. The cartridge or the kit of claim 59 or 68, wherein the nucleobase of the dATP analog is of Formula 5 :wherein RBis hydrogen, halo, alkyl, acyl, trihaloalkyl, sulfinyl, sulfonyl, hydroxyl (-Oil), an amine, cyano, group comprising a cyano moiety, alkynyl, or a group comprising an alkynyl moiety.

70. The cartridge or the kit of claim 69, wherein RBis I, Br, Cl, or F.

71. The cartridge or the kit of claim 69, wherein RBa sulfonyl of the formula - S(O)2-R30where R30is a Cl to C3 alkyl.IP-2872-PCT PATENT72. The cartridge or the kit of claim 59 or 69, wherein the nucleobase of the dATP

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