Compositions and methods for reducing basecall errors by removing deaminated nucleotides from a nucleic acid library

Abstract

The present disclosure provides a composition comprising a reagent for use in a nucleic acid library preparation workflow for removing deaminated bases, and a method for using the reagent. The compositions and methods described herein reduce base calling errors such as the shift from C:G to T:A in nucleic acid sequencing workflows.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 349,016, filed on June 3, 2022, the entire content of which is incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (ELEM_011_001WO_SeqList_ST26.xml, size: 58,832 bytes, and creation date: June 1, 2023) is incorporated herein by reference in its entirety.

[0003] The present disclosure provides compositions comprising reagents for use in a nucleic acid library preparation workflow for removing deaminated bases, and methods for using the reagents. The compositions and methods described herein reduce base - call errors such as the shift from C:G to T:A in nucleic acid sequencing workflows.

Background Art

[0004] Polynucleotide sequencing technology has applications in biomedical research and medical settings. Improved methods for polynucleotides require enhanced surface chemistry, polynucleotide amplification on supports, and base - calling. Currently, these elements pose barriers in existing sequencing technologies, which result in limitations in throughput and poor signal - to - noise ratios, ultimately leading to increased costs associated with polynucleotide sequencing.

[0005] There is a need for new polynucleotide sequencing methods with improved surface chemistry, amplification on supports, and base - calling. The present disclosure provides methods and compositions for improving polynucleotide sequencing by improving base - calling and then increasing sequencing quality.

Summary of the Invention

[0006] In one aspect, the present disclosure provides a method for reducing deaminated nucleotide bases in a nucleic acid library, the method comprising: providing a plurality of linear nucleic acid library molecules, wherein each library molecule in the plurality of library molecules comprises at least one universal adapter sequence having a binding sequence for a surface capture primer and a target sequence linked to one universal adapter sequence having a binding sequence for a sequencing primer, and at least one of the library molecules carries one or more deaminated nucleotide bases; contacting the plurality of nucleic acid library molecules with a reagent that removes deaminated nucleotide bases, thereby generating at least one library molecule carrying a non-nucleobase site; circularizing the plurality of nucleic acid library molecules to generate a plurality of covalently closed circular library molecules; distributing the plurality of covalently closed circular library molecules onto a support having a plurality of immobilized surface capture primers under conditions suitable for hybridizing each covalently closed circular library molecule to a surface capture primer; performing a rolling circle amplification reaction to generate a plurality of nucleic acid concatemer template molecules immobilized on the support; and sequencing the plurality of nucleic acid concatemer template molecules to determine the sequence of at least a portion of the concatemer template molecules.

[0007] In some embodiments, the method further comprises: (g) contacting the plurality of covalently closed circular library molecules with a reagent that removes deaminated nucleotide bases, thereby generating at least one circular library molecule carrying a non-nucleobase site.

[0008] In some embodiments, the reagent for removing the deaminated nucleotide base includes DNA glycosylase (UDG) and (i) AP lyase, (ii) Endo IV endonuclease, (iii) FPG glycosylase / AP lyase, and / or (iv) Endo VIII glycosylase / AP lyase, or a combination thereof.

[0009] In some embodiments, the reagent for removing the deaminated nucleotide base in step (g) includes DNA glycosylase (UDG) and (i) AP lyase, (ii) Endo IV endonuclease, (iii) FPG glycosylase / AP lyase, and / or (iv) Endo VIII glycosylase / AP lyase, or a combination thereof.

[0010] In some embodiments, the plurality of immobilized surface capture primers are tethered to a polymer coating on a support.

[0011] In some embodiments, the rolling circle amplification reaction in step (e) includes a strand displacement polymerase and a plurality of nucleotides including dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0012] In some embodiments, the plurality of immobilized surface capture primers are located at predetermined positions on a support.

[0013] In some embodiments, the plurality of immobilized surface capture primers are located at random positions on a support.

[0014] In some embodiments, the plurality of immobilized concatemer template molecules on the support are in fluid communication with each other to allow a solution of a reagent to flow onto the support. In some embodiments, the solution of the reagent includes an enzyme, nucleotides, and divalent cations.

[0015] In some embodiments, multiple immobilized concatemeric template molecules react with a reagent essentially simultaneously in a massively parallel manner.

[0016] In some embodiments, the density of the plurality of immobilized concatemeric template molecules on the polymer-coated support is greater than 1 mm 2 10 per 2 ~10 12 It is.

[0017] In some embodiments, sequencing the plurality of immobilized concatemers includes contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases, and (ii) a plurality of soluble sequencing primers, where the contacting is performed under conditions suitable for forming a plurality of multiplexed polymerases, each of the multiplexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising the concatemer molecules hybridized to the soluble sequencing primers, and contacting the multiplexed sequencing polymerases with the plurality of nucleotides and at least one soluble sequencing primer. the plurality of nucleotides comprises at least one nucleotide, the at least one nucleotide analogue being labeled with a fluorophore and having a removable chain-terminating moiety at a sugar 3' position, contacting the plurality of nucleotides with a multiplexed sequencing polymerase under conditions suitable for binding of at least one nucleotide to the multiplexed sequencing polymerase, the plurality of nucleotides comprising at least one nucleotide analogue, the at least one nucleotide analogue being labeled with a fluorophore and having a removable chain-terminating moiety at a sugar 3' position; incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of nascent extended sequencing primers; detecting the incorporated nucleotide and identifying the nucleobase of the incorporated nucleotide.

[0018] In some embodiments, the plurality of nucleotides include a removable chain-terminating moiety at the 3'-sugar group, and the removable chain-terminating moiety includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0019] In some embodiments, the removable chain-terminating moiety is cleavable by a chemical compound to generate an extendable 3'-OH moiety on the sugar group.

[0020] In some embodiments, the plurality of nucleotides include one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0021] In some embodiments, the plurality of nucleotides include a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0022] In some embodiments, sequencing a plurality of immobilized concatemers comprises contacting the plurality of immobilized concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, the contacting being performed under conditions suitable to form a plurality of first composite polymerases, each of the plurality of first composite polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising a concatemer molecule hybridized to a soluble sequencing primer, contacting; contacting the plurality of composite sequencing polymerases with a plurality of detectably labeled multivalent molecules and complementary nucleotide units of the multivalent molecules, under conditions suitable to form a plurality of multivalent composite polymerases by binding to at least two of the plurality of first composite polymerases, the conditions inhibiting incorporation of the complementary nucleotide units into the sequencing primers of the plurality of multivalent composite polymerases, wherein each individual multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit, forming; detecting the plurality of multivalent composite polymerases; identifying the nucleobases of the complementary nucleotide units bound to the plurality of first composite polymerases in the plurality of multivalent composite polymerases, thereby determining the sequence of the nucleic acid template.

[0023] In some embodiments, the method comprises dissociating a plurality of multivalent composite polymerases, removing the plurality of first sequencing polymerases and their associated multivalent molecules, and retaining the plurality of nucleic acid duplexes; contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contacting is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second composite polymerases, each of the plurality of second composite polymerases comprising a second sequencing polymerase bound to a retained nucleic acid duplex; contacting the plurality of second composite polymerases with a plurality of nucleotides comprising at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the 3'-position of the sugar, wherein the contacting is performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second composite polymerases of step (f), thereby forming a plurality of nucleotide composite polymerases, and the conditions are suitable for facilitating incorporation of the bound complementary nucleotides into the sequencing primer of the nucleotide composite polymerase.

[0024] In some embodiments, the method further comprises detecting complementary nucleotides incorporated into the sequencing primer of the nucleotide composite polymerase.

[0025] In some embodiments, the method further comprises detecting complementary nucleotides incorporated into the sequencing primer of the nucleotide composite polymerase and identifying the nucleobases of the complementary nucleotides incorporated into the sequencing primer of the nucleotide composite polymerase.

[0026] In another aspect, provided herein is a method for sequencing by forming at least one avidity complex, the method comprising generating a nucleic acid concatemer by performing rolling circle amplification on a closed circular nucleic acid molecule comprising at least one non-basic site, wherein the non-basic site is generated by contacting the closed circular nucleic acid molecule or the corresponding linear nucleic acid molecule with a reagent that removes deaminated nucleotide bases; binding a first universal sequencing primer, a first sequencing polymerase, and a first detectably labeled multivalent molecule to a first portion of the concatemer molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; binding a second universal sequencing primer, a second sequencing polymerase, and the first detectably labeled multivalent molecule to a second portion of the same concatemer molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex, and the first detectably labeled multivalent molecule comprises a core attached to a plurality of nucleotide arms, each nucleotide arm being attached to a nucleotide unit; the concatemer molecule comprising a target sequence (110) and two or more tandem repeat sequences of a universal primer binding site that binds to the first and second universal sequencing primers; contacting being performed under conditions suitable for inhibiting polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes; detecting the first and second binding complexes on the same concatemer molecule; identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule; and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule.

[0027] In some embodiments, the plurality of nucleotide arms attached to the core of each multivalent molecule have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0028] In some embodiments, the plurality of multivalent molecules includes a mixture of two or more types of multivalent molecules, and each of the two or more types of multivalent molecules has a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0029] In some embodiments, the plurality of nucleotides includes a removable chain-terminating moiety at the 3'-sugar group, and the removable chain-terminating moiety includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

[0030] In some embodiments, the removable chain-terminating moiety is cleavable with a chemical compound to generate an extendable 3'-OH moiety on the sugar group.

[0031] In some embodiments, the plurality of nucleotides includes one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0032] In some embodiments, the plurality of nucleotides includes a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

[0033] In some embodiments, the support includes a glass substrate.

[0034] In some embodiments, the support comprises a plastic substrate.

[0035] In some embodiments, the support is passivated with at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less. In some embodiments, the at least one hydrophilic polymer coating comprises molecules selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

[0036] In some embodiments, the method further comprises determining a percent-based call error from the sequencing of step (f).

[0037] In some embodiments, the method further comprises determining a quality score for the sequencing data from the percent-based call error. In some embodiments, the quality score is a Phred quality score.

[0038] The features of the present invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth exemplary embodiments in which the principles of the present disclosure are utilized, and to the appended drawings.

Brief Description of the Drawings

[0039]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

DETAILED DESCRIPTION OF THE INVENTION

[0040] Definitions: The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the entire specification.

[0041] Unless otherwise defined, all technical and scientific terms used in this specification have the meaning commonly understood by one of ordinary skill in the art. Generally, the terms used in connection with the techniques of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, production of genetically engineered cells, and hybridization described herein are well known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). Also see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with, and the laboratory procedures and techniques described herein, are those well known and commonly used in the art.

[0042] Unless the context clearly requires otherwise herein, the singular forms of terms include the plural, and the plural forms of terms include the singular. The use of the singular forms of "a", "an", and "the", and any word in the singular, is inclusive of the plural reference unless clearly and unambiguously limited to one reference.

[0043] The use of alternative terms (e.g., "or") is understood to mean either one or both of the alternative forms, or any combination thereof.

[0044] As used herein, the term "and / or" should be understood to mean a specific disclosure that each of the particular features or components either has or does not have the other. For example, when used in a phrase such as "A and / or B" herein, the term "and / or" is intended to include "A and B", "A or B", "A" (A alone), and "B" (B alone). In a similar manner, when used in a phrase such as "A, B, and / or C", the term "and / or" is intended to include each of the following aspects: "A, B, and C", "A, B, or C", "A or C", "A or B", "B or C", "A and B", "B and C", "A and C", "A" (alone), "B" (alone), and "C" (alone).

[0045] As used in this specification and the appended claims, the terms "comprising", "including", "having", and "containing", and their grammatical variations as used herein, are intended to be non-limiting such that one item or a plurality of items within the listing do not exclude other items that may be substituted or added to the listed items. It is understood that any aspect described herein in terms of the term "comprising" also provides a similar aspect described in terms of the terms "consisting of" and / or "consisting essentially of".

[0046] As used herein, the terms "about" or "approximately" refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, and the acceptable error range depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations of the mean per practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) depending on the limitations of the measurement system. For example, about 5 mg can include any number from 4.5 mg to 5.5 mg. Further, particularly with respect to biological systems or processes, this term can mean up to one order of magnitude or up to five-fold of the value. When a particular value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be considered to be within the acceptable error range for this particular value or composition. Also, when ranges and / or sub-ranges of values are provided, the ranges and / or sub-ranges can include the endpoints of the ranges and / or sub-ranges.

[0047] The terms "peptide", "polypeptide", and "protein" and other related terms used herein are used synonymously and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides can include natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized types. Also, polypeptides include precursor molecules that have not yet undergone post-translational modifications such as proteolytic cleavage, cleavage by ribosomal skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass native and artificial proteins of protein sequences, protein fragments, and polypeptide analogs (such as mutant proteins, variants, chimeric proteins, and fusion proteins), as well as proteins that have been modified post-translationally or otherwise covalently or non-covalently.

[0048] The term "cellular biological sample" refers to a single cell, multiple cells, tissue, organ, organism, or a section of any of these cellular biological samples. The cellular biological sample may be extracted (e.g., by biopsy) from an organism or obtained from a cell culture growing in a liquid or in a culture dish. The cellular biological sample includes a fresh sample, a frozen sample, a fresh frozen sample, or an archived (e.g., formalin-fixed paraffin-embedded; FFPE) sample. The cellular biological sample may be embedded in wax, resin, epoxy, or agar. The cellular biological sample may be fixed in any one or a combination of any two or more of, for example, acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton, or glutaraldehyde. The cellular biological sample may or may not be sectioned. The cellular biological sample may or may not be stained, destained, or unstained.

[0049] The nucleic acid of interest can be extracted from a cell or a cellular biological sample using any of several techniques known to those skilled in the art. For example, a typical DNA extraction procedure includes (i) collecting a cell sample or tissue sample from which the DNA is to be extracted, (ii) disrupting the cell membrane (i.e., lysing the cells) to release the DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, and then centrifuging to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell lysis. Various suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp™ kit (for isolation of genomic DNA from human samples) and the DNAeasy™ kit (for isolation of genomic DNA from animal or plant samples) from Qiagen® (Germantown, MD), or the Maxwell® and ReliaPrep™ series of kits from Promega® (Madison, WI).

[0050] As used herein, the term "polymerase" and variations thereof include enzymes that contain a domain that binds to nucleotides (or nucleosides), and the polymerase can form a complex with a template nucleic acid and complementary nucleotides. The polymerase can have one or more activities, and the one or more activities include, but are not limited to, base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. The polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including their analogs) into a nucleic acid strand. Typically, although not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, the polymerase contains one or more active sites, and at the one or more active sites, catalysis of nucleotide binding and / or nucleotide polymerization can occur. In some embodiments, the polymerase includes other enzyme activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand displacement activity. The polymerase can include naturally occurring polymerases and any of their subunits and truncated, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives, or fragments (e.g., catalytically active fragments) thereof that retain the ability to catalyze nucleotide polymerization, but are not limited thereto. The polymerase includes catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes that contain nucleotide binding domains. In some embodiments, the polymerase may be isolated from cells or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, the polymerase can be expressed in prokaryotic, eukaryotic, viral, or phage organisms. In some embodiments, the polymerase can be a post-translationally modified protein or a fragment thereof. The polymerase can be derived from prokaryotes, eukaryotes, viruses, or phages.Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0051] The term "strand displacement" refers to the ability of a polymerase to locally separate the strands of a double-stranded nucleic acid and synthesize a new strand in a template-based manner. Strand displacement polymerases displace the complementary strand from the template strand and catalyze the synthesis of a new strand. Strand displacement polymerases include mesophilic and thermophilic polymerases. Strand displacement polymerases include variants that include wild-type enzymes, as well as exonuclease-minus mutants, mutant versions, chimeric enzymes, and cleaved enzymes. Examples of strand displacement polymerases include phi29 DNA polymerase, the large fragment of Bst DNA polymerase, the large fragment of Bsu DNA polymerase (exo-), Bca DNA polymerase (exo-), the Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be the wild-type phi29 DNA polymerase (e.g., MagniPhi(™) from Expedeon(™)), or the variant EquiPhi29(™) DNA polymerase (e.g., from Thermo Fisher Scientific(™)), or the chimeric QualiPhi(™) DNA polymerase (e.g., from 4basebio(™)).

[0052] The terms "nucleic acid", "polynucleotide", and "oligonucleotide" as used herein, and other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, and nucleotides include natural or unnatural bases and / or sugars. Nucleic acids include naturally occurring nucleoside linkages, e.g., phosphodiester linkages. Nucleic acids include non-natural nucleoside linkages, and non-natural nucleoside linkages include phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, a nucleic acid includes one type of polynucleotide, or a mixture of two or more different types of polynucleotides.

[0053] As used herein, the terms "operably linked" and "operably connected," or related terms, refer to the juxtaposition of components. Juxtaposed components can be joined together covalently. For example, two nucleic acid components can be enzymatically ligated together, and the bond that joins the two components together includes a phosphodiester bond. A first and a second nucleic acid component can be joined together, and the first nucleic acid component can confer a function on the second nucleic acid component. For example, the bond between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) can be ligated into a vector, and the bond enables the expression or function of the transgene sequence contained within the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects the expression of the transgene. In some embodiments, the vector includes at least one host cell regulatory sequence, and the at least one host cell regulatory sequence includes, for example, a promoter sequence, an enhancer, a transcription initiation sequence and / or a translation initiation sequence, a transcription termination sequence and / or a translation termination sequence, and a polypeptide secretion signal sequence. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of expression of the transgene.

[0054] The terms “combined,” “linked,” “attached,” “added,” and variations thereof include any kind of fusion, bonding, adhesion, or association between any combination of compounds or molecules having sufficient stability to withstand use in a particular procedure. The procedure can include, but is not limited to, nucleotide binding, nucleotide incorporation, deblocking (e.g., removal of a chain terminating moiety), washing, removal, flow, detection, imaging, and / or identification. Such bonding can include, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonding or association including van der Waals forces, and mechanical bonding. In some embodiments, such bonding occurs within a molecule, for example, to join the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonding can occur between combinations of different molecules, or between a molecule and a non-molecule, including, but not limited to, bonding between a nucleic acid molecule and a solid surface, bonding between a protein and a detectable reporter moiety, and bonding between a nucleotide and a detectable reporter moiety. Some examples of bonding can be found, for example, in Hermanson, G., “Bioconjugate Techniques,” Second Edition (2008), Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences,” London: Macmillan (1998), Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences,” London: Macmillan (1998).

[0055] As used herein, the term "primer" and related terms refer to oligonucleotides capable of hybridizing to a DNA and / or RNA polynucleotide template to form a double-stranded molecule. Primers may be single-stranded along their entire length or may have single-stranded and double-stranded portions. Primers include natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can have any length but typically can range from 4 to 50 nucleotides. A typical primer includes a 5' end and a 3' end. The 3' end of a primer can include a 3'OH moiety that functions as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer may lack a 3'OH moiety or may include a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any single nucleotide or two or more nucleotides along the length of a primer can be labeled with a detectable reporter moiety. Primers may be in solution (e.g., soluble primers) or may be immobilized on a support (e.g., capture primers).

[0056] The terms "template nucleic acid", "template polynucleotide", "target nucleic acid", "target polynucleotide", "template strand" and other variations refer to a nucleic acid strand that functions as a basic nucleic acid molecule for any of the amplification and / or sequencing methods described herein. The template nucleic acid may be single-stranded or double-stranded, or the template nucleic acid may have single-stranded or double-stranded portions. The template nucleic acid may be obtained from a natural source or in recombinant form, or may be chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, concatemeric, circular, or in other forms.

[0057] The term "adapter" and related terms refer to an oligonucleotide that can be operably linked (added) to a target polynucleotide, and the adapter confers a function to the co-ligated adapter-target molecule. The adapter includes DNA, RNA, chimeric DNA / RNA, or analogs thereof. The adapter can include at least one ribonucleoside residue. The adapter can be single-stranded or double-stranded, or can have single-stranded and / or double-stranded portions. The adapter can be configured to be in a linear, stem-loop, hairpin, or Y-shaped form. The adapter can be of any length including from 4 to 100 or more nucleotides. The adapter can have blunt ends, overhang ends, or a combination of both. Overhang ends include 5' overhang and 3' overhang ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, can have a 5' phosphate group or can lack a 5' phosphate group. The adapter can include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tailed adapter), or the adapter can be tail-less. At least a portion of the adapter includes a known and predetermined sequence. The adapter can include a sequence that is complementary to at least a portion of a primer, e.g., an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter can include a random sequence or a degenerate sequence. The adapter can include at least one inosine residue. The adapter can include at least one phosphorothioate, phosphorothiolate, and / or phosphoramidate bond. The adapter can include at least one barcode sequence, and at least one barcode sequence can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. The adapter can include at least one unique identifier sequence (e.g., a molecular tag), and at least one unique identifier sequence can be used to uniquely identify the nucleic acid molecule to which the adapter is added.In some embodiments, the unique identification sequence comprises two to twelve (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) or more nucleotides having a known sequence. For example, the unique identification sequence comprises a known random sequence, in which the nucleotide at each position is randomly selected from nucleotides having bases A, G, C, T, or U. The adapter can comprise at least one restriction enzyme recognition sequence, and the at least one restriction enzyme recognition sequence comprises any one or two or more combinations selected from the group consisting of type I, type II, type III, type IV, Hs type, or type IIB.

[0058] The term "universal sequence" and related terms refer to a sequence in a nucleic acid molecule that is common among two or more polynucleotide molecules. For example, an adapter having a universal sequence can be operably linked to a plurality of polynucleotides, whereby the population of co-ligated molecules bears the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0059] When used in reference to nucleic acids, the terms "hybridize", "hybridizing", "hybridization", or other related terms refer to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a double-stranded region. Hybridization can include Watson-Crick or Hoogsteen bonds to form a double-stranded duplex nucleic acid or a double-stranded region within a nucleic acid molecule. The double-stranded nucleic acid, or the two different regions of a single nucleic acid, may be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other over their entire length. Complementary base pairing may be standard A-T or C-G base pairing, or other forms of base pairing interactions. The double-stranded nucleic acid may contain mismatched base-pairing nucleotides.

[0060] When used in reference to nucleic acids, the terms "extend", "extending", "extension", and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Incorporation of nucleotides includes polymerization of one or more nucleotides to the 3'OH terminus of the end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, although not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending a nucleic acid molecule can be used, and suitable methods include primer extension catalyzed by DNA polymerase or RNA polymerase.

[0061] The term "nucleotide" and related terms refer to a molecule containing an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of this term. In some embodiments, a nucleotide contains a monophosphate, diphosphate, or triphosphate, or the corresponding phosphate analog. The term "nucleoside" refers to a molecule containing an aromatic base and a sugar. Nucleotides and nucleosides may be unlabeled or labeled with a detectable reporter moiety.

[0062] Nucleotides (and nucleosides) typically contain a heterocyclic base comprising a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, which are commonly found in nucleic acids and include naturally occurring, substituted, modified, or engineered variants, or analogs thereof. The bases of nucleotides (or nucleosides) are capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with appropriate complementary bases. Exemplary bases are purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N 6 -Δ 2 -isopentenyladenine (6iA), N 6 -Δ 2 -isopentenyl-2-methylthioadenine (2ms6iA), N 6 -methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine, and O 6 -methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4-Methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methylcytosine; bases (Y); and methylated, glycosylated, and acylated base moieties, among others, but not limited to these. Additional exemplary bases can be found in Fasman, 1989, “Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0063] Nucleotides (and nucleosides) typically include a sugar moiety such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), an acyclic moiety (Martinez, et al., 1999 Nucleic Acids Research 27:1271-1274, Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7:3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36:2627-2638, Kim, et al., 1993 J. Med. Chem. 36:30-7, Eschenmosser 1999 Science 284:2118-2124, and U.S. Patent No. 5,558,991). The sugar moiety includes ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthi ribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthi ribosyl carbocyclic; acyclic, or other modified sugars.

[0064] In some embodiments, the nucleotide comprises a chain of 1, 2, or 3 phosphorus atoms, which chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side chain groups containing O, S, or BH3. In some embodiments, the chain includes a phosphate group substituted with analogs including phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0065] As used herein, "nucleotide unit" or "nucleotide moiety" refers to a nucleotide (e.g., dATP, dTTP, dGTP, dCTP, or dUTP), or an analog thereof, that includes a base, a sugar, and at least one phosphate group. Nucleotide units can be attached to the multivalent molecules used in the sequencing reactions described herein. Generally, all nucleotide units attached to the same multivalent molecule will have the same identity (e.g., all A, all T, all C, or all G), although one of ordinary skill in the art will understand that there may be situations where a multivalent molecule containing nucleotide units of different identities is advantageous.

[0066] The term "rolling circle amplification" generally refers to an amplification method using a circularized nucleic acid template molecule containing a target sequence of interest, an amplification primer binding sequence, and optionally one or more adapter sequences, such as a sequencing primer binding sequence and / or a sample index sequence. The rolling circle amplification reaction can be carried out under isothermal amplification conditions and includes a circularized nucleic acid template molecule, an amplification primer, a strand displacement polymerase, and a plurality of nucleotides, generating a concatemer containing tandem repeat sequences of the circular template molecule and any adapter sequences present within the original circularized nucleic acid template molecule. The concatemer can self-destruct to form nucleic acid nanoballs. The shape and size of the nanoballs can be further compacted by including a pair of inverted repeat sequences within the circular template molecule or by carrying out the rolling circle amplification reaction with one or more compaction oligonucleotides. One of the advantages of using rolling circle amplification to generate cloned amplicons for a sequencing workflow is that the repeat copies of the target sequence in the nanoballs can be sequenced simultaneously to increase the signal intensity. In some embodiments, the rolling circle amplification reaction can be carried out in the presence of a plurality of compaction oligonucleotides having at least four consecutive guanines. The rolling circle amplification reaction generates a concatemer containing repeat copies of a universal binding sequence for the compaction oligonucleotide. At least one compaction oligonucleotide can form a guanine quadruplex, can hybridize to the universal binding sequence for the compaction oligonucleotide, and the resulting concatemer can fold to form an intramolecular G quadruplex structure. The concatemer can self-destruct to form compact nanoballs.The formation of guanine quadruplexes and G-quadruplexes in nanoballs can increase the stability of the nanoballs and allow them to maintain their compact size and shape, which can withstand the repeated flow of reagents for performing any of the sequencing workflows described herein.

[0067] When used with respect to nucleic acids, the terms "amplify", "amplifying", "amplification" and other related terms refer to producing multiple copies of an original polynucleotide template molecule, where the copies include a sequence that is complementary to the template sequence and / or the copies include a sequence that is the same as the template sequence. In some embodiments, the copies include a sequence that is substantially identical to the template sequence and / or a sequence that is substantially identical to a sequence that is complementary to the template sequence.

[0068] The terms "reporter moiety" or "reporter moieties" and related terms refer to a compound that generates a detectable signal or causes it to be generated. Reporter moieties are often referred to as "labels". Any suitable reporter moiety can be used, and suitable reporter moieties include luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. Reporter moieties generate a detectable signal resulting from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or proximity event). Proximity events include two reporter moieties coming into proximity with each other, associating with each other, or binding to each other. It is well known to those skilled in the art to select reporter moieties such that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from that of other reporter moieties to enable monitoring the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties having spectrally distinct emission profiles or minimal overlapping spectral emission profiles can be selected. Reporter moieties can be bound (e.g., operably bound) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerase or reverse transcriptase), or supports (e.g., surfaces).

[0069] The reporter moiety (or label) includes a fluorescent label or fluorophore. Exemplary fluorescent moieties that can function as a fluorescent label or fluorophore include fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamide fluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY 530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl azide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanine and derivatives such as indolium-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridinium-based cyanine dyes, thiazolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, Cy5,Lanthanide chelates and derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycobiliproteins and phycobilin dyes, malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition, Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition or their derivatives, or any combination thereof, but not limited thereto. Cyanine dyes can exist in either sulfonated or non-sulfonated forms and consist of two indolenine, benzindolium, pyridinium, thiazolium, and / or quinolinium groups separated by a polymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which is 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium, or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which is1-(6-((2,5-Dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium, or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate (which may be included), and Cy7 (1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-triene-1-yl]-3H-indolium, or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-triene-1-yl]-3H-indolium-5-sulfonate (which may be included), where "Cy" represents "cyanine" and the first number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzoderivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.,

[0070] In some embodiments, the reporter moiety can be a FRET pair, whereby multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET can include Förster (excitation transfer) or Dexter (electron exchange) transfer.,

[0071] As used herein, the term "support" refers to a substrate designed for the deposition of biomolecules or biological samples for assays and / or analysis. Examples of biomolecules to be deposited on the support include nucleic acids (e.g., DNA, RNA, and combinations thereof), polypeptides, sugars, lipids, single cells, or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0072] In some embodiments, the support is a solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, including a capillary or the inner surface of a capillary.

[0073] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can have a regular or irregular texture, including ridges, etching, pores, three-dimensional scaffolds, or any combination thereof.

[0074] In some embodiments, the support includes beads having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0075] The support can be manufactured from any material, including but not limited to glass, fused silica, silicon, polymers (such as polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. In some embodiments, the support comprises a polymer, such as a synthetic polymer. In some embodiments, the support comprises glass. In some embodiments, the support comprises plastic. Various compositions of both glass and plastic substrates are contemplated.

[0076] In some embodiments, the present disclosure provides a plurality of (e.g., two or more) nucleic acid template molecules immobilized on a support. In some embodiments, the plurality of immobilized nucleic acid template molecules have the same sequence. In some embodiments, the plurality of immobilized nucleic acid template molecules have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid template molecules are immobilized at different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at sites on the support.

[0077] The term "array" refers to a support that includes a plurality of sites located at predetermined positions on the support, forming an array of the sites. The sites can be dispersed and separated by interstitial regions. In some embodiments, the predetermined sites on the support may be arranged in one dimension, for example, in a row or a column. In some embodiments, the predetermined sites on the support may be arranged in two dimensions, for example, in a row or a column. In some embodiments, the plurality of predetermined sites are arranged on the support in an organized manner. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, which includes patterns such as a straight line, a hexagonal pattern, a lattice pattern, a pattern having reflection symmetry, or a pattern having rotational symmetry. The pitch between different pairs of sites may be the same or may vary. In some embodiments, the support includes at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, or at least 10 15 sites or more, and the sites are located at predetermined positions on the support. In some embodiments, the plurality of predetermined sites on the support (for example, 10 2 ~10 15In one or more sites, nucleic acid template molecules are immobilized to form a nucleic acid template array. In some embodiments, at a plurality of predetermined sites, nucleic acid template molecules are immobilized by hybridization to immobilized surface capture primers. In some embodiments, the nucleic acid template molecules are covalently attached to the surface capture primers. In some embodiments, at a plurality of predetermined sites, such as 10 2 ~10 15 sites (e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, or 10 15 sites or more, nucleic acid template molecules are immobilized. In some embodiments, the immobilized nucleic acid template molecules are clonally amplified to generate immobilized nucleic acid clusters at a plurality of predetermined sites. In some embodiments, individual immobilized nucleic acid clusters include linear clusters. In some embodiments, individual immobilized nucleic acid clusters include single-stranded or double-stranded concatemers.

[0078] In some embodiments, a support that includes a plurality of sites located at random positions on the support is referred to herein as a support having randomly positioned sites on top. The positions of the randomly positioned sites on the support are not predetermined positions. The plurality of randomly positioned sites are arranged on the support in a disordered and / or unpredictable manner. In some embodiments, the support has at least 10 2 sites, at least 10 3sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, or at least 10 15 sites or more, and the sites are randomly located on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 10 2 ~10 15 sites or more (e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 sites, or more)) have nucleic acid template molecules immobilized thereon. In some embodiments, the nucleic acid template molecules are immobilized at a plurality of randomly located sites by hybridization to immobilized surface capture primers. In some embodiments, the nucleic acid template molecules are covalently attached to the surface capture primers. In some embodiments, a plurality of randomly located sites, e.g., 10 2 ~10 15 sites (e.g., 10 2 sites, 10 3 sites, 10 4 sites, 105 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 sites, or more), a nucleic acid template is immobilized. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate an immobilized nucleic acid cluster at a plurality of randomly located sites. In some embodiments, individual immobilized nucleic acid clusters include linear clusters. In some embodiments, individual immobilized nucleic acid clusters include single-stranded concatemers or double-stranded concatemers.

[0079] In some embodiments, a plurality of immobilized surface capture primers on a support (e.g., located at predetermined or random positions on the support) are in fluid communication with each other, allowing a solution of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, and buffers, etc.) to flow over the support, whereby the plurality of immobilized surface capture primers on the support can react in a super-parallel manner essentially simultaneously with the reagents. In some embodiments, the fluid communication of the plurality of immobilized surface capture primers can be used to perform a nucleic acid amplification reaction (e.g., RCA, MDA, PCR, and / or bridge amplification) essentially simultaneously at the plurality of immobilized surface capture primers.

[0080] In some embodiments, the plurality of immobilized nucleic acid clusters on the support are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes, nucleotides, and divalent cations, etc.) to flow over the support, whereby the plurality of immobilized nucleic acid clusters on the support can react in a substantially parallel manner essentially simultaneously with the reagents. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid clusters is used to perform a nucleotide binding assay and / or to perform a nucleotide polymerization reaction (e.g., primer extension or sequencing) substantially simultaneously on the plurality of immobilized nucleic acid clusters, and optionally, for detection and imaging for massively parallel sequencing.

[0081] In some embodiments, the term "immobilized" and related terms refer to nucleic acid molecules that are attached to the support via covalent or non-covalent interactions, or attached to a coating on the support, or embedded within a matrix formed by a coating on the support, where the nucleic acid molecules include surface capture primers, nucleic acid template molecules, and extension products of the capture primers. Extension products of the capture primers include, but are not limited to, nucleic acid concatemers (e.g., nucleic acid clusters). The nucleic acid molecules can be immobilized at predetermined or random positions on the support. The nucleic acid molecules can be immobilized at predetermined or random positions on or within an immobilized coating on the support.

[0082] In some embodiments, the term "immobilized" and related terms refer to enzymes (e.g., polymerases) that are attached to the support via covalent or non-covalent interactions, or attached to a coating on the support, or embedded within a matrix formed by a coating on the support. The enzymes can be immobilized at predetermined or random positions on the support. The enzymes can be immobilized at predetermined or random positions on or within an immobilized coating on the support.

[0083] In some embodiments, one or more nucleic acid template molecules are immobilized on a support, e.g., immobilized at a site on the support. In some embodiments, one or more nucleic acid template molecules are clonally amplified. In some embodiments, one or more nucleic acid template molecules are clonally amplified outside the support (e.g., in solution). In some embodiments, after clonal amplification, one or more nucleic acid template molecules are deposited on the support and immobilized on the support. In some embodiments, the clonal amplification reaction of one or more nucleic acid template molecules is performed on the support, resulting in immobilization on the support. In some embodiments, one or more nucleic acid template molecules are clonally amplified using a nucleic acid amplification reaction (e.g., in solution or on a support). In some embodiments, the nucleic acid amplification reaction includes any one or any combination of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-stranded binding (SSB) protein-dependent amplification. Other suitable methods of nucleic acid amplification are well known in the art.

[0084] The term "surface primer" and related terms refer to single-stranded oligonucleotides that are immobilized on a support and contain sequences capable of hybridizing to at least a portion of a nucleic acid template molecule. Surface capture primers can be used to immobilize template molecules to a support via hybridization. Surface capture primers can be immobilized on a support in a manner that resists primer removal during flow, washing, aspiration, and changes in temperature, pH, salt, chemistry, and / or enzymatic conditions. Typically, but not necessarily, the 5' end of a surface capture primer can be immobilized (or embedded within a coating on the support) to the support or a coating on the support. Alternatively, the inner portion or 3' end of a surface capture primer can be immobilized to the support.

[0085] The sequences of surface capture primers can be wholly or partially complementary to at least a portion of a nucleic acid template molecule along their lengths. In some embodiments, a support can include a plurality of immobilized surface capture primers having the same sequence. In some embodiments, a support can include a plurality of immobilized surface capture primers having two or more different sequences. Surface capture primers can be of any length, for example, 4 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, or longer. Those skilled in the art will appreciate that the preferred length of a surface capture primer depends, for example, on the template molecule, the properties of the surface capture primer, etc.

[0086] A surface capture primer can have a terminal 3' nucleotide having a sugar 3'OH moiety that is extendable for nucleotide polymerization (e.g., polymerase-catalyzed polymerization). A surface capture primer can have a terminal 3' nucleotide having a 3'-sugar position bonded to a chain-terminating moiety that inhibits nucleotide polymerization. The 3'-chain-terminating moiety can be removed (e.g., deblocked) using a deblocking agent to convert the 3' terminus to an extendable 3'OH terminus. Examples of chain-terminating moieties include, but are not limited to, an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. Examples of azide-type chain-terminating moieties include, but are not limited to, azide, azido, and azidomethyl groups. Examples of deblocking agents include, but are not limited to, for azide, azido, and azidomethyl groups which are chain-terminating groups, phosphine compounds such as tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfotriphenylphosphine (BS-TPP). Examples of deblocking agents include, but are not limited to, for alkyl, alkenyl, alkynyl, and allyl which are chain-terminating groups, tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) having piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). Examples of deblocking agents include, but are not limited to, for aryl and benzyl which are chain-terminating groups, Pd / C. Examples of deblocking agents include, but are not limited to, for amine, amide, keto, isocyanate, phosphate, thio, and disulfide which are chain-terminating groups, phosphine, beta-mercaptoethanol, or dithiothreitol (DTT). Examples of deblocking agents include, but are not limited to, for carbonate chain-terminating groups, potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, and Zn(AcOH) in acetic acid.Examples of deblocking agents include, but are not limited to, tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, and triethylamine trihydrofluoride for the chain-terminating groups urea and silyl.

[0087] The term "sequencing" and related terms refer to methods for obtaining nucleotide sequence information from nucleic acid molecules, typically by determining the identity of at least some of the nucleotides (including their nucleobase components) within the nucleic acid molecule. In some embodiments, the sequence information for a given region of a nucleic acid molecule includes identifying every single nucleotide within the region to be sequenced. In some embodiments, the sequencing information determines only some of the nucleotides within the region, and the identity of some nucleotides remains unknown, e.g., undetermined or misdetermined. Any suitable sequencing method well known in the art can be used. In an exemplary embodiment, sequencing can include a label-free method. In an exemplary embodiment, sequencing can include an ion-based sequencing method. In some embodiments, sequencing can include a labeled, e.g., dye-containing nucleotide, or a fluorescence-based nucleotide sequencing method. In some embodiments, sequencing can include a polony-based sequencing or bridge sequencing method. In some embodiments, sequencing uses a polymerase and a multivalent molecule to generate at least one avidity complex, where each individual multivalent molecule includes a plurality of nucleotide units tethered to a core. In some embodiments, sequencing uses a polymerase and free nucleotides to perform sequencing by synthesis. In some embodiments, sequencing uses a ligase enzyme and a plurality of sequence-specific oligonucleotides to perform sequencing by ligation.

[0088] In some embodiments, base calling from sequencing data can be evaluated for accuracy and quality. The Q-score is a measure of data quality. In some embodiments, the Q-score can be defined as a Phred quality score. In some embodiments, the Q-score is based on a logarithmic scale. In a particular embodiment, Q = -10 log(P), where P is the error probability. For example, Q10 represents a 10% error, Q20 represents a 1% error, Q30 represents a 0.1% error, and Q40 represents a 0.01% error. In another example, Q10 is 1 error out of 10, Q20 is 1 error out of 100, Q30 is 1 error out of 1,000, Q40 is 1 error out of 10,000, and Q50 is 1 error out of 100,000.

[0089] Introduction In a paired-end sequencing workflow, a low-quality T base call was observed when sequencing the first strand (e.g., R1 read), and a low-quality A base call was observed when sequencing the corresponding position on the complementary second strand (e.g., R2 read). Many of the low-quality T base calls on the first strand alignment align with C bases within a known reference sequence. Without wishing to be bound by theory, it was hypothesized that some of the bases in the library molecules were deaminated, resulting in a base substitution that included a shift from C:G to T:A.

[0090] Deamination is generally the removal of an amino group from a molecule. With respect to nucleotide bases, cytosine (C) can be deaminated to produce uracil (U) where uracil can base pair with adenine (A), guanine (G) can be deaminated to produce xanthine where xanthine can base pair with cytosine (C), and adenine (A) can be deaminated to produce hypoxanthine where hypoxanthine can base pair with cytosine (C).

[0091] The workflow for preparing nucleic acid library molecules includes a number of steps that can cause deamination of nucleotide bases. For example, deamination can be caused by the presence of deaminase enzymes at any stage of the library preparation workflow. As another example, any of the library preparation buffers having a low pH can cause base deamination. In another example, the high temperatures used in PCR can cause deamination of the bases. In another example, the mechanical shearing forces used to fragment the input nucleic acid can generate harmful free radicals that result in deamination. Exemplary mechanical forces include, but are not limited to, ultrasonic forces, acoustic forces, spraying forces, shearing forces, and cavitation forces. In another example, certain chemicals such as bisulfite can cause deamination. It will be appreciated by those skilled in the art that nucleotide base deamination can be produced by many other conditions.

[0092] The present disclosure provides compositions and methods for removing deaminated bases in nucleic acid library molecules. The compositions and methods described herein can be applied to any type of nucleic acid library molecule, including, for example, linear or circularized library molecules, and library molecules for sequencing in a massively parallel fashion.

[0093] Preparation of Linear Library Molecules with Reduced Deaminated Nucleotide Bases In one aspect, the present disclosure provides a method for preparing nucleic acid library molecules having reduced deaminated nucleotide bases. In some embodiments, linear and / or circularized nucleic acid libraries can be prepared, and the library molecules can be treated with a reagent that removes deaminated bases.

[0094] In some embodiments, a method for preparing nucleic acid library molecules having reduced deaminated nucleotide bases generally includes preparing a plurality of linear nucleic acid library molecules, wherein at least one of the linear library molecules includes at least one deaminated base; contacting the plurality of linear library molecules with a reagent that removes the deaminated base, thereby generating at least one linear library molecule having a non-basic site. In some embodiments, the method further includes circularizing individual linear library molecules, including at least one linear library molecule having a non-basic site, to generate a plurality of circularized nucleic acid library molecules. In some embodiments, the plurality of circularized nucleic acid library molecules may include a circularized molecule having at least one non-basic site. In some embodiments, the plurality of circularized nucleic acid library molecules may not include a circularized molecule having at least one non-basic site. In some embodiments, the method further includes contacting the plurality of circularized library molecules with a reagent that removes the deaminated base to generate at least one circularized library molecule having at least one non-basic site.

[0095] In some embodiments, a nucleic acid library can be prepared by fragmenting an input nucleic acid. In some embodiments, the input nucleic acid comprises DNA, RNA, or cDNA. In some embodiments, the input nucleic acid comprises nucleic acids having the same or different sequences. In some embodiments, the input nucleic acid comprises single-stranded or double-stranded nucleic acids. In some embodiments, the input nucleic acid can be fragmented using mechanical force, enzymatic (e.g., restriction endonucleases), or chemical fragmentation methods. In some embodiments, the mechanical force includes ultrasonic force, acoustic force, spraying force, shearing force, or cavitation force. In some embodiments, the mechanical force can generate free radicals that can deaminate nucleotide bases. In some embodiments, nucleic acid fragments can be generated from RNA using reverse transcriptase to produce RNA hybridized to cDNA. In some embodiments, double-stranded DNA can be prepared by reacting DNA polymerase with RNA hybridized to cDNA. In some embodiments, DNA fragments can be generated by performing PCR using a template polynucleotide and a pair of PCR primers. In some embodiments, the input nucleic acid can be fragmented using an enzyme that generates single-stranded nicks and another enzyme that catalyzes double-stranded cleavage. An exemplary enzyme mixture is FRAGMENTASE™ (e.g., from New England Biolabs™). In yet another embodiment, the nucleic acid fragments include cell-free DNA (e.g., double-stranded cfDNA) that has not been subjected to a fragmentation procedure. The cell-free DNA can be 50-200 bp in length, e.g., about 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 110 bp, 120 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp, or 200 bp. In some embodiments, the nucleic acid fragments can be size-selected. In some embodiments, the nucleic acid fragments lack size selection. Those skilled in the art will recognize that nucleic acid fragments can be generated using any of these methods.In some embodiments, the fragments can be of a length of 50 to 1000 bp, or more than 1000 bp, for example, about 50 bp, 75 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, or more than 1000 bp. The nucleic acid fragments can include a heterogeneous mixture of fragments having 5' overhang ends, 3' overhang ends, and / or blunt ends.

[0096] In some embodiments, the nucleic acid library is prepared by modifying (e.g., end-repairing) the 5' end and / or 3' end of the fragmented nucleic acid to convert overhang ends to blunt ends and / or generate desired overhang ends. For example, the nucleic acid fragments can be treated with at least one enzyme to remove 3' overhang ends. Enzymes suitable for use include, for example, but not limited to, DNA polymerase I, the large (Klenow) fragment, T4 DNA polymerase, and / or mung bean nuclease. In some embodiments, the nucleic acid fragments can be treated with at least one enzyme to fill in 5' overhang ends. Enzymes suitable for use include, for example, but not limited to, T4 DNA polymerase, Tfi DNA polymerase, Tli DNA polymerase, Taq DNA polymerase, the large (Klenow) fragment, phi29 DNA polymerase, and / or Mako DNA polymerase. Any of these polymerases can be a thermostable or thermolabile enzyme. In some embodiments, the nucleic acid fragments can be treated with at least one enzyme to remove 5' overhang ends, for example, using SI nuclease. In some embodiments, the nucleic acid fragments can be treated with at least one enzyme to remove 5' or 3' overhang ends, for example, using mung bean nuclease.

[0097] In some embodiments, a nucleic acid library can be prepared by phosphorylating the 5′ end of fragmented nucleic acids or by removing 5′ or 3′ phosphates. For example, nucleic acid fragments can be treated with T4 polynucleotide kinase to phosphorylate the 5′ end of at least one strand of double-stranded DNA. In some embodiments, nucleic acid fragments can be treated with a phosphatase, such as, but not limited to, shrimp alkaline phosphatase, calf intestinal alkaline phosphatase, bacterial alkaline phosphatase, antarctic phosphatase, and / or placental alkaline phosphatase, to remove 5′ or 3′ phosphates.

[0098] In some embodiments, a nucleic acid library can be prepared by adding a non-template tail. In some embodiments, an A-tail (e.g., a polyA tail) containing one or more non-template adenosine nucleotides can be added to the ends of linear library molecules using a DNA polymerase, such as, but not limited to, Taq DNA polymerase (or a derivative thereof), Tfi (exo-minus) DNA polymerase, large fragment Klenow (e.g., 3′-5′ exo-minus), or T4 DNA polymerase.

[0099] In some embodiments, a single non-template A-tail (e.g., a polyA-tail) can be added to the 3′ end of linear library molecules using proofreading of a DNA polymerase, Tfi (exo-) DNA polymerase, or Pfu DNA polymerase, both in the presence of dATP.

[0100] In some embodiments, the nucleic acid library can be prepared by adding at least one adapter to one end of a nucleic acid fragment. In some embodiments, the adapter comprises an oligonucleotide that can be operably linked (added) to the nucleic acid fragment, and the adapter confers a function to the co-ligated adapter fragment molecule. The adapter includes DNA, RNA, chimeric DNA / RNA, or analogs thereof. The adapter can include at least one ribonucleoside residue. The adapter can be single-stranded or double-stranded, or can have single-stranded and / or double-stranded portions. The adapter can be configured to be in a linear, stem-loop, hairpin, or Y-shaped form. The adapter can be of any length including 4 to 100 or more nucleotides, for example, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more. The adapter can have blunt ends, overhang ends, or a combination of both. The overhang ends include 5' overhang and 3' overhang ends. In some embodiments, the 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, may have a 5' phosphate group or may lack a 5' phosphate group. In some embodiments, the adapter may include a 5' tail that does not hybridize to the nucleic acid fragment (e.g., a tailed adapter), or the adapter may be tail-less. At least a portion of the adapter includes a known and predetermined sequence. The adapter can include a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter can include, for example, a random sequence or a degenerate sequence. In some embodiments, the adapter can include at least one inosine residue. In some embodiments, the adapter can include at least one phosphorothioate, phosphorothiolate, and / or phosphoramidate bond.In some embodiments, the adapter can include at least one barcode / index array, and the at least one barcode / index array can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. In some embodiments, the adapter can include at least one unique identification sequence (e.g., molecular tag), and the at least one unique identification sequence can be used to uniquely identify a nucleic acid molecule to which the adapter is added. In some embodiments, the unique identification sequence includes two to twelve (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) or more nucleotides having a known sequence. For example, the unique identification sequence includes a known random sequence, and in the known random sequence, the nucleotide at each position is randomly selected from nucleotides having bases A, G, C, T, or U. The adapter can include, for example, at least one restriction enzyme recognition sequence. In some embodiments, the adapter can include any one or any combination of two or more of the restriction enzyme recognition sequences. In some embodiments, the restriction enzyme recognition sequence is selected from the group consisting of type I, type II, type III, type IV, Hs type, or type IIB.

[0101] In some embodiments, the adapter is universal, for example, it can have a universal sequence. In some embodiments, the universal sequence and related terms refer to a sequence in a nucleic acid molecule (e.g., an adapter) that is common among two or more polynucleotide molecules. For example, an adapter having a universal sequence can be operably linked to a plurality of polynucleotides, whereby the population of co-ligated molecules carries the same universal adapter sequence. Examples of universal adapter sequences include, but are not limited to, amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or immobilized capture primers).

[0102] In some embodiments, the adapter comprises a double-stranded nucleic acid Y-shaped adapter. In certain embodiments, each individual double-stranded adapter comprises first and second oligonucleotide strands that hybridize together, and both the first and second oligonucleotide strands comprise complementary regions and mismatch regions, thereby forming a Y-shaped adapter having a double-stranded annealing portion and a mismatch portion having two single strands.

[0103] In some embodiments, the 5' ends of the first and / or second oligonucleotide strands forming the Y-shaped adapter can be phosphorylated. In some embodiments, the first oligonucleotide strand comprises a first universal adapter sequence or its complementary sequence that comprises a binding sequence for a first sequencing primer. In some embodiments, the second oligonucleotide strand comprises a second universal adapter sequence or its complementary sequence that comprises a binding sequence for a second sequencing primer. In some embodiments, in a population of Y-shaped adapters, each of the first oligonucleotide strands forming the Y-shaped adapter has the same sequence. In some embodiments, in a population of Y-shaped adapters, each of the second oligonucleotide strands forming the Y-shaped adapter has the same sequence. In some embodiments, the double-stranded annealing region of the Y-shaped adapter comprises at least four consecutive base pair nucleotides. In some embodiments, the double-stranded annealing region comprises a terminus that can be ligated to a nucleic acid fragment having a target sequence via an enzymatic ligation reaction. In some embodiments, the double-stranded annealing region comprises a terminus that is a blunt end or the terminus can have a 5' or 3' overhang region. In some embodiments, the first and second oligonucleotide strands of the mismatch portion can be of the same length. In some embodiments, the first and second oligonucleotide strands of the mismatch portion can be of different lengths.

[0104] In some embodiments, the first strand of the double-stranded annealing region of the Y-shaped adapter comprises at least a portion of the binding sequence for a first sequencing primer (e.g., a reverse or forward sequencing primer) or its complementary sequence. In some embodiments, the second strand of the double-stranded annealing region of the Y-shaped adapter comprises at least a portion of the binding sequence for a second sequencing primer (e.g., a forward or reverse sequencing primer) or its complementary sequence.

[0105] In some embodiments, the first strand of the mismatch region comprises at least a portion of the binding sequence for a first sequencing primer (e.g., a reverse or forward sequencing primer) or its complementary sequence. In some embodiments, the first strand of the mismatch region further comprises at least a portion of the binding sequence for a first surface primer or its complementary sequence. In some embodiments, the first strand of the mismatch region comprises a universal adapter sequence that includes a first sample index sequence.

[0106] In some embodiments, the second strand of the mismatch region comprises at least a portion of the binding sequence for a second sequencing primer (e.g., a reverse or forward sequencing primer) or its complementary sequence. In some embodiments, the second strand of the mismatch region further comprises at least a portion of the binding sequence for a second surface primer or its complementary sequence. In some embodiments, the second strand of the mismatch region further comprises a universal adapter sequence that includes a second sample index sequence.

[0107] In some embodiments, only the first strand of the mismatch region comprises a universal adapter, e.g., comprising a first sample index array. In some embodiments, only the second strand of the mismatch region comprises a universal adapter, e.g., comprising a second sample index array. In some embodiments, both the first strand of the mismatch region comprises a first sample index array and the second strand of the mismatch region comprises a second sample index array.

[0108] In some embodiments, a double-stranded nucleic acid fragment having blunt ends or 5' overhang ends or 3' overhang ends can be added to one or both ends using at least one adapter in a ligation reaction. The adapter ligation reaction can comprise a linear double-stranded adapter and / or a double-stranded Y-shaped adapter. In some embodiments, the ligation reaction can be performed using T4 DNA ligase, T3 DNA ligase, or T7 DNA ligase.

[0109] In some embodiments, an adapter sequence can be added to one or both ends of a nucleic acid fragment using a single-stranded-tailed primer. In some embodiments, the adapter sequence is added using at least one primer extension reaction. In some embodiments, the adapter sequence is added using PCR. In some embodiments, the 5' end of the tailed primer contains the adapter sequence to be added to the nucleic acid fragment. In some embodiments, the 5' end of the tailed primer does not hybridize to the nucleic acid fragment. In some embodiments, the 3' end of the tailed primer contains a sequence capable of hybridizing to at least a portion of the nucleic acid fragment. In some embodiments, the adapter sequence can be added to the nucleic acid fragment by performing at least one primer extension reaction using a single-stranded-tailed primer, a polymerase, and a plurality of nucleotides. In some embodiments, the primer extension reaction can add the adapter sequence to, for example, one end of the nucleic acid fragment using one type of tailed primer. In some embodiments, the primer extension reaction can add the adapter sequence to, for example, both ends of the nucleic acid fragment using two types of tailed primers. In some embodiments, multiple primer extension reactions can be used using PCR. In certain embodiments, the heat from the PCR reaction can generate nucleic acid library molecules containing at least one deaminated nucleotide base.

[0110] In some embodiments, the nucleic acid linear library molecules can be generated using any of the methods described above. In some embodiments, individual library molecules include a sequence of interest operably linked on both sides by at least one nucleic acid adapter sequence. In some embodiments, at least one library molecule carries at least one deaminated nucleotide base. In some embodiments, the library molecules can be treated with a reagent that removes the deaminated base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates an abasic site at a uracil base in the nucleic acid molecule. For example, without limitation, DNA glycosylase (UDG) can generate an abasic site at a uracil base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates a gap at the abasic site in the nucleic acid strand. For example, without limitation, the gap can be generated by contacting the abasic site with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site to release the abasic deoxyribose and generate a gap. In certain embodiments, the abasic site can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase, or a combination thereof. In some embodiments, generating an abasic site and removing the abasic site to generate a gap can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. For example, without limitation, a suitable DNA glycosylase-lyase endonuclease VIII can be USER™ (Uracil-Specific Excision Reagent Enzyme from New England Biolabs™) or thermolabile USER™ (similarly from New England Biolabs™).

[0111] Preparation of Cyclized Library Molecules with Reduced Deaminated Nucleotide Bases In another aspect, the present disclosure provides a method for preparing nucleic acid library molecules with reduced deaminated nucleotide bases. In some embodiments, linear library molecules can be cyclized to generate a cyclized nucleic acid library. In some embodiments, the cyclized library molecules can be treated with a reagent that removes deaminated bases.

[0112] In some embodiments, the linear library molecules can be generated using any of the adapters and any of the adapter addition methods described herein. In some embodiments, the linear library molecules can be cyclized using an intramolecular ligation method, a padlock probe, or telomerase. Other suitable methods for cyclizing linear molecules are known in the art.

[0113] In some embodiments, the ends of single-stranded library molecules are subjected to intramolecular ligation using a single-stranded ligase (e.g., CircLigase from Epicentre™ or Lucigen™) to generate covalently closed circular library molecules. The covalently closed circular library molecules can be subjected to a rolling circle amplification reaction.

[0114] In some embodiments, single-stranded library molecules can be circularized using padlock probes. In some embodiments, a padlock probe typically comprises a single-stranded linear oligonucleotide having a 5' portion, an internal linker portion, and a 3' portion. In a padlock probe, the 5' and 3' portions are separately complementary to target sequences in the linear library molecule, and the linker portion is designed to have little or no complementarity to the target sequence. The 5' and 3' portions hybridize to the target sequences, causing the padlock probe to form a circularized molecule. Ligation of the padlock probe forms a covalently closed single-stranded circular nucleic acid molecule when it is hybridized to the target sequence. In some embodiments, the internal linker portion can be engineered to include one or more universal adapter sequences, barcode adapters, or unique identifier adapters. In some embodiments, the covalently closed circular library molecules can be subjected to rolling circle amplification reactions.

[0115] In some embodiments, the circular DNA molecule may be generated without a nucleic acid ligase. For example, without limitation, a telomerase enzyme may identify a target enzyme recognition sequence within a nucleic acid molecule (e.g., a linear library molecule), cleave the enzyme recognition sequence to generate ends with exposed 5' and 3' cleavage termini, and religate the single exposed 5' and 3' cleavage termini at the target site to form a single linear molecule from the cleaved 5' and 3' termini. When this reaction is carried out on both ends of a double-stranded nucleic acid molecule having target enzyme recognition sequences added at each end, the result is a circularized nucleic acid molecule. In some embodiments, an adapter carrying the enzyme recognition sequence may be added to the double-stranded library molecule via ligation or PCR using a tailed PCR primer. Some enzymes or enzyme combinations are compatible with this reaction, including, for example, without limitation, telomerase. One suitable type of telomerase is TelN telomerase, e.g., from E. coli phage Nl. In some embodiments, the covalently closed circular library molecule may be subjected to a rolling circle amplification reaction.

[0116] In some embodiments, the linear library molecule can be generated using any of the adapters and any of the adapter addition methods described herein. In some embodiments, the linear library molecule can be circularized using a single-stranded splint or a double-stranded splint adapter. For example, the linear library molecule can hybridize to a single-stranded splint that can bring about the ends of linear molecules juxtaposed to each other for ligation. In another example, the linear library molecule can hybridize to a double-stranded splint adapter having a short splint strand and a long splint strand, respectively. The long splint strand holds the linear library molecule in a circularized form, and the ends of the short splint strand are juxtaposed to the ends of the linear library molecule for ligation.

[0117] For example, FIG. 1 shows a linear single-stranded library molecule (100) that hybridizes with a double-stranded (ds) splint molecule adapter (200), thereby circularizing the library molecule to form a library-splint complex (500) having two nicks. The library molecule (100) includes a first left universal adapter sequence (120), a first left unique identifier sequence (180), a first left index sequence (160), a second left universal adapter sequence (140), an insert sequence of interest (110), a second right universal adapter sequence (150), a first right index sequence (170), and a first right universal adapter sequence (130). The double-stranded splint molecule includes a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)). The first splint strand includes a first sequence (320) that hybridizes with a sequence on one end of the linear single-stranded library molecule, and a second sequence (330) that hybridizes with a sequence on the other end of the linear single-stranded library molecule. The internal region (310) of the first splint strand hybridizes with the second splint strand (400). The second splint strand (400) includes three sub-regions. The first sub-region includes a universal binding sequence for a third surface primer. The second sub-region includes a universal binding sequence for a fourth surface primer. The third sub-region includes a sample index sequence having 5 to 20 bases and / or a unique identifier sequence having 2 to 10 or more bases (e.g., NN). The internal region (310) of the first splint strand (300) includes three sub-regions. The fourth sub-region hybridizes with the first sub-region of the second splint strand (400). The fifth sub-region hybridizes with the second sub-region of the second splint strand (400). The sixth sub-region hybridizes with the third sub-region of the second splint strand (400).

[0118] In some embodiments, the linear library molecules can be circularized using a double-stranded split adapter (200), which includes a first split strand (long split strand (300)) and a second split strand (short split strand (400)), and the first and second split strands hybridize together to form a double-stranded split adapter (200) having a double-stranded region and two adjacent single-stranded regions (see, e.g., FIGS. 1-2). The second split strand (400) carries the newly introduced adapter sequence(s), e.g., a new universal linker sequence and / or a new index sequence. The first split strand includes a first region (320), an internal region (310), and a second region (330). The internal region of the first split strand (310) hybridizes to the second split strand (400). The two adjacent single-stranded regions of the double-stranded split adapter (e.g., (320) and (330)) are designed to hybridize to the universal adapter sequences at the ends of a single-stranded linear library molecule (100) having the target sequence (110). For example, the first region of the first split strand (320) hybridizes to one end of the library molecule, and the second region of the first split strand (330) hybridizes to the other end of the library molecule, thereby circularizing the library molecule to produce a library-split complex (500) containing two nicks (see, e.g., FIGS. 1-2). The nicks can be enzymatically ligated to produce a covalently closed circular molecule (600), in which the second split strand (400) is covalently linked to the library molecule at both ends, thereby introducing the new adapter sequence into the library molecule.

[0119] In some embodiments, the covalently closed circular library molecules can be generated using any of the methods described above. In some embodiments, individual covalently closed circular library molecules include a sequence of interest operably linked on both sides to at least one nucleic acid adapter sequence. In some embodiments, at least one covalently closed circular library molecule carries at least one deaminated nucleotide base. In some embodiments, the covalently closed circular library molecules can be treated with a reagent that removes the deaminated base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates an abasic site at a uracil base in a nucleic acid molecule. For example, without limitation, DNA glycosylase (UDG) can generate an abasic site at a uracil base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates a gap at an abasic site in a nucleic acid strand. For example, without limitation, the gap can be generated by contacting with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site to release abasic deoxyribose and generate a gap. The abasic site can be removed, for example, without limitation, using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase, or a combination thereof. In some embodiments, generating an abasic site and removing the abasic site to generate a gap can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. For example, without limitation, a suitable DNA glycosylase-lyase endonuclease VIII can be USER™ (Uracil-Specific Excision Reagent Enzyme from New England Biolabs™) or thermolabile USER™ (similarly from New England Biolabs™).

[0120] Method for forming a plurality of library-sprint complexes Use of double-stranded sprint adapters In some embodiments, using any of the methods described above, nucleic acid linear library molecules can be generated that include, in order from 5' to 3': (i) a first left universal adapter sequence (120) having a binding sequence for a first surface primer, (ii) a first left sample index sequence (160), (iii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer, (iv) a target sequence (110), (v) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer, (vi) a first right index sequence (170), and (vii) a first right universal adapter sequence (130) having a binding sequence for a second surface primer (see, for example, FIG. 1).

[0121] In some embodiments, at least one of the linear library molecules bears at least one deaminated nucleotide base. In some embodiments, prior to cyclization, the linear library molecules can be treated with a reagent that removes the deaminated base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates an abasic site at a uracil base in a nucleic acid molecule. For example, DNA glycosylase (UDG) can generate an abasic site at a uracil base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates a gap at the abasic site in a nucleic acid strand. For example, but not limited to, the gap can be generated by contacting with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site to release abasic deoxyribose and generate a gap. The abasic site can be removed, for example, but not limited to, using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase, or a combination thereof. In some embodiments, generating an abasic site and removing the abasic site to generate a gap can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. For example, but not limited to, a suitable DNA glycosylase-lyase endonuclease VIII can be USER™ (Uracil-Specific Excision Reagent Enzyme from New England Biolabs™) or thermolabile USER™ (also from New England Biolabs™).

[0122] In some embodiments, the present disclosure provides a method for forming a plurality of library-sprint complexes (500), comprising: (a) providing a plurality of double-stranded sprint adapters (200), wherein each double-stranded sprint adapter (200) in the plurality of double-stranded sprint adapters (200) comprises a first sprint strand (300) hybridized to a second sprint strand (400), the double-stranded sprint adapter comprising a double-stranded region and two adjacent single-stranded regions, the first sprint strand comprising a first region (320), an internal region (310), and a second region (330), and the internal region of the first sprint strand (310) being hybridized to the second sprint strand (400). Exemplary double-stranded sprint adapters (200) are shown in FIGS. 1-2.

[0123] In some embodiments, the method for forming a plurality of library-sprint complexes (500) further comprises step (b): hybridizing the plurality of double-stranded sprint adapters to a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule comprises a target sequence (110) flanked on one side by at least a first left universal adapter sequence (120) and on the other side by at least a first right universal adapter sequence (130) (e.g., FIGS. 1-2). Hybridizing is performed under conditions suitable for hybridizing the first region of the first sprint strand (320) to at least the first left universal adapter sequence (120) of the library molecule. The hybridization conditions are suitable for hybridizing the second region of the first sprint strand (330) to at least the first right universal sequence (130) of the library molecule, thereby circularizing the plurality of library molecules to form a plurality of library-sprint complexes (500).

[0124] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of a first sprint strand (320) comprises a first universal adapter sequence that can hybridize to a first universal binding sequence at one end of a linear nucleic acid library molecule. In some embodiments, the first region of the first sprint strand (320) comprises a first universal adapter sequence that comprises a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the 5' end of the first sprint strand (300) is phosphorylated. In some embodiments, the 5' end of the first sprint strand (300) lacks a phosphate group. In some embodiments, the 3' end of the first sprint strand (300) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0125] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a second region of a first sprint strand (330) comprises a second universal adapter sequence capable of hybridizing to a second universal binding sequence at the other end of a linear nucleic acid library molecule. In some embodiments, the second region of the first sprint strand (330) comprises a second universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the 5' end of the second sprint strand (400) is phosphorylated. In some embodiments, the 5' end of the second sprint strand (400) lacks a phosphate group. In some embodiments, the 3' end of the second sprint strand (400) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0126] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of a first sprint strand (320) hybridizes to at least a first left universal adapter sequence (120) of a library molecule, and a second region of a first sprint strand (330) hybridizes to at least a first right universal sequence (130) of a library molecule, thereby circularizing the library molecule to generate a library-sprint complex (500). The library-sprint complex (500) comprises a first nick between the 5' end of the library molecule and the 3' end of the second sprint strand (e.g., FIGS. 1-2). The library-sprint complex (500) also comprises a second nick between the 5' end of the second sprint strand and the 3' end of the library molecule (e.g., FIGS. 1-2). In some embodiments, the first and second nicks are enzymatically ligatable.

[0127] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of a first sprint strand (320) can hybridize to a sense or antisense strand of a double-stranded nucleic acid library molecule. In the library-sprint complex (500), a second region of the first sprint strand (330) can hybridize to a sense or antisense strand of the double-stranded nucleic acid library molecule. In some embodiments, the double-stranded nucleic acid library molecule can be denatured to produce single-stranded sense and antisense library strands.

[0128] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a second sprint strand (400) does not hybridize to a target sequence (110), and an internal region of the first sprint strand (310) does not hybridize to the target sequence (110).

[0129] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first region of the first sprint strand (320) does not hybridize to a target sequence (110), and a second region of the first sprint strand (330) does not hybridize to the target sequence (110).

[0130] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the 5' end of a single-stranded library molecule (100) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the single-stranded library molecule includes a terminal 3' OH group or a terminal 3' blocking group.

[0131] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the nucleic acid library molecule (100) further comprises a second left universal adapter sequence (140). In some embodiments, the nucleic acid library molecule (100) further comprises a second right universal adapter sequence (150). In some embodiments, the nucleic acid library molecule (100) can further comprise additional left universal adapter sequences and / or right universal adapter sequences.

[0132] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the nucleic acid library molecule (100) further comprises a first left index sequence (160). In some embodiments, the nucleic acid library molecule (100) further comprises a first right index sequence (170). In some embodiments, the first left index sequence (160) comprises a sample index sequence. In some embodiments, the first right index sequence (170) comprises a different sample index sequence. The sample index sequences can be used to distinguish target sequences obtained from different sample sources in a multiplex assay. A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Tables 1-2 in FIG. 7. In some embodiments, the first left index sequence (160) may comprise a random sequence (e.g., NNN) or may lack a random sequence. In some embodiments, the first right index sequence (170) comprises a random sequence (e.g., NNN). Alternatively, in some embodiments, the first right index sequence (170) lacks a random sequence.

[0133] In some embodiments, multiplex workflows are enabled by preparing a sample-indexed library using one or both index arrays (e.g., a left index array and / or a right index array). In some embodiments, a first left index array (160) and / or a first right index array (170) can be used to prepare separate sample-indexed libraries using input nucleic acids isolated from different sources. In some embodiments, the sample-indexed libraries can be pooled together to generate a multiplex library mixture, and the pooled library can be amplified and / or sequenced. In some embodiments, the sequence of the insert region can be used, together with the first left index array (160) and / or the first right index array (170), to identify the source of the input nucleic acid. In some embodiments, any number of sample-indexed libraries can be pooled together, e.g., 2 to 10, 10 to 50, 50 to 100, 100 to 200, or more than 200 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more than 200) sample-indexed libraries can be pooled. Exemplary nucleic acid sources include, for example, but are not limited to, naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include, for example, but are not limited to, single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include, for example, but are not limited to, fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) sources. It will be appreciated by those skilled in the art that nucleic acids can be isolated from many other sources. In some embodiments, nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0134] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the nucleic acid library molecule (100) further comprises a first left unique identification sequence (180). In some embodiments, the nucleic acid library molecule (100) further comprises a first right unique identification sequence (190). In some embodiments, each of the first left unique identification sequence (180) and the first right unique identification sequence (190) comprises a sequence used to uniquely identify an individual target sequence (e.g., an insert sequence) to which a unique adapter has been added among other sequences of the target molecule. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular tagging.

[0135] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the nucleic acid library molecule (100) comprises any one of a first left universal adapter sequence (120), a second left universal adapter sequence (140), a first left index sequence (160), a first left unique identification sequence (180), a first right universal adapter sequence (130), a second right universal adapter sequence (150), a first right index sequence (170), and / or a first right unique identification sequence (190). In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the nucleic acid library molecule (100) comprises any combination of two or more of a first left universal adapter sequence (120), a second left universal adapter sequence (140), a first left index sequence (160), a first left unique identification sequence (180), a first right universal adapter sequence (130), a second right universal adapter sequence (150), a first right index sequence (170), and / or a first right unique identification sequence (190).

[0136] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the first left universal adapter array (120) and / or the second left universal adapter array (140) comprises a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) can further comprise an additional left universal adapter array.

[0137] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the first right universal adapter array (130) and / or the second right universal adapter array (150) comprises a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, in a method for forming a plurality of library-sprint complexes (500), the first right universal adapter array (130) and / or the second right universal adapter array (150) comprises any combination of two or more of a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (100) can further comprise an additional right universal adapter array.

[0138] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a second sprint strand (400) includes at least two sub-regions including a first and a second sub-region (e.g., FIG. 1). In some embodiments, the first sub-region includes a universal binding sequence for a third surface primer, and the second sub-region includes a universal binding sequence for a fourth surface primer. In certain embodiments, the first and second sub-regions do not hybridize to the first and second surface primers (e.g., exhibit very little or no hybridization to them). In some embodiments, the second sprint strand (400) further includes an optional third sub-region, and the optional third sub-region includes a sample index sequence having 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), and / or an identification sequence having 2 to 10 or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) bases (e.g., NN) (e.g., FIG. 1). In some embodiments, the second sprint strand (400) includes only one sub-region and lacks the second and third sub-regions. In certain embodiments, the first sub-region includes a sample index sequence having 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases). In some embodiments, the sample index sequence can be used to distinguish sequences of interest obtained from different sample sources in a multiplex assay. In some embodiments, the identification sequence includes a random sequence. In some embodiments, the identification sequence can be designed to exhibit reduced hybridization to the first, second, third, and fourth surface primers or no hybridization to them. A non-limiting exemplary arrangement of sub-regions in the second sprint strand (400) in the 5' to 3' direction includes 5'-[second sub-region]-[first sub-region]-3'.Another non-limiting exemplary arrangement of sub-regions in the second sprint strand (400) in the 5' to 3' direction is 5'-[third sub-region]-[second sub-region]-[first sub-region]-3'. In some embodiments, the second sprint strand (400) can be 20 to 100 (e.g., about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100) nucleotides in length. In some embodiments, the second sprint strand (400) can be 30 to 80 (e.g., about 30, 35, 40, 45, 50, 60, 70, or 80) nucleotides in length. In some embodiments, the second sprint strand (400) can be 40 to 60 (e.g., about 40, 45, 50, or 60) nucleotides in length. In some embodiments, the second sprint strand (400) includes one or more phosphorothioate bonds at the 5' and / or 3' termini to confer exonuclease resistance. In some embodiments, the second sprint strand (400) includes one or more phosphorothioate bonds at internal positions to confer endonuclease resistance. In some embodiments, the second sprint strand (400) includes one or more 2'-O-methylcytosine bases at the 5' and / or 3' termini or at internal positions. In some embodiments, the 5' terminus of the second sprint strand (400) is phosphorylated. In some embodiments, the 5' terminus of the second sprint strand (400) is non-phosphorylated. In some embodiments, the 3' terminus of the second sprint strand (400) includes a terminal 3' OH group or a terminal 3' blocking group.

[0139] In some embodiments, in a method for forming a plurality of library-sprint complexes (500), a first sprint strand (300) includes an internal region (310) that includes at least two sub-regions including a fourth and a fifth sub-region (e.g., FIG. 1). In some embodiments, the fourth sub-region hybridizes to a first sub-region of a second sprint strand (400). In some embodiments, the fifth sub-region hybridizes to a second sub-region of the second sprint strand (400). The fourth and fifth sub-regions do not hybridize to the first and second surface primers (e.g., show very little or no hybridization to them). In some embodiments, the internal region (310) of the first sprint strand further includes an optional sixth sub-region that hybridizes to a third sub-region of the second sprint strand (400) (e.g., FIG. 1). Non-limiting exemplary arrangements of the sub-regions of the first sprint strand (300) in the 3' to 5' direction include 3'-[fourth sub-region]-[fifth sub-region]-5'. Another non-limiting exemplary arrangement of the sub-regions of the first sprint strand (300) in the 3' to 5' direction includes 3'-[fourth sub-region]-[fifth sub-region]-[sixth sub-region]-5'. In some embodiments, the first sprint strand (300) can be 50 to 150 (e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150) nucleotides in length. In some embodiments, the first sprint strand (300) can be 60 to 100 (e.g., about 60, 70, 80, 90, or 100) nucleotides in length. In some embodiments, the first sprint strand (300) can be 70 to 90 (e.g., about 70, 75, 80, 85, or 90) nucleotides in length. In some embodiments, the first sprint strand (300) includes one or more phosphorothioate bonds at the 5' and / or 3' termini to confer exonuclease resistance. In some embodiments, the first sprint strand (300) includes one or more phosphorothioate bonds at internal positions to confer endonuclease resistance.In some embodiments, the first splint strand (300) comprises one or more 2'-O-methylcytosine bases at the 5' and / or 3' terminus, or at an internal position.

[0140] In another aspect, the present disclosure provides a method for forming a plurality of library-sprint complexes (500), the method comprising: (a) providing a plurality of double-stranded sprint adapters (200), each double-stranded sprint adapter (200) including a first sprint strand (300) hybridized to a second sprint strand (400). In some embodiments, the first sprint strand (300) includes a first region (320), an internal region (310), and a second region (330) that are arranged in a 5' to 3' order. In some embodiments, the internal region of the first sprint strand (310) is hybridized to the second sprint strand (400). In certain embodiments, the second sprint strand is a region arranged in a 5' to 3' order and includes: (i) a second sub-region having a universal binding sequence for a fourth surface primer, and (ii) a first sub-region having a universal binding sequence for a third surface primer. In some embodiments, the method for forming a plurality of library-sprint complexes (500) further comprises step (b): hybridizing the plurality of double-stranded sprint adapters to a plurality of single-stranded nucleic acid library molecules (100). In certain embodiments, each library molecule is a region arranged in a 5' to 3' order and includes: (i) a first left universal adapter sequence (120) having a binding sequence for a first surface primer, (ii) a second left universal adapter sequence (140) having a binding sequence for a first sequencing primer, (iii) a target sequence (110), (iv) a second right universal adapter sequence (150) having a binding sequence for a second sequencing primer, and (v) a first right universal adapter sequence (130) having a binding sequence for a second surface primer (130). In certain embodiments, the hybridization is performed under conditions suitable for hybridizing the first sprint strand (300) to the library molecule (100), thereby circularizing the library molecule to generate a library-sprint complex (500).In some embodiments, a first region (320) of the first split strand hybridizes to a binding sequence for a first surface primer (120), and a third region (330) of the first split strand hybridizes to a binding sequence for a second surface primer (130). In certain embodiments, the library-split complex (500) includes a first nick between the 5' end of a library molecule and the 3' end of the second split strand (300). In certain embodiments, the library-split complex (500) also includes a second nick between the 5' end of the second split strand (300) and the 3' end of the library molecule (100). In certain embodiments, the first and second nicks are enzymatically ligatable. In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further includes a first left index sequence (160) and / or a first right index sequence (170) (see, e.g., FIGS. 1 and 2). A list of exemplary first left index sequences (160) and first right index sequences (170) is provided in Tables 1-2 in FIG. 7. In some embodiments, the first left index sequence (160) includes a short random sequence (e.g., NNN). In some embodiments, the first left index sequence (160) lacks a short random sequence (e.g., NNN). In some embodiments, the first right index sequence (170) includes a short random sequence (e.g., NNN). In some embodiments, the first right index sequence (170) lacks a short random sequence (e.g., NNN). In some embodiments, the plurality of single-stranded nucleic acid library molecules (100) further includes a first left unique identification sequence (180) and / or a first right unique identification sequence (190). In certain embodiments, each of the unique identification sequences includes a sequence used to uniquely identify an individual target sequence (e.g., an insert sequence) to which a unique adapter has been added among a population of other sequences of the target molecule. In some embodiments, the first left unique identification sequence (180) and / or the first right unique identification sequence (190) can be used for molecular tagging. (See, e.g., FIG. 1).

[0141] Multiplex workflows are enabled by preparing a sample-indexed library using one or both index arrays (e.g., a left index array and / or a right index array). In some embodiments, a first left index array (160) and / or a first right index array (170) can be used to prepare separate sample-indexed libraries using input nucleic acids isolated from different sources. In some embodiments, the sample-indexed libraries can be pooled together to generate a multiplex library mixture, and the pooled library can be amplified and / or sequenced. In some embodiments, the sequence of the insert region can be used, together with the first left index array (160) and / or the first right index array (170), to identify the source of the input nucleic acid. In some embodiments, the sample-indexed libraries can be pooled together, for example, 2 to 10 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10) libraries. In some embodiments, 10 to 50 (e.g., about 10, 20, 30, 40, or 50) sample-indexed libraries are pooled together. In some embodiments, 50 to 100 (e.g., about 50, 60, 70, 80, 90, or 100) sample-indexed libraries are pooled together. In some embodiments, 100 to 200 (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) sample-indexed libraries are pooled together. In some embodiments, more than 200 sample-indexed libraries can be pooled. Exemplary nucleic acid sources include, but are not limited to, naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include, but are not limited to, fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) sources.Those skilled in the art will recognize that nucleic acids can be isolated from many other sources. In some embodiments, nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0142] In some embodiments, a plurality of single-stranded nucleic acid library molecules (100) further comprise a first left unique identifier sequence (180) and / or a first right unique identifier sequence (190) (see, for example, FIG. 1). In some embodiments, the first left unique identifier sequence (180) and the first right unique identifier sequence (190) each comprise sequences that are used to uniquely identify an individual sequence of interest (e.g., an insert sequence) to which a unique adapter has been added, among a population of other sequences of the molecule of interest. In some embodiments, the first left unique identifier sequence (180) and / or the first right unique identifier sequence (190) can be used for molecular tagging.

[0143] In some embodiments, any of the methods for forming a plurality of library-sprint complexes (500) described herein can further comprise at least one enzymatic reaction, such as a phosphorylation reaction, a ligation reaction, an exonuclease reaction, or a combination thereof. The enzymatic reactions can be performed sequentially or essentially simultaneously. In some embodiments, the enzymatic reactions can be performed in a single reaction vessel. Alternatively, in some embodiments, a first enzymatic reaction can be performed in a first reaction vessel and then transferred to a second reaction vessel, where a second enzymatic reaction can be performed in the second reaction vessel, and then transferred to a third reaction vessel, where a third enzymatic reaction can be performed in the third reaction vessel, and so on.

[0144] In some embodiments, any of the methods for forming the plurality of library-sprint complexes (500) described herein involves performing separate and sequential phosphorylation and ligation reactions, which further includes performing the separate and sequential phosphorylation and ligation reactions in separate reaction vessels. In some embodiments, the method for forming the plurality of library-sprint complexes (500) further includes step (c1): contacting, in a first reaction vessel, a plurality of double-stranded sprint adapters (200) and a plurality of single-stranded nucleic acid library molecules (100) with a T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded sprint adapters (200) and / or the plurality of single-stranded nucleic acid library molecules (100). In some embodiments, the phosphorylation reaction is transferred to a second reaction vessel. In some embodiments, the method for forming the plurality of library-sprint complexes (500) further includes step (d1): contacting, in a second reaction vessel, a plurality of phosphorylated double-stranded sprint adapters (200) and a plurality of phosphorylated single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatically ligating the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600) each hybridized to a first sprint strand (300). In some embodiments, the ligase enzyme includes T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0145] In some embodiments, any of the methods for forming the plurality of library-sprint complexes (500) described herein includes performing sequential phosphorylation and ligation reactions, and further includes performing the sequential phosphorylation and ligation reactions sequentially within the same reaction vessel. In some embodiments, the method for forming the plurality of library-sprint complexes (500) includes step (c2): contacting, within a first reaction vessel, a plurality of double-stranded sprint adapters (200) and a plurality of single-stranded nucleic acid library molecules (100) with a T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded sprint adapters (200) and the plurality of single-stranded nucleic acid library molecules (100). In some embodiments, the method for forming the plurality of library-sprint complexes (500) includes step (d2): contacting, within the same first reaction vessel, the phosphorylated double-stranded sprint adapters (200) and the phosphorylated single-stranded nucleic acid library molecules (100) with a ligase under conditions suitable for enzymatically ligating the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600) each hybridized to a first sprint strand (300). In some embodiments, the ligase enzyme includes T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0146] In some embodiments, any of the methods for forming a plurality of library-sprint complexes (500) described herein comprises performing phosphorylation and ligation reactions that are essentially simultaneous, further comprising performing the phosphorylation and ligation reactions that are essentially simultaneous together within the same reaction vessel. In some embodiments, the method for forming a plurality of library-sprint complexes (500) comprises step (c3): contacting, within a first reaction vessel, a plurality of double-stranded sprint adapters (200) and a ligase enzyme with a plurality of single-stranded nucleic acid library molecules (100), (i) a T4 polynucleotide kinase enzyme and (ii) a ligase enzyme, under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded sprint adapters (200) and the plurality of single-stranded nucleic acid library molecules (100). In some embodiments, the conditions are suitable for enzymatically ligating the first and second nicks, thereby generating a plurality of covalently closed circular library molecules (600) each hybridized to a first sprint strand (300). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0147] In some embodiments, any of the methods for forming the plurality of library-sprint complexes (500) described herein may include an optional step of enzymatically removing a plurality of first sprint strands (300) from a plurality of covalently closed circular library molecules (600), the method further including contacting the plurality of covalently closed circular library molecules (600) with at least one exonuclease enzyme to remove the plurality of first sprint strands (300) and retain the plurality of covalently closed circular library molecules (600). In some embodiments, the exonuclease reaction can be performed in the same reaction buffer used for performing phosphorylation and / or ligation reactions. In some embodiments, the exonuclease reaction can be performed in a reaction buffer different from the reaction buffer used for performing phosphorylation and / or ligation reactions. In some embodiments, after performing the phosphorylation reaction in a first reaction vessel (c1) and performing the ligation reaction in a second reaction vessel (d1), the exonuclease reaction can be performed in a third reaction vessel. In some embodiments, after performing the phosphorylation reaction in a first reaction vessel (c2) and performing sequential ligation reactions in the first reaction vessel (d2), the exonuclease reaction can be performed in the first reaction vessel. In some embodiments, after performing essentially simultaneous phosphorylation and ligation reactions in a first reaction vessel (c3), the exonuclease reaction can be performed in the first reaction vessel. In some embodiments, the at least one exonuclease enzyme includes any combination of two or more of exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0148] In some embodiments, the covalently closed circular library molecule (600) can be generated using any of the methods described above. In some embodiments, an individual covalently closed circular library molecule comprises a sequence of interest operably linked on both sides by at least one nucleic acid adapter sequence. In some embodiments, at least one covalently closed circular library molecule has at least one deaminated nucleotide base. In some embodiments, the covalently closed circular library molecule can be treated with a reagent that removes the deaminated base. In some embodiments, the reagent that removes the deaminated base comprises a compound that generates an abasic site at a uracil base in a nucleic acid molecule. For example, but not limited to, DNA glycosylase (UDG) can generate an abasic site at a uracil base. In some embodiments, the reagent that removes the deaminated base comprises a compound that generates a gap at an abasic site in a nucleic acid strand. For example, but not limited to, a gap can be generated by contacting with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site to release abasic deoxyribose and generate a gap. The abasic site can be removed using, for example, but not limited to, AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase, and combinations thereof. In some embodiments, generating an abasic site and removing the abasic site to generate a gap can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. Suitable uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII include, for example, but not limited to, USER™ (Uracil-Specific Excision Reagent Enzyme from New England Biolabs™) or thermolabile USER™ (similarly from New England Biolabs™).

[0149] In some embodiments, in any of the methods for forming the plurality of library-sprint complexes (500) described herein, the first sub-region of the second sprint strand (400) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1). In some embodiments, the second sub-region of the second sprint strand (400) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2). In some embodiments, the second sprint strand (400) comprises the first and second sub-regions comprising the sequence 5'-AGTCGTCGCAGCCTCACCTGATCCATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 3). See Figure 3. In some embodiments, the 5' end of the second sprint strand (400) can be phosphorylated. In some embodiments, the 5' end of the second sprint strand (400) can be non-phosphorylated.

[0150] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, a first region of a first sprint strand (320) comprises a first universal adapter sequence that comprises a universal binding sequence (or its complementary sequence) for a first surface primer. In some embodiments, the first region (320) comprises the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 4). For example, without limitation, the first region of the first sprint strand (320) can hybridize to a P5 surface primer or a complementary sequence of a P5 surface primer. For example, without limitation, the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 204; short P5), or the P5 surface primer comprises the sequence 5'-AATGATACGGCGACCACCGAGATC-3' (SEQ ID NO: 205; long P5). In some embodiments, a second region of a first sprint strand (330) comprises a second universal adapter sequence that comprises a universal binding sequence (or its complementary sequence) for a second surface primer. In some embodiments, the second region (330) comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5). For example, without limitation, the second region of the first sprint strand (330) can hybridize to a P7 surface primer or a complementary sequence of a P7 surface primer. For example, without limitation, the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5; short P7), or the P7 surface primer comprises the sequence 5'-CAAGCAGAAGACGGCATACGAGAT-3' (SEQ ID NO: 206; long P7). In some embodiments, the first sprint strand (300) comprises an internal region (310) that comprises a fourth sub-region having the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 6). In some embodiments, the first sprint strand (300) comprises an internal region (310) that comprises a fifth sub-region having the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7).In some embodiments, the first splint strand (300) includes a first region (320), an internal region (310) having fourth and fifth sub-regions, and a second region (330) having the sequence 5'-TCGGTGGTCGCCGTATCATTACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 8). See FIG. 3. In some embodiments, the 5' end of the first splint strand (300) can be phosphorylated. In some embodiments, the 5' end of the first splint strand (300) can be non-phosphorylated. In some embodiments, the first sub-region of the second splint strand (400) can hybridize to the fourth sub-region of the first splint strand (300). In some embodiments, the second sub-region of the second splint strand (400) can hybridize to the fifth sub-region of the first splint strand (300).

[0151] In some embodiments, in any of the methods for forming a plurality of library-splint complexes (500) described herein, the first region of the first splint strand (320) includes a sequence that can bind to the first left universal adapter sequence (120) of a library molecule, and the first region of the first splint strand (320) includes the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 6) or its complementary sequence.

[0152] In some embodiments, in any of the methods for forming a plurality of library-splint complexes (500) described herein, the second region of the first splint strand (330) includes a sequence that can bind to the first right universal adapter sequence (130) of a library molecule, and the second region of the first splint strand (330) includes the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7) or its complementary sequence.

[0153] In some embodiments, in any of the methods for forming the plurality of library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first sprint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 204).

[0154] In some embodiments, in any of the methods for forming the plurality of library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (120) that binds to a first region of the first sprint strand (320), and the left universal binding sequence (120) comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1) or its complementary sequence.

[0155] In some embodiments, in any of the methods for forming the plurality of library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (140) for a sequencing primer, and the left universal binding sequence comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 207).

[0156] In some embodiments, in any of the methods for forming the plurality of library-sprint complexes (500) described herein, the library molecule comprises a left universal binding sequence (140) for a sequencing primer, and the left universal binding sequence comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 208).

[0157] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules include a left universal binding sequence (140) for a sequencing primer, and the left universal binding sequence includes the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 209).

[0158] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules include a left universal binding sequence (150) for a sequencing primer, and the left universal binding sequence includes the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 210).

[0159] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules include a left universal binding sequence (150) for a sequencing primer, and the left universal binding sequence includes the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 211).

[0160] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecules include a left universal binding sequence (150) for a sequencing primer, and the left universal binding sequence includes the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 212).

[0161] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first sprint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 213).

[0162] In some embodiments, in any of the methods for forming a plurality of library-sprint complexes (500) described herein, the library molecule comprises a right universal binding sequence (130) that binds to a first region of the first sprint strand (330), and the right universal binding sequence (130) comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2) or its complementary sequence.

[0163] Method for rolling circle amplification Use of circularized library molecules generated via a ds-sprint adapter The present disclosure provides a method for performing a rolling circle amplification reaction in a covalently closed circular library molecule (600). In some embodiments, the rolling circle amplification reaction can be performed after both phosphorylation and ligation reactions. In some embodiments, the rolling circle amplification reaction is after the ligation reaction. In some embodiments, the rolling circle amplification reaction can be performed in a covalently closed circular library molecule (600) that is no longer hybridized to the first splint strand (300) after an exonuclease reaction. In some embodiments, the rolling circle amplification reaction can be performed in a covalently closed circular library molecule (600) hybridized to the first splint strand (300). In some embodiments, the covalently closed circular library molecule (600) can be dispensed onto a support and then subjected to a rolling circle amplification reaction. In some embodiments, the covalently closed circular library molecule (600) can be subjected to a rolling circle amplification reaction in solution and then dispensed onto a support. In some embodiments, the rolling circle amplification reaction may use the retained first splint strand (300) as an amplification primer, or the first splint strand (300) can be removed (e.g., via exonuclease digestion) and replaced with a soluble amplification primer.

[0164] Rolling Circle Amplification on a Support Use of Circularized Library Molecules Generated via a ds-Splint Adapter In some embodiments, a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules lacking a hybridized first sprint strand (300). In certain embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) include a second sprint strand region (400) that includes a universal binding sequence for a third surface primer, and the method includes: (a) distributing the plurality of covalently closed circular library molecules (600) onto a support on which a plurality of third surface primers are immobilized under conditions suitable for hybridizing individual covalently closed circular library molecules (600) to the individual immobilized third surface primers, thereby immobilizing the plurality of covalently closed circular library molecules (600).

[0165] In some embodiments, the plurality of third surface primers immobilized on the support include the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7). Individual third surface primers can hybridize to a covalently closed circular library molecule (600) having a second sprint strand region (400) that includes a universal binding sequence for the third surface primer. In certain embodiments, the universal binding sequence for the third surface primer includes a second sub-region that includes the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2).

[0166] In some embodiments, a plurality of covalently-closed cyclic library molecules (600) can be dispensed onto a support coated with one or more compounds to produce an immobilized layer on the support (e.g., FIG. 8). In some embodiments, the immobilized layer forms a porous layer. In some embodiments, the immobilized layer forms a semi-porous layer. In some embodiments, a surface primer, concatemer template molecule, polymerase, or a combination thereof can attach to the immobilized layer for immobilization to the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. Generally, the support can include one or more layers of a low-binding chemical modification layer, e.g., a silane layer, a polymer film, covalently or non-covalently attached, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can include, at least in part of the support, a functionalized polymer coating layer covalently bonded via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating includes poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support includes a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In certain embodiments, the hydrophilic polymer coating layer can include polyethylene glycol (PEG). In certain embodiments, the hydrophilic polymer coating layer can include branched PEG having at least four branches. In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity that can be measured as a water contact angle.In certain embodiments, the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less). In some embodiments, the density of the covalently closed cyclic library molecules (600) immobilized on the support or immobilized on a coating on the support is about 10 2 to 10 2 ~10 6 , (e.g., 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 ) per mm. In some embodiments, the covalently closed cyclic library molecules (600) immobilized on the support or immobilized on a coating on the support are about 10 2 to 10 6 ~10 9 (e.g., about 10 6 , 10 7 , 10 8 , or 10 9 ) per mm. In some embodiments, the density of the covalently closed cyclic library molecules (600) immobilized on the support or immobilized on a coating on the support is about 10 2 to 10 9 ~10 12 (e.g., about 10 9 , 10 10 , 10 11 , or 10 12 ) per mm. In some embodiments, the plurality of covalently closed cyclic library molecules (600) are immobilized on the support or on a coating on the support at a predetermined site on the support (or the coating on the support). In some embodiments, the plurality of covalently closed cyclic library molecules (600) are immobilized on the support or on a coating on the support at a random site on the support (or the coating on the support).

[0167] In some embodiments, the dispensing in step (a) can be performed in the presence of a high-efficiency hybridization buffer, the high-efficiency hybridization buffer having: (i) a first polar aprotic solvent having a dielectric constant of 40 or less (e.g., less than 10, or about 10, 15, 20, 30, or 40) and a polarity index of 4 to 9 (e.g., 4, 5, 6, 7, 8, or 9); (ii) a second polar aprotic solvent having a dielectric constant of 115 or less (e.g., less than 10, 10, 15, 20, 30, 40, 50, 75, 100, 105, 105, 110, or 115) and present in an amount effective to denature double-stranded nucleic acid in the hybridization buffer formulation; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8 (e.g., 4, 5, 6, 7, or 8); and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprising acetonitrile at 25 to 50% by volume of the hybridization buffer (e.g., 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, or 50% by volume); (ii) the second polar aprotic solvent comprising formamide at 5 to 10% by volume of the hybridization buffer (e.g., 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, or 10% by volume); (iii) the pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5); and (iv) the crowding agent comprising polyethylene glycol (PEG) at 5 to 35% by volume of the hybridization buffer (e.g., 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, or 35% by volume). In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0168] In some embodiments, a method for performing a rolling circle amplification reaction comprises step (b): contacting a plurality of immobilized covalently closed circular library molecules (600) with a plurality of strand-displacing polymerases and a plurality of nucleotides (e.g., bases A, G, C, T, and / or U) under conditions suitable for performing a rolling circle amplification reaction on a support, using a plurality of third surface primers as immobilized amplification primers and using a plurality of covalently closed circular library molecules (600) as template molecules, thereby further generating a plurality of nucleic acid concatemer molecules immobilized to the third surface primers. In some embodiments, the plurality of nucleotides comprises any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual immobilized concatemers are covalently linked to individual third surface primers. In some embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) comprise a second sprint strand region (400) that also includes a universal binding sequence for a fourth surface primer, whereby the rolling circle amplification reaction generates concatemer molecules having multiple copies of the universal binding sequences for the third and fourth surface primers. In some embodiments, the method comprises dispensing covalently closed circular library molecules (600) onto a support comprising a plurality of immobilized third and fourth surface primers, performing a rolling circle amplification reaction under conditions suitable for hybridizing at least one second sprint strand region (400) of a concatemer molecule to the immobilized third and fourth surface primers to generate a concatemer molecule, thereby pressing at least one portion of the concatemer molecule against the support. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0169] In some embodiments, the plurality of fourth surface primers immobilized on the support comprise the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1) or its complementary sequence). Each individual fourth surface primer is capable of hybridizing to a portion of a concatemer molecule having a second splint strand region (400) that comprises a universal binding sequence (or its complementary sequence) for the fourth surface primer, and the universal binding sequence for the fourth surface primer comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1).

[0170] Rolling circle amplification in solution using soluble amplification primers Use of circularized library molecules generated via ds-splint adapters In some embodiments, in a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules lacking a hybridized first sprint strand (300), individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) comprise a first left universal adapter sequence (120) or a second left universal adapter sequence (140) of a universal binding sequence for a forward amplification primer. In some embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) comprise a second sprint strand region (400) that comprises a universal binding sequence for a third surface primer. In some embodiments, the method comprises: (a) hybridizing a plurality of soluble forward amplification primers in solution to a first or second left universal adapter sequence that comprises a universal binding sequence for a forward amplification primer; and (b) performing a first rolling circle amplification reaction by contacting the plurality of covalently closed circular library molecules (600) with a plurality of strand displacement polymerases and a plurality of nucleotides (e.g., bases A, G, C, T, and / or U) under conditions suitable for performing a rolling circle amplification reaction in solution using the plurality of forward amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules. In certain embodiments, the nucleic acid concatemer molecules remain hybridized to the covalently closed circular library molecules (600). In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (c): dispensing the plurality of concatemer molecules onto a support on which a plurality of third surface primers are immobilized under conditions suitable for hybridizing at least a portion of the concatemer to the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules. In some embodiments, the plurality of immobilized concatemer molecules remain hybridized to the covalently closed circular library molecules (600).In some embodiments, a method for performing a rolling circle amplification reaction further includes step (d): contacting a plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides (e.g., including bases A, G, C, T, and / or U) under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the first and / or second rolling circle amplification reaction can be performed with a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual immobilized concatemers are hybridized to individual third surface primers. In some embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) include a second sprint strand region (400) that also includes a universal binding sequence for a fourth surface primer, whereby the first rolling circle amplification reaction in solution generates concatemer molecules having multiple copies of the universal binding sequences for the third and fourth surface primers. In some embodiments, the method includes dispensing the concatemer molecules onto a support including a plurality of immobilized third and fourth surface primers and incubating the concatemer molecules under conditions suitable for hybridizing at least one second sprint strand region (400) of the concatemer molecules to the immobilized third and fourth surface primers, thereby pressing at least one portion of the concatemer molecules against the support. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0171] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support to which a plurality of third surface primers are immobilized thereon and contain the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7). In some embodiments, an individual third surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence for the third surface primer, and the universal binding sequence for the third surface primer contains the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2 or its complementary sequence).

[0172] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support to which a plurality of fourth surface primers are immobilized thereon and contain the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1 or its complementary sequence). In some embodiments, an individual fourth surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence (or its complementary sequence) for the fourth surface primer, and the universal binding sequence for the fourth surface primer contains the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1).

[0173] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support coated with one or more compounds to produce an immobilized layer on the support (e.g., FIG. 8). In some embodiments, the immobilized layer forms a porous or semi-porous layer. In some embodiments, a surface primer, concatemer template molecule, and / or polymerase can attach to the immobilized layer for immobilization to the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. Generally, the support can include a low binding chemically modified layer attached covalently or non-covalently, such as one or more layers of a silane layer, polymer film, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can include, at least in part of the support, a functionalized polymer coating layer covalently bound via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating includes poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support includes a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In some embodiments, the hydrophilic polymer coating layer can include polyethylene glycol (PEG). In some embodiments, the hydrophilic polymer coating layer can include branched PEG having at least four branches. In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity as measured by the water contact angle, and the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less).In some embodiments, the density of concatenamer molecules immobilized on a support or immobilized on a coating on the support is 1 mm. 2 per about 10 2 ~10 6 (e.g., about 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 ). In some embodiments, the density of concatenamer molecules immobilized on a support or immobilized on a coating on the support is 1 mm 2 per about 10 6 ~10 9 (e.g., about 10 6 , 10 7 , 10 8 , or 10 9 ). In some embodiments, the density of concatenamer molecules immobilized on a support or immobilized on a coating on the support is 1 mm 2 per about 10 9 ~10 12 (e.g., about 10 9 , 10 10 , 10 11 , or 10 12 ). In some embodiments, a plurality of concatenamer molecules are immobilized on a support or are immobilized on a coating on the support at a predetermined site on the support (or the coating on the support). In some embodiments, a plurality of concatenamer molecules are immobilized on a coating on the support at a random site on the support (or the coating on the support).

[0174] In some embodiments, the dispensing of step (c) can be performed in the presence of a high-efficiency hybridization buffer, the high-efficiency hybridization buffer having: (i) a first polar aprotic solvent having a dielectric constant of 40 or less (e.g., less than 10, or about 10, 15, 20, 30, or 40) and a polarity index of 4-9 (e.g., 4, 5, 6, 7, 8, or 9); (ii) a second polar aprotic solvent having a dielectric constant of 115 or less (e.g., less than 10, 10, 15, 20, 30, 40, 50, 75, 100, 105, 105, 110, or 115) and present in an amount effective to denature double-stranded nucleic acid in the hybridization buffer formulation; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4-8 (e.g., 4, 5, 6, 7, or 8); and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer (e.g., 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, or 50% by volume); (ii) the second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer (e.g., 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, or 10% by volume); (iii) the pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5); and (iv) the crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer (e.g., 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, or 35% by volume). In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0175] In some embodiments, in a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules lacking a hybridized first sprint strand (300), individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) include a first right universal adapter sequence (130) or a second right universal adapter sequence (150) of a universal binding sequence for a forward amplification primer. In some embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) include a second sprint strand region (400) that includes a universal binding sequence for a third surface primer, and the method comprises: (a) hybridizing a plurality of soluble forward amplification primers in solution to a first or second left universal adapter sequence that includes a universal binding sequence for a forward amplification primer; and (b) performing a first rolling circle amplification reaction by contacting the plurality of covalently closed circular library molecules (600) with a plurality of strand displacement polymerases and a plurality of nucleotides (e.g., bases A, G, C, T, and / or U) under conditions suitable for performing a rolling circle amplification reaction in solution using the plurality of forward amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules that remain hybridized to the covalently closed circular library molecules (600). In some embodiments, the method for performing the rolling circle amplification reaction further comprises step (c): dispensing the plurality of concatemer molecules onto the support on which a plurality of third surface primers are immobilized, under conditions suitable for hybridizing at least a portion of the concatemer to the plurality of immobilized third surface primers, thereby immobilizing the plurality of concatemer molecules. In certain embodiments, the plurality of immobilized concatemer molecules remain hybridized to the covalently closed circular library molecules (600).In some embodiments, a method for performing a rolling circle amplification reaction further includes step (d): contacting a plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides (e.g., including bases A, G, C, T, and / or U) under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the first and / or second rolling circle amplification reaction can be performed with a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual immobilized concatemers are hybridized to individual third surface primers. In some embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) include a second sprint strand region (400) that also includes a universal binding sequence for a fourth surface primer, whereby the first rolling circle amplification reaction in solution generates concatemer molecules having multiple copies of the universal binding sequences for the third and fourth surface primers. In some embodiments, the method includes dispensing the concatemer molecules onto a support that includes a plurality of immobilized third and fourth surface primers, and incubating the concatemer molecules under conditions suitable for hybridizing at least one second sprint strand region (400) of the concatemer molecules to the immobilized third and fourth surface primers, thereby pressing at least one portion of the concatemer molecules against the support. In some embodiments, the immobilized concatemers can be subjected to a sequencing reaction.

[0176] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support to which a plurality of third surface primers are immobilized thereon and contain the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7). In some embodiments, an individual third surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence for the third surface primer, and the universal binding sequence for the third surface primer contains the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2 or its complementary sequence).

[0177] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support to which a plurality of fourth surface primers are immobilized thereon and contain the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1 or its complementary sequence). In some embodiments, an individual fourth surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence (or its complementary sequence) for the fourth surface primer, and the universal binding sequence for the fourth surface primer contains the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1).

[0178] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support coated with one or more compounds to produce an immobilized layer on the support (e.g., FIG. 8). In some embodiments, the immobilized layer forms a porous or semi-porous layer. In some embodiments, a surface primer, concatemer template molecule, and / or polymerase can attach to the immobilized layer for immobilization to the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. Generally, the support can include a low binding chemically modified layer attached covalently or non-covalently, e.g., one or more layers of a silane layer, polymer film, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can include, at least in part of the support, a functionalized polymer coating layer covalently bound via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating includes poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support includes a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In some embodiments, the hydrophilic polymer coating layer can include polyethylene glycol (PEG). The hydrophilic polymer coating layer can include branched PEG having at least four branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches). In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity that can be measured as a water contact angle, and the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less).In some embodiments, the density of concatemer molecules immobilized on a support or immobilized on a coating on the support is 1 mm. 2 per about 10 2 ~10 6 (e.g., about 10 2 , 10 3 , 10 4 , 10 5 or 10 6 ). In some embodiments, the density of concatemer molecules immobilized on a support or immobilized on a coating on the support is 1 mm 2 per about 10 6 ~10 9 (e.g., about 10 6 , 10 7 , 10 8 , or 10 9 ). In some embodiments, the density of concatemer molecules immobilized on a support or immobilized on a coating on the support is 1 mm 2 per about 10 9 ~10 12 (e.g., about 10 9 , 10 10 , 10 11 , or 10 12 ). In some embodiments, a plurality of concatemer molecules are immobilized on a support or are immobilized on a coating on the support at a predetermined site on the support (or the coating on the support). In some embodiments, a plurality of concatemer molecules are immobilized on a coating on the support at a random site on the support (or the coating on the support).

[0179] In some embodiments, the dispensing of step (c) can be performed in the presence of a high-efficiency hybridization buffer, the high-efficiency hybridization buffer having (i) a first polar aprotic solvent having a dielectric constant of 40 or less (e.g., less than 10, or about 10, 15, 20, 30, or 40) and a polarity index of 4-9 (e.g., 4, 5, 6, 7, 8, or 9), (ii) a second polar aprotic solvent having a dielectric constant of 115 or less (e.g., less than 10, 10, 15, 20, 30, 40, 50, 75, 100, 105, 105, 110, or 115) and present in an amount effective to denature double-stranded nucleic acids in the hybridization buffer formulation, (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4-8 (e.g., 4, 5, 6, 7, or 8), and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises (i) the first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer (e.g., 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, or 50% by volume), (ii) the second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer (e.g., 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, or 10% by volume), (iii) the pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5), and (iv) the crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer (e.g., 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, or 35% by volume). In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0180] Rolling circle amplification in solution using a first sprint strand Use of circularized library molecules generated via a ds-sprint adapter In some embodiments, in a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules hybridized to a first sprint strand (300), each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules (600) comprises a second sprint strand region (400) comprising a universal binding sequence for a third surface primer. In some embodiments, the method comprises: (a) contacting, in solution, a plurality of covalently closed circular library molecules (600) hybridized to a first sprint strand (300) with a plurality of strand displacement polymerases and a plurality of nucleotides (e.g., comprising bases A, G, C, T, and / or U) under conditions suitable for performing a first rolling circle amplification reaction using the first sprint strand (300) as an amplification primer, thereby generating a plurality of concatemer molecules still hybridized to the covalently closed circular library molecules (600). See Figure 2.

[0181] In some embodiments, a method for performing a rolling circle amplification reaction further comprises step (b): dispensing, on a support on which a plurality of third surface primers are immobilized, a plurality of concatemer molecules hybridized to the covalently closed circular library molecules (600) under conditions suitable for hybridizing at least a portion of the concatemers to the plurality of immobilized third surface primers, thereby further immobilizing the plurality of concatemer molecules hybridized to the covalently closed circular library molecules (600).

[0182] In some embodiments, the plurality of concatemer molecules of step (b) can be dispensed onto a support having a plurality of third surface primers immobilized thereon and include the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7). In some embodiments, an individual third surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence for the third surface primer. In certain embodiments, the universal binding sequence for the third surface primer includes the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2, or its complementary sequence).

[0183] In some embodiments, the plurality of concatemer molecules of step (b) can be dispensed onto a support having a plurality of fourth surface primers immobilized thereon and include the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1 or its complementary sequence). In some embodiments, an individual fourth surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence for the fourth surface primer (or its complementary sequence). In certain embodiments, the universal binding sequence for the fourth surface primer includes the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1).

[0184] In some embodiments, the plurality of concatemer molecules of step (b) can be dispensed onto a support coated with one or more compounds to produce a passivated layer on the support (e.g., FIG. 8). In some embodiments, the passivated layer forms a porous or semi-porous layer. In some embodiments, a surface primer, concatemer template molecule, and / or polymerase can adhere to the passivated layer for immobilization to the support. In some embodiments, the support comprises a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. In some embodiments, the support can comprise a low binding chemical modification layer attached covalently or non-covalently, such as one or more layers of a silane layer, polymer film, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can comprise, at least in part of the support, a functionalized polymer coating layer covalently bonded via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In some embodiments, the hydrophilic polymer coating layer can comprise polyethylene glycol (PEG). In some embodiments, the hydrophilic polymer coating layer can comprise branched PEG having at least four branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches).In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity that can be measured as a water contact angle, and the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less). In some embodiments, the density of the concatemer molecules immobilized on the support or immobilized on the coating on the support is about 10 2 per 1 mm 2 ~10 6 (e.g., about 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 ). In some embodiments, the density of the concatemer molecules immobilized on the support or immobilized on the coating on the support is about 10 2 per 1 mm 6 ~10 9 (e.g., about 10 6 , 10 7 , 10 8 , or 10 9 ). In some embodiments, the density of the concatemer molecules immobilized on the support or immobilized on the coating on the support is about 10 2 per 1 mm 9 ~10 12 (e.g., about 10 9 , 10 10 , 10 11 , or 10 12 ). In some embodiments, the plurality of concatemer molecules are immobilized on the support or are immobilized on the coating on the support at a predetermined site on the support (or the coating on the support). In some embodiments, the plurality of concatemer molecules are immobilized on the coating on the support at a random site on the support (or the coating on the support).

[0185] In some embodiments, the dispensing in step (b) can be performed in the presence of a high-efficiency hybridization buffer, which comprises: (i) a first polar aprotic solvent having a dielectric constant of 40 or less (e.g., less than 10, or about 10, 15, 20, 30, or 40) and a polarity index of 4 to 9 (e.g., 4, 5, 6, 7, 8, or 9); (ii) a second polar aprotic solvent having a dielectric constant of 115 or less (e.g., less than 10, 10, 15, 20, 30, 40, 50, 75, 100, 105, 105, 110, or 115) and present in an amount effective to denature double-stranded nucleic acid in the hybridization buffer formulation; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8 (e.g., 4, 5, 6, 7, or 8); and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25 to 50% by volume of the hybridization buffer (e.g., 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, or 50% by volume); (ii) the second polar aprotic solvent comprises formamide at 5 to 10% by volume of the hybridization buffer (e.g., 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, or 10% by volume); (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5); and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5 to 35% by volume of the hybridization buffer (e.g., 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, or 35% by volume). In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0186] In some embodiments, a method for performing a rolling circle amplification reaction further includes step (c): contacting a plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides (e.g., including bases A, G, C, T, and / or U) under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (600) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules.

[0187] In some embodiments, the first and / or second rolling circle amplification reaction can be performed with a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual covalently closed circular library molecules (600) in the plurality of covalently closed circular library molecules (600) include a second splint strand region (400) that also includes a universal binding sequence for a fourth surface primer, whereby the first rolling circle amplification reaction in solution generates a concatemer molecule having a plurality of copies of the universal binding sequences for the third and fourth surface primers. In some embodiments, the method includes dispensing the concatemer molecules onto a support including a plurality of immobilized third and fourth surface primers and incubating the plurality of immobilized nucleic acid concatemer molecules under conditions suitable for hybridizing at least one second splint strand region (400) of the concatemer molecule to the immobilized third and fourth surface primers, thereby pressing at least one portion of the concatemer molecule against the support. In some embodiments, individual immobilized concatemers are hybridized to individual third surface primers. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0188] Method for forming a plurality of library-splint complexes Use of single-stranded splint strands In some embodiments, any of the methods described above can be used to generate nucleic acid linear library molecules each containing an array arranged in the 5' to 3' order.

[0189] In some embodiments, the nucleic acid linear library molecule has, from 5' to 3': (i) a first left universal adapter sequence (720) having a binding sequence for a first surface primer, (ii) a first left index sequence (760), (iii) a second left universal adapter sequence (740) having a binding sequence for a first sequencing primer, (iv) a sequence of interest (710), (v) a second right universal adapter sequence (750) having a binding sequence for a second sequencing primer, (vi) a first right index sequence (770), and (vii) a first right universal adapter sequence (730) having a binding sequence for a second surface primer (see, for example, FIGS. 4-5).

[0190] In some embodiments, at least one of the linear library molecules bears at least one deaminated nucleotide base. In some embodiments, prior to cyclization, the linear library molecules can be treated with a reagent that removes the deaminated base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates an abasic site at a uracil base in a nucleic acid molecule. For example, without limitation, DNA glycosylase (UDG) can generate an abasic site at a uracil base. In some embodiments, the reagent that removes the deaminated base includes a compound that generates a gap at an abasic site in a nucleic acid strand. For example, without limitation, the gap can be generated by contacting with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site to release abasic deoxyribose and generate a gap. In some embodiments, the abasic site can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase, and combinations thereof. In some embodiments, generating an abasic site and removing the abasic site to generate a gap can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. For example, without limitation, generating an abasic site and removing the abasic site can be performed with USER™ (Uracil-Specific Excision Reagent Enzyme from New England Biolabs™) or thermolabile USER™ (also from New England Biolabs™).

[0191] In another aspect, the present disclosure is a method for forming a plurality of library-sprint complexes (900), comprising: (a) providing a plurality of single-stranded nucleic acid library molecules (700), wherein individual library molecules in the plurality of library molecules include a target sequence (710) flanked on one side by at least a first left universal adapter sequence (720) and on the other side by at least a first right universal adapter sequence (730) (see, e.g., FIGS. 4-5).

[0192] In some embodiments, the method for forming a plurality of library-sprint complexes (900) further comprises step (b): providing a plurality of single-stranded sprint strands (800), wherein individual single-stranded sprint strands (800) in the plurality of single-stranded sprint strands (800) include a first region (810) capable of hybridizing with at least the first left universal adapter sequence (720) of an individual library molecule and a second region (820) capable of hybridizing with at least the first right universal adapter sequence (730) of an individual library molecule. Exemplary single-stranded sprint strands (800) are shown in FIGS. 4-5. In some embodiments, the single-stranded sprint strand (800) can be 20-150 nucleotides in length, or 60-100 nucleotides in length, or 70-90 nucleotides in length, or 60-80 nucleotides in length.

[0193] In some embodiments, a method for forming a plurality of library-sprint complexes (900) further includes step (c): hybridizing a plurality of single-stranded sprint strands (800) with a plurality of single-stranded nucleic acid library molecules (700). In certain embodiments, the hybridizing is performed under conditions suitable for hybridizing individual library molecules with individual single-stranded sprint strands, whereby a first region of one of the single-stranded sprint strands (810) anneals to at least a first left universal adapter sequence (720) of the library molecule, and a second region of the single-stranded sprint strand (820) anneals to at least a first right universal sequence (730) of the library molecule, thereby circularizing the individual library molecules to form a plurality of library-sprint complexes (900). In some embodiments, the library-sprint complex (900) includes a nick between the 5' end and the 3' end of the library molecule (e.g., FIGS. 4-5). In some embodiments, the nick is enzymatically ligatable.

[0194] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the first region of the single-stranded sprint strand (810) includes a first universal adapter sequence capable of hybridizing to a first universal binding sequence (720) at one end of a linear nucleic acid library molecule. In some embodiments, the first region of the single-stranded sprint strand (810) includes a first universal adapter sequence that includes a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0195] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), a second region of a single-stranded sprint strand (820) comprises a second universal adapter sequence capable of hybridizing to a second universal binding sequence at the other end of a linear nucleic acid library molecule. In some embodiments, a second region of a single-stranded sprint strand (820) comprises a second universal adapter sequence that comprises a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, or a universal binding sequence for a compaction oligonucleotide.

[0196] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), a single-stranded sprint strand (800) comprises one or more phosphorothioate linkages at its 5' and / or 3' ends to confer exonuclease resistance. In some embodiments, a single-stranded sprint strand (800) comprises one or more phosphorothioate linkages at internal positions to confer endonuclease resistance. In some embodiments, a single-stranded sprint strand (800) comprises one or more 2'-O-methylcytosine bases at its 5' and / or 3' ends or at internal positions. In some embodiments, the 5' end of a single-stranded sprint strand (800) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of a single-stranded sprint strand (800) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0197] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), a first region of a single-stranded sprint strand (810) can hybridize to a sense or antisense strand of a double-stranded nucleic acid library molecule. In some embodiments, in a library-sprint complex (900), a second region of a single-stranded sprint strand (820) can hybridize to a sense or antisense strand of a double-stranded nucleic acid library molecule. In some embodiments, the double-stranded nucleic acid library molecule can be denatured to produce single-stranded sense and antisense library strands. In certain embodiments, the double-stranded nucleic acid library molecule can be denatured to produce the single-stranded nucleic acid library molecule (700) of step (a).

[0198] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the first and second regions of the single-stranded sprint strand (210 and 220, respectively) do not hybridize to the target sequence (710).

[0199] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the nucleic acid library molecule (700) further comprises a second left universal adapter sequence (740). In some embodiments, the nucleic acid library molecule (700) further comprises a second right universal adapter sequence (750). In some embodiments, the nucleic acid library molecule (700) can further comprise additional left and / or right universal adapter sequences.

[0200] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the nucleic acid library molecules (700) further comprise a first left index sequence (760) and / or a first right index sequence (770). In some embodiments, the first left index sequence (760) comprises a sample index sequence. The first left index sequence (760) can be 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length. In some embodiments, the first right index sequence (770) comprises another sample index sequence. In some embodiments, the first right index sequence (770) can be 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length. In some embodiments, the sequences of the left sample index sequence and the right sample index sequence (e.g., (760) and (770)) may be the same as or different from each other. In some embodiments, the sample index sequence can be used to distinguish target sequences obtained from different sample sources in a multiplex assay. A list of exemplary first left index sequences (760) and first right index sequences (770) is provided in Table 2 in FIG. 7. In some embodiments, the first left index sequence (760) may comprise a short random sequence (e.g., NNN) or may lack a random sequence. In some embodiments, the first right index sequence (770) may comprise a short random sequence (e.g., NNN) or may lack a short random sequence. In some embodiments, the short random sequence (e.g., NNN) can be 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length.

[0201] In some embodiments, the multiplex workflow is enabled by preparing a sample-indexed library using one or both index arrays (e.g., a left index array and / or a right index array). In some embodiments, a first left index array (760) and / or a first right index array (770) can be used to prepare separate sample-indexed libraries using input nucleic acids isolated from different sources. In some embodiments, the sample-indexed libraries can be pooled together to generate a multiplex library mixture, and the pooled libraries can be circularized, amplified, and / or sequenced. In some embodiments, the sequence of the insert region can be used, together with the first left index array (760) and / or the first right index array (770), to identify the source of the input nucleic acid. In some embodiments, the sample-indexed libraries can be pooled together, for example, where 2 to 10 (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10) libraries can be pooled together. In some embodiments, 10 to 50 (e.g., about 10, 20, 30, 40, or 50) sample-indexed libraries are pooled together. In some embodiments, 50 to 100 (e.g., about 50, 60, 70, 80, 90, or 100) sample-indexed libraries are pooled together. In some embodiments, 100 to 200 (e.g., about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) sample-indexed libraries are pooled together. Exemplary nucleic acid sources include naturally occurring sources, recombinant sources, or chemically synthesized sources. Exemplary nucleic acid sources include, but are not limited to, single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include, but are not limited to, fresh sources, frozen sources, fresh frozen sources, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) sources. It will be appreciated by those skilled in the art that nucleic acids can be isolated from many other sources.In some embodiments, the nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0202] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the nucleic acid library molecule (700) further comprises an optional first left unique identifier sequence (780) and / or an optional first right unique identifier sequence (790). In some embodiments, the first left unique identifier sequence (780) and the first right unique identifier sequence (790) each comprise a sequence used to uniquely identify an individual target sequence (e.g., an insert sequence) to which a unique adapter has been added among a population of other sequences of the target molecule. In some embodiments, the first left unique identifier sequence (780) and / or the first right unique identifier sequence (790) can be used for molecular tagging. In some embodiments, the unique identifier sequence comprises 2 to 12 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12) nucleotides having a known sequence. For example, the unique identifier sequence includes, but is not limited to, a known random sequence, in which the nucleotide at each position is randomly selected from nucleotides having bases A, G, C, T, or U. In some embodiments, the unique identifier sequences (780) and / or (790) can be used for the molecular tagging procedure.

[0203] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the nucleic acid library molecule (700) comprises any combination of any one or two or more of the first left universal adapter sequence (720), the second left universal adapter sequence (740), the first left index sequence (760), the first left unique identifier sequence (780), the first right universal adapter sequence (730), the second right universal adapter sequence (750), the first right index sequence (770), and / or the first right unique identifier sequence (790) in any order.

[0204] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the first left universal adapter array (720) and / or the second left universal adapter array (740) includes a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (700) can further include an additional left universal adapter array.

[0205] In some embodiments, in a method for forming a plurality of library-sprint complexes (900), the first right universal adapter array (730) and / or the second right universal adapter array (750) includes a universal binding sequence for a forward or reverse sequencing primer, a universal binding sequence for a first or second surface primer, a universal binding sequence for a forward amplification primer or a reverse amplification primer, and / or a universal binding sequence for a compaction oligonucleotide. In some embodiments, the nucleic acid library molecule (700) can further include an additional right universal adapter array.

[0206] In another aspect, the present disclosure provides a method for forming a plurality of library-sprint complexes (900), comprising: (a) providing a plurality of single-stranded sprint strands (800), wherein each single-stranded sprint strand (800) comprises: (i) a first region (810) having a universal binding sequence that hybridizes to a sequence (e.g., 120) at one end of a linear single-stranded library molecule, and (ii) a second region (820) having a universal binding sequence that hybridizes to a sequence (e.g., 130) at the other end of the linear single-stranded library molecule, and the regions are arranged in the 5' to 3' order. In some embodiments, the method for forming a plurality of library-sprint complexes (900) further comprises step (b): hybridizing the plurality of single-stranded sprint strands (800) to a plurality of single-stranded nucleic acid library molecules (700), wherein each library molecule comprises regions arranged in the 5' to 3' order: (i) a first left universal adapter sequence (720) having a binding sequence for a first surface primer, (ii) a second left universal adapter sequence (740) having a binding sequence for a first or second sequencing primer, (iii) a target sequence (710), (iv) a second right universal adapter sequence (750) having a binding sequence for a second sequencing primer, and (v) a first right universal adapter sequence (730) having a binding sequence for a second surface primer. In certain embodiments, the hybridizing is performed under conditions suitable for hybridizing the single-stranded sprint strands (800) to the library molecules (700), thereby circularizing the library molecules to generate library-sprint complexes (900), wherein the first region (810) of the single-stranded sprint strand hybridizes to the binding sequence (720) for the first surface primer, and the second region (820) of the single-stranded sprint strand hybridizes to the binding sequence (730) for the second surface primer. In certain embodiments, the library-sprint complex (900) comprises a nick between the 5' end and the 3' end of the library molecule.In certain embodiments, the nick is enzymatically ligatable (see, e.g., FIG. 4). In some embodiments, the plurality of single-stranded nucleic acid library molecules (700) further comprises a first left index sequence (760) and / or a first right index sequence (770) (see, e.g., FIG. 4). In some embodiments, in a given library-sprint complex (300) of the plurality of library-sprint complexes (900), the sequences of the first left index (760) and the first right index (770) are the same as or different from each other. In some embodiments, the first left index sequence (760) can be 3 to 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length. The first right index sequence (770) can be 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length. In some embodiments, the first left index sequence (760) and / or the first right index sequence (770) can include a short random sequence (e.g., NNN). In some embodiments, the short random sequence can be 3 to 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) nucleotides in length. A list of exemplary first left index sequences (760) and first right index sequences (770) is provided in Tables 1-2 in FIG. 7. In some embodiments, the plurality of single-stranded nucleic acid library molecules (700) further comprises a first left unique identification sequence (780) and / or a first right unique identification sequence (790) that can be used for molecular tagging (see, e.g., FIG. 4).

[0207] In some embodiments, in the method, the first left universal adapter sequence (720) in the library molecule comprises the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1).

[0208] In some embodiments, in the method, the first left universal adapter sequence (720) in the library molecule comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 204).

[0209] In some embodiments, in the method, the second left universal adapter sequence (740) in the library molecule comprises the sequence 5'-CGTGCTGGATTGGCTCACCAGACACCTTCCGACAT-3' (SEQ ID NO: 209).

[0210] In some embodiments, in the method, the second left universal adapter sequence (740) in the library molecule comprises the sequence 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 207).

[0211] In some embodiments, in the method, the second left universal adapter sequence (740) in the library molecule comprises the sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 208).

[0212] In some embodiments, in the method, the second right universal adapter sequence (750) in the library molecule comprises the sequence 5'-ATGTCGGAAGGTGTGCAGGCTACCGCTTGTCAACT-3' (SEQ ID NO: 212).

[0213] In some embodiments, in the method, the second right universal adapter sequence (750) in the library molecule comprises the sequence 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 210).

[0214] In some embodiments, in the method, the second right universal adapter sequence (750) in the library molecule comprises the sequence 5'-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3' (SEQ ID NO: 211).

[0215] In some embodiments, in the method, the first right universal adapter sequence (730) in the library molecule comprises the sequence 5'-AGTCGTCGCAGCCTCACCTGATC-3' (SEQ ID NO: 2).

[0216] In some embodiments, in the method, the first right universal adapter sequence (730) in the library molecule comprises the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 213).

[0217] In some embodiments, in the method, the first region of the single-stranded splint strand (810) comprises a universal binding sequence for the first left universal adapter sequence (720) of the library molecule, and the first region (810) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATG-3' (SEQ ID NO: 6).

[0218] In some embodiments, in the method, the second region of the single-stranded splint strand (820) comprises a universal binding sequence for the first right universal adapter sequence (730) of the library molecule, and the second region (820) comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7).

[0219] In some embodiments, in the method, the single-stranded splint strand (800) comprises the sequence 5'-ACCCTGAAAGTACGTGCATTACATGGATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 8). See, for example, FIG. 6.

[0220] In some embodiments, in the method, the first region of the single-stranded splint strand (810) comprises a universal binding sequence for the first left universal adapter sequence (720) of the library molecule, and the first region (810) comprises 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 4).

[0221] In some embodiments, in the method, a second region of the single-stranded splint strand (820) includes a universal binding sequence for a first right universal adapter sequence (730) of the library molecule, and the second region (820) includes the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5).

[0222] In some embodiments, in the method, the single-stranded splint strand (800) includes the sequence 5'-TCGGTGGTCGCCGTATCATTCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 214).

[0223] In some embodiments, any of the methods for forming a plurality of library-splint complexes (900) described herein can further include at least one enzymatic reaction including a phosphorylation reaction, a ligation reaction, and / or an exonuclease reaction. In some embodiments, the enzymatic reactions can be performed sequentially. In some embodiments, the enzymatic reactions can be performed essentially simultaneously. In some embodiments, the enzymatic reactions can be performed in a single reaction vessel. Alternatively, in some embodiments, a first enzymatic reaction can be performed in a first reaction vessel and then transferred to a second reaction vessel, where a second enzymatic reaction can be performed in the second reaction vessel, and then transferred to a third reaction vessel, where a third enzymatic reaction can be performed, and so on.

[0224] In some embodiments, any of the methods for forming the plurality of library-sprint complexes (900) described herein includes performing separate and sequential phosphorylation and ligation reactions, further including performing the separate and sequential phosphorylation and ligation reactions in separate reaction vessels. In some embodiments, the method for forming the plurality of library-sprint complexes (900) includes step (c1): in a first reaction vessel, contacting a plurality of single-stranded sprint strands (800) and a plurality of single-stranded nucleic acid library molecules (700) with a T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded sprint strands (800) and / or the plurality of single-stranded nucleic acid library molecules (700), and transferring the phosphorylation reaction to a second reaction vessel. In some embodiments, the method for forming the plurality of library-sprint complexes (900) includes step (d1): in a second reaction vessel, contacting a plurality of phosphorylated single-stranded sprint strands (800) and a plurality of phosphorylated single-stranded nucleic acid library molecules (700) with a ligase under conditions suitable for enzymatically ligating the nicks, thereby generating a plurality of covalently closed circular library molecules (1000) each hybridized to a single-stranded sprint strand (800). In some embodiments, the ligase enzyme includes T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0225] In some embodiments, any of the methods for forming the plurality of library-sprint complexes (900) described herein comprises performing sequential phosphorylation and ligation reactions, wherein the sequential phosphorylation and ligation reactions are performed sequentially within the same reaction vessel. In some embodiments, the method for forming the plurality of library-sprint complexes (900) further comprises step (c2): contacting, within a first reaction vessel, the plurality of single-stranded sprint strands (800) and the plurality of single-stranded nucleic acid library molecules (700) with a T4 polynucleotide kinase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded sprint strands (800) and the plurality of single-stranded nucleic acid library molecules (700). In some embodiments, the method for forming the plurality of library-sprint complexes (900) further comprises step (d2): contacting, within the same first reaction vessel, the phosphorylated single-stranded sprint strands (800) and the phosphorylated single-stranded nucleic acid library molecules (700) with a ligase under conditions suitable for enzymatically ligating the nicks, thereby generating a plurality of covalently closed circular library molecules (1000), each hybridized to a single-stranded sprint strand (800). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0226] In some embodiments, any of the methods for forming the plurality of library-sprint complexes (900) described herein comprises performing phosphorylation and ligation reactions that are essentially simultaneous, further comprising performing the phosphorylation and ligation reactions that are essentially simultaneous together in the same reaction vessel. In some embodiments, the method for forming the plurality of library-sprint complexes (900) comprises step (c3): in a first reaction vessel, contacting a plurality of single-stranded sprint strands (800) and a plurality of single-stranded nucleic acid library molecules (700) with (i) a T4 polynucleotide kinase enzyme and (ii) a ligase enzyme under conditions suitable for phosphorylating the 5' ends of the plurality of single-stranded sprint strands (800) and the plurality of single-stranded nucleic acid library molecules (700), the conditions being suitable for enzymatically ligating nicks, thereby generating a plurality of covalently closed circular library molecules (1000) each hybridized to a single-stranded sprint strand (800). In some embodiments, the ligase enzyme comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase.

[0227] In some embodiments, any of the methods for forming a plurality of library-sprint complexes (900) described herein may further include an optional step of enzymatically removing a plurality of single-stranded sprint strands (800) from a plurality of covalently closed circular library molecules (1000), the step of contacting the plurality of covalently closed circular library molecules (1000) with at least one exonuclease enzyme to remove the plurality of single-stranded sprint strands (800) and retain the plurality of covalently closed circular library molecules (1000). In some embodiments, the exonuclease reaction may be performed in the same reaction buffer used for performing the phosphorylation and / or ligation reactions, or in a different reaction buffer. In some embodiments, after performing the phosphorylation reaction in a first reaction vessel (see step c1 above) and performing the ligation reaction in a second reaction vessel (see step d1 above), the exonuclease reaction may be performed in a third reaction vessel. In some embodiments, after performing the phosphorylation reaction in a first reaction vessel (see step c2 above) and performing sequential ligation reactions in the first reaction vessel (see step d2 above), the exonuclease reaction may be performed in the first reaction vessel. In some embodiments, after performing essentially simultaneous phosphorylation and ligation reactions in a first reaction vessel (see step c3 above), the exonuclease reaction may be performed in the first reaction vessel. In some embodiments, the at least one exonuclease enzyme includes any combination of two or more of exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0228] In some embodiments, the covalently closed circular library molecule (1000) can be generated using any of the methods described above. In some embodiments, an individual covalently closed circular library molecule comprises a sequence of interest operably linked on both sides by at least one nucleic acid adapter sequence. In some embodiments, at least one covalently closed circular library molecule carries at least one deaminated nucleotide base. In some embodiments, the covalently closed circular library molecule can be treated with a reagent that removes the deaminated base. In some embodiments, the reagent that removes the deaminated base comprises a compound that generates an abasic site at a uracil base in a nucleic acid molecule. For example, without limitation, DNA glycosylase (UDG) can generate an abasic site at a uracil base. In some embodiments, the reagent that removes the deaminated base comprises a compound that generates a gap at the abasic site in a nucleic acid strand. For example, without limitation, the gap can be generated by contacting with an enzyme or a mixture of enzymes having lyase activity that breaks the phosphodiester backbone on the 5' and 3' sides of the abasic site to release the abasic deoxyribose and generate a gap. In some embodiments, the abasic site can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase, and combinations thereof. In some embodiments, generating an abasic site and removing the abasic site to generate a gap can be achieved using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. For example, without limitation, generating an abasic site and removing the abasic site to generate a gap can be achieved using USER™ (Uracil-Specific Excision Reagent Enzyme from New England Biolabs™) or thermolabile USER™ (similarly from New England Biolabs™).

[0229] Method for Rolling Circle Amplification Use of Circularized Library Molecules Generated via an ss-Sprinter Chain In another aspect, the present disclosure provides a method for performing a rolling circle amplification reaction in a covalently closed circular library molecule (1000). In some embodiments, the rolling circle amplification reaction can be performed after a phosphorylation and ligation reaction, or after a ligation reaction. In some embodiments, the rolling circle amplification reaction can be performed in a covalently closed circular library molecule (1000) hybridized to a single-stranded sprinter chain (800). In some embodiments, the rolling circle amplification reaction can be performed, for example, in a covalently closed circular library molecule (1000) that is no longer hybridized to the single-stranded sprinter chain (800) after an exonuclease reaction. In some embodiments, the covalently closed circular library molecule (1000) can be dispensed onto a support and then subjected to a rolling circle amplification reaction. In some embodiments, the covalently closed circular library molecule (1000) can be subjected to a rolling circle amplification reaction in solution and then dispensed onto a support. In some embodiments, the retained single-stranded sprinter chain (800) may be used as an amplification primer for the rolling circle amplification reaction, or the single-stranded sprinter chain (800) can be removed (e.g., via exonuclease digestion) and replaced with a soluble amplification primer.

[0230] Rolling Circle Amplification on a Support Use of Circularized Library Molecules Generated via an ss-Sprinter Chain In some embodiments, a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules lacking a hybridized single-stranded sprint strand (800). In some embodiments, each covalently closed circular library molecule (1000) in the plurality of covalently closed circular library molecules (1000) contains a universal binding sequence for a first surface primer, and step (a) is to place the plurality of covalently closed circular library molecules (1000) on a support on which a plurality of first surface primers are immobilized, and distribute each covalently closed circular library molecule (1000) under conditions suitable for hybridizing it to an individual immobilized first surface primer, thereby including immobilizing the plurality of covalently closed circular library molecules (1000) on the support.

[0231] In some embodiments, in a method for performing a rolling circle amplification reaction, the plurality of first surface primers immobilized on the support contain the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7).

[0232] In some embodiments, in a method for performing a rolling circle amplification reaction, the plurality of first surface primers immobilized on the support contain the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5).

[0233] In some embodiments, each first surface primer can hybridize to a covalently closed circular library molecule (1000) having a universal binding sequence for the first surface primer.

[0234] In some embodiments, a method for performing a rolling circle amplification reaction further includes step (b): contacting a plurality of immobilized covalently closed circular library molecules (1000) with a plurality of strand displacement polymerases and a plurality of nucleotides, and using a plurality of first surface primers as immobilized amplification primers and a plurality of covalently closed circular library molecules (1000) as template molecules under conditions suitable for performing a rolling circle amplification reaction on a support, thereby generating a plurality of nucleic acid concatemer molecules immobilized to the first surface primers. In some embodiments, the plurality of nucleotides includes any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual immobilized concatemers are covalently linked to individual first surface primers. In some embodiments, individual covalently closed circular library molecules (1000) in the plurality of covalently closed circular library molecules (1000) include universal binding sequences (e.g., (720) and (730), respectively) for the first and second surface primers, whereby the rolling circle amplification reaction generates concatemer molecules having multiple tandem copies of the universal binding sequences for the first and second surface primers. In some embodiments, the support further includes a plurality of second surface primers. In some embodiments, the immobilized second surface primer functions to hold at least a portion of the concatemer molecule against the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0235] In some embodiments, the plurality of second surface primers immobilized on the support includes the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1 or its complementary sequence).

[0236] In some embodiments, the plurality of second surface primers immobilized on the support comprises the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 204 or its complementary sequence).

[0237] In some embodiments, an individual second surface primer can hybridize to a portion of a concatemer molecule having a universal binding sequence for the second surface primer. In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule against the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0238] In some embodiments, in a method for performing rolling circle amplification reactions, a plurality of covalently closed circular library molecules (1000) can be dispensed onto a support coated with one or more compounds to produce an immobilized layer on the support (e.g., FIG. 8). In some embodiments, the immobilized layer forms a porous or semi-porous layer. In some embodiments, one or more types of surface primers, concatemer template molecules, and / or polymerases can adhere to the immobilized layer for immobilization to the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. In some embodiments, the support can include a low binding chemical modification layer attached covalently or non-covalently, e.g., one or more layers of a silane layer, a polymer film, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can include, at least in part of the support, a functionalized polymer coating layer covalently bonded via chemical groups on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating includes poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support includes a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In some embodiments, the hydrophilic polymer coating layer can include polyethylene glycol (PEG). In some embodiments, the hydrophilic polymer coating layer can include branched PEG having at least four branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches).In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity that can be measured as a water contact angle, and the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less). In some embodiments, the density of the covalently closed cyclic library molecules (1000) immobilized on the support or immobilized on the coating on the support is about 10. 2 ~10 6 (e.g., about 10 2 、10 3 、10 4 、10 5 、or 10 6 ). In some embodiments, the covalently closed cyclic library molecules (600) immobilized on the support or immobilized on the coating on the support are about 10 per 1 mm 2 . 6 ~10 9 (e.g., about 10 6 、10 7 、10 8 、or 10 9 ). In some embodiments, the density of the covalently closed cyclic library molecules (600) immobilized on the support or immobilized on the coating on the support is about 10 per 1 mm 2 . 9 ~10 12 (e.g., about 10 9 、10 10 、10 11 、or 10 12 ). In some embodiments, a plurality of covalently closed cyclic library molecules (1000) are immobilized on the support or on the coating on the support at a predetermined site on the support (or the coating on the support). In some embodiments, a plurality of covalently closed cyclic library molecules (600) are immobilized on the support or on the coating on the support at a random site on the support (or the coating on the support).

[0239] In some embodiments, in a method for performing a rolling circle amplification reaction, the dispensing in step (a) (e.g., dispensing a plurality of covalently closed circular library molecules (1000) onto a support) can be performed in the presence of a high-efficiency hybridization buffer, the high-efficiency hybridization buffer comprising: (i) a first polar aprotic solvent having a relative permittivity of 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a relative permittivity of 115 or less and present in an amount effective to denature double-stranded nucleic acids in the hybridization buffer formulation; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprising acetonitrile in an amount of 25 to 50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprising formamide in an amount of 5 to 10% by volume of the hybridization buffer; (iii) the pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5; and (iv) the crowding agent comprising polyethylene glycol (PEG) in an amount of 5 to 35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0240] Rolling circle amplification in solution using a soluble amplification primer Use of circularized library molecules generated via ss-sprint strands In some embodiments, in a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules (1000) lacking a hybridized single-stranded sprint strand (800), individual covalently closed circular library molecules (1000) in the plurality of covalently closed circular library molecules (1000) include a universal binding sequence for a forward amplification primer and a universal binding sequence for a first surface primer. In some embodiments, the method comprises: (a) hybridizing a plurality of covalently closed circular library molecules and a plurality of soluble forward amplification primers in solution; and (b) contacting the plurality of covalently closed circular library molecules (1000) with a plurality of strand displacement polymerases and a plurality of nucleotides, using the plurality of forward amplification primers, under conditions suitable for performing a rolling circle amplification reaction in solution using the plurality of covalently closed circular library molecules (1000) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules having a portion that remains hybridized to those covalently closed circular library molecules (1000). In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (c): distributing the plurality of concatemer molecules onto a support having a plurality of first surface primers immobilized thereon, under conditions suitable for hybridizing at least a portion of the concatemer to the plurality of immobilized first surface primers, thereby further immobilizing the plurality of concatemer molecules. In some embodiments, the plurality of immobilized concatemer molecules remain hybridized to those covalently closed circular library molecules (1000).In some embodiments, a method for performing a rolling circle amplification reaction further comprises step (d): contacting a plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (1000) as template molecules, thereby extending the plurality of immobilized nucleic acid concatemer molecules. In some embodiments, the first and / or second rolling circle amplification reaction can be performed with a plurality of nucleotides comprising any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual immobilized concatemers are hybridized to individual first surface primers. In some embodiments, individual covalently closed circular library molecules (1000) in the plurality of covalently closed circular library molecules (1000) comprise universal binding sequences for the first and second surface primers (e.g., (720) and (730), respectively), whereby the rolling circle amplification reaction in solution generates concatemer molecules having a plurality of tandem copies of the universal binding sequences for the first and second surface primers. In some embodiments, the support further comprises a plurality of second surface primers. In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule against the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0241] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7). In some embodiments, individual first surface primers can hybridize to a covalently closed circular library molecule (1000) having a universal binding sequence for the first surface primer.

[0242] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5). In some embodiments, individual first surface primers can hybridize to a covalently closed circular library molecule (1000) having a universal binding sequence for the first surface primer.

[0243] In some embodiments, a plurality of second surface primers immobilized on a support comprise the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1 or its complementary sequence). In some embodiments, individual second surface primers can hybridize to a portion of a concatemer molecule having a universal binding sequence for the second surface primer.

[0244] In some embodiments, a plurality of second surface primers immobilized on a support comprise the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 204 or its complementary sequence). In some embodiments, individual second surface primers can hybridize to a portion of a concatemer molecule having a universal binding sequence for the second surface primer.

[0245] In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule against the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0246] In some embodiments, the plurality of concatemer molecules of step (c) can be dispensed onto a support coated with one or more compounds to produce a passivated layer on the support (e.g., FIG. 8). In some embodiments, the passivated layer forms a porous or semi-porous layer. In some embodiments, one or more types of surface primers, concatemer template molecules, and / or polymerases can adhere to the passivated layer for immobilization on the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. In some embodiments, the support can include a low binding chemical modification layer attached covalently or non-covalently, such as one or more layers of a silane layer, a polymer film, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize the plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can include, at least in part of the support, a functionalized polymer coating layer covalently bonded via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating includes poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support includes a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In some embodiments, the hydrophilic polymer coating layer can include polyethylene glycol (PEG). In some embodiments, the hydrophilic polymer coating layer can include branched PEG having at least four branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches).In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity that can be measured as the water contact angle, and the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less). In some embodiments, the density of the concatenamer molecules immobilized on the support or on the coating on the support is about (e.g., about 10. 2 , 10 3 , 10 4 , 10 5 , or 10 6 ). In some embodiments, the density of the concatenamer molecules immobilized on the support or on the coating on the support is about 10 per mm 2 (e.g., about 10 6 ~10 9 (e.g., about 10 6 , 10 7 , 10 8 , or 10 9 ). In some embodiments, the density of the concatenamer molecules immobilized on the support or on the coating on the support is about 10 per mm 2 (e.g., about 10 9 ~10 12 (e.g., about 10 9 , 10 10 , 10 11 , or 10 12 ). In some embodiments, the plurality of concatenamer molecules are immobilized on the support or on the coating on the support at a predetermined site on the support (or the coating on the support). In some embodiments, the plurality of concatenamer molecules are immobilized on the coating on the support at a random site on the support (or the coating on the support).

[0247] In some embodiments, the dispensing of step (c) can be performed in the presence of a high-efficiency hybridization buffer, which comprises: (i) a first polar aprotic solvent having a dielectric constant of 40 or less (e.g., less than 10, or about 10, 15, 20, 30, or 40) and a polarity index of 4 to 9 (e.g., 4, 5, 6, 7, 8, or 9); (ii) a second polar aprotic solvent having a dielectric constant of 115 or less (e.g., less than 10, 10, 15, 20, 30, 40, 50, 75, 100, 105, 105, 110, or 115) and present in an amount effective to denature double-stranded nucleic acids in the hybridization buffer formulation; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8 (e.g., 4, 5, 6, 7, or 8); and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25 to 50% by volume of the hybridization buffer (e.g., 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, or 50% by volume); (ii) the second polar aprotic solvent comprises formamide at 5 to 10% by volume of the hybridization buffer (e.g., 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, or 10% by volume); (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5); and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5 to 35% by volume of the hybridization buffer (e.g., 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, or 35% by volume). In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0248] Rolling circle amplification in solution using single-stranded sprint strands Use of circularized library molecules generated via ss-sprint strands In some embodiments, in a method for performing a rolling circle amplification reaction in a plurality of covalently closed circular library molecules hybridized to a single-stranded splint strand (800), individual covalently closed circular library molecules (1000) in the plurality of covalently closed circular library molecules (1000) include a universal binding sequence for a first surface primer, and the method comprises: (a) contacting a plurality of covalently closed circular library molecules (1000) hybridized to a single-stranded splint strand (800) with a plurality of strand-displacing polymerases and a plurality of nucleotides in solution under conditions suitable for performing a first rolling circle amplification reaction using the single-stranded splint strand (800) as an amplification primer, thereby generating a plurality of concatemer molecules still hybridized to their covalently closed circular library molecules (1000) (see, e.g., FIGS. 4-5).

[0249] In some embodiments, a method for performing a rolling circle amplification reaction comprises step (b): distributing a plurality of concatemer molecules hybridized to their covalently closed circular library molecules (1000) onto a support having a plurality of first surface primers immobilized thereon under conditions suitable for hybridizing at least a portion of the concatemer to the plurality of immobilized first surface primers, thereby further immobilizing the plurality of concatemer molecules. In some embodiments, the plurality of immobilized concatemer molecules are still hybridized to their covalently closed circular library molecules (1000).

[0250] In some embodiments, a method for performing a rolling circle amplification reaction comprises step (c): contacting the plurality of immobilized concatemer molecules with a plurality of strand-displacing polymerases and a plurality of nucleotides on the support under conditions suitable for performing a second rolling circle amplification reaction on the support using the plurality of covalently closed circular library molecules (1000) as template molecules, thereby further extending the plurality of immobilized nucleic acid concatemer molecules.

[0251] In some embodiments, the first and / or second rolling circle amplification reactions can be performed with a plurality of nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual immobilized concatemers are hybridized to individual first surface primers. In some embodiments, individual covalently closed circular library molecules (1000) in the plurality of covalently closed circular library molecules (1000) include universal binding sequences for the first and second surface primers (e.g., (720) and (730), respectively), whereby the rolling circle amplification reaction in solution generates concatemer molecules having multiple tandem copies of the universal binding sequences for the first and second surface primers. In some embodiments, the support further includes a plurality of second surface primers. In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule against the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0252] In some embodiments, in a method for performing a rolling circle amplification reaction, the plurality of first surface primers immobilized on a support includes the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 7). In some embodiments, individual first surface primers can hybridize to covalently closed circular library molecules (1000) having a universal binding sequence for the first surface primer.

[0253] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of first surface primers immobilized on a support comprise the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5). In some embodiments, individual first surface primers can hybridize to a covalently closed circular library molecule (1000) having a universal binding sequence for the first surface primer.

[0254] In some embodiments, a plurality of second surface primers immobilized on a support comprise the sequence 5'-CATGTAATGCACGTACTTTCAGGGT-3' (SEQ ID NO: 1 or its complementary sequence). In some embodiments, individual second surface primers can hybridize to a portion of a concatemer molecule having a universal binding sequence for the second surface primer.

[0255] In some embodiments, a plurality of second surface primers immobilized on a support comprise the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 204 or its complementary sequence). Individual second surface primers can hybridize to a part of a concatemer molecule having a universal binding sequence for the second surface primer.

[0256] In some embodiments, the immobilized second surface primer functions to hold at least one portion of the concatemer molecule against the support. In some embodiments, the immobilized second surface primer has a non-extendable 3' end and is not used for amplification. In some embodiments, the immobilized concatemer can be subjected to a sequencing reaction.

[0257] In some embodiments, the plurality of concatemer molecules of step (b) can be dispensed onto a support coated with one or more compounds to produce a passivated layer on the support (e.g., FIG. 8). In some embodiments, the passivated layer forms a porous or semi-porous layer. In some embodiments, a surface primer, a concatemer template molecule, and / or a polymerase can attach to the passivated layer for immobilization on the support. In some embodiments, the support includes a low non-specific binding surface that enables improved nucleic acid hybridization and amplification performance on the support. In some embodiments, the support can include a low-binding chemical modification layer attached covalently or non-covalently, such as one or more layers of a silane layer, a polymer film, and one or more covalently or non-covalently attached oligonucleotides that can be used to immobilize a plurality of nucleic acid concatemer molecules to the support. In some embodiments, the support can include, at least in part of the support, a functionalized polymer coating layer covalently bonded via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating includes poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support includes a surface coating having at least one hydrophilic polymer coating layer and at least one layer of a plurality of oligonucleotides. In some embodiments, the hydrophilic polymer coating layer can include polyethylene glycol (PEG). In some embodiments, the hydrophilic polymer coating layer can include branched PEG having at least four branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches).In some embodiments, the low non-specific binding coating has a certain degree of hydrophilicity that can be measured as a water contact angle, and the water contact angle is 45 degrees or less (e.g., 5 degrees or less, 10 degrees or less, 15 degrees or less, 20 degrees or less, 25 degrees or less, 30 degrees or less, 35 degrees or less, 40 degrees or less, or 45 degrees or less). In some embodiments, the density of the concatemer molecules immobilized on the support or on the coating on the support is about 10 2 per mm 2 ~10 6 (e.g., about 10 2 , 10 3 , 10 4 , 10 5 , or 10 6 ). In some embodiments, the density of the concatemer molecules immobilized on the support or on the coating on the support is about 10 2 per mm 6 ~10 9 (e.g., about 10 6 , 10 7 , 10 8 , or 10 9 ). In some embodiments, the density of the concatemer molecules immobilized on the support or on the coating on the support is about 10 2 per mm 9 ~10 12 (e.g., about 10 9 , 10 10 , 10 11 , or 10 12 ). In some embodiments, the plurality of concatemer molecules are immobilized on the support or on the coating on the support at a predetermined site on the support (or on the coating on the support). In some embodiments, the plurality of concatemer molecules are immobilized on the coating on the support at random sites on the support (or on the coating on the support).

[0258] In some embodiments, the dispensing of step (b) can be performed in the presence of a high-efficiency hybridization buffer, which has a dielectric constant of 40 or less (e.g., less than 10, or about 10, 15, 20, 30, or 40) and a polarity index of 4 to 9 (e.g., 4, 5, 6, 7, 8, or 9), a first polar aprotic solvent; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less (e.g., less than 10, 10, 15, 20, 30, 40, 50, 75, 100, 105, 105, 110, or 115) and present in an amount effective to denature double-stranded nucleic acid in the hybridization buffer formulation; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4 to 8 (e.g., 4, 5, 6, 7, or 8); and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25 to 50% by volume of the hybridization buffer (e.g., 25% by volume, 30% by volume, 35% by volume, 40% by volume, 45% by volume, or 50% by volume); (ii) the second polar aprotic solvent comprises formamide at 5 to 10% by volume of the hybridization buffer (e.g., 5% by volume, 6% by volume, 7% by volume, 8% by volume, 9% by volume, or 10% by volume); (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5 to 6.5 (e.g., about 5.0, 5.5, 6.0, or 6.5); and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5 to 35% by volume of the hybridization buffer (e.g., 5% by volume, 10% by volume, 15% by volume, 20% by volume, 25% by volume, 30% by volume, or 35% by volume). In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0259] Method for sequencing In another aspect, provided is a method for sequencing any of the immobilized concatemer molecules described herein. In some embodiments, any of the methods for performing a rolling circle amplification reaction described herein can be used to generate a plurality of concatemer molecules immobilized on a support, and the immobilized concatemers can be subjected to a sequencing reaction. In some embodiments, the sequencing reaction uses nucleotide analogs that are detectably labeled. In some embodiments, the sequencing reaction uses a two-step sequencing reaction that includes binding to a detectably labeled multivalent molecule and incorporating a nucleotide analog. The terms concatemer molecule and template molecule are used interchangeably.

[0260] In some embodiments, the use of at least one reagent to remove deaminated nucleotide bases from linear library molecules and / or circular library molecules can improve the quality of sequencing data, for example, by reducing the level of low-quality T base calls or A base calls.

[0261] In some embodiments, the immobilized concatemer can self-destruct into compact nucleic acid nanoballs. In some embodiments, including one or more compaction oligonucleotides during the RCA reaction can further compact the size and / or shape of the nanoballs. In some embodiments, an increase in the number of tandem repeat units in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers (e.g., sequencing primers having a universal sequence) that function as multiple initiation sites for a polymerase-catalyzed sequencing reaction. In some embodiments, when the sequencing reaction uses detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., those having nucleotide units), the signal emitted by the nucleotides or nucleotide units involved in the parallel sequencing reaction along the concatemer results in increased signal intensity for each concatemer. In certain embodiments, multiple portions of a given concatemer can be sequenced simultaneously. Further, in some embodiments, multiple binding complexes can form along a particular concatemer molecule, each binding complex including a sequencing polymerase bound to a multivalent molecule. In certain embodiments, the multiple binding complexes remain stable without dissociating, resulting in an increased duration, e.g., the increased duration increases the signal intensity and reduces the imaging time.

[0262] Method for sequencing using nucleotide analogs In another aspect, the present disclosure provides a method for sequencing, comprising step (a): contacting a sequencing polymerase with (i) a nucleic acid concatemer molecule and (ii) a nucleic acid primer, wherein the contacting is performed under conditions suitable for binding the sequencing polymerase to the nucleic acid concatemer molecule hybridized to the nucleic acid primer, and the nucleic acid concatemer molecule hybridized to the nucleic acid primer forms a nucleic acid duplex. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase. In some embodiments, the primer comprises a 3'-extensible terminus.

[0263] In some embodiments, the method for sequencing further comprises step (b): contacting the sequencing polymerase with a plurality of nucleotides and at least one nucleotide under conditions suitable for binding the at least one nucleotide to the sequencing polymerase bound to the nucleic acid duplex and suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, the sequencing polymerase contacts the plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain-terminating moiety at the 2' or 3' position of the sugar. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain-terminating moiety.

[0264] In some embodiments, the method for sequencing further comprises step (c): incorporating at least one nucleotide into the 3′ end of an extendable primer under conditions suitable for incorporation of the at least one nucleotide. In some embodiments, the conditions suitable for binding of the nucleotide to the polymerase and for nucleotide incorporation may be the same or different. In some embodiments, the conditions suitable for nucleotide incorporation include including at least one catalytic cation including magnesium and / or manganese. In some embodiments, the at least one nucleotide binds to a sequencing polymerase and is incorporated into the 3′ end of an extendable primer. In some embodiments, incorporating the nucleotide into the 3′ end of the primer in step (c) includes a primer extension reaction.

[0265] In some embodiments, the method for sequencing further comprises step (d): repeating at least once the incorporation of at least one nucleotide of step (c) into the 3′ end of an extendable primer. In some embodiments, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base by a linker, and the linker is cleavable / removable from the base. In some embodiments, at least one of the nucleotides in the plurality of nucleotides is not labeled with a detectable reporter moiety. In some embodiments, a particular detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) so as to enable detection and identification of the nucleotide base. In some embodiments, the method further comprises detecting at least one incorporated nucleotide in step (c) and / or (d). In some embodiments, the method further comprises identifying at least one incorporated nucleotide in step (c) and / or (d). In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying nucleotides bound to the sequencing polymerase, thereby determining the sequence of the concatemer molecule. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying nucleotides incorporated into the 3′ end of the primer, thereby determining the sequence of the concatemer molecule.

[0266] In some embodiments, in a method for sequencing, a plurality of sequencing polymerases bound to a nucleic acid duplex comprises a plurality of composite polymerases having at least first and second composite polymerases, (a) the first composite polymerase comprises a first sequencing polymerase bound to a first nucleic acid duplex comprising a first nucleic acid template sequence hybridized to a first nucleic acid primer, (b) the second composite polymerase comprises a second sequencing polymerase bound to a second nucleic acid duplex comprising a second nucleic acid template sequence hybridized to a second nucleic acid primer, (c) the first and second nucleic acid template sequences comprise the same or different sequences, (d) the first and second nucleic acid concatemers are clonally amplified, (e) the first and second primers comprise an extendable 3' end or a non-extendable 3' end, and (f) the plurality of composite polymerases are immobilized on a support. In some embodiments, the density of the plurality of composite polymerases is about 10 2 to 10 2 per mm 15 of immobilized composite polymerase on the support (e.g., 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 ).

[0267] Two-step method for nucleic acid sequencing In another aspect, the present disclosure provides a two-step method for sequencing nucleic acid molecules. In some embodiments, the first step generally comprises binding a multivalent molecule to a composite polymerase to form a multivalent composite polymerase and detecting the multivalent composite polymerase.

[0268] In some embodiments, the first step is to contact (a) a plurality of first sequencing polymerases with (i) a plurality of nucleic acid concatemer molecules and (ii) a plurality of nucleic acid primers, the contacting being performed under conditions suitable for binding the plurality of first sequencing polymerases to the plurality of nucleic acid concatemer molecules and the plurality of nucleic acid primers, thereby forming a plurality of first composite polymerases, each of the plurality of first composite polymerases comprising a first sequencing polymerase bound to a nucleic acid duplex, the nucleic acid duplex comprising a nucleic acid concatemer molecule hybridized to a nucleic acid primer, the contacting including. In some embodiments, the first polymerase includes a recombinant mutant sequencing polymerase.

[0269] In some embodiments, in a method for sequencing concatemer molecules, the primer includes a 3'-extendable end or a 3'-non-extendable end. In some embodiments, the plurality of nucleic acid concatemer molecules include amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, the plurality of nucleic acid concatemer molecules include one copy of a target sequence of interest. In some embodiments, the plurality of nucleic acid molecules include two or more tandem copies (e.g., concatemers) of a target sequence of interest. In some embodiments, the nucleic acid concatemer molecules in the plurality of nucleic acid concatemer molecules include the same target sequence of interest or different target sequences of interest. In some embodiments, the plurality of nucleic acid concatemer molecules and / or the plurality of nucleic acid primers are in solution or immobilized on a support. In some embodiments, when the plurality of nucleic acid concatemer molecules and / or the plurality of nucleic acid primers are immobilized on a support, the binding to the first sequencing polymerase generates a plurality of immobilized first composite polymerases. In some embodiments, the plurality of nucleic acid concatemer molecules and / or nucleic acid primers are on the support 10 2 ~10 15 pieces (e.g., 10 2 、10 3 、104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 ) are immobilized on different sites. In some embodiments, the binding of the plurality of nucleic acid concatemer molecules and nucleic acid primers is 10 on the support 2 - 10 15 (e.g., 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 ) to generate a plurality of first composite polymerases immobilized on different sites. In some embodiments, the plurality of immobilized first composite polymerases on the support are immobilized at predetermined or random sites on the support. In some embodiments, the plurality of immobilized first composite polymerases are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes including sequencing polymerase, multivalent molecules, nucleotides, and / or divalent cations) to flow over the support, whereby the plurality of immobilized composite polymerases on the support react with the solution of reagents in a super-parallel manner.

[0270] In some embodiments, the method for sequencing further includes step (b): contacting a plurality of first complex polymerases with a plurality of multivalent molecules to form a plurality of multivalent complex polymerases (e.g., binding complexes). In some embodiments, individual multivalent molecules in the plurality of multivalent molecules include a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide (e.g., nucleotide unit) (e.g., FIGS. 9-13). In some embodiments, the contacting in step (b) is performed under conditions suitable for binding complementary nucleotide units of the multivalent molecules to at least two of the plurality of first complex polymerases, thereby forming a plurality of multivalent complex polymerases. In some embodiments, the conditions are suitable for inhibiting polymerase-catalyzed incorporation of complementary nucleotide units into the primers of the plurality of multivalent complex polymerases. In some embodiments, the plurality of multivalent molecules includes at least one multivalent molecule having a plurality of nucleotide arms (e.g., FIGS. 9-13), and each of the plurality of nucleotide arms is attached to a nucleotide analog (e.g., nucleotide analog unit), and the nucleotide analog includes a chain-terminating moiety at the sugar 2' and / or 3' position. In some embodiments, the plurality of multivalent molecules includes at least one multivalent molecule including a plurality of nucleotide arms, and each of the plurality of nucleotide arms is attached to a nucleotide unit lacking a chain-terminating moiety. In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules is labeled with a detectable reporter moiety. In some embodiments, any portion of the multivalent molecule, including the core, nucleotide arm, or nucleobase, can be labeled. In some embodiments, the detectable reporter moiety includes a fluorophore. In some embodiments, the contacting in step (b) is performed in the presence of at least one non-catalytic cation including strontium, barium, and / or calcium.

[0271] In some embodiments, the method for sequencing further includes step (c): detecting a plurality of multivalent composite polymerases. In some embodiments, detecting comprises detecting a multivalent molecule bound to a composite polymerase, wherein the complementary nucleotide units of the multivalent molecule are bound to a primer, but the incorporation of the complementary nucleotide units is inhibited. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety to enable detection. In some embodiments, the labeled multivalent molecule comprises a fluorophore attached to the core, linker, and / or nucleotide units of the multivalent molecule.

[0272] In some embodiments, the method for sequencing further includes step (d): identifying the bases of the complementary nucleotide units bound to a plurality of first composite polymerases, thereby determining the sequence of the concatemer molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety corresponding to a specific nucleotide unit attached to a nucleotide arm to enable identification of the complementary nucleotide units (e.g., the nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to a plurality of first composite polymerases.

[0273] In some embodiments, the second stage of the two-stage sequencing method generally includes nucleotide incorporation. In some embodiments, the method for sequencing further includes step (e): dissociating a plurality of multivalent composite polymerases, removing a plurality of first sequencing polymerases and their bound multivalent molecules, and retaining a plurality of nucleic acid duplexes.

[0274] In some embodiments, the method for sequencing comprises step (f): contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, the contacting being performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second complex polymerases, each of the plurality of second complex polymerases comprising a second sequencing polymerase bound to a nucleic acid duplex, and further comprising contacting. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0275] In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is 100% identical to the amino acid sequence as the plurality of second sequencing polymerases of step (f). In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is different from the amino acid sequence of the plurality of second sequencing polymerases of step (f).

[0276] In some embodiments, the method for sequencing comprises step (g): contacting a plurality of second composite polymerases with a plurality of nucleotides, the contacting being performed under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second composite polymerases, thereby further comprising contacting to form a plurality of nucleotide composite polymerases. In some embodiments, the contacting of step (g) is performed under conditions suitable for promoting polymerase-catalyzed incorporation of the bound complementary nucleotides into the primer of the nucleotide composite polymerase, thereby forming a plurality of nucleotide composite polymerases. In some embodiments, incorporating the nucleotides in step (g) into the 3' end of the primer includes a primer extension reaction. In some embodiments, the contacting of step (g) is performed in the presence of at least one catalytic cation including magnesium and / or manganese. In some embodiments, the contacting of step (g) is performed in the presence of magnesium and / or manganese. In some embodiments, the plurality of nucleotides includes natural nucleotides (e.g., non-analog nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides includes 2' and / or 3' chain terminating moieties that are removable or non-removable. In some embodiments, the plurality of nucleotides includes a plurality of nucleotides labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety includes a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base by a linker that is cleavable / removable from the base or non-removable from the base. In some embodiments, at least one of the nucleotides in the plurality of nucleotides is not labeled with a detectable reporter moiety.In some embodiments, a specific detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to enable detection and identification of the nucleotide base.

[0277] In some embodiments, a method for sequencing further includes step (h): detecting complementary nucleotides incorporated within a primer of a nucleotide complexed polymerase. In some embodiments, a plurality of nucleotides are labeled with a detectable reporter moiety to enable detection. In some embodiments, in a method for sequencing concatemer molecules, the detection step is omitted.

[0278] In some embodiments, a method for sequencing further includes step (i): identifying the base of a complementary nucleotide incorporated within a primer of a nucleotide complexed polymerase. In some embodiments, the identification of the incorporated complementary nucleotide in step (i) can be used to confirm the identity of the complementary nucleotides of the multivalent molecule bound to the plurality of first complexed polymerases in step (d). In some embodiments, the identifying in step (i) can be used to determine the sequence of a nucleic acid concatemer molecule. In some embodiments, in a method for sequencing concatemer molecules, the identifying step is omitted.

[0279] In some embodiments, a method for sequencing further includes step (j): removing a chain termination moiety from an incorporated nucleotide when step (g) is performed by contacting a plurality of second complexed polymerases with a plurality of nucleotides including at least one nucleotide having a 2’ and / or 3’ chain termination moiety.

[0280] In some embodiments, the method for sequencing further comprises step (k): repeating steps (a) through (j) at least once (e.g., 1, 2, 3, 4, 5, or more than 6 times). In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying a multivalent molecule that binds to the sequencing polymerase in steps (c) and (d) but is not incorporated within the 3' end of the primer. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined (or confirmed) by detecting and discriminating nucleotides incorporated within the 3' end of the primer in steps (h) and (i).

[0281] In some embodiments, in any of the methods for sequencing a nucleic acid molecule, the binding of a plurality of first complex polymerases and a plurality of multivalent molecules forms at least one avidity complex, and the method comprises the following steps: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and (b) binding a second nucleic acid primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the concatemer template molecule comprises a tandem repeat sequence of a sequence of interest and at least one universal sequencing primer binding site. The first and second nucleic acid primers can bind to the sequencing primer binding site along the concatemer template molecule. Exemplary multivalent molecules are shown in FIGS. 9-13.

[0282] In some embodiments, in any of the methods for sequencing a nucleic acid molecule, the method comprises binding a plurality of first complex polymerases to a plurality of multivalent molecules to form at least one avidity complex, and the method comprises the following steps: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers with different portions of a concatemer nucleic acid concatemer molecule to form at least first and second complex polymerases on the same concatemer molecule; and (b) contacting a plurality of multivalent molecules with at least first and second complex polymerases on the same concatemer template molecule under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first and second complex polymerases, wherein at least a first nucleotide unit of the single multivalent molecule hybridizes to a first portion of the concatemer template molecule, thereby binding to a first complex polymerase comprising a first primer that forms a first binding complex (e.g., a first ternary complex), and at least a second nucleotide unit of the single multivalent molecule hybridizes to a second portion of the concatemer template molecule, thereby binding to a second complex polymerase comprising a second primer that forms a second binding complex (e.g., a second ternary complex), and the contacting is performed under conditions suitable for inhibiting the polymerase-catalyzed incorporation of the bound first and second nucleotide units in the first and second binding complexes, and the first and second binding complexes that bind to the same multivalent molecule form an avidity complex; (c) detecting the first and second binding complexes on the same concatemer template molecule; and (d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule. In some embodiments, the plurality of sequencing polymerases comprises any wild-type or mutant sequencing polymerase described herein.The concatemer template molecule includes a tandem repeat sequence of a target sequence and at least one universal sequencing primer binding site. In some embodiments, multiple nucleic acid primers can bind to the sequencing primer binding site along the concatemer template molecule. Exemplary multivalent molecules are shown in FIGS. 9-13.

[0283] Sequencing by Binding In another aspect, the present disclosure provides a method for sequencing any of the immobilized concatemer molecules described herein. In some embodiments, the sequencing method includes a sequencing by binding (SBB) procedure using unlabeled chain-terminating nucleotides. In some embodiments, the sequencing by binding (SBB) method includes (a) sequentially contacting a primed template nucleic acid with at least two separate mixtures under ternary complex stabilizing conditions, wherein the at least two separate mixtures each comprise a polymerase and a nucleotide. In some embodiments, the sequential contacting results in the primed template nucleic acid contacting nucleotide homologs for the base types of the first, second, and third bases in the template under ternary complex stabilizing conditions. In some embodiments, SBB further includes (b) examining the at least two separate mixtures to determine whether a ternary complex has formed. In some embodiments, SBB further includes (c) identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first, second, or third base type. In certain embodiments, based on the absence of a ternary complex in step (b), the next correct nucleotide is presumed to be a nucleotide homolog of the fourth base type. In some embodiments, SBB further includes (d) adding the next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby producing an extended primer. In some embodiments, SBB further includes (e) repeating steps (a)-(d) at least once on the primed template nucleic acid comprising the extended primer. In certain specific embodiments, each of steps (a), (b), (c), (d), and (e) is performed, for example, in order.Exemplary sequencing methods by ligation are described in U.S. Patent Nos. 10,246,744 and 10,731,141 (the contents of both patents are hereby incorporated by reference in their entireties).

[0284] Sequencing polymerase The present disclosure provides a method for sequencing a nucleic acid molecule, wherein any of the sequencing methods described herein uses at least one type of sequencing polymerase and a plurality of nucleotides, or at least one type of sequencing polymerase, a plurality of nucleotides, and a plurality of multivalent molecules. In some embodiments, the sequencing polymerase(s) is / are capable of incorporating complementary nucleotides opposite the nucleotides in the concatemer template molecule. In some embodiments, the sequencing polymerase(s) is / are capable of binding to the complementary nucleotide units of the multivalent molecule opposite the nucleotides in the concatemer template molecule. In some embodiments, the plurality of sequencing polymerases includes recombinant mutant polymerases.

[0285] Examples of polymerases suitable for use in sequencing with nucleotides and / or multivalent molecules include Klenow DNA polymerase; Thermus aquaticus DNA polymerase I (Taq polymerase); KlenTaq polymerase; Candidatus altiarchaeales archaea; Candidatus Hadarchaeum Yellowstonense; Hadesarchaea archaea; Euryarchaeota archaea; Thermoplasmata archaea; Thermococcus polymerases, such as Thermococcus litoralis, bacteriophage T7 DNA polymerase; human alpha, delta, and epsilon DNA polymerases; bacteriophage polymerases, such as T4, RB69, and phi29 bacteriophage DNA polymerases; Pyrococcus furiosus DNA polymerase (Pfu polymerase); Bacillus subtilis DNA polymerase III; E. coli DNA polymerase III alpha and epsilon; 9 degree N polymerase; reverse transcriptases, such as HIV type M or O reverse transcriptase; avian myeloblastosis virus reverse transcriptase; Moloney murine leukemia virus (MMLV) reverse transcriptase; or telomerase, but are not limited thereto. Further non-limiting examples of DNA polymerases include those from various archaeal families, such as Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus, Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus, Thermococcus, and Vulcanisaeta, or variants thereof, which include such polymerases known in the art, such as 9 degrees N, VENT®, DEEP VENT®, THERMINATOR®, Pfu, KOD, Pfx, Tgo, and RB69 polymerase.

[0286] Nucleotide The present disclosure provides a method for sequencing a nucleic acid molecule using nucleotides, wherein at least one nucleotide among a plurality of nucleotides comprises a base, a sugar, and at least one phosphate group. In some embodiments, at least one nucleotide among the plurality of nucleotides comprises an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and one or more phosphate groups (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups). The plurality of nucleotides can comprise at least one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP. The plurality of nucleotides can comprise a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, dUTP, and combinations thereof. In some embodiments, at least one nucleotide among the plurality of nucleotides is not a nucleotide analog. In some embodiments, at least one nucleotide among the plurality of nucleotides comprises a nucleotide analog.

[0287] In some embodiments, in any of the methods for sequencing a nucleic acid molecule described herein, at least one nucleotide among the plurality of nucleotides comprises a chain of 1, 2, or 3 phosphorus atoms, the chain typically being attached to the 5'-carbon of the sugar moiety via an ester or phosphoramidate bond. In some embodiments, at least one nucleotide among the plurality of nucleotides is an analog having a phosphorus chain, in which the phosphorus atoms are bonded together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise a substituted side chain group containing O, S, or BH3. In some embodiments, the chain comprises a phosphate group substituted with an analog containing phosphoramidate, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0288] In some embodiments, in any of the methods for sequencing nucleic acids described herein, at least one nucleotide in a plurality of nucleotides comprises a terminator nucleotide analog, and the terminator nucleotide analog has a chain-terminating moiety (e.g., a blocking moiety) at the sugar 2'-position, sugar 3'-position, or both sugar 2' and 3'-positions. In some embodiments, the chain-terminating moiety can inhibit polymerase-catalyzed incorporation of subsequent nucleotide units or free nucleotides in the nascent strand during a primer extension reaction. In some embodiments, the chain-terminating moiety is attached to the 3'-sugar hydroxyl position, and at the 3'-sugar hydroxyl position, the sugar comprises a ribose or deoxyribose sugar moiety. In some embodiments, the chain-terminating moiety is removable / cleavable from the 3'-sugar hydroxyl position to produce a nucleotide having a 3'-OH sugar group. In certain embodiments, the 3'-OH sugar group is extendable with a subsequent nucleotide in a polymerase-catalyzed nucleotide incorporation reaction. In some embodiments, the chain-terminating moiety comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the chain-terminating moiety is cleavable / removable from the nucleotide, for example, but not limited to, by reacting the chain-terminating moiety with a chemical agent, a pH change, light, or heat. In some embodiments, the chain-terminating moieties alkyl, alkenyl, alkynyl, and allyl are cleavable using tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) having piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the chain-terminating moieties aryl and benzyl are cleavable with H2 Pd / C. In some embodiments, the chain-terminating moieties amine, amide, keto, isocyanate, phosphate, thio, and / or disulfide are cleavable with a phosphine or a thiol group comprising, for example, but not limited to, beta-mercaptoethanol or dithiothreitol (DTT).In some embodiments, the chain-terminating moiety carbonate can be cleaved using potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the urea and silyl, which are chain-terminating moieties, can be cleaved with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0289] In some embodiments, in any of the methods for sequencing a nucleic acid molecule described herein, at least one nucleotide in a plurality of nucleotides comprises a terminator nucleotide analog, and the terminator nucleotide analog has a chain-terminating moiety (e.g., a blocking moiety) at the sugar 2'-position, the sugar 3'-position, or both the sugar 2'- and 3'-positions. In some embodiments, the chain-terminating moiety comprises an azide, azido, and azidomethyl group. In some embodiments, the chain-terminating moiety comprises a 3'-O-azide or 3'-O-azidomethyl group. In some embodiments, the azide, azido, and azidomethyl groups, which are chain-terminating moieties, are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxyproyl)phosphine (THPP). In some embodiments, the cleavage agent comprises 4-dimethylaminopyridine (4-DMAP).

[0290] In some embodiments, in any of the methods for sequencing a nucleic acid molecule described herein, the nucleotide comprises a chain-terminating moiety selected from the group consisting of 3'-deoxynucleotide, 2',3'-dideoxynucleotide, 3'-methyl, 3'-azide, 3'-azidomethyl, 3'-O-azidoalkyl, 3'-O-ethynyl, 3'-O-aminoalkyl, 3'-O-fluoroalkyl, 3'-fluoromethyl, 3'-difluoromethyl, 3'-trifluoromethyl, 3'-sulfonyl, 3'-malonyl, 3'-amino, 3'-O-amino, 3'-sulfhydryl, 3'-aminomethyl, 3'-ethyl, 3'-butyl, 3'-tert-butyl, 3'-fluorenylmethyloxycarbonyl, 3'-tert-butyloxycarbonyl, 3'-O-alkylhydroxylamino group, 3'-phosphorothioate, and 3-O-benzyl, or derivatives thereof.

[0291] In some embodiments, in any of the methods for sequencing a nucleic acid molecule described herein, the plurality of nucleotides comprises a plurality of nucleotides labeled with a detectable reporter moiety. In some embodiments, the detectable reporter moiety comprises a fluorophore. In some embodiments, the fluorophore is attached to the nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base by a linker, and the linker is cleavable / removable from the base. In some embodiments, at least one of the nucleotides in the plurality of nucleotides is not labeled with a detectable reporter moiety. In some embodiments, a particular detectable reporter moiety (e.g., a fluorophore) attached to a nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) such that detection and identification of the nucleotide base is enabled.

[0292] In some embodiments, in any of the methods for sequencing nucleic acids described herein, the cleavable linker on the nucleotide base comprises a cleavable moiety. In certain embodiments, the cleavable moiety comprises an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group. In some embodiments, the cleavable linker on the base is cleavable / removable from the base by reacting the cleavable moiety with a chemical agent, a pH change, light, or heat. In some embodiments, the cleavable moieties alkyl, alkenyl, alkynyl, and allyl are cleavable using tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) having piperidine, or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In some embodiments, the cleavable moieties aryl and benzyl are cleavable with H2 Pd / C. In some embodiments, the cleavable moieties amine, amide, keto, isocyanate, phosphate, thio, and / or disulfide are cleavable with a phosphine, or a thiol group including beta-mercaptoethanol or dithiothreitol (DTT). In some embodiments, the cleavable moiety carbonate is cleavable using potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or Zn in acetic acid (AcOH). In some embodiments, the cleavable moieties urea and silyl are cleavable with tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, or triethylamine trihydrofluoride.

[0293] In some embodiments, in any of the methods for sequencing nucleic acids described herein, the cleavable linker on the nucleotide base comprises a cleavable moiety. In some embodiments, the cleavable moiety comprises an azide, azido, or azidomethyl group. In some embodiments, the azide, azido, and azidomethyl groups, which are cleavable moieties, are cleavable / removable with a phosphine compound. In some embodiments, the phosphine compound comprises a derivatized trialkylphosphine moiety or a derivatized triarylphosphine moiety. In some embodiments, the phosphine compound comprises tris(2-carboxyethyl)phosphine (TCEP), or bis-sulfotriphenylphosphine (BS-TPP), or tris(hydroxyproyl)phosphine (THPP). In some embodiments, the cleaving agent comprises 4-dimethylaminopyridine (4-DMAP).

[0294] In some embodiments, in any of the methods for sequencing nucleic acids described herein, the chain terminating moiety (e.g., at the sugar 2' and / or 3' positions), and the cleavable linker on the nucleotide base have the same or different cleavable moieties. In some embodiments, the chain terminating moiety (e.g., at the sugar 2' and / or 3' positions), and the detectable reporter moiety attached to the base are chemically cleavable / removable with the same chemical agent. In some embodiments, the chain terminating moiety (e.g., at the sugar 2' and / or 3' positions), and the detectable reporter moiety attached to the base are chemically cleavable / removable with different chemical agents.

[0295] Multivalent molecule The present disclosure provides a method for sequencing nucleic acid molecules using multivalent molecules. In some embodiments, the multivalent molecule includes a plurality of nucleotide arms, the plurality of nucleotide arms are attached to a core, and have any configuration including a starburst, helter-skelter, or bottlebrush configuration (e.g., FIGS. 9-13). In some embodiments, the multivalent molecule includes (1) a core and (2) a plurality o...

Claims

1. A method for reducing deaminated nucleotide bases in a nucleic acid library, a) To provide a plurality of linear nucleic acid library molecules, wherein the linear nucleic acid library molecules are in single-stranded or double-stranded form, and each linear nucleic acid library molecule in the plurality of linear nucleic acid library molecules contains a target sequence linked to at least one universal adapter sequence having a binding sequence for a surface capture primer and one universal adapter sequence having a binding sequence for a sequencing primer, and at least one of the linear nucleic acid library molecules possesses one or more deaminated nucleotide bases, b) Contacting the plurality of linear nucleic acid library molecules with a reagent that removes deaminated nucleotide bases, thereby generating at least one linear nucleic acid library molecule having a non-basic site, wherein the reagent contains an enzyme or enzyme mixture having lyase activity, thereby generating a gap in the non-basic site. c) Cyclizing the plurality of linear nucleic acid library molecules to generate a plurality of covalently bound closed cyclic library molecules, d) Distributing the plurality of covalent closed cyclic library molecules onto a support having a plurality of immobilized surface capture primers under conditions suitable for hybridizing each covalent closed cyclic library molecule to the surface capture primer, e) Performing a rolling circle amplification reaction to generate a plurality of nucleic acid concatemer template molecules immobilized on the support, f) A method comprising sequencing the plurality of immobilized concatemer template molecules to determine the sequence of at least a portion of the immobilized concatemer template molecules.

2. (g) The method according to claim 1, comprising contacting the plurality of covalently bound closed cyclic library molecules with a reagent that removes deaminated nucleotide bases, thereby generating at least one cyclic library molecule having a non-basic site, wherein the reagent comprises an enzyme or mixture of enzymes having lyase activity, thereby generating a gap in the non-basic site.

3. The method according to claim 12, wherein the reagent for removing the deaminated nucleotide base in step (b) comprises DNA glycosylase (UDG) and (i) AP lyase, (ii) Endo IV endonuclease, (iii) FPG glycosylase / AP lyase, and / or (iv) Endo VIII glycosylase / AP lyase, or a combination thereof.

4. The method according to claim 2, wherein the reagent for removing the deaminated nucleotide base in step (g) comprises DNA glycosylase (UDG), and (i) AP lyase, (ii) Endo IV endonuclease, (iii) FPG glycosylase / AP lyase, and / or (iv) Endo VIII glycosylase / AP lyase, or a combination thereof.

5. The method according to claim 1, wherein the plurality of immobilized surface capture primers are tethered to a polymer coating on the support.

6. The method according to claim 1, wherein the rolling circle amplification reaction of step (e) comprises a chain substitution polymerase and a plurality of nucleotides including dATP, dGTP, dCTP, dTTP, and / or dUTP.

7. The method according to claim 1, wherein the plurality of immobilized surface capture primers are located at predetermined positions on the support, or the plurality of immobilized surface capture primers are located at random positions on the support.

8. The method according to claim 1, wherein the plurality of immobilized concatemer template molecules on the support are in fluid communication with one another, enabling the reagent solution to flow onto the support.

9. The method according to claim 8, wherein the solution of the reagent comprises an enzyme, a nucleotide, and a divalent cation.

10. The method according to claim 8, wherein the plurality of immobilized concatemer template molecules react with the reagent in an essentially simultaneous and ultra-parallel manner.

11. Sequencing the aforementioned multiple immobilized concatemer template molecules is a) Contacting the plurality of immobilized concatemer template molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contact is carried out under conditions suitable for forming a plurality of complex polymerases, each of the plurality of complex polymerases comprising a sequencing polymerase bound to a nucleic acid double helix, and the nucleic acid double helix comprising an immobilized concatemer template molecule hybridized to a soluble sequencing primer. b) Contacting the plurality of complex polymerases with a plurality of nucleotides under conditions suitable for binding at least one nucleotide to the complex polymerase, wherein the plurality of nucleotides include at least one nucleotide analog, the at least one nucleotide analog is labeled with a fluorophore and has a removable chain termination at the sugar 3' position, c) Incorporating at least one nucleotide into the 3' end of the hybridized sequencing primer, thereby generating a plurality of newly generated, elongated sequencing primers, d) The method according to claim 1, comprising detecting the incorporated nucleotide and identifying the nucleic acid base of the incorporated nucleotide.

12. The method according to claim 11, wherein the plurality of nucleotides include a removable chain termination portion at the 3' sugar group, and the removable chain termination portion includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

13. The method according to claim 12, wherein the removable chain termination portion can be cleaved with a chemical compound such that it generates an extendable 3'OH portion on the sugar group.

14. The method according to claim 11, wherein the plurality of nucleotides comprises one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, or comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

15. Sequencing the aforementioned multiple immobilized concatemer template molecules is a) Contacting the plurality of immobilized concatemer template molecules with (i) a plurality of first sequencing polymerases and (ii) a plurality of soluble sequencing primers, wherein the contact is performed under conditions suitable for forming a plurality of first complex polymerases, each of the plurality of first complex polymerases comprising a first sequencing polymerase bound to a nucleic acid double helix, and the nucleic acid double helix comprising an immobilized concatemer template molecule hybridized to a soluble sequencing primer. b) Forming a plurality of polyvalent polymerases by conjugating a plurality of detectably labeled polyvalent molecules and complementary nucleotide units of the polyvalent molecules to at least two of the plurality of first polyvalent polymerases, and thereby contacting them under conditions suitable for forming a plurality of polyvalent polymerases, wherein the conditions inhibit the incorporation of the complementary nucleotide units into the sequencing primers of the plurality of polyvalent polymerases, and the plurality of polyvalent molecules form a core attached to a plurality of nucleotide arms, each nucleotide arm attached to a nucleotide unit. c) To detect the multiple multivalent complex polymerases, d) The method according to claim 1, comprising identifying the nucleic acid bases of the complementary nucleotide units bound to the plurality of first complex polymerases in the plurality of multivalent complex polymerases, and thereby determining the sequence of the nucleic acid template.

16. e) Dissociating the plurality of multivalent complex polymerases, removing the plurality of first sequencing polymerases and the multivalent molecules to which they are bound, and retaining the plurality of nucleic acid double helixes, f) Contacting the plurality of nucleic acid double helixes of step (e) with a plurality of second sequencing polymerases, wherein the contact is carried out under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of nucleic acid double helixes, thereby forming a plurality of second complex polymerases, each of which contains a second sequencing polymerase bound to a nucleic acid double helix. g) The method according to claim 15, further comprising contacting the plurality of second complex polymerases with a plurality of nucleotides comprising at least one nucleotide analog having a fluorophore-labeled chain termination portion at the sugar 3' position, wherein the contact is carried out under conditions suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the second complex polymerases of step (f), thereby forming a plurality of nucleotide complex polymerases, wherein the conditions are suitable for promoting the incorporation of the bound complementary nucleotides into the sequencing primers of the nucleotide complex polymerases.

17. h) The method according to claim 16, further comprising detecting the complementary nucleotide incorporated within the sequencing primer of the nucleotide complex polymerase.

18. The method according to claim 17, further comprising: i) identifying the nucleic acid base of the complementary nucleotide incorporated within the sequencing primer of the nucleotide complex polymerase.

19. A method for sequencing by forming at least one avidity complex, a) Generating nucleic acid concatemer molecules by performing rolling circle amplification on a plurality of closed circular nucleic acid molecules, each comprising at least one covalent closed circular nucleic acid molecule containing at least one nonbasic site, wherein the nonbasic site is generated by contacting a closed circular nucleic acid molecule or a corresponding linear nucleic acid molecule containing a target sequence operably bound on both sides by at least one adapter sequence with a reagent that removes deaminated nucleotide bases. b) Binding a first universal sequencing primer, a first sequencing polymerase, and a first detectably labeled polyvalent molecule to a first portion of a concatemer molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first polyvalent molecule binds to the first sequencing polymerase. c) Forming a second binding complex, wherein a second universal sequencing primer, a second sequencing polymerase, and the first detectably labeled polyvalent molecule are bound to a second portion of the same concatemer molecule, the second nucleotide unit of the first polyvalent molecule being bound to the second sequencing polymerase, the first and second binding complexes containing the same polyvalent molecule forming an avidity complex, the first detectably labeled polyvalent molecule comprising a core attached to a plurality of nucleotide arms containing spacers, linkers, and nucleotide units, the concatemer molecule comprising the sequence of interest (110) and two or more tandem repeat sequences of universal primer binding sites that bind to the first and second universal sequencing primers, and the contact is carried out under conditions suitable for inhibiting polymerase catalyst integration of the bound first and second nucleotide units in the first and second binding complexes. d) To detect the first and second binding complexes on the same concatemer molecule, e) A method comprising: identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer molecule; identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer molecule.

20. The plurality of nucleotide arms attached to the core of each of the polyvalent molecules have the same type of nucleotide unit, and the type of nucleotide unit is selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, and / or The method according to claim 15 or 19, wherein the plurality of polyvalent molecules comprises a mixture of two or more types of polyvalent molecules, and each of the two or more types of polyvalent molecules has a nucleotide unit selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

21. The method according to claim 16, wherein the plurality of nucleotides include a removable chain termination portion at the 3' sugar group, and the removable chain termination portion includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azide group, an azido group, an O-azidomethyl group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, or a silyl group.

22. The method according to claim 21, wherein the removable chain termination portion can be cleaved with a chemical compound such that it generates an extendable 3'OH portion on the sugar group.

23. The plurality of nucleotides include one type of nucleotide selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP, or The method according to claim 16, wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from the group consisting of dATP, dGTP, dCTP, dTTP, and dUTP.

24. The method according to claim 1, wherein the support comprises a glass substrate or a plastic substrate.

25. The method according to claim 1, wherein the support is passivated with at least one hydrophilic polymer coating having a water contact angle of 45 degrees or less.

26. The method according to claim 25, wherein the at least one hydrophilic polymer coating comprises a molecule selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, and dextran.

27. The method according to claim 1, further comprising determining a percentage-based call error from the sequencing of step (f).

28. The method according to claim 27, further comprising determining a quality score for sequencing data from the percentage-based call error, wherein the quality score is a Fred quality score.

29. The annularization is i) Intramolecular ligation, ii) Padlock probe, iii) Telomerase, iv) Single-strand splint adapter, or v) Double-strand splint adapter The method according to claim 1, including the method described in claim 1.

Images