Streamlined nucleic acid library preparation and sequencing workflows

Abstract

The present disclosure provides compositions and methods for preparing a plurality of linear nucleic acid library molecules. In some embodiments, two or more of the library preparation steps can be conducted in a single reaction vessel (e.g., one-pot library prep workflow) resulting in a streamlined workflow that requires reduced hands-on manipulation and time. In some embodiments, the plurality of linear library molecules can be circularized using soluble or immobilized splint oligonucleotides. In some embodiments, the circular library molecules can be subjected to rolling circle amplification and the resulting concatemer template molecules can be sequenced.

Description

STREAMLINED NUCLEIC ACID LIBRARY PREPARATIONAND SEQUENCING WORKFLOWSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, U. S. Provisional Application No.63 / 713,405, filed on October 29, 2024, and U. S. Provisional Application No. 63 / 894,080 filed on October 6, 2025, the contents of each of which are incorporated by reference in their entirety herein.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (EL EM- 038_001WO_SeqList_ST26. xml; file creation date: October 28, 2025; file size: 27,312 bytes) are herein incorporated by reference in their entirety.TECHNICAL FIELD

[0003] The present disclosure relates to compositions and methods for preparing a plurality of linear nucleic acid library molecules for high throughput sequencing methods. Two or more of the library preparation steps can be conducted in a single reaction vessel (e.g., one-pot library prep workflow) resulting in a streamlined workflow that requires reduced hands-on manipul ation and time. A plurality of linear library molecules can be circularized using soluble or immobilized splint oligonucleotides, subjected to rolling circle amplification and the resulting concatemer template molecules sequenced.BACKGROUND

[0004] Massively parallel sequencing methods have applications in biomedical research and healthcare settings as they allow for analyzing large quantities of nucleic acids with different sequences from biological samples. Efficient preparation of libraries having target sequences, and attachment of libraries to substrates suitable for sequencing, such as flow cell surfaces, is important for downstream amplification and sequencing workflows. Accordingly, there is a need for methods for producing libraries containing target sequences, unique index sequences and sequencing primer binding sequences which are compatible with substrate attachment and downstream next generation sequencing technologies. Provided herein are compositions, methods and kits addressing this need.SUMMARY

[0005] The disclosure provides a method for preparing a plurality of DNA library molecules comprising: (a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) conducting a tagmentation reaction by contacting a plurality of input double-stranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising a sequence complementary to at least a portion of the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end of the individual double-stranded DNA fragment, thereby forming a plurality of double-stranded 5 ’-adaptor-tagged molecules, wherein individual 5 ’adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individual 5 ’-adaptor-tagged molecule; (c) denaturing the plurality of doublestranded 5 ’-adaptor-tagged molecules, thereby generating a plurality of single-stranded 5’-adaptor-tagged molecules and a plurality of single- stranded non-transfer strands that are not hybridized to the portion of the transfer strand; (d) contacting the plurality of single-stranded 5 ’-adaptor-tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual single- stranded 5 ’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules comprising a 3’ templateindependent polynucleotide; (e) hybridizing the plurality of single-stranded linear DNA library molecules to a plurality of polyN splint capture primers thereby forming a plurality of open circle library splint complexes, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety that can bind to a receptor moiety of the capture support, of step (a), and wherein individual polyN splint capture primers comprise 3’ ends that are extendible; (f) distributing the plurality of open circle library splint complexes onto the capture support, thereby immobilizing the plurality of open circle library splint complexes to the capture support, wherein the distributing is conducted under a condition suitable for binding the affinity moiety to a receptor moiety; (g) contacting the plurality of open circle library splint complexes with aclosure reagent to generate a plurality of covalently closed circular library molecules, wherein individual covalently closed circular library molecules are hybridized to polyN splint capture primers immobilized to the capture support; (h) contacting the plurality of covalently closed circular library molecules with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the polyN splint capture primers using the plurality of covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support; and (i) sequencing at least a portion of the plurality of concatemer template molecules.

[0006] The disclosure provides a method for preparing a plurality of DNA library molecules comprising: (a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) conducting a tagmentation reaction by contacting a plurality of input double-stranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising a sequence complementary to at least a portion of the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end of the individual double-stranded DNA fragment, thereby forming a plurality of double-stranded 5 ’-adaptor-tagged molecules, wherein individual 5 ’-adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individual 5 ’-adaptor-tagged molecule; (c) denaturing the plurality of doublestranded 5 ’-adaptor- tagged molecules, thereby generating a plurality of single-stranded 5’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are not hybridized to the portion of the transfer strand; (d) contacting the plurality of single-stranded 5 ’-adaptor-tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual single-stranded 5 ’-adaptor-tagged molecule, thereby generating a plurality of single- stranded linear DNA library molecules comprising a 3’ templateindependent polynucleotide; (e) hybridizing the plurality of single-stranded linear DNA library molecules to a plurality of polyN splint capture primers, thereby forming a plurality of open circle library splint complexes, wherein individual polyN splint capture primerscomprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety that can bind to a receptor moiety of the capture support of step (a), and wherein individual polyN splint capture primers comprise 3’ ends that are extendible; (f) contacting the plurality of open circle library splint complexes with a closure reagent generate a plurality of covalently closed circular library molecule complexes, wherein an individual covalently closed circular library molecule is hybridized to a polyN splint capture primer; (g) distributing the plurality of covalently closed circular library molecule complexes onto the capture support, thereby immobilizing the plurality of covalently closed circular library molecule complexes to the capture support, wherein the distributing is conducted under a condition suitable for binding the affinity moiety of individual polyN splint capture primers to a receptor moiety; (h) contacting the plurality of covalently closed circular library molecules with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the polyN splint capture primers using the covalently closed circular library molecules as template molecules thereby, generating a plurality of concatemer template molecules which are immobilized to the capture support; and (i) sequencing at least a portion of the plurality of concatemer template molecules.

[0007] The disclosure provides a method for preparing a plurality of DNA library molecules comprising (a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) conducting a tagmentation reaction by contacting a plurality of input double-stranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising a sequence complementary' to at least a portion of the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end the individual double-stranded DNA fragment, thereby forming a plurality of double-stranded 5 ’-adaptor-tagged molecules, wherein individual 5 ’adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individual 5 ’-adaptor-tagged molecule; (c) denaturing the plurality of doublestranded 5 ’-adaptor- tagged molecules, thereby generating a plurality of single-stranded 5’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are nothybridized to the portion of one of the transfer strands; (d) contacting the plurality of single-stranded 5 ’-adaptor-tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual single-stranded 5 ’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules comprising a 3’ template-independent polynucleotide; (e) distributing a plurality of polyN splint capture primers onto the capture support, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety that can bind to a receptor moiety of the capture support of step (a), thereby immobilizing plurality of the polyN splint capture primers to the capture support, and wherein f individual polyN splint capture primers comprise 3’ ends that are extendible; (f) distributing the plurality of single-stranded linear DNA library molecules onto the capture support under a condition suitable for hybridizing an individual single-stranded linear DNA library molecules to an individual polyN splint capture primer, thereby forming a plurality of open circle library splint complexes; (g) contacting the plurality of open circle library splint complexes immobilized to the capture support with a closure reagent to generate a plurality of covalently closed circular library molecules, wherein individual covalently closed circular library molecules are hybridized to individual polyN splint capture primers; (h) contacting the plurality of covalently closed circular library molecules with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the polyN splint capture primers using the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support; and (i) sequencing at least a portion of the plurality of concatemer template molecules.

[0008] In some embodiments, individual open circle library splint complexes comprise a nick and / or a 5’ flap structure. In some embodiments, the closure reagent comprises a reagent that closes the nick and / or 5’ flap structure.

[0009] The disclosure provides a method for preparing a plurality of DNA library molecules comprising: (a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; (b) conducting a tagmentation reaction by contacting a plurality of input double-stranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzymeand a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising a sequence complementary' to the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end of the individual doublestranded DNA fragment, thereby forming a plurality of double-stranded 5 ’-adaptor-tagged molecules, wherein individual 5 ’adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individual 5 ’-adaptor- tagged molecule; (c) denaturing the plurality of double-stranded 5’-adaptor-tagged molecules, thereby generating a plurality of single-stranded 5 ’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are not hybridized to the portion of the transfer strand; (d) distributing a plurality of polyN splint capture primers onto the capture support, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety binds to receptor moiety of the capture support of step (a), thereby immobilizing the plurality of polyN splint capture primers to the support, and wherein the 3’ ends of individual polyN splint capture primers comprise at least one scissile moiety near the 3’ ends,, and wherein a terminal 3’ ends of individual polyN splint capture primers comprise blocking moieties that inhibit polymerase-catalyzed extension of the 3’ ends; (e) distributing the plurality of single-stranded 5 ’-adaptor-tagged molecules onto the capture support under a condition suitable for hybridizing individual single-stranded 5 ’-adaptor-tagged molecules to individual polyN splint capture primers immobilized to the capture support, thereby forming a plurality of immobilized 5’-adaptor-tagged molecules, wherein individual immobilized 5 ’-adaptor-tagged molecule do not form open circle library splint complexes while hybridized to an immobilized capture primer; (f) contacting the plurality of immobilized 5 ’-adaptor-tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual 5’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules immobilized to the capture support, wherein individual single-stranded linear DNA library molecules comprise a 3’ template-independent polynucleotide, wherein individual single- stranded linear DNA library molecules are hybridized to individual immobilized polyN splint capture primers to form a plurality of open circle library splint complexes comprising a nick, wherein the plurality of open circle library splint complexesare immobilized to the capture support; (g) contacting the plurality of open circle library splint complexes with a closure reagent that can close the nick to generate a plurality of covalently closed circular library molecules, wherein individual covalently closed circular library molecules are hybridized to individual polyN splint capture primers immobilized to the capture support; (h) contacting the plurality of polyN splint capture primers that are hybridized to the covalently closed circular library molecules with an unblocking reagent that converts the scissile moiety into an abasic site and the blocking moieties of the polyN splint capture primers into 3’ extendible ends; (i) contacting the plurality of covalently closed circular library with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ extendable ends of the polyN splint capture primers using the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support; and (j) sequencing at least a portion of the plurality of concatemer template molecules.

[0010] In some embodiments, individual single-stranded linear DNA library molecules comprise at least one universal adaptor sequence and a transfer end sequence from the transfer strand.

[0011] In some embodiments, the tagmentation reaction is conducted in solution.

[0012] In some embodiments, the at least one scissile moiety can be converted into at least one abasic site.

[0013] In some embodiments, the receptor moiety comprises streptavidin or avidin, or a derivative thereof. In some embodiments, the affinity moiety comprises biotin, desthiobiotin or iminobiotin. In some embodiments, the tagmentation reaction comprises a Tn5 transposase-based tagmentation reaction.

[0014] In some embodiments, at least one universal adaptor sequence comprises any one or any combination of two or more of: (a) a universal sequence for binding a forward sequencing primer (FWD seq); (b) a universal sequence for binding a reverse sequencing primer (REV seq); (c) at least one sample index sequence; (d) a universal sequence for binding a polyN splint capture primer; and / or (e) a universal sequence for binding a pinning primer.

[0015] In some embodiments, the at least one sample index sequence comprises a random sequence, optionally wherein the random sequence is 3-7 nucleotides long.

[0016] In some embodiments, the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and / or dUTP.

[0017] In some embodiments, the plurality of concatemer template molecules can be sequenced essentially simultaneously or using batch sequencing.

[0018] The disclosure provides a method for preparing a plurality of nucleic acid library molecules, comprising: (a) providing a plurality of double-stranded input polynucleotides; (b) generating a plurality of double-stranded polynucleotide fragments from the plurality of double-stranded input polynucleotides; (c) joining at least one universal adaptor sequence to only one end of individual double-stranded polynucleotide fragments, thereby generating a plurality of double-stranded library molecules, wherein individual double-stranded library molecules comprise an insert sequence joined to at least one universal adaptor sequence; (d) generating a plurality of single-stranded library molecules from the plurality of doublestranded library molecules; (e) generating a plurality of single-stranded open circle library molecules from the plurality of single-stranded linear library molecules, wherein individual single-stranded open circle library molecules comprise a nick; (f) immobilizing the plurality of open circle library molecules to a support, thereby generating a plurality of immobilized open circle library molecules each having a nick, and ligating the nicks, thereby generating a plurality of immobilized covalently closed circle library molecules; (g) generating a plurality of immobilized concatemer template molecules by conducting rolling circle amplification on the support using the immobilized covalently closed circular library molecules as template mol ecules to generate the plurality of immobilized concatemer template molecules; and (h) sequencing the plurality of immobilized concatemer template molecules.

[0019] In some embodiments, the plurality of single-stranded library molecules are generated by denaturing the plurality of double- stranded library molecules.

[0020] In some embodiments, the at least one universal adaptor sequence comprises anyone or any combination of two or more of: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence; (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer. In some embodiments, the at least one sample index sequence comprises a random sequence, optionally wherein the random sequence is 3-7 nucleotides long.

[0021] The disclosure provides a method for preparing a plurality of nucleic acid library molecules, comprising (a) providing a plurality of input polynucleotides comprising aplurality of double-stranded input polynucleotides; (b) generating a plurality of doublestranded polynucleotide fragments from the plurality of double-stranded input polynucleotides; (c) generating a plurality of single-stranded library molecules from the plurality of double-stranded library molecules; (d) joining at least one universal adaptor sequence to only one end of an individual single-stranded polynucleotide fragment, thereby generating a plurality of single-stranded library molecules, wherein individual single-stranded library molecules comprise an insert sequence joined to at least one universal adaptor sequence; (e) generating a plurality of single-stranded open circle library molecules from the plurality of single- stranded linear library molecules, wherein individual single-stranded open circle library molecules comprise a nick; (f) immobilizing the plurality of single-stranded open circle library molecules to a support, thereby generating a plurality of immobilized open circle library molecules each having a nick, and ligating the nicks, thereby generating a plurality of immobilized covalently closed circle library / molecules; (g) generating a plurality of immobilized concatemer template molecules by conducting rolling circle amplification on the support using the plurality of immobilized covalently closed circular library molecules as template molecules to generate the plurality of immobilized concatemer template molecules; and (h) sequencing the plurality of immobilized concatemer template molecules.

[0022] In some embodiments, the at least one universal adaptor sequence that is joined to only one end of individual single-stranded polynucleotide fragments comprises any one or any combination of two or more of: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence; (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer. In some embodiments, the at least one sample index sequence comprises a random sequence, optionally wherein the random sequence is 3-7 nucleotides long.

[0023] In some embodiments, the sequencing comprises: (a) contacting a first plurality of polymerases to (i) the plurality of concatemer template molecules and (ii) a plurality of sequencing primers, wherein the contacting is conducted under a condition suitable to bind the first plurality of polymerases to the plurality of concatemer template molecules and the plurality of nucleic acid primers, thereby forming a first plurality of complexed polymerases each comprising a polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises an concatemer template molecule hybridized to sequencing primer; (b) contacting the first plurality of complexed polymerases with a plurality of multivalent molecules to form a plurality of multivalent-binding complexes, wherein individualmultivalent molecules in the plurality comprise a core attached to multiple nucleotide arms and individual nucleotide arms are attached to a nucleotide moiety, wherein the contacting is conducted under a condition suitable for binding complementary nucleotide moieties of the multivalent molecules to at least two of the first plurality of complexed polymerases, thereby forming a plurality of multivalent-binding complexes, and the condition is suitable for inhibiting incorporation of the complementary nucleotide moieties into the nucleic acid primers of the plurality of multivalent-binding complexes; (c) detecting the plurality of multivalent-binding complexes; and (d) identifying the nucleobase of the complementary' nucleotide moieties in the plurality of multivalent-binding complexes, thereby determining the sequence of the nucleic acid template molecules.

[0024] In some embodiments, the method comprises (e) dissociating the plurality of multivalent-binding complexes by removing the first plurality of polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes; (f) contacting the plurality of the nucleic acid duplexes retained at step (e) with a second plurality of a polymerases under a condition suitable for binding the second plurality of polymerases to the plurality of the nucleic acid duplexes, thereby forming a second plurality of complexed polymerases, individual complexed polymerases comprising a polymerase bound to a nucleic acid duplex; and (g) contacting the second plurality of second polymerases with a plurality of nucleotides, wherein the contacting is conducted under a condition suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the complexed polymerases, thereby forming a plurality of nucleotide-binding complexes, and the condition is suitable for promoting nucleotide incorporation of the bound complementary nucleotides into the nucleic acid primers of the nucleotide-binding complexes.

[0025] In some embodiments, the method further comprises (h) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases.

[0026] In some embodiments, the method further comprises (h) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases; and (i) identifying the nucleobases of the complementary nucleotides which are incorporated into the primers of the nucleotide-complexed polymerases.

[0027] In some embodiments, the complementary' nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases are not detected or identified.

[0028] In some embodiments, contacting the first plurality of complexed polymerases with the plurality of multivalent molecules of step (b) is conducted in the presence of a non-catalytic divalent cation that inhibits polymerase-catalyzed nucleotide incorporation, optionally wherein the non-catalytic divalent cation comprises strontium or barium.

[0029] In some embodiments, contacting the second plurality of complexed polymerases with the plurality of nucleotides of step (g) is conducted in the presence of a catalytic divalent cation that promotes polymerase-catalyzed nucleotide incorporation, optionally wherein the catalytic divalent cation comprises magnesium or manganese.

[0030] In some embodiments, the plurality of concatemer template molecules in step (a) comprise clonally amplified concatemer template molecules. In some embodiments, individual concatemer template molecules in the plurality of step (a) comprise a concatemer template molecule having two or more tandem copies of a target sequence. In some embodiments, the concatemer template molecules in the plurality of concatemer template molecules in step (a) comprise the same target sequence or different target sequences.

[0031] In some embodiments, individual multivalent molecules in the plurality of multivalent molecules comprise: (a) a core; and (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide moiety, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide moiety. In some embodiments, the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits. In some embodiments, the plurality of nucleotide arms attached to a given core have the same type of nucleotide moieties, and wherein the types of nucleotide moieties comprise dATP, dGTP, dCTP, dTTP or dUTP.

[0032] In some embodiments, the plurality of multivalent molecules comprise one type of a multivalent molecule wherein each multivalent molecule in the plurality has the same type of nucleotide moiety selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP. In some embodiments, the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules each type having nucleotide moieties selected from a group consisting of dATP, dGTP, dCTP, dTTP and / or dUTP.

[0033] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore. In some embodiments, at least one multivalent molecule in the pluralityof multivalent molecules comprises one or more nucleotide moieties that are labeled with a fluorophore.

[0034] In some embodiments, individual nucleotides in the plurality of nucleotides in step (g) comprise an aromatic base, a five carbon sugar, and 1-10 phosphate groups.

[0035] In some embodiments, the plurality of nucleotides of step (g) comprise one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP, or comprise a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and / or dUTP.

[0036] In some embodiments, at least one of the nucleotides in the plurality of nucleotides in step (g) is labeled with a fluorophore. In some embodiments, the plurality of nucleotides in step (g) lack a fluorophore label.

[0037] In some embodiments, at least one of the nucleotides in the plurality of nucleotides of step (g) comprises a removable chain terminating moiety attached to the 3’ carbon position of the sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3 ’OH moiety on the sugar group.

[0038] In some embodiments, the method further comprises forming a plurality of binding complexes, comprising the steps: (a) binding a first sequencing primer, a first 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 moiety of the first multivalent molecule binds to the first polymerase; and (b) binding a second sequencing primer, a second polymerase, and the first multivalent molecule to a second portion of the immobilized concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule binds to the second polymerase, wherein the first and second binding complexes which include the same multivalent molecule form an avidity complex.

[0039] In some embodiments, the method further comprises (a) contacting the first plurality of polymerases and the plurality of sequencing primers with different portions of a concatemer template molecule to form at least first and second complexed polymerases on the concatemer template molecule; (b) contacting a plurality of multivalent molecules to the at least first and second complexed polymerases, under conditions suitable to bind a singlemultivalent molecule from the plurality to the first and second complexed polymerases, wherein at least a first nucleotide moiety of the single multivalent molecule is bound to the first complexed polymerase which includes a first sequencing primer hybridized to a first portion of the concatemer template molecule, thereby forming a first binding complex, and wherein at least a second nucleotide moiety of the single multivalent molecule is bound to the second complexed polymerase which includes a second sequencing primer hybridized to a second portion of the concatemer template molecule, thereby forming a second binding complex, and wherein the contacting is conducted under a condition suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide moieties in the first and second binding complexes, and wherein the first and second binding complexes which are bound to the same multivalent molecule form an avidity complex; (c) detecting the first and second binding complexes on the concatemer template molecule; and (d) identifying the first nucleotide moiety in the first binding complex thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide moiety in the second binding complex thereby determining the sequence of the second portion of the concatemer template molecule.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0041] FIG, 1 is a schematic of various exemplary configurations of multivalent molecules. Left (Class I): schematics of multivalent molecules having a “starburst” or “helter-skelter” configuration. Center (Class II): a schematic of a multivalent molecule having a dendrimer configuration. Right (Class III): a schematic of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. Nucleotide moieties are designated ‘N’, biotin is designated ‘B’, and streptavidin is designated ‘SA’.

[0042] FIG. 2 is a schematic of an exemplary multivalent molecule comprising a generic core attached to a plurality of nucleotide-arms.

[0043] FIG. 3 is a schematic of an exemplary multivalent molecule comprising a dendrimer core attached to a plurality of nucleotide-arms.

[0044] FIG. 4 shows a schematic of an exemplary multivalent molecule comprising a core attached to a plurality of nucleotide-arms, where the nucleotide arms comprise biotin, spacer, linker and a nucleotide moiety.

[0045] FIG. 5 is a schematic of an exemplary nucleotide-arm comprising a core attachment moiety, spacer, linker and nucleotide moiety.

[0046] FIG. 6 shows the chemical structure of an exemplary spacer (top), and the chemical structures of various exemplary linkers, including an 11 -atom Linker, 16-atom Linker, 23-atom Linker and an N3 Linker (bottom).

[0047] FIG. 7 shows the chemical structures of various exemplary linkers, including Linkers 1-9.

[0048] FIG. 8 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0049] FIG. 9 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0050] FIG. 10 shows the chemical structures of various exemplary linkers joined / attached to nucleotide moieties.

[0051] FIG. 11 shows the chemical structure of an exemplary biotinylated nucleotide-arm. In this example, the nucleotide moiety is connected to the linker via a propargyl amine attachment at the 5 position of a pyrimidine base or the 7 position of a purine base.

[0052] FIG. 12 is a schematic of an exemplary low binding support comprising a glass substrate and alternating layers of hydrophilic coatings which are covalently or non-covalently adhered to the glass, and which further comprises chemically-reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides). Alternatively, the support can be made of any material such as glass, plastic or a polymer material.

[0053] FIG. 13 is a schematic of a guanine tetrad (e.g., G-tetrad).

[0054] FIG. 14 is a schematic of an exemplary intramolecular G-quadruplex structure.

[0055] FIG. 15 is a schematic showing an exemplary' workflow for preparing a plurality of nucleic acid library molecules and sequencing the plurality of nucleic acid library molecules. For example, one end of an individual double-stranded polynucleotide is appended to at least one universal adaptor sequence, thereby generating a double-stranded library molecule.

[0056] FIG. 16 is a schematic showing an exemplary workflow for preparing a plurality of nucleic acid library molecules and sequencing the plurality of nucleic acid library molecules.For example, one end of an individual single-stranded polynucleotide is appended to at least one universal adaptor sequence, thereby generating a single-stranded library molecule.

[0057] FIG. 17 shows an embodiment of a method for generating polynucleotide fragments and appending universal adaptors using transposase-mediated tagmentation. The partially double-stranded oligonucleotide bound to the transposase comprises a transfer strand (e.g., long strand) comprising from 5’ to 3’, a universal sequence for binding a reverse sequencing primer (REV seq), a universal sequence for binding a forward sequencing primer (FWD Seq) and a transposon end sequence, for example the Mosaic End (ME) sequence, as well as a non-transfer strand (e.g., short strand) comprising a sequence complementary to the transposon end sequence.

[0058] FIG. 18 is a schematic showing an exemplary method for generating a plurality of polynucleotide fragments by conducting amplification using a plurality of random-sequence primers, strand displacing polymerases and a plurality of chain terminating nucleotides (solid rectangles).

[0059] FIG. 19 is a schematic showing an exemplary' method for generating a plurality of polynucleotide fragments by conducting amplification using a plurality of tailed randomsequence primers, strand displacing polymerases and a plurality of chain terminating nucleotides (solid rectangles).

[0060] FIG. 20 is a schematic of two exemplary methods for appending at least one universal adaptor sequence to one end of a polynucleotide fragment using a splint-mediated and ligation workflow.

[0061] FIG. 21 is a schematic of an exemplary method to append at least one universal adaptor sequence to one end of a double-stranded polynucleotide fragment using a Moloney Murine Leukemia Virus MMLV (MMLV) reverse transcriptase (MMLV-rt) enzyme and guide adaptor oligonucleotides (GAOs). In FIG. 21 the Xs indicate at least one universal adaptor sequence.

[0062] FIG. 22 is a schematic of an exemplary method to append at least one universal adaptor sequence to one end of a single-stranded polynucleotide fragment using a MML V reverse transcriptase enzyme and guide adaptor oligonucleotides (GAOs). In FIG. 22, the Xs indicate at least one universal adaptor sequence.

[0063] FIGS. 23A-23B show a schematic of an exemplary attenuator-mediated workflow. FIG. 23A is a schematic showing an exemplary method to append at least one universal adaptor sequence to one end of a single-stranded polynucleotide (e.g., ssDNA) fragment using a non-template tailing enzyme and a plurality of double-stranded (ds) attenuator-adaptor molecules each having a 3’ overhang end. The non-template tailing enzyme adds a tail to the 3’ end of the single-stranded polynucleotide. In FIG, 23B, the 3’ non-template tail (CCCCCC) is hybridized to the double-stranded attenuator-adaptor molecule.

[0064] FIGS, 24A-24B show schematics of an exemplary attenuator-mediated workflow. FIG. 24A is a schematic of an exemplary method to append at least one universal adaptor sequence to one end of a single-stranded polynucleotide (e.g., ssDNA) fragment using a non-template tailing enzyme and a plurality of single- stranded (ss) attenuator-adaptor molecules. The non-template tailing enzyme adds a tail to the 3’ end of the single-stranded polynucleotide. In FIG. 24B, the 3’ non-template tail (CCCCCC) is hybridized to the single-stranded attenuator-adaptor molecule.

[0065] FIG, 25 is a schematic of an exemplary method for circularizing a linear library molecule. One end of the linear library molecule includes a homopolymer-A sequence and at least one uracil base which is cleaved with a cleaving reagent (e.g., USER II) comprising an Antarctic Thermolabile Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease III.

[0066] FIG. 26 is a schematic of an exemplary method for circularizing a linear library molecule. One end of the linear library molecule includes a homopolymer-A sequence and at least one uracil base which can form a 5’ flap structure when the linear library molecule is hybridized to a splint oligonucleotide.

[0067] FIG, 27A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence, and (ii) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule from Part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0068] FIG, 27B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single- stranded linear library molecule comprising, in a 5' to 3’ direction, (i) an insert sequence, and (ii) a universal sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule from Part (i) with a soluble splint oligonucleotide and a target-specific bait / probe.Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0069] FIG. 28A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a forward sequencing primer (FWD seq), and (ii) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0070] FIG. 28B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, and (ii) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary' a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0071] FIG. 29A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows a single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (i) a universal sequence for binding a forward sequencing primer (FWD seq), and (ii) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a targetspecific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a targetspecific bait / splint capture primer.

[0072] FIG. 29B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) aninsert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), and (iii) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a targetspecific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a targetspecific bait / splint capture primer.

[0073] FIG. 30A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) is a schematic of an exemplary single-stranded linear library’ molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), (iii) a random sequence (nnnn) with a sample index sequence, and (iv) an insert sequence. Part, (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0074] FIG. 30B is a series of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary’ single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iv) a random sequence (nnnn) with a sample index sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0075] FIG. 31A is a series of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a sample index sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii ) a universal sequence for binding a forward sequencing primer (FWD seq),and (iv) an insert sequence. Part (ii) shows an exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0076] FIG. 31B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a sample index sequence, (iii) a universal sequence for binding a reverse sequencing primer (REV seq), and (iv) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0077] FIG. 32A is a series of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a second sample index sequence (Index-2), (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a forward sequencing primer (FWD seq), (iv) a random sequence (nnnn) with a first sample index sequence (Index-1), and (v) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0078] FIG. 32B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a second sample index sequence (Index-2), (iii) a universal sequence for binding a reverse sequencing primer (REV seq), (iv) a universal sequence for binding a forward sequencing primer (FWD seq), and (v) a random sequence (nnnn) with a first sample index sequence (Index-1). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a solublesplint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule with a target-specific bait / splint capture primer.

[0079] FIG. 33A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a pinning primer (pinning pbs), (iii) a universal sequence for binding a foiward sequencing primer (FWD seq), (iv) a random sequence (nnnn) with a sample index sequence, and (v) an insert sequence. Part (ii) shows an exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0080] FIG. 33B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a pinning primer (pinning pbs), (iv) a universal sequence for binding a forward sequencing primer (FWD seq), and (v) a random sequence (nnnn) with a sample index sequence. Part (ii) shows an exemplary' method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0081] FIG. 34A is a series of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a second sample index sequence (Index-2), (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a pinning primer (pinning pbs), (iv) a universal sequence for binding a forward sequencing primer (FWD seq), (v) a random sequence (nnnn) with a first sample index sequence (Index- 1), and (vi) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splintoligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0082] FIG. 34B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii ) a second sample index sequence (Index-2), (iii) a universal sequence for binding a reverse sequencing primer (REV seq), (iv) a universal sequence for binding a pinning primer (pinning pbs), (v) a universal sequence for binding a forward sequencing primer (FWD seq), and (vi) a random sequence (nnnn) with a first sample index sequence (Index-1), Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0083] FIG. 35A is a series of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a random sequence (nnnn) with a first sample index sequence (Index-1), (iii) a universal sequence for binding a pinning primer (pinning pbs), (iv) a second sample index sequence (Index-2), (v) a universal sequence for binding a forward sequencing primer (FWD seq), and (vi) an insert sequence. Part (ii) shows an exemplary’ method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii ) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer.

[0084] FIG. 35B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary’ single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a random sequence (nnnn) with a first sample index sequence (Index- 1), (iv) a universal sequence for binding a pinning primer (pinning pbs), (v) a second sample index sequence (Index-2), and (vi) a universal sequence for binding a forward sequencing primer (FWD seq).Part (ii) shows an exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and a target-specific bait / probe. Part (iii) shows a further exemplary' method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule with a target-specific bait / splint capture primer.

[0085] FIG. 36A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iii) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) Shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the immobilized capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0086] FIG. 36B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary’ single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), and (iii) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0087] FIG. 37A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) auniversal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (iii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iv) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii ) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the immobilized capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0088] FIG. 37B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), and (iv) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii ) or (iii).

[0089] FIG. 38A is a series of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), (iii) a random sequence (nnnn) with a sample index sequence, and (iv) an insert sequence. Part, (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to acoating on the support. Part (iii) shows a further exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the immobilized capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0090] FIG. 38B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (iii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iv) a random sequence (nnnn) with a sample index sequence. Part (ii) shows an exemplary’ method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0091] FIG. 39A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (iii) a universal sequence for binding a forward sequencing primer (FWD seq), (iv) a random sequence (nnnn) with a sample index sequence, and (v) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the immobilized capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii ).

[0092] FIG. 39B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (iv) a universal sequence for binding a forward sequencing primer (FWD seq), and (v) a random sequence (nnnn) with a sample index sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0093] FIG. 40A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a second sample index sequence (Index-2), (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (iv) a universal sequence for binding a forward sequencing primer (FWD seq), (v) a random sequence (nnnn) with a first sample index sequence (Index- 1), and (vi) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5' end of the immobilized capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii),

[0094] FIG. 40B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) aninsert sequence, (ii) a second sample index sequence (Index-2), (iii) a universal sequence for binding a reverse sequencing primer (REV seq), (iv) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (v) a universal sequence for binding a forward sequencing primer (FWD seq), and (vi) a random sequence (nnnn) with a first sample index sequence (Index- 1). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0095] FIG. 41A is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a random sequence (nnnn) with a first sample index sequence (Index- 1), (iii ) a universal sequence for binding a capture primer (Capture primer binding site [pbs]), (iv) a universal sequence for binding a pinning primer (pinning pbs), (v) a second sample index sequence (Index-2), (vi) a universal sequence for binding a forward sequencing primer (FWD seq), and (vii) an insert sequence. Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii ) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the immobilized capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0096] FIG. 41B is a series of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a random sequence (nnnn) with a first sample index sequence (Index- 1), (iv) a universalsequence for binding a capture primer (Capture primer binding site [pbs]), (v) a universal sequence for binding a pinning primer (pinning pbs), (vi) a second sample index sequence (Index-2), and (vii) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a soluble splint oligonucleotide and an immobilized capture primer. The 5’ end of the capture primer can be immobilized to a support or immobilized to a coating on the support. Part (iii) shows a further exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii) or (iii).

[0097] FIG. 42A is a pair of schematics showing exemplary methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iii) an insert sequence. Part ii) shows an exemplary' method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii).

[0098] FIG. 42B is a pair of schematics showing exemplary' methods for generating an open circle library molecule from a single-stranded linear library molecule. Part (i) shows an exemplary' single- stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), and (iii) a universal sequence for binding a forward sequencing primer (FWD seq). Part (ii) shows an exemplary method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with an immobilized splint capture primer. The 5’ end of the immobilized splint capture primer can be immobilized to a support or immobilized to a coating on the support. For the sake of simplicity, the support is not shown in Part (ii).

[0099] FIG. 43A is a schematic showing an exemplary method for generating a capture support with at least one pinning primer.

[0100] FIG. 43B is a schematic showing an exemplary single-stranded linear library molecule which comprises at least one universal adaptor sequence and an insert sequence.The linear library molecule shown in FIG. 43B has a 5’ to 3’ orientation and comprises: (i) at least one universal adaptor sequence, and (ii) an insert sequence. The exemplary at least one universal adaptor sequence depicted in FIG. 43B comprises a universal sequence for binding a reverse sequencing primer (REV seq), a universal sequence for binding a forward sequencing primer (FWD seq), and a random sequence (nnnn) with a sample index sequence. The linear library molecule can hybridize to a target-specific bait / probe and a soluble splint oligonucleotide. Various embodiments of soluble splint oligonucleotides are shown below.

[0101] FIG. 43C is a schematic showing an exemplary method for contacting a linear library molecule with a soluble splint oligonucleotide (left) and a target-specific bait / probe (right).

[0102] FIG. 43D is a schematic showing an exemplary method for contacting the captured library-splint complex (FIG. 43D, left) with a capture support thereby immobilizing the captured library-splint complex (FIG. 43D, right).

[0103] FIG. 43E is a schematic showing an exemplary method for generating an immobilized circle-bait complex and conducting rolling circle amplification reaction (RCA) to generate an immobilized concatemer template molecule.

[0104] FIG. 43F is a schematic showing an exemplary' capture support having an immobilized pinning primer that hybridizes to at least a portion of an immobilized single-stranded (ss) concatemer template molecule which is joined to an immobilized target-specific bait / probe.

[0105] FIGS. 44A and 44B depict an exemplary’ method of generating an immobilized concatemer template molecule from a linear library molecule. FIG. 44A shows an exemplary method for hybridizing and circularizing a linear library molecule using an immobilized splint capture primer that is immobilized to a support. FIG. 44B shows an exemplary method for generating a concatemer template molecule immobilized to the support by conducting rolling circle amplification.

[0106] FIG. 45 shows an exemplary support with an immobilized splint capture primer and a pinning primer immobilized thereon, and a concatemer template molecule. The support can be coated with at least one layer of a hydrophilic polymer. A plurality of immobilized splint capture primers and a plurality of pinning primers can be embedded and immobilized to the hydrophilic polymer coating. The concatemer template molecule can be joined to the immobilized splint capture primer. The pinning primer can hybridize to at least a portion of the concatemer template molecule.

[0107] FIG. 46 is a schematic showing an exemplary method for generating an immobilized concatemer template molecule and conducting pairwise sequencing. A covalently closed circular library molecule can be contacted with a capture support. The covalently closed circular library molecule can be hybridized to a target-specific bait / probe having an affinity moiety. The capture support can comprise a hydrophilic polymer coating embedded with a plurality of receptor moieties wherein individual receptor moieties can bind an affinity moiety. The covalently closed circular library molecule can be contacted with a capture support thereby immobilizing the covalently closed circular library molecule to the capture support. The target-specific bait / probe, which is hybridized to the covalently closed circular library molecule, can be used to initiate rolling circle amplification to generate an immobilized concatemer template molecule. The immobilized concatemer template molecule can be subjected single pass sequencing or pairwise sequencing. Exemplary methods for conducting pairwise sequencing are depicted in schematics of FIGS. 47-54.

[0108] FIG. 47 is a schematic showing an exemplary on-support rolling circle amplification reaction using a covalently closed circular library molecule, the immobilized target-specific bait-probe shown in FIG. 46, and a mixture of nucleotides including nucleotides having a scissile moiety that can be cleaved to generate an abasic site. The rolling circle amplification reaction generates an immobilized single-stranded concatemer template molecule having at least one nucleotide with a scissile moiety which can be cleaved to generate an abasic site in the immobilized concatemer template molecule. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustration purposes. The skilled artisan will appreciate that many other arrangements are possible.

[0109] FIG. 48 is a schematic showing an exemplary single-stranded concatemer template molecule immobilized to an immobilized target-specific bait-probe. The immobilized concatemer template molecule comprises at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the immobilized concatemer template molecule. The immobilized concatemer template molecule can be generated by conducting an on-support rolling circle amplification reaction. The arrangement of the various primer binding sequences is for illustration purposes. The skilled artisan will appreciate that many other arrangements are possible.

[0110] FIG. 49 is a schematic showing an exemplary forward sequencing reaction conducted on the immobilized concatemer template molecule shown in FIG. 48. The forward sequencing reaction can be conducted with a plurality of soluble forward sequencing primersand generates a plurality of extended forward sequencing primer strands. The immobilized concatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon.

[0111] FIG. 50 is a schematic showing an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer thereby generating a forward extension strand.

[0112] FIG. 51 is a schematic showing an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a soluble forward sequencing primer thereby generating a forward extension strand.

[0113] FIG. 52 is a schematic showing an exemplary method for generating abasic sites in the immobilized single-stranded concatemer template molecules at the nucleotides having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gapcontaining concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized target-specific baits / probes.

[0114] FIG. 53 is a schematic showing an exemplary retained forward extension strand after removal of the gap-containing concatemer template molecule shown in FIG. 52.

[0115] FIG. 54 is a schematic showing an exemplary reverse sequencing reaction conducted on the retained forward extension strand shown in FIG. 53. The reverse sequencing reaction can be conducted with a plurality of soluble reverse sequencing primers. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the immobilized target-specific bait / probe. Therefore, the extended reverse sequencing primer strands are not immobilized to the support. For the sake of simplicity, FIGS. 48-52 show an exemplary immobilized concatemer template molecule with two copies of the sequence of interest and various universal primer binding sites. The skilled artisan will appreciate that the immobilized concatemer template molecule can include three or more tandem copies containing the sequence of interest and various universal primer binding sites.

[0116] FIG. 55 is a schematic showing an exemplary method for generating an immobilized concatemer template molecule and conducting pairwise sequencing. A covalently closed circular library molecule can be contacted with an immobilized splint capture primer that is immobilized on a support. The immobilized splint capture primer can comprise a 5’ portion that can hybridize with at least a portion of a universal adaptor sequence of the covalently closed circular library molecule (e g., a universal sequence forbinding a reverse sequencing primer (REV seq)). The 3’ portion of the immobilized splint capture primer can comprise a random sequence (NNN) that can hybridize with at least a portion of the insert sequence of the covalently closed circular library molecule. The support can comprise at least one layer of a low binding hydrophilic polymer coating comprising a plurality of immobilized splint capture primers and a plurality of immobilized pinning primers (not shown). The covalently closed circular library molecule can be contacted with the immobilized splint capture primer, thereby immobilizing the covalently closed circular library molecule to the support. The immobilized splint capture primer, which is hybridized to the covalently closed circular library molecule, can be used to initiate rolling circle amplification to generate an immobilized concatemer template molecule. The immobilized concatemer template molecule can be subjected single pass sequencing or pairwise sequencing. In some embodiments, methods for conducting pairwise sequencing are depicted in FIGS. 56-63.

[0117] FIG. 56 shows an exemplary on-support rolling circle amplification reaction using a covalently closed circular library molecule, the immobilized splint capture primer shown in FIG. 55, and a mixture of nucleotides including nucleotides having a scissile moiety that can be cleaved to generate an abasic site. The rolling circle amplification reaction generates an immobilized single-stranded concatemer template molecule having at least one nucleotide with a scissile moiety which can be cleaved to generate an abasic site in the immobilized concatemer template mol ecule. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustration purposes. The skilled artisan will appreciate that many other arrangements are possible.

[0118] FIG. 57 shows an exemplary single-stranded concatemer template molecule joined to an immobilized splint capture primer. The immobilized concatemer template molecule comprises at least one nucleotide having a scissile moiety that can be cleaved to generate an abasic site in the immobilized concatemer template molecule. The immobilized concatemer template molecule can be generated by conducting an on-support rolling circle amplification reaction. The arrangement of the various primer binding sequences is for illustration purposes. The skilled artisan will appreciate that many other arrangements are possible.

[0119] FIG. 58 shows an exemplary' forward sequencing reaction conducted on the immobilized concatemer template molecule shown in FIG. 57. The forward sequencing reaction can be conducted with a plurality of soluble forward sequencing primers and generates a plurality of extended forward sequencing primer strands. The immobilizedconcatemer template molecule can have two or more extended forward sequencing primer strands hybridized thereon.

[0120] FIG. 59 shows an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a strand displacing polymerase in the absence of a soluble primer thereby generating a forward extension strand.

[0121] FIG. 60 shows an exemplary method for replacing the extended forward sequencing primer strands by conducting a primer extension reaction with a soluble forward sequencing primer thereby generating a forward extension strand.

[0122] FIG. 61 shows an exemplary method for generating abasic sites in the immobilized single-stranded concatemer template molecules at the nucleotides having the scissile moiety and generating gaps at the abasic sites to generate a plurality of gapcontaining concatemer template molecules while retaining the plurality of forward extension strands.

[0123] FIG. 62 shows an exemplary retained forward extension strand after removal of the gap-containing concatemer template molecule shown in FIG. 61.

[0124] FIG. 63 shows an exemplary reverse sequencing reaction conducted on the retained forward extension strand shown in FIG. 62. The reverse sequencing reaction can be conducted with a plurality of soluble reverse sequencing primers. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereon. The extended reverse sequencing primer strands are not hybridized to the immobilized splint capture primer. Therefore, the extended reverse sequencing primer strands are not immobilized to the support. For the sake of simplicity, FIGS. 56-63 show an exemplary immobilized concatemer template molecule with two copies of the sequence of interest and various universal primer binding sites. The skilled artisan will appreciate that the immobilized concatemer template molecule can include three or more tandem copies containing the sequence of interest and various universal primer binding sites.

[0125] FIG. 64 is a graph showing the nucleotide base diversity of a right sample index sequence including universal right sample index and the 3-mer random sequence (NNN). The graph shows a nucleotide diversity of the 3-mer random sequence (NNN) of approximately 30% for A and T base calls, and approximately 20% for C and G base calls.

[0126] FIG. 65 is a graph showing the nucleotide base diversity of a left sample index sequence which lacks a 3-mer random sequence (NNN). The graph shows a nucleotide diversity of approximately 40% for A and T base calls, approximately 15% for C base calls, and approximately 5% for G base calls.

[0127] FIG. 66 is a schematic showing four exemplary' DNA tagmentation workflows for preparing a plurality of nucleic acid library molecules and sequencing the plurality of library molecules. FIG. 66 shows workflows (A), (B), (C) and (D). Both ends of tagmented DNA can be appended to at least one universal adaptor sequence thereby generating a plurality of library molecules. Some of the workflow steps can be conducted in-solution (above the dashed line) and other steps can be conducted on a capture support (below the dashed line).DETAILED DESCRIPTIONDefinitions

[0128] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0129] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of molecular biology, nucleic acid chemistry', protein chemistry', genetics, microbiology, transgenic cell production, and hybridization described herein are those well-known and commonly used in the art.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 that are cited and discussed throughout the instant specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0130] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.

[0131] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[0132] It is understood the use of the alternative term (e.g., “or”) is taken to mean either one or both or any combination thereof of the alternatives.

[0133] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include: “A and B”; “A or B”; “A” (A alone); and “B” (B alone). In a similar manner, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and B”; “B and C”; “A and C”; “A” (A alone); “B” (B alone); and “C” (C alone).

[0134] As used herein and in the appended claims, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and / or “consisting essentially of” are also provided.

[0135] As used herein, the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg.Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. Also, where ranges and / or subranges of values are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0136] The term “polymerase” and its variants, as used herein, comprises an enzyme comprising a domain that binds a nucleotide (or nucleoside) where the polymerase can form a complex having a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities including, but not limited to, base analog detection activities, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. A polymerase can be any enzyme that can catalyze polymerization of nucleotides (including analogs thereof) into anucleic acid strand. Typically but not necessarily such nucleotide polymerization can occur in a template-dependent fashion. Typically, a polymerase comprises one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization can occur.Polymerases can include other enzymatic activities, such as for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacing activity. A polymerase can include, without limitation, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze nucleotide polymerization (e.g., catalytically active fragment). The polymerase includes catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases, and other enzymes comprising a nucleotide binding domain. A polymerase can be isolated from a cell, or generated using recombinant DNA technology or chemical synthesis methods. A polymerase can be expressed in prokaryote, eukaryote, viral, or phage organisms. A polymerase can be a post-translationally modified protein or fragment thereof. A polymerase can be derived from a prokaryote, eukaryote, virus or phage. A polymerase comprises DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0137] As used herein, the term “strand displacing” refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acids and synthesize a new strand in a template-based manner. Strand displacing polymerases displace a complementary strand from a template strand and catalyze new strand synthesis. Strand displacing polymerases include mesophilic and thermophilic polymerases. Strand displacing polymerases include wild type enzymes, and variants including exonuclease minus mutants, mutant versions, chimeric enzymes and truncated enzymes. Examples of strand displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (exo-), Bea DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase and KOD DNA polymerase. The phi29 DNA polymerase can be wild type phi29 DNA polymerase (e.g., MagniPhi® from Expedeon®), or variant EquiPhi29TMDNA polymerase (e.g,, from Thermo Fisher Scientific®), or chimeric QualiPhi® DNA polymerase (e.g., from 4basebio).

[0138] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides andare 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 the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids comprise polymers of nucleotides, where the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids comprise naturally-occurring intemucleosidic linkages, for example phosphdiester linkages. Nucleic acids can lack a phosphate group. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides.

[0139] The term “operably linked” and “operably joined” or related terms as used herein refers to juxtaposition of components. The juxtapositioned components can be linked together covalently. For example, two nucleic acid components can be enzymatically ligated together where the linkage that joins together the two components comprises phosphodiester linkage. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function on a second nucleic acid component. For example, linkage 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 a nucleic acid sequence of interest) can be ligated to a vector where the linkage permits expression or functioning of the transgene sequence contained in the vector. In some embodiments, a transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, enhancer, transcription and / or translation initiation sequence, transcription and / or translation termination sequence, polypeptide secretion signal sequences, and the like. In some embodiments, the host cell regulatory sequence controls expression of the level, timing and / or location of the transgene. However, the skilled artisan will appreciate that two components need not be directly linked in order to be considered operably linked.

[0140] The terms “linked”, “joined”, “attached”, “appended” and variants thereof comprise any type of fusion, bond, adherence or association between any combination of compounds or molecules that is of sufficient stability to withstand use in a particular procedure. The procedure can include but are not limited to: nucleotide binding; nucleotideincorporation; de-blocking (e.g., removal of chain-terminating moiety); washing; removing; flowing; detecting; imaging and / or identifying. Such linkage can comprise, for example, covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. Such linkage can occur intramolecularly, for example linking together the ends of a single-stranded or double-stranded linear nucleic acid molecule to form a circular molecule. Alternatively, such linkage can occur between a combination of different molecules, or between a molecule and a non-molecule, including but not limited to: linkage between a nucleic acid molecule and a solid surface; linkage between a protein and a detectable reporter moiety; linkage between a nucleotide and detectable reporter moiety; and the like. Some examples of linkages 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).

[0141] The term “primer” and related terms used herein refers to an oligonucleotide that is capable of hybridizing with a DNA and / or RNA polynucleotide template to form a duplex molecule. Primers can comprise natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers may have any length, but typically range from 4-50 nucleotides. A typical primer comprises a 5’ end and 3’ end. The 3’ end of the primer can include a 3’ OH moiety which serves as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3’ end of the primer can lack a 3’ OH moiety, or can include a terminal 3’ blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide, or more than one nucleotide, along the length of the primer can be labeled with a detectable reporter moiety. A primer can be in solution (e g., a soluble primer) or can be immobilized to a support (e.g., a capture primer).

[0142] The term “template nucleic acid”, “template polynucleotide”, “target nucleic acid” “target polynucleotide”, “template strand” and other variations refer to a nucleic acid strand that serves as the basis nucleic acid molecule for any of the methods described herein. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from a naturally-occurring source, recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or other forms. The template nucleic acids can include an insert portion having an insertsequence, sometimes also referred to herein as a “target sequence” or similar. The template nucleic acids can also include at least one adaptor sequence. The insert portion can be isolated in any form, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotides, whole genomic DNA, obtained from fresh frozen paraffin embedded tissue, needle biopsies, circulating tumor cells, cell free circulating DNA, or any type of nucleic acid library. The insert portion can be isolated from any source including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic samples, perspiration, semen, environmental samples, culture samples, or synthesized nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. The insert portion can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and composition analysis.

[0143] The term “adaptor” and related terms refers to oligonucleotides that can be operably linked to a target polynucleotide (e.g. “insert sequence”), where the adaptor confers a function to the co-joined adaptor-target molecule. Adaptors can comprise DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adaptors can include at least one ribonucleoside residue. Adaptors can be single-stranded, double-stranded, or have single-stranded and / or double-stranded portions. Adaptors can be configured to be linear, stem-looped, hairpin, or Y-shaped forms. Adaptors can be any length, including 4-100 nucleotides or longer. Adaptors 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 adaptor, or one strand of a double-stranded adaptor, can have a 5’ phosphate group or lack a 5’ phosphate group.Adaptors can include a 5’ tail that does not hybridize to a target polynucleotide (e.g., tailed adaptor), or adaptors can be non-tailed. An adaptor 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., soluble or immobilized capture primers).Adaptors can include a random sequence or degenerate sequence. Adaptors can include at least one inosine residue. Adaptors can include at least one phosphorothioate, phosphorothiolate and / or phosphoramidate linkage. Adaptors can include a barcode sequencewhich can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. Adaptors can include a unique identification sequence (e.g., unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify a nucleic acid molecule to which the adaptor is appended. In some embodiments, a unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false-positive variant calls and / or increase sensitivity of variant detection.Adaptors can include at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from a group consisting of type I, type II, type III, type IV, type Hs and type IIB.

[0144] In some embodiments, any of the amplification primer sequences, sequencing primer sequences, capture primer sequences, target capture sequences, circularization anchor sequences, sample barcode sequences, spatial barcode sequences, or anchor region sequences can be about 3-50 nucleotides in length, about 5-40 nucleotides in length, about 5-25 nucleotides in length, about 3-10 nucleotides in length, about 3-7 nucleotides in length, about 4-8 nucleotides in length, or about 10-20 nucleotides in length.

[0145] The term “universal sequence” and related terms refers to a sequence in a nucleic acid molecule that is common among two or more nucleic acid molecules. For example, an adaptor having a universal sequence can be operably joined to a plurality of polynucleotides so that the population of co-joined molecules carry the same universal sequence. Examples of universal sequences include an amplification primer sequence, a sequencing primer sequence or a capture primer sequence (e.g., soluble or immobilized capture primers). The skilled artisan will appreciate that adaptors can contain a mixture of both universal sequences, such as a common universal amplification sequence, as well as non-universal sequences that vary between adaptors in a plurality of adaptors (for example, an index sequence).

[0146] When used in reference to nucleic acid molecules, the terms “hybridize” or “hybridizing” or “hybridization” or other related terms refers to hydrogen bonding between two different nucleic acids to form a duplex 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 duplex region. Hybridization can comprise Watson-Crick or Hoogstein binding to form a duplex double-stranded 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 wholly complementary, or partially complementary. Complementary nucleic acid strands need not hybridize with each other across their entire length. The complementary base pairing can be the standard A- T or C-G base pairing, or canbe other forms of base-pairing interactions. Duplex nucleic acids can include mismatched base-paired nucleotides.

[0147] When used in reference to nucleic acids, the terms ‘’extend”, “extending”, “extension” and other variants, refers to incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises polymerization of one or more nucleotides into the terminal 3’ OH end of a nucleic acid strand, resulting in extension of the nucleic acid strand. Nucleotide incorporation can be conducted with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent fashion. Any suitable method of extending a nucleic acid molecule may be used, including primer extension catalyzed by a DNA polymerase or RNA polymerase.

[0148] The term “nucleotides” and related terms refers to a molecule comprising an aromatic base, a five carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Canonical or non-canonical nucleotides are consistent with use of the term. The phosphate in some embodiments comprises a monophosphate, diphosphate, or triphosphate, or corresponding phosphate analog. The term “nucleoside” refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be non-labeled or labeled with a detectable reporter moiety.

[0149] Nucleotides (and nucleosides) typically comprise a hetero cyclic base including substituted or unsubstituted nitrogen-containing parent heteroaromatic ring which are commonly found in nucleic acids, including naturally-occurring, substituted, modified, or engineered variants, or analogs of the same. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoogstein hydrogen bonds with an appropriate complementary' base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-A2-isopentenyladenine (6iA), N6-A2-isopentenyl-2-m ethylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2 -thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-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, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methyl indole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additionalexemplary bases can be found in Fasman, 1989, in ‘’Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, CRC Press, Boca Raton, Fla.

[0150] Nucleotides (and nucleosides) typically comprise a sugar moiety, such as carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (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. Pat. No. 5,558,991). The sugar moiety comprises: ribosyl; 2'-deoxyribosyl; 3 '-deoxyribosyl; 2', 3 '-dideoxyribosyl; 2',3 '-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-mercaptoriboxyl; 2'-alkylthioribosyl; 3 '-alkoxyribosyl; 3 '-azidoribosyl; 3 '-aminoribosyl; 3 '-fluororibosyl; 3'-mercaptoriboxyl; 3 '-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0151] In some embodiments, nucleotides comprise a chain of one, two or three phosphorus atoms where the chain is typically attached to the 5’ carbon of the sugar moiety via an ester or phosphoramide linkage. 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 groups including O, S or BH3. In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoroamidite groups.

[0152] The term “reporter moiety”, “reporter moieties” or related terms refers to a compound that generates, or causes to generate, a detectable signal. A reporter moiety is sometimes called a “label”. Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. A reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other. It is well known to one skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selectedhaving spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. Reporter moieties can be linked (e.g., operably linked) to nucleotides, nucleosides, nucleic acids, enzymes (e.g., polymerases or reverse transcriptases), or support (e.g., surfaces).

[0153] A reporter moiety (or label) comprises a fluorescent label or a fluorophore.Exemplary fluorescent moieties which may serve as fluorescent labels or fluorophores include, but are not limited to fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, 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 hydrazine, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, A CE-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, pyridium based cyanine dyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes, imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BOOT, Europium chelates, Terbium chelates, Alexa Fluor® dyes, DyLight® dyes, Atto™ dyes, LightCycler® Red dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Green™dyes, WellRED dyes, IRD dyes, phycoerythrin 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 derivatives thereof, or any combination thereof. Cyanine dyes may exist in either sulfonated or non-sulfonated forms, and consist of two indolenin, benzo-indolium, pyridium, thiozolium, and / or quinolinium groups separated by apolymethine bridge between two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3, (which may comprise l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{ l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-l,3-dihydro-2H-indol -2-ylidene }prop-l-en-l-yl)-3,3-dimethyl-3H-indolium or l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-2-(3-{l-[6-(2,5-dioxopyrrolidin-l-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene}prop-l-en-l-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may comprise l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium or l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-2-((lE,3E)-5-((E)-l-(6-((2,5-dioxopyrrolidin-l-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)pent -l,3-dien-l-yl)-3,3-dimethyl-3H-indol-l-ium-5-sulfonate), and Cy7 (which may comprise l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium or l-(5-carboxypentyl)-2-[(lE,3E,5E,7Z)-7-(l-ethyl-5-sulfo-l,3-dihydro-2H-indol-2-ylidene)hepta-l,3,5-trien-l-yl]-3H-indolium-5-sulfonate), where “Cy” stands for 'cyanine', and the first digit identifies the number of carbon atoms between two indolenine groups. Cy2 which is an oxazole derivative rather than indolenin, and the benzo-derivatized Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule. Suitable dyes are described, for example, in U. S.Publication No. 2024-0240249, the contents of which are incorporated by reference in their entirety herein.

[0154] In some embodiments, the reporter moiety can be a fluorescence resonance energy transfer (FRET) pair, such that multiple classifications can be performed under a single excitation and imaging step. As used herein, FRET may comprise excitation exchange (Forster) transfers, or electron-exchange (Dexter) transfers.

[0155] When used in reference to nucleic acids, the terms “amplify”, “amplifying”, “amplification”, and other related terms include producing multiple copies of an original polynucleotide template molecule, where the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is the same as the template sequence. The copies can comprise a sequence that is identical to, or substantially identical to, a template sequence, or is identical to, or substantially identical to, a sequence that is complementary to the template sequence.

[0156] As used herein, “transposase” refers to an enzyme that is capable of binding a double-stranded DNA fragment called a transposon end to form a transpososome complex, where the transposase enzyme catalyzes insertion of the transposon end into a double-stranded target DNA using a “cut and paste” mechanism. The transposase enzyme can insert the transposon end into random sites in the target DNA, The transposase enzyme recognizes a transposon end having a specific sequence. The double-stranded transposon end comprises a transfer strand hybridized to a complementary non-transfer strand. Upon completion of the insertion reaction, the transposase covalently joins the transfer strand to one strand of the target DNA and the non-transfer strand is hybridized to the newly joined transfer strand. The non-transfer strand is not joined to the target DNA. The insert reaction generates a gap between the 3’ end of the cut target DNA and the 5’ end of the non-transfer strand (e.g., see FIG. 17).

[0157] Different transposase enzymes recognize and bind their specific transposon end having a specific sequence. For example, Tn5 transposase enzyme binds a transposon end where the transfer strand comprises the sequence 5 ’-AG AT GTGT ATA AG AG AC AG-3’ (SEQ ID NO:1) and the non-transfer strand comprises the sequence 5’-CTGTCTCTTATACACATCT-3’ (SEQ ID NO:2).

[0158] A recombinant transposon end comprises a transfer strand joined to additional sequences, including for example at least one universal adaptor sequence. The non-transfer strand optionally comprises additional sequences. The transposase can insert the recombinant transposon end into a target DNA to generate one DNA strand covalently joined to the transposon end and the universal adaptor sequence. The transposase enzyme can catalyze the insertion reaction in an in vitro transposition reaction. Exemplary transposases include the Tn5 transposase, the Tn7 transposase complex, the sleeping beauty transposase and the piggyBac transposase. The term “transposase” encompasses wild type transposases, as well as analogs or derivatives thereof, such as hyperactive mutants of wild type transposases.

[0159] As used herein, a “tagmentation reaction” refers to an in vitro enzymatic reaction in which a transposase is loaded with recombinant transposon ends to form transposomes which are contacted with double-strand target DNA. The transposase binds to and cuts the target DNA, and inserts the recombinant transposon ends at the breakpoints. The tagmentation reaction can produce a library of DNA fragments flanked at least on one side by a recombinant transposon end,

[0160] A “transposome” refers to a transposase that is bound to a double-stranded, or partially double-stranded, transposon end or recombinant transposon end. As shown in FIG.17, an exemplaty partially double-stranded recombinant transposon end that forms a part of the transposome can include a transfer strand, i.e. the strand that is physically joined to the target DNA during the transfer step, and a non-transfer strand. The transfer strand cancomprise the transfer end sequence, a short sequence derived from the inverted terminal repeat at the end of the transposon that is involved in the strand transfer step, as well as additional sequences to be appended to the end of the target DNA. The additional sequences can comprise binding sequences for forward or reverse sequencing primers, splint capture primers, pinning primers, sample index sequences, or any combination thereof. The nontransfer strand can include a sequence complementary to part or all of the non-transfer strand.

[0161] A “template-independent tailing enzyme” and similar terms refers to an enzyme that adds nucleotides to the end of a DNA or RNA molecule without using a complementary (template) strand to guide base incorporation. Exemplary template-independent tailing enzymes include a terminal deoxynucleotidyl transferase (TdT), an A. coli RNA-specific Poly(A) polymerase, an 5. pombe RNA-specific poly(U) polymerase or a yeast poly(a) polymerase.

[0162] “Flap endonuclease 1 (FEN1)” refers to an enzyme that recognizes and cleaves single-stranded DNA flaps that arise during DNA replication, long-patch base excision repair, and certain recombination processes. It is “structure-specific,” meaning it cuts DNA based on the shape of the molecule rather than its exact sequence.

[0163] A “pinning primer” refers to a primer immobilized to a support that comprises a sequence complementary to a sequence of a nucleic acid molecule that is the subject of one of the sequencing workflows described herein. The pinning primer binds to the nucleic acid molecule, “pinning” it to the support,

[0164] A “sample index sequence” and similar terms refers to a sequence that is appended to nucleic acids to be sequenced that varies between nucleic acids from different sources (i.e., different samples), but is the same for multiple nucleic acids from the same source. The sample index can be used to distinguish polynucleotides from different sample sources in a multiplex assay.

[0165] A “unique molecular identifier” or UMI refers to a sequence that is appended to nucleic acids to be sequenced that varies between individual nucleic acids, for example nucleic acids from the same sample, and can be used to identify individual nucleic acids.

[0166] A “batch specific sequencing primer” and similar terms refers to a primer with a sequence that is tailored to sequence a specific batch of samples or nucleic acid library derived therefrom. In general, if a sequencing library is indexed with sequences unique to a batch, the sequencing primer is typically also designed to match the batch, and to anneal to sequencing primer binding sites specific to the batch.

[0167] The term “support” as used herein refers to a substrate that is designed for deposition of biological molecules or biological samples for assays and / or analyses.Examples of biological molecules to be deposited onto a support include nucleic acids (e.g., DNA, RNA), polypeptides, saccharides, lipids, a single cell or multiple cells. Examples of biological samples include but are not limited to saliva, phlegm, mucus, blood, plasma, serum, urine, stool, sweat, tears and fluids from tissues or organs. Suitable supports are described, for example, in WO 2020 / 102594, the contents of which are incorporated by reference in their entirety herein.

[0168] The support can be 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 comprising a capillary / or interior surface of a capillary.

[0169] The surface of the support can be substantially smooth. In some embodiments, the support can be regularly or irregularly textured, including bumps, etched, pores, three-dimensional scaffolds, or any combination thereof.

[0170] The support can comprise a bead having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disc-shaped, rod-like, conical, triangular, cubical, polygonal, tubular or wire-like.

[0171] The support can be fabricated from any material, including but not limited to glass, fused-silica, silicon, a polymer (e.g., polystyrene (PS), niacroporous polystyrene (MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefin polymers (COP), cyclic olefin copolymers (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0172] The support can have a plurality (e.g., two or more) of nucleic acid templates immobilized thereon. The plurality of immobilized nucleic acid templates can have the same sequence or have different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a different site on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized to a site on the support.

[0173] The term “array” refers to a support comprising a plurality of sites located at predetermined locations on the support to form an array of sites. The sites can be discrete and separated by interstitial regions. In some embodiments, the pre-determined sites on thesupport can be arranged in one dimension in a row or a column, or arranged in two dimensions in rows and columns. In some embodiments, the plurality of pre-determined sites is arranged on the support in an organized fashion. In some embodiments, the plurality of pre-determined sites is arranged in any organized pattern, including rectilinear, hexagonal patterns, grid patterns, patterns having reflective symmetry, patterns having rotational symmetry, or the like. The pitch between different pairs of sites can be that same or can vary'. In some embodiments, the support comprises at least 102sites, at least 103sites, at least 104sites, at least 105sites, at least 106sites, at least 107sites, at least 108sites, at least 109sites, at least 1010sites, at least 1011sites, at least 1012sites, at least 1013sites, at least 1014sites, at least 1015sites, or more, where the sites are located at pre-determined locations on the support. In some embodiments, a plurality of pre-determined sites on the support (e.g., 102-1013sites or more) are immobilized with nucleic acid templates to form a nucleic acid template array. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to the surface capture primer. In some embodiments, the nucleic acid templates that are immobilized at a plurality of pre-determined sites, for example immobilized at 102- 1013sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobilized nucleic acid clusters at the plurality of pre-determined sites. In some embodiments, individual immobilized nucleic acid clusters comprise linear clusters, or comprise single-stranded or double-stranded concatemers.

[0174] In some embodiments, a support compri sing a plurality of sites located at random locations on the support is referred to herein as a support having randomly located sites thereon. The location of the randomly located sites on the support are not pre-determined. The plurality of randomly-located sites is arranged on the support in a disordered and / or unpredictable fashion. In some embodiments, the support comprises at least 102sites, at least 103sites, at least 104sites, at least 105sites, at least 106sites, at least 107sites, at least 108sites, at least 109sites, at least 1010sites, at least 1011sites, at least 1012sites, at least 1013sites, at least 1014sites, at least 1015sites, or more, where the sites are randomly located on the support. In some embodiments, a plurality of randomly located sites on the support (e.g., 102- 1013sites or more) are immobilized with nucleic acid templates to form a support immobilized with nucleic acid templates. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites by hybridization to immobilized surface capture primers, or the nucleic acid templates are covalently attached to theimmobilized surface capture primers. In some embodiments, the nucleic acid templates that are immobilized at a plurality of randomly located sites, for example immobilized at 102-1013sites or more. In some embodiments, the immobilized nucleic acid templates are clonally-amplified to generate immobili zed nucleic acid clusters at the plurality of randomly located sites. In some embodiments, individual immobilized nucleic acid clusters (also referred to as “polonies”) comprise linear clusters, or comprise single-stranded or doublestranded concatemers.

[0175] In some embodiment, the plurality of immobilized surface capture primers on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and the like) onto the support so that the plurality of immobilized surface capture primers on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized surface capture primers can be used to conduct nucleic acid amplification reactions (e.g., RCA, MDA, PCR and bridge amplification) essentially simultaneously on the plurality of immobilized surface capture primers.

[0176] In some embodiment, the plurality of immobilized nucleic acid clusters on the support are in fluid communication with each other to permit flowing a solution of reagents (e.g., enzymes, nucleotides, divalent cations, and the like) onto the support so that the plurality of immobilized nucleic acid clusters on the support can be essentially simultaneously reacted with the reagents in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized nucleic acid clusters can be used to conduct nucleotide binding assays and / or conduct nucleotide polymerization reactions (e.g., primer extension or sequencing) essentially simultaneously on the plurality of immobilized nucleic acid clusters, and optionally to conduct detection and imaging for massively parallel sequencing.

[0177] When used in reference to immobilized enzymes, the term “immobilized” and related terms refer to enzymes (e.g., polymerases) that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support.

[0178] When used in reference to immobilized nucleic acids, the term “immobilized” and related terms refer to nucleic acid molecules that are attached to a support through covalent bond or non-covalent interaction, or attached to a coating on the support, or buried within a matrix formed by a coating on the support, where the nucleic acid molecules include surfacecapture primers, nucleic acid template molecules and extension products of capture primers. Extension products of capture primers includes nucleic acid concatemer template molecules (e.g., nucleic acid clusters).

[0179] In some embodiments, one or more nucleic acid template molecules are immobilized on the support, for example immobilized at the sites on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified. In some embodiments, the one or more nucleic acid templates are clonally-amplified off the support (e.g., in-solution) and then deposited onto the support and immobilized on the support. In some embodiments, the clonal amplification reaction of the one or more nucleic acid templates is conducted on the support resulting in immobilization on the support. In some embodiments, the one or more nucleic acid templates are clonally-amplified (e g., in solution or on the support) using a nucleic acid amplification reaction, including 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.

[0180] As used herein, the term “binding complex” refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or a nucleotide moiety of a multivalent molecule, where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotide or nucleotide moiety may or may not be bound to the 3’ end of the nucleic acid primer at a position that is opposite a complementary nucleotide in the nucleic acid template molecule. A “ternary complex” is an example of a binding complex which is formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide moiety of a multivalent molecule, where the free nucleotide or nucleotide moiety is bound to the 3’ end of the nucleic acid primer (as part of the nucleic acid duplex) at a position that is opposite a complementary nucleotide in the nucleic acid template molecule.

[0181] The term “persistence time” and related terms refers to the length of time that a binding complex, which is formed between the target nucleic acid, a primer, a polymerase, a conjugated or unconjugated nucleotide, remains stable without any binding component dissociates from the binding complex. The persi stence time is indicative of the stability of the binding complex and strength of the binding interactions. Persistence time can be measuredby observing the onset and / or duration of a binding complex, such as by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides may be present in a binding complex, thus allowing the signal from the label to be detected during the persistence time of the binding complex. One exemplary label is a fluorescent label.

[0182] As used herein, the term ‘‘soluble splint oligonucleotide” and related terms refers to an oligonucleotide comprising an anchor sequence at one end and a bridging sequence at the other end, and lacking an affinity moiety. In some embodiments, the anchor sequence is located at the 5’ or 3’ end of the soluble splint oligonucleotide. In some embodiments, the bridging sequence is located at the 5’ or 3’ end of the soluble splint oligonucleotide. In some embodiments, the anchor or bridging sequence comprises a random sequence (NNN) that can hybridize to at least a portion of an insert sequence of a single-stranded linear library molecule. In some embodiments, the anchor or bridging sequence comprises a sequence that can hybridize to at least a portion of a universal adaptor sequence of a single-stranded linear library molecule. In some embodiments, the anchor sequence can hybridize to a region at one end of the single-stranded linear library molecule, and the bridging sequence can hybridize to a region at the other end of the same single-stranded linear library molecule thereby forming an open circle library molecule have a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, a single-stranded circular library molecule can be generated by contacting a single- stranded linear library molecule with a soluble splint oligonucleotide to generate a single-stranded open circle library molecule having a nick. Exemplary soluble splint oligonucleotides are shown in FIGS, 27A-41B. In some embodiments, the ends of a soluble splint oligonucleotide can hybridize to the ends of a single-stranded linear library’ molecule comprising an insert sequence joined at one end to at least one universal adaptor sequence thereby forming an open circle library molecule having a nick.

[0183] As used herein, the term “target-specific bait / splint capture primer” and related terms refers to an oligonucleotide comprising a target- specific sequence at one end and an affinity moiety at the other end. In some embodiments, the affinity moiety of the targetspecific bait / splint capture primer can bind a receptor moiety on a capture support for immobilization to the capture support. In some embodiments, the target-specific sequence can be located near the 5’ or 3’ end of the target-specific bait / splint capture primer. In some embodiments, the target-specific sequence can hybridize with at least a portion of the insert region of a linear library molecule. In some embodiments, the affinity moiety can be located at the 5’ or 3’ of the target-specific bait / splint capture primer. In some embodiments, the endsof the target-specific bait / splint capture primers can hybridize to the ends of the single-stranded linear library molecule thereby forming an open circle library molecule have a nick. In some embodiments, a single-stranded circular library molecule can be generated by contacting a single-stranded linear library molecule with a target-specific bait / splint capture primer to generate a single-stranded open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. Exemplary target-specific bait / splint capture primers are shown in FIGS. 27A-35B. In some embodiments, the ends of a targetspecific bait / splint capture primer can hybridize to the ends of a single-stranded linear library molecule comprising an insert sequence joined at one end to at least one universal adaptor sequence thereby forming an open circle library molecule having a nick.

[0184] As used herein, the term “immobilized splint capture primers” and related terms refers to an oligonucleotide comprising a sequence at one end that can hybridize with at least a portion of universal adaptor sequence of an single- stranded linear library molecule and a random sequence (NNN) at the other end. The immobilized splint capture primer lacks an affinity moiety that can bind a receptor moiety on a capture support for immobilization to the capture support. In some embodiments, the adaptor-specific sequence can be located near the 5’ or 3’ end of the immobilized splint capture primer. In some embodiments, the random sequence ( NNN) can be located near the 5’ or 3’ end of the immobilized splint capture primer. In some embodiments, the adaptor-specific sequence at one end of the immobilized splint capture primer can hybridize with at least a portion of a universal adaptor sequence at one end of a linear library molecule, and the random sequence (NNN) at the other end of the immobilized splint capture primer can hybridize with at least a portion of the insert sequence at the other end of the same linear library molecule, thereby forming an open circle library’ molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, one end of the immobilized splint capture primer is immobilized to a support or a coating on the support via chemically-reactive functional groups that serve as attachment sites for the immobilized splint capture primer (e g., FIG. 12). In some embodiments, the 5’ end of the immobilized splint capture primer can be immobilized to the support or the coating on the support. Exemplary immobilized splint capture primer are shown in FIGS, 36A-41B. In some embodiments, the ends of an immobilized splint capture primer can hybridize to the ends of a single-stranded linear library molecule comprising an insert sequence joined at one end to at least one universal adaptor sequence thereby forming an open circle library molecule having a nick.

[0185] As used herein, the term “target-specific bait / probe” and related terms refers to an oligonucleotide comprising a target-specific sequence at one end and an affinity moiety at the other end. In some embodiments, the target-specific sequence can hybridize with at least a portion of the insert region of a single-stranded linear library molecule. In some embodiments, the affinity moiety of the target-specific bait / probe can bind a receptor moiety on a capture support for immobilization of the target-specific bait / probe to the capture support. In some embodiments, the target-specific sequence can be located near the 5’ or 3’ end of the target-specific bait / probe. In some embodiments, the affinity moiety can be located at the 5’ or 3’ of the target-specific bait / probe. In some embodiments, an open circle library molecule that is hybridized to a soluble splint oligonucleotide can be immobilized to a capture support by hybridization to a target-specific bait / probe. Exemplary target-specific baits / probes are shown in FIGS. 27A-35B. In some embodiments, a target-specific baits / probe hybridize to a single- stranded linear library molecule comprising an insert sequence joined at one end to at least one universal adaptor sequence.

[0186] As used herein, the term “immobilized capture primer” and related terms refers to an oligonucleotide comprising a sequence that can hybridize with at least a portion of a universal adaptor sequence of a single-stranded linear library molecule. The immobilized capture primer lacks an affinity moiety. In some embodiments, one end of the immobilized capture primer is immobilized to a support or a coating on the support via chemically-reactive functional groups that serve as attachment sites for the immobilized capture primer (e g., FIG.12). In some embodiments, the adaptor-specific sequence can be located near the 5’ end, the 3’ end or an internal portion of the immobili zed capture primer. In some embodiments, the 5’ end of the immobilized capture primer can be immobilized to the support or the coating on the support. In some embodiments, an open circle library molecule that is hybridized to a soluble splint oligonucleotide can be immobilized to a support by hybridization to an immobilized capture primer. Exemplary immobilized capture primers are shown in FIGS. 36A-41B. In some embodiments, an immobilized capture primer can hybridize to a single-stranded linear library molecule comprising an insert sequence joined at one end to at least one universal adaptor sequence.

[0187] As used herein, the term “polyN splint capture primer” and related terms refers to an oligonucleotide comprising an anchor sequence at one end, a bridging sequence at the other end, and an affinity moiety. In some embodiments, the anchor sequence is located at a 5’ or a 3’ end of the polyN splint capture primer. In some embodiments, the bridging sequence is located at a 3’ or a 5’ end of the polyN splint capture primer. The anchorsequence can hybridize to a universal adaptor sequence of a single-stranded linear DNA library molecule, while the bridging sequence can hybridize to a template-independent homopolymer tail of the same single-stranded linear DNA library molecule. In some embodiments, the affinity moiety that can bind to a receptor moiety of the capture support for immobilization of the polyN splint capture primer. In some embodiments, the 3’ end of a polyN splint capture primer comprises a 3 ’OH extendible moiety. In some embodiments, the anchor sequence of a polyN splint capture primer can hybridize to a universal adaptor sequence of a single-stranded linear DNA library molecule, and the bridging sequence of the same polyN splint capture primer can hybridize to the template-independent homopolymer tail of the same single-stranded linear DNA library molecule thereby forming an open circle library molecule having a nick.Methods for Preparing Double-Stranded Library Molecules

[0188] The present disclosure provides methods for preparing a plurality of doublestranded (ds) nucleic acid library molecules. In some embodiments, only one side of an insert sequence is joined to at least one universal adaptor sequence. FIG. 15 is a schematic showing an embodiment of a workflow for preparing a plurality of nucleic acid library molecules and sequencing the plurality of library molecules. In some embodiments, one end of an individual double-stranded polynucleotide is appended to at least one universal adaptor sequence, thereby generating a plurality of double-stranded (ds) library molecules. In some embodiments, both ends of an individual double-stranded polynucleotides are appended to at least one universal adaptor sequence, thereby generating a plurality of double-stranded library molecules.

[0189] In some embodiments, the methods for preparing a plurality of nucleic acid library molecules comprise step (a): providing a plurality of input polynucleotides. The plurality of input polynucleotides can comprise a plurality of double-stranded polynucleotides from any source. Various embodiments of input polynucleotides are described below.

[0190] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (b): generating a plurality of double-stranded polynucleotide fragments from the plurality of input polynucleotides. In some embodiments, the plurality of polynucleotide fragments can be generated by fragmenting the plurality of input polynucleotides using any method including and without limitation mechanical, enzymatic and / or chemical fragmentation methods.

[0191] In some embodiments, the plurality of polynucleotide fragments can be generated by conducting a tagmentation reaction using a plurality of transposomes and transposases (e.g., FIG. 17).

[0192] In some embodiments, the plurality of polynucleotide fragments can be generated by conducting a Primary Template-Directed Amplification (PTA) workflow using a plurality of random-sequence primers and chain terminating nucleotides (e.g., FIG. 18), or using a plurality of tailed random-sequence primers and chain terminating nucleotides (e.g., FIG. 19). Various embodiments for generating polynucleotide fragments are described below.

[0193] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (c): joining at least one universal adaptor sequence to one end of individual double-stranded polynucleotide fragments, thereby generating a plurality of double-stranded library molecules wherein individual double-stranded library molecules comprise an insert sequence (e.g., one of the polynucleotide fragments) joined to at least one universal adaptor sequence. In some embodiments, only one side of the individual doublestranded polynucleotide fragments is joined to at least one universal adaptor sequence. In some embodiments, the universal adaptor sequence comprises any one or any combination of two or more of: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii ) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence with an optional random sequence (nnnn); (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer. In some embodiments, two or more universal adaptor sequences can be joined in any order to one side of the double-stranded polynucleotide fragments. In some embodiments, two or more universal adaptor sequences can be joined in any order to both sides of the doublestranded polynucleotide fragments.

[0194] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise adding a non-template DNA tail to the 3’ end of the double-stranded polynucleotide fragments using MMLV reverse transcriptase (e.g., FIG. 21). In some embodiments, the non-template DNA tail comprises a polyC tail, polyG tail, polyT tail or poly A tail.

[0195] Various embodiments for joining at least one universal adaptor sequence to one end of individual double-stranded polynucleotide fragments are described below.

[0196] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (d): generating a plurality of single-stranded library molecules from the plurality of double-stranded library molecules. The plurality of double-stranded librarymolecules can be treated or denatured using any suitable methods, for example with heat or NaOH to generate a plurality of single- stranded library molecules. The plurality of single-stranded library molecules can be linear, i.e. single-stranded linear library molecules.

[0197] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (e): generating a plurality of single-stranded circular library molecules from the plurality of single-stranded linear library molecules.

[0198] In some embodiments, the plurality of single-stranded circular library molecules can be generated by contacting the plurality of single-stranded linear library molecules with any of the soluble splint oligonucleotides described herein (e.g., FIGS. 27A-41B ) to generate a plurality of single-stranded open circle library molecules.

[0199] In some embodiments, the plurality of single-stranded circular library molecules can be generated by contacting the plurality of single-stranded linear library molecules with any of the target-specific bait / splint capture primers described herein (e.g., FIGS. 27A-35B) to generate a plurality of single-stranded open circle library molecules.

[0200] In some embodiments, the plurality of single-stranded circular library molecules can be generated by contacting the plurality of single-stranded linear library molecules with any of the immobilized splint capture primers described herein (e.g., FIGS. 36A-41B) to generate a plurality of single-stranded open circle library molecules.

[0201] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (f): immobilizing the plurality of open circle library molecules to a support.

[0202] In some embodiments, an open circle library molecule that is hybridized to a soluble splint oligonucleotide can be immobilized to a support by hybridization to any of the target-specific baits / probes described herein (e.g., FIGS. 27A-35B).

[0203] In some embodiments, an open circle library molecule that is hybridized to a soluble splint oligonucleotide can be immobilized to a support by hybridization to any of the immobilized capture primers described herein (e.g., FIGS. 36A-41B).

[0204] In some embodiments, the nicks of individual immobilized single-stranded open circle library molecules can be ligated on-support to generate a plurality of immobilized covalently closed circular library molecules.

[0205] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (g): generating a plurality of concatemer template molecules immobilized to the support by conducting rolling circle amplification on the support using the covalently closed circular library molecules as template molecules to generate the plurality ofconcatemer template molecules. Various embodiments of rolling circle amplification are described below.

[0206] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (h): sequencing the plurality of concatemer template molecules. In some embodiments, the plurality of concatemer template molecules can be sequenced to determine the sequence of the insert region. The sequencing can be conducted using any suitable sequencing method. For example, the sequencing method can employ a plurality of sequencing primers, a plurality of sequencing polymerases, and a plurality of nucleotide reagents. In some embodiments, the plurality of nucleotide reagents comprise a plurality of nucleotides, a plurality of chain terminating nucleotides, a plurality of phosphate-chain labeled nucleotides and / or a plurality of multivalent molecules. Various embodiments for sequencing are described below.

[0207] In some embodiments, the nucleotide reagents comprise multivalent molecules. In some embodiments, individual multivalent molecules comprise a central core attached to multiple polymer arms having a nucleotide moiety at the ends of the arms. In some embodiments, the sequencing reaction employs binding non-labeled nucleotides without incorporation. In some embodiments, the sequencing reaction employs incorporating nonlabeled nucleotide analogs. In some embodiments, the sequencing reaction employs incorporating detectably labeled nucleotides having removable chain terminating moieties. In some embodiments, the sequencing reaction employs a two-stage sequencing reaction comprising binding detectably labeled multivalent molecules without incorporation, and incorporating nucleotide analogs. An exemplary nucleotide arm is shown in FIG. 5, and exemplary multivalent molecules are shown in FIGS. 1-4. In some embodiments, any of the detectably labeled nucleotide reagents comprise at least one fluorophore.

[0208] In some embodiments, at least some of the library preparation steps can be conducted in one reaction vessel (e.g., one-pot), including generating the polynucleotide fragments, the adaptor-joining step, the library denaturation step, and the library circularization step. In some embodiments, at least some of the library preparation steps can be conducted in one reaction vessel (e.g., one-pot), including the adaptor-joining step, the library denaturation step, and the library circularization step. In some embodiments, the single reaction vessel comprises a well located at or near the inlet port at one end of a sequencing flowcell. In some embodiments, the sequencing flowcell comprises one or more inlet ports that are fluidically connected to the body of the flowcell to permit distribution of the prepared library molecules to the body of the flowcell.Methods for Preparing Single-Strand Library Molecules

[0209] The present disclosure provides methods for preparing a plurality of single-stranded (ss) nucleic acid library molecules. In some embodiments, only one side of an insert sequence is joined to at least one universal adaptor sequence. FIG. 16 is a schematic showing an embodiment of a workflow for preparing a plurality of nucleic acid library molecules and sequencing the plurality of library molecules. In some embodiments, one end of an individual single-stranded polynucleotides is appended to at least one universal adaptor sequence, thereby generating a plurality of single-stranded library molecules.

[0210] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (a): providing a plurality of input polynucleotides. In some embodiments, the plurality of input polynucleotides comprises a plurality' of double-stranded polynucleotides. The plurality of double-stranded polynucleotides can be from any source. Various embodiments of input polynucleotides are described below.

[0211] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (b): generating a plurality of double-stranded polynucleotide fragments from the plurality of input polynucleotides. The plurality of polynucleotide fragments can be generated by fragmenting the plurality of input polynucleotides using any suitable method known in the art, including and without limitation mechanical, enzymatic and / or chemical fragmentation methods.

[0212] In some embodiments, the plurality of polynucleotide fragments can be generated by conducting a tagmentation reaction using a plurality of transposomes and transposases (e.g., FIG. 17).

[0213] In some embodiments, the plurality of polynucleotide fragments can be generated by conducting a Primary Template-Directed Amplification (P TA) workflow using a plurality of random-sequence primers and chain terminating nucleotides (e.g., FIG. 18), or using a plurality of tailed random-sequence primers and chain terminating nucleotides (e.g., FIG. 19). Various embodiments for generating polynucleotide fragments are described below.

[0214] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (c): generating a plurality of single-stranded library molecules from the plurality of double-stranded library molecules. In some embodiments, the plurality of double-stranded library molecules can be treated or denatured using any suitable methods, for example with heat or NaOH, to generate a plurality of single-stranded library molecules.

[0215] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (d): joining at least one universal adaptor sequence to one end of an individual single-stranded polynucleotide fragment, thereby generating a plurality of single-stranded library molecules wherein individual single- stranded library molecules comprise an insert sequence (e.g., one of the polynucleotide fragments) joined to at least one universal adaptor sequence. In some embodiments, only one side of the individual polynucleotide fragment is joined to at least one universal adaptor sequence. In some embodiments, both sides of the individual polynucleotide fragment are joined to at least one universal adaptor sequence. In some embodiments, a universal adaptor sequence comprises any one or any combination of two or more of: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence with an optional random sequence (nnnn); (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer. In some embodiments, two or more universal adaptor sequences can be joined in any order to one side of the single-stranded polynucleotide fragments. The plurality of single-stranded library molecules can be single-stranded linear library molecules.

[0216] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual single-stranded polynucleotide fragments comprise conducting tagmentation using a plurality of transposomes and transposases (e.g., FIG. 17).

[0217] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise conducting a Primary Template-Directed Amplification (PTA) workflow using tailed random-sequence primers (e.g,, FIG. 19).

[0218] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise conducting a splint-mediated and ligation workflow (e.g., FIG. 20 parts (i) and (ii)).

[0219] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise adding a non-template DNA tail to the 3’ end of the single-stranded polynucleotide fragments using MMLV reverse transcriptase (e.g., FIGS. 22, 23A-23B, 24A-24B). In some embodiments, the non-template DNA tail comprises a polyC tail, polyG tail, polyT tail or poly A tail.

[0220] Various embodiments for joining at least one universal adaptor sequence to one end of individual single-stranded polynucleotide fragments are described below.

[0221] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (e): generating a plurality of single-stranded open circle library molecules from the plurality of single-stranded linear library molecules.

[0222] In some embodiments, the plurality of single-stranded circular library molecules can be generated by contacting the plurality of single-stranded linear library molecules with any of the soluble splint oligonucleotides described herein (e.g., FIGS. 27A-41B) to generate a plurality of single-stranded open circle library molecules.

[0223] In some embodiments, the plurality of single-stranded circular library molecules can be generated by contacting the plurality of single-stranded linear library molecules with any of the target-specific bait / splint capture primers described herein (e.g., FIGS. 27A-35B) to generate a plurality of single- stranded open circle library molecules.

[0224] In some embodiments, the plurality of single-stranded circular library molecules can be generated by contacting the plurality of single-stranded linear library molecules with any of the immobilized splint capture primers described herein (e.g., FIGS. 36A-41B) to generate a plurality of single-stranded open circle library molecules.

[0225] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (f): immobilizing the plurality of single-stranded open circle library molecules to a support.

[0226] In some embodiments, an open circle library molecule that is hybridized to any of the soluble splint oligonucleotides described herein (e.g., FIGS. 27A-35B) can be immobilized to a support by hybridizing the open circle library’ molecule to a target-specific bait / probe.

[0227] In some embodiments, an open circle library molecule that is hybridized to any of the soluble splint oligonucleotides described herein (e.g., FIGS. 36A-41B) can be immobilized to a support by hybridizing the open circle library molecule to an immobilized capture primer.

[0228] In some embodiments, the nicks of individual immobilized single-stranded open circle library molecules can be ligated on-support to generate a plurality of covalently closed circular library molecules immobilized to the support.

[0229] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (g): generating a plurality of concatemer template molecules immobilized to the support by conducting rolling circle amplification on the support using the covalently closed circular library molecules as template molecules to generate the plurality ofconcatemer template molecules. Various embodiments of rolling circle amplification are described below.

[0230] In some embodiments, methods for preparing a plurality of nucleic acid library molecules comprise step (h): sequencing the plurality of concatemer template molecules. In some embodiments, the plurality of concatemer template molecules can be sequenced to determine the sequence of the insert region. The sequencing can be conducted using any suitable sequencing method described herein.Sources of Input Polynucleotides

[0231] Input polynucleotides isolated from any source can be manipulated using molecular biology techniques to generate polynucleotide fragments. The polynucleotide fragments can be joined to at least one universal adaptor sequence to generate a plurality of nucleic acid library molecules. In some embodiments, individual library molecules comprise an insert sequence and at least one universal adaptor sequence. The insert sequence is derived from the input polynucleotide.

[0232] The input polynucleotides can be isolated from any source including a biological sample (e.g., fresh or live sample) such as a single cell, a plurality of cells or tissue. In some embodiments, the input polynucleotides can be isolated from healthy or diseases cells or tissues. In some embodiments, the input polynucleotides can be isolated from an archived sample such as a fresh frozen paraffin embedded (FFPE) sample, or from needle biopsies, circulating tumor cells, cell free circulating DNA (e.g., from tumor cells or a fetus). In some embodiments, the input polynucleotides can be isolated by lysing cells or tissues to release their DNA and RNA, and the desired nucleic acid can be separated from non-desired macromolecules such as proteins.

[0233] The input polynucleotides can be isolated in any form, including chromosomal, genomic (e.g., whole genomic), organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned or amplified. In some embodiments, the input polynucleotides can be methylated or non-methylated polynucleotides.

[0234] The input polynucleotides can be isolated from any organism including viruses, fungi, prokaryotes or eukaryotes. In some embodiments, the input polynucleotides can be isolated from any organism including human, simian, ape, canine, feline, bovine, equine, murine, porcine, caprine, lupine, ranine, piscine, plant, insect or bacteria. In some embodiments, the input polynucleotides can be isolated from organisms borne in air, water, soil or food.

[0235] The input polynucleotides can be isolated from any biological fluid, including blood, urine, serum, lymph, tumor, saliva, anal secretions, vaginal secretions, amniotic samples, perspiration, semen, environmental samples or culture samples. In some embodiments, the input polynucleotides can be isolated from any organ, including head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestines, bladder, prostate, testicles, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary, thymus, skin, heart, larynx, or other organs.

[0236] The input polynucleotides can be prepared using recombinant nucleic acid technology including but not limited to any combination of vector cloning, transgenic host cell preparation, host cell culturing and / or PCR amplification.

[0237] In some embodiments, the input polynucleotides are prepared using chemical synthesis procedures using native nucleotides with or without nucleotide analogs or modified nucleotide linkages that confer certain properties, including resistance to enzymatic digestion, or increased thermal stability. Examples of nucleotide analogs and modified nucleotide linkages that inhibit nuclease digestion include phosphorothioate, 2’-O-methyl RNA, inverted dT, and 2’ 3’ dideoxy-dT. In some embodiments, the input polynucleotides include locked nucleic acids (LNA) having increased thermal stability.Fragmenting Polynucleotides

[0238] The present disclosure provides methods for preparing nucleic acid library molecules by fragmenting input polynucleotides thereby generating a plurality of polynucleotide fragments. The input polynucleotides can be fragmented using mechanical force, enzymatic fragmentation methods or chemical fragmentation methods. In some embodiments, the polynucleotide fragments can be generated using procedures that yield a population of fragments having overlapping sequences or non-overlapping sequences. In some embodiments, the polynucleotide fragments can be single-stranded or double-stranded polynucleotides. In some embodiments, the double-stranded polynucleotide fragments comprise ends that are blunt-ended, have a 5’ overhang, a 3’ overhang end, or any combination thereof. In some embodiments, the polynucleotide fragments can be subjected to enzymatic reactions for end-repair and / or A-tailing. The polynucleotide fragments can be contacted with a plurality of enzymes under a condition suitable to generate blunt-ended 5’ phosphorylated ends.Mechanical Fragmentation Methods

[0239] The disclosure provides methods for generating a plurality of polynucleotide fragments using mechanical fragmentation. Mechanical fragmentation typically generates randomly fragmented polynucleotides. Mechanical fragmentation methods include mechanical shearing such as fluid shear, constant shear and pulsatile shear. Mechanical fragmentation methods also include mechanical stress including sonication, nebulization and acoustic cavitation. In some embodiments focused acoustic energy can be used to randomly fragment polynucleotides. A commercially-available apparatus (e.g., Covaris®) can be used to fragment polynucleotides using focused acoustic energy.Enzymatic Fragmentation Methods

[0240] The disclosure provides methods for generating a plurality of polynucleotide fragments using enzymatic fragmentation. Enzymatic fragmentation procedures can be conducted under conditions suitable to generate randomly or non-randomly fragmented polynucleotides. For example, restriction endonuclease enzyme digestion can be conducted to completion to generate non-randomly fragmented polynucleotides. Alternatively, partial or incomplete restriction enzyme digestion can be conducted to generate randomly-fragmented polynucleotides. Enzymatic fragmentation using restriction endonuclease enzymes includes any one or any combination of two or more restriction enzymes selected from a group consisting of type I, type II, type Ils, type IIB, type III, and type IV restriction enzymes. Enzymatic fragmentation includes digestion of polynucleotides with a rare-cutting restriction enzyme, comprising Not I, Asc I, Bae I, AspC I, Pac I, Fse I, Sap I, Sfi I or Psr I. Enzymatic fragmentation can include use of any combination of a nicking restriction endonuclease, endonuclease and / or exonuclease. Enzymatic fragmentation can be achieved by conducting a nick translation reaction. In a nick translation reaction, a DNA polymerase uses its 5’ to 3’ exonuclease activity to remove nucleotides ahead of a single-strand break (nick) in doublestranded DNA, while simultaneously using its 5’ to 3’ polymerase activity to add nucleotides, moving the position of the nick before the DNA is fragmented at the position of the nick.

[0241] In some embodiments, enzymatic fragmentation can be achieved by reacting polynucleotides with an enzyme mixture, for example 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®).Tagmentation Methods

[0242] The disclosure provides methods for generating a plurality of polynucleotide fragments using a tagmentation reaction. Polynucleotide fragments can be generated by conducting a tagmentation method using a transposase-based tagmentation method. FIG. 17 is a shows an embodiment of a method for generating polynucleotide fragments and appending universal adaptors using transposase-mediated tagmentation. In some embodiments, polynucleotide fragments can be generated using a transposase-based tagmentation method using NEXTERA® (from Epicentre®). In some embodiments, input polynucleotide can be fragmented using a Tn5 transposase-based workflow that employs a transposon end sequence comprising a double-stranded DNA having sequences that can bind a transposase enzyme to form a DNA-transposase complex (e.g., a transpososome), where the complex can transpose / insert the transposon end sequences into DNA in an in vitro tagm en tati on reacti on,

[0243] In some embodiments, the transposase enzyme comprises a Tn5 transposase. In some embodiments, the transposon end sequence comprises a first and second DNA strand. In some embodiments, the first DNA strand (the transfer strand) comprises a transfer end sequence. In some embodiments the transfer end sequence comprises a sequence of 5'AGATGTGTATAAGAGACAG 3' (SEQ ID NO: 1). In some embodiments, the transfer end sequence comprises a sequence of SEQ ID NO: 1, or a sequence having 1, 2, or 3 nucleotide insertions, deletions or substitutions relative thereto. In some embodiments, the transfer end sequence consists of SEQ ID NO: 1. In some embodiments, the transfer strand comprises, from 5’ to 3’, a sequence of the at least one universal adaptor and the transfer end sequence. In some embodiments, the second DNA strand (the non-transfer strand) comprises a non-transfer end sequence. In some embodiments, the non-transfer end sequence is complementary to all or a portion of the transfer end sequence. In some embodiments, the non-transfer end sequence comprises a sequence of 5' CTGTCTCTTATACACATCT 3' (SEQ ID NO: 2). In some embodiments, the non-transfer end sequence comprises a sequence of SEQ ID NO: 2, or a sequence having 1, 2, or 3 nucleotide insertions, deletions or substitutions relative thereto. In some embodiments, the non-transfer end sequence consists of SEQ ID NO: 2. In some embodiments, the non-transfer end sequence is phosphorylated at its 5’ end.

[0244] When it is desirable to fragment input polynucleotides and append universal adaptors using the Tn5 transposase-based workflow (e.g., tagmentation reaction), the first transposon DNA strand further comprises a universal adaptor sequence. In someembodiments, the second transposon DNA strand further comprises a universal adaptor sequence. In some embodiments, the first and second adaptor sequences are fully complementary along their lengths, thereby forming a linear double-stranded transposon-end-adaptor molecule. In some embodiments, the first and second adaptor sequences are partially complementary' along their lengths thereby forming a Y-shaped double-stranded transposon-end-adaptor molecule. In some embodiments, the Y-shaped double-stranded transposon-end-adaptor molecule can be a full length or stubby Y-shaped adaptor.

[0245] In some embodiments, the first transposon DNA strand further comprises a universal adaptor sequence and the second transposon DNA strand lacks an adaptor sequence. In some embodiments, the first strand of a universal adaptor sequence comprises any one or any combination of two or more adaptor sequences: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence with an optional random sequence (nnnn); (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer.

[0246] In some embodiments, a plurality of Tn5 transposases and a plurality of doublestranded transposon-end-adaptor molecules can be mixed together under conditions suitable to bind / load the double-stranded transposon-end-adaptor molecules onto the transposase enzymes to form a plurality of DNA-transposase complexes (transposomes).

[0247] In some embodiments, input polynucleotides (e.g., double-stranded polynucleotides) can be mixed with a plurality of transpososomes under a condition suitable for transposing (inserting) the transposon end sequences into random sites in the input polynucleotides, thereby fragmenting the input polynucleotides and covalently attaching the transfer strand to the 5’ end of one strand of the input polynucleotide. The non-transfer strand is hybridized to the transfer end sequence with a gap (e.g., a 9 base gap) at the 3’ end of the input DNA polynucleotide. In some embodiments, the gap can be subjected to a polymerase-catalyzed fill-in reaction and enzymatic ligation to generate tagmented double-stranded DNA with no gaps and with adaptors appended to both ends. In some embodiments, the gap is not subjected to a polymerase-catalyzed fill-in reaction and enzymatic ligation thereby generating tagmented single-stranded DNA carrying an adaptor appended to one end. The transposase-based tagmentation workflow can be conducted using any of the methods described in U. S. patent Nos. 10,184,122, 10,287,574, 11,028,438, and published U. S. application No.2019 / 0194737, the contents of all of which incorporated by reference in their entireties.Generating Polynucleotide Fragments with Amplification

[0248] The disclosure provides methods for generating a plurality of polynucleotide fragments by conducting PCR using multiple sequence-specific primers (e.g., random primers, or target-specific primers). In some embodiments, polynucleotide fragments can be generated by conducting PCR using sequence-specific primers that hybridize to target regions in genomic DNA samples to generate insert regions having known fragment lengths and sequences.

[0249] In some embodiments, polynucleotide fragments can be generated by conducting amplification using random-sequence primers. FIG. 18 is a schematic showing an embodiment of a method for generating a plurality of polynucleotide fragments by conducting amplification using a plurality of random-sequence primers, strand displacing polymerases and a plurality of chain terminating nucleotides (solid rectangles). Exemplary' methods are described in U. S, patent No. 11,905,553, entitled ‘Method for Nucleic Acid Amplification’. The contents of the aforementioned issued patent is hereby expressly incorporated by reference in its entirety. In some embodiments, this amplification method can be conducted using a Primary Template-Directed Amplification (PTA) workflow which is part of a ResolveDNA kit (commercially-available from BioSkryb Genomics). In some embodiments, the amplification method comprises contacting input polynucleotides with a plurality of random-sequence primers, a plurality of strand-displacing polymerases and a plurality of nucleotides which includes a plurality of chain terminating nucleotides, under a condition suitable for conducting nucleic acid amplification to generate a plurality of amplification products. The nucleic acid amplification reaction progresses along the input polynucleotides until a chain terminating nucleotide is incorporated, which terminates polymerization. In some embodiments, the amplification products can be about 50-2000 nucleotides in length. The skilled artisan will appreciate that amplification product length can be controlled by adjusting the percentage of nucleotides in the plurality that are chain terminating.

[0250] In some embodiments, polynucleotide fragments can be generated by conducting amplification using tailed random-sequence primers. FIG. 19 is a schematic showing an embodiment of a method for generating a plurality of polynucleotide fragments by conducting amplification using a plurality of tailed random-sequence primers, strand displacing polymerases and a plurality of chain terminating nucleotides (solid rectangles). Exemplary methods are described in U. S. patent No. 11,905,553, entitled ‘Method for Nucleic Acid Amplification’. The contents of the aforementioned issued patent are herebyexpressly incorporated by reference in their entirety. This amplification method can be conducted using a Primary Template-Directed Amplification (PT A) workflow which is part of a ResolveDNA™ kit (commercially-available from BioSkryb Genomics®). In some embodiments, the amplification method comprises contacting the input polynucleotides with a plurality of tailed random-sequence primers, a plurality of strand-displacing polymerases and a plurality of nucleotides which includes a plurality of chain terminating nucleotides, under a condition suitable for conducting nucleic acid amplification to generate a plurality of amplification products. As above, the nucleic acid amplification reaction progresses along the input polynucleotides until a chain terminating nucleotide is incorporated which terminates polymerization, thereby generating a plurality of polynucleotide fragments each carrying a 5’ tail sequence and a 3’ region having a sequence that is complementary to the input polynucleotides. In some embodiments, the amplification products can be about 50-2000 nucleotides in length. Adaptor sequences, such as any of the adaptor sequences described herein, can be introduced on the tail of the tailed random-sequence primers.

[0251] In some embodiments, individual tailed random-sequence primers comprise a 5’ tail carrying at least one universal adaptor sequence and a 3 ’ region comprising a random sequence. In some embodiments, the 5’ tail comprises at least one sample index sequence. In some embodiments, the 5’ tail comprises any one or any combination of two or more universal adaptor sequences including: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence; (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer.

[0252] The Primary Template-Directed Amplification (PTA) methods described above (e.g., FIGS. 18 and 19) can employ a strand-displacing polymerase that lacks exonuclease proofreading activity or includes exonuclease proofreading activity. In some embodiments, the polymerase having strand-displacing activity comprises bacteriophage phi29 DNA polymerase, genetically modified phi29 DNA polymerase, Klenow Fragment of DNA polymerase I, phage M2 DNA polymerase, phage phiPRDl DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo(-) Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, DNA polymerase VENT®, DNA polymerase DEEP VENT®, DNA polymerase THERMIN AT OR®,, DNA polymerase ISOPOL, DNA polymerase I, T5 DNA polymerase SEQUENASE®,, T7 DNA polymerase, or T4 DNA polymerase.

[0253] In some embodiments, the Primary Template-Directed Amplification (PTA) methods described above (e.g., FIGS. 18 and 19) employ chain terminating nucleotides comprising X blocked deoxynucleotides, X fluoro nucleotides and / or 2'-O-methyl modified nucleotides. In some embodiments, the chain terminating nucleotides comprise dideoxynucleotides and / or inverted dideoxynucleotides. In some embodiments, the chain terminating nucleotides comprise 3' biotinylated nucleotides, 3' amino nucleotides, O'phosphorylated nucleotides, 3'-O-methyl nucleotides, 3' carbon spacer nucleotides including 3' C3 spacer nucleotides, 3' C18 nucleotides, 3' Hexanediol spacer nucleotides and / or acyclonucleotides. In some embodiments, the chain terminating nucleotides comprise nucleotides with modification to the alpha group, C3 spacer nucleotides, locked nucleic acids (LNA), inverted nucleic acids and / or 3' phosphorylated nucleotides.Polynucleotide Fragment Size and Size Selection

[0254] The polynucleotide fragments can be any length, for example the insert region can be about 50-250, or about 250-500, or about 500-750, or about 750-1000, or about 1000-1500, or about 1500-2000 bases or base pairs in length. In some embodiments, the polynucleotide fragments are 2000-5000 bases or base pairs in length.

[0255] The polynucleotide fragments can be subjected to a size selection process.Alternatively, or the polynucleotide fragments are not size selected. For example, the polynucleotide fragments can be size selected by gel electrophoresis and gel slice extraction. The polynucleotide fragments can be size selected using a solid phase adherence / immobilization method which typically employs micro paramagnetic beads coated with a chemical functional group that interacts with nucleic acids under certain ionic strength conditions with or without polyethylene glycol or polyalkylene glycol. Commercially-available solid phase adherence beads include SPRI® (Solid Phase Reversible Immobilization) beads from Beckman Coulter® (AMPURE® XP paramagnetic beads, catalog No. B23318), MAGNA PURE® magnetic glass particles (Roche Diagnostics®, catalog No. 03003990001), MAGNASIL® paramagnetic beads from Promega® (catalog No. MD1360), MAGTRATION paramagnetic beads and system from Precision System Science® (catalog Nos. Al 120 and A1060), MAG-BIND® from Omega Bio-Tek® (catalog No.M1378-01), MAGPREP® silica from Millipore® (catalog No. 101193), SNARE™ DN A purification systems from Bangs Laboratories® (catalog Nos. BP691, BP692 and BP693), and CHEMAGEN® M-PVA beads from PerkinElmer® (catalog No. CMG-200).Joining Universal Adaptors to Double-Stranded Polynucleotide Fragments

[0256] The present disclosure provides methods for preparing nucleic acid library molecules by joining at least one universal adaptor sequence to one end of individual doublestranded polynucleotide fragments, thereby generating a plurality of double-stranded library molecules wherein individual double-stranded library molecules comprise an insert sequence (e.g., one of the polynucleotide fragments) joined to at least one universal adaptor sequence. In some embodiments, only one side of the individual polynucleotide fragments is joined to at least one universal adaptor sequence, for example the 5 ’end. In some embodiments, a universal adaptor sequence comprises any one or any combination of two or more of: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence with an optional random sequence (nnnn); (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer. In some embodiments, both sides of the individual polynucleotide fragments are joined to at least one universal adaptor sequence (i.e., both the 5’ end and the 3’ end).

[0257] FIG. 21 is a shows an embodiment for a method to append at least one universal adaptor sequence to one end of a double-stranded polynucleotide fragment using a MMLV reverse transcriptase enzyme and guide adaptor oligonucleotides (GAOs). This method is described in Ohtsubo, et al., 2018 DNA Research 25(5):477-487; and Ohtsubo, et al., 2017 Nature Scientific Reports 7:41769 (DOI: 10.1038 / srep41769; and WO 2017 / 21744. The contents of the aforementioned publications are hereby expressly incorporated by reference in their entireties. In some embodiments, the method comprises contacting a plurality of doublestranded polynucleotide fragments with a plurality of a reverse transcriptase enzyme and a first plurality of nucleotides under a condition suitable for catalyzing addition of nontemplate DNA tails to the 3’ ends of the double-stranded polynucleotide fragments thereby generating a plurality of double-stranded 3 ’-tailed polynucleotide fragments wherein one strand of individual double-stranded 3 ’-tailed polynucleotide fragments carries a nontemplate 3’ tail. In some embodiments, the reverse transcriptase enzyme comprises MMLV (Moloney murine leukemia virus) reverse transcriptase. In some embodiments, the first plurality' of nucleotides comprises one type of a nucleotide, for example dATP, dGTP, dTTP or dCTP. The schematic of FIG 21 shows a non-template 3’ poly-C tail appended to the 3’ ends of the polynucleotide fragments. In some embodiments, the plurality of double-stranded 3’-tailed polynucleotide fragments can be denatured to generate a plurality of single-stranded 3 ’-tailed polynucleotide fragments. In some embodiments, the plurality of single-stranded 3’-tailed polynucleotide fragments can be hybridized to a plurality of guide adaptor oligonucleotides (GAOs) comprising (i) a sequence that can hybridize to the non-template 3’ tail (e.g., a poly-G sequence, or GGGG) and (ii) at least one universal adaptor sequence (e.g., XXX in FIG. 21), thereby forming a plurality of nucleic acid duplexes at one end of the 3’-tailed polynucleotide fragments. In some embodiments, the duplexes are formed at the 3’ ends of the polynucleotide fragments by hybridizing the non-template 3’ tails with the guide adaptor oligonucleotides. The skilled artisan will appreciate that the non-template 3’ tail in FIG. 21 is represented as a poly-C tail that hybridizes to a poly-G sequence in the GAO for illustrative purposes. Poly-A, poly-T and poly-G tails, with corresponding complementary GAO sequences, may also be suitable for the methods disclosed herein. In some embodiments, the terminal 3’ ends of the plurality of guide adaptor oligonucleotides (G AOs) comprise blocking moieties that inhibits polymerase-catalyzed extension (e.g., a nonextendible terminal 3’ end) (solid rectangles in FIG. 21). In some embodiments, the non-template 3’ tail can be extended by contacting the nucleic acid duplexes with a plurality of MMLV reverse transcriptase enzyme and a second plurality of nucleotides under a condition suitable for catalyzing template-dependent extension (dashed arrow in FIG. 21) thereby generating a plurality of polynucleotide fragments appended on one side to a non-template 3’ tail and a newly generated universal adaptor sequence(s). In some embodiments, the newly generated universal adaptor sequence(s) is complementary to the universal adaptor sequence carried by the guide adaptor oligonucleotides (GAOs). The newly generated universal adaptor sequence(s) that is generated by the template-dependent extension reaction is covalently joined to the non-template 3 ’-tail. In some embodiments, the second plurality of nucleotides comprises any combination of two or more nucleotides including dATP, dGTP, dTTP and / or dCTP In some embodiments, the plurality of guide adaptor oligonucleotides (G AOs) can be removed, while retaining the plurality of single-stranded polynucleotide fragments appended on one side to a non-template 3’ tail and a newly generated universal adaptor sequence(s).Joining Universal Adaptors to Single-Stranded Polynucleotide Fragments

[0258] The present disclosure provides methods for preparing nucleic acid library molecules by joining at least one universal adaptor sequence to one end of individual single-stranded polynucleotide fragments, thereby generating a plurality of single-stranded library molecules wherein individual single-stranded library molecules comprise an insert sequence (e.g., one of the polynucleotide fragments) joined to at least one universal adaptor sequence. In some embodiments, only one side of the individual polynucleotide fragments is joined toat least one universal adaptor sequence. In some embodiments, a universal adaptor sequence comprises any one or any combination of two or more of (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence with an optional random sequence (nnnn); (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer. In some embodiments, both sides of the individual polynucleotide fragments are joined to at least one universal adaptor sequence.Tagmentation-Based Methods

[0259] The disclosure provides methods for joining at least one universal adaptor sequence to one end of individual single- stranded polynucleotide fragments comprise conducting a tagmentation reactions using a plurality of transposomes and transposases (e.g., FIG. 17). FIG. 17 is a shows an embodiment of a method for generating polynucleotide fragments and appending universal adaptors using transposase-mediated tagmentation.

[0260] In some embodiments, polynucleotide fragments can be generated using a transposase-based tagmentation method using NEX TERA® (from Epicentre®). In some embodiments, input polynucleotide can be fragmented using a Tn5 transposase-based workflow that employs a transposon end sequence comprising a double-stranded DNA having sequences that can bind a transposase enzyme to form a DNA-transposase complex (e.g., a transpososome), where the complex can transpose / insert the transposon end sequences into DNA in an in vitro tagmentation reaction.

[0261] In some embodiments, the transposase enzyme comprises a Tn5 transposase. In some embodiments the transposon end sequence comprises a first and second DNA strand. In some embodiments, the first DNA strand (the transfer strand) comprises a transfer end sequence. In some embodiments the transfer end sequence comprises a sequence of5 ' AG AT GTGT AT A AG AG AC AG 3' (SEQ ID NO: 1). In some embodiments, the transfer end sequence comprises a sequence of SEQ ID NO: 1, or a sequence having 1, 2, or 3 nucleotide insertions, deletions or substitutions relative thereto. In some embodiments, the transfer end sequence consists of SEQ ID NO. 1. In some embodiments, the transfer strand comprises, from 5’ to 3’, a sequence of the at least one universal adaptor and the transfer end sequence.

[0262] In some embodiments, the second DNA strand (the non-transfer strand) comprises a non-transfer end sequence. In some embodiments, the non-transfer end sequence is complementary to all or a portion of the transfer end sequence. In some embodiments, thenon-transfer end sequence comprises a sequence of 5' CTGTCTCTTATACACATCT 3' (SEQ ID NO: 2). In some embodiments, the non-transfer end sequence comprises a sequence of SEQ ID NO: 2, or a sequence having 1, 2, or 3 nucleotide insertions, deletions or substitutions relative thereto. In some embodiments, the non-transfer end sequence consists of SEQ ID NO: 2. In some embodiments, the non-transfer end sequence is phosphorylated at its 5’ end.

[0263] When it is desirable to fragment input polynucleotides and append universal adaptors using the Tn5 transposase-based workflow (e.g., tagmentation workflow), the first transposon DNA strand further comprises a universal adaptor sequence. In some embodiments, the second transposon DNA strand further comprises a universal adaptor sequence. In some embodiments, the first and second adaptor sequences are fully complementary along their lengths thereby forming a linear double-stranded transposon-end-adaptor molecule. In some embodiments, the first and second adaptor sequences are partially complementary along their lengths thereby forming a Y-shaped double-stranded transposonend-adaptor molecule. In some embodiments, the Y-shaped double-stranded transposon-end-adaptor molecule can be a full length or stubby Y-shaped adaptor.

[0264] In some embodiments, the first transposon DNA strand further comprises of a universal adaptor sequence and the second transposon DNA strand lacks an adaptor sequence. In some embodiments, the first strand of a universal adaptor sequence comprises any one or any combination of two or more adaptor sequences: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence with an optional random sequence (nnnn); (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer.

[0265] In some embodiments, a plurality of Tn5 transposases and a plurality of doublestranded transposon-end-adaptor molecules can be mixed together under conditions suitable to bind / load the double-stranded transposon-end-adaptor molecules onto the transposase enzymes to form a plurality of DNA-transposase complexes ( transpososomes).

[0266] In some embodiments, input polynucleotides (e.g., double-stranded polynucleotides) can be mixed with a plurality of transpososomes under a condition suitable for transposing (inserting) the transposon end sequences into random sites in the input polynucleotides, thereby fragmenting the input polynucleotides and covalently attaching the transfer strand to the 5’ end of one strand of the input polynucleotide. The and the non-transfer strand is hybridized to the transferred end sequence with a gap (e.g., a 9 base gap) atthe 3’ end of the input DNA polynucleotide. In some embodiments, the gap is not subjected to a polymerase-catalyzed fill-in reaction and enzymatic ligation thereby generating tagmented single-stranded DNA carrying an adaptor appended to one end. The transposase-based tagmentation workflow can be conducted using any of the methods described in U. S. patent Nos. 10,184,122, 10,287,574, 11,028,438, and published U. S. application No.2019 / 0194737, the contents of all of which are incorporated by reference in their entireties.Primary' Template-Directed Amplification (PTA) Methods

[0267] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise conducting a Primary’ Template-Directed Amplification (PTA) workflow using tailed random-sequence primers (e.g., FIG. 19). FIG. 19 is a schematic showing an embodiment of a method for generating a plurality of polynucleotide fragments by conducting amplification using a plurality of tailed randomsequence primers, strand displacing polymerases and a plurality' of chain terminating nucleotides (solid rectangles). Exemplary methods are described in U. S. patent No.11,905,553, entitled ‘Method for Nucleic Acid Amplification’. The contents of the aforementioned issued patent are hereby expressly incorporated by reference in their entirety. This amplification method can be conducted using a Primary Template-Directed Amplification (PTA) workflow which is part of a ResolveDNA™ kit (commercially-available from BioSkryb Genomics®).

[0268] In some embodiments, the method comprises contacting input polynucleotides with a plurality of tailed random-sequence primers, a plurality of strand-displacing polymerases and a plurality of nucleotides which includes a plurality of chain terminating nucleotides, under a condition suitable for conducting nucleic acid amplification to generate a plurality of amplification products. In some embodiments, the nucleic acid amplification reaction progresses along the input polynucleotides until a chain terminating nucleotide is incorporated which terminates polymerization, thereby generating a plurality of polynucleotide fragments each carrying a 5’ tail sequence and a 3’ region having a sequence that is complementary to the input polynucleotides. In some embodiments, the amplification products can be about 50-2000 nucleotides in length. Adaptor sequences, such as any of the adaptor sequences described herein, can be introduced on the tail of the tailed randomsequence primers.

[0269] In some embodiments, individual tailed random-sequence primers comprise a 5’ tail carrying at least one universal adaptor sequence and a 3 ’ region comprising a randomsequence. In some embodiments, the 5’ tail comprises at least one sample index sequence. In some embodiments, the 5’ tail comprises any one or any combination of two or more universal adaptor sequences including: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence; (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer.

[0270] In some embodiments, the strand-displacing polymerase lacks exonuclease proofreading activity or includes exonuclease proofreading activity. In some embodiments, the polymerase having strand-displacing activity comprises bacteriophage phi29 DNA polymerase, genetically modified phi29 DNA polymerase, Klenow Fragment of DNA polymerase I, phage M2 DNA polymerase, phage phiPRDl DNA polymerase, Bst DNA polymerase, Bst large fragment DNA polymerase, exo(-) Bst polymerase, exo(-)Bca DNA polymerase, Bsu DNA polymerase, DNA polymerase VENT, DNA polymerase DEEP VENT, DNA polymerase THERMINATOR, DNA polymerase ISOPOL, DNA polymerase I, T5 DNA polymerase SEQUENASE, T7 DNA polymerase, or T4 DNA polymerase. In some embodiments, the chain terminating nucleotides comprise 2’ blocked deoxynucleotides, 2' fluoro nucleotides and / or 2'-O-methyl modified nucleotides. In some embodiments, the chain terminating nucleotides comprise dideoxynucleotides and / or inverted dideoxynucleotides. In some embodiments, the chain terminating nucleotides comprise 3' biotinylated nucleotides, 3' amino nucleotides, 3 '-phosphorylated nucleotides, 3'-O-methyl nucleotides, 3' carbon spacer nucleotides including 3' C3 spacer nucleotides, 3 ' C 18 nucleotides, 3' Hexanediol spacer nucleotides and / or acyclonucleotides. In some embodiments, the chain terminating nucleotides comprise nucleotides with modification to the alpha group, C3 spacer nucleotides, locked nucleic acids (LNA), inverted nucleic acids and / or 3' phosphorylated nucleotides.Splint-Mediated and Ligation Methods

[0271] The disclosure provides methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise conducting a splint-mediated and ligation workflow. FIG. 20 is a schematic of two exemplary embodiments of a method for appending at least one universal adaptor sequence to one end of a polynucleotide fragment using a splint-mediated and ligation workflow. Additional splint-mediated ligation methods are described in U. S. Publication No. 2023-0279382, U. S. Publication No. US 2023-0279483, U. S. Publication No. US 2024-0011022, U. S. Publication No. US 2024-0191225and WO 2025 / 191535, the contents of each of which are incorporated by reference in their entirety herein.

[0272] In some embodiments, a plurality of polynucleotide fragments can be contacted with a plurality of a universal adaptors and a plurality of splint oligonucleotides, under a condition suitable for hybridizing the 5’ ends of individual splint oligonucleotides to the 5’ ends of the polynucleotide fragments, and suitable for hybridizing the 3’ ends of individual splint oligonucleotides to the 3’ end of the universal adaptors, thereby forming a nick between the 5’ end of the polynucleotide fragment and the 3’ end of the universal adaptor. In some embodiments, the nick is contacted with a ligase enzyme to generate a polynucleotide fragment appended at its 5’ end to a universal adaptor (FIG. 20 part (i)). In some embodiments, individual splint oligonucleotides comprise, in a 5’ to 3’ orientation, a random sequence (NNNN) that can hybridize to an end portion of a polynucleotide fragment, and a universal adaptor sequence that can hybridize to an end portion of the universal adaptor. For example, the splint oligonucleotide comprises a random sequence (NNNN) and a universal adaptor sequence for binding a forward sequencing primer (FWD seq). In some embodiments, the random sequence (NNNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length.

[0273] In some embodiments, a plurality of polynucleotide fragments can be contacted with a plurality of a universal adaptors and a plurality of splint oligonucleotides, under a condition suitable for hybridizing the 5’ ends of individual splint oligonucleotides to the 5’ ends of the universal adaptors, and suitable for hybridizing the 3’ ends of individual splint oligonucleotides to the 3’ end of the polynucleotide fragments, thereby forming a nick between the 3’ end of the polynucleotide fragment and the 5’ end of the universal adaptor. In some embodiments, the nick is contacted with a ligase enzyme to generate a polynucleotide fragment appended at its 3’ end to a universal adaptor (FIG. 20 part (ii)). In some embodiments, individual splint oligonucleotides comprise, in a 5’ to 3’ orientation, a universal adaptor sequence that can hybridize to an end portion of the universal adaptor, and a random sequence (NNNN) that can hybridize to an end portion of a polynucleotide fragment. For example, the splint oligonucleotide comprises a universal adaptor sequence for binding a reverse sequencing primer (REV seq) and a random sequence (NNNN). In some embodiments, the random sequence (NNNN) comprises any combination of dATP, dGTP,dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length.Adding Non-Tem pl ate Tails

[0274] In some embodiments, methods for joining at least one universal adaptor sequence to one end of individual polynucleotide fragments comprise adding a non-template DNA tail to the 3’ ends of the single-stranded polynucleotide fragments using a reverse transcriptase, such as MMLV reverse transcriptase (e.g., FIGS. 22, 23A-23B, 24A-24B).

[0275] FIG. 22 shows an embodiment for a method to append at least one universal adaptor sequence to one end of a single-stranded polynucleotide fragment using a reverse transcriptase such as MMLV reverse transcriptase enzyme and guide adaptor oligonucleotides (GAOs). This method is described in Ohtsubo, et al., 2018 DNA Research 25(5):477-487; Ohtsubo, et al., 2017 Nature Scientific Reports 7:41769 (DOI:10.1038 / srep41769; and WO 2017 / 21744. The contents of the aforementioned publications and patent applications are hereby expressly incorporated by reference in their entireties. In some embodiments, the method comprises denaturing a plurality of double-stranded polynucleotide fragments into a plurality of single-stranded polynucleotide fragments. In some embodiments, the plurality of single-stranded polynucleotide fragments is contacted with a plurality of a reverse transcriptase enzyme and a first plurality of nucleotides under a condition suitable for catalyzing addition of non-template DNA tails to the 3’ ends of the single-stranded polynucleotide fragments, thereby generating a plurality of single-stranded 3 ’ -tailed polynucleotide fragments each carrying a non-template 3’ tail. In some embodiments, the plurality of reverse transcriptase enzyme comprises MMLV (Moloney murine leukemia virus) reverse transcriptase. In some embodiments, the first plurality of nucleotides comprises one type of a nucleotide, for example dATP, dGTP, dTTP or dCTP The schematic of FIG. 22 shows a non-template 3’ poly-C tail appended to the 3’ ends of the polynucleotide fragments. In some embodiments, the plurality of single-stranded 3 ’-tailed polynucleotide fragments can be hybridized to a plurality of guide adaptor oligonucleotides (GAOs) comprising (i) a sequence that can hybridize to the non-template 3’ tail (GGGG) and (ii) at least one universal adaptor sequence (e.g., XXX in FIG. 22), thereby forming a plurality of nucleic acid duplexes at one end of the 3 ’ -tailed polynucleotide fragments. In some embodiments, the duplexes are formed at the 3’ ends of the polynucleotide fragments by hybridizing the non-template 3’ tails with the guide adaptor oligonucleotides. The skilled artisan will appreciate that the non-template 3’ tail in FIG. 22 is represented as a poly-C tailthat hybridizes to a poly-G sequence in the GAO for illustrative purposes. Poly-A, poly-T and poly-G tails, with corresponding complementary GAO sequences, may also be suitable for the methods disclosed herein. In some embodiments, the terminal 3’ end of the plurality of guide adaptor oligonucleotides (GAOs) comprises a blocking moiety that inhibits polymerase-catalyzed extension (e.g., a non-extendible terminal 3’ end) (solid rectangles in FIG. 22). In some embodiments, the non-template 3’ tail can be extended by contacting the nucleic acid duplexes with a plurality of MMLV reverse transcriptase enzyme and a second plurality of nucleotides under a condition suitable for catalyzing template-dependent extension (dashed arrow in FIG. 22) thereby generating a plurality of polynucleotide fragments appended on one side with a non-template 3’ tail and a universal adaptor sequence(s). In some embodiments, the universal adaptor sequence(s) is complementary to the universal adaptor sequence carried by the guide adaptor oligonucleotides (GAOs). The newly generated universal adaptor sequence(s) that is generated by the template-dependent extension reaction is covalently joined to the non-template 3 ’-tail. In some embodiments, the second plurality of nucleotides comprises any combination of two or more nucleotides including dATP, dGTP, dTTP and / or dCTP. In some embodiments, the plurality of guide adaptor oligonucleotides (GAOs) can be removed, while retaining the plurality of single-stranded polynucleotide fragments appended on one side to a non-template 3’ tail and a newly generated universal adaptor sequence(s).

[0276] FIG. 23 A shows an embodiment for a method to append at least one universal adaptor sequence to one end of a single-stranded polynucleotide fragment using a non-template tailing enzyme and a plurality of double-stranded attenuator-adaptor molecules each having a 3’ overhang end. The schematics shown in FIGS. 23A-23B include steps of the same attenuator-mediated workflow. Exemplary methods are described in U. S. patent Nos.9,896,709; 10,731,194; 11,118,207; and 12,104,194, all entitled ‘Methods and Compositions for Size-Controlled Homopolymer Tailing of Substrate Polynucleotides by a Nucleic Acid Polymerase’. The contents of the aforementioned issued patents are hereby expressly incorporated by reference in their entireties. In some embodiments, the attenuator-mediated method can be conducted using reagents contained in an Adaptase® kit (e.g., xGen Adaptase Module kit commercially-available from Integrated DNA Technologies (IDT)® (catalog # 10009826) and following the manufacturer's instructions.

[0277] In some embodiments, the method shown in FIG. 23 A comprises contacting a plurality of single-stranded polynucleotide fragments with a plurality of double-stranded attenuator-adaptor molecules, a plurality of non-template (e.g., template-independent) tailingenzymes and a plurality of nucleotides under a condition suitable for catalyzing addition of non-template DNA tails to the 3’ ends of the single-stranded polynucleotide fragments, thereby generating a plurality of single-stranded 3 ’-tailed polynucleotide fragments each carrying a non-template 3’ tail. In some embodiments, the double-stranded attenuator-adaptor molecule comprises an oligonucleotide that associates with the non-template 3’ tail sequence, where the double-stranded attenuator-adaptor molecule controls the number of added nucleotides that form part of the non-template 3’ tail. In some embodiments, individual double-stranded attenuator-adaptor molecules comprise a first strand comprising at least one universal adaptor sequence (e.g., top strand in FIG. 23 A) and a second strand comprising a homo-polymeric sequence (e.g., GGGGGG in FIG. 23 A) and a sequence that is complementary to the at least one universal adaptor sequence of the first strand. In some embodiments, the homo-polymeric sequence is part of a 3’ overhang end. In some embodiments, the 5’ end of the first strand is phosphorylated. In some embodiments, the 3’ end of the second strand comprises a blocking moiety that inhibits polymerase-catalyzed extension (e.g., a non-extendible terminal 3’ end) (solid rectangles in FIG. 23 A). The non-template 3’ tail is covalently joined to the 3’ ends of individual single- stranded polynucleotide fragments, for example by hybridizing with the homo-polymeric sequence of the 3’ overhang. In some embodiments, the plurality of a template-independent tailing enzyme comprises a terminal deoxynucleotidyl transferase (TdT), an E. coli RNA-specific Poly (A) polymerase, an S. pombe RNA-specific poly(U) polymerase or a yeast poly(a) polymerase. In some embodiments, the plurality of nucleotides comprises one type of a nucleotide, for example dATP, dGTP, dTTP or dCTP. In some embodiments, the condition is suitable for hybridizing at least a portion of the double-stranded attenuator-adaptor molecule to the non-template 3’ tail. In some embodiments, the plurality of nucleotides comprises one type of a nucleotide, for example dATP, dGTP, dTTP or dCTP.

[0278] FIG. 23B continues the method shown in FIG. 23 A, wherein the 3’ non-template tail (CCCCCC) is hybridized to the double-stranded attenuator-adaptor molecule. Exemplary methods are described in U. S. patent Nos. 9,896,709; 10,731,194; 11,118,207; and 12,104,194, all entitled ‘Methods and Compositions for Size-Controlled Homopolymer Tailing of Substrate Polynucleotides by a Nucleic Acid Polymerase’. The contents of the aforementioned issued patents are hereby expressly incorporated by reference in their entireties. In some embodiments, the method (shown in FIG. 23B) further comprises hybridizing the non-template 3’ tails (which are covalently joined to the 3’ ends of individual single- stranded polynucleotide fragments) to the homo-polymeric sequence of the 3’overhang ends of the attenuator-adaptor molecules, thereby forming a nick between the 3’ ends of the non-template 3’ tails and the 5’ ends of the first strands of the double-stranded attenuator-adaptor molecules. In some embodiments, the method comprises contacting the nick with a ligase enzyme thereby generating a single-stranded polynucleotide fragment covalently joined at one end to a homo-polymeric sequence and at least one universal adaptor sequence, wherein the homo-polymeric sequence and the at least one universal adaptor sequence is hybridized to the second strand of the attenuator-adaptor molecule. In some embodiments, the ligase enzyme comprises a bacteriophage DNA ligase, including a T3, T4 or T7 DNA ligase. In some embodiments, the ligase comprises a thermal stable DNA ligase including a Taq DNA ligase, a Tfu DNA ligase or a DNA ligase from Thermococcus nautili. In some embodiments, the ligase comprises a recombinant thermal tolerant T4 DNA ligase (e.g., Hi-T4 DNA ligase from New England Biolabs®, catalog # M2622S). In some embodiments, the method comprises removing the second strand of the attenuator-adaptor molecule by denaturation and retaining the single-stranded polynucleotide fragment covalently joined at one end to a homo-polymeric sequence and at least one universal adaptor sequence.

[0279] FIG. 24A shows an embodiment for a method to append at least one universal adaptor sequence to one end of a single-stranded polynucleotide fragment using a nontemplate tailing enzyme and a plurality of single-stranded attenuator-adaptor molecules. FIGs 24A-24B include steps of the same attenuator-mediated workflow. Exemplary' methods are described in U. S. patent Nos. 9,896,709; 10,731,194; 11,118,207; and 12,104,194, all entitled ‘Methods and Compositions for Size-Controlled Homopolymer Tailing of Substrate Polynucleotides by a Nucleic Acid Polymerase’. The contents of the aforementioned issued patents are hereby expressly incorporated by reference in their entireties. In some embodiments, the attenuator-mediated method can be conducted using reagents contained in an Adaptase kit (e.g., xGen Adaptase Module kit commercially-available from Integrated DNA Technologies (IDT) (catalog # 10009826) and following the manufacturer’s instructions.

[0280] In some embodiments, the method shown in FIG. 24A comprises contacting a plurality' of single-stranded polynucleotide fragments with a plurality of single-stranded attenuator-adaptor molecules, a plurality of non-template (e.g., template-independent) tailing enzymes and a plurality of nucleotides under a condition suitable for catalyzing addition of a non-template DNA tails to the 3’ ends of the single- stranded polynucleotide fragments thereby generating a plurality of single-stranded 3 ’-tailed polynucleotide fragments eachcarrying a non-template 3’ tail. In some embodiments, the single-stranded attenuator-adaptor molecule comprises an oligonucleotide that associates with the non-template 3’ tail sequence where the single-stranded attenuator-adaptor molecule controls the number of added nucleotides that form part of the non-template 3’ tail. In some embodiments, individual single-stranded attenuator-adaptor molecules comprise a sequence that is complementary' to least one universal adaptor sequence and homo-polymeric sequence (e g., GGGGGG in FIG.24 A). In some embodiments, the 5’ end of single-stranded attenuator-adaptor molecule is phosphorylated. In some embodiments, the terminal 3’ end of the single-stranded attenuatoradaptor molecule comprises a blocking moiety that inhibits polymerase-catalyzed extension (e.g., a non-extendible terminal 3’ end) (solid rectangles in FIG. 24A). The non-template 3’ tails are covalently joined to the 3’ ends of individual single-stranded polynucleotide fragments. In some embodiments, the plurality of a template-independent tailing enzyme comprises terminal deoxynucleotidyl transferase (TdT), E. coli RNA-specific Poly(A) polymerase, S. pombe RNA-specific poly(U) polymerase or yeast poly(a) polymerase. In some embodiments, the plurality of nucleotides comprises one type of a nucleotide, for example dATP, dGTP, dTTP or dCTP. In some embodiments, the condition is suitable for hybridizing at least a portion of the single-stranded attenuator-adaptor molecule to the non-template 3’ tail. In some embodiments, the plurality of nucleotides comprises one type of a nucleotide, for example dATP, dGTP, dTTP or dCTP.

[0281] FIG. 24B shows a continuation of the method shown in FIG. 24A, wherein the 3’ non-template tail (CCCCCC) is hybridized to the single-stranded attenuator-adaptor molecule. Exemplary methods are described in U. S. patent Nos. 9,896,709; 10,731,194; 11,118,207; and 12,104,194, all entitled ‘Methods and Compositions for Size-Controlled Homopolymer Tailing of Substrate Polynucleotides by a Nucleic Acid Polymerase’, The contents of the aforementioned issued patents are hereby expressly incorporated by reference in their entireties.

[0282] In some embodiments, the method (shown in FIG. 24B) further comprises hybridizing the non-template 3’ tails (which are covalently joined to the 3’ ends of individual single- stranded polynucleotide fragments) to the homo-polymeric sequence of the single-stranded attenuator-adaptor molecules, thereby forming single-stranded polynucleotide fragments having a duplex portion at one end, wherein the duplex portion comprises the homo-polymeric sequence hybridized to the single-stranded attenuator-adaptor molecule that forms a 3’ recessed region. In some embodiments, the method comprises contacting the recessed 3’ region with a DNA polymerase and a plurality of nucleotides to conduct anextension reaction, using the single-stranded attenuator-adaptor molecule as a template molecule, thereby generating a universal adaptor sequence (dashed arrow in FIG. 24B) which is covalently joined to the homo-polymeric sequence. In some embodiments, the extension reaction generates a plurality of single-stranded polynucleotide fragments each covalently joined at one end to a homo-polymeric sequence and at least one universal adaptor sequence. In some embodiments, the DNA polymerase comprises a polymerase enzyme having proofreading activity (e.g., high fidelity DNA polymerases), including and without limitation Q5® DNA polymerase, Kapa® HiFi Polymerase, Pfu Ultra polymerase, Phusion® polymerase, Platinum® Taq HiFi DNA polymerase, and AccuPrime™ Pfx DNA polymerase.

[0283] In some embodiments, the method comprises removing the single-stranded attenuator-adaptor molecules by denaturation and retaining the single-stranded polynucleotide fragments covalently joined at one end to a homo-polymeric sequence and at least one universal adaptor sequence.Methods for Circularizing Single-Stranded Linear Library Molecules and Immobilizing on a Capture Support Comprising Receptor Moieties

[0284] The present disclosure provides methods for generating a plurality of single-stranded circular library molecules from the plurality of single-stranded linear library molecules.Circularizing Single-Stranded Library Molecules with Soluble Splint Oligonucleotides or Target-Specific Bait / Splint Capture Primers

[0285] The disclosure provides methods of circularizing the single-stranded library molecules described herein. The plurality of single- stranded linear library molecules can be contacted with a plurality of soluble splint oligonucleotides or target-specific bait / splint capture primers to generate a plurality of single-stranded open circle library molecules insolution wherein individual open circle library molecules have a nick (e.g., FIGS. 27A-35B). In some embodiments, the nicks of individual single-stranded open circle library molecules are enzymatically ligatable.

[0286] In some embodiments, the single-stranded linear library molecules comprise an insert sequence and at least one universal adaptor sequence joined to either the 5’ end or the 3’ end of the insert sequence. FIGS. 25-35B show various embodiments of single-stranded linear library molecules and various embodiments of soluble splint oligonucleotides and target-specific bait / splint capture primers.

[0287] FIG. 25 shows an embodiment of a method for circularizing a single-stranded linear library molecule. In some embodiments, one end of the single-stranded linear library molecule includes a homopolymer (e.g., homopolymer-A) sequence and at least one uracil base which is cleaved with a cleaving reagent (e.g., USER® II) comprising an Antarctic Themiolabile Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease III. In some embodiments, the single-stranded linear library molecule comprises an insert region and a universal adaptor comprising a terminal end having a homopolymer sequence (e.g., AAAA) and at least one uracil. The single-stranded linear library molecule can be contacted with a cleaving reagent that can cleave the uracil. In some embodiments, the cleaving reagent comprises USER II (commercially-available from New England Biolabs) which includes an Antarctic Thermolabile Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease III. The cleaving reagent removes the homopolymer sequence (e.g., AAAA) and uracil. The single- stranded linear library molecule can be circularized by hybridizing the single-stranded linear library molecule to a splint oligonucleotide, thereby forming an open circle library molecule having a nick. The nick can be contacted with a ligase enzyme to form a covalently closed circular library molecule. In some embodiments, the splint oligonucleotide comprises a sequence that hybridizes to an end portion of the universal adaptor (e.g., universal adaptor sequence for binding a reverse sequencing primer (REV seq)) and a random sequence (e.g., NNN) that can hybridize to an end portion of the insert region of the same single-stranded linear library molecule. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the ligase enzyme comprises a bacteriophage DNA ligase, including a T3, T4 or T7 DNA ligase. In some embodiments, the ligase comprises a thermal stable DNA ligase including a Taq DNA ligase, a Tfu DNA ligase or a DNA ligase from Thermococcus nautili. In some embodiments, the ligase comprises a recombinant thermal tolerant T4 DNA ligase (e.g., Hi-T4 DNA ligase from New England Biolabs, catalog # M2622S).

[0288] FIG. 26 shows an embodiment of a method for circularizing a single-stranded linear library molecule. In some embodiments, one end of the linear library molecule includes a homopolymer (e.g. homopolymer-A) sequence and at least one uracil base which can form a 5’ flap structure when the linear library molecule is hybridized to a splint oligonucleotide. In some embodiments, the linear library molecule comprises an insert region and a universal adaptor comprising a terminal end having a homopolymer sequence (e.g., AAAA) and at least one uracil. The linear library molecule can be circularized by hybridizing the linearlibrary molecule to a splint oligonucleotide thereby forming an open circle library molecule having a 5’ overhang flap structure that is cleavable with a flap cleaving reagent such as a structure specific 5’ flap endonuclease. The 5’ overhang flap structure cleaved by contacting the 5’ flap with a flap cleaving reagent to form an open circle library molecule having a nick. In some embodiments, the flap cleaving reagent comprises a FEN1 enzyme. The nick can be contacted with a ligase enzyme to form a covalently closed circular library molecule. In some embodiments, the splint oligonucleotide comprises a sequence that hybridizes to an end portion of the universal adaptor (e g., universal adaptor sequence for binding a reverse sequencing primer (REV seq)) and a random sequence (e.g., NNN) that can hybridize to an end portion of the insert region of the same linear library molecule. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the ligase enzyme comprises a bacteriophage DNA ligase, including a T3, T4 or 17 DNA ligase. In some embodiments, the ligase comprises a thermal stable DNA ligase including a Taq DNA ligase, a Tfu DNA ligase or a DNA ligase from Thermococcus nautili. In some embodiments, the ligase comprises a recombinant thermal tolerant T4 DNA ligase (e.g., Hi-T4 DNA ligase from New England Biolabs, catalog # M2622S).

[0289] FIG. 27A part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence, and (ii) an insert sequence. In some embodiments, one side of the insert sequence is joined to a universal sequence while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated. In some embodiments, the universal sequence comprises any one or any combination of two or more of a universal sequence for binding a forward sequencing primer, one or more sample index sequences, a universal sequence for binding a pinning primer, a universal sequence for binding a reverse sequencing primer and / or a universal sequence for binding a capture primer.

[0290] FIG. 27 A part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule shown in part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence, and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the insertsequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a non-extendible 3’ end). In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 3’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence of the linear library molecule and inhibit hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specific bait / probe.

[0291] As used herein, a “target-specific bait / probe” refers to an oligonucleotide comprising an affinity moiety at the one end (e.g. the 5’ end) and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0292] FIG. 27 A part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule shown in part (i) with a target-specific bait / splint capture primer. A “target-specific bait / splint capture primer” refers to a primer comprising an affinity moiety, and a sequence complementary to and capable of hybridizing to at least a portion of the insert sequence of the library molecule. The target-specific bait / splint capture primer can also comprise a sequence complementary to at least a portion of a universal sequence, for example a universal sequence appended to a library molecule using an adapter.

[0293] In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the universal sequence of a single-stranded linear library molecule, and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof) and a universal adaptor sequence (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprise an affinity moiety at the 5’ end. In some embodiments, the target-specific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprisinga 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0294] FIG, 27B part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, and (ii) a universal sequence. In some embodiments, one side of the insert sequence is joined to a universal sequence while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated. In some embodiments, the universal sequence comprises any one or any combination of two or more of a universal sequence for binding a forward sequencing primer, one or more sample index sequences, a universal sequence for binding a pinning primer, a universal sequence for binding a reverse sequencing primer and / or a universal sequence for binding a capture primer.

[0295] FIG. 27B part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule shown in part (i) with a soluble splint oligonucleotide and a target-specific bait / probe as described herein. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence of the same linear library molecule as the insert sequence, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library- molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. Insome embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a nonextendible 3’ end). In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 5’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence of the linear library molecule and inhibit hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specifi c bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0296] FIG. 27B part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule shown in part (i) with a target-specific bait / splint capture primer as described herein. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to at least a portion of the universal sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a targetspecific sequence (or a complementary sequence thereof) and a universal adaptor sequence (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the target-specific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0297] FIG. 28A part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a forward sequencing primer (FWD seq), and (ii) an insert sequence. In some embodiments, one side of the insert sequence is joined to a universal sequence for binding a forward sequencing primer (FWD seq) while the other side of the insert sequence is not joined to a uni versal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated.

[0298] FIG. 28A part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule shown in part (i) with a soluble splint oligonucleotide and a target-specific bait / probe as described herein. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq), and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonucleaseunder a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the noncleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a non-extendible 3’ end). In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 3’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence for binding a forward sequencing primer (FWD seq) of the linear library molecule and inhibit hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specifi c bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0299] FIG. 28A part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single- stranded linear library molecule shown in part (i) with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the targetspecific bait / splint capture primer to at least a portion of the universal sequence for binding aforward sequencing primer (FWD seq), and suitable for hybridizing a 3’ portion of the targetspecific bait / splint capture primer to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof) and a universal sequence for binding a forward sequencing primer (FWD seq) (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the targetspecific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture pri m er can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0300] FIG. 28B part (i) is a schematic showing one embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, and (ii) a universal sequence for binding a forward sequencing primer (FWD seq). In some embodiments, one side of the insert sequence is joined to a universal sequence for binding a forward sequencing primer (FWD seq) while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated.

[0301] FIG. 28B part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a solublesplint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a nonextendible 3’ end). In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 5’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence for binding a forward sequencing primer (FWD seq) of the linear library molecule and inhibits hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specific bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, theextendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0302] FIG. 28B part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the targetspecific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof) and a universal sequence for binding a forward sequencing primer (FWD seq) (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the target-specific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the noncleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library’ molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ fl p endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure andligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0303] FIG. 29A part (i) is a schematic showing one embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (i) a universal sequence for binding a forward sequencing primer (FWD seq), and (ii) an insert sequence. In some embodiments, one side of the insert sequence is joined to the universal sequence for binding a reverse sequencing primer (REV seq) and the universal sequence for binding a forward sequencing primer (FWD seq), while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated.

[0304] FIG. 29A part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule shown in part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence for binding a reverse sequencing primer (REV seq), and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the noncleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a non-extendible 3’ end). In some embodiments, the 3’portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 3’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence for binding a reverse sequencing primer (REV seq) of the linear library molecule and inhibits hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specific bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target- specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0305] FIG. 29A part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the universal sequence for binding a reverse sequencing primer (REV seq), and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary’ sequence thereof) and a universal sequence for binding a reverse sequencing primer (REV seq) (or a complementary’ sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the targetspecific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0306] FIG. 29B part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), and (iii) a universal sequence for binding a forward sequencing primer (FWD seq). In some embodiments, one side of the insert sequence is joined to a universal sequence for binding a reverse sequencing primer (REV seq) and a universal sequence for binding a forward sequencing primer (FWD seq), while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single- stranded library molecule is phosphorylated.

[0307] FIG. 29B part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhangflap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a nonextendible 3’ end). In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 5’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence for binding a forward sequencing primer (FWD seq) of the linear library molecule and inhibits hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specific bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0308] FIG. 29B part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specificbait / splint capture primer to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the target- specific bait / splint capture primer to at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof) and a universal sequence for binding a forward sequencing primer (FWD seq) (or a complementary’ sequence thereof. In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the targetspecific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0309] FIG. 30A part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), (iii) a random sequence (nnnn) with a sample index sequence, and (iv) an insert sequence. In some embodiments, one side of the insert sequence is joined to the sample index sequence, the random sequence, the universal sequence for binding a forward sequencing primer (FWD seq and the universal sequence for binding a reverse sequencing primer (REV seq). while the other side of the insert sequence is not joined to a universalsequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated.

[0310] FIG. 30A part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence for binding a reverse sequencing primer (REV seq), and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FENl). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a nonextendible 3’ end). In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 3’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP, In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the universal sequence for binding a reverse sequencing primer (REV seq) of the linear library molecule and inhibit hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to atarget-specific bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0311] FIG. 30A part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the universal sequence for binding a reverse sequencing primer (REV seq), and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof) and a universal sequence for binding a reverse sequencing primer (REV seq) (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprise an affinity moiety at the 5’ end. In some embodiments, the targetspecific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circlelibrary molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0312] FIG. 30B part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iv) a random sequence (nnnn) with a sample index sequence. In some embodiments, one side of the insert sequence is joined to (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (ii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iv) a random sequence (nnnn) with a sample index sequence, while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated.

[0313] FIG. 30B part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the sample index sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flapendonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a non-extendible 3’ end). In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 5’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the sample index sequence of the linear library molecule and inhibit hybridization of another oligonucleotide to the same sample index sequence. In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the sample index sequence and and at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the linear library molecule. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specific bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0314] FIG. 30B part (iii) is a schematic showing one embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to the sample index sequence and optionally, at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the same linear library molecule, therebygenerating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the targetspecific bait / splint capture primer comprises an oligonucleotide comprising a target- specific sequence (or a complementary sequence thereof) and a sample index sequence and optionally the random sequence (nnnn) (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof), a sample index sequence and optionally the random sequence (nnnn) (or a complementary sequence thereof), and a universal sequence for binding a forward sequencing primer (FWD seq) (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the target-specific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0315] FIG. 31 A part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) a sample index sequence, (ii) a universal sequence for binding a reverse sequencing primer (REV seq), (iii) a universal sequence for binding a forward sequencing primer (FWD seq), and (iv) an insert sequence. In some embodiments, one side of the insert sequence is joined to the universal sequence for binding a forward sequencing primer (FWD seq), the universal sequence for binding a reverse sequencing primer (REV seq) and the sample index sequence, while the other side of theinsert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single- stranded library molecule is phosphorylated.

[0316] FIG. 31 A part (ii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the sample index sequence, and optionally, at least a portion of the universal sequence for binding a reverse sequencing primer (REV seq), and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library / molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a non-extendible 3’ end). In some embodiments, the 3’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 3’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the random sequence (NNN) can be 3-20 nucleotides in length. In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a sequence that can hybridize to the sample index sequence of the linear library molecule and inhibit hybridization of another oligonucleotide to the same universal sequence. In some embodiments, the 5’ portion of the soluble splint oligonucleotidecomprises a sequence that can hybridize to the sample index sequence and the universal sequence for binding a reverse sequencing primer (REV seq) of the linear library molecule. In some embodiments, the method comprises hybridizing at least a portion of the insert sequence to a target-specific bait / probe. In some embodiments, the target-specific bait / probe comprises an oligonucleotide comprising an affinity moiety at the 5’ end and a target-specific sequence. In some embodiments, the target-specific bait / probe comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ end of the target-specific bait / probe can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the single-stranded linear library molecule can hybridize first to the soluble splint oligonucleotide and then the target-specific bait / probe, or the reverse order. In some embodiments, the single-stranded linear library molecule can hybridize to the soluble splint oligonucleotide and the target-specific bait / probe essentially simultaneously.

[0317] FIG. 31A part (iii) shows an embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a target-specific bait / splint capture primer. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the target-specific bait / splint capture primer to at least a portion of the sample index sequence, and optionally, at least a portion of the universal sequence for binding a reverse sequencing primer (REV seq), and suitable for hybridizing a 3’ portion of the target-specific bait / splint capture primer to at least a portion of the insert sequence of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the targetspecific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary' sequence thereof) and a sample index sequence (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an oligonucleotide comprising a target-specific sequence (or a complementary sequence thereof), a sample index sequence (or a complementary sequence thereof), and a universal sequence for binding a reverse sequencing primer (REV seq) (or a complementary sequence thereof). In some embodiments, the target-specific bait / splint capture primer comprises an affinity moiety at the 5’ end. In some embodiments, the targetspecific bait / splint capture primer comprises a terminal 3’ end that permits polymerase-catalyzed extension (e.g., an extendible 3’ end). In some embodiments, the extendible 3’ endof the target-specific bait / splint capture primer can be used to initiate rolling circle amplification and generate a concatemer template molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FEN1). In some embodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the non-cleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule.

[0318] FIG. 3 IB part (i) shows an embodiment of a single-stranded linear library molecule comprising, in a 5’ to 3’ direction, (i) an insert sequence, (ii) a sample index sequence, (iii) a universal sequence for binding a reverse sequencing primer (REV seq), and (iv) a universal sequence for binding a forward sequencing primer (FWD seq). In some embodiments, one side of the insert sequence is joined to the sample index sequence, the universal sequence for binding a reverse sequencing primer (REV seq), and the universal sequence for binding a forward sequencing primer (FWD seq), while the other side of the insert sequence is not joined to a universal sequence. In some embodiments, the 5’ end of the single-stranded library molecule is phosphorylated.

[0319] FIG. 3 IB part (ii) is a schematic showing one embodiment of a method for generating an open circle library molecule by hybridizing the single-stranded linear library molecule of part (i) with a soluble splint oligonucleotide and a target-specific bait / probe. In some embodiments, the hybridizing is conducted under a condition suitable for hybridizing a 5’ portion of the soluble splint oligonucleotide to at least a portion of the insert sequence, and suitable for hybridizing a 3’ portion of the soluble splint oligonucleotide to at least a portion of the universal sequence for binding a forward sequencing primer (FWD seq) of the same linear library molecule, thereby generating an open circle library molecule having a nick. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the nick can be closed using a ligase enzyme to generate a covalently closed circular library molecule. In some embodiments, the open circle library molecule comprises a 5’ overhang flap structure that is cleavable with a structure specific 5’ flap endonuclease (e.g., FENI). In someembodiments, the 5’ overhang flap structure can be contacted with a 5’ flap endonuclease under a condition suitable for cleaving the 5’ overhang flap structure to generate a newly cleaved 5’ end and a non-cleaved 3’ end, wherein the newly cleaved 5’ end and the noncleaved 3’ end of the same open circle library molecule can form a nick while being hybridized to the soluble splint oligonucleotide. In some embodiments, the nick is enzymatically ligatable. In some embodiments, the open circle library molecule having a 5’ overhang flap structure can be contacted with a flap cleaving reagent comprising a 5’ flap endonuclease and a DNA ligase under a condition suitable for cleaving the 5’ overhang flap structure and ligating together the newly cleaved 5’ end and a non-cleaved 3’ end to generate a covalently closed circular library molecule. In some embodiments, the terminal 3’ end of the soluble splint oligonucleotide comprises a blocking moiety (solid rectangle) that inhibits polymerase-catalyzed extension (e.g., a non-extendible 3’ end). In some embodiments, the 5’ portion of the soluble splint oligonucleotide comprises a random sequence (e.g., NNN) which can hybridize to the 5’ portion of the insert sequence. In some embodiments, the random sequence (NNN) comprises any combination of dATP, dGTP, dCTP, dTTP and / or diTP. In some embodiments, the ...

Claims

CLAIMSWhat is claimed is:1, A method for preparing a plurality of DNA library molecules comprising:a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) conducting a tagmentation reaction by contacting a plurality of input doublestranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising a sequence complementary' to at least a portion of the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end of the individual double-stranded DNA fragment, thereby forming a plurality of double-stranded 5 ’-adaptor-tagged molecules, wherein individual 5 ’adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individual 5 ’-adaptor-tagged molecule;c) denaturing the plurality of double-stranded 5 ’-adaptor-tagged molecules, thereby generating a plurality of single-stranded 5 ’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are not hybridized to the portion of the transfer strand;d) contacting the plurality of single-stranded 5 ’-adaptor- tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual single-stranded 5 ’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules comprising a 3’ template-independent polynucleotide;e) hybridizing the plurality of single-stranded linear DNA library molecules to a plurality of polyN splint capture primers thereby forming a plurality of open circle library splint complexes, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence atone end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety that can bind to a receptor moiety of the capture support of step (a), and wherein individual polyN splint capture primers comprise 3’ ends that are extendible;f) distributing the plurality of open circle library splint complexes onto the capture support, thereby immobilizing the plurality of open circle library splint complexes to the capture support, wherein the distributing is conducted under a condition suitable for binding the affinity moiety to a receptor moiety;g) contacting the plurality of open circle library splint complexes with a closure reagent to generate a plurality of covalently closed circular library molecules, wherein individual covalently closed circular library molecules are hybridized to polyN splint capture primers immobilized to the capture support;h) contacting the plurality of covalently closed circular library molecules with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the polyN splint capture primers using the plurality of covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support; and i) sequencing at least a portion of the plurality of concatemer template molecules.

2. A method for preparing a plurality of DNA library molecules comprising:a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) conducting a tagmentation reaction by contacting a plurality of input doublestranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising a sequence complementary to at least a portion of the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end of the individual double-stranded DNA fragment, thereby forming a plurality of double- stranded 5 ’-adaptor-tagged molecules,wherein individual 5 ’-adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single- stranded gap at a 3’ end of the individual 5 ’-adaptor-tagged molecule;c) denaturing the plurality of double-stranded 5 ’-adaptor-tagged molecules, thereby generating a plurality of single-stranded 5 ’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are not hybridized to the portion of the transfer strand;d) contacting the plurality of single-stranded 5 ’-adaptor-tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual single-stranded 5 ’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules comprising a 3’ template-independent polynucleotide;e) hybridizing the plurality of single-stranded linear DNA library molecules to a plurality of polyN splint capture primers, thereby forming a plurality of open circle library splint complexes, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety that can bind to a receptor moiety of the capture support of step (a), and wherein individual polyN splint capture primers comprise 3’ ends that are extendible;f) contacting the plurality of open circle library splint complexes with a closure reagent generate a plurality of covalently closed circular library molecule complexes, wherein an individual covalently closed circular library molecule is hybridized to a polyN splint capture primer;g) distributing the plurality of covalently closed circular library molecule complexes onto the capture support, thereby immobilizing the plurality of covalently closed circular library molecule complexes to the capture support, wherein the distributing is conducted under a condition suitable for binding the affinity moiety of individual polyN splint capture primers to a receptor moiety; h) contacting the plurality of covalently closed circular library molecules with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the polyN splint capture primers using the covalently closed circular library molecules astemplate molecules thereby, generating a plurality of concatemer template molecules which are immobilized to the capture support; andi) sequencing at least a portion of the plurality of concatemer template molecules.

3. A method for preparing a plurality of DNA library molecules comprising:a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) conducting a tagmentation reaction by contacting a plurality of input doublestranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a n on-transfer strand comprising a sequence complementary to at least a portion of the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual double-stranded DNA fragment comprises a transfer strand covalently attached to a 5’ end the individual double-stranded DNA fragment, thereby forming a plurality of double-stranded 5 ’-adaptor-tagged molecules, wherein individual 5 ’adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individual 5 ’-adaptor-tagged molecule;c) denaturing the plurality of double-stranded 5 ’-adaptor-tagged molecules, thereby generating a plurality of single-stranded 5 ’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are not hybridized to the portion of one of the transfer strands;d) contacting the plurality of single-stranded 5 ’-adaptor-tagged molecules with a template-independent DNA polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual single-stranded 5 ’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules comprising a 3’ template-independent polynucleotide;e) distributing a plurality of polyN splint capture primers onto the capture support, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequenceat another end, and an affinity moiety, wherein the affinity moiety' that can bind to a receptor moiety of the capture support of step (a), thereby immobilizing plurality of the polyN splint capture primers to the capture support, and wherein f individual polyN splint capture primers comprise 3’ ends that are extendible; f) distributing the plurality of single-stranded linear DNA library molecules onto the capture support under a condition suitable for hybridizing an individual single-stranded linear DNA library molecules to an individual polyN splint capture primer, thereby forming a plurality of open circle library splint complexes;g) contacting the plurality of open circle library splint complexes immobilized to the capture support with a closure reagent to generate a plurality of covalently closed circular library molecules, wherein individual covalently closed circular library molecules are hybridized to individual polyN splint capture primers; h) contacting the plurality of covalently closed circular library molecules with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ ends of the polyN splint capture primers using the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support; andi) sequencing at least a portion of the plurality of concatemer template molecules.4, The method of any one of claims 1-3, wherein individual open circle library splint complexes comprise a nick and / or a 5’ flap structure, and wherein the closure reagent comprises a reagent that closes the nick and / or 5’ flap structure.

5. A method for preparing a plurality of DNA library molecules comprising:a) providing a capture support comprising (i) a support coated with at least one layer of hydrophilic polymer coating and (ii) a plurality of receptor moieties embedded in the at least one layer of hydrophilic polymer coating; b) conducting a tagmentation reaction by contacting a plurality of input doublestranded DNA molecules with a plurality of transposomes, wherein individual transposomes comprise a transposase enzyme and a transposon end sequence comprising (i) a transfer strand comprising a transfer end sequence and at least one universal adaptor sequence, and (ii) a non-transfer strand comprising asequence complementary to the transfer end sequence, thereby generating a plurality of double-stranded DNA fragments, wherein an individual doublestranded DNA fragment comprises a transfer strand covalently attached to a 5’ end of the individual double-stranded DNA fragment, thereby forming a plurality of double-stranded 5’ -adaptor-tagged molecules, wherein individual 5 ’adaptor-tagged molecules comprise the non-transfer strand hybridized to a portion of the transfer strand with a single-stranded gap at a 3’ end of the individuals ’ -adaptor-tagged molecule;c) denaturing the plurality of double-stranded 5 ’-adaptor-tagged molecules, thereby generating a plurality of single-stranded 5 ’-adaptor-tagged molecules and a plurality of single-stranded non-transfer strands that are not hybridized to the portion of the transfer strand;d) distributing a plurality of polyN splint capture primers onto the capture support, wherein individual polyN splint capture primers comprise a single-stranded oligonucleotide comprising an anchor sequence at one end, a bridging sequence at another end, and an affinity moiety, wherein the affinity moiety binds to receptor moiety of the capture support of step (a), thereby immobilizing the plurality of polyN splint capture primers to the support, and wherein the 3’ ends of individual polyN splint capture primers comprise at least one scissile moiety near the 3’ ends,, and wherein a terminal 3’ ends of individual polyN splint capture primers comprise blocking moieties that inhibit polymerase-catalyzed extension of the 3’ ends;e) distributing the plurality of single-stranded 5 ’-adaptor-tagged molecules onto the capture support under a condition suitable for hybridizing individual single-stranded 5 ’-adaptor-tagged molecules to individual polyN splint capture primers immobilized to the capture support, thereby forming a plurality of immobilized 5 ’-adaptor-tagged molecules, wherein individual immobilized 5 ’-adaptor-tagged molecule do not form open circle library splint complexes while hybridized to an immobilized capture primer;f) contacting the plurality of immobilized 5 ’-adaptor-tagged molecules with a template-independent DN A polymerase and a plurality of nucleotides under a condition suitable for appending a template-independent polynucleotide to a 3’ end of an individual 5 ’-adaptor-tagged molecule, thereby generating a plurality of single-stranded linear DNA library molecules immobilized to the capturesupport, wherein individual single-stranded linear DNA library molecules comprise a 3’ template-independent polynucleotide, wherein individual single-stranded linear DNA library molecules are hybridized to individual immobilized polyN splint capture primers to form a plurality of open circle library / splint complexes comprising a nick, wherein the plurality of open circle library splint complexes are immobilized to the capture support;g) contacting the plurality of open circle library splint complexes with a closure reagent that can close the nick to generate a plurality of covalently closed circular library molecules, wherein individual covalently closed circular library molecules are hybridized to individual polyN splint capture primers immobilized to the capture support;h) contacting the plurality of polyN splint capture primers that are hybridized to the covalently closed circular library molecules with an unblocking reagent that converts the scissile moiety into an abasic site and the blocking moieties of the polyN splint capture primers into 3’ extendible ends;i) contacting the plurality of covalently closed circular library with a rolling circle amplification reagent and conducting a rolling circle amplification reaction under a condition suitable to extend the 3’ extendable ends of the polyN splint capture primers using the covalently closed circular library molecules as template molecules, thereby generating a plurality of concatemer template molecules which are immobilized to the capture support; andj) sequencing at least a portion of the plurality of concatemer template molecules.

6. The method of any one of claims 1-5, wherein individual single-stranded linear DNA library molecules comprise at least one universal adaptor sequence and a transfer end sequence from the transfer strand.

7. The method of any one of claims 1-6, wherein the tagmentation reaction is conducted in solution.

8. The method of any one of claims 5-7, wherein the at least one scissile moiety can be converted into at least one abasic site.

9. The method of any one of claims 1-8, wherein the receptor moiety comprises streptavidin or avidin, or a derivative thereof.

10. The method of any one of claims 1-9, wherein the affinity moiety comprises biotin, desthiobiotin or iminobiotin.

11. The method of any one of claims 1-10, wherein the tagmentation reaction comprises a Tn5 transposase-based tagmentation reaction.

12. The method of any one of claims 1-11, wherein at least one universal adaptor sequence comprises any one or any combination of two or more of:a) a universal sequence for binding a forward sequencing primer (FWD seq); b) a universal sequence for binding a reverse sequencing primer (REV seq); c) at least one sample index sequence;d) a universal sequence for binding a polyN splint capture primer; and / or e) a universal sequence for binding a pinning primer.

13. The method of claim 12, wherein the at least one sample index sequence comprises a random sequence, optionally wherein the random sequence is 3-7 nucleotides long.

14. The method of any one of claims 1-13, wherein the rolling circle amplification reagent comprises: (i) a plurality of strand-displacing polymerases; and (ii) a plurality of nucleotides comprising dATP, dGTP, dCTP, dTTP and / or dUTP.

15. The method of any one of claims 1-14, wherein the plurality of concatemer template molecules can be sequenced essentially simultaneously or using batch sequencing.

16. A method for preparing a plurality of nucleic acid library molecules, comprising:a) providing a plurality of double-stranded input polynucleotides;b) generating a plurality of double-stranded polynucleotide fragments from the plurality of double-stranded input polynucleotides;c) joining at least one universal adaptor sequence to only one end of individual double-stranded polynucleotide fragments, thereby generating a plurality ofdouble-stranded library molecules, wherein individual double-stranded library molecules comprise an insert sequence joined to at least one universal adaptor sequence;d) generating a plurality of single-stranded library molecules from the plurality of double-stranded library molecules;e) generating a plurality of single-stranded open circle library molecules from the plurality of single- stranded linear library molecules, wherein individual single-stranded open circle library molecules comprise a nick;f) immobilizing the plurality of open circle library molecules to a support, thereby generating a plurality of immobilized open circle library molecules each having a nick, and ligating the nicks, thereby generating a plurality of immobilized covalently closed circle library molecules;g) generating a plurality of immobilized concatemer template molecules by conducting rolling circle amplification on the support using the immobilized covalently closed circular library molecules as template molecules to generate the plurality of immobilized concatemer template molecules; and h) sequencing the plurality of immobilized concatemer template molecules.

17. The method of claim 16, wherein the plurality of single-stranded library molecules are generated by denaturing the plurality of double-stranded library molecules.

18. The method of claim 16 or 17, wherein the at least one universal adaptor sequence comprises any one or any combination of two or more of: (i) a universal sequence for binding a forward sequencing primer (FWD seq); (ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence; (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer.

19. The method of claim 18, wherein the at least one sample index sequence comprises a random sequence, optionally wherein the random sequence is 3-7 nucleotides long.

20. A method for preparing a plurality of nucleic acid library molecules, comprising:a) providing a plurality of input polynucleotides comprising a plurality of double-stranded input polynucleotides;b) generating a plurality of double-stranded polynucleotide fragments from the plurality of double-stranded input polynucleotides;c) generating a plurality of single-stranded library molecules from the plurality of double-stranded library molecules;d) joining at least one universal adaptor sequence to only one end of an individual single-stranded polynucleotide fragment, thereby generating a plurality of single- stranded library molecules, wherein individual single-stranded library molecules comprise an insert sequence joined to at least one universal adaptor sequence;e) generating a plurality' of single-stranded open circle library molecules from the plurality of single- stranded linear library molecules, wherein individual single-stranded open circle library molecules comprise a nick;f) immobilizing the plurality of single- stranded open circle library molecules to a support, thereby generating a plurality of immobilized open circle library molecules each having a nick, and ligating the nicks, thereby generating a plurality of immobilized covalently closed circle library molecules;g) generating a plurality of immobilized concatemer template molecules by conducting rolling circle amplification on the support using the plurality of immobilized covalently closed circular library molecules as template molecules to generate the plurality of immobilized concatemer template molecules; andh) sequencing the plurality of immobilized concatemer template molecules.

21. The method of claim 20, wherein the at least one universal adaptor sequence that is joined to only one end of individual single-stranded polynucleotide fragments comprises any one or any combination of two or more of: (i) a universal sequence for binding a foiward sequencing primer (FWD seq); ( ii) a universal sequence for binding a reverse sequencing primer (REV seq); (iii) at least one sample index sequence; (iv) a universal sequence for binding a capture primer; and / or (v) a universal sequence for binding a pinning primer.

22. The method of claim 21, wherein the at least one sample index sequence comprises a random sequence, optionally wherein the random sequence is 3-7 nucleotides long.

23. The method of any one of claims 1-22, wherein the sequencing comprises: a) contacting a first plurality of polymerases to (i) the plurality of concatemer template molecules and (ii) a plurality of sequencing primers, wherein the contacting is conducted under a condition suitable to bind the first plurality of polymerases to the plurality of concatemer template molecules and the plurality of nucleic acid primers, thereby forming a first plurality of complexed polymerases each comprising a polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises an concatemer template molecule hybridized to sequencing primer;b) contacting the first plurality of complexed polymerases with a plurality of multivalent molecules to form a plurality of multivalent-binding complexes, wherein individual multivalent molecules in the plurality comprise a core attached to multiple nucleotide arms and individual nucleotide arms are attached to a nucleotide moiety, wherein the contacting is conducted under a condition suitable for binding complementary nucleotide moieties of the multivalent molecules to at least two of the first plurality of complexed polymerases, thereby forming a plurality of multivalent-binding complexes, and the condition is suitable for inhibiting incorporation of the complementary nucleotide moieties into the nucleic acid primers of the plurality of multivalent-binding complexes;c) detecting the plurality of multivalent-binding complexes; andd) identifying the nucleobase of the complementary nucleotide moieties in the plurality of multivalent-binding complexes, thereby determining the sequence of the nucleic acid templ ate molecul es.

24. The method of claim 23, comprising:e) dissociating the plurality of multivalent-binding complexes by removing the first plurality of polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes;f) contacting the plurality of the nucleic acid duplexes retained at step (e) with a second plurality of a polymerases under a condition suitable for binding the second plurality of polymerases to the plurality of the nucleic acid duplexes,thereby forming a second plurality of complexed polymerases, individual complexed polymerases comprising a polymerase bound to a nucleic acid duplex; andg) contacting the second plurality of second polymerases with a plurality of nucleotides, wherein the contacting is conducted under a condition suitable for binding complementary nucleotides from the plurality of nucleotides to at least two of the complexed polymerases, thereby forming a plurality of nucleotide- binding complexes, and the condition is suitable for promoting nucleotide incorporation of the bound complementary nucleotides into the nucleic acid primers of the nucleotide-binding complexes.

25. The method of claim 24, further comprising: (h) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide- complexed polymerases.

26. The method of claim 25, further comprising:h) detecting the complementary nucleotides which are incorporated into the nucleic acid primers of the nucleotide-complexed polymerases; andi) identifying the nucleobases of the complementary nucleotides which are incorporated into the primers of the nucleotide-complexed polymerases.

27. The method of claim 26, wherein the complementary nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases are not detected or identified.

28. The method of any one of claims 23-27, wherein contacting the first plurality of complexed polymerases with the plurality of multivalent molecules of step (b) is conducted in the presence of a non-catalytic divalent cation that inhibits polymerase- catalyzed nucleotide incorporation, optionally wherein the non-catalytic divalent cation comprises strontium or barium.

29. The method of any one of claim 24-27, wherein the contacting the second plurality of complexed polymerases with the plurality of nucleotides of step (g) is conducted in the presence of a catalytic divalent cation that promotes polymerase-catalyzed nucleotideincorporation, optionally wherein the catalytic divalent cation comprises magnesium or manganese.

30. The method of any one of claims 23-29, wherein the plurality of concatemer template molecules in step (a) comprise clonally amplified concatemer template molecules.

31. The method of any one of claims 23-30, wherein individual concatemer template molecules in the plurality of step (a) comprise a concatemer template molecule having two or more tandem copies of a target sequence.

32. The method of any one of claims 23-31, wherein the concatemer template molecules in the plurality of concatemer template molecules in step (a) comprise the same target sequence or different target sequences.

33. The method of any one of claims 23-32, wherein individual multivalent molecules in the plurality of multivalent molecules comprise: (a) a core; and (b) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide moiety, wherein the core is attached to the plurality of nucleotide arms via their core attachment moiety, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide moiety.

34. The method of claim 33, wherein the linker comprises an aliphatic chain having 2-6 subunits or an oligo ethylene glycol chain having 2-6 subunits.

35. The method of claim 33 or 34, wherein the plurality of nucleotide arms attached to a given core have the same type of nucleotide moieties, and wherein the types of nucleotide moieties comprise dATP, dGTP, dCTP, dTTP or dUTP.

36. The method of claim 33 or 34, wherein the plurality of multivalent molecules comprise one type of a multivalent molecule wherein each multivalent molecule in the plurality has the same type of nucleotide moiety selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

37. The method of claim 33 or 34, wherein the plurality of multivalent molecules comprise a mixture of any combination of two or more types of multivalent molecules each typehaving nucleotide moieties selected from a group consisting of dATP, dGTP, dCTP dTTP and / or dUTP.

38. The method of any one of claims 23-37, wherein at least one multivalent molecule in the plurality of multivalent molecules is labeled with a fluorophore.

39. The method of any one of claims 23-38, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises a core that is labeled with a fluorophore.

40. The method of any one of claims 23-39, wherein at least one multivalent molecule in the plurality of multivalent molecules comprises one or more nucleotide moieties that are labeled with a fluorophore.

41. The method of any one of claims 24-40, wherein individual nucleotides in the plurality of nucleotides in step (g) comprise an aromatic base, a five carbon sugar, and 1-10 phosphate groups.

42. The method of any one of claims 24-41, wherein the plurality of nucleotides of step (g) comprise one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP, or comprise a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and / or dUTP.

43. The method of any one of claims 24-42, wherein at least one of the nucleotides in the plurality of nucleotides in step (g) is labeled with a fluorophore.

44. The method of any one of claims 24-42, wherein the plurality of nucleotides in step (g) lack a fluorophore label.

45. The method of any one of claims 24-44, wherein at least one of the nucleotides in the plurality of nucleotides of step (g) comprises a removable chain terminating moiety attached to the 3’ carbon position of the sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group,aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3 ’OH moiety on the sugar group.

46. The method of any one of claims 23-45, further comprising forming a plurality of binding complexes, comprising the steps:a) binding a first sequencing primer, a first 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 moiety of the first multivalent molecule binds to the first polymerase; andb) binding a second sequencing primer, a second polymerase, and the first multivalent molecule to a second portion of the immobilized concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide moiety of the first multivalent molecule binds to the second polymerase,wherein the first and second binding complexes which include the same multivalent molecule form an avidity complex.

47. The method of any one of claims 23-46, further comprising:a) contacting the first plurality of polymerases and the plurality of sequencing primers with different portions of a concatemer template molecule to form at least first and second complexed polymerases on the concatemer template molecule;b) contacting a plurality of multivalent molecules to the at least first and second complexed polymerases, under conditions suitable to bind a single multivalent molecule from the plurality to the first and second complexed polymerases, wherein at least a first nucleotide moiety of the single multivalent molecule is bound to the first complexed polymerase which includes a first sequencing primer hybridized to a first portion of the concatemer template molecule, thereby forming a first binding complex, and wherein at least a second nucleotide moiety of the single multivalent molecule is bound to the secondcomplexed polymerase which includes a second sequencing primer hybridized to a second portion of the concatemer template molecule, thereby forming a second binding complex, and* wherein the contacting is conducted under a condition suitable to inhibit polymerase-catalyzed incorporation of the bound first and second nucleotide moieties in the first and second binding complexes, and• wherein the first and second binding complexes which are bound to the same multivalent molecule form an avidity complex;c) detecting the first and second binding complexes on the concatemer template molecule; andd) identifying the first nucleotide moiety in the first binding complex thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide moiety in the second binding complex thereby determining the sequence of the second portion of the concatemer template molecule.

Images