Methods and compositions for tracing the origin of nucleic acid fragments for nucleic acid sequencing

Abstract

The present invention provides methods and compositions for tracking the source of nucleic acid fragments by target-specific barcode labeling when the original nucleic acid target is broken into small fragments. The nucleic acid target is captured in vitro on a solid support with a clone-localized nucleic acid barcode template. Many nucleic acid targets can be processed simultaneously in a massively parallel manner without segmentation. These nucleic acid target tracking methods can be used for a variety of applications in whole genome sequencing and targeted sequencing, such as to accurately identify genomic variants, haplotype phasing and compilation.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent claims priority to provisional applications US 62 / 628,079 filed on February 8, 2018 and US 62 / 656,796 filed on April 12, 2018. They are all included here. All publications, patents and other documents mentioned herein will be incorporated by reference in their entirety. Technical Field

[0003] The present invention generally relates to methods and compositions for nucleic acid sequencing. In particular, the methods and compositions provided herein relate to preparing nucleic acid libraries and generating sequencing data therefrom. Background Art

[0004] Nucleic acid sequencing can provide information for a variety of biomedical applications, including diagnostics, prognostics, pharmacogenomics, and forensic biology. Sequencing can involve basic low-throughput methods, including Maxam-Gilbert sequencing (chemically modified nucleotides) and Sanger sequencing (chain end) methods, or next-generation high-throughput methods, including massively parallel pyrosequencing, synthesis sequencing, ligation sequencing, semiconductor sequencing, and others. For most sequencing methods, samples (such as nucleic acid targets) need to be processed into sequencing libraries before sequencing on a sequencing instrument. For example, samples may be fragmented, amplified, or attached to identifiers. Unique identifiers are often used to identify the source of a specific sample.

[0005] Most commercially available sequencing technologies have limited sequencing read lengths. In particular, second-generation sequencing technologies can only sequence a few hundred bases and barely reach a thousand bases. However, the nucleic acid sequence of a gene can span a few kilobases as well as tens or hundreds of kilobases, which means that to successfully determine the haplotypes of all genes, sequencing read lengths of tens of kilobases is required.

[0006] To overcome the problem of short sequence read length, many methods have been developed to target-specifically label long nucleic acid targets when they are broken down into small fragments for preparing sequencing libraries. These methods include: long fragment reads of the entire genome, Illumina's synthetic long reads, 10x genome concatenated reads (Zheng et al., 2016), Illumina's single-tube method (Zhang et al., 2017), and our own single-tube method (WO2017 / 151828). These target-specific tags are short nucleic acid sequences, called barcodes. The source of these short nucleic acid fragments can be identified based on their unique associated barcodes. In this method, the wider the diversity of the barcode group used, the better the specificity of identification it provides. The 10x genome concatenated read method is widely used. However, to maintain the clonality of the specific target barcoding reaction, an oil-in-water emulsion method is required. This requirement greatly increases the complexity and cost of its sample preparation procedure. Several methods, including those belonging to Total Genomics (U.S. Patent 9328382), Illumina's single-tube method (Zhang et al., 2017), and our method (WO2017 / 151828) use transposase-based systems and eliminate the need to partition nucleic acid targets with emulsion droplets in the reaction. These methods can, in principle, achieve target-specific barcoding in a single-tube reaction format. The present invention provides a novel transposase-based single-tube barcoding method that significantly improves reaction efficiency and simplifies the workflow. Summary of the invention

[0007] In one aspect, the present invention describes a method for tracking the source of nucleic acid fragments by barcode tags. The method includes providing a ligase, a plurality of solid supports having a cloned barcode template or a semi-cloned barcode template fixed thereon, and a plurality of transpososomes, each of which includes a transposable DNA and a transposase, wherein at least one of the transposable DNAs in the transpososome is capable of directly or indirectly connecting to the barcode template on the solid support. The nucleic acid target is contacted with the ligase, the solid support, and the transpososome in a reaction vessel to attach the barcode information on the solid support to the nucleic acid target by simultaneous chain transfer and ligation reactions. The nucleic acid target is broken into fragments by a broken chain transfer complex, wherein at least one fragment is attached to the barcode template on the solid support.

[0008] In one aspect, a method for tracking the source of nucleic acid fragments by barcode tags is described herein. The method includes providing a plurality of solid supports on which a clonal barcode template or a semi-clonal barcode template is fixed, and a plurality of transposons, each of which includes a transposable DNA and a transposase, wherein at least one transposable DNA in the transposons can be captured directly or indirectly by the barcode template on the solid support through hybridization or affinity groups. The nucleic acid target is contacted with the solid support and the transposons in a reaction vessel to attach the barcode information on the solid support to the nucleic acid target through simultaneous chain transfer and capture reactions. The above reactions occur substantially simultaneously, and there is no need to additionally separate each nucleic acid target from another nucleic acid target in the total plurality of nucleic acid targets. The nucleic acid target is broken into fragments by a broken chain transfer complex, wherein at least one fragment is attached to the barcode template on the solid support.

[0009] In some cases, the clonal barcode template or semi-clonal barcode template fixed on the solid support is prepared by direct synthesis, clonal amplification or a combination thereof. Clonal amplification can be emulsion PCR, bridge PCR, isothermal PCR, template walking, nanosphere generation and a combination thereof. In certain cases, the barcoded solid support is prepared by clonal amplification methods without separating amplified and unamplified populations. In further cases, the barcoded solid support is prepared by clonal amplification, and has only or mainly enriched amplified populations.

[0010] In one aspect, the reaction is carried out in a buffer system with controlled viscosity, with a viscosity of about 1-200 mPas at about 20° C., preferably, a viscosity of 1.5-30 mPas at about 20° C., by adding a substance selected from the group consisting of polyethylene glycol, pluronic, cellulose, agarose, their derivatives, other polymers and combinations thereof to reduce diffusion and increase suspension.

[0011] On the one hand, the transposome can be replaced by separate transposable DNA and transposase without the need to pre-assemble them into a transposome.

[0012] In another aspect, after the initial reaction, a second transposome is added to the reaction vessel; wherein the previously added transposome is referred to as the first transposome, and the first transposome and the second transposome may be of the same type or different types, or different transposome sequences of the same type. In another aspect, the second transposome is added after the nucleic acid target is broken into fragments.

[0013] On the other hand, the nucleic acid target can be pre-attached to the solid support by non-specific binding.

[0014] In another aspect, the capture reaction in the container is performed by ligation, or by hybridization, or by affinity tag, or by antibody and antigen reaction, or by click chemistry, or a combination thereof.

[0015] On the other hand, the capture reaction involves first hybridization and then ligation.

[0016] In another aspect, the barcoded solid support has transposable DNA or transposomes pre-attached to the ends of some of the barcode templates immobilized on the solid support.

[0017] In another aspect, the transposase is selected from the group consisting of wild-type or mutant or tagged Tn, Mu, Ty and Tc transposases, and combinations thereof. In particular, the transposase is wild-type or mutant or tagged MuA transposase, or Tn5 transposase, or a combination thereof.

[0018] In another aspect, the transposable DNA contains a transposon, wherein the transposon is selected from wild-type or mutant Tn, Mu, Ty and Tc transposon DNA and combinations thereof. In particular, the transposon is a wild-type or mutant MuA transposon, or a Tn5 transposon, or a combination thereof.

[0019] In another aspect, the transferable DNA further comprises an adaptor sequence.

[0020] On the other hand, the transposable DNA capable of being captured by the barcode template does not have a complementary sequence to the barcode template, and the capture of the transposable DNA onto the barcode template is facilitated by a linker.

[0021] In another aspect, the transposome comprises at least one type of transposase, at least one type of transposable DNA, or a combination thereof.

[0022] In one aspect, a method for tracking the source of nucleic acid fragments by barcode tags is described herein. The method includes providing a ligase, a plurality of solid carriers on which a cloned barcode template or a semi-cloned barcode template is fixed, and a nucleic acid target is captured on the solid carrier by non-specific binding, and a plurality of transposons are provided, each transposon comprising a transposable DNA and a transposase, wherein at least one transposable DNA in the transposon can be directly or indirectly connected to the barcode template on the solid carrier. The non-specifically bound nucleic acid target on the solid carrier is contacted with a ligase and a transposon in a reaction vessel to attach the barcode information on the solid carrier to the nucleic acid target by simultaneous chain transfer and ligation reactions. The nucleic acid target is broken into fragments by a broken chain transfer complex, wherein at least one fragment is attached to the barcode template on the solid carrier.

[0023] In one aspect, the present invention describes a method for tracking the source of nucleic acid fragments by barcode tags. The method includes providing a plurality of solid carriers on which a clonal barcode template or a semi-clonal barcode template is fixed, capturing a nucleic acid target on the solid carrier by non-specific binding, and providing a plurality of transpososomes, each of which includes a transposable DNA and a transposase, wherein at least one transposable DNA in the transpososome can be directly or indirectly captured specifically on the barcode template on the solid carrier. The non-specifically bound nucleic acid target on the solid carrier is contacted with the transpososome in a reaction vessel to attach the barcode information on the solid carrier to the nucleic acid target by simultaneous chain transfer and capture reactions. The nucleic acid target is broken into fragments by a broken chain transfer complex, wherein at least one fragment is attached to the barcode template on the solid carrier.

[0024] On the one hand, the present invention describes a method for tracking the source of nucleic acid fragments by barcode tags. The method includes providing a plurality of solid carriers on which a clonal barcode template or a semi-clonal barcode template is fixed, and providing a plurality of transposons, each of which includes a transposable DNA and a transposase, wherein at least one transposable DNA in the transposons can be directly or indirectly connected to the barcode template on the solid carrier. The nucleic acid target contacts the transposons to form a stable chain transfer complex. The chain transfer complex is captured on the solid carrier by non-specific binding. The barcode information on the solid carrier is attached to the nucleic acid target by connection. The nucleic acid target is broken into fragments by breaking the chain transfer complex, wherein at least one fragment is attached to the barcode template on the solid carrier. In some embodiments, the captured nucleic acid target with the chain transfer complex on the solid carrier is first broken into fragments by breaking the chain transfer complex. The nucleic acid fragment is retained on the solid carrier by non-specific binding. The barcode information on the solid carrier is then attached to the nucleic acid fragment by connection, wherein at least one fragment is attached to the barcode template on the solid carrier.

[0025] On the one hand, the present invention describes a method for tracking the source of nucleic acid fragments by barcode tags. The method includes providing a plurality of solid carriers on which a clonal barcode template or a semi-clonal barcode template is fixed, and providing a plurality of transposons, each of which includes a transposable DNA and a transposase, wherein at least one transposable DNA in the transposons can be directly or indirectly connected to the barcode template on the solid carrier. The nucleic acid target is captured on the solid carrier by non-specific binding. The transposons contact the non-specifically bound nucleic acid target to form a stable chain transfer complex on the solid carrier. The barcode information on the solid carrier is attached to the nucleic acid target by connection. The nucleic acid target is broken into fragments by breaking the chain transfer complex, wherein at least one fragment is attached to the barcode template on the solid carrier. In some embodiments, the captured nucleic acid target with the chain transfer complex on the solid carrier is first broken into fragments by breaking the chain transfer complex. The nucleic acid fragment is retained on the solid carrier by non-specific binding. The barcode information on the solid carrier is then attached to the nucleic acid fragment by connection, wherein at least one fragment is attached to the barcode template on the solid carrier.

[0026] In one aspect, a method for determining the connection information of a nucleic acid target is described herein. The method comprises: generating barcode-tagged fragments of a nucleic acid target according to any of the methods described in the present invention; determining the sequences of the nucleic acid fragments and the barcodes; and determining the connection information of the nucleic acid target based on the barcode sequence when at least two fragments from the same nucleic acid target receive the same barcode information.

[0027] In one aspect, methods are described herein for generating soluble libraries of barcoded fragments of nucleic acid targets. In some embodiments, the soluble library includes sequence information for the entire genome. In some embodiments, the soluble library includes sequence information for the target region. In some embodiments, the soluble library is used for sequencing to determine the phasing information of the nucleic acid target. In some embodiments, the soluble library is used for sequencing to determine the identity of the replicate reads.

[0028] In one aspect, described herein is a method for sequencing error correction for barcode designs having degenerate bases at some or all positions without prior knowledge of the specific barcode sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A method for generating clonal barcoded nucleic acid fragments using simultaneous strand transfer and ligation reactions to a barcoded solid support in an open monolithic reaction without the need for fragmentation of the nucleic acid target is shown.

[0030] Figure 2Different designs of transposable DNA are shown, (A) a transposon-complementary strand with a 3' overhang, (B) a transposon-complementary strand with a separate complementary adapter oligonucleotide, (C) a transposon-binding strand with a 5' overhang, and (D) a transposon with a blunt end at the non-binding end.

[0031] Figure 3 Examples of different free linker designs are shown: (A) single-strand linker, (B) double-strand linker, (C) partial double-strand linker.

[0032] Figure 4 A method for generating clonal barcoded nucleic acid fragments using different transposomes simultaneously in an open monolithic reaction using simultaneous strand transfer and ligation reactions onto a barcoded solid support without the need for fragmentation of the nucleic acid target is shown.

[0033] Figure 5 The use of Exonuclease I to remove single-stranded polynucleotides from a solid support is shown.

[0034] Figure 6 A method for generating clonal barcoded nucleic acid fragments using different transposomes simultaneously in an open monolithic reaction using simultaneous strand transfer and ligation reactions onto a barcoded solid support without the need for fragmentation of the nucleic acid target is shown.

[0035] Figure 7 A method for generating clonal barcoded nucleic acid fragments using different transposons simultaneously in an open monolithic reaction using simultaneous strand transfer and ligation reactions to a barcoded solid support without the need to fragment the nucleic acid target is shown. The sequence of the operation flow is different from Figure 6 shown.

[0036] Figure 8 A method with an alternative workflow to generate clonal barcoded nucleic acid fragments using simultaneous strand transfer and ligation reactions onto a barcoded solid support is shown.

[0037] Fig. 9 A method with an alternative workflow to generate clonal barcoded nucleic acid fragments using simultaneous strand transfer and ligation reactions onto a barcoded solid support is shown.

[0038] Fig.10 A method for generating clonal barcoded nucleic acid fragments utilizing non-specific binding and attachment to a barcoded solid support is shown.

[0039] Fig.11 A method with an alternative workflow for generating clonal barcoded nucleic acid fragments utilizing non-specific binding and attachment to a barcoded solid support is shown.

[0040] Fig.12 A method for introducing adaptors onto immobilized barcoded fragments using transposomes is shown.

[0041] Fig.13 A method for introducing adapters onto immobilized barcoded fragments using fragmentation and ligation reactions is shown.

[0042] Fig.14 Methods for releasing one or more copies of immobilized barcoded fragments by primer extension and / or PCR amplification are shown.

[0043] Fig.15 is an example of an Illumina sequencing library generated from barcoded fragments.

[0044] Fig.16 Shown are electropherograms of a barcoded Illumina sequencing library run on a TapeStation (A) and sequenced read count histograms for reads with the same barcode (B) based on read distance to the next alignment.

[0045] Fig.17 Shown is an electropherogram of another barcoded Illumina sequencing library run on TapeStation (A) and sequenced read count histograms for reads with the same barcode (B) based on read distance to the next alignment.

[0046] Fig.18 A method for generating clonal barcoded nucleic acid fragments using simultaneous strand transfer and hybridization reactions to a barcoded solid support in an open monolithic reaction without the need for fragmentation of the nucleic acid target is shown.

[0047] Fig.19 Three different transposase-based methods to generate clonal barcoded fragments for sequencing library construction are shown.

[0048] Fig. 202% agarose gel EX images of three amplified sequencing libraries using three different transposase-based methods (M1, method 1; M2, method 2; M3, method 3) are shown. The fragment sizes of the 100 bp DNA ladder from top to bottom are 3000 bp, 2000 bp, 1500 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp and 100 bp, respectively.

[0049] The transposases in all figures are shown as tetramers in transposomes based on the MuA transposition system. However, other transposases may also be used. DETAILED DESCRIPTION

[0050] As used in the present invention and the claims attached thereto, a barcode template and a solid carrier having a cloned barcode template or a semi-cloned barcode template fixed thereon, i.e., a barcode solid carrier, are described in patent application WO2017 / 151828, which is incorporated herein by reference in its entirety. In some embodiments, all solid carriers are attached with a barcode template. In some embodiments, only a portion of the solid carriers have an attached barcode template. The proportion of solid carriers with barcodes can be between 1% and 99%. When the solid carrier is physically separable, such as a bead or microparticle, a barcode solid carrier can be prepared by a clonal amplification method, whether or not the amplified solid carrier is enriched from an unamplified solid carrier.

[0051] The term "adapter" as used herein is a nucleic acid sequence that may include a primer binding sequence, a barcode, a linker sequence, a sequence complementary to a linker sequence, a capture sequence, a sequence complementary to a capture sequence, a restriction site, an affinity group, a unique molecular identifier, and combinations thereof.

[0052] The term "transposase" as used herein refers to a component of a functional nucleic acid protein complex that is capable of transposition and mediates transposition, including but not limited to Tn, Mu, Ty and Tc transposases. The term "transposase" also refers to an integrase of retrotransposon or retroviral origin. It also refers to wild-type proteins, mutant proteins, and fusion proteins with tags (such as GST tags, His-tags, etc.), and combinations thereof.

[0053] The term "transposon" as used herein refers to a nucleic acid fragment recognized by a transposase or an integrase, which is a basic component of a functional nucleic acid-protein complex capable of transposition. Together with the transposase, it forms a transposome and performs a transposition reaction. It includes both wild-type transposons and mutant transposons.

[0054] As used herein, "transposable DNA" refers to a nucleic acid fragment comprising at least one transposon unit. It may also include affinity groups, non-natural nucleotides and other modifications. In addition to the transposon sequence, the transposable DNA sequence may also include an adapter sequence.

[0055] As used herein, the term "transposome" refers to a stable nucleic acid and protein complex formed by non-covalent binding of a transposase to a transposon. It may include multimeric units having identical or different monomeric units.

[0056] As used herein, "transposition reaction" refers to a reaction in which a transposon is inserted into a target nucleic acid. The main components of a transposition reaction are a transposon, a transposase or an integrase, and a target nucleic acid.

[0057] As used herein, a "strand transfer reaction" refers to a reaction between a nucleic acid and a transposome in which a stable strand transfer complex is formed.

[0058] The term "strand transfer complex (STC)" as used herein refers to a nucleic acid-protein complex of a transposon and its target nucleic acid, into which the transposon is inserted, wherein the 3' end of the transposon binding strand is covalently linked to both strands of its target nucleic acid. It is a very stable form of nucleic acid and protein complex, with extremely high heat and high salt resistance in vitro (Burton and Baker, 2003).

[0059] As used herein, "transposase binding region" refers to a nucleotide sequence within the transposon end sequence to which the transposase specifically binds when mediating transposition. The transposase binding region may include more than one site for binding to a transposase subunit.

[0060] As used herein, "transposon-bound strand" refers to the double-stranded transposon DNA strand that is attached to the target nucleic acid at the insertion site by the transposase.

[0061] As used herein, "transposon complementary strand" refers to the complementary strand of the transposon binding strand in a double-stranded transposon DNA.

[0062] As used herein, "solid support" is selected from the group consisting of beads, microparticles, wells, tubes, slides, plates, flow cells, and combinations thereof, wherein when the solid support is physically separable, such as beads or microparticles, the barcode template is clonally or semi-clonally immobilized on the entire surface, and when the solid support is a continuous flat surface (e.g., wells, tubes, slides, plates, flow cells), the barcode template is immobilized on the surface as separable clonal clusters or semi-clonal clusters.

[0063] The "ligase" used herein is selected from wild-type, mutant or tagged DNA ligase, or RNA ligase, and combinations thereof; the ligase is used in a ligation reaction.

[0064] As used herein, "capture reaction" refers to specific capture, which is performed by ligation, hybridization, affinity binding with affinity groups (eg, biotin and streptavidin, antibodies and antigens), click chemistry, or a combination thereof.

[0065] As used herein, "reaction vessel" refers to a substance having a continuous open space to hold a liquid; the reaction vessel is selected from a test tube, a well, a plate, a well in a multi-well plate, a slide, a spot on a slide, a droplet, a tube, a channel, a bottle, a chamber, and a flow cell.

[0066] The methods and materials of the present invention are exemplified by using in vitro MuA transposition (Haapa et al., 1999 and Savilahti et al., 1995). Other transposition systems or combinations of these different transposition systems may be used, such as Ty1 (Devine and Boeke, 1994), Tn7 (Craig, 1996), Tn10 and IS10 (Kleckner et al., 1996), Mariner transposase (Lampe et al., 1996), Tc1 (Vos et al., 1996), Tn5 (Park et al., 1992), P elements (Kaufman and Rio, 1992) and Tn3 (Ichikawa and Ohtsubo, 1990), bacterial insertion sequences (Ohtsubo and Sekine, 1996), retroviruses (Varmus and Brown, 1989) and yeast retrotransposons (Boeke, 1989).

[0067] The present invention generally relates to methods and compositions for nucleic acid sequencing. In particular, the present invention provides methods and compositions related to the preparation of nucleic acid libraries and the sequencing data generated thereby.

[0068] On the one hand, the methods and compositions relate to haplotype phasing of target nucleic acids. In some embodiments, the nucleic acid target is DNA. In some embodiments, the nucleic acid target is genomic DNA. In some embodiments, the nucleic acid target is amplified DNA. In some embodiments, the DNA is modified DNA. The modifications include non-natural nucleotides, affinity groups, chemical treatments (such as bisulfite treatment or formalin fixation paraffin embedding), and protein attachments (such as histones, transcription factors). In some embodiments, the nucleic acid target is synthetic DNA. In some embodiments, the nucleic acid target is RNA. In some embodiments, the nucleic acid target is mRNA. In some embodiments, the nucleic acid target is complementary DNA (cDNA). In some embodiments, the target nucleic acid is from a single cell. In some embodiments, the target nucleic acid is cell-free DNA. The length of the nucleic acid target can vary greatly. It ranges from about 50bp to 1Mb, or even more. The longer the length of the nucleic acid target, the better the effect of the phasing application. In one reaction, the number of nucleic acid targets can be from 1 billion to billions, or even more. In some embodiments, the reaction vessel is a tube, a well, a plate, a well in a multiwell plate, a slide, a spot on a slide, a droplet, a tube, a channel, a bottle, a chamber, or a flow cell. The reaction occurs in an integral form without segmenting each nucleic acid target from another nucleic acid target in the total plurality of nucleic acid targets. Examples of the segmentation include emulsions, wells, droplets, diluents, etc. The present invention greatly simplifies the operating process and is easy to scale and automate without the need for segmentation.

[0069] Strand transfer reaction to nucleic acid target and specific capture of barcoded template

[0070] The present invention provides methods and compositions for simultaneously capturing nucleic acid targets onto solid supports of clonal barcodes by utilizing the strand transfer reaction of a transposition system and a specific capture reaction (e.g., using ligase and / or hybridization). The captured nucleic acid targets can be fragmented by a fragmentation strand transfer complex, which generates small fragments ( Figure 1 ).

[0071] In one embodiment, the transposable DNA may comprise only one transposon sequence. Thus, the transposon sequence in the transposable DNA is not linked to another transposon sequence via a nucleotide sequence, i.e., the transposable DNA comprises only one transposase binding region ( Figure 2 ). In addition, the 5' end of the binding strand of the transposable DNA has a phosphate and can be connected to the 3' end of any DNA strand with an -OH group through single-stranded end-to-end ligation, double-stranded end-to-end ligation, or through a linker molecule. Figure 2Some examples of connectable transposable DNAs are shown. In the presence of a transposase on the transposable DNA, the 5' end of the transposon binding strand can be connected to the polynucleotide on the solid support. In some cases, the 3' end of the transposon complementary strand and the 5' end of the transposable DNA binding strand have different lengths. In some cases, the 3' end of the transposon complementary strand and the 5' end of the transposable DNA binding strand have the same length. In some cases, the 3' end of the transposon complementary strand is modified, for example, by dideoxynucleotides, C3-spacers (spacer), phosphate groups, thiophosphorothioate groups, azido groups or amino linkers, to block self-connection. In some cases, the 3' end of the transposon complementary strand can have a single nucleotide protrusion or a single nucleotide recess or mismatched nucleotides to block self-connection. In some cases, the single nucleotide protrusion on the double-stranded barcode template and the single nucleotide protrusion of the double-stranded transposon are complementary, for promoting connection. In some cases, more than one nucleotide on the double-stranded barcode template and the double-stranded transposon is prominent, which can be used to promote connection. In some cases, the barcode template on the solid support is double-stranded. In some cases, the barcode template on the solid support is single-stranded. In some cases, the barcode template on the solid support is partially single-stranded and partially double-stranded. In some cases, some sequence variation in the transposon sequence is used as an additional sample identifier. In some cases, the transposable DNA includes an adapter. In some cases, sequence variation in the adapter is used as an additional sample identifier. Samples reacted with different transposons and / or transposable DNA sequences can be mixed together after the reaction to simplify downstream processes if necessary.

[0072] In one embodiment, there is no complementary capture sequence between the barcode template on the solid support and the transposable DNA being captured.Adaptor-based capture methods can be used to facilitate the capture reaction. Figure 3 Some examples of connection and / or capture based on joints are shown. In one embodiment, the joint molecule is single-stranded. In one embodiment, the joint molecule is double-stranded. In one embodiment, the joint molecule is partially double-stranded. In some cases, the joint oligonucleotide may be combined with the transposable DNA in advance. In some cases, the joint oligonucleotide can be combined with the immobilized polynucleotide on the solid support in advance. In some cases, only when the capture reaction just connects the 5' end of the binding chain of the transposable DNA chain to the 3' end of the immobilized polynucleotide on the solid support, the joint oligonucleotide can be added. The free joint method often generates less PCR byproducts than the pre-joint joint method. The length of the joint molecule varies, such as 5b (p), 10b (p), 20b (p), 30b (p), 40b (p), 50b (p), 100b (p), 200b (p) or more.

[0073] A method for cloning, fragmenting and barcoding a nucleic acid target is described as follows ( Figure 1 ). In a reaction vessel, a double-stranded nucleic acid target, an assembled transposome, a ligase, and a clonal or semi-clonal barcode solid carrier are mixed together without being separated by an emulsion or diluent. The transposable DNA in the transposome can be connected to the barcode template on the solid carrier. The chain transfer reaction between the transposome and the nucleic acid target and the ligation reaction between the transposable DNA and the barcode template occur simultaneously in the same solution. The stable STC formed during the chain transfer reaction will keep the nucleic acid target together. Through the ligatable transposable DNA in the STC, the barcode sequence will be clonally attached to the nucleic acid target (together with the STC). After the reaction, the STC is broken with an SDS solution to break the nucleic acid target into small fragments. Many small fragments contain barcode sequences, and fragments from the same nucleic acid target will have the same barcode. The key to the efficiency of this clonal barcode labeling method is the simultaneous chain transfer reaction and barcode ligation reaction. The yield of barcode-tagged fragments generated by the present invention is much higher than the method in patent application WO2017 / 151828, in which the nucleic acid target first forms an STC with the transposome in solution, and then the transposable DNA in the STC is connected to the barcode template on the solid support. The reaction efficiency of the present invention is also much higher than the method in U.S. Patent 9,328,382B2 and the single-tube article (Zhang et al., 2017), in which the transposome is first fixed on the barcode bead, and then the free nucleic acid target in the solution is captured only by the chain transfer reaction. In the present invention, the ligation reaction and chain transfer reaction used are both used to capture nucleic acid targets. The explanation for the low yield of the method using ligation only for capture is that a nucleic acid target filled with STC may produce steric hindrance, which limits the efficiency of ligation. The explanation for the low efficiency of the method using chain transfer only for capture is that the transposable DNA is fixed on the surface of the beads with a fixed spatial arrangement and position, which may inhibit the formation of the transposome and / or the efficiency of the chain transfer reaction with the free nucleic acid target. In order to fully utilize the simultaneous chain transfer and ligation reactions, the reaction conditions including buffer composition, pH and temperature were optimized.

[0074] Multiple nucleic acid targets can be used in one reaction vessel. The reaction occurs in an integral form without segmenting each nucleic acid target from another nucleic acid target in the total multiple nucleic acid targets. The present invention greatly simplifies the operation process and is easy to scale and automate without segmentation. Multiple nucleic acid targets are uniformly dissolved in a solution so that they are uniformly captured on a solid support after the reaction. In some embodiments, limiting the diffusion rate in the reaction solution can be used to promote capture uniformity on a solid support. The solid support can be a continuous surface, such as in a hole, tube, slide, plate or flow cell with a full clone or semi-clone fixed barcode template cluster. It can also be physically separated into single beads or microparticles. Beads and microparticles can have a size ranging from 50nm to 100μm, preferably 1μm to 15μm. Each bead or microparticle has multiple barcode templates with unique sequences. The main advantage of the present invention is that the labeling of target-specific barcodes can occur in an open overall reaction without using holes, micropores, spots, nanochannels, droplets, emulsion droplets, capsules or diluents to segment nucleic acid targets. For better results, the size of the beads or microparticles should be controlled between 50nm and 100μm (diameter), preferably between 1μm and 15μm, although it can be less than 50nm or greater than 100μm. In order to make the reaction uniform, the beads or microparticles need to remain suspended during the reaction, and the viscosity of the solution is controlled by using polyethylene glycol, pluronic, cellulose, agarose, or their derivatives, or other polymers or combinations thereof, with a viscosity of from 1 to 200mPa·s at 20°C, most preferably 1.5-30mPa·s at 20°C. For barcode clusters on solid surfaces (such as the surface of a flow cell), the size of the clusters should be controlled between 50nm and 200μm (diameter), preferably between 100nm and 10μm. The larger the separation distance of the clusters, the smaller the chance that a nucleic acid target molecule is tagged by two or more barcodes.

[0075] Due to the very stable nature of the STC structure (Surette et al. 1987, Mizuuchi et al. 1992, Savilahti et al. 1995, Burton and Baker 2003, Au et al. 2004, Amini et al. 2014) and the clonal barcode template on the solid support, the barcode-tagged fragments generated by the present invention can maintain the identity of their source nucleic acid target in the barcode sequence. Fragments from the same nucleic acid target share the same barcode sequence. It is well known that such barcode-tagged fragments can be used for haplotype phasing, de novo assembly and other applications (Zheng et al., 2016, Zhang et al., 2017).

[0076] In one aspect, the nucleic acid target can first be non-specifically bound to a barcoded solid support, which is then mixed with a transposome and a ligase to covalently attach the barcode information to the nucleic acid target through a simultaneous strand transfer reaction and ligation reaction.

[0077] In some embodiments, the transpososome is not preassembled prior to the reaction. The transposase and transposable DNA are used directly in the reaction with the nucleic acid target, ligase, and solid support. In some embodiments, the transposable DNA can be directly connected to the barcode template ( Figure 2 In some embodiments, linker units can be added in simultaneous chain transfer and ligation reactions to facilitate ligation ( Figure 3 In some embodiments, all transposable DNA sequences are identical. In some embodiments, the transposable DNA sequences are different. Figure 4 In some embodiments, only one transposable DNA in a transposome can be linked to a barcode template ( Figure 4 ). In some embodiments, all transposable DNAs in the transposome can be connected to a barcode template. In some embodiments, the monomer unit sequence of all transposable DNAs in the same transposome is the same. In some embodiments, the monomer unit sequence of the transposable DNA in the same transposome is different. In some embodiments, different transposases are used in the reaction. Different methods can be used to break the chain transfer complex, such as protease treatment, high temperature treatment or protein denaturing agents (such as SDS solution, guanidine hydrochloride, urea, etc.) or a combination thereof. In some embodiments, after barcode labeling, single-stranded nuclease can be used to remove unwanted single-stranded polynucleotides on the solid support ( Figure 5 ).

[0078] In one aspect, the transposomes can be used sequentially in the reaction. In some embodiments, the transposomes are identical. In other embodiments, the transposomes are different. In some embodiments ( Figure 6), the first transposome is mixed with a nucleic acid target, a ligase, and a barcoded solid support to generate an immobilized nucleic acid STC complex I on the solid support. A second transposome is then added to attack the immobilized STC I and form STCⅡ. In some embodiments, the second transposome may have different types of transposons and transposases. In some embodiments, the transposable DNA in the second transposome may have a different transposon sequence from the same type of transposon in the first transposome. In some embodiments, the second transposome may have a different transposable DNA sequence, but has the same transposon sequence as the first transposome. In some embodiments, a capture reaction, such as ligation and / or hybridization, occurs again simultaneously with a second chain transfer reaction. In some embodiments, the reaction buffer is optimized for the first and second simultaneous chain transfer reactions with the transposome and a capture reaction using hybridization and / or ligation. Between different steps, there is no need to change the buffer of any reaction, which can significantly simplify the operational process. After breaking the STC, a target-specific barcode can be attached to the fragment. In some embodiments, the second transposome can be added only after the first STC is destroyed ( Figure 7 ). The first and second transposomes can be of the same type or of different types. This method has better chain transfer efficiency because the steric effect from the first STC is eliminated before the second chain transfer reaction. In some embodiments, the second chain transfer reaction is used to generate a shorter fragment size. In some embodiments, the second transposome is used to introduce an adapter or primer sequence different from the first transposome to facilitate downstream amplification and sequencing.

[0079] In one aspect, a nucleic acid target is reacted with a first transposome to form a stable STC I. The nucleic acid with STC I is then reacted with a second transposome, a ligase, and a cloned barcoded solid support to generate target-specific barcoded fragments ( Figure 8 ).

[0080] In one aspect, the transposome can first be attached to a barcoded solid support. To generate target-specific barcoded fragments, such as Fig. 9 As shown, the nucleic acid target, the transposome in solution, the ligase, and the barcoded solid support to which the transposome is attached are then mixed together in a reaction vessel. In some embodiments, the transposome in solution is the same as the transposome pre-attached to the solid support. In some embodiments, the transposome in solution is different from the transposome pre-attached to the solid support.

[0081] On the one hand, first the transposable DNA can be attached to a barcode solid carrier. In order to generate target-specific barcode labeling fragments, the nucleic acid target, the transposable body in solution, the ligase and the barcode solid carrier attached to the transposable DNA are then mixed together in a reaction vessel. In some embodiments, the transposable body in solution has the same transposon as the transposable DNA pre-attached on the solid carrier. In some embodiments, the transposable body in solution has different transposons from the transposable DNA pre-attached on the solid carrier. In some embodiments, the transposable body in solution is replaced with a separate transposable DNA and a transposase.

[0082] on the one hand, Figure 1 , 4 The simultaneous chain transfer reaction and ligation reaction described in 6, 7, 8 and 9 can be carried out by Fig.18 Ligase can be added at a later stage in the protocol, either before STC fragmentation or after STC fragmentation, to covalently link the hybridized transposable DNA to the barcode template on the solid support.

[0083] on the one hand, Fig.18 The hybridization reaction in the simultaneous strand transfer reaction and hybridization reaction described in can be replaced by other capture reactions, such as affinity tags (eg, biotin and streptavidin), antigen and antibody, click chemistry, or a combination thereof.

[0084] Cloning and capturing nucleic acid targets by nonspecific binding to solid supports for barcoding

[0085] The present invention provides methods and compositions for capturing nucleic acid targets by non-specific binding to solid supports of cloned barcodes. The captured nucleic acid targets can be covalently attached to barcode templates on the solid supports, and small fragments can be generated from nucleic acid targets attached with target-specific barcodes.

[0086] In one aspect, the nucleic acid target reacts with the transposome and forms a strand transfer complex. Using a cloned or semi-cloned barcode template immobilized on a surface, the nucleic acid target with STC non-specifically binds to a solid support ( Fig.10 ). In some embodiments, the nucleic acid target can first be non-specifically bound to the barcoded solid support. Then, the bound nucleic acid reacts with the transposome in the solution to form an STC on the solid support. On the one hand, by SDS treatment, the STC is broken and the nucleic acid target is broken into small fragments. Under this condition, the small fragments are still attached to the solid support by non-specific binding ( Fig.10 ). A ligase is added to covalently attach the small fragment to the barcode template on the solid support, generating a small fragment with a target-specific barcode sequence attached ( Fig.10On the other hand, the nucleic acid target with STC that is non-specifically bound to the solid support is first connected to the barcode template on the solid support through the ligatable transposable DNA in the STC. Then, the STC is broken by SDS treatment to generate small fragments with target-specific barcode sequences attached ( Fig.11 ).

[0087] In one aspect, the nucleic acid target can first be non-specifically bound to a barcoded solid support. The bound nucleic acid then reacts with a transposome and a ligase in solution to form an STC and simultaneously ligates the transposable DNA to the barcoded template on the solid support.

[0088] Many conditions can cause nucleic acids and nucleic acid and protein complexes to bind non-specifically to solid supports. Most notably, salt-containing polyethylene glycols (Lis and Scheif, 1975), polyamines and cobalamines (Pelta et al., 1996), and alcohols (Crouse and Amorese, 1987) are widely used to precipitate and / or concentrate nucleic acids.

[0089] In one aspect, the ligation reaction described in the present invention can be replaced by other capture reactions, such as hybridization, affinity tags (eg, biotin and streptavidin), antigen and antibody, click chemistry, or a combination thereof.

[0090] Release cloned barcoded nucleic acid fragments to generate sequencing libraries

[0091] The barcoded fragments are immobilized on a solid support. They can be used to make sequencing libraries. In some embodiments, they can be further manipulated for other applications, such as methylation studies via bisulfite treatment. In some embodiments, additional sequencing adapters can be attached to the barcoded fragments using a transposase-based tagging method ( Fig.12 In some embodiments, the barcoded fragments can be further fragmented using physical shearing methods and / or enzymatic fragmentation methods, which can then be ligated to other sequencing adapters ( Fig.13 ). The immobilized barcoded fragments can be released from the solid support in a variety of ways. In one embodiment, the oligonucleotide sequence attached to the solid support can include a cleavable linker or rare restriction site. The barcoded fragments can be released from the solid support using a cleavage reaction or restriction enzyme digestion. In some cases, primer extension can be performed to make one or more copies of the barcoded fragments ( Fig.14 A). In some embodiments, the primer is a random primer. In some embodiments, the primer is a target-specific primer ( Fig.14A, 14C). The target of the specific primer can be an exon, intron, gene, exome, etc. Patent application WO 2017 / 151828 describes a more detailed application of targeted sequencing. Further PCR amplification is performed using primers specific to the sequencing platform, such as the P5 and P7 primers of the SBS library of Illumina ( Fig.15 ) or P1 and A primers of lonTorrent libraries may generate libraries that are easy to sequence for specific sequencing platforms. When preparing libraries by releasing barcode-tagged fragments from solid supports, primers with sample-specific indexes can be used. In some cases, the sequence in the barcode template can be used as a sample-specific index. The released barcode-tagged fragments with sample-specific indexes can be mixed with tagged fragments of other samples with their own sample-specific indexes for further downstream operation processes to increase sample preparation and simplify the process. When at least two fragments from the same nucleic acid target receive the same barcode information, the constructed library can be sequenced to determine the sequence of the barcode and the nucleic acid fragment, and determine the connection information of the nucleic acid target based on the barcode sequence. The connection information can be used for haplotype phasing, structural variation detection, CNV detection, etc. The barcode information can also be used to distinguish the source of duplicate reads from amplification or sequencing.

[0092] Assembly of barcoded sequencing reads into long reads

[0093] The present invention provides methods and compositions for cloning barcode label nucleic acid samples in an open overall reaction, without having a complex separation or segmentation scheme like other methods. The barcode labeling fragments can come from the entire genome sample, or a part of the genome, or a specific target region, or a metagenome sample. The sequencing reads generated from these barcode labeling fragments contain barcode information that can be used to identify the original targets of these fragments, and these short sequencing reads with the same barcode can be combined together and clustered along the original nucleic acid target. Depending on which transposase system is used, in these reads with the same barcode, the starting ends of two original adjacent reads from the same nucleic acid target will share some reverse complementary sequence bases (5 bases for the MuA transposase system and 9 bases for the Tn5 transposase system). These overlapping sequences can further connect the barcode reads together. In principle, when all labeling fragments are captured and sequenced by barcode solid carriers, it can completely reconstruct the original nucleic acid target. They provide useful long-range connection information and can be used for haplotype phasing. The longer the original nucleic acid target is, the longer the connection information will be, and the more useful it is for phasing applications. These barcode reads can be used to develop analysis pipelines for complete genome assembly or structural variation analysis to perform de novo sequencing and resequencing. In one case, all sequencing reads can be used for standard shotgun assembly analysis to first establish many initial overlapping groups. The initial overlapping groups can then be phased into longer overlapping groups using barcode information. These barcode labeling methods can also be used for phasing a gene, multiple genes, or exon groups of the target. These barcode labeling methods can also be used as tools to distinguish repeated reads in target sequencing applications. This method improves the detection limit of sequencing assays on mixed samples, such as somatic mutation detection in cancer biopsy samples or circulating tumor cells / DNA.

[0094] Sequencing error correction method with barcode design having random degenerate bases

[0095] Any sequencing technology will produce sequencing errors. Most NGS sequencing technologies have a raw read sequencing error rate of 0.1%-1%. For barcode designs using random degenerate bases, especially for barcodes that use all four possible degenerate bases A, C, G, or T for a given position, it is difficult to distinguish sequencing errors from true base variations by position design.

[0096] The present invention provides a method that can detect and correct sequencing errors at degenerate bases without the need for a sequence reference at that position. Preferably, the total sequence diversity of the barcode set is much greater than the actual number of unique barcode sequences used in a given experiment. The sequencing read count should be at least twice the number of unique barcodes used in the experiment. The deeper the sequencing depth, the better the correction function.

[0097] For example, a random 10-mer barcode design with four degenerate bases A, C, G, and T at all positions NNNNNNNNNN would have a maximum of 4 10 =1,048,576 different unique barcode sequences. If 1,000 unique barcode sequences are randomly selected from the barcode library and used for sequencing library construction, sequencing generates 20,000 barcode reads. Among the 1,000 unique barcode sequences, most should have more than 1 read and each barcode has an average of 20 reads, and if there are no sequencing errors, a total of 1,000 unique barcode sequences should be detected. However, due to sequencing errors, the actual number of unique barcodes identified after sequencing will exceed 1,000. Because most sequencing errors are randomly generated, a barcode sequence containing one or more sequencing errors is likely to become a new unique barcode sequence outside of the original 1000 barcode sequences. And, a barcode with a sequencing error is also likely to be associated with only one sequencing read. In addition, because the chance of generating multiple sequencing errors in a 10-mer sequence is very low, the sequence of a barcode with a sequencing error is likely to have a difference of 1 or 2 bases from its original correct barcode sequence. Such erroneous barcodes can be identified by comparing the sequence homology of unique barcodes with only one sequencing read to those unique barcodes with multiple sequencing reads. One would expect to find barcodes with 1 base mismatch or 2 base mismatch among those unique barcodes with multiple sequencing reads, which are likely to be the actual correct barcode sequence for unique barcodes with only one sequencing read if this is due to a sequencing error in sequencing.

[0098] The degenerate bases in the barcode can be all 4 bases from A, C, G and T (U), or any 3 bases, or any 2 bases. In the barcode design, some positions can be degenerate bases. Other features can be used in the barcode design to limit the length of homopolymers. The correction method described in the present invention can still be used for the design of these barcodes.

[0099] This barcode design with degenerate bases can be used not only for sequencing applications of sequencing reads, but can also be associated with other information and / or characteristics, such as color, index, ID, cluster, position, container, or separation information. In some embodiments, it can be used for DNA or RNA. In some embodiments, it can be used for proteins, antibodies, and chemicals, etc.

[0100] Although the present invention has been described by way of embodiments, it should be understood that numerous other possible modifications and variations may be made without departing from the spirit and scope of the invention as described herein.

[0101] Furthermore, generally speaking, with respect to the processes, systems, methods, etc. described herein, it should be understood that although the steps of these processes, etc. have been described as occurring according to a certain ordered sequence, these processes may be practiced by performing the described steps sequentially in an order different from that described herein. It should also be understood that certain steps may be performed simultaneously, other steps may be added, or certain steps described in the present invention may be omitted. In other words, the description of the processes herein is provided for the purpose of illustrating certain embodiments and should in no way be construed as limiting the claimed invention.

[0102] Moreover, it should be understood that the above description is intended to be illustrative rather than restrictive. After reading the above description, many embodiments and applications other than the examples provided will be apparent to those skilled in the art. The scope of the present invention should not be determined with reference to the above description, but should be determined with reference to the full scope of equivalents given by the attached claims and such claims. It can be expected and predicted that future developments in the art discussed herein, and the disclosed systems and methods will be incorporated into such future embodiments. In short, it should be understood that the present invention can be modified and varied, which is limited only by the attached claims.

[0103] Finally, all defined terms used in this application are intended to be given their broadest reasonable construction consistent with the definitions provided herein. Unless expressly indicated to the contrary herein, all undefined terms used in the claims are intended to be given their broadest reasonable construction consistent with their ordinary meaning as understood by those skilled in the art. In particular, unless an express limitation to the contrary is recited in a claim, singular articles such as "a," "an," "the," and "said" are understood to recite one or more of the indicated elements.

[0104] Example 1

[0105] This example describes a method for target-specific barcoding of genomic DNA by simultaneous strand transfer and ligation to barcoded beads in an open monolithic reaction without genomic DNA fragmentation ( Figure 4). Clonal barcode beads were prepared using the method described in patent application WO 2017 / 151828. The length of each barcode sequence is 18 bases. All beads were collected directly after the BEAMing reaction, including beads without clonally amplified barcode templates (Diehl et al., 2005). Two MuA transposons were pre-assembled with two different MuA transposable DNAs, respectively. The MuA transposable DNA in one MuA transposon has a 5' end transposon ligation chain that can be connected and can be hybridized and / or connected to the barcode template on the barcode beads. The double-stranded barcode template on the beads was denatured to a single strand. 20 million denatured beads were incubated with 5 ng of genomic DNA extracted from human embryonic kidney cells 293FT, two pre-assembled MuA transposons and T4 DNA ligase in the reaction buffer, and chain transfer reactions and ligation reactions were able to be performed substantially simultaneously at 37°C for 30 minutes. The reaction was terminated with a 0.5% SDS solution. The washed beads were treated with exonuclease I to remove single-stranded polynucleotides and then subjected to 15 cycles of PCR amplification to release the immobilized barcoded DNA fragments. PCR products were purified using 0.8XAMPure XP beads to remove small primer dimers and PCR byproducts and screened on the TapeStation using the High Sensitivity D5000 Band Check ( Fig.16 A). Purified PCR products were sequenced on an Illumina MiniSeq instrument. Reads with the same barcode sequence were sorted for each barcode pair based on their alignment position to the reference genome. The read distance to the next alignment was calculated, and the read count frequency along the read distance was plotted on Fig.16 B. When the barcode reads retain the connection information of the reads from the tagged DNA fragments, the proximal reads are expected to increase. The read distances of the original DNA fragments before tagging will also increase as the distances of the distal reads increase. A bimodal distribution of the read count frequency plot is expected, Fig.16 This can be accurately observed in B. Despite the very limited sequencing depth in the multi-sample MiniSeq run, the strong enrichment of short-range proximal reads demonstrates successful barcoded read continuity.

[0106] Example 2

[0107] This example describes a method for target-specific barcoding of genomic DNA ( Figure 7), the genomic DNA is transferred and linked to barcode beads in an open overall reaction using simultaneous strand transfer without genomic DNA segmentation. A MuA transposon contains a MuA transposable DNA having a 5' end transposon linker strand that can be linked to the barcode template of the barcode beads. 20 million barcode beads were incubated with 5ng of genomic DNA extracted from human embryonic kidney cells 293FT, the ligatable MuA transposon and T4 DNA ligase in buffer and reacted at 37°C for 30 minutes. The reaction was terminated with a 0.5% SDS solution. The washed beads were then reacted with another MuA transposon and exonuclease I. The reaction was terminated again with 0.5% SDS. The beads with barcode-tagged fragments were subjected to 15 cycles of PCR amplification to release the immobilized barcode-tagged DNA fragments. This method produces fewer PCR byproducts than the method in Example 1. PCR products were purified using 0.8X AMPure XP beads to remove small primer dimers and PCR byproducts and screened on the TapeStation using the High Sensitivity D5000 ( Fig.17 A). Purified PCR products were sequenced on an Illumina MiniSeq instrument. Reads with the same barcode sequence were sorted for each barcode based on their alignment position to the reference genome. The read distance to the next alignment was calculated, and the read count frequency along the read distance was plotted on Fig.17 B. When the barcode reads retain the connection information of the reads from the tagged DNA fragments, the proximal reads are expected to increase. The read distances of the original DNA fragments before tagmentation will also increase as the distances of the distal reads increase. A bimodal distribution of the read count frequency plot is expected, Fig.17 This can be accurately observed in B. Despite the very limited sequencing depth in the multi-sample MiniSeq run, the strong enrichment of short-range proximal reads demonstrates successful barcoded read continuity.

[0108] Example 3

[0109] Barcoded Illumina sequencing libraries were generated using 10 ng of genomic DNA from HapMap sample NA12878 as described above. Figure 4 As shown. 2×75bp paired-end sequencing was performed on the Illumina NextSeq system. More than 600 million paired-end reads were collected for haplotype phasing analysis using the HapCUT2 algorithm (Edge P et al., 2017). After removing duplicate reads, there was a genome coverage depth of approximately 22 times. The maximum phasing block size was 9.5Mb, and the phasing block size of N50 was 1.7Mb, with a switching error rate of 0.14%.

[0110] Example 4

[0111] An 18-base barcode design with 14 degenerate bases was used to generate clonal barcode template beads. Using these barcode-templated beads, Figure 7 According to the method shown in, a barcode tagging sequencing library was prepared from 0.5 ng of Escherichia coli DH10B genomic DNA sample mixed with lambda DNA. The library was combined with other libraries and sequenced on the Illumina MiniSeq system according to the following sequencing cycle conditions: read 1 (71 cycles), index 1 (18 cycles), index 2 (8 cycles) and read 2 (71 cycles). Based on the present invention, a sequence error correction algorithm was developed and applied to these sequence data. First, the algorithm can sort out unique barcodes and their associated tagging reads. Then, it compares unique barcodes with only a single tagging read (single-read barcode) with unique barcodes with 2 or more tagging reads (multi-read barcodes) that have only a mismatch of 1 base. When only one such multi-read barcode is identified, its sequence will be regarded as the original sequence of the unique barcode with a single associated tagging read, that is, the correct order of this barcode, so as to perform barcode error correction.

[0112] Table 1 below shows the results of one example.

[0113] Table 1

[0114]

[0115] Of the total 550,271 barcodes with unique sequences identified during the sequencing run, 211,484 barcodes were associated with only a single tagged read. These unique barcodes with a single tagged read were corrected. Of these, 110,140 were able to identify barcode partners that differed by only 1-base from 338,787 unique barcodes with multiple tagged reads. These barcode sequences will be corrected accordingly.

[0116] These data will be further used for de novo assembly analysis using an improved assembler based on SPAdes (Bankevich A et al, 2012). Table 2 below lists a first-level summary of the de novo assembly results. By further optimizing the assembler, we expect to obtain better assembly results.

[0117] Table 2

[0118]

[0119]

[0120] Example 5

[0121] We compared three different transposase-based approaches to generate clonal barcoded fragments for sequencing library construction ( Fig.19 ). Method 1 is a simultaneous chain transfer and capture reaction disclosed in the present invention. 1 ng of E. coli genomic DNA is mixed with a ligatable transposome, DNA ligase and 20 million beads, of which 1 million clonal barcode-templated beads are in the same reaction buffer to produce clonal barcode-tagged fragments and soluble sequencing libraries by further PCR amplification. Methods 2 and 3 are two methods that use chain transfer and capture reactions to generate clonal barcode-tagged fragments, respectively. Method 2 is to first generate STCs in a solution by mixing a ligatable transposome with 10 ng of E. coli genomic DNA; then 1 / 10 of these solutions containing 1 ng of original E. coli genomic DNA are added. th The STCs were mixed with DNA ligase and 20 million beads, of which one million cloned barcode templated beads were in the ligation buffer to capture the STCs in the solution onto the beads. Method 3 is to first mix the ligatable transposome and DNA ligase with 20 million beads to immobilize the ligatable transposome on the barcode template beads, of which one million cloned barcode template beads were in the ligation buffer. The reacted beads were washed to remove the ligase and then reacted with 1 ng of Escherichia coli genomic DNA in a chain transfer reaction buffer. Finally, the same number of beads from the three methods were PCR amplified to generate a soluble sequencing library through the same number of PCR cycles. The PCR products were loaded onto a 2% agarose E-gel EX to compare their yields ( Fig. 20 As expected, method 1 ( Fig. 20 , lane M1) produced the most library products, which demonstrated that method 1 was superior to the other two methods ( Fig. 20 , lane M2 and lane M3).

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Claims

1. A method for tracing the source of a nucleic acid fragment by using a barcode label, the method comprising: a. providing a plurality of solid carriers having clonal barcode templates or semi-clonal barcode templates fixed thereon; b. providing a plurality of transposable bodies, each transposable body comprising a transposable DNA and a transposase, wherein at least one transposable DNA in the transposable body can be directly or indirectly captured by the barcode template on the solid support; c. providing a nucleic acid target; d. wherein steps a, b and c occur at the same time in one reaction vessel, and the barcode information on the solid support is attached to the nucleic acid target by simultaneous strand transfer and capture reactions, without the need to additionally segment each nucleic acid target from another nucleic acid target in the total plurality of nucleic acid targets to form a strand transfer complex; e. fragmenting the nucleic acid target into fragments by a fragmentation strand transfer complex, wherein at least one fragment is attached with barcode information.

2. The method of claim 1, wherein the solid support is selected from the group consisting of beads, microparticles, wells, tubes, slides, plates, flow cells, and combinations thereof.

3. The method of claim 2, wherein when the solid support is a bead or microparticle, the barcode template is clonally or semi-clonally immobilized on the entire surface.

4. The method of claim 2, wherein when the solid support is a well, a tube, a slide, a plate or a flow cell, the barcode template is immobilized on the surface as separable clonal clusters or semi-clonal clusters.

5. The method according to any one of claims 1 to 4, wherein the clonal barcode template or semi-clonal barcode template immobilized on the solid support is prepared by direct synthesis, clonal amplification, or a combination thereof.

6. The method of claim 5, wherein the clonal amplification is selected from the group consisting of emulsion PCR, bridge PCR, isothermal amplification, template walking, nanosphere generation, and combinations thereof.

7. The method of claim 5, wherein the barcoded solid support is prepared by a clonal amplification method without separating amplified and non-amplified populations.

8. The method of claim 5, wherein the barcoded solid support is prepared by a clonal amplification method to have only or mainly enriched amplified populations.

9. The method according to any one of claims 2 to 4, wherein the beads and microparticles have a size range of 50 nm to 100 μm.

10. The method of claim 1, wherein the reaction is carried out in a buffer system with controlled viscosity, by adding substances to reduce diffusion and increase suspension, the substances being selected from the group consisting of polyethylene glycol, pluronic, cellulose, agarose, their derivatives, other polymers, and combinations thereof.

11. The method according to claim 10, wherein at the working concentration, the viscosity of the buffer system is 1-200 mPa·s at 20°C.

12. The method according to claim 1, wherein the transposome can be replaced by separate transposable DNA and transposase without pre-assembling them into a transposome.

13. The method of claim 1, further comprising adding a second transposome after step d.

14. The method of claim 1, further comprising adding a second transposome after step e.

15. The method according to claim 1, wherein the nucleic acid target can be pre-attached to a solid support by non-specific binding.

16. The method according to claim 1, wherein the capture reaction in the reaction vessel is performed by ligation, or by hybridization, or by affinity tag, or by antibody and antigen reaction, or by click chemistry, or a combination thereof.

17. The method of claim 1, wherein the capture reaction comprises first hybridization and then ligation.

18. The method of claim 1, wherein the solid support has a clonal barcode template or a semi-clonal barcode template immobilized thereon, and has a transposable DNA or a transposome pre-attached to the ends of some of the barcode templates immobilized on the solid support.

19. The method of any one of claims 1-4, wherein the transposable DNA further comprises an adapter sequence.

20. The method of claim 1, wherein the transposable DNA capable of being captured by the barcode template does not have a complementary sequence to the barcode template, and capture of the transposable DNA onto the barcode template is facilitated by a linker.

21. A method for generating a soluble library of barcoded fragments of a nucleic acid target, the method comprising: a. generating barcoded fragments of nucleic acid targets according to the method of claim 1; b. physically or enzymatically further fragmenting the barcoded fragments; c. ligating the adapter sequence to the barcoded fragments on the solid support; and d. Release the barcoded nucleic acid fragments from the solid support by primer extension or amplification to generate a soluble library using at least one primer targeting the attached additional sequence.

22. The method of claim 21, wherein the primers used in the primer extension or amplification are selected from the group consisting of random degenerate primers, common adapter primers, gene-specific primers, exome-specific primers, or a combination thereof.

23. The method of claim 21, wherein the soluble library is used for sequencing to determine the phasing information of the nucleic acid target.

24. The method of claim 21, wherein the soluble library is used for sequencing to determine identifiers of duplicate reads.

25. The method of any one of claims 1-4, wherein the nucleic acid target comprises a plurality of nucleic acid molecules derived from DNA or RNA in native, modified, amplified or otherwise chemically processed form or in the form of a strand transfer complex.

26. The method according to any one of claims 1 to 4, wherein the fragmentation of the strand transfer complex is performed using protease treatment, high temperature treatment, or a protein denaturant or a combination thereof.

27. The method according to any one of claims 1 to 4, further comprising providing a ligase, wherein the ligase is selected from the group consisting of a wild-type, mutant or tagged DNA ligase, or RNA ligase, and a combination thereof.

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