Pcr-free library preparation using double stranded splint adapters and methods of use

Abstract

The present disclosure provides compositions, including kits, comprising nucleic acid double-stranded splint adapters, as well as methods, such as PCR-free workflows, employing the double-stranded splint adapters. The double-stranded splint adaptor (200) can be used in a one-pot multienzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor (200) comprises a first splint chain (long splint chain (300)) and a second splint chain (short splint chain (400)), wherein the first splint chain and the second splint chain hybridize together to form the double-stranded splint adaptor (200) having a double-stranded region and two flanked single-stranded regions. The second splint chain (400) carries a new adapter sequence to be introduced, such as, for example, a universal binding sequence and / or an index sequence.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 358,491, filed on July 5, 2022, and U.S. Provisional Patent Application No. 63 / 508,833, filed on June 16, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the Electronic Sequence Listing (ELEM-013_001WO_SeqListing_ST26.xml; size 50,242 bytes; and creation date June 30, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure provides compositions comprising nucleic acid double-stranded splint adapters and methods for preparing nucleic acid libraries using double-stranded splint adapters. The double-stranded splint adapters can hybridize with library molecule portions to form a library-splint complex with a gap, wherein the gap can be connected to form a covalently closed circular molecule, which can be subjected to downstream amplification and sequencing workflows. Background Art

[0006] The transition from traditional Sanger sequencing methods to next-generation sequencing methods has reduced the cost of sequencing, but next-generation sequencing methods still have significant limitations. In one aspect, the sequencing reads generated by available sequencing platforms are numerous but relatively short and may need to be reassembled into a complete target sequence by calculation. Available assembly methods may be slow, laborious, expensive, computationally demanding and / or unsuitable for groups of similar individuals (e.g., viruses). This is especially true for sequencing of complex genomes. Assembly is challenging, in part due to the ever-expanding sequencing data sets associated with the assembly of short reads. Such data sets can put a lot of pressure on computer clusters. For example, de novo assembly may require that the sequencing reads (or k-mers derived therefrom) be stored in random access memory (RAM) simultaneously. For large data sets, this requirement is not trivial. In addition, even when assembly is possible, key haplotype information is usually not recoverable. In fact, the inherent limitations of available technologies have hindered improvements to overcome the shortcomings of current sequencing technologies. Therefore, improved sequencing methods and related assembly technologies are needed, which reduce the time and / or computational requirements required to obtain accurate sequences. Summary of the invention

[0007] The present disclosure provides a method for forming a plurality of library-splint complexes (500), the method comprising: (a) providing a plurality of double-stranded splint adaptors (200), wherein each of the plurality of double-stranded splint adaptors (200) comprises a first splint strand (300) hybridized to a second splint strand (400), wherein the double-stranded splint adaptors comprise a double-stranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region (320), an internal region (310) and a second region (330), and wherein the internal region of the first splint strand is The portion region (310) is hybridized with the second splint chain (400); and a plurality of double-stranded splint adapters are hybridized with a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule comprises a target sequence (110) flanked on a first side by a universal adapter sequence for a forward sequencing primer binding site (120) and on a second side by a universal adapter sequence for a reverse sequencing primer binding site (130), thereby circularizing the plurality of library molecules to form a plurality of library-splint complexes (500) each having two gaps.

[0008] In some embodiments, the method further comprises: (c) contacting the plurality of library-splint complexes (500) with a ligase to generate a plurality of covalently closed circular library molecules (600).

[0009] In some embodiments, hybridization is performed under conditions suitable for hybridizing the first region (320) of the first splint strand to the universal adapter sequence for the forward sequencing primer binding site (120) of the library molecule. In some embodiments, the conditions are suitable for hybridizing the second region (330) of the first splint strand to the universal adapter sequence for the reverse sequencing primer binding site (130) of the library molecule.

[0010] In some embodiments, the inner region (310) of the first splint chain (300) includes at least three sub-regions. In some embodiments, the at least three sub-regions include sub-region (311), sub-region (312) and sub-region (313). In some embodiments, sub-region (311) includes a universal adapter sequence for a surface capture primer binding site, a universal adapter sequence for a surface pinning primer binding site, a sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI). In some embodiments, sub-region (312) includes a universal adapter sequence for a surface capture primer binding site, a universal adapter sequence for a surface pinning primer binding site, a sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI). In some embodiments, sub-region (313) includes a universal adapter sequence for a surface capture primer binding site, a universal adapter sequence for a surface pinning primer binding site, a sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI).

[0011] In some embodiments, subregions (311), (312) or (313) contain a sample index sequence, and wherein the sample index sequence includes: a sample index sequence lacking a short random sequence (NNN); a sample index sequence and a short random sequence (NNN); a sample index flanked on both sides by nucleotide bases convertible to an abasic base; at least one nucleotide base convertible to an abasic base; at least one deoxyinosine; an 18-carbon spacer; and / or an 18-carbon spacer and at least one deoxyinosine.

[0012] In some embodiments, the method further comprises: distributing the plurality of covalently closed circular library molecules (600) onto a support having a plurality of surface capture primers immobilized to the support under conditions suitable for hybridizing each covalently closed circular library molecule (600) to each immobilized surface capture primer, thereby immobilizing the plurality of covalently closed circular library molecules (600) to the support. In some embodiments, the support further comprises a plurality of surface pinning primers immobilized to the support.

[0013] In some embodiments, the method further comprises: contacting a plurality of immobilized covalently closed circular library molecules (600) with a plurality of chain displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on a support using a plurality of surface capture primers as immobilized amplification primers and a plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules immobilized to the surface capture primers.

[0014] In some embodiments, the method further comprises: iii) sequencing the plurality of nucleic acid concatemer molecules immobilized to the surface capture primers, wherein the sequencing comprises (i) sequencing the sample index and (ii) sequencing the target sequence (110).

[0015] In some embodiments, the method further comprises: iv) sequencing the plurality of nucleic acid concatemer molecules fixed to the surface capture primers, wherein the sequencing comprises (A) sequencing one or more short random sequences NNN, (B) sequencing one or more sample indexes, and (C) sequencing the target sequence (110).

[0016] In some embodiments, the interior region (310) of the first splint chain (300) includes a sample index.

[0017] In some embodiments, the inner region (310) of the first splint chain (300) includes a sample index and a short random sequence (NNN).

[0018] In some embodiments, the library molecule (100) comprises one or more nucleotide sequences selected from Table 1.

[0019] In some embodiments, the first splint strand (300) comprises one or more nucleotide sequences selected from Table 2.

[0020] In some embodiments, the second splint strand (400) comprises one or more nucleotide sequences selected from Table 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the present disclosure are utilized, and in the accompanying drawings:

[0022] Figure 1 is a schematic diagram showing an exemplary linear nucleic acid library molecule (100) comprising an insert region (110) (e.g., a sequence of interest) flanked on one side by a universal adapter sequence for a forward sequencing primer binding site (120), and the insert region (110) is flanked on the other side by a universal adapter sequence for a reverse sequencing primer binding site (130).

[0023] Figure 2 is a schematic diagram of an exemplary double-stranded splint adaptor (200) comprising a first splint strand (long strand (300)) hybridized to a second splint strand (short strand (400)).

[0024] Figure 3 is a schematic diagram showing an exemplary library circularization workflow, which includes hybridizing a linear single-stranded library molecule (100) to a double-stranded splint adaptor (200), thereby circularizing the library molecule to form a library-splint complex (500) with two gaps.

[0025] Figure 4 is a schematic diagram showing an exemplary ligation reaction, which includes performing an enzymatic ligation reaction on a gap in the library-splint complex (500), thereby closing the gap to form a covalently closed circular library molecule (600) hybridized to the first splint strand (300).

[0026] Figure 5 is a schematic diagram showing an exemplary covalently closed circular library molecule (600) hybridized to an amplification primer. The dashed line represents the nascent extension product.

[0027] Figure 6Schematic diagram showing several embodiments of nucleic acid sequences of a first splint chain (300; long splint chain), the first splint chain comprising: an outer first region (320); an outer second region (330); and three internal subregions, the three internal subregions including subregion (311), subregion (312), and subregion (313). The schematic diagram also shows an exemplary nucleic acid sequence of a second splint chain (400; short splint chain), the second splint chain comprising: three subregions, the three subregions including: subregion (411); subregion (412); and subregion (413).

[0028] Figure 7 is a schematic diagram showing an exemplary nucleic acid sequence of a first splint chain (300; long splint chain), the first splint chain comprising: an outer first region (320); an outer second region (330); and two inner subregions, including subregion (311) and subregion (313). The schematic diagram also shows an exemplary nucleic acid sequence of a second splint chain (400; short splint chain), the second splint chain comprising: three subregions, including: subregion (411); subregion (412); and subregion (413). The loop at subregion (412) represents a loop formed due to the lack of subregion (312) of the first splint chain (300).

[0029] FIG. 8A to FIG. 8D is a set of schematic diagrams showing various embodiments of double-stranded nucleic acid adaptors, each carrying a universal adaptor sequence for a forward sequencing primer binding site (120). Fig. 8A is a schematic diagram of a double-stranded adaptor carrying the full-length sequence of the universal adaptor sequence for the forward sequencing primer binding site (120). Figure 8B is a schematic diagram of a double-stranded adaptor carrying a truncated sequence of the universal adaptor sequence for the forward sequencing primer binding site (120). Figure 8C is a schematic diagram of a double-stranded adapter with a 5' overhang, wherein one of the adapter strands carries the full-length sequence of the universal adapter sequence for the forward sequencing primer binding site (120), and the other adapter strand carries a truncated sequence of the universal adapter sequence for the forward sequencing primer binding site (120). Fig.8D is a schematic diagram of a double-stranded adapter with a 3' overhang, wherein one of the adapter strands carries a truncated sequence of the universal adapter sequence for the forward sequencing primer binding site (120) and the other adapter strand carries the full-length sequence of the universal adapter sequence for the forward sequencing primer binding site (120).

[0030] 9A to 9D is a set of schematic diagrams showing various embodiments of double-stranded nucleic acid adaptors, each carrying a universal adaptor sequence for a reverse sequencing primer binding site (130). Fig.9Ais a schematic diagram of a double-stranded adaptor carrying the full-length sequence of the universal adaptor sequence for the reverse sequencing primer binding site (130). Fig. 9B is a schematic diagram of a double-stranded adaptor carrying a truncated sequence of the universal adaptor sequence for the reverse sequencing primer binding site (130). Fig. 9C is a schematic diagram of a double-stranded adapter with a 5' overhang, wherein one of the adapter strands carries the full-length sequence of the universal adapter sequence for the reverse sequencing primer binding site (130), and the other adapter strand carries a truncated sequence of the universal adapter sequence for the reverse sequencing primer binding site (130). Fig.9D is a schematic diagram of a double-stranded adapter with a 3' overhang, wherein one of the adapter strands carries a truncated sequence of the universal adapter sequence for the reverse sequencing primer binding site (130) and the other adapter strand carries the full-length sequence of the universal adapter sequence for the reverse sequencing primer binding site (130).

[0031] FIG. 10A to FIG. 10D is a set of schematic diagrams showing various embodiments of Y-shaped adaptors, each of which comprises two oligonucleotides hybridized together and having a double-stranded annealing region and a mismatch portion. Fig. 10A is a schematic diagram of a Y-shaped adaptor comprising a first oligonucleotide carrying the full-length sequence of a universal adaptor sequence for a forward sequencing primer binding site (120) and a second oligonucleotide carrying the full-length sequence of a universal adaptor sequence for a reverse sequencing primer binding site (130). Fig. 10B is a schematic diagram of a Y-shaped adaptor comprising a first oligonucleotide carrying a truncated sequence of a universal adaptor sequence for a forward sequencing primer binding site (120) and a second oligonucleotide carrying a truncated sequence of a universal adaptor sequence for a reverse sequencing primer binding site (130). Fig. 10C is a schematic diagram of a Y-shaped adaptor comprising a first oligonucleotide carrying a full-length sequence of a universal adaptor sequence for a forward sequencing primer binding site (120) and a second oligonucleotide carrying a truncated sequence of a universal adaptor sequence for a reverse sequencing primer binding site (130). Fig. 10D is a schematic diagram of a Y-shaped adaptor comprising a first oligonucleotide carrying a truncated sequence of a universal adaptor sequence for a forward sequencing primer binding site (120) and a second oligonucleotide carrying a full-length sequence of a universal adaptor sequence for a reverse sequencing primer binding site (130).

[0032] FIG. 11A to FIG. 11B is a set of schematic diagrams illustrating embodiments of transposomes. Fig.11ASchematic diagram of two transpososomes, wherein the first transpososome (top) comprises a transposase bound to a double-stranded polynucleotide comprising a transposon end sequence and a universal adapter sequence for a forward sequencing primer binding site (120). The transposon end sequence specifically binds the transposase. The second transposome (bottom) comprises a transposase bound to a double-stranded polynucleotide comprising a transposon end sequence and a universal adapter sequence for a reverse sequencing primer binding site (130). The transposon end sequence specifically binds the transposase. Fig. 11B is a schematic diagram of an exemplary transposome comprising a transposase bound to a first double-stranded polynucleotide comprising a transposon end sequence and a universal adapter sequence for a forward sequencing primer binding site (120), and the transposase bound to a second double-stranded polynucleotide comprising a transposon end sequence and a universal adapter sequence for a reverse sequencing primer binding site (130). The transposon end sequence specifically binds to the transposase.

[0033] Fig.12 is a schematic diagram showing an example of a transposome comprising a transposase bound to a Y-shaped adaptor. Each Y-shaped adaptor comprises two oligonucleotides hybridized together and having a double-stranded annealing region and a mismatch portion.

[0034] Fig.13 Schematic diagram showing an exemplary adapter ligation workflow for generating double-stranded linear nucleic acid library molecules. Double-stranded nucleic acid fragments are enzymatically ligated on one side to a double-stranded adapter carrying a universal adapter sequence for a forward sequencing primer binding site (120). Double-stranded nucleic acid fragments are enzymatically ligated on the other side to a double-stranded adapter carrying a universal adapter sequence for a reverse sequencing primer binding site (130).

[0035] Fig.14 Schematic diagram of an exemplary adapter connection workflow for generating double-stranded linear nucleic acid library molecules. The double-stranded nucleic acid fragments are enzymatically connected to a first Y-shaped adapter on one side, the first Y-shaped adapter comprising a first oligonucleotide carrying a full-length sequence of a universal adapter sequence for a forward sequencing primer binding site (120), and a second oligonucleotide carrying a full-length sequence of a universal adapter sequence for a reverse sequencing primer binding site (130). The double-stranded nucleic acid fragments are enzymatically connected to a second Y-shaped adapter on the other side, the second Y-shaped adapter comprising a first oligonucleotide carrying a full-length sequence of a universal adapter sequence for a forward sequencing primer binding site (120), and a second oligonucleotide carrying a full-length sequence of a universal adapter sequence for a reverse sequencing primer binding site (130).

[0036] Fig.15It is a schematic diagram showing an exemplary adapter connection workflow for generating double-stranded linear nucleic acid library molecules. The double-stranded nucleic acid fragment is enzymatically connected to a first Y-shaped adapter on one side, and the first Y-shaped adapter includes a first oligonucleotide carrying a full-length sequence of a universal adapter sequence for a forward sequencing primer binding site (120), and a second oligonucleotide carrying a full-length sequence of a universal adapter sequence for a reverse sequencing primer binding site (130). The double-stranded nucleic acid fragment is enzymatically connected to a second Y-shaped adapter on the other side, and the second Y-shaped adapter includes a first oligonucleotide carrying a full-length sequence of a universal adapter sequence for a forward sequencing primer binding site (120), and a second oligonucleotide carrying a full-length sequence of a universal adapter sequence for a reverse sequencing primer binding site (130). The resulting double-stranded linear nucleic acid library molecule is primer-extended using a primer having a region hybridized with one of the mismatched portions (e.g., (130)), and the primer also carries a sample index sequence and a universal surface primer binding site (SurfPBS) at the other end. A primer extension reaction is performed using the hybridized primer as a template to generate an extended library molecule comprising: a forward sequencing primer binding site (120); an insert sequence (110); a reverse sequencing primer binding site (130); a sample index sequence; and a universal surface primer binding site (SurfPBS).

[0037] Fig.16 It is a schematic diagram showing an exemplary adapter connection workflow for generating double-stranded linear nucleic acid library molecules. The double-stranded nucleic acid fragment is enzymatically connected to the first Y-shaped adapter on one side, and the first Y-shaped adapter includes a first oligonucleotide carrying a full-length sequence of a universal adapter sequence for a forward sequencing primer binding site (120), and a second oligonucleotide carrying a full-length sequence of a universal adapter sequence for a reverse sequencing primer binding site (130). The double-stranded nucleic acid fragment is enzymatically connected to the second Y-shaped adapter on the other side, and the second Y-shaped adapter includes a first oligonucleotide carrying a full-length sequence of a universal adapter sequence for a forward sequencing primer binding site (120), and a second oligonucleotide carrying a full-length sequence of a universal adapter sequence for a reverse sequencing primer binding site (130). The resulting double-stranded linear nucleic acid library molecule is hybridized with a linear double-stranded adapter having a 3' overhang and a blunt end. The 3' overhang includes a sequence that can be hybridized with a mismatched portion having a reverse sequencing primer binding site (130), which generates a partially double-stranded region with a gap (solid triangle). Gaps can be connected and unconnected strands can be removed.

[0038] Fig.17Schematic diagram of an exemplary tagmentation workflow using multiple transposomes. Input double-stranded DNA is contacted with multiple transposomes, wherein each transposome comprises a transposase bound to a first double-stranded polynucleotide comprising a transposon end sequence and a universal adapter sequence for a forward sequencing primer binding site (120), and the transposase is bound to a second double-stranded polynucleotide comprising a transposon end sequence and a universal adapter sequence for a reverse sequencing primer binding site (130).

[0039] Fig.18 Schematic diagram of an exemplary library circularization workflow, which includes hybridizing a linear single-stranded library molecule (100) with a first splint strand (200), thereby circularizing the library molecule to form a library single splint complex (700) having a gap between the ends of the library molecule. The linear nucleic acid library molecule (100) includes an insertion region (110) (e.g., a target sequence) flanked on one side by a universal adapter sequence for a forward sequencing primer binding site (120), and the insertion region (110) is flanked on the other side by a universal adapter sequence for a reverse sequencing primer binding site (130). The first splint strand (200) includes a first region (320) that hybridizes to the universal adapter sequence for the forward sequencing primer binding site (120) on one end of the linear single-stranded library molecule (100), and the first splint strand includes a second region (330) that hybridizes to the universal adapter sequence for the reverse sequencing primer binding site (130) on the other end of the linear single-stranded library molecule. The gap is closed using a polymerase-catalyzed filling reaction and an enzymatic ligation reaction to generate a covalently closed circular molecule.

[0040] Fig.19 is a graph showing the nucleotide base diversity of a sample index sequence including a short 3-mer random sequence (NNN). The graph shows that the nucleotide diversity of the 3-mer random sequence (NNN) for A and T base recognition is about 30%, and the nucleotide diversity for C and G base recognition is about 20%.

[0041] Fig. 20 is a graph showing the nucleotide base diversity of sample index sequences lacking a short 3-mer random sequence (NNN). The graph shows that the nucleotide diversity for A and T base recognition is approximately 40%, the nucleotide diversity for C base recognition is approximately 15%, and the nucleotide diversity for G base recognition is approximately 5%.

[0042] Fig.21 is a schematic diagram of an exemplary low binding support comprising alternating layers of a glass substrate and a hydrophilic coating 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 and circularization oligonucleotides).

[0043] Fig. 22 Schematic diagrams of various exemplary configurations of multivalent molecules. Left: Schematic diagrams of multivalent molecules having starburst or helter-skelter configurations. Center: Schematic diagrams of multivalent molecules having dendrimer configurations. Right: Schematic diagrams of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. The nucleotide unit is designated as 'N', biotin is designated as 'B', and streptavidin is designated as 'SA'.

[0044] Fig.23 is a schematic diagram of an exemplary multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0045] Fig.24 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms.

[0046] Fig.25 A schematic diagram of an exemplary multivalent molecule comprising a core attached to a plurality of nucleotide arms, wherein the nucleotide arms comprise biotin, spacers, linkers, and nucleotide units is shown.

[0047] Fig.26 is a schematic diagram of an exemplary nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit.

[0048] Fig. 27 Shown are the chemical structures of exemplary spacers (top) and various exemplary linkers (bottom), including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker.

[0049] Fig.28 The chemical structures of various exemplary linkers, including Linkers 1-9, are shown.

[0050] Fig.29 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0051] Fig.30 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0052] Fig.31 The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0053] Fig.32 The chemical structure of an exemplary biotinylated nucleotide arm is shown. In this example, the nucleotide unit is linked to the linker via a propargylamine attachment at position 5 of the pyrimidine base or position 7 of the purine base. DETAILED DESCRIPTION

[0054] definition:

[0055] The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0056] Unless otherwise defined, the technology and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. In general, the terms related to the technology of molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization described herein are well-known and commonly used terms in the art. The technology and procedures described herein are usually performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, referring to Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with laboratory procedures and techniques described herein is well-known and commonly used nomenclature in the art.

[0057] Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a / an" and "the" and any word used in the singular include plural referents unless expressly and unequivocally limited to one referent.

[0058] It should be understood that the use of alternative terms (eg, "or") is taken to mean one or both of the alternatives or any combination thereof.

[0059] As used herein, the term "and / or" should be deemed to mean a specific disclosure of each of the specified features or components with or without another. For example, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include: "A and B"; "A or B"; "A" (only A); and "B" (only B). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" herein is intended to cover each of the following aspects: "A, B, and C"; "A, B, or C"; "A or C"; "A or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (only A); "B" (only B); and "C" (only C).

[0060] As used herein and in the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be non-limiting, such that one or more items in a list are not exclusive of other items that can be substituted or added to the listed items. It should be understood that whenever aspects are described herein with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0061] As used herein, the terms "about" and "approximately" refer to values ​​or compositions within an acceptable error range for a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" or "approximately" may mean within one or more than one standard deviation. Alternatively, "about" or "approximately" may 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 may include any numerical value between 4.5 mg and 5.5 mg. In addition, particularly for biological systems or processes, these terms may represent up to an order of magnitude or up to 5 times of a value. When a particular value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for the particular value or composition. In addition, in the case of providing a range and / or sub-range of a value, these ranges and / or sub-ranges may include the endpoints of these ranges and / or sub-ranges.

[0062] The terms "peptide", "polypeptide" and "protein" and other related terms used herein are used interchangeably and refer to polymers of amino acids and are not limited to any particular length. Polypeptides can include natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. Polypeptides also include precursor molecules that have not yet undergone post-translational modifications, such as proteolytic cleavage, cleavage due to ribosome skipping, hydroxylation, methylation, lipidation, acetylation, SUMOylation, ubiquitination, glycosylation, phosphorylation and / or disulfide bond formation. These terms cover natural and artificial proteins, protein fragments and polypeptide analogs (such as mutant proteins, variants, chimeric proteins and fusion proteins) of protein sequences, as well as proteins that are covalently or non-covalently modified after translation or otherwise.

[0063] The term "cell biological sample" refers to a single cell, a plurality of cells, a tissue, an organ, an organism or a section of any of these cell biological samples. Cell biological samples can be extracted from an organism (e.g., a biopsy), or obtained from a cell culture grown in a liquid or a culture dish. Cell biological samples include fresh, frozen, freshly frozen or archived samples (e.g., formalin-fixed paraffin embedded; FFPE). Cell biological samples can be embedded in wax, resin, epoxy resin or agar. Cell biological samples can be fixed in any one of, for example, acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton or glutaraldehyde or any combination of two or more. Cell biological samples can be sectioned or unsectioned. Cell biological samples can be stained, decolorized or unstained.

[0064] The target nucleic acid can be extracted from a cell or cell biological sample using any of a variety of techniques known to those skilled in the art. For example, a typical DNA extraction procedure includes: (i) collecting a cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components, (iii) treating the dissolved sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell membrane lysis. A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolating genomic DNA from human samples) and the DNAeasy kit (for isolating genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the DNAeasy kit (for isolating genomic DNA from animal or plant samples) from Promega (Madison, WI). and ReliaPrep TM Series of test kits.

[0065] As used herein, the term "polymerase" and variants thereof include enzymes comprising a domain that binds nucleotides (or nucleosides), wherein the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities, including but not limited to: base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. The polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid chain. Typically, but not necessarily, this nucleotide polymerization can occur in a template-dependent manner. Typically, the polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization can occur. In some embodiments, the polymerase includes other enzymatic activities, such as, for example, 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has a strand displacement activity. Polymerases may include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases and other enzymes comprising nucleotide binding domains. In some embodiments, polymerases may be isolated from cells or generated using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases may be expressed in prokaryotes, eukaryotes, viruses or bacteriophage organisms. In some embodiments, polymerases may be post-translationally modified proteins or fragments thereof. Polymerases may be derived from prokaryotes, eukaryotes, viruses or bacteriophages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0066] The term "strand displacement" refers to the ability of a polymerase to locally separate a double-stranded nucleic acid chain and synthesize a new chain in a template-based manner. The strand displacement polymerase displaces the complementary chain from the template chain and catalyzes the synthesis of the new chain. Strand displacement polymerases include mesophilic and thermophilic polymerases. Strand displacement polymerases include wild-type enzymes and variants (including exonucleases minus mutants, mutant versions, chimeric enzymes, and truncated enzymes). Examples of strand displacement polymerases include, but are not limited to, phi29 DNA polymerase, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment (exo-), Bca DNA polymerase (exo-), Klenow fragment of Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV virus reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., from Expedeon TM MagniPhiTM ), or variant EquiPhi29 TM DNA polymerase (e.g., from Thermo Fisher Scientific TM ), or chimeric QualiPhi TM DNA polymerase (e.g., from 4basebio TM ).

[0067] As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" and other related terms are used interchangeably and refer to polymers of nucleotides and are not limited to any specific length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs) and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, wherein the nucleotides may include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside bonds, such as and not limited to phosphodiester bonds. Nucleic acids may include non-natural internucleoside bonds, including thiophosphates, sulfur-containing phosphates and / or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids include a mixture of one type of polynucleotide or two or more different types of polynucleotides.

[0068] As used herein, the terms "operably connected" and "operably joined" or related terms refer to the juxtaposition of components. The juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond joining the two components together includes a phosphodiester bond. The first nucleic acid component and the second nucleic acid component can be linked together, wherein the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between the primer binding sequence and the target sequence forms a nucleic acid library molecule having a portion that can bind to the primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a target nucleic acid sequence) can be linked to a vector, wherein the connection allows the transgenic sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects the expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls the expression of the level, timing, and / or position of the transgene.

[0069] The terms "connected", "joined", "attached", "attached" and their variants include any type of fusion, bonding, adhesion or association between any combination of compounds or molecules that has sufficient stability to withstand use in a particular procedure. The procedure may include, but is not limited to: nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of chain terminating moieties); washing; removal; flow; detection; imaging and / or recognition. Such bonds may include, for example, covalent bonds, ionic bonds, hydrogen bonds, dipole-dipole bonds, hydrophilic bonds, hydrophobic bonds or affinity bonds, bonds or associations involving van der Waals forces, mechanical bonds, and the like. In some embodiments, such bonds occur within a molecule, such as connecting the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds may occur between combinations of different molecules, or between molecules and non-molecules, including but not limited to: bonds between nucleic acid molecules and solid surfaces; connections between proteins and detectable reporter gene moieties; connections between nucleotides and detectable reporter gene moieties; and the like. Some examples of bonds can be found, for example, in Hermanson, G., “Bioconjugate Techniques”, 2nd edition (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0070] As used herein, the term "primer" and related terms refer to oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form duplex molecules. Primers can be single-stranded or have single-stranded and double-stranded portions along their entire length. Primers can include natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can have any length, but generally range from 4 to 50 nucleotides. Typical primers include 5' ends and 3' ends. The 3' end of a primer may include a 3'OH portion that serves as a nucleotide polymerization start site in a primer extension reaction catalyzed by a polymerase. Alternatively, the 3' end of a primer may lack a 3'OH portion or may include a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any nucleotide along the length of a primer or more than one nucleotide may be labeled with a detectable reporter gene portion. Primers may be in solution (e.g., soluble primers) or may be fixed to a support (e.g., capture primers).

[0071] The terms "template nucleic acid," "template polynucleotide," "target nucleic acid," "target polynucleotide," "template strand," and other variants refer to a nucleic acid strand used as a base nucleic acid molecule in any amplification and / or sequencing method described herein. The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from a naturally occurring source, a recombinant form, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, concatemeric, circular, or in other forms.

[0072] When used to refer to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization" or other related terms refer 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 with a duplex region. Hybridization may include Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. A double-stranded nucleic acid or two different regions of a single nucleic acid may be fully complementary or partially complementary. Complementary nucleic acid chains do not need to hybridize to each other across their entire length. Complementary base pairing may be standard AT or CG base pairing or may be other forms of base pairing interactions. A duplex nucleic acid may include mismatched base-paired nucleotides.

[0073] When used in reference to nucleic acids, the terms "extend," "extending," "extension," and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation includes polymerization of one or more nucleotides into the terminal 3'OH end of a nucleic acid chain, resulting in extension of the nucleic acid chain. Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0074] The term "nucleotide" and related terms refer to molecules comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and at least one phosphate group. Regular or non-regular nucleotides are consistent with the use of the term. In certain embodiments, nucleotides comprise monophosphate, diphosphate or triphosphate, or corresponding phosphate analogs. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be unlabeled or labeled with a detectable reporter moiety.

[0075] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings commonly found in nucleic acids, including naturally occurring, substituted, modified or engineered variants or analogs thereof. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoostein 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), ethyleneadenine, N 6 -Δ 2 -Isopentenyl adenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6 -methylguanine; 7-deazapurines, such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated and acylated base moieties; etc. Additional exemplary bases can be found in Fasman, 1989, in "Practical Handbook of Biochemistry and Molecular Biology", pages 385-394, CRC Press, Boca Raton, Fla.

[0076] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), an acyclic moiety (Martinez et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7:3013-3016) and other sugar moieties (Joeng et al., 1993 J. Med. Chem. 36:2627-2638; Kim et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). Sugar moieties include: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-thiolribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-thiolribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0077] In some embodiments, the nucleotide comprises a chain with one, two or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester bond or a phosphoramide bond. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together by intervening O, S, NH, methylene or ethylene. In some embodiments, the phosphorus atoms in the chain include substituted side groups, including O, S or BH 3 In some embodiments, the chain includes phosphate groups substituted with analogs including phosphoramide, phosphorothioate, phosphorodithioate, and O-methylphosphoramidite groups.

[0078] As used herein, a "nucleotide unit" or "nucleotide moiety" refers to a nucleotide (e.g., dATP, dTTP, dGTP, dCTP, or dUTP) or an analog thereof comprising a base, a sugar, and at least one phosphate group. A nucleotide unit can be attached to a multivalent molecule used in a sequencing reaction as described herein. Generally, all nucleotide units attached to the same multivalent molecule will have the same identity (e.g., all A, all T, all C, or all G), although those skilled in the art will appreciate that there may be situations in which it would be advantageous for a multivalent molecule to contain nucleotide units of different identities.

[0079] The term "rolling circle amplification" generally refers to an amplification method using a circularized nucleic acid template molecule, which contains a target sequence, an amplification primer binding sequence, and optionally one or more adapter sequences, such as a sequencing primer binding sequence and / or a sample index sequence. The rolling circle amplification reaction can be performed under isothermal amplification conditions and includes a circularized nucleic acid template molecule, an amplification primer, a strand displacement polymerase, and a plurality of nucleotides to generate a concatemer containing a tandem repeat sequence of a circular template molecule and any adapter sequence present in the original circularized nucleic acid template molecule. The concatemer can self-collapse to form a nucleic acid nanoball. The shape and size of the nanoball can be further compressed by including a pair of inverted repeat sequences in the circular template molecule, or by performing a rolling circle amplification reaction with one or more compacting oligonucleotides. One of the advantages of using rolling circle amplification to generate clonal amplicons for sequencing workflows is that repeated copies of the target sequence in the nanoball can be sequenced simultaneously to increase signal intensity. In some embodiments, the rolling circle amplification reaction can be carried out in the presence of multiple compacted oligonucleotides with at least four consecutive guanines (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 guanines). The rolling circle amplification reaction can generate a concatemer comprising repeated copies of a universal binding sequence for the compacted oligonucleotide. At least one compacted oligonucleotide can form a guanine quadruplex and hybridize with a universal binding sequence for the compacted oligonucleotide, and the resulting concatemer can fold to form an intramolecular G-quadruplex structure. The concatemer can self-collapse to form a compact nanosphere. Without wishing to be bound by theory, it is assumed that the formation of guanine quadruplexes and G-quadruplexes in the nanosphere can increase the stability of the nanosphere to maintain its compact size and shape, so that it can withstand the repeated flow of reagents used to perform any of the sequencing workflows described herein.

[0080] When used in reference to nucleic acids, the terms "amplify," "amplifying," "amplification," and other related terms include generating multiple copies of an original polynucleotide template molecule, wherein the copies comprise a sequence complementary to the template sequence, and / or the copies comprise a sequence identical to the template sequence. In some embodiments, the copies comprise a sequence substantially identical to the template sequence, and / or a sequence substantially identical to a sequence complementary to the template sequence.

[0081] The term "reporter moiety", "reporter moieties" or related terms refers to a compound that generates or causes a detectable signal. The reporter moiety is sometimes referred to as a "label". Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophore, radioisotope, electrochemistry, mass spectrometry, Raman, hapten, affinity tag, atom or enzyme. The reporter moiety generates a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity or adjacent event). The adjacent event comprises two reporter moieties approaching, associating or binding to each other. It is well known to those skilled in the art that the reporter moiety is selected so that each reporter moiety absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties to allow monitoring of the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected that have spectrally distinct emission curves or have minimally overlapping spectral emission curves. The reporter moiety can be attached (eg, operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (eg, a polymerase or reverse transcriptase), or support (eg, a surface).

[0082] The reporter gene part (or label) includes a fluorescent marker or fluorophore. Exemplary fluorescent moieties that can be used as fluorescent markers or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthol fluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, maleimide fluorescein, SAMSA-fluorescein, thiosemicarbazide fluorescein, hydrazine carbonate methylthioacetylaminofluorescein, rhodamine and rhodamine derivatives, such as TRITC, TMR, lissamine rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPYFLC3-SE, BODIPY 530 / 550C3, BODIPY 530 / 550C3-SE, BODIPY 530 / 550C3 hydrazide, BODIPY 493 / 503C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FLIA, BODIPY 530 / 551IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl triazoide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives such as indolium cyanine dyes, benzindolium cyanine dyes, pyridinium cyanine dyes, thiazolium cyanine dyes, quinolinium cyanine dyes, imidazolium cyanine dyes, Cy3, Cy5, lanthanide chelates and derivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, AlexaFluor dyes, DyLight dyes, Atto dyes, LightCyclerRed dyes, CALFlour dyes, JOE and its derivatives, Oregon Green dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, diphenylethylene, DEG dyes, NR dyes, near infrared dyes and other dyes known in the art, such as Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition; Lakowicz,Principles of Fluorescence Spectroscopy, 2nd edition, Plenum Press New York (1999) or those described in Hermanson, Bioconjugate Techniques, 2nd edition, or derivatives thereof, or any combination thereof. Cyanine dyes may exist in sulfonated or non-sulfonated form and consist of two indolenine, benzindolium, pyridinium, thiazolium and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which may include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2- (3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)- ​​... 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolyl-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolyl-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium 1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (which may include 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for 'cyanine' and the first number indicates the number of carbon atoms between the two indolenine groups. Cy2 is an oxazole derivative rather than an indolenine, and the benzo-derived Cy3.5, Cy5.5 and Cy7.5 are exceptions to this rule. ,

[0083] In some embodiments, the reporter moiety can be a FRET pair, for example, allowing multiple classifications to be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0084] As used herein, the term "support" refers to a substrate designed for the deposition of biological molecules or biological samples for measurement and / or analysis. Examples of biological molecules to be deposited on the support include nucleic acids (e.g., DNA, RNA), polypeptides, carbohydrates, lipids, single cells or multiple cells. Examples of biological samples include, but are not limited to: saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0085] In some embodiments, the support is solid, semi-solid, or a combination of the two. 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 the inner surface of a capillary.

[0086] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can have a regular or irregular texture, including, for example, bumps, etchings, holes, three-dimensional scaffolds, or any combination thereof.

[0087] In some embodiments, the support comprises beads having any shape, including spheres, hemispheres, cylinders, barrels, rings, disks, rods, cones, triangles, cubes, polygons, tubes, or wires.

[0088] The support may be made of any material, including, but not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0089] In some aspects, the present disclosure provides multiple (e.g., two or more) nucleic acid template molecules fixed to a support. In some embodiments, multiple nucleic acid template molecules fixed have the same sequence. In some embodiments, multiple nucleic acid template molecules fixed have different sequences. In some embodiments, each nucleic acid template molecule in a plurality of nucleic acid template molecules is fixed to different sites on a support. In some embodiments, two or more separate nucleic acid template molecules in a plurality of nucleic acid templates are fixed to sites on a support.

[0090] The term "array" refers to a support comprising a plurality of sites located at predetermined positions on the support to form an array of sites. These sites can be discrete and separated by interstitial distinctions. In some embodiments, the predetermined sites on the support can be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, the plurality of predetermined sites are arranged in an organized manner on the support. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including straight lines, hexagonal patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, etc. The spacing between different pairs of sites may be the same or may be different. In some embodiments, the support comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 In some embodiments, the plurality of predetermined sites (e.g., 10 2 Up to 10 15 sites or more, e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 In some embodiments, the nucleic acid template molecules are fixed at a plurality of predetermined sites by hybridizing with the fixed surface capture primers, or the nucleic acid template molecules are covalently attached to the surface capture primers. In some embodiments, the nucleic acid template molecules are fixed at a plurality of predetermined sites, for example, fixed at 10 2 Up to 10 15 sites or more (e.g., 10 2 Up to 10 15 sites or more, e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 In some embodiments, the fixed nucleic acid template molecules are clonally amplified to generate fixed nucleic acid clusters at multiple predetermined sites. In some embodiments, each fixed nucleic acid cluster comprises a linear cluster, or comprises a single-stranded or double-stranded concatemer.

[0091] In some embodiments, a support comprising a plurality of sites located at random positions on the support is referred to herein as a support having randomly positioned sites thereon. In such embodiments, the positions of the randomly positioned sites on the support are not predetermined. Thus, the plurality of randomly positioned sites are arranged on the support in a disordered and / or unpredictable manner. In some embodiments, the support comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 In some embodiments, the plurality of randomly positioned sites (e.g., 10 2 Up to 10 15 sites or more, e.g., 10 2 Up to 10 15 sites or more, e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 In some embodiments, the nucleic acid template molecules are immobilized at multiple randomly positioned sites by hybridization with immobilized surface capture primers, or the nucleic acid template molecules are covalently attached to the surface capture primers. In some embodiments, the nucleic acid templates are immobilized at multiple randomly positioned sites, for example, at 10 2 Up to 10 15 sites or more, e.g., 10 2 Up to 10 15 sites or more, e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 In some embodiments, the fixed nucleic acid template is cloned and amplified to generate fixed nucleic acid clusters at multiple randomly positioned sites. In some embodiments, each fixed nucleic acid cluster comprises a linear cluster, or comprises a single-stranded or double-stranded concatemer.

[0092] In some embodiments, a plurality of fixed surface capture primers on a support (e.g., located at predetermined or random positions on the support) are fluidically connected to each other to allow a solution of a reagent (e.g., a nucleic acid template molecule, a soluble primer, an enzyme, a nucleotide, a divalent cation, a buffer, etc.) to flow onto the support so that a plurality of fixed surface capture primers on the support can react with the reagent in a massively parallel manner substantially at the same time. In some embodiments, the fluidic connection of a plurality of fixed surface capture primers can be used to perform a nucleic acid amplification reaction (e.g., RCA, MDA, PCR, and bridge amplification) substantially simultaneously on the plurality of fixed surface capture primers.

[0093] In some embodiments, multiple fixed nucleic acid clusters on the support are fluidically connected to each other to allow a solution of a reagent (e.g., an enzyme, a nucleotide, a divalent cation, etc.) to flow onto the support so that multiple fixed nucleic acid clusters on the support can react with the reagent in a large-scale parallel manner substantially at the same time. In some embodiments, the fluidic connection of multiple fixed nucleic acid clusters can be used to perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​multiple fixed nucleic acid clusters substantially at the same time, and optionally perform detection and imaging of large-scale parallel sequencing.

[0094] In certain embodiments, the term "fixed" and related terms refer to nucleic acid molecules attached to a support or a coating on a support or embedded in a matrix formed by a coating on a support by covalent bonds or non-covalent interactions, wherein the nucleic acid molecules include surface capture primers, nucleic acid template molecules and extension products of capture primers. The extension products of capture primers may include nucleic acid concatemers (e.g., nucleic acid clusters). Nucleic acid molecules can be fixed at predetermined random positions on a support. Nucleic acid molecules can be fixed at predetermined or random positions on or within a coating passivated on a support.

[0095] In some embodiments, the term "immobilized" and related terms refer to an enzyme (e.g., a polymerase) that is attached to a support, or to a coating on a support, or embedded in a substrate formed by a coating on a support, by covalent bonds or non-covalent interactions. The enzyme can be immobilized at a predetermined or random position on the support. The enzyme can be immobilized at a predetermined or random position on or within a passivated coating on a support.

[0096] In some embodiments, one or more nucleic acid template molecules are fixed on a support, for example, fixed at a site on a support. In some embodiments, one or more nucleic acid template molecules are cloned and amplified. In some embodiments, one or more nucleic acid template molecules are cloned and amplified from a support (for example, in a solution), and then deposited on a support and fixed on a support. In some embodiments, the cloned amplification reaction of one or more nucleic acid template molecules is carried out on a support, resulting in fixation on a support. In some embodiments, one or more nucleic acid template molecules are cloned and amplified (for example, in a solution or on a support) using a nucleic acid amplification reaction, and the nucleic acid amplification reaction includes any one or any combination of the following: 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), loop-to-loop amplification, helicase-dependent amplification, recombinase-dependent amplification and / or single-stranded binding (SSB) protein-dependent amplification.

[0097] The term "surface primer" and related terms refer to a single-stranded oligonucleotide that is fixed to a support and comprises a sequence that can hybridize with at least a portion of a nucleic acid template molecule. Surface capture primers can be used to fix template molecules to a support via hybridization. Surface capture primers can be fixed to a support in a manner that resists primer removal during flow, washing, suction, and changes in temperature, pH, salt, chemicals, and / or enzyme conditions. Typically, but not necessarily, the 5' end of the surface capture primer can be fixed to a support or a coating on a support (or embedded in a coating on a support). Alternatively, the interior or 3' end of the surface capture primer can be fixed to a support.

[0098] The sequence of the surface capture primer can be fully or partially complementary to at least a portion of the nucleic acid template molecule along its length. The support can include a plurality of fixed surface capture primers having the same sequence or having two or more different sequences. The surface capture primer can be any length, such as 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides, or longer lengths.

[0099] The surface capture primer can have a terminal 3' nucleotide, which has a sugar 3'OH part that can be extended for nucleotide polymerization (e.g., polymerase-catalyzed polymerization). The surface capture primer can have a terminal 3' nucleotide, which has a 3' sugar position connected to a chain termination part that inhibits nucleotide polymerization. The 3' chain termination part can be removed (e.g., deblocked) using a deblocking agent to convert the 3' end into an extendable 3' OH end. The example of the chain termination part includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azido group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group, an acetal group or a silyl group. The azido type chain termination part includes an azido group, an azido group and an azidomethyl group. Examples of deblocking agents include phosphine compounds such as tris(2-carboxyethyl)phosphine (TCEP) and bissulfotriphenylphosphine (BS-TPP) for chain-terminating groups azido, azido and azidomethyl. Examples of deblocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh 3 ) 4 ) with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) for chain terminating groups alkyl, alkenyl, alkynyl and allyl. Examples of deblocking agents include Pd / C for chain terminating groups aryl and benzyl. Examples of deblocking agents include phosphines, β-mercaptoethanol or dithiothreitol (DTT) for chain terminating groups amine, amide, ketone, isocyanate, phosphate, thio and disulfide. Examples of deblocking agents include potassium carbonate (K2CO3) in MeOH. 2 CO 3 ), triethylamine in pyridine and Zn in acetic acid (AcOH) for carbonate chain-terminating groups. Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, and ammonium fluoride and triethylamine trihydrofluoride for chain-terminating groups urea and silyl.

[0100] The term "sequencing" and related terms refer to methods for obtaining nucleotide sequence information from nucleic acid molecules, typically by determining the identity of at least some nucleotides (including their nucleobase components) within the nucleic acid molecule. In some embodiments, the sequence information of a given region of a nucleic acid molecule includes identifying each nucleotide in the region being sequenced. In some embodiments, the sequencing information only determines some nucleotides in the region, while the identities of some nucleotides remain undetermined or incorrectly determined. Any suitable sequencing method can be used. In an exemplary embodiment, sequencing may include a label-free or ion-based sequencing method. In some embodiments, sequencing may include labeled or dye-containing nucleotides or fluorescence-based nucleotide sequencing methods. In some embodiments, sequencing may include sequencing based on a polymerase colony (polony) or a bridge sequencing method. In some embodiments, sequencing uses a polymerase and a multivalent molecule to generate at least one affinity complex, wherein each multivalent molecule comprises a plurality of nucleotide units tethered to a core. In some embodiments, sequencing uses a polymerase and free nucleotides to perform sequencing while synthesis. In some embodiments, sequencing uses a ligase and a plurality of sequence-specific oligonucleotides to perform sequencing while ligating.

[0101] In some aspects, the present disclosure provides various reagents and methods for nucleic acid denaturation (dehybridization) and sequencing using these reagents. Various reagents can include at least one pH buffer. The full names of exemplary non-limiting pH buffers are listed herein.

[0102] The term "Tris" refers to the pH buffer tris(hydroxymethyl)-aminomethane. The term "Tris-HCl" refers to the pH buffer tris(hydroxymethyl)-aminomethane hydrochloride. The term "Tris-acetate" refers to a pH buffer comprising the acetate salt of tris(hydroxymethyl)-aminomethane.

[0103] The term "Tricine" refers to the pH buffer N-[tris(hydroxymethyl)methyl]glycine.

[0104] The term "Bicine" refers to the pH buffer N,N-bis(2-hydroxyethyl)glycine.

[0105] The term "Bis-Tris propane" refers to the pH buffer 1,3-bis[tris(hydroxymethyl)methylamino]propane

[0106] The term "HEPES" refers to the pH buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0107] The term "MES" refers to the pH buffer 2-(N-morpholino)ethanesulfonic acid).

[0108] The term "MOPS" refers to the pH buffer 3-(N-morpholino)propane sulfonic acid.

[0109] The term "MOPSO" refers to the pH buffer 3-(N-morpholino)-2-hydroxypropanesulfonic acid.

[0110] The term "BES" refers to the pH buffer N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid.

[0111] The term "TES" refers to the pH buffer 2-[(2-hydroxy-1,1 bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid).

[0112] The term "CAPS" refers to the pH buffer 3-(cyclohexylamino)-1-propanesulfonic acid.

[0113] The term "TAPS" refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid.

[0114] The term "TAPSO" refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid.

[0115] The term "ACES" refers to the pH buffer N-(2-acetamido)-2-aminoethanesulfonic acid.

[0116] The term "PIPES" refers to the pH buffer piperazine-1,4-bis(2-ethanesulfonic acid).

[0117] The term "ethanolamine" refers to a pH buffering agent also known as 2-aminoethanol.

[0118] Introduction: Double-stranded splint adapter

[0119] In some aspects, the present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods employing double-stranded splint adaptors.

[0120] The double-stranded splint adapter (200) can be used in a one-pot multi-enzyme reaction to introduce one or more new adapter sequences into a library molecule. In some embodiments, the double-stranded splint adapter (200) comprises a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)). In certain embodiments, the first splint strand and the second splint strand are hybridized together to form a double-stranded splint adapter (200) having a double-stranded region and two flanking single-stranded regions (see, for example, Figure 2 and 3). In some embodiments, the first splint chain (long splint chain (300)) and the second splint chain (short splint chain (400)) hybridize to each other along their entire length. In some embodiments, the first splint chain (long splint chain (300)) and the second splint chain (short splint chain (400)) include portions that are not hybridized together. The second splint chain (400) can carry a new adapter sequence to be introduced, such as, for example, a new universal binding sequence and / or a new index sequence. The first splint chain may include a first region (320), an internal region (310), and a second region (330). The internal region (310) of the first splint chain can hybridize with the second splint chain (400). In some embodiments, the two flanking single-stranded regions (e.g., (320) and (330)) of the double-stranded splint adapter are designed to hybridize with the universal adapter sequence at the end of the single-stranded linear library molecule (100) having the target sequence (110). For example, the first region (320) of the first splint strand can hybridize to one end (120) of the library molecule, and the second region (330) of the first splint strand can hybridize to the other end (130) of the library molecule, thereby looping the library molecule to generate a library-splint complex (500) including two gaps (see, e.g., Figure 3 ).

[0121] In some embodiments, the linear nucleic acid library molecule (100) comprises an insertion region (110) flanked on one side by a universal adapter sequence for a forward sequencing primer binding site (120) (e.g., a target sequence), and the insertion region (110) is flanked on the other side by a universal adapter sequence for a reverse sequencing primer binding site (130). In some embodiments, the double-stranded splint adapter (200) comprises a first splint strand (long strand (300)) hybridized with a second splint strand (short strand (400)). The first splint strand may comprise a first region (320) hybridized with a universal adapter sequence for a forward sequencing primer binding site (120) on one end of the linear single-stranded library molecule (100), and the first splint strand may comprise a second region (330) hybridized with a universal adapter sequence for a reverse sequencing primer binding site (130) on the other end of the linear single-stranded library molecule. The internal region (310) of the first splint strand may hybridize with the second splint strand (400). The gaps can be enzymatically ligated to generate a covalently closed circular molecule (600) in which the second splint strand (400) is covalently joined to the library molecule at both ends, thereby introducing a new adapter sequence into the library molecule (e.g., see Figure 4 ). The ligation reaction can join the sequence from the second splint strand (400) to the end of the library molecule (100).

[0122] In some embodiments, any of the subregions of the second splint strand (400) can be designed to hybridize with an amplification primer. In some embodiments, the amplification primer has an extendable 3' end that can be used to initiate a primer extension reaction, e.g., Figure 5 In some embodiments, the amplification primer can be a soluble primer or fixed to a support (e.g., a surface capture primer). In some embodiments, the amplification primer can be used to perform a rolling circle amplification reaction to generate a nucleic acid concatemer molecule complementary to the covalently closed circular library molecule (600).

[0123] Therefore, the double-stranded splint adapters and methods described herein can be used to convert any linear library molecule into a covalently closed circular molecule. The second splint chain (400) may include at least one new universal adapter sequence (e.g., a new surface primer sequence) so that the covalently closed circular molecule (600) can be bound to a support having a plurality of surface capture primers fixed thereon. The new universal adapter sequence in the second splint chain (400) allows the covalently closed circular molecule (600) to be used in an amplification and sequencing workflow.

[0124] The methods described herein also provide the advantage of using a ligation reaction rather than a gap-filling reaction to introduce new adapter sequences. The ligation reaction can produce efficient circularization with as little as 0.25 pmol of library molecules.

[0125] The methods described herein can be performed manually or easily automated because annealing and multi-enzyme reactions can be performed in a single reaction vessel (one pot) by combining some enzymatic reactions (e.g., phosphorylation and ligation) and by adding subsequent enzymes (e.g., exonucleases), without the need for intervening alcohol precipitation or organic extraction.

[0126] Double-stranded splint adapter

[0127] In some aspects, the present disclosure provides a nucleic acid double-stranded splint adaptor (200) comprising: (i) a first splint strand (long splint strand (300)) hybridized with (ii) a second splint strand (short splint strand (400)) (e.g., see Figure 2 and 3). The first splint chain may include a first region (320), an internal region (310) and a second region (330). The internal region (310) of the first splint chain can be hybridized with the second splint chain (400) to form a double-stranded splint adapter (200) having a double-stranded region and two flanking single-stranded regions. The two flanking single-stranded regions of the double-stranded splint adapter (200) can be designed to hybridize with the end sequence of the linear nucleic acid library molecule (100). The end sequence of the linear nucleic acid library molecule can include the first (120) and second (130) sequence universal adapter sequences, respectively. In some embodiments, the first universal adapter sequence and the second universal adapter sequence of the linear library molecule respectively include binding sequences for the forward (120) and reverse (130) sequencing primer binding sites.

[0128] like Figure 2 As shown, in some embodiments, the first splint chain comprises a first region (320) hybridized to a sequence at one end of the linear single-stranded library molecule and a second region (330) hybridized to a sequence at the other end of the linear single-stranded library molecule. The inner region (310) of the first splint chain can hybridize with the second splint chain (400). The inner region (310) of the first splint chain (300) may include at least three sub-regions, including sub-regions (311), (312) and (313). The second splint chain (400) may include at least three sub-regions, including sub-regions (411), (412) and (413). For example, sub-region (311) hybridizes with sub-region (411). In another example, sub-region (312) hybridizes with sub-region (412). In another example, sub-region (313) hybridizes with sub-region (413). The sub-region of the second splint strand (400) may include any one or more of the following, in any combination and in any order: a universal primer binding sequence for a surface capture primer, a universal primer binding sequence for a surface pinning primer, a sample index sequence, a short random sequence, and / or a unique molecular index (UMI) sequence.

[0129] In some embodiments, the first region (320) of the first splint strand comprises a first universal adaptor sequence that can hybridize to a first universal binding sequence at one end of a linear nucleic acid library molecule (e.g., see Figure 2 ). The second region (330) of the first splint strand may comprise a second universal adapter sequence that may hybridize to a second universal binding sequence at the other end of the linear nucleic acid library molecule (e.g., see Figure 2). In some embodiments, the first region (320) of the first splint strand comprises a first universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer. In some embodiments, the second region (330) of the first splint strand comprises a second universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer. In some embodiments, the 5' end of the first splint strand (300) is phosphorylated or non-phosphorylated. In some embodiments, the 3' end of the first splint strand (300) comprises a terminal 3' OH group or a terminal 3' blocking group.

[0130] In some embodiments, the second cleat chain (400) comprises at least three sub-regions, including a first sub-region, a second sub-region, and a third sub-region (see, for example, Figure 2 and 3 ). The first sub-region (411) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN), and / or a unique molecular index (UMI). The second sub-region (412) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN), and / or a unique molecular index (UMI). The third sub-region (413) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplexed assays. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify the nucleic acid molecule (e.g., a molecular marker) to which the adaptor is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence. In some embodiments, the second splint strand (400) is designed to exhibit reduced or no hybridization with the insert sequence (110) of the library molecule (100).

[0131] Exemplary arrangements of sequences in the sub-region of the second splint strand (400) in a 3' to 5' orientation include: [(411) comprises a universal sequence for binding a surface pinning primer] - [(412) comprises a sample index sequence and an optional short random sequence NNN] - [(413) comprises a universal sequence for binding a surface capture primer].

[0132] Exemplary arrangements of sequences in the sub-region of the second splint strand (400) in a 3' to 5' orientation include: [(411) comprises a universal sequence for binding a surface pinning primer] - [(412) comprises a universal sequence for binding a surface capture primer] - [(413) comprises a sample index sequence and an optional short random sequence NNN].

[0133] Exemplary arrangements of sequences in the sub-region of the second splint strand (400) in a 3' to 5' orientation include: [(411) comprises a sample index sequence and an optional short random sequence NNN] - [(412) comprises a universal sequence for binding a surface pinning primer] - [(413) comprises a universal sequence for binding a surface capture primer].

[0134] In some embodiments, the second splint chain (400) includes an additional sub-region carrying a second sample index sequence and an optional short random sequence NNN. For example, the additional sub-region can be located between sub-regions (411) and (412), or between sub-regions (412) and (413).

[0135] In some embodiments, the length of the second splint chain (400) can be 20 to 100 (e.g., about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100) nucleotides. In some embodiments, the length of the second splint chain (400) can be 30 to 80 (e.g., about 30, 35, 40, 45, 50, 60, 70 or 80) nucleotides, with a length of 80 nucleotides. The length is 40 to 6060 (e.g., about 40, 45, 50 or 60) nucleotides. In some embodiments, the 5' end of the second splint chain (400) is phosphorylated; alternatively, the 5' end of the second splint chain (400) is non-phosphorylated. In some embodiments, the 3' end of the second splint chain (400) includes a terminal 3'OH group; alternatively, the 3' end of the second splint chain (400) includes a terminal 3' blocking group. In some embodiments, the second splint strand (400) comprises one or more phosphorothioate bonds at the 5' end and / or the 3' end to confer resistance to exonucleases. In some embodiments, the second splint strand (400) comprises one or more phosphorothioate bonds at an internal position, for example, to confer resistance to endonucleases. In some embodiments, the second splint strand (400) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or the 3' end or at an internal position.

[0136] In some embodiments, the first splint chain (300) includes an inner region (310) including at least three sub-regions, including a fourth sub-region (311), a fifth sub-region (312), and a sixth sub-region (313). The fourth sub-region (311) can be hybridized with the first sub-region (411) of the second splint chain (400). The fourth sub-region (311) can be completely or partially complementary to the first sub-region (411) of the second splint chain (400). The fifth sub-region (312) can be hybridized with the second sub-region (412) of the second splint chain (400). The fifth sub-region (312) can be completely or partially complementary to the second sub-region (412) of the second splint chain (400). The sixth sub-region (313) can be hybridized with the third sub-region (413) of the second splint chain (400). The sixth sub-region (313) can be completely or partially complementary to the third sub-region (413) of the second splint chain (400). The fourth sub-region, the fifth sub-region and the sixth sub-region do not hybridize (or at least show very little hybridization) to the target sequence, the surface capture primer or the surface pinning primer.

[0137] In some embodiments, one of the subregions of the first splint strand (300) comprises an index or random sequence, such as a sample index, a short random sequence (e.g., NNN), and / or a unique molecule index (UMI). For example, subregions (311), (312), or (313) comprise a sample index, a short random sequence (e.g., NNN), and / or a unique molecule index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. In some embodiments, a unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify a nucleic acid molecule (e.g., a molecular tag) to which an adaptor is attached. In some embodiments, a unique molecular index (UMI) comprises a random sequence.

[0138] like Figure 6 As shown, in some embodiments, the sub-region (312) includes any one of the following or any combination of two or more: a sample index sequence (represented by "S"); a random sequence (represented by "N"); at least one nucleotide that can be converted into an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer (e.g., an 18-carbon spacer). Figure 6An alternative exemplary nucleic acid sequence of a second splint strand (400; short splint strand) is depicted, the second splint strand comprising: three subregions including: subregion (411); subregion (412); and subregion (413).

[0139] In some embodiments, the first splint chain subregion comprising an index or random sequence comprises any one of the following or any combination of two or more: a sample index sequence (represented by "S"); a random sequence (represented by "N"); at least one nucleotide that can be converted into an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer. For non-limiting examples, see Figure 6 .

[0140] In some embodiments, the double-stranded splint adapter (200) comprises a first splint strand (300) partially hybridized with a second splint strand (400), wherein the first splint strand (300) comprises a universal long splint strand. In some embodiments, the universal long splint strand comprises at least one subregion that is only partially hybridized with the second splint strand (400). For example, but not limited to, the universal long splint strand (300) comprises a subregion carrying at least one nucleotide that can be converted into an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer. An exemplary universal long splint chain (300) is as follows Figure 6 shown.

[0141] In some embodiments, Figure 7 As depicted in , the first cleat chain lacks a sub-region (312). Figure 7Also shown is an exemplary nucleic acid sequence of a second splint strand (400; short splint strand), the second splint strand comprising: three sub-regions, the three sub-regions comprising: sub-region (411); sub-region (412); and sub-region (413). In certain embodiments, when the first splint strand (300) hybridizes with the second splint strand (400), the sub-region (412) of the second splint strand (400) loops out because the first splint strand (300) lacks the sub-region (312).

[0142] In some embodiments, the first splint strand (300) lacks subregions (311), (312) or (313), so that the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400) has a portion of the second splint strand looped out. For example, but not limited to, the first splint strand (300) lacks subregion (312), and in the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400), the subregion (412) of the second splint strand (400) loops out (for example, see Figure 7 ). In some embodiments, the first cleat chain (300) lacking sub-regions is an example of a universal long cleat chain (300).

[0143] In some embodiments, the length of the first splint chain (300) may be 50 to 150 nucleotides, or 60 to 100 nucleotides, or 70 to 90 nucleotides in length. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at the 5' end and / or the 3' end to confer resistance to nucleic acid exonucleases. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at an internal position to confer resistance to nucleic acid endonucleases. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or the 3' end. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at an internal position.

[0144] Tables 1 to 6 below list various embodiments of universal adaptor sequences in the library molecule (100), sequences in the first splint strand (300), sequences in the second splint strand (400), sequences of immobilized surface primers, and sequences of sequencing primers.

[0145] In some embodiments, any universal adapter sequence in the library molecules (100) listed in Table 1 can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In some embodiments, the length of the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0146] In some embodiments, the sequencing primer comprises a sequence complementary to any one of the sequences listed in Table 1 below.

[0147] In some embodiments, the sequencing primer comprises a sequence complementary to any one of the sequences listed in Table 1 below, wherein the sequencing primer is truncated at the 5' end and / or the 3' end, and wherein the truncation can be 1 to 12 nucleotides. In some embodiments, the length of the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0148] In some embodiments, any sequence in the first cleat strand (300) and / or the second cleat strand (400) comprises a sequence complementary to any sequence listed in Tables 1 to 6 below.

[0149] In some embodiments, any sequence in the first splint strand (300) and / or the second splint strand (400) listed in Tables 2 to 3 can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In some embodiments, the length of the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0150] Table 1: Common sequences in library molecules (100)

[0151]

[0152] Table 2: First Clamp Chain (300): Sequence in Long Clamp Chain

[0153]

[0154]

[0155] Table 3: Second Cleat Chain (400): Sequence in Short Cleat Chain

[0156]

[0157]

[0158] Table 4: Immobilized surface primers

[0159]

[0160]

[0161] Table 5:

[0162]

[0163] Table 6:

[0164]

[0165] Library-splint complex

[0166] In some aspects, the present disclosure provides a library-splint complex (500) comprising: (i) a single-stranded nucleic acid library molecule (100) comprising an insert region (110) flanked on one side by a universal adapter sequence for a forward sequencing primer binding site (120) (e.g., a target sequence), and the insert region (110) is flanked on the other side by a universal adapter sequence for a reverse sequencing primer binding site (130); and (ii) a double-stranded splint adapter (200) comprising a first splint strand (long splint strand (300)) hybridized to a second splint strand (short splint strand (400)), wherein the first splint strand comprises a first region (320), an internal region (310), and a second region (330). In certain embodiments, the internal region (310) of the first splint strand hybridizes with the second splint strand (400) to form a double-stranded splint adapter (200) having a double-stranded region and two flanking single-stranded regions.

[0167] In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a universal adapter sequence for a surface capture primer binding site. In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a universal adapter sequence for a surface pinning primer binding site. In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a universal adapter sequence for a sample index sequence. In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a unique molecular index (UMI) sequence.

[0168] In the library-splint complex (500), the first region (320) of the first splint strand can hybridize with the universal adapter sequence for the forward sequencing primer binding site (120) of the library molecule, and the second region (330) of the first splint strand can hybridize with the universal adapter sequence for the reverse sequencing primer binding site (130) of the library molecule, thereby circularizing the library molecule to generate the library-splint complex (500) (see, for example, Figure 3 and 4 ).

[0169] In the library-splint complex (500), the second splint strand (400) can bring the library molecule (100) into proximity with at least a universal adapter sequence for a surface pinning primer binding site, a universal adapter sequence for a surface capture primer binding site, and a sample index sequence. In some embodiments, the second splint strand (400) brings the library molecule (100) into proximity with other sequences including a short random sequence (e.g., NNN) and / or a unique molecular index (UMI) sequence.

[0170] The library-splint complex (500) may comprise a first gap between the 5' end of the library molecule and the 3' end of the second splint strand. In certain embodiments, the library-splint complex (500) further comprises a second gap between the 5' end of the second splint strand and the 3' end of the library molecule (e.g., see Figure 3 and 4 In some embodiments, the first and second gaps are enzymatically ligatable. The ligation reaction will join the sequence from the second splint strand (400) to the end of the library molecule (100).

[0171] In the library-splint complex (500), the first region (320) of the first splint strand can hybridize with the sense strand or the antisense strand of the double-stranded nucleic acid library molecule. In the library-splint complex (500), the second region (330) of the first splint strand can hybridize with the sense strand or the antisense strand of the double-stranded nucleic acid library molecule. The double-stranded nucleic acid library molecules can be denatured to produce single-stranded sense and antisense library strands.

[0172] In the library-splint complex (500), the first region (320) of the first splint strand does not hybridize to the target sequence (110), and the second region (330) of the first splint strand does not hybridize to the target sequence (110).

[0173] In the library-splint complex (500), the second splint strand (400) does not hybridize to the target sequence (110), and the internal region (310) of the first splint strand does not hybridize to the target sequence (110).

[0174] In some embodiments, in the library-splint complex (500), the inner region (310) of the first splint chain (300) hybridizes with the second splint chain (400). The inner region (310) of the first splint chain (300) may include at least three sub-regions, including sub-regions (311), (312) and (313). The second splint chain (400) may include at least three sub-regions, including sub-regions (411), (412) and (413). For example, sub-region (311) hybridizes with sub-region (411). In another example, sub-region (312) hybridizes with sub-region (412). In another example, sub-region (313) hybridizes with sub-region (413).

[0175] In some embodiments, any library-splint complex (500) described herein comprises a plurality of library-splint complexes (500), wherein the sequence of interest (110) of each library-splint complex in the plurality of library-splint complexes comprises the same sequence of interest or a different sequence of interest.

[0176] In some embodiments, in the library-splint complex (500), the 5' end of the single-stranded library molecule (100) is phosphorylated. In some embodiments, in the library-splint complex (500), the 5' end of the single-stranded library molecule (100) lacks a phosphate group. In some embodiments, the 3' end of the single-stranded library molecule includes a terminal 3' OH group or a terminal 3' blocking group.

[0177] In some embodiments, in the library-splint complex (500), the length of the first splint chain (300) can be 50 to 150 (e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150) nucleotides. In some embodiments, in the library-splint complex (500), the length of the first splint chain (300) can be 60 to 100 (e.g., about 60, 70, 80, 90, or 100) nucleotides. In some embodiments, in the library-splint complex (500), the length of the first splint chain (300) can be 70 to 90 (e.g., about 70, 75, 80, 85, or 90) nucleotides. In some embodiments, the first splint chain (300) comprises one or more phosphorothioate bonds at the 5' end and / or the 3' end to confer exonuclease resistance. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at an internal position to confer endonuclease resistance. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or 3' end. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at an internal position. In some embodiments, the 5' end of the first splint chain (300) is phosphorylated. In some embodiments, the 5' end of the first splint chain (300) lacks a phosphate group. In some embodiments, the 3' end of the first splint chain (300) comprises a terminal 3'OH group or a terminal 3' blocking group.

[0178] In some embodiments, in the library-splint complex (500), the first splint chain (300) includes an internal region (310), which includes at least three sub-regions, including a fourth sub-region (311), a fifth sub-region (312), and a sixth sub-region (313). The fourth sub-region (311) can hybridize with the first sub-region (411) of the second splint chain (400). The fourth sub-region (311) can be completely or partially complementary to the first sub-region (411) of the second splint chain (400). The fifth sub-region (312) can hybridize with the second sub-region (412) of the second splint chain (400). The fifth sub-region (312) can be completely or partially complementary to the second sub-region (412) of the second splint chain (400). The sixth sub-region (313) can hybridize with the third sub-region (413) of the second splint chain (400). The sixth subregion (313) may be fully or partially complementary to the third subregion (413) of the second splint strand (400). The fourth, fifth and sixth subregions do not hybridize (e.g., at least exhibit very little hybridization) with the target sequence, the surface capture primer or the surface pinning primer.

[0179] In some embodiments, one of the subregions of the first splint strand (300) comprises an index or random sequence, such as a sample index, a short random sequence (e.g., NNN), and / or a unique molecule index (UMI). For example, but not limited to, subregions (311), (312), or (313) comprise a sample index, a short random sequence (e.g., NNN), and / or a unique molecule index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplexed assays. In some embodiments, a unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify a nucleic acid molecule (e.g., a molecular tag) to which an adaptor is attached. In some embodiments, a unique molecular index (UMI) comprises a random sequence.

[0180] In some embodiments, the first splint chain subregion comprising an index or random sequence comprises any one of the following or any combination of two or more: a sample index sequence (represented by "S"); a random sequence (represented by "N"); at least one nucleotide that can be converted to an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer.

[0181] In some embodiments, the double-stranded splint adapter (200) comprises a first splint strand (300) partially hybridized with a second splint strand (400), wherein the first splint strand (300) comprises a universal long splint strand. In some embodiments, the universal long splint strand comprises at least one sub-region that is only partially hybridized with the second splint strand (400). For example, the universal long splint strand (300) may comprise a sub-region carrying at least one nucleotide that can be converted into an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer. An exemplary universal long splint chain (300) is as follows Figure 6 shown.

[0182] In some embodiments, in the library-splint complex (500), the first splint strand (300) lacks subregion (311), (312) or (313), so that the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400) has a portion of the second splint strand looped out. For example, the first splint strand (300) lacks subregion (312), and in the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400), the subregion (412) of the second splint strand (400) loops out (e.g., see Figure 7 ). In some embodiments, the first cleat chain (300) lacking sub-regions is an example of a universal long cleat chain (300).

[0183] In some embodiments, in the library-splint complex (500), the second splint strand (400) comprises at least three sub-regions, including a first sub-region, a second sub-region, and a third sub-region (e.g., see Figure 3 and 4 ). The first sub-region (411) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN), and / or a unique molecular index (UMI). The second sub-region (412) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN), and / or a unique molecular index (UMI). The third sub-region (413) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplexed assays. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify the nucleic acid molecule (e.g., a molecular marker) to which the adaptor is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence. The second splint strand (400) is designed to exhibit reduced or no hybridization with the insert sequence (110) of the library molecule (100).

[0184] In some embodiments, in the library-splint complex (500), the sequences in the sub-region of the second splint chain (400) are arranged in a 3' to 5' orientation including: [(411) comprises a universal sequence for binding to a surface pinning primer] - [(412) comprises a sample index sequence and an optional short random sequence NNN] - [(413) comprises a universal sequence for binding to a surface capture primer].

[0185] In some embodiments, in the library-splint complex (500), the sequences in the sub-region of the second splint strand (400) are arranged in a 3' to 5' orientation including: [(411) comprising a universal sequence for binding a surface pinning primer] - [(412) comprising a universal sequence for binding a surface capture primer] - [(413) comprising a sample index sequence and an optional short random sequence NNN].

[0186] In some embodiments, in the library-splint complex (500), the sequences in the sub-region of the second splint chain (400) are arranged in a 3' to 5' orientation including: [(411) comprises a sample index sequence and an optional short random sequence NNN] - [(412) comprises a universal sequence for binding to a surface pinning primer] - [(413) comprises a universal sequence for binding to a surface capture primer].

[0187] In some embodiments, in the library-splint complex (500), the second splint strand (400) comprises an additional sub-region carrying a second sample index sequence and an optional short random sequence NNN. For example, but not limited to, the additional sub-region can be located between sub-regions (411) and (412), or between sub-regions (412) and (413).

[0188] In some embodiments, in the library-splint complex (500), the length of the second splint chain (400) can be 20 to 100 (e.g., about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100) nucleotides. In some embodiments, the length of the second splint chain (400) can be 30 to 80 (e.g., about 30, 35, 40, 45, 50, 60, 70 or 80) nucleotides, with a length of 80 nucleotides. The length is 40 to 6060 (e.g., about 40, 45, 50 or 60) nucleotides. In some embodiments, the 5' end of the second splint chain (400) is phosphorylated; alternatively, the 5' end of the second splint chain (400) is non-phosphorylated. In some embodiments, the 3' end of the second splint chain (400) comprises a terminal 3'OH group; alternatively, the 3' end of the second splint chain (400) comprises a terminal 3' blocking group. In some embodiments, the second splint chain (400) comprises one or more thiophosphate bonds at the 5' end and / or the 3' end to confer resistance to nucleic acid exonucleases. In some embodiments, the second splint chain (400) comprises one or more thiophosphate bonds at an internal position, for example, to confer resistance to nucleic acid endonucleases. In some embodiments, the second splint chain (400) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or the 3' end. In some embodiments, the second splint chain (400) comprises one or more 2'-O-methylcytosine bases at an internal position.

[0189] In the library-splint complex (500), the second splint strand (400) brings the library molecule (100) into proximity with at least a universal adapter sequence for a surface pinning primer binding site, a universal adapter sequence for a surface capture primer binding site, and a sample index sequence. In some embodiments, the second splint strand (400) brings the library molecule (100) into proximity with other sequences including a short random sequence (e.g., NNN) and / or a unique molecular index (UMI) sequence.

[0190] Tables 1 to 4 above list various embodiments of sequences of various molecules forming the library-splint complex (500), and the various molecules include library molecules (100), first splint chains (300) and second splint chains (400). In some embodiments, the sequences in the first splint chain (300) and / or the second splint chain (400) can be sequences complementary to the sequences listed in Tables 2 to 3. In some embodiments, any sequence in the first splint chain (300) and / or the second splint chain (400) listed in Tables 2 to 3 can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated. In some embodiments, in the library molecule, the sequence of the forward sequencing primer binding site (120) listed in Table 1 and / or the sequence of the reverse sequencing primer binding site (130) can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0191] In some aspects, the present disclosure provides a reaction mixture comprising a plurality of any library-splint complexes (500) described herein. In some embodiments, the reaction mixture comprises a plurality of any library-splint complexes (500) described herein and T4 polynucleotide kinase. In some embodiments, the reaction mixture comprises a plurality of any library-splint complexes (500) described herein and lacks T4 polynucleotide kinase. In some embodiments, the reaction mixture comprises a plurality of any library-splint complexes (500) described herein and a ligase. In some embodiments, the reaction mixture comprises a plurality of any library-splint complexes (500) described herein and a T4 polynucleotide kinase and a ligase. In some embodiments, the reaction mixture comprises a plurality of any library-splint complexes (500) described herein and a ligase and lacks T4 polynucleotide kinase. In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase or Taq ligase.

[0192] Covalently closed ring molecule

[0193] In some aspects, the present disclosure provides a covalently closed circular library molecule (600) comprising: a target sequence (110), a forward sequencing primer binding site (120), a reverse sequencing primer binding site (130), and the sequences form a second splint strand (400). In some embodiments, the covalently closed circular library molecule (600) comprises a target sequence (110) flanked on one side by a forward sequencing primer binding site (120) and on the other side by a reverse sequencing primer binding site (130), wherein the forward sequencing primer binding site (120) and the reverse sequencing primer binding site (130) are covalently joined to the second splint strand (400). An exemplary covalently closed circular library molecule (600) is shown in Figure 4 and 5 middle.

[0194] In some embodiments, any covalently closed circular library molecule (600) described herein further comprises a plurality of covalently closed circular library molecules (600), wherein the target sequence (110) of each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules comprises the same target sequence. Alternatively, in some embodiments, the target sequence (110) comprises a different target sequence.

[0195] In some embodiments, in the covalently closed circular library molecule (600), the second splint strand (400) comprises at least three sub-regions, including a first sub-region, a second sub-region, and a third sub-region (e.g., see Figure 4 and 5 ). The first sub-area (411) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI). The second sub-area (412) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI). The third sub-area (413) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (e.g., NNN) and / or a unique molecular index (UMI). In some embodiments, the sample index comprises 5 to 20 bases, which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex determinations. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases, which can be used to uniquely identify a nucleic acid molecule (e.g., a molecular tag) to which an adapter is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence. The second splint strand (400) is designed to exhibit reduced or no hybridization to the insert sequence (110) of the library molecule (100).

[0196] In some embodiments, in the covalently closed circular library molecule (600), the sequences in the sub-region of the second splint chain (400) are arranged in a 3' to 5' orientation and include: [(411) comprises a universal sequence for binding to a surface pinning primer] - [(412) comprises a sample index sequence and an optional short random sequence NNN] - [(413) comprises a universal sequence for binding to a surface capture primer].

[0197] In some embodiments, in the covalently closed circular library molecule (600), the sequences in the sub-region of the second splint strand (400) are arranged in a 3' to 5' orientation including: [(411) comprises a universal sequence for binding a surface pinning primer] - [(412) comprises a universal sequence for binding a surface capture primer] - [(413) comprises a sample index sequence and an optional short random sequence NNN].

[0198] In some embodiments, in the covalently closed circular library molecule (600), the sequences in the sub-region of the second splint chain (400) are arranged in a 3' to 5' orientation including: [(411) comprises a sample index sequence and an optional short random sequence NNN] - [(412) comprises a universal sequence for binding to a surface pinning primer] - [(413) comprises a universal sequence for binding to a surface capture primer].

[0199] In some embodiments, in the covalently closed circular library molecule (600), the second splint chain (400) comprises an additional subregion carrying a second sample index sequence and an optional short random sequence NNN. For example, the additional subregion can be located between subregions (411) and (412), or between subregions (412) and (413).

[0200] In some embodiments, in the covalently closed circular library molecule (600), the length of the second splint chain (400) can be 20 to 100 nucleotides, or 30 to 80 nucleotides, or 40 to 60 nucleotides. In some embodiments, the second splint chain (400) comprises one or more thiophosphate bonds at the 5' end and / or 3' end to confer resistance to exonucleases. In some embodiments, the second splint chain (400) comprises one or more thiophosphate bonds at an internal position to confer resistance to endonucleases. In some embodiments, the second splint chain (400) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or 3' end or at an internal position.

[0201] In some embodiments, the covalently closed circular library molecule (600) is hybridized with the first splint chain (300) or the first splint chain (300) is not present. In some embodiments, the length of the first splint chain (300) can be 50 to 150 (e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150) nucleotides. In some embodiments, the length of the first splint chain (300) can be 60 to 100 (e.g., about 60, 70, 80, 90 or 100) nucleotides. In some embodiments, the length of the first splint chain (300) can be 70 to 90 (e.g., about 70, 75, 80, 85 or 90) nucleotides. In some embodiments, the first splint chain (300) comprises one or more phosphorothioate bonds at the 5' end and / or 3' end, for example, to confer exonuclease resistance. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at an internal position, for example to confer endonuclease resistance. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or the 3' end. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at an internal position. In some embodiments, the 5' end of the first splint chain (300) is phosphorylated; alternatively, the 5' end of the first splint chain (300) lacks a phosphate group. In some embodiments, the 3' end of the first splint chain (300) includes a terminal 3'OH group; alternatively, the 3' end of the first splint chain (300) includes a terminal 3' blocking group.

[0202] In some embodiments, in the covalently closed circular library molecule (600), the first splint chain (300) comprises an internal region (310), which includes at least three sub-regions, including a fourth sub-region (311), a fifth sub-region (312), and a sixth sub-region (313). The fourth sub-region (311) can hybridize with the first sub-region (411) of the second splint chain (400). The fourth sub-region (311) can be completely or partially complementary to the first sub-region (411) of the second splint chain (400). The fifth sub-region (312) can hybridize with the second sub-region (412) of the second splint chain (400). The fifth sub-region (312) can be completely or partially complementary to the second sub-region (412) of the second splint chain (400). The sixth sub-region (313) can hybridize with the third sub-region (413) of the second splint chain (400). The sixth subregion (313) may be fully or partially complementary to the third subregion (413) of the second splint strand (400). The fourth, fifth, and sixth subregions do not hybridize (eg, show very little hybridization) with the target sequence, the surface capture primer, or the surface pinning primer.

[0203] In some embodiments, in the covalently closed circular library molecule (600), one of the subregions of the first splint strand (300) comprises an index or a random sequence, such as a sample index, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI). For example, subregions (311), (312), or (313) comprise a sample index, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI). In some embodiments, the sample index comprises 5 to 20 bases, which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplexed assays. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases, which can be used to uniquely identify a nucleic acid molecule (e.g., a molecular tag) to which an adapter is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence.

[0204] In some embodiments, in the covalently closed circular library molecule (600), the subregion (312) comprises any one of the following or any combination of two or more: a sample index sequence (represented by "S"); a random sequence (represented by "N"); at least one nucleotide that can be converted to an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer.

[0205] In some embodiments, when the covalently closed circular library molecule (600) is hybridized with the first splint strand (300), the first splint strand partially hybridizes with the sequence from the second splint strand (400), wherein the first splint strand (300) comprises a universal long splint strand. In some embodiments, the universal long splint strand may comprise at least one subregion that is only partially hybridized with the sequence from the second splint strand (400). For example, the universal long splint strand (300) may comprise a subregion carrying at least one nucleotide that can be converted to an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, for example, a PEG2, PEG3 or PEG4 spacer. Exemplary universal long splint chains (300) are as follows Figure 6shown.

[0206] In some embodiments, in the covalently closed circular library molecule (600), the first splint strand (300) lacks subregion (311), (312) or (313), so that the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400) has a portion of the second splint strand looped out. For example, the first splint strand (300) may lack subregion (312), and in the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400), the subregion (412) of the second splint strand (400) loops out (e.g., see Figure 7 ). In some embodiments, the first cleat chain (300) lacking sub-regions is an example of a universal long cleat chain (300).

[0207] In some embodiments, an exemplary covalently closed circular molecule (600) comprises: (i) a target sequence (110); (ii) a universal adapter sequence having a binding sequence for a forward sequencing primer (140); (iii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (150); (iv) a universal adapter sequence having a binding sequence for a surface pinning primer (subregion 411); (v) a universal adapter sequence having a binding sequence for a surface capture primer (subregion 413); and (vi) a sample index, a short random sequence (NNN) and / or a unique molecular index (UMI) (subregion 412), wherein the covalently closed circular molecule (600) is optionally hybridized to a first splint chain (300).

[0208] In some embodiments, an exemplary covalently closed circular molecule (600) comprises: (i) a target sequence (110); (ii) a universal adapter sequence having a binding sequence for a forward sequencing primer (140); (iii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (150); (iv) a universal adapter sequence having a binding sequence for a surface pinning primer (subregion 411); (v) a universal adapter sequence having a binding sequence for a surface capture primer (subregion 412); and (vi) a sample index, a short random sequence (NNN) and / or a unique molecular index (UMI) (subregion 413), wherein the covalently closed circular molecule (600) is optionally hybridized to the first splint chain (300).

[0209] In some embodiments, an exemplary covalently closed circular molecule (600) comprises: (i) a target sequence (110); (ii) a universal adapter sequence having a binding sequence for a forward sequencing primer (140); (iii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (150); (iv) a universal adapter sequence having a binding sequence for a surface pinning primer (subregion 412); (v) a universal adapter sequence having a binding sequence for a surface capture primer (subregion 413); and (vi) a sample index, a short random sequence (NNN) and / or a unique molecular index (UMI) (subregion 411), wherein the covalently closed circular molecule (600) is optionally hybridized to a first splint chain (300).

[0210] In some embodiments, any covalently closed circular molecule (600) described herein comprises a plurality of covalently closed circular molecules (600), wherein the target sequence (110) of each covalently closed circular molecule (600) in the plurality of covalently closed circular molecules comprises the same target sequence. In some embodiments, the target sequence (110) of each covalently closed circular molecule (600) in the plurality of covalently closed circular molecules comprises a different target sequence.

[0211] Tables 1 to 4 above list various embodiments of sequences forming covalently closed circular library molecules (600), including library molecules (100), first splint chains (300) and second splint chains (400). In some embodiments, the sequences in the first splint chain (300) and / or the second splint chain (400) include sequences complementary to the sequences listed in Tables 2 to 3. In some embodiments, any sequence in the first splint chain (300) and / or the second splint chain (400) listed in Tables 2 to 3 can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated. In some embodiments, in the library molecule, the sequence of the forward sequencing primer binding site (120) listed in Table 1 and / or the sequence of the reverse sequencing primer binding site (130) can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0212] The present disclosure provides a reaction mixture comprising a plurality of any covalently closed circular molecules (600) described herein and at least one exonuclease. In some embodiments, the exonuclease comprises any one of exonuclease I, thermolabile exonuclease I and / or T7 exonuclease or any combination of two or more thereof.

[0213] By using a double-stranded splint adapter carrying at least one sample index sequence to prepare a covalently closed circular library molecule (600) with a sample index, a multiple workflow can be achieved. The sample index sequence can be used to prepare a separate batch of covalently closed circular library molecules (600) with a sample index using input nucleic acids isolated from different sources. The covalently closed circular library molecules (600) with a sample index can be pooled together to generate a multiple covalently closed circular library molecule (600) mixture, and the pooled covalently closed circular library molecules (600) can be amplified and / or sequenced. The sequence of the insertion region together with the sample index sequence can be used to identify the source of the input nucleic acid. In some embodiments, any number of covalently closed circular library molecules (600) can be pooled together, for example, 2 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 batches of covalently closed circular library molecules (600) with a sample index can be pooled. Exemplary nucleic acid sources include naturally occurring, recombinant or chemically synthesized sources. Exemplary nucleic acid sources include individual cells, multiple cells, tissues, biological fluids, environmental samples or whole organisms. Exemplary nucleic acid sources include fresh, frozen, fresh frozen or archived sources (e.g., formalin-fixed paraffin embedded; FFPE). Technicians will recognize that nucleic acids can be separated from many other sources. Nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0214] Kits containing double-stranded splint adapters

[0215] The present disclosure provides a kit for introducing one or more new adapter sequences into a linear nucleic acid library molecule. In some embodiments, the kit can be used to loop a single-stranded nucleic acid library molecule having a universal adapter sequence flanking a forward sequencing primer binding site (120) on one side and a target sequence (110) flanking a universal adapter sequence for a reverse sequencing primer binding site (130) on the other side. In some embodiments, the looped library molecule can be converted into a covalently closed circular molecule, which can undergo a rolling circle amplification (RCA) reaction to generate a nucleic acid concatemer. The concatemer can be fixed to a support for large-scale parallel sequencing.

[0216] The present disclosure provides a kit including a double-stranded nucleic acid splint adapter (200), which comprises: (i) a first splint chain (long splint chain (300)), and (ii) a second splint chain (short splint chain (400)). The first splint chain comprises a first region (320), an internal region (310) and a second region (330). The internal region (310) of the first splint chain can be hybridized with the second splint chain (400) to form a double-stranded splint adapter (200) having a double-stranded region and two flanking single-stranded regions. The two flanking single-stranded regions of the double-stranded splint adapter (200) are designed to hybridize with the terminal sequence of a linear nucleic acid library molecule (100). The terminal sequences of the linear nucleic acid library molecules respectively comprise the first (120) and second (130) sequence universal adapter sequences. In some embodiments, the first universal adapter sequence and the second universal adapter sequence of the linear library molecule comprise binding sequences for the forward (120) and reverse (130) sequencing primer binding sites, respectively.

[0217] At least a portion of the internal region (310) of the first splint strand can hybridize with the second splint strand (400) to form a double-stranded splint adapter (200) having a double-stranded region and two flanking single-stranded regions. The second splint strand (400) includes at least one new adapter sequence. The formation of the library-splint complex (500) brings the library molecule (100) into proximity with at least one new universal adapter sequence, including, for example, a universal sequence for a surface pinning primer binding site, a universal sequence for a surface capture primer binding site, a sample index sequence, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI) sequence. An exemplary double-stranded splint adapter is shown in Figure 2 and 3 The kit may include a container containing a first splint chain (300) and a second splint chain (400) in hybrid or non-hybrid form. The kit may include a first container and a second container, the first container containing the first splint chain (300) and the second container containing the second splint chain (400).

[0218] In some embodiments, in the kit, the first region (320) of the first splint strand comprises a first universal adapter sequence that can hybridize to a first universal binding sequence at one end of a linear nucleic acid library molecule (e.g., see Figure 2 and 3 The second region (330) of the first splint strand comprises a second universal adapter sequence that can hybridize to a second universal binding sequence at the other end of the linear nucleic acid library molecule (e.g., see Figure 2 and 3). In some embodiments, the first region (320) of the first splint strand comprises a first universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer. In some embodiments, the second region (330) of the first splint strand comprises a second universal adapter sequence comprising a universal binding sequence for a forward or reverse sequencing primer. In some embodiments, the 5' end of the first splint strand (300) is phosphorylated; alternatively, the 5' end of the first splint strand (300) is non-phosphorylated. In some embodiments, the 3' end of the first splint strand (300) comprises a terminal 3' OH group; alternatively, the 5' end of the first splint strand (300) is a terminal 3' blocking group.

[0219] In some embodiments, in the kit, the second splint chain (400) comprises at least three sub-regions, including a first sub-region, a second sub-region, and a third sub-region (eg, see Figure 2 and 3 ). The first sub-region (411) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN), and / or a unique molecular index (UMI). The second sub-region (412) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (NNN), and / or a unique molecular index (UMI). The third sub-region (413) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplexed assays. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify the nucleic acid molecule (e.g., a molecular marker) to which the adaptor is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence. The second splint strand (400) is designed to exhibit reduced or no hybridization with the insert sequence (110) of the library molecule (100).

[0220] Exemplary arrangements of sequences in the sub-region of the second splint strand (400) in a 3' to 5' orientation include: [(411) comprises a universal sequence for binding a surface pinning primer] - [(412) comprises a sample index sequence and an optional short random sequence NNN] - [(413) comprises a universal sequence for binding a surface capture primer].

[0221] Exemplary arrangements of sequences in the sub-region of the second splint strand (400) in a 3' to 5' orientation include: [(411) comprises a universal sequence for binding a surface pinning primer] - [(412) comprises a universal sequence for binding a surface capture primer] - [(413) comprises a sample index sequence and an optional short random sequence NNN].

[0222] Exemplary arrangements of sequences in the sub-region of the second splint strand (400) in a 3' to 5' orientation include: [(411) comprises a sample index sequence and an optional short random sequence NNN] - [(412) comprises a universal sequence for binding a surface pinning primer] - [(413) comprises a universal sequence for binding a surface capture primer].

[0223] In some embodiments, in the kit, the length of the second splint chain (400) can be 20 to 100 (e.g., about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100) nucleotides. In some embodiments, the length of the second splint chain (400) can be 30 to 80 (e.g., about 30, 35, 40, 45, 50, 60, 70 or 80) nucleotides, with a length of 80 nucleotides. The length is 40 to 6060 (e.g., about 40, 45, 50 or 60) nucleotides. In some embodiments, the 5' end of the second splint chain (400) is phosphorylated; alternatively, the 5' end of the second splint chain (400) is non-phosphorylated. In some embodiments, the 3' end of the second splint chain (400) includes a terminal 3'OH group; alternatively, the 3' end of the second splint chain (400) includes a terminal 3' blocking group. In some embodiments, the second splint strand (400) comprises one or more phosphorothioate bonds at the 5' end and / or the 3' end to confer resistance to exonucleases. In some embodiments, the second splint strand (400) comprises one or more phosphorothioate bonds at an internal position, for example, to confer resistance to endonucleases. In some embodiments, the second splint strand (400) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or the 3' end or at an internal position.

[0224] In some embodiments, in the kit, the first splint chain (300) comprises an internal region (310), which includes at least three sub-regions, including a fourth sub-region (311), a fifth sub-region (312) and a sixth sub-region (313). The fourth sub-region (311) can be hybridized with the first sub-region (411) of the second splint chain (400). The fourth sub-region (311) can be completely or partially complementary to the first sub-region (411) of the second splint chain (400). The fifth sub-region (312) can be hybridized with the second sub-region (412) of the second splint chain (400). The fifth sub-region (312) can be completely or partially complementary to the second sub-region (412) of the second splint chain (400). The sixth sub-region (313) can be hybridized with the third sub-region (413) of the second splint chain (400). The sixth subregion (313) may be fully or partially complementary to the third subregion (413) of the second splint strand (400). The fourth, fifth and sixth subregions do not hybridize (or at least show very little hybridization) with the target sequence, the surface capture primer or the surface pinning primer.

[0225] In some embodiments, in the kit, one of the subregions of the first splint strand (300) comprises an index or random sequence, such as a sample index, a short random sequence (e.g., NNN), and / or a unique molecule index (UMI). For example, subregions (311), (312), or (313) comprise a sample index, a short random sequence (e.g., NNN), and / or a unique molecule index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. In some embodiments, a unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify a nucleic acid molecule (e.g., a molecular tag) to which an adaptor is attached. In some embodiments, a unique molecular index (UMI) comprises a random sequence.

[0226] In some embodiments, in the kit, the first splint chain subregion comprising an index or random sequence comprises any one of the following or any combination of two or more: a sample index sequence (represented by "S"); a random sequence (represented by "N"); at least one nucleotide that can be converted to an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer.

[0227] In some embodiments, in the kit, the double-stranded splint adapter (200) comprises a first splint strand (300) partially hybridized with a second splint strand (400), wherein the first splint strand (300) comprises a universal long splint strand. In some embodiments, the universal long splint strand comprises at least one subregion that is only partially hybridized with the second splint strand (400). For example, but not limited to, the universal long splint strand (300) comprises a subregion carrying at least one nucleotide that can be converted into an abasic base (represented by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (represented by "I"); and / or a spacer. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer. An exemplary universal long splint chain (300) is as follows Figure 6 shown.

[0228] In some embodiments, in the kit, the first splint strand (300) lacks subregion (311), (312) or (313), so that the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400) has a portion of the second splint strand looped out. For example, but not limited to, the first splint strand (300) lacks subregion (312), and in the duplex formed by hybridization between the first splint strand (300) and the second splint strand (400), the subregion (412) of the second splint strand (400) loops out (for example, see Figure 7 ). In some embodiments, the first cleat chain (300) lacking sub-regions is an example of a universal long cleat chain (300).

[0229] In some embodiments, in the kit, the length of the first splint chain (300) can be 50 to 150 nucleotides, or 60 to 100 nucleotides, or 70 to 90 nucleotides. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at the 5' end and / or 3' end to confer resistance to exonucleases. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at an internal position to confer resistance to endonucleases. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or 3' end or at an internal position.

[0230] Tables 1 to 4 above list various embodiments of universal adapter sequences that can be included in the library molecule (100), the first splint chain (300), the second splint chain (400), and the fixed surface primers in the kit. In some embodiments, in any kit described herein, the sequence in the first splint chain (300) and / or the second splint chain (400) includes a sequence complementary to the sequence listed in Tables 2 to 3. In some embodiments, in any kit described herein, any sequence in the first splint chain (300) and / or the second splint chain (400) listed in Tables 2 to 3 can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0231] In some embodiments, the kit comprises a nucleic acid double-stranded splint adapter (200) and further comprises T4 polynucleotide kinase. In some embodiments, the kit further comprises a ligase, optionally wherein the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase or Taq ligase. In some embodiments, the kit further comprises at least one endonuclease, comprising any one of exonuclease I, thermolabile exonuclease I and / or T7 exonuclease or any combination of two or more thereof.

[0232] In some embodiments, the kit comprises at least one buffer for hybridizing a plurality of double-stranded splint adapters (200) and a plurality of nucleic acid library molecules (100). In some embodiments, the kit comprises a buffer for performing multiple enzymatic reactions in a single reaction vessel, comprising any combination of: (i) phosphorylating the 5' end of the first and / or second splint strands (e.g., (300) and / or (400)), (ii) ligating a gap in the library-splint complex (500), and / or (iii) exonuclease digestion of the first splint strand (300) from the covalently closed circular molecule (600). Alternatively, the kit comprises two or more separate buffers, for example, wherein a first buffer can be used to perform a phosphorylation reaction, a second buffer can be used to perform a ligation reaction, and a third buffer can be used to perform an exonuclease digestion reaction.

[0233] In some embodiments, the kit comprises one or more containers containing any double-stranded splint adapter (200) described herein or any of the first splint strand (300) and the second splint strand (400) described herein. The kit may further comprise one or more containers containing T4 polynucleotide kinase, at least one ligase and / or at least one exonuclease. The kit may comprise any of these components in any combination and may be contained in a single container, or may be contained in separate containers, or any combination thereof.

[0234] The kit can include instructions for using the kit, for example, to perform a reaction to introduce one or more new adaptor sequences into a linear nucleic acid library molecule.

[0235] Methods for forming multiple library-splint complexes

[0236] In some aspects, the present disclosure provides a method for forming a plurality of library-splint complexes (500), the method comprising: (a) providing a plurality of double-stranded splint adaptors (200), wherein each of the plurality of double-stranded splint adaptors (200) comprises a first splint strand (300) hybridized to a second splint strand (400), wherein the double-stranded splint adaptors comprise a double-stranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region (320), an internal region (310), and a second region (330), and wherein the internal region (310) of the first splint strand hybridizes to the second splint strand (400). Exemplary double-stranded splint adaptors (200) are shown in Figure 2 and 3 In some embodiments, a portion of the interior region (310) of the first splint strand (300) does not hybridize with a portion of the second splint strand (400).

[0237] In some embodiments, the method for forming a plurality of library-splint complexes (500) further comprises step (b): hybridizing a plurality of double-stranded splint adaptors to a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule comprises a sequence of interest (110) flanked on one side by a universal adaptor sequence for a forward sequencing primer binding site (120) and on the other side by a universal adaptor sequence for a reverse sequencing primer binding site (130) (e.g., Figure 3 ). The hybridization can be performed under conditions suitable for hybridizing the first region (320) of the first splint strand to the portion (120) of the library molecule, and the conditions are suitable for hybridizing the second region (330) of the first splint strand to the portion (130) of the library molecule, thereby looping the plurality of library molecules to form a plurality of library-splint complexes (500).

[0238] In some embodiments, in a method for forming a plurality of library-splint complexes (500), a first region (320) of a first splint strand comprises a sequence that hybridizes to a universal adapter sequence for a forward sequencing primer binding site (120) at one end of a linear nucleic acid library molecule.

[0239] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the second region (330) of the first splint strand comprises a sequence that can hybridize to a universal adapter sequence for a reverse sequencing primer binding site (130) at the other end of the linear nucleic acid library molecule.

[0240] In some embodiments, the 5' end of the second splint chain (400) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the second splint chain (400) includes a terminal 3' OH group or a terminal 3' blocking group.

[0241] In some embodiments, in a method for forming a plurality of library-splint complexes (500), a first region (320) of a first splint strand hybridizes with a universal adapter sequence for a forward sequencing primer binding site (120) of a library molecule, and a second region (330) of a first splint strand hybridizes with a universal adapter sequence for a reverse sequencing primer binding site (130) of a library molecule, thereby circularizing the library molecule to generate a library-splint complex (500). The library-splint complex (500) may include a first gap (e.g., Figure 3 and 4 The library-splint complex (500) may further comprise a second gap (eg, Figure 3 and 4In some embodiments, the first and second gaps are enzymatically ligatable. The ligation reaction will join the sequence from the second splint strand (400) to the end of the library molecule (100).

[0242] In some embodiments, in a method for forming a plurality of library-splint complexes (500), a first region (320) of a first splint strand can hybridize with a sense strand or an antisense strand of a double-stranded nucleic acid library molecule. In the library-splint complex (500), a second region (330) of a first splint strand can hybridize with a sense strand or an antisense strand of a double-stranded nucleic acid library molecule. Double-stranded nucleic acid library molecules can be denatured to produce single-stranded sense and antisense library strands.

[0243] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the second splint strand (400) does not hybridize to the sequence of interest (110), and the internal region (310) of the first splint strand does not hybridize to the sequence of interest (110).

[0244] In some embodiments, in a method for forming a plurality of library-splint complexes (500), a first region (320) of a first splint strand does not hybridize to a sequence of interest (110), and a second region (330) of a first splint strand does not hybridize to a sequence of interest (110).

[0245] In some embodiments, in the method for forming a plurality of library-splint complexes (500), the 5' end of the single-stranded library molecule (100) is phosphorylated; alternatively, the 5' end of the single-stranded library molecule (100) lacks a phosphate group. In some embodiments, the 3' end of the single-stranded library molecule includes a terminal 3' OH group; alternatively, the 3' end of the single-stranded library molecule includes a terminal 3' blocking group.

[0246] In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a universal adapter sequence for a surface capture primer binding site. In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a universal adapter sequence for a surface pinning primer binding site. In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a universal adapter sequence for a sample index sequence. In some embodiments, the single-stranded nucleic acid library molecule (100) lacks a unique molecular index (UMI) sequence.

[0247] In some embodiments, in the method for forming a plurality of library-splint complexes (500), the second splint strand (400) comprises at least three sub-regions, including a first sub-region, a second sub-region, and a third sub-region (e.g., see Figure 2 and 3). The first sub-region (411) includes a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence and / or a unique molecular index (UMI). The second sub-region (412) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence and / or a unique molecular index (UMI). The third sub-region (413) may include a universal binding sequence for the fixed surface pinning primer, a fixed surface capture primer, at least one sample index sequence, a short random sequence (e.g., NNN) and / or a unique molecular index (UMI). In some embodiments, the sample index includes 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplexed assays. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases) that can be used to uniquely identify the nucleic acid molecule (e.g., a molecular marker) to which the adaptor is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence. The second splint strand (400) is designed to exhibit reduced or no hybridization with the insert sequence (110) of the library molecule (100).

[0248] In some embodiments, in a method for forming multiple library-splint complexes (500), the sequences in the sub-regions of the second splint chain (400) are arranged in a 3' to 5' orientation and include: [(411) comprises a universal sequence for binding to a surface pinning primer] - [(412) comprises a sample index sequence and an optional short random sequence NNN] - [(413) comprises a universal sequence for binding to a surface capture primer].

[0249] In some embodiments, in a method for forming multiple library-splint complexes (500), the sequences in the sub-regions of the second splint chain (400) are arranged in a 3' to 5' orientation including: [(411) comprising a universal sequence for binding to a surface pinning primer] - [(412) comprising a universal sequence for binding to a surface capture primer] - [(413) comprising a sample index sequence and an optional short random sequence NNN].

[0250] In some embodiments, in a method for forming multiple library-splint complexes (500), the sequences in the sub-regions of the second splint chain (400) are arranged in a 3' to 5' orientation including: [(411) comprising a sample index sequence and an optional short random sequence NNN] - [(412) comprising a universal sequence for binding to a surface pinning primer] - [(413) comprising a universal sequence for binding to a surface capture primer].

[0251] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the second splint strand (400) comprises an additional subregion carrying a second sample index sequence and an optional short random sequence NNN. For example, the additional subregion may be located between subregions (411) and (412), or between subregions (412) and (413).

[0252] In some embodiments, in the method of forming multiple library-splint complexes (500), the length of the second splint chain (400) can be 20 to 100 (e.g., about 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100) nucleotides. In some embodiments, the length of the second splint chain (400) can be 30 to 80 (e.g., about 30, 35, 40, 45, 50, 60, 70 or 80) nucleotides, with a length of 80 nucleotides. The length is 40 to 6060 (e.g., about 40, 45, 50 or 60) nucleotides. In some embodiments, the 5' end of the second splint chain (400) is phosphorylated; alternatively, the 5' end of the second splint chain (400) is non-phosphorylated. In some embodiments, the 3' end of the second splint chain (400) comprises a terminal 3'OH group; alternatively, the 3' end of the second splint chain (400) comprises a terminal 3' blocking group. In some embodiments, the second splint chain (400) comprises one or more thiophosphate bonds at the 5' end and / or the 3' end to confer resistance to nucleic acid exonucleases. In some embodiments, the second splint chain (400) comprises one or more thiophosphate bonds at an internal position, for example, to confer resistance to nucleic acid endonucleases. In some embodiments, the second splint chain (400) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or the 3' end or at an internal position.

[0253] In some embodiments, in the method for forming a plurality of library-splint complexes (500), the length of the first splint chain (300) can be 50 to 150 nucleotides, or 60 to 100 nucleotides, or 70 to 90 nucleotides in length. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at the 5' end and / or 3' end to confer resistance to nucleic acid exonucleases. In some embodiments, the first splint chain (300) comprises one or more thiophosphate bonds at an internal position to confer resistance to nucleic acid endonucleases. In some embodiments, the first splint chain (300) comprises one or more 2'-O-methylcytosine bases at the 5' end and / or 3' end or at an internal position. In some embodiments, the 5' end of the first splint chain (300) is phosphorylated or lacks a phosphate group. In some embodiments, the 3' end of the first splint chain (300) comprises a terminal 3'OH group or a terminal 3' blocking group.

[0254] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the first splint chain (300) comprises an internal region (310) comprising at least three sub-regions, including a fourth sub-region (311), a fifth sub-region (312), and a sixth sub-region (313). The fourth sub-region (311) may hybridize with the first sub-region (411) of the second splint chain (400). The fourth sub-region (311) may be completely or partially complementary to the first sub-region (411) of the second splint chain (400). The fifth sub-region (312) may hybridize with the second sub-region (412) of the second splint chain (400). The fifth sub-region (312) may be completely or partially complementary to the second sub-region (412) of the second splint chain (400). The sixth sub-region (313) may hybridize with the third sub-region (413) of the second splint chain (400). The sixth subregion (313) may be fully or partially complementary to the third subregion (413) of the second splint strand (400). The fourth, fifth and sixth subregions do not hybridize (or at least show very little hybridization) with the target sequence, the surface capture primer or the surface pinning primer.

[0255] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the first splint strand (300) comprises a subregion (312) comprising a sample index, a short random sequence (e.g., NNN), and / or a unique molecular index (UMI). In some embodiments, the sample index comprises 5 to 20 bases (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. In some embodiments, the unique molecular index (UMI) comprises 3 to 20 bases (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases), which can be used to uniquely identify a nucleic acid molecule (e.g., a molecular marker) to which an adaptor is attached. In some embodiments, the unique molecular index (UMI) comprises a random sequence.

[0256] In some embodiments, in a method for forming a plurality of library-splint complexes (500), the sub-region (312) includes any one of the following or any combination of two or more: a sample index sequence (denoted by "S"); a random sequence (denoted by "N"); at least one nucleotide that can be converted to an abasic base (denoted by a solid black rectangle) (e.g., uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine); deoxyinosine (denoted by "I"); and / or a spacer. In some embodiments, the spacer includes an 18-carbon spacer (e.g., including a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 including a trimethylene glycol spacer. In some embodiments, the spacer includes a polyethylene glycol spacer, e.g., a PEG2, PEG3, or PEG4 spacer.

[0257] In some embodiments, in a method of forming a plurality of library-splint complexes (500), the first splint strand (300) lacks subregion (311), (312) or (313), such that a duplex formed by hybridization between the first splint strand (300) and the second splint strand (400) has a portion of the second splint strand looped out. For example, the first splint strand (300) lacks subregion (312), and in a duplex formed by hybridization between the first splint strand (300) and the second splint strand (400), subregion (412) of the second splint strand (400) loops out (e.g., see Figure 7 ).

[0258] Tables 1 to 4 above list various embodiments of sequences of various molecules that form library-splint complexes (500), and the various molecules include library molecules (100), first splint chains (300), and second splint chains (400). In some embodiments, in any method for forming a plurality of library-splint complexes (500) described herein, the sequence in the first splint chain (300) and / or the second splint chain (400) includes a sequence complementary to the sequence listed in Tables 2 to 3. In some embodiments, in any method for forming a plurality of library-splint complexes (500) described herein, any sequence in the first splint chain (300) and / or the second splint chain (400) listed in Tables 2 to 3 can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In some embodiments, the length of the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated. In certain embodiments, in the library molecule, the sequence of the forward sequencing primer binding site (120) listed in Table 1 and / or the sequence of the reverse sequencing primer binding site (130) can be truncated at the 5' end and / or the 3' end, wherein the truncation can be 1 to 12 nucleotides. In certain embodiments, the length of the truncation can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In certain embodiments, the 5' end is truncated. In certain embodiments, the 3' end is truncated.

[0259] In some embodiments, any method for forming a plurality of library-splint complexes (500) described herein may further include at least one enzymatic reaction, including a phosphorylation reaction, a ligation reaction, and / or a nuclease exonuclease reaction. The enzymatic reactions may be performed sequentially or substantially simultaneously. The enzymatic reactions may be performed in a single reaction vessel. Alternatively, a first enzymatic reaction may be performed in a first reaction vessel, then transferred to a second reaction vessel for performing a second enzymatic reaction, and then transferred to a third reaction vessel for performing a third enzymatic reaction.

[0260] In some embodiments, any method for forming a plurality of library-splint complexes (500) described herein further comprises performing separate and sequential phosphorylation and ligation reactions, the phosphorylation and ligation reactions being performed in separate reaction vessels. In some embodiments, the method for forming a plurality of library-splint complexes (500) further comprises step (c1): contacting a plurality of double-stranded splint adapters (200) and a plurality of single-stranded nucleic acid library molecules (100) with T4 polynucleotide kinase in a first reaction vessel under conditions suitable for phosphorylating the 5' ends of a plurality of double-stranded splint adapters (200) and / or a plurality of single-stranded nucleic acid library molecules (100); and transferring the phosphorylation reaction to a second reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (500) further comprises step (d1): contacting a plurality of phosphorylated double-stranded splint adaptors (200) and a plurality of phosphorylated single-stranded nucleic acid library molecules (100) with a ligase in a second reaction vessel under conditions suitable for enzymatically ligating the first gap and the second gap, thereby generating a plurality of covalently closed circular library molecules (600), each of which is hybridized to the first splint strand (300). In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. An exemplary method for generating covalently closed circular library molecules (600) is shown in Figure 4 The enzymatic ligation reaction can join the sequence from the second splint strand (400) to the end of the library molecule (100). The double-stranded adapter (200) can join the new universal adapter sequence and the sample index sequence to both ends of the library molecule (100) without the need for primer extension or PCR reaction. Therefore, the PCR-free workflow using the double-stranded adapter (200) can be used to generate covalently circularized library molecules (600) with the adapter sequence required for downstream workflows (such as amplification and sequencing).

[0261] like Figure 4 As shown, in some embodiments, in the covalently closed circular library molecule (600), a sub-region of the second splint strand (400) is covalently bonded to a universal forward sequencing primer binding site (120) and a universal reverse sequencing primer binding site (130). In some embodiments, the first splint strand (300) has an extendable 3' end, which can be used to initiate a primer extension reaction. In some embodiments, the first splint strand (300) can be used as an amplification primer to perform a rolling circle amplification reaction to generate a nucleic acid concatemer molecule complementary to the covalently closed circular library molecule (600).

[0262] In some embodiments, any of the methods for forming a plurality of library-splint complexes (500) described herein further comprises performing consecutive phosphorylation and ligation reactions, wherein the phosphorylation and ligation reactions are performed consecutively in the same reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (500) further comprises step (c2): contacting a plurality of double-stranded splint adapters (200) and a plurality of single-stranded nucleic acid library molecules (100) with T4 polynucleotide kinase in a first reaction vessel under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded splint adapters (200) and the plurality of single-stranded nucleic acid library molecules (100). In some embodiments, the method for forming a plurality of library-splint complexes (500) further comprises step (d2): contacting the phosphorylated double-stranded splint adaptor (200) and the phosphorylated single-stranded nucleic acid library molecule (100) with a ligase in the same first reaction vessel under conditions suitable for enzymatically ligating the first gap and the second gap, thereby generating a plurality of covalently closed circular library molecules (600), each of which is hybridized to the first splint strand (300). In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. An exemplary method for generating covalently closed circular library molecules (600) is shown in Figure 4 middle.

[0263] In some embodiments, any of the methods for forming a plurality of library-splint complexes (500) described herein further comprises performing substantially simultaneous phosphorylation and ligation reactions, the phosphorylation and ligation reactions being performed together in the same reaction vessel. In some embodiments, the method for forming a plurality of library-splint complexes (500) further comprises step (c3): contacting a plurality of double-stranded splint adaptors (200) and a plurality of single-stranded nucleic acid library molecules (100) with (i) T4 polynucleotide kinase and (ii) ligase in a first reaction vessel under conditions suitable for phosphorylating the 5' ends of the plurality of double-stranded splint adaptors (200) and the plurality of single-stranded nucleic acid library molecules (100), and the conditions being suitable for enzymatically ligating the first gap and the second gap, thereby generating a plurality of covalently closed circular library molecules (600), each of which is hybridized to the first splint strand (300). In some embodiments, the ligase comprises T7 DNA ligase, T3 ligase, T4 ligase, or Taq ligase. An exemplary method for generating covalently closed circular library molecules (600) is shown in Figure 4 middle.

[0264] In some embodiments, any method for forming a plurality of library-splint complexes (500) described herein further comprises the following optional step: enzymatically removing a plurality of first splint chains (300) from a plurality of covalently closed circular library molecules (600), comprising: contacting a plurality of covalently closed circular library molecules (600) with at least one exonuclease to remove a plurality of first splint chains (300) and retain a plurality of covalently closed circular library molecules (600). In some embodiments, the exonuclease reaction may be performed in the same reaction buffer used for the phosphorylation and / or ligation reaction, or in a different reaction buffer. In some embodiments, the exonuclease reaction may be performed in a third reaction vessel after the phosphorylation reaction (c1) is performed in the first reaction vessel and the ligation reaction (d1) is performed in the second reaction vessel. In some embodiments, the exonuclease reaction may be performed in the first reaction vessel after the phosphorylation reaction (c2) is performed in the first reaction vessel and the sequential ligation reaction (d2) is performed in the first reaction vessel. In some embodiments, the exonuclease reaction may be performed in the first reaction vessel after substantially simultaneous phosphorylation and ligation reactions (c3) are performed in the first reaction vessel. In some embodiments, the at least one exonuclease comprises any combination of two or more of Exonuclease I, Thermolabile Exonuclease I, and / or T7 Exonuclease.

[0265] By using a double-stranded splint adapter carrying at least one sample index sequence to prepare a covalently closed circular library molecule (600) with a sample index, a multiple workflow can be achieved. The sample index sequence can be used to prepare a separate batch of covalently closed circular library molecules (600) with a sample index using input nucleic acids isolated from different sources. The covalently closed circular library molecules (600) with a sample index can be pooled together to generate a multiple covalently closed circular library molecule (600) mixture, and the pooled covalently closed circular library molecules (600) can be amplified and / or sequenced. The sequence of the insertion region together with the sample index sequence can be used to identify the source of the input nucleic acid. In some embodiments, any number of covalently closed circular library molecules (600) can be pooled together, for example, 2 to 10, or 10 to 50, or 50 to 100, or 100 to 200, or more than 200 batches of covalently closed circular library molecules (600) with a sample index can be pooled. Exemplary nucleic acid sources include naturally occurring, recombinant or chemically synthesized sources. Exemplary nucleic acid sources include individual cells, multiple cells, tissues, biological fluids, environmental samples or whole organisms. Exemplary nucleic acid sources include fresh, frozen, fresh frozen or archived sources (e.g., formalin-fixed paraffin embedded; FFPE). Technicians will recognize that nucleic acids can be separated from many other sources. Nucleic acid library molecules can be prepared in single-stranded or double-stranded form.

[0266] Rolling circle amplification method

[0267] The present disclosure provides a method for performing a rolling circle amplification reaction on a covalently closed circular library molecule (600). The rolling circle amplification reaction can be performed after phosphorylation and ligation reaction, or after ligation reaction. In some embodiments, the rolling circle amplification reaction can be performed after the exonuclease reaction, at which time the covalently closed circular library molecule (600) is no longer hybridized with the first splint chain (300). In some embodiments, the rolling circle amplification reaction can be performed on the covalently closed circular library molecule (600) hybridized with the first splint chain (300). In some embodiments, the covalently closed circular library molecule (600) can be distributed on a support, and then a rolling circle amplification reaction is performed. In some embodiments, the covalently closed circular library molecule (600) can be subjected to a rolling circle amplification reaction in a solution, and then distributed on a support. In some embodiments, the rolling circle amplification reaction can use the retained first splint chain (300) as an amplification primer (for example, Figure 4 ), or the first splint strand (300) can be removed (e.g., via exonuclease digestion) and replaced with a soluble amplification primer (e.g., Figure 5 ).

[0268] Rolling circle amplification on support

[0269] In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules (600) lacking a hybridized first splint chain (300), and wherein each of the plurality of covalently closed circular library molecules (600) comprises a universal binding sequence for a surface capture primer, the method comprising step (a): distributing the plurality of covalently closed circular library molecules (600) onto a support having a plurality of surface capture primers fixed thereto under conditions suitable for hybridizing each covalently closed circular library molecule (600) to each fixed surface capture primer, thereby fixing the plurality of covalently closed circular library molecules (600) to the support.

[0270] In some embodiments, the plurality of surface capture primers comprises any one of the sequences listed in Table 4 or a complementary sequence thereof.

[0271] Each surface capture primer can hybridize to a covalently closed circular library molecule (600) having a universal binding sequence for the surface capture primer.

[0272] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (b): contacting a plurality of immobilized covalently closed circular library molecules (600) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on a support using a plurality of surface capture primers as immobilized amplification primers and using a plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules immobilized to the surface capture primers. In some embodiments, the plurality of nucleotides comprises any combination of two or more of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, each immobilized concatemer is covalently bonded to each surface capture primer. In some embodiments, each covalently closed circular library molecule (600) in a plurality of covalently closed circular library molecules comprises a universal binding sequence for a surface capture primer and a surface pinning primer, so that a rolling circle amplification reaction generates a concatemer molecule having a plurality of tandem copies of a sequence carried by the covalently closed circular library molecule (600), the concatemer molecule comprising a universal binding sequence for a surface capture primer and a surface pinning primer. In some embodiments, the support further comprises a plurality of surface pinning primers. In some embodiments, the fixed surface pinning primer is used to pin at least a portion of the concatemer molecule to the support. In some embodiments, the fixed surface pinning primer has a non-extendable 3' end and cannot be used for amplification. In some embodiments, a plurality of surface pinning primers comprise any one of the sequences listed in Table 4 or their complementary sequences. In some embodiments, the fixed concatemers can be subjected to sequencing reactions.

[0273] Each surface pinning primer can hybridize with a portion of a concatemer molecule with a universal binding sequence for the surface pinning primer. In certain embodiments, the fixed surface pinning primer is used to pin at least a portion of the concatemer molecule to a support. In certain embodiments, the fixed surface pinning primer has a non-extendable 3' end and cannot be used for amplification. In certain embodiments, the fixed concatemer can be subjected to sequencing reactions.

[0274] In some embodiments, in a method for performing a rolling circle amplification reaction, a plurality of covalently closed circular library molecules (600) may be distributed onto a support that is coated with one or more compounds to create a passivation layer (e.g., Fig.21). In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, one or more types of surface primers, concatemer template molecules and / or polymerases can be attached to the passivation layer to be fixed to the support. In some embodiments, the support comprises a low non-specific binding surface, which enables the improvement of nucleic acid hybridization and amplification performance on the support. In general, the support may comprise one or more covalently or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid concatemer molecules to the support. In some embodiments, the support may comprise a functionalized polymer coating, which is covalently bound to at least a portion of the support via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of a hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise a branched PEG having at least 4 branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees. In some embodiments, the density of the covalently closed circular library molecules (600) immobilized to the support or to the coating on the support is 100 μg / cm2 per mm 2 Approx. 10 2 Up to 10 6 (For example, about 10 2 , 10 3 , 10 4 , 10 5 or 10 6 In some embodiments, the density of the covalently closed circular library molecules (600) immobilized to the support or to a coating on the support is 100 μm / mm 2 Approx. 10 6 Up to 10 9 (For example, about 10 6 , 10 7 , 10 8 or 10 9 In some embodiments, the density of the covalently closed circular library molecules (600) immobilized to the support or to a coating on the support is 100 μm / mm 2 Approx. 10 9 Up to 10 12 (For example, about 10 9 , 10 10 , 10 11 or 10 12In some embodiments, a plurality of covalently closed circular library molecules (600) are immobilized to a support or to a coating on a support at predetermined sites on the support (or coating on a support). In some embodiments, a plurality of covalently closed circular library molecules (600) are immobilized to a support or to a coating on a support at random sites on the support (or coating on a support).

[0275] In-solution rolling circle amplification using soluble amplification primers

[0276] In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules (600) lacking a hybridized first splint strand (300), wherein each of the plurality of covalently closed circular library molecules (600) comprises a universal binding sequence for a forward amplification primer and a universal binding sequence for a surface capture primer, the method comprising: (a) hybridizing the plurality of covalently closed circular library molecules with a plurality of soluble forward amplification primers in a solution; and (b) performing a first rolling circle amplification reaction by contacting the plurality of covalently closed circular library molecules (600) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction in a solution using the plurality of forward amplification primers and the plurality of covalently closed circular library molecules (600) as template molecules, thereby generating a plurality of nucleic acid concatemer molecules having portions that are still hybridized to the covalently closed circular library molecules (600). In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (c): distributing the plurality of concatemer molecules to a support on which a plurality of surface capture primers are fixed, under conditions suitable for hybridizing at least a portion of the concatemer molecules with a plurality of fixed surface capture primers, thereby fixing the plurality of concatemer molecules. The plurality of fixed concatemer molecules are still hybridized with their covalently closed circular library molecules (600). In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (d): contacting the fixed plurality of concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides, thereby extending a plurality of fixed nucleic acid concatemer molecules, under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (600) as template molecules. In some embodiments, the first rolling circle amplification reaction and / or the second rolling circle amplification reaction can be performed with a plurality of nucleotides comprising any combination of two or more of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, each fixed concatemer hybridizes with each surface capture primer. In some embodiments, each covalently closed circular library molecule (600) in a plurality of covalently closed circular library molecules comprises a universal binding sequence for a surface capture primer and a surface pinning primer, so that a rolling circle amplification reaction in a solution generates a concatemer molecule having a plurality of tandem copies of a sequence carried by the covalently closed circular library molecule (600), the concatemer molecule comprising a universal binding sequence for a surface capture primer and a surface pinning primer. In some embodiments, the support further comprises a plurality of surface pinning primers. In some embodiments, the fixed surface pinning primer is used to pin at least a portion of the concatemer molecule to the support. In some embodiments, the fixed surface pinning primer has a non-extendable 3' end and cannot be used for amplification. In some embodiments, the fixed concatemer can be subjected to a sequencing reaction.In some embodiments, the soluble forward amplification primer has the same sequence as the surface capture primer.

[0277] In some embodiments, the plurality of surface capture primers comprises any one of the sequences listed in Table 4, or a complement thereof. In some embodiments, the plurality of surface pinning primers comprises any one of the sequences listed in Table 4, or a complement thereof.

[0278] In some embodiments, in the method for performing a rolling circle amplification reaction, the plurality of surface capture primers immobilized on the support comprises the sequence 5'-GATCAGGTGAGGCTGCGACGACT-3' (SEQ ID NO: 34) (or its complementary sequence).

[0279] In some embodiments, in the method for performing a rolling circle amplification reaction, the plurality of surface capture primers immobilized on the support comprises the sequence 5'-ATTACATGGATCAGGTGAGGCT-3' (SEQ ID NO: 35) (or its complement).

[0280] In some embodiments, the fixed surface pinning primer is used to pin at least a portion of the concatemer molecule to a support. In some embodiments, the fixed surface pinning primer has a non-extendable 3' end and cannot be used for amplification. In some embodiments, the fixed concatemer can be subjected to a sequencing reaction.

[0281] In some embodiments, the plurality of concatemer molecules of step (c) may be distributed onto a support that is coated with one or more compounds to produce a passivation layer on the support, such as glass (see, e.g., Fig.21). In alternative embodiments, the support may be made of any material (such as glass, plastic or polymer material). In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, one or more types of surface primers, concatemer template molecules and / or polymerases can be attached to the passivation layer to be fixed to the support. In some embodiments, the support comprises a low non-specific binding surface, which enables to improve the nucleic acid hybridization and amplification performance on the support. In general, the support may comprise one or more covalently or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid concatemer molecules to the support. In some embodiments, the support may comprise a functionalized polymer coating, which is covalently bound to at least a portion of the support via a chemical group on the support, a primer grafted to the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of a hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise a branched PEG having at least 4 branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches). In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees (e.g., no more than 5 degrees, no more than 10 degrees, no more than 15 degrees, no more than 20 degrees, no more than 25 degrees, no more than 30 degrees, no more than 35 degrees, no more than 40 degrees, or no more than 45 degrees). In some embodiments, the density of concatemer molecules immobilized to the support or to the coating on the support is per mm 2 Approx. 10 2 Up to 10 6 (For example, about 10 2 , 10 3 , 10 4 , 10 5 or 10 6 In some embodiments, the density of concatemer molecules immobilized to the support or to a coating on the support is 100 μm / mm 2 Approx. 10 6 Up to 10 9 (For example, about 10 6 , 10 7 , 10 8 or 10 9 In some embodiments, the density of concatemer molecules immobilized to the support or to a coating on the support is 100 μm / mm 2Approx. 10 9 Up to 10 12 (For example, about 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, a plurality of concatemer molecules are immobilized to a support or to a coating on a support at predetermined sites on the support (or coating on a support). In some embodiments, a plurality of concatemer molecules are immobilized to a coating on a support at random sites on the support (or coating on a support).

[0282] In-solution rolling amplification using the first splint strand

[0283] In some embodiments, a method for performing a rolling circle amplification reaction on a plurality of covalently closed circular library molecules hybridized to a first splint strand (300), wherein each covalently closed circular library molecule (600) in the plurality of covalently closed circular library molecules comprises a universal binding sequence for a surface capture primer, the method comprising: (a) contacting the plurality of covalently closed circular library molecules (600) hybridized to the first splint strand (300) with a plurality of strand displacement polymerases and a plurality of nucleotides in a solution under conditions suitable for performing a first rolling circle amplification reaction using the first splint strand (300) as an amplification primer, thereby generating a plurality of concatemer molecules (e.g., see Figure 4 ).

[0284] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (b): distributing the plurality of concatemer molecules hybridized with the covalently closed circular library molecules (600) to a support on which a plurality of surface capture primers are immobilized, under conditions suitable for hybridizing at least a portion of the concatemer with a plurality of immobilized surface capture primers, thereby immobilizing the plurality of concatemer molecules. The plurality of immobilized concatemer molecules are still hybridized with the covalently closed circular library molecules (600).

[0285] In some embodiments, the method for performing a rolling circle amplification reaction further comprises step (c): contacting a plurality of immobilized concatemer molecules with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a second rolling circle amplification reaction on a support using a plurality of covalently closed circular library molecules (600) as template molecules, thereby extending a plurality of immobilized nucleic acid concatemer molecules.

[0286] In some embodiments, the first rolling circle amplification reaction and / or the second rolling circle amplification reaction can be carried out with multiple nucleotides including any combination of two or more of dATP, dGTP, dCTP, dTTP and / or dUTP. In some embodiments, each fixed concatemer hybridizes with each surface capture primer. In some embodiments, each covalently closed circular library molecule (600) in a plurality of covalently closed circular library molecules comprises a universal binding sequence for surface capture primers and surface pinning primers, so that the rolling circle amplification reaction in the solution generates a concatemer molecule with multiple tandem copies of the sequence carried by the covalently closed circular library molecule (600), and the concatemer molecule comprises a universal binding sequence for surface capture primers and surface pinning primers. In some embodiments, the support further comprises a plurality of surface pinning primers. In some embodiments, the fixed surface pinning primer is used to pin at least a portion of the concatemer molecule to the support. In some embodiments, the fixed surface pinning primer has an unextendable 3' end and cannot be used for amplification. In some embodiments, the fixed concatemer can be subjected to sequencing reaction.

[0287] In some embodiments, the plurality of surface capture primers comprises any one of the sequences listed in Table 4, or a complement thereof. In some embodiments, the plurality of surface pinning primers comprises any one of the sequences listed in Table 4, or a complement thereof.

[0288] In some embodiments, the fixed surface pinning primer is used to pin at least a portion of the concatemer molecule to a support. In some embodiments, the fixed surface pinning primer has a non-extendable 3' end and cannot be used for amplification. In some embodiments, the fixed concatemer can be subjected to a sequencing reaction.

[0289] In some embodiments, the plurality of concatemer molecules of step (b) can be distributed onto a support that is coated with one or more compounds to produce a passivation layer on the support (e.g., Fig.21). In some embodiments, the passivation layer forms a porous or semi-porous layer. In some embodiments, surface primers, concatemer template molecules and / or polymerases can be attached to the passivation layer to be fixed to the support. In some embodiments, the support comprises a low non-specific binding surface, which enables the improvement of nucleic acid hybridization and amplification performance on the support. In some embodiments, the support may comprise one or more covalently or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalently or non-covalently attached oligonucleotides that can be used to fix multiple nucleic acid concatemer molecules to the support. In some embodiments, the support may comprise a functionalized polymer coating, which is covalently bound to at least a portion of the support via chemical groups on the support, primers grafted to the functionalized polymer coating, and a water-soluble protective coating on the primers and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one layer of a hydrophilic polymer coating and at least one layer of a plurality of oligonucleotides. The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise

[0290] At least 4 branches (e.g., 4, 5, 6, 7, 8, 9, 10 or more branches) of branched PEG. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, wherein the water contact angle is no more than 45 degrees (e.g., no more than 5 degrees, no more than 10 degrees, no more than 15 degrees, no more than 20 degrees, no more than 25 degrees, no more than 30 degrees, no more than 35 degrees, no more than 40 degrees, or no more than 45 degrees). In some embodiments, the density of concatemer molecules immobilized to the support or to the coating immobilized on the support is 100% per mm 2 Approx. 10 2 Up to 10 6 (For example, about 10 2 , 10 3 , 10 4 , 10 5 or 10 6 In some embodiments, the density of concatemer molecules immobilized to the support or to a coating on the support is 100 μm / mm 2 Approx. 10 6 Up to 10 9 (For example, about 10 6 , 10 7 , 10 8 or 10 9 In some embodiments, the density of concatemer molecules immobilized to the support or to a coating on the support is 100 μm / mm 2 Approx. 10 9 Up to 1012 (For example, about 10 9 , 10 10 , 10 11 or 10 12 In some embodiments, a plurality of concatemer molecules are immobilized to a support or to a coating on a support at predetermined sites on the support (or coating on a support). In some embodiments, a plurality of concatemer molecules are immobilized to a coating on a support at random sites on the support (or coating on a support).

[0291] Compacted oligonucleotides

[0292] In some aspects, the present disclosure provides compositions and methods for rolling circle amplification in the presence or absence of multiple compaction oligonucleotides.The compaction oligonucleotide is single-stranded and can include a 5' region, an optional internal region and a 3' region.The 5' region and 3' region of the compaction oligonucleotide can be hybridized with concatemers to pull the distal end portion of the concatemer together, causing the compaction of the concatemer to form a nanostructure.For example, but not limited to, the 5' region of the compaction oligonucleotide can be designed to hybridize with the first part (for example, the first universal adapter sequence) of the concatemer molecule, and the 3' region of the compaction oligonucleotide can be designed to hybridize with the second part (for example, the second universal adapter sequence) of the concatemer molecule.Compared with the concatemer generated in the absence of the compaction oligonucleotide, the inclusion of the compaction oligonucleotide during the rolling circle amplification can promote the formation of a nanostructure with a more compact size and shape.The compact and stable features of the nucleic acid nanostructure are, for example, improved by increasing signal intensity to improve sequencing accuracy, and they maintain their shape and size during multiple sequencing cycles.In certain embodiments, the 3' end of the compaction oligonucleotide is designed to block primer extension.

[0293] In certain embodiments, compaction oligonucleotide is a single-stranded oligonucleotide comprising a combination of DNA, RNA or DNA and RNA.Compaction oligonucleotide can be any length, including 20 to 150 nucleotides, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 nucleotides, or any range therebetween.In certain embodiments, the length of compaction oligonucleotide can be 30 to 100 nucleotides.In certain embodiments, the length of compaction oligonucleotide can be 40 to 80 nucleotides.

[0294] In some embodiments, the compacted oligonucleotide comprises a 5' region and a 3' region, and an intervening region between the optional 5' region and the 3' region. The intervening region can be any length, for example, a length of about 2 to 20 nucleotides, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, or any range therebetween. In some embodiments, the intervening region comprises a homopolymer with continuous identical bases (e.g., AAA, GGG, CCC, TTT or UUU). In some embodiments, the intervening region comprises a non-homopolymer sequence.

[0295] In some embodiments, the 5' region of the compacted oligonucleotide can be fully complementary or partially complementary to the first portion of the concatemer molecule along its length. For example, but not limited to, the 5' region of the compacted oligonucleotide comprises a sequence that can hybridize to the universal adapter sequence listed in Tables 1 to 3, including a surface capture primer binding site, a surface pinning primer binding site, a forward sequencing primer binding site, or a reverse sequencing primer binding site.

[0296] In some embodiments, the 3' region of the compacted oligonucleotide can be fully complementary or partially complementary to the second portion of the concatemer molecule along its length. For example, but not limited to, the 3' region of the compacted oligonucleotide comprises a sequence that can hybridize to the universal adapter sequence listed in Tables 1 to 3, including a surface capture primer binding site, a surface pinning primer binding site, a forward sequencing primer binding site, or a reverse sequencing primer binding site.

[0297] The 5' region of the compaction oligonucleotide can hybridize with the first universal sequence portion of the concatemer molecule. The 3' region of the compaction oligonucleotide can hybridize with the second universal sequence portion of the concatemer molecule. The 5' region and the 3' region of the compaction oligonucleotide can hybridize with the concatemer to pull the distal portions of the concatemer together, causing compaction of the concatemer to form a nanostructure.

[0298] In some embodiments, the 5' region of the compacted oligonucleotide may have the same sequence as the 3' region. In some embodiments, the 5' region of the compacted oligonucleotide may have a sequence different from the 3' region. In some embodiments, the 3' region of the compacted oligonucleotide may have a sequence that is the reverse sequence of the 5' region.

[0299] In some embodiments, the compacted oligonucleotide comprises one or more modified bases or bonds at its 5' end or 3' end to confer certain functions. In some embodiments, the compacted oligonucleotide comprises at least one thiophosphate bond at its 5' end and / or 3' end, for example, to confer exonuclease resistance. In some embodiments, at least one nucleotide at or near the 3' end comprises a 2' fluoro base that confers exonuclease resistance. In some embodiments, the 3' end of the compacted oligonucleotide comprises at least one 2'-O-methyl RNA base that blocks polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide comprises 3' reverse dT at its 3' end, which blocks polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide comprises 3' phosphorylation that blocks polymerase-catalyzed extension. In some embodiments, the internal region of the compacted oligonucleotide comprises at least one locked nucleic acid (LNA), which increases the thermal stability of the duplex formed by hybridizing the compacted oligonucleotide with the concatemer molecule.

[0300] The compacted oligonucleotide may include at least one region with continuous guanines. For example, the compacted oligonucleotide may include at least one region with 2, 3, 4, 5, 6 or more continuous guanines. In some embodiments, the compacted oligonucleotide includes four continuous guanines that can form a guanine quadruplex structure. For example, the guanine quadruplex structure can be stabilized via Hoogsteen hydrogen bonding. The guanine quadruplex structure can be stabilized by a central cation (e.g., potassium, sodium, lithium, rubidium or cesium).

[0301] Rolling circle amplification reactions can be performed in the presence of multiple compacted oligonucleotides with at least four consecutive guanines. The resulting concatemers contain repeated copies of the universal binding sequence for the compacted oligonucleotides. At least one compacted oligonucleotide can form a guanine quadruplex and hybridize with the universal binding sequence for the compacted oligonucleotide, and the resulting concatemers can fold to form an intramolecular G-quadruplex structure. The concatemers can self-collapse to form a compact nanostructure. The formation of guanine quadruplexes and G-quadruplexes in the nanostructure can increase the stability of the nanostructure to maintain its compact size and shape, which can withstand changes in pH, temperature and / or repeated flow of reagents.

[0302] Methods used for sequencing

[0303] The present disclosure provides methods for sequencing any fixed concatemer molecules described herein. Any method for performing rolling circle amplification reactions described herein can be used to produce multiple concatemer molecules fixed to a support, and the fixed concatemer molecules can be subjected to sequencing reactions. In some embodiments, the sequencing reaction uses detectably labeled nucleotide analogs. In some embodiments, the sequencing reaction uses a two-stage sequencing reaction, including combining a detectably labeled multivalent molecule and incorporating a nucleotide analog. The terms concatemer molecule and template molecule are used interchangeably herein.

[0304] In some embodiments, any rolling circle amplification reaction described herein (e.g., RCA performed on a support or in solution) can be used to generate immobilized concatemers, each concatemer containing tandem repeat units of a sequence of interest and any adapter sequence present in the covalently closed circular library molecule (600).

[0305] In some embodiments, an exemplary tandem repeat unit comprises: (i) a universal adapter sequence having a binding sequence for a surface capture primer (subregion 413); (ii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (130); (iii) a target sequence (110); (iv) a universal adapter sequence having a binding sequence for a forward sequencing primer (120); (v) a universal adapter sequence having a binding sequence for a surface pinning primer (subregion 411); and (vi) a sample index, a short random sequence (NNN) and / or a unique molecular index (UMI) (subregion 412).

[0306] In some embodiments, an exemplary tandem repeat unit comprises: (i) a universal adapter sequence having a binding sequence for a surface capture primer (subregion 412); (ii) a sample index, a short random sequence (NNN) and / or a unique molecular index (UMI) (subregion 413); (iii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (130); (iv) a target sequence (110); (v) a universal adapter sequence having a binding sequence for a forward sequencing primer (120); and (vi) a universal adapter sequence having a binding sequence for a surface pinning primer (subregion 411).

[0307] In some embodiments, an exemplary tandem repeat unit comprises: (i) a universal adapter sequence having a binding sequence for a surface capture primer (subregion 413); (ii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (150); (iii) a target sequence (110); (iv) a universal adapter sequence having a binding sequence for a forward sequencing primer (140); (v) a sample index, a short random sequence (NNN) and / or a unique molecular index (UMI) (subregion 412); and (vi) a universal adapter sequence having a binding sequence for a surface pinning primer (subregion 411).

[0308] Fixed concatemers can collapse into compact nucleic acid nanoballs by themselves. One or more compacting oligonucleotides are included during the RCA reaction to further compact the size and / or shape of the nanoballs. The increase in the number of tandem repeat units in a given concatemer increases the number of sites along the concatemer for hybridization with multiple sequencing primers (e.g., sequencing primers with universal sequences), and multiple sequencing primers serve as multiple starting sites for sequencing reactions catalyzed by polymerase. When sequencing reactions employ detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., with nucleotide units), the signal emitted by the nucleotides or nucleotide units participating in the parallel sequencing reactions along the concatemers can produce an increased signal intensity for each concatemer. Multiple parts of a given concatemer can be sequenced simultaneously. In addition, multiple binding complexes can be formed along specific concatemer molecules, each binding complex comprising a sequencing polymerase bound to a multivalent molecule, wherein multiple binding complexes remain stable and do not dissociate, resulting in an increase in duration, which increases signal intensity and reduces imaging time.

[0309] Sequencing methods using engineered polymerases

[0310] In some aspects, the present disclosure provides concatemer template molecules that can be sequenced using any nucleic acid sequencing method using labeled or unlabeled chain-terminating nucleotides, wherein the chain-terminating nucleotides include 3'-O-azido groups (or 3'-O-methylazido groups) or any other type of bulky blocking groups at the 3' position of the sugar. In some embodiments, concatemer template molecules can be sequenced using affinity sequencing methods (SBA) using labeled multivalent molecules and unlabeled chain-terminating nucleotides. In some embodiments, concatemer template molecules can be sequenced using synthetic sequencing (SBS) methods that use labeled chain-terminating nucleotides. In some embodiments, concatemer template molecules can be sequenced using a combined sequencing method (SBB) using unlabeled chain-terminating nucleotides. In some embodiments, concatemer template molecules can be sequenced using nucleotides labeled with phosphate chains.

[0311] Methods for sequencing using nucleotide analogs

[0312] In some aspects, the present disclosure provides a method for sequencing, comprising the step (a): contacting a sequencing polymerase with (i) a nucleic acid concatemer molecule and (ii) a nucleic acid primer, wherein the contact is performed under conditions suitable for binding the sequencing polymerase to the nucleic acid concatemer molecule (the nucleic acid concatemer molecule hybridizes with the nucleic acid primer), wherein the nucleic acid concatemer molecule hybridized with the nucleic acid primer forms a nucleic acid duplex. In some embodiments, the sequencing polymerase comprises a recombinant mutant sequencing polymerase. In some embodiments, the primer comprises a 3' extendable end.

[0313] In some embodiments, the method for sequencing further comprises step (b): contacting the sequencing polymerase with the plurality of nucleotides under conditions suitable for binding at least one nucleotide to a sequencing polymerase that binds to a nucleic acid duplex and for polymerase-catalyzed incorporation of the nucleotides. In some embodiments, the sequencing polymerase is contacted with the plurality of nucleotides in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the plurality of nucleotides comprises at least one nucleotide analog having a chain termination moiety at the 2' or 3' position of the sugar. In some embodiments, the plurality of nucleotides comprises at least one nucleotide lacking a chain termination moiety.

[0314] In some embodiments, the method for sequencing further comprises step (c): incorporating at least one nucleotide into the 3' end of the extendable primer under conditions suitable for incorporating at least one nucleotide. In some embodiments, the conditions suitable for nucleotide binding polymerase and incorporating nucleotides can be the same or different. In some embodiments, the conditions suitable for incorporating nucleotides include adding at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, at least one nucleotide binds to the sequencing polymerase and is incorporated into the 3' end of the extendable primer. In some embodiments, incorporating the nucleotide into the 3' end of the primer in step (c) comprises a primer extension reaction.

[0315] In some embodiments, the method for sequencing further comprises step (d): repeating at least one nucleotide incorporated into the 3' end of the extendable primer in step (c) at least once. In some embodiments, a plurality of nucleotides include a plurality of nucleotides labeled with a detectable reporter gene portion. The detectable reporter gene portion includes a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to a nucleotide base with a joint that can be cut / removed from the base. In some embodiments, at least one of the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, a specific detectable reporter gene portion (e.g., a fluorophore) attached to a nucleotide can correspond to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to allow detection and identification of nucleotide bases. In some embodiments, the method further comprises detecting the at least one incorporated nucleotide at step (c) and / or (d). In some embodiments, the method further comprises identifying the at least one incorporated nucleotide at step (c) and / or (d). In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying the nucleotides bound to the sequencing polymerase, thereby determining the sequence of the concatemer molecule. In some embodiments, the sequence of a nucleic acid concatemer molecule can be determined by detecting and identifying the nucleotide incorporated into the 3' end of the primer, thereby determining the sequence of the concatemer molecule.

[0316] In some embodiments, in a method for sequencing, a plurality of sequencing polymerases bound to a nucleic acid duplex comprises a plurality of composite polymerases having at least a first composite polymerase and a second composite polymerase, wherein (a) the first composite polymerase comprises a first sequencing polymerase bound to a first nucleic acid duplex, wherein the first nucleic acid duplex comprises a first nucleic acid template sequence hybridized to a first nucleic acid primer, (b) the second composite polymerase comprises a second sequencing polymerase bound to a second nucleic acid duplex, wherein the second nucleic acid duplex comprises a second nucleic acid template sequence hybridized to a second nucleic acid primer, (c) the first nucleic acid template sequence and the second nucleic acid template sequence comprise the same or different sequences, (d) the first nucleic acid concatemer and the second nucleic acid concatemer are clonally amplified, (e) the first primer and the second primer comprise an extendable 3' end or a non-extendable 3' end, and (f) the plurality of composite polymerases are immobilized on a support. In some embodiments, the density of the plurality of composite polymerases is 1000 Å per mm 2 Approx. 10 2 Up to 10 15 (For example, 10 2 Up to 10 15 or more, for example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 A composite polymerase (a) is immobilized to a support.

[0317] Two-stage method for sequencing nucleic acids

[0318] In some aspects, the present disclosure provides a two-stage method for sequencing nucleic acid molecules. In some embodiments, the first stage generally includes combining a multivalent molecule with a complex polymerase to form a multivalent-complex polymerase, and detecting the multivalent-complex polymerase.

[0319] In some embodiments, the first stage comprises the step (a): contacting a plurality of first sequencing polymerases with (i) a plurality of nucleic acid concatemer molecules and (ii) a plurality of nucleic acid primers, wherein the contacting is performed under conditions suitable for binding the plurality of first sequencing polymerases to the plurality of nucleic acid concatemer molecules and the plurality of nucleic acid primers, thereby forming a plurality of first composite polymerases, each first composite polymerase comprising a first sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a nucleic acid concatemer molecule hybridized to a nucleic acid primer. In some embodiments, the first polymerase comprises a recombinant mutant sequencing polymerase.

[0320] In some embodiments, in a method for sequencing a concatemer molecule, the primer comprises a 3' extendable end or a 3' non-extendable end. In some embodiments, a plurality of nucleic acid concatemer molecules comprise amplified template molecules (e.g., clonally amplified template molecules). In some embodiments, a plurality of nucleic acid concatemer molecules comprise a copy of a target sequence of interest. In some embodiments, a plurality of nucleic acid molecules comprise two or more tandem copies of a target sequence of interest (e.g., concatemers). In some embodiments, the nucleic acid concatemer molecules in a plurality of nucleic acid concatemer molecules comprise the same target sequence of interest or different target sequences of interest. In some embodiments, a plurality of nucleic acid concatemer molecules and / or a plurality of nucleic acid primers are in solution or fixed to a support. In some embodiments, when a plurality of nucleic acid concatemer molecules and / or a plurality of nucleic acid primers are fixed to a support, binding to a first sequencing polymerase generates a plurality of fixed first composite polymerases. In some embodiments, a plurality of nucleic acid concatemer molecules and / or nucleic acid primers are fixed to a support 10 2 Up to 10 15 In some embodiments, the combination of the plurality of concatemer molecules and the nucleic acid primers with the plurality of first sequencing polymerases generates a plurality of first composite polymerases, which are immobilized to 10 2 Up to 1015 different sites (e.g., 10 2 Up to 10 15 sites or more, e.g., 10 2 sites, 10 3 sites, 10 4 sites, 10 5 sites, 10 6 sites, 10 7 sites, 10 8 sites, 10 9 sites, 10 10 sites, 10 11 sites, 10 12 sites, 10 13 sites, 10 14 sites, 10 15 In some embodiments, the plurality of immobilized first composite polymerases on the support are immobilized to predetermined or random sites on the support. In some embodiments, the plurality of immobilized first composite polymerases are in fluid communication with each other to allow a solution of reagents (e.g., enzymes including sequencing polymerases, multivalent molecules, nucleotides, and / or divalent cations) to flow onto the support, such that the plurality of immobilized composite polymerases on the support react with the reagent solution in a massively parallel manner.

[0321] In some embodiments, the method for sequencing further comprises step (b): contacting the plurality of first complex polymerases with the plurality of multivalent molecules to form a plurality of multivalent-complex polymerases (e.g., binding complexes). In some embodiments, each multivalent molecule in the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms, and each nucleotide arm is attached to a nucleotide (e.g., a nucleotide unit) (e.g., Figures 22 to 25 In some embodiments, the contacting of step (b) is performed under conditions suitable for binding the complementary nucleotide units of the multivalent molecule to at least two of the plurality of first composite polymerases, thereby forming a plurality of multivalent-complex polymerases. In some embodiments, the conditions are suitable for inhibiting polymerase-catalyzed incorporation of the complementary nucleotide units into primers of the plurality of multivalent-complex polymerases. In some aspects, the plurality of multivalent molecules comprises at least one multivalent molecule having a plurality of nucleotide arms (e.g., Figures 22 to 25), each nucleotide arm is attached with a nucleotide analog (e.g., a nucleotide analog unit), wherein the nucleotide analog includes a chain termination portion at the sugar 2' and / or 3' position. In some embodiments, a plurality of multivalent molecules include at least one multivalent molecule, the multivalent molecule comprising a plurality of nucleotide arms, each nucleotide arm being attached with a nucleotide unit lacking a chain termination portion. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a detectable reporter gene portion. Any portion of the multivalent molecule can be labeled, including the core, nucleotide arms, or nucleobases. In some embodiments, the detectable reporter gene portion includes a fluorophore. In some embodiments, the contacting of step (b) is performed in the presence of at least one non-catalytic cation comprising strontium, barium, and / or calcium.

[0322] In some embodiments, the method for sequencing further comprises step (c): detecting a plurality of multivalent-complex polymerases. In some embodiments, the detection comprises detecting a multivalent molecule bound to a complex polymerase, wherein the complementary nucleotide unit of the multivalent molecule is bound to a primer, but the incorporation of the complementary nucleotide unit is inhibited. In some embodiments, the multivalent molecule is labeled with a detectable reporter gene moiety to allow detection. In some embodiments, the labeled multivalent molecule comprises a fluorophore attached to a core, a linker, and / or a nucleotide unit of the multivalent molecule.

[0323] In some embodiments, the method for sequencing further comprises step (d): identifying the bases of complementary nucleotide units bound to the plurality of first composite polymerases, thereby determining the sequence of the concatemer molecule. In some embodiments, the multivalent molecule is labeled with a detectable reporter moiety corresponding to a specific nucleotide unit attached to the nucleotide arm to allow identification of complementary nucleotide units (e.g., nucleotide bases adenine, guanine, cytosine, thymine, or uracil) bound to the plurality of first composite polymerases.

[0324] In some embodiments, the second stage of the two-stage sequencing method generally comprises nucleotide incorporation. In some embodiments, the method for sequencing further comprises step (e): dissociating the plurality of multivalent-complex polymerases and removing the plurality of first sequencing polymerases and their bound multivalent molecules, and retaining the plurality of nucleic acid duplexes.

[0325] In some embodiments, the method for sequencing further comprises step (f): contacting the plurality of retained nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases, wherein the contact is performed under conditions suitable for binding the plurality of second sequencing polymerases to the plurality of retained nucleic acid duplexes, thereby forming a plurality of second composite polymerases, each second composite polymerase comprising a second sequencing polymerase bound to a nucleic acid duplex. In some embodiments, the second sequencing polymerase comprises a recombinant mutant sequencing polymerase.

[0326] In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is 100% identical to the amino acid sequence of the plurality of second sequencing polymerases of step (f). In some embodiments, the plurality of first sequencing polymerases of step (a) have an amino acid sequence that is different from the amino acid sequence of the plurality of second sequencing polymerases of step (f).

[0327] In some embodiments, the method for sequencing further comprises step (g): contacting a plurality of second composite polymerases with a plurality of nucleotides, wherein the contact is carried out under conditions suitable for combining complementary nucleotides from a plurality of nucleotides with at least two of the composite polymerases, thereby forming a plurality of nucleotide-composite polymerases. In some embodiments, the contact of step (g) is carried out under conditions suitable for promoting the incorporation of the complementary nucleotides of the binding into the primer of the nucleotide composite-polymerase through polymerase catalysis, thereby forming a plurality of nucleotide-composite polymerases. In some embodiments, the incorporation of nucleotides into the 3' end of the primer in step (g) includes a primer extension reaction. In some embodiments, the contact of step (g) is carried out in the presence of at least one catalytic cation comprising magnesium and / or manganese. In some embodiments, the contact of step (g) is carried out in the presence of magnesium and / or manganese. In some embodiments, the plurality of nucleotides include natural nucleotides (e.g., non-analog nucleotides) or nucleotide analogs. In some embodiments, the plurality of nucleotides include removable or non-removable 2' and / or 3' chain termination parts. In some embodiments, a plurality of nucleotides include a plurality of nucleotides labeled with a detectable reporter gene portion. The detectable reporter gene portion may include a fluorophore. In some embodiments, the fluorophore is attached to a nucleotide base. In some embodiments, the fluorophore is attached to the nucleotide base with a linker that can be cut / removed from the base or cannot be removed from the base. In some embodiments, the specific detectable reporter gene portion (e.g., fluorophore) attached to the nucleotide can correspond to the nucleotide base (e.g., dATP, dGTP, dCTP, dTTP or dUTP) to allow detection and identification of the nucleotide base. In some embodiments, at least one of the plurality of nucleotides is not labeled with a detectable reporter gene portion. In some embodiments, the plurality of nucleotides is not labeled with a detectable reporter gene portion.

[0328] In some embodiments, the method for sequencing further comprises step (h): when the nucleotide is labeled with a detectable reporter moiety, step (h) comprises detecting complementary nucleotides in a primer incorporated into a nucleotide-complex polymerase. In some embodiments, the plurality of nucleotides are labeled with a detectable reporter moiety to allow detection. In some embodiments, in the method for sequencing concatemer molecules, when the nucleotide is not labeled, the detection step is omitted.

[0329] In some embodiments, the method for sequencing further comprises step (i): when the nucleotide is labeled with a detectable reporter gene portion, step (i) comprises identifying the base of the complementary nucleotide in the primer incorporated into the nucleotide-complex polymerase. In some embodiments, the complementary nucleotide identified in step (i) can be used to confirm the identity of the complementary nucleotide of the multivalent molecule bound to the plurality of first complex polymerases in step (d). In some embodiments, the identification of step (i) can be used to determine the sequence of the nucleic acid concatemer molecule. In some embodiments, in the method for sequencing the concatemer molecule, when the nucleotide is not labeled, the identification step is omitted.

[0330] In some embodiments, the method for sequencing further comprises step (j): removing the chain terminating moiety from the incorporated nucleotides when step (g) is performed by contacting a plurality of second composite polymerases with a plurality of nucleotides including at least one nucleotide having a 2' and / or 3' chain terminating moiety.

[0331] In some embodiments, the method for sequencing further comprises step (k): repeating steps (a) to (j) at least once. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined by detecting and identifying the multivalent molecule that binds the sequencing polymerase in steps (c) and (d) but is not incorporated into the 3' end of the primer. In some embodiments, the sequence of the nucleic acid concatemer molecule can be determined (or confirmed) by detecting and identifying the nucleotide that is incorporated into the 3' end of the primer in steps (h) and (i).

[0332] In some embodiments, in any method for sequencing a nucleic acid molecule, the combination of a plurality of first complex polymerases and a plurality of multivalent molecules forms at least one affinity complex, the method comprising the following steps: (a) binding a first nucleic acid primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of a concatemer template molecule, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; and (b) binding a second nucleic acid primer, a second sequencing polymerase, and a first multivalent molecule to a second portion of the same concatemer template molecule, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex. In some embodiments, the first sequencing polymerase comprises any wild-type or mutant polymerase described herein. In some embodiments, the second sequencing polymerase comprises any wild-type or mutant polymerase described herein. The concatemer template molecule comprises a tandem repeat sequence of a target sequence and at least one universal sequencing primer binding site. The first nucleic acid primer and the second nucleic acid primer can bind to the sequencing primer binding site along the concatemer template molecule. Exemplary multivalent molecules are shown in Figures 22 to 25 .

[0333] In some embodiments, in any method for sequencing a nucleic acid molecule, wherein the method comprises binding a plurality of first composite polymerases to a plurality of multivalent molecules to form at least one affinity complex, the method comprises the following steps: (a) contacting a plurality of sequencing polymerases and a plurality of nucleic acid primers to different portions of a concatemer nucleic acid concatemer molecule to form at least a first composite polymerase and a second composite polymerase on the same concatemer template molecule; (b) contacting the plurality of multivalent molecules with at least the first composite polymerase and the second composite polymerase on the same concatemer template molecule under conditions suitable for binding a single multivalent molecule from the plurality of multivalent molecules to the first composite polymerase and the second composite polymerase, wherein at least a first nucleotide unit of the single multivalent molecule binds to the first composite polymerase comprising a first primer that hybridizes to a first portion of the concatemer template molecule, thereby forming a first binding complex (e.g., a first primer). ternary complex), and wherein at least a second nucleotide unit of the single multivalent molecule is bound to a second composite polymerase comprising a second primer that hybridizes to a second portion of the concatemer template molecule, thereby forming a second binding complex (e.g., a second ternary complex), wherein the contacting is performed under conditions suitable for inhibiting the incorporation of the first nucleotide unit and the second nucleotide unit bound in the first binding complex and the second binding complex catalyzed by the polymerase, and wherein the first binding complex and the second binding complex bound to the same multivalent molecule form an affinity complex; and (c) detecting the first binding complex and the second binding complex on the same concatemer template molecule, and (d) identifying the first nucleotide unit in the first binding complex, thereby determining the sequence of the first portion of the concatemer template molecule, and identifying the second nucleotide unit in the second binding complex, thereby determining the sequence of the second portion of the concatemer template molecule. In some embodiments, the plurality of sequencing polymerases comprises any wild-type or mutant sequencing polymerase described herein. The concatemer template molecule comprises a tandem repeat sequence of a target sequence and at least one universal sequencing primer binding site. Multiple nucleic acid primers can bind to the sequencing primer binding sites along the concatemer template molecule. Exemplary multivalent molecules are shown in Figures 22 to 25 .

[0334] Combined sequencing

[0335] In some aspects, the disclosure provides methods for sequencing any of the immobilized concatemer molecules described herein, wherein the sequencing method comprises a sequencing by binding (SBB) procedure employing unlabeled chain terminating nucleotides. In some embodiments, a sequencing by binding (SBB) method comprises the following steps: (a) sequentially contacting a primed template nucleic acid with at least two separate mixtures under ternary complex stabilizing conditions, wherein the at least two separate mixtures each comprise a polymerase and nucleotides, wherein the sequential contacting results in the primed template nucleic acid contacting nucleotide homologs of a first base type, a second base type, and a third base type in the template under ternary complex stabilizing conditions; (b) examining the at least two separate mixtures to determine whether a ternary complex is formed; and (c) identifying the next correct nucleotide of the primed template nucleic acid molecule, wherein if a ternary complex is detected in step (b), the next correct nucleotide is identified as a homolog of the first base type, the second base type, or the third base type, and wherein based on the absence of a ternary complex in step (b), the next correct nucleotide is inferred to be a nucleotide homolog of the fourth base type; (d) adding the next correct nucleotide to a primer of the primed template nucleic acid after step (b), thereby generating an extended primer; and (e) repeating steps (a) to (d) at least once for the primed template nucleic acid comprising the extended primer. Exemplary combined sequencing methods are described in U.S. Pat. Nos. 10,246,744 and 10,731,141 (the contents of which are incorporated herein by reference in their entirety). Methods for sequencing using phosphate-tagged nucleotides

[0336] The present disclosure provides a method for sequencing using a fixed sequencing polymerase bound to an unfixed template molecule, wherein a sequencing reaction is performed with a nucleotide labeled with a phosphate chain. In some embodiments, a covalently closed circular library molecule (600) can serve as an unfixed template molecule. In some embodiments, the sequencing method includes step (a): providing a support on which a plurality of sequencing polymerases are fixed. In some embodiments, the sequencing polymerase includes a processive DNA polymerase. In some embodiments, the sequencing polymerase includes a wild-type or mutant DNA polymerase, including, for example, but not limited to, a Phi29 DNA polymerase. In some embodiments, the support includes a plurality of separate compartments, and the sequencing polymerase is fixed to the bottom of the compartment. In some embodiments, a separate compartment includes a silica bottom through which light can penetrate. In some embodiments, a separate compartment includes a silica bottom configured with a nanophoton confinement structure including a hole in a metal coating (e.g., an aluminum coating). In some embodiments, the small aperture of the hole in the metal coating is, for example, about 70 nm. In some embodiments, the height of the nanophoton confinement structure is about 100 nm. In some embodiments, the nanophoton confinement structure includes a zero mode waveguide (ZMW). In some embodiments, the nanophotonic confinement structure contains a liquid.

[0337] In some embodiments, the sequencing method further comprises step (b): contacting a plurality of immobilized sequencing polymerases with a plurality of single-stranded circular nucleic acid template molecules (e.g., covalently closed circular library molecules (600)) and a plurality of oligonucleotide sequencing primers under conditions suitable for each immobilized sequencing polymerase to bind to the single-stranded circular template molecule and suitable for each sequencing primer to hybridize to each single-stranded circular template molecule, thereby generating a plurality of polymerase / template / primer complexes. In some embodiments, a separate sequencing primer hybridizes to a universal sequencing primer binding site on a single-stranded circular template molecule.

[0338] In some embodiments, the sequencing method further comprises step (c): contacting a plurality of polymerase / template / primer complexes with a plurality of nucleotides labeled with phosphate chains, each nucleotide labeled with a phosphate chain comprising an aromatic base, a pentose (e.g., ribose or deoxyribose) and a phosphate chain comprising 3 to 20 phosphate groups (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 phosphate groups), wherein the terminal phosphate group is connected to a detectable reporter gene portion (e.g., a fluorophore). The first phosphate group, the second phosphate group, and the third phosphate group may be referred to as α, β, and γ phosphate groups. In some embodiments, the specific detectable reporter gene portion attached to the terminal phosphate group corresponds to a nucleotide base (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) to allow detection and identification of the nucleobase. In some embodiments, a plurality of polymerase / template / primer complexes are contacted with a plurality of nucleotides labeled with a phosphate chain under conditions suitable for polymerase-catalyzed nucleotide incorporation. In some embodiments, a sequencing polymerase is capable of binding to a nucleotide labeled with a complementary phosphate chain and incorporating a complementary nucleotide relative to a nucleotide in the template molecule. In some embodiments, the polymerase-catalyzed nucleotide incorporation reaction cleaves between an alpha phosphate group and a beta phosphate group, thereby releasing a polyphosphate chain attached to a fluorophore.

[0339] In some embodiments, the sequencing method further comprises step (d): detecting a fluorescent signal emitted by a nucleotide labeled with a phosphate chain, which is bound by a sequencing polymerase and incorporated into the end of a sequencing primer. In some embodiments, step (d) further comprises identifying the nucleotide labeled with a phosphate chain bound by a sequencing polymerase and incorporated into the end of a sequencing primer.

[0340] In some embodiments, the sequencing method further comprises step (d): repeating steps (c) to (d) at least once. In some embodiments, the sequencing method using nucleotides labeled with phosphate chains can be performed according to the methods described in U.S. Patent Nos. 7,170,050; 7,302,146; and / or 7,405,281.

[0341] Traditional pooling workflow for multiplexing

[0342] In one embodiment, a method for preparing a multiplex mixture of target sequences isolated from multiple sample sources comprises: (a) providing two or more populations (100) of single-stranded nucleic acid library molecules, each population of library molecules (100) being contained in a separate compartment, wherein the nucleic acid library molecules in a given population comprise (i) a target sequence (110); (ii) a universal adapter sequence having a binding sequence for a forward sequencing primer (120); and (iii) a universal adapter sequence having a binding sequence for a reverse sequencing primer (130) (e.g., Figure 1 ).

[0343] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (b): providing a plurality of double-stranded splint adapters (200), wherein each double-stranded splint adapter (200) comprises a first splint strand (300) hybridized to a second splint strand (400) (e.g., Figure 2 ). In some embodiments, the second splint chain (400) comprises at least one sample index sequence and an optional short random sequence NNN.

[0344] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (c): in a separate compartment, contacting a population of single-stranded nucleic acid library molecules (100) with a quota of multiple double-stranded splint adaptors (200), wherein the contact is performed under conditions suitable for hybridizing a portion of the first splint strand (300) with a portion of the library molecule (100), thereby looping the library molecules to generate a population of library-splint complexes (500), such that a region (320) of each first splint strand is aligned with a forward sequencing primer binding site (100) of each library molecule (100). 20), and the region (330) of each first splint strand is hybridized with the universal adapter sequence for the reverse sequencing primer binding site (130) of each library molecule (100), wherein each of the library-splint complexes (500) comprises a first gap between the 5' end of the library molecule and the 3' end of the second splint strand (300), wherein each of the library-splint complexes (500) comprises a second gap between the 5' end of the second splint strand (300) and the 3' end of the library molecule (100), and wherein the first gap and the second gap are enzymatically ligatable (e.g., see Figure 3 and 4 ).

[0345] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (d): contacting the population of library-splint complexes (500) with a ligase in a separate compartment under conditions suitable for enzymatically ligating the first gap and the second gap, thereby generating a population of covalently closed circular library molecules (600) each hybridized to the first splint strand (300) (e.g., see Figure 4 ).

[0346] In some embodiments, the method for preparing a multiplex mixture of target sequences isolated from multiple sample sources further comprises step (e): pooling together populations of covalently closed circular library molecules (600) from separate compartments to generate a multiplex mixture of covalently closed circular library molecules (600) comprising a multiplex mixture of target sequences isolated from multiple sample sources.

[0347] In some embodiments, the target sequence can be separated from two or more different sample sources (e.g., 2 to 10, 10 to 50, 50 to 100, 100 to 250, any range therebetween, or more than 250 different sample sources). Exemplary nucleic acid sources include naturally occurring, recombinant, or chemically synthesized sources. Exemplary nucleic acid sources include single cells, multiple cells, tissues, biological fluids, environmental samples, or whole organisms. Exemplary nucleic acid sources include fresh, frozen, fresh frozen, or archived sources (e.g., formalin-fixed paraffin embedded; FFPE). Technicians will recognize that nucleic acids can be separated from many other sources. The target sequences in a given population have identical or different sequences.

[0348] In some embodiments, the number of the population of single-stranded nucleic acid library molecules (100) of step (a) can be 2 to 10, 10 to 50, 50 to 100, 100 to 250, any range therebetween, or more than 250 different populations of single-stranded nucleic acid library molecules (100). In some embodiments, any number of different populations of covalently closed circular library molecules (600) can be pooled together in step (e), for example, 2 to 10, 10 to 50, 50 to 100, 100 to 200, any range therebetween, or more than 200 different populations of covalently closed circular library molecules (600) can be pooled together.

[0349] One skilled in the art will recognize that any number of separate compartments (e.g., 2 to 10, 10 to 50, 50 to 100, 100 to 250, any range therebetween, or more separate compartments) (e.g., a multiwell plate such as, for example, a 96-well plate) may be used in step (a).

[0350] In some embodiments, the 3' end of the first splint strand (300) hybridized to the covalently closed circular library molecule (600) of step (d) or (e) comprises an extendable 3' OH end, which can serve as a starting point for a primer extension reaction (eg, a rolling circle amplification reaction).

[0351] In some embodiments, in step (d) or (e), the population of covalently closed circular library molecules (600) hybridized to the first splint strand (300) can be optionally reacted with at least one exonuclease to remove the plurality of first splint strands (300) and retain the plurality of covalently closed circular library molecules (600). In some embodiments, the at least one exonuclease comprises exonuclease I, thermolabile exonuclease I, and / or T7 exonuclease.

[0352] In some embodiments, the single-stranded nucleic acid library molecule (100) of step (a) further comprises any one or any combination of two or more of the following: a universal binding sequence for a forward amplification primer; a universal binding sequence for a reverse amplification primer; and / or a universal binding sequence for a compacted oligonucleotide.

[0353] Sample indexing for improved base calling

[0354] Typically, a nucleic acid library needs to be prepared to be distributed onto a support (e.g., a coated flow cell), where the library molecules are converted into template molecules, which are immobilized at high density to the support for massively parallel sequencing. For template molecules immobilized at high density at random positions on the support, the challenge of parsing high-density fluorescent images to achieve accurate base recognition during a sequencing run becomes challenging.

[0355] The nucleotide diversity of a fixed population of template molecules refers to the relative proportions of nucleotides A, G, C, and T present in each sequencing cycle. In some embodiments, the optimal high-diversity template molecules include a target sequence (insert) region in which all four nucleotides are present in approximately equal proportions in each cycle of the sequencing run. In some embodiments, the low-diversity template molecules include a target sequence (insert) region having a high proportion of certain nucleotides and a low proportion of other nucleotides. In order to overcome the problem of low-diversity template molecules, a small amount of high-diversity molecules prepared from PhiX phage can be mixed with the target template molecules (e.g., PhiX spike-in library) and sequenced together, for example, on the same flow cell. Although the PhiX spike-in library provides nucleotide diversity, it also takes up space on the flow cell, thereby replacing the template molecules carrying the target sequence and reducing the amount of sequencing data that can be obtained from the template molecules (e.g., reducing sequencing throughput).

[0356] Another approach to overcome the problem of low diversity template molecules is to prepare template molecules with at least one sample index sequence that is designed to be color balanced. However, it may be necessary to design a large number of sample index sets, such as a set of single index sample sequences or paired index sample sequences for 16-plex, 24-plex, 96-plex or larger multiplexes. For large sample index sets where all sample index sequences are color balanced, it may be challenging to design sample index sequences as single or paired sample indexes (e.g., see Fig.19 and 20 ).

[0357] As described herein, an alternative approach to overcome the challenges of sequencing low-diversity template molecules (e.g., at high density on a support) is to prepare a template molecule having at least one sample index sequence, the sample index sequence comprising a short random sequence (e.g., NNN) directly linked to the sample index sequence, wherein the short random sequence (NNN) provides nucleotide diversity and color balance. In some embodiments, the sample index sequence comprises a short random sequence (NNN) directly linked to the sample index sequence. In some embodiments, the short random sequence (NNN) is located upstream or downstream of the sample index sequence. Some exemplary sample index sequences include, but are not limited to: NNNGTAGGAGCC; NNNCCGCTGCTA; NNNAACAACAAG; NNNGGTGGTCTA; NNNTTGGCCAAC; NNNCAGGAGTGC; and NNNATCACACTA (e.g., see Table 3).

[0358] The skilled person will recognize that the universal sample index can be of any length and have any sequence that can be used to distinguish target sequences obtained from different sample sources in multiplexed assays. In a population of a given sample index, such as but not limited to NNNGTAGGAGCC, the population contains a mixture of individual sample index molecules that each carry the same universal sample index sequence (e.g., GTAGGAGCC) and a different short random sequence (e.g., NNN), where there may be up to 64 different short random sequences in the population of a given sample index.

[0359] In a population of template molecules with a sample index, a short random sequence of the sample index (NNN) can provide a high nucleotide diversity that includes approximately equal proportions of all four nucleotides (e.g., A, G, C, T, and / or U) that will be represented in each cycle of a sequencing run (see Fig.19 and 20The high nucleotide diversity of the short random sequences can also provide color balancing during each cycle of the sequencing run. An advantage of designing a sample index to include a short random sequence (e.g., NNN) is that in a low-plex population of template molecules (e.g., 2-plex or 4-plex), a universal sample index sequence that identifies two or four different samples does not need to exhibit nucleotide diversity (e.g., see Fig.19 and 20 ). Additionally, the nucleotide diversity of short random sequences (e.g., NNN) can eliminate the need to include a PhiX spike-in library, or allow for the use of reduced amounts of a PhiX spike-in library to be distributed onto a flow cell and sequenced.

[0360] The template molecule may include a first sample index sequence comprising a short random sequence (NNN) and a second sample index sequence lacking the short random sequence. In some embodiments, only sequencing data from sample index sequences having a short random sequence (NNN) are used for polymerase community mapping and template alignment because the short random sequence (e.g., NNN) provides sufficient nucleotide diversity and color balance. Two types of sample indexes can be used to distinguish target sequences obtained from different sample sources in multiplex assays.

[0361] The order in which the target sequence region and the sample index region are sequenced can also be used to improve the challenge of sequencing low-diversity template molecules. For example, but not limited to, the sample index region can be sequenced before the target sequence region is sequenced, and the sample index sequence can be associated with the target sequence region. For example, but not limited to, the sample index region can be sequenced first, including sequencing a short random sequence (e.g., NNN), and optionally sequencing at least a portion of the universal sample index, and then sequencing the target sequence region. In a population of template molecules with sample indexes, a short random sequence (e.g., NNN) can provide a nucleotide diversity that may not be provided by the target sequence region of the template molecule. The sequence of the sample index can provide improved nucleotide diversity and color balance for polymerase community mapping and template alignment.

[0362] In addition, when the sample index region is first sequenced, the length of the sample index region after sequencing can be relatively short (e.g., less than 30 nucleotides in length), so that the product dehybridization of the sample index region after sequencing is more complete. Milder dehybridization conditions can be used to remove most or all of the products of the sequencing sample index region, which reduces the residual signal level from any sequencing products that remain hybridized with the template molecule. In contrast, the target sequence region is typically much longer than the sample index region (e.g., more than 100 nucleotides in length). In some embodiments, when the target sequence region is sequenced before the sample index region, the products of the sequenced target sequence region must undergo more stringent dehybridization conditions to remove any remaining products that hybridize with the template molecule, which may damage the template molecule.

[0363] In some aspects, the disclosure provides template molecules, each template molecule comprising at least one sample index sequence, which can be used to distinguish target sequences obtained from different sample sources in multiple determinations, wherein at least one sample index sequence includes a short random sequence (e.g., NNN) connected to a universal sample index sequence. At least one sample index sequence can include sequence diversity for improving base recognition. At least one sample index sequence can be used to improve the accuracy of base recognition.

[0364] In some embodiments, the short random sequence (e.g., NNN) is located upstream of the universal sample index sequence, such that during a sequencing run, the random sequence portion is sequenced before the universal sample index sequence. In some embodiments, the short random sequence is located downstream of the universal sample index sequence, such that during a sequencing run, the random portion is sequenced after the universal sample index sequence.

[0365] In some embodiments, in the random sequence, each base "N" at a given position is independently selected from A, G, C, T or U. In some embodiments, the random sequence lacks a continuous repeating sequence with 2 or 3 identical nucleobases, such as and not limited to AA, TT, CC, GG, UU, AAA, TTT, CCC, GGG or UUU. In some embodiments, in a population of template molecules, the universal sample index sequence includes a short random sequence with a high diversity sequence, which includes approximately equal proportions of all four nucleotides (e.g., A, G, C, T and / or U), which will be represented in each cycle of the sequencing run.

[0366] In some embodiments, the short random sequence (eg, NNN) comprises 3 to 20 nucleotides, 3 to 10 nucleotides, 3 to 8 nucleotides, 3 to 6 nucleotides, 3 to 5 nucleotides, or 3 to 4 nucleotides, or any range therebetween.

[0367] In some embodiments, short random sequences (e.g., NNN) include, but are not limited to: AGC, AGT, GAC, GAT, CAT, CAG, TAG, TAC. The skilled person will recognize that more random sequences (e.g., 64 possible combinations) can be prepared, wherein each base "N" at a given position in the random sequence is independently selected from A, G, C, T or U.

[0368] In some embodiments, the universal sample index sequence comprises 5 to 20 nucleotides, 7 to 18 nucleotides, or 9 to 16 nucleotides, or any range therebetween.

[0369] In some embodiments, each sample index sequence in the population of sample indexes includes a universal sample index sequence and a short random sequence (e.g., NNN). In some embodiments, the short random sequence in the population of sample index sequences has all four nucleotide bases (e.g., A, G, C, and T / U) accounting for about 25% or about 20% to 30% of the overall base composition to provide nucleotide diversity in each sequencing cycle during sequencing of the short random sequence (e.g., NNN).

[0370] In some embodiments, in a population of sample index sequences, the proportion of adenine (A) at any given position in the short random sequence is about 20% to 30%, about 15% to 35%, about 10% to 40%, or any range therebetween. In some embodiments, in a population of sample index sequences, the proportion of guanine (G) at any given position in the short random sequence is about 20% to 30%, about 15% to 35%, about 10% to 40%, or any range therebetween. In some embodiments, in a population of sample index sequences, the proportion of cytosine (C) at any given position in the short random sequence is about 20% to 30%, about 15% to 35%, about 10% to 40%, or any range therebetween. In some embodiments, in a population of sample index sequences, the proportion of thymine (T) or uracil (U) at any given position in the short random sequence is about 20% to 30%, about 15% to 35%, about 10% to 40%, or any range therebetween.

[0371] In some embodiments, in the population of sample index sequences, the ratio of adenine (A) to thymine (T) or the ratio of adenine (A) to uracil (U) at any given position in the short random sequence is about 10% to 65%. In some embodiments, in the population of sample index sequences, the ratio of guanine (G) to cytosine (C) at any given position in the short random sequence is about 10% to 65%.

[0372] In some embodiments, the sequence diversity of the short random sequences in the population of sample index sequences ensures that sequencing cycles with fewer than four different nucleotide bases do not occur during sequencing of at least the short random sequences (eg, NNN).

[0373] In some embodiments, the random sequence (e.g., NNN) provides a balanced ratio of the nucleobases adenine, cytosine, guanine, thymine, and / or uracil (see Fig.19 ). In some embodiments, in a population of template molecules having a sample index, the random sequence (e.g., NNN) together with at least a portion of the universal sample index sequence provides a balanced ratio of nucleobases adenine, cytosine, guanine, thymine and / or uracil represented in each cycle of a sequencing run.

[0374] In some embodiments, the sequencing reaction comprises the use of a polymerase and nucleotides (e.g., nucleotide analogs) labeled with different fluorophores corresponding to nucleobases. In some embodiments, sequencing a random sequence (e.g., NNN) using labeled nucleotides provides a ratio-balanced fluorescent color corresponding to the nucleobases adenine, cytosine, guanine, thymine, and / or uracil in each cycle of the sequencing run. In some embodiments, sequencing a random sequence (e.g., NNN) and at least a portion of a universal sample index sequence using labeled nucleotides provides a ratio-balanced fluorescent color corresponding to the nucleobases adenine, cytosine, guanine, thymine, and / or uracil (e.g., see Fig.19). The labeled nucleotides emit a fluorescence signal during the sequencing reaction. In some embodiments, the sequencing reaction is performed on a sequencing device having a detector that captures a fluorescence image of the sequencing reaction from the fixed template molecule. The sequencing device can be configured to forward the fluorescence imaging data captured by the detector to a computer system that is programmed to determine the position of the template molecule fixed on the circulation pool (e.g., mapping). The computer system can generate a map of the position of the fixed template molecule based only on the random sequence (e.g., NNN), or based on the fluorescence imaging data of at least a portion of the random sequence (e.g., NNN) and the universal sample index sequence. Therefore, a small number of sequencing cycles for sequencing a portion of the random sequence (e.g., NNN) and an optional universal sample index sequence can be used to generate a map of the position of the fixed template molecule. The computer system can be configured to extract only the fluorescence color and intensity of the random sequence (e.g., NNN) or the random sequence (e.g., NNN) and at least a portion of the universal sample index sequence. The computer system can be configured to use the position of a given fixed template molecule and the fluorescence color and intensity associated with the given template molecule (which is established when the random sequence is sequenced) for base calling when sequencing the insertion region (110). The computer system can be configured to detect lags and leads while sequencing the random sequence (e.g., NNN) and the universal sample index sequence and the insertion region (110). In some embodiments, the balanced ratio of fluorescence colors provided by the random sequence (e.g., NNN) at each sequencing cycle can improve the quality of the data processed from the fluorescence image captured by the detector, and can in turn improve the ability of the computer system to determine the position of the fixed template molecule on the flow cell as well as the color and intensity, all of which can improve the accuracy and quality score of the base calls of the insertion region (110) after sequencing.

[0375] In some embodiments, the sequencing reaction comprises the use of a polymerase and a multivalent molecule labeled with different fluorophores corresponding to the nucleobases of the nucleotide units (e.g., adenine, guanine, cytosine, thymine, or uracil) that are attached to the nucleotide arms in a given multivalent molecule. In some embodiments, the core of each multivalent molecule is attached to a fluorophore corresponding to the nucleotide unit (e.g., adenine, guanine, cytosine, thymine, or uracil) that is attached to the nucleotide arms in a given multivalent molecule (e.g., see Figures 22 to 25). In some embodiments, at least one nucleotide arm of the multivalent molecule comprises a linker and / or a nucleotide base attached to a fluorophore, and wherein the fluorophore attached to a given linker or nucleotide base corresponds to the nucleotide base (e.g., adenine, guanine, cytosine, thymine, or uracil) of the nucleotide arm. In some embodiments, sequencing a random sequence (e.g., NNN) using a labeled multivalent molecule provides a balanced ratio of fluorescent colors corresponding to the nucleobases adenine, cytosine, guanine, thymine, and / or uracil in each cycle of the sequencing run. In some embodiments, sequencing a random sequence (e.g., NNN) and at least a portion of a universal sample index sequence using a labeled multivalent molecule provides a balanced ratio of fluorescent colors corresponding to the nucleobases adenine, cytosine, guanine, thymine, and / or uracil (e.g., see Fig.19 ). The labeled multivalent molecules emit fluorescence signals during the sequencing reaction. In some embodiments, the sequencing reaction is performed on a sequencing device having a detector that captures a fluorescence image of the sequencing reaction from the fixed template molecule. The sequencing device can be configured to forward the fluorescence imaging data captured by the detector to a computer system that is programmed to determine the position of the template molecule (polymerase community) fixed on the circulation pool (e.g., mapping). The computer system can generate a map of the position of the fixed template molecule based only on the random sequence (e.g., NNN), or based on the fluorescence imaging data of at least a portion of the random sequence (e.g., NNN) and the universal sample index sequence. Therefore, a small number of sequencing cycles for sequencing a portion of the random sequence (e.g., NNN) and an optional universal sample index sequence can be used to generate a map of the position of the fixed template molecule. The computer system can be configured to extract only the fluorescence color and intensity of the random sequence (e.g., NNN) or the random sequence (e.g., NNN) and the universal sample index sequence. The computer system can be configured to use the position of a given fixed template molecule and the fluorescence color and intensity associated with the given template molecule (which is established when the random sequence is sequenced) for base calling when sequencing the insertion region (110). The computer system can be configured to detect lags and leads while sequencing the random sequence (e.g., NNN) and the universal sample index sequence and the insertion region (110). In some embodiments, the balanced ratio of fluorescence colors provided by the random sequence (e.g., NNN) at each sequencing cycle can improve the quality of the data processed from the fluorescence image captured by the detector, and can in turn improve the ability of the computer system to determine the position of the fixed template molecule on the flow cell as well as the color and intensity, all of which can improve the accuracy and quality score of the base calls of the insertion region (110) after sequencing.

[0376] First embodiment: Order of sequencing sample index sequences

[0377] In some embodiments, the covalently closed circular library molecule (600) comprises: (i) a first subregion (411) comprising a universal binding sequence for an immobilized surface pinning primer; (ii) a second subregion (412) comprising a short random sequence (NNN) and a sample index sequence; (iii) a third subregion (413) comprising a universal binding sequence for an immobilized surface capture primer; (iv) a universal binding sequence for a reverse sequencing primer (130); (v) an insert sequence (110); and (vi) a universal binding sequence for a forward sequencing primer (120) (e.g., see Figure 4 ). In some embodiments, a plurality of covalently closed circular library molecules (600) are subjected to rolling circle amplification by hybridizing the plurality of covalently closed circular library molecules (600) with a plurality of surface capture primers fixed to a support, wherein the surface capture primers initiate amplification, thereby generating a plurality of fixed concatemers, each concatemer having a tandem repeat sequence of its homologous covalently closed circular library molecule (600). In some embodiments, the rolling circle amplification is performed in the presence of a plurality of compacted oligonucleotides, which can hybridize with the concatemers at their universal binding sequences for surface pinning primers, for surface capture primers, for reverse sequencing primers, and / or for forward sequencing primers. In some embodiments, a plurality of fixed capture primers lack uracil bases. In some embodiments, the rolling circle amplification reaction comprises a plurality of nucleotides, including dATP, dGTP, dCTP, dTTP, and dUTP, to generate a plurality of fixed concatemers, wherein each concatemer molecule comprises randomly distributed uracil bases. In some embodiments, at least a portion of the fixed concatemers are sequenced.

[0378] In some embodiments, the sequencing sequence includes: (1) sequencing the short random sequence (NNN) and the sample index sequence (412) of the concatemer; and (2) sequencing the insertion region (110) of the concatemer. In some embodiments, the sequencing sequence further includes: (3) performing a paired rotation reaction so that the fixed concatemer molecule is replaced by a fixed second strand complementary to the concatemer molecule; and (4) sequencing the insertion region (110) on the second strand. In some embodiments, sequencing the short random sequence (NNN) and the sample index sequence (412) can provide sufficient nucleotide diversity and color balance for polymerase community mapping and concatemer alignment.

[0379] In some embodiments, a method for sequencing a concatemer molecule fixed to a support includes step (a): hybridizing the concatemer molecule with a first plurality of soluble sequencing primers that hybridize to at least a portion of the third sub-region (413), and sequencing a short random sequence (e.g., NNN) and a universal sample index sequence of the third sub-region (413), thereby generating a plurality of sample index extension products hybridized to the fixed concatemer molecule, wherein the plurality of sample index extension products are complementary to the short random sequence (e.g., NNN) and the universal sample index sequence.

[0380] In some embodiments, the method for sequencing further comprises step (b): removing the first plurality of sample index extension products and retaining the immobilized concatemer molecules.

[0381] In some embodiments, the method for sequencing further comprises step (c): hybridizing the retained fixed concatemer molecules with a second plurality of soluble sequencing primers, the second plurality of soluble sequencing primers hybridizing with a universal binding sequence for the forward sequencing primer (120), and sequencing the insertion region (110), thereby generating a plurality of insertion sequence extension products hybridized to the fixed concatemer molecules.

[0382] In some embodiments, the method for sequencing further comprises step (d): replacing a plurality of insertion sequence extension products hybridized to the fixed concatemer molecule by performing a primer extension reaction using a strand displacement polymerase and a plurality of nucleotides to generate a second strand extension product hybridized to the fixed concatemer molecule (including the fixed capture primer).

[0383] In some embodiments, the method for sequencing further comprises step (e): removing the fixed concatemer molecules by generating abasic sites at uracil sites in the fixed concatemer molecules and generating gaps at the abasic sites, thereby generating concatemer molecules containing gaps, while retaining the second strand extension products generated in step (d), wherein each second strand extension product is retained by hybridization with the fixed capture primer. In some embodiments, paired rotation is achieved by performing steps (d) and (e).

[0384] In some embodiments, the method for sequencing further comprises step (f): hybridizing the retained second strand extension product with a third plurality of soluble sequencing primers, which hybridize to a universal binding sequence for the reverse sequencing primer (130), and sequencing at least a portion of the insertion region (110).

[0385] In some embodiments, the method for sequencing further comprises: assigning (i) the sequence of the insert region (110) to (ii) the sample index sequence, thereby identifying the insert region as being obtained from the first source. In some embodiments, the assignment may be performed after steps (c) and / or (f).

[0386] In some embodiments, the removal of the plurality of sequencing extension products of step (b) can be performed using a denaturing reagent comprising an SSC (e.g., saline-sodium citrate) buffer with or without formamide at a temperature that promotes nucleic acid denaturation (such as, for example, 50° C. to 90° C.). In some embodiments, the removal of the plurality of sequencing extension products of step (b) can be performed using a dehybridization reagent at a temperature that promotes nucleic acid denaturation (such as, for example, 50° C. to 90° C.). In some embodiments, the dehybridization reagent comprises a pH buffer, a reducing agent, a monovalent salt, and a crowding agent. In some embodiments, the dehybridization reagent further comprises a chaotropic agent.

[0387] In some embodiments, the sequencing of steps (a), (c) and (f) comprises performing any sequencing method described herein, which uses a sequencing polymerase and a detectably labeled nucleotide analog. In some embodiments, the sequencing of steps (a), (c) and (f) comprises performing any two-stage sequencing method described herein, which employs a sequencing polymerase, a detectably labeled multivalent molecule and a labeled nucleotide analog or an unlabeled nucleotide analog. In some embodiments, the sequencing of steps (a), (c) and (f) comprises performing any combined sequencing method or any sequencing method employing nucleotides labeled with phosphate bonds described herein.

[0388] In some embodiments, the density of the plurality of concatemer molecules immobilized to the support is 100 μm / mm. 2 Approx. 10 2 Up to 10 15 (For example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 In some embodiments, the plurality of concatemer molecules are immobilized at random positions on the support. In some embodiments, the plurality of concatemer molecules are immobilized on the support in a predetermined pattern.

[0389] Second embodiment: Order of sequencing sample index sequences

[0390] In some embodiments, the covalently closed circular library molecule (600) comprises: (i) a first subregion (411) comprising a universal binding sequence for an immobilized surface pinning primer; (ii) a second subregion (412) comprising a short random sequence (NNN) and a sample index sequence; (iii) a third subregion (413) comprising a universal binding sequence for an immobilized surface capture primer; (iv) a universal binding sequence for a reverse sequencing primer (130); (v) an insert sequence (110); and (vi) a universal binding sequence for a forward sequencing primer (120) (e.g., see Figure 4 ). In some embodiments, a plurality of covalently closed circular library molecules (600) are subjected to rolling circle amplification by hybridizing the plurality of covalently closed circular library molecules (600) with a plurality of surface capture primers fixed to a support, wherein the surface capture primers initiate amplification, thereby generating a plurality of fixed concatemers, each concatemer having a tandem repeat sequence of its homologous covalently closed circular library molecule (600). In some embodiments, the rolling circle amplification is performed in the presence of a plurality of compacted oligonucleotides, which can hybridize with the concatemers at their universal binding sequences for surface pinning primers, for surface capture primers, for reverse sequencing primers, and / or for forward sequencing primers. In some embodiments, a plurality of fixed capture primers lack uracil bases. In some embodiments, the rolling circle amplification reaction comprises a plurality of nucleotides, including dATP, dGTP, dCTP, dTTP, and dUTP, to generate a plurality of fixed concatemers, wherein each concatemer molecule comprises randomly distributed uracil bases. In some embodiments, at least a portion of the fixed concatemers are sequenced.

[0391] In some embodiments, the sequencing sequence includes: (1) sequencing the insertion region (110) of the concatemer; and (2) sequencing the short random sequence (NNN) and the sample index sequence (412) of the concatemer. In some embodiments, the sequencing sequence further includes: (3) performing a paired rotation reaction so that the fixed concatemer molecule is replaced by a fixed second strand complementary to the concatemer molecule; and (4) sequencing the insertion region (110) on the second strand. In some embodiments, sequencing the short random sequence (NNN) and the sample index sequence (412) can provide sufficient nucleotide diversity and color balance for polymerase community mapping and concatemer alignment.

[0392] In some embodiments, a method for sequencing concatemer molecules fixed to a support comprises step (a): hybridizing the fixed concatemer molecules with a first plurality of soluble sequencing primers, the first plurality of soluble sequencing primers hybridizing with a universal binding sequence for a forward sequencing primer (120), and sequencing the insertion region (110), thereby generating a plurality of insertion sequence extension products hybridized to the fixed concatemer molecules.

[0393] In some embodiments, the method for sequencing further comprises step (b): removing the plurality of insert extension products and retaining the immobilized concatemer molecules.

[0394] In some embodiments, the method for sequencing further includes step (c): hybridizing the retained concatemer molecules with a second plurality of soluble sequencing primers that hybridize to at least a portion of the third sub-region (413), and sequencing the short random sequence (e.g., NNN) and the universal sample index sequence of the third sub-region (412), thereby generating a plurality of sample index extension products hybridized to the fixed concatemer molecules, wherein the plurality of sample index extension products are complementary to the short random sequence (e.g., NNN) and the universal sample index sequence.

[0395] In some embodiments, the method for sequencing further includes step (d): replacing multiple sample index extension products hybridized to the fixed multimer molecule by performing a primer extension reaction using a strand displacement polymerase and multiple nucleotides to generate a second strand extension product hybridized to the fixed multimer molecule (including the fixed capture primer).

[0396] In some embodiments, the method for sequencing further comprises step (e): removing the fixed concatemer molecules by generating abasic sites at uracil sites in the fixed concatemer molecules and generating gaps at the abasic sites, thereby generating concatemer molecules containing gaps, while retaining the second strand extension products generated in step (d), wherein each second strand extension product is retained by hybridization with the fixed capture primer. In some embodiments, paired rotation is achieved by performing steps (d) and (e).

[0397] In some embodiments, the method for sequencing further comprises step (f): hybridizing the retained second strand extension product with a third plurality of soluble sequencing primers, which hybridize to a universal binding sequence for the reverse sequencing primer (130), and sequencing at least a portion of the insertion region (110).

[0398] In some embodiments, the method for sequencing further comprises: assigning (i) the sequence of the insert region (110) to (ii) the sample index sequence, thereby identifying the insert region as being obtained from the first source. In some embodiments, the assignment may be performed after steps (c) and / or (f).

[0399] In some embodiments, the removal of the plurality of sequencing extension products of step (b) can be performed using a denaturing reagent comprising an SSC (e.g., saline-sodium citrate) buffer with or without formamide at a temperature that promotes nucleic acid denaturation (such as, for example, 50° C. to 90° C.). In some embodiments, the removal of the plurality of sequencing extension products of step (b) can be performed using a dehybridization reagent at a temperature that promotes nucleic acid denaturation (such as, for example, 50° C. to 90° C.). In some embodiments, the dehybridization reagent comprises a pH buffer, a reducing agent, a monovalent salt, and a crowding agent. In some embodiments, the dehybridization reagent further comprises a chaotropic agent.

[0400] In some embodiments, the sequencing of steps (a), (c) and (f) comprises performing any sequencing method described herein, which uses a sequencing polymerase and a detectably labeled nucleotide analog. In some embodiments, the sequencing of steps (a), (c) and (f) comprises performing any two-stage sequencing method described herein, which employs a sequencing polymerase, a detectably labeled multivalent molecule and a labeled nucleotide analog or an unlabeled nucleotide analog. In some embodiments, the sequencing of steps (a), (c) and (f) comprises performing any combined sequencing method or any sequencing method employing nucleotides labeled with phosphate bonds described herein.

[0401] In some embodiments, the density of the plurality of concatemer molecules immobilized to the support is 100 μm / mm. 2 Approx. 10 2 Up to 10 15 (For example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 In some embodiments, the plurality of concatemer molecules are immobilized at random positions on the support. In some embodiments, the plurality of concatemer molecules are immobilized on the support in a predetermined pattern.

[0402] Third Example: Sequencing Order

[0403] In some embodiments, the covalently closed circular library molecule (600) comprises: (i) a first subregion (411) comprising a universal binding sequence for an immobilized surface pinning primer; (ii) a second subregion (412) comprising a short random sequence (NNN) and a sample index sequence; (iii) a third subregion (413) comprising a universal binding sequence for an immobilized surface capture primer; (iv) a universal binding sequence for a reverse sequencing primer (130); (v) an insert sequence (110); and (vi) a universal binding sequence for a forward sequencing primer (120) (e.g., see Figure 4 ). In some embodiments, a plurality of covalently closed circular library molecules (600) are subjected to rolling circle amplification by hybridizing the plurality of covalently closed circular library molecules (600) with a plurality of surface capture primers fixed to a support, wherein the surface capture primers initiate amplification, thereby generating a plurality of fixed concatemers, each concatemer having a tandem repeat sequence of its homologous covalently closed circular library molecule (600). In some embodiments, the rolling circle amplification is performed in the presence of a plurality of compacted oligonucleotides, which can hybridize with the concatemers at their universal binding sequences for surface pinning primers, for surface capture primers, for reverse sequencing primers, and / or for forward sequencing primers. In some embodiments, a plurality of fixed capture primers lack uracil bases. In some embodiments, the rolling circle amplification reaction comprises a plurality of nucleotides, including dATP, dGTP, dCTP, dTTP, and dUTP, to generate a plurality of fixed concatemers, wherein each concatemer molecule comprises randomly distributed uracil bases. In some embodiments, at least a portion of the fixed concatemers are sequenced.

[0404] In some embodiments, the sequencing order includes: (1) sequencing the first 3 to 5 bases of the insertion region (110) of the concatemer; (2) sequencing the short random sequence (NNN) and the sample index sequence (412) of the concatemer; and (3) sequencing the remaining portion of the insertion region (110) of the concatemer. In some embodiments, the sequencing order further includes: (4) performing a paired rotation reaction so that the fixed concatemer molecule is replaced by a fixed second strand complementary to the concatemer molecule; and (5) sequencing the insertion region (110) on the second strand. In some embodiments, sequencing the first 3 to 5 bases of the insertion region (110) of the concatemer can provide sufficient nucleotide diversity and color balance for polymerase community mapping and concatemer alignment. In some embodiments, sequencing the short random sequence (NNN) and the sample index sequence (412) can provide sufficient nucleotide diversity and color balance for polymerase community mapping and concatemer alignment.

[0405] In some embodiments, a method for sequencing concatemer molecules immobilized to a support comprises step (a): hybridizing the concatemer molecules with a first plurality of soluble sequencing primers, the first plurality of soluble sequencing primers hybridizing with a universal binding sequence for a forward sequencing primer (120), and sequencing the first 3 to 5 bases of an insertion region (110), thereby generating a plurality of insertion extension products hybridized to the immobilized concatemer molecules, wherein the plurality of insertion extension products are complementary to a target sequence (110). The sequence of the first 3 to 5 bases of the insertion region (110) can provide sufficient sequence diversity and color balance for polymerase community mapping and concatemer alignment.

[0406] In some embodiments, the method for sequencing further comprises step (b): removing the plurality of insert extension products and retaining the immobilized concatemer molecules.

[0407] In some embodiments, the method for sequencing further includes step (c): hybridizing the retained concatemer molecules with a second plurality of soluble sequencing primers that hybridize to at least a portion of the third sub-region (413), and sequencing the short random sequence (e.g., NNN) and the universal sample index sequence of the third sub-region (412), thereby generating a plurality of sample index extension products hybridized to the fixed concatemer molecules, wherein the plurality of sample index extension products are complementary to the short random sequence (e.g., NNN) and the universal sample index sequence.

[0408] In some embodiments, the method for sequencing further comprises step (d): removing the plurality of sample index extension products and retaining the immobilized concatemer molecules.

[0409] In some embodiments, the method for sequencing further comprises step (e): hybridizing the retained concatemer molecules with a third plurality of soluble sequencing primers, the third plurality of soluble sequencing primers hybridizing with universal binding sequences for the forward sequencing primer (120), and sequencing the remainder of the insertion region (110) or sequencing the entire length of the insertion region (110), thereby generating a plurality of insertion extension products hybridized to the immobilized concatemer molecules, wherein the plurality of insertion extension products are complementary to the target sequence (110). In some embodiments, in step (e), the first 3 to 5 bases of the insertion region (110) may be sequenced using labeled nucleotides and / or labeled multivalent molecules. In some embodiments, in step (e), the first 3 to 5 bases of the insertion region (110) may be sequenced (e.g., dark sequencing) using unlabeled nucleotides and / or unlabeled multivalent molecules. In some embodiments, in step (e), the remainder of the insertion region (110) may be sequenced using labeled nucleotides and / or labeled multivalent molecules. In some embodiments, in step (e), the entire length of the insertion region (110) can be sequenced using labeled nucleotides and / or labeled multivalent molecules.

[0410] In some embodiments, the method for sequencing further comprises step (f): replacing a plurality of insertion sequence extension products hybridized to the fixed concatemer molecule by performing a primer extension reaction using a strand displacement polymerase and a plurality of nucleotides to generate a second strand extension product hybridized to the fixed concatemer molecule (including the fixed capture primer).

[0411] In some embodiments, the method for sequencing further comprises step (g): removing the fixed concatemer molecules by generating abasic sites at uracil sites in the fixed concatemer molecules and generating gaps at the abasic sites, thereby generating concatemer molecules containing gaps, while retaining the second strand extension products generated in step (f), wherein each second strand extension product is retained by hybridization with the fixed capture primer. In some embodiments, paired rotation is achieved by performing steps (f) and (g).

[0412] In some embodiments, the method for sequencing further comprises step (h): hybridizing the retained second strand extension product with a third plurality of soluble sequencing primers, which hybridize to a universal binding sequence for the reverse sequencing primer (130), and sequencing at least a portion of the insertion region (110).

[0413] In some embodiments, the method for sequencing further comprises: assigning (i) the sequence of the insert region (110) to (ii) the sample index sequence, thereby identifying the insert region as being obtained from the first source. In some embodiments, the assignment may be performed after steps (c), (e) and / or (h).

[0414] In some embodiments, the removal of the plurality of sequencing extension products of steps (b) and (d) can be performed using a denaturing reagent comprising an SSC (e.g., saline-sodium citrate) buffer with or without formamide at a temperature that promotes nucleic acid denaturation (such as, for example, 50° C. to 90° C.). In some embodiments, the removal of the plurality of sequencing extension products of steps (b) and (d) can be performed using a dehybridization reagent at a temperature that promotes nucleic acid denaturation (such as, for example, 50° C. to 90° C.). In some embodiments, the dehybridization reagent comprises a pH buffer, a reducing agent, a monovalent salt, and a crowding agent. In some embodiments, the dehybridization reagent further comprises a chaotropic agent.

[0415] In some embodiments, the sequencing of steps (a), (c) and (e) comprises performing any sequencing method described herein, which uses a sequencing polymerase and a detectably labeled nucleotide analog. In some embodiments, the sequencing of steps (a), (c) and (e) comprises performing any two-stage sequencing method described herein, which employs a sequencing polymerase, a detectably labeled multivalent molecule, and a labeled or unlabeled nucleotide analog. In some embodiments, the sequencing of steps (a), (c) and (e) comprises performing any combined sequencing method or any sequencing method employing nucleotides labeled with phosphate bonds described herein.

[0416] In some embodiments, the density of the plurality of concatemer molecules immobilized to the support is 100 μm / mm. 2 Approx. 10 2 Up to 10 15 (For example, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 In some embodiments, the plurality of concatemer molecules are immobilized at random positions on the support. In some embodiments, the plurality of concatemer molecules are immobilized on the support in a predetermined pattern.

[0417] Fourth Example: Sequencing Order

[0418] In some embodiments, the covalently closed circular library molecule (600) comprises: (i) a first subregion (411) comprising a universal binding sequence for an immobilized surface pinning primer; (ii) a second subregion (412) comprising a short random sequence (NNN) and a sample index sequence; (iii) a third subregion (413) comprising a universal binding sequence for an immobilized surface capture primer; (iv) a universal binding sequence for a reverse sequencing primer (130); (v) an insert sequence (110); and (vi) a universal binding sequence for a forward sequencing primer (120) (e.g., see Figure 4 ). In some embodiments, a plurality of covalently closed circular library molecules (600) are subjected to rolling circle amplification by hybridizing the plurality of covalently closed circular library molecules (600) with a plurality of surface capture primers fixed to a support, wherein the surface capture primers initiate amplification, thereby generating a plurality of fixed concatemers, each concatemer having a tandem repeat sequence of its homologous covalently closed circular library molecule (600). In some embodiments, the rolling circle amplification is performed in the presence of a plurality of compacted oligonucleotides, which can hybridize...

Claims

1. A method for forming a plurality of library-splint complexes (500), the method comprising: a) providing a plurality of double-stranded splint adaptors (200), wherein each double-stranded splint adaptor (200) of the plurality of double-stranded splint adaptors comprises a first splint strand (300) hybridized to a second splint strand (400), wherein the double-stranded splint adaptor comprises a double-stranded region and two flanking single-stranded regions, wherein the first splint strand comprises a first region (320), an internal region (310) and a second region (330), and wherein the internal region (310) of the first splint strand hybridizes to the second splint strand (400); as well as b) hybridizing the plurality of double-stranded splint adaptors to a plurality of single-stranded nucleic acid library molecules (100), wherein each library molecule comprises a sequence of interest (110) flanked on a first side by a universal adaptor sequence for a forward sequencing primer binding site (120) and on a second side by a universal adaptor sequence for a reverse sequencing primer binding site (130), The plurality of library molecules are thereby circularized to form a plurality of library-splint complexes (500) each having two gaps.

2. The method according to claim 1, further comprising: (c) contacting the plurality of library-splint complexes (500) with a ligase to generate a plurality of covalently closed circular library molecules (600).

3. A method according to claim 1 or 2, wherein the hybridization is performed under conditions suitable for hybridizing the first region (320) of the first splint strand to the universal adapter sequence for the forward sequencing primer binding site (120) of the library molecule.

4. The method of claim 3, wherein the conditions are suitable for hybridizing the second region (330) of the first splint strand to the universal adapter sequence for the reverse sequencing primer binding site (130) of the library molecule.

5. The method according to any one of claims 1 to 4, wherein the inner zone (310) of the first clamping plate chain (300) comprises at least three sub-zones.

6. The method according to claim 5, wherein the at least three sub-areas include a sub-area (311), a sub-area (312), and a sub-area (313).

7. The method according to claim 6, wherein the sub-region (311) comprises a universal adapter sequence for a surface capture primer binding site, a universal adapter sequence for a surface pinning primer binding site, a sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI).

8. The method according to claim 6 or 7, wherein the sub-region (312) comprises a universal adapter sequence for a surface capture primer binding site, a universal adapter sequence for a surface pinning primer binding site, a sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI).

9. The method according to any one of claims 6 to 8, wherein the sub-region (313) comprises a universal adapter sequence for a surface capture primer binding site, a universal adapter sequence for a surface pinning primer binding site, a sample index sequence, a short random sequence (NNN) and / or a unique molecular index (UMI).

10. The method according to any one of claims 6 to 9, wherein the sub-region (311), (312) or (313) comprises a sample index sequence, and wherein the sample index sequence comprises: Sample index sequence lacking short random sequence (NNN); Sample index sequence and short random sequence (NNN); A sample index flanked on both sides by nucleotide bases that can be converted to abasic bases; at least one nucleotide base that can be converted into an abasic base; At least one deoxyinosine; · 18 carbon spacer; and / or • An 18-carbon spacer and at least one deoxyinosine.

11. The method according to any one of claims 6 to 10, further comprising: i) distributing the plurality of covalently closed circular library molecules (600) onto a support having a plurality of surface capture primers immobilized to the support under conditions suitable for hybridizing each covalently closed circular library molecule (600) to each immobilized surface capture primer, thereby immobilizing the plurality of covalently closed circular library molecules (600) to the support; 12. The method of claim 11, wherein the support further comprises a plurality of surface-pinned primers immobilized to the support.

13. The method according to claim 11, further comprising: ii) contacting the plurality of immobilized covalently closed circular library molecules (600) with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for performing a rolling circle amplification reaction on the support using the plurality of surface capture primers as immobilized amplification primers and using the plurality of covalently closed circular library molecules (600) as template molecules, Thereby, a plurality of nucleic acid concatemer molecules are generated which are immobilized to the surface capture primers.

14. The method according to claim 13, further comprising: iii) sequencing the plurality of nucleic acid concatemer molecules fixed to the surface capture primers, wherein the sequencing comprises (i) sequencing the sample index and (ii) sequencing the target sequence (110).

15. The method according to claim 13, further comprising: iv) sequencing the multiple nucleic acid concatemer molecules fixed to the surface capture primers, wherein the sequencing includes (A) sequencing one or more short random sequences NNN, (B) sequencing one or more sample indexes, and (C) sequencing the target sequence (110).

16. The method according to any one of claims 7 to 15, wherein the inner area (310) of the first clamping plate chain (300) contains a sample index.

17. The method according to any one of claims 7 to 15, wherein the inner region (310) of the first splint chain (300) comprises a sample index and a short random sequence (NNN).

18. The method according to any one of claims 1 to 17, wherein the library molecule (100) comprises one or more nucleotide sequences selected from Table 1.

19. The method according to any one of claims 1 to 18, wherein the first splint strand (300) comprises one or more nucleotide sequences selected from Table 2.

20. The method according to any one of claims 1 to 19, wherein the second splint strand (400) comprises one or more nucleotide sequences selected from Table 3.

Images