Method for position-coordinated barcoding in solution for long DNA molecule sequencing

Abstract

The methods and compositions disclosed herein relate to the use of massively parallel short reading sequencing to prepare libraries for intact sequencing of long molecules. The methods disclosed herein generate a nested set of nucleic acid constructs for each genomic fragment and generate a plurality of nested sets for a plurality of genomic fragments. The nucleic acid construct may be single-stranded or double-stranded. Each nucleic acid construct in each nested group comprises a barcode and a target sequence portion, and the nucleic acid constructs within each nested group have different lengths. The nucleic acid constructs in each nested group share a unique barcode sequence.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 369,346, filed on July 25, 2022. The entire contents of this provisional application are hereby incorporated by reference herein for all purposes. Background Art

[0003] Sequencing long genomic DNA can be challenging. Many sequencing platforms, such as the DNBseq sequencer and the Illumina sequencer, are not designed for sequencing long DNA molecules. For example, it can be difficult to generate DNBs from long DNA molecules and obtain enough template copies for high-quality sequencing in the DNBseq sequencer. Illumina sequencers often require bridge amplification, which tends to be inefficient for long DNA molecules. In addition, the length of the reads possible using these systems is often less than 500 bases, so the middle of these molecules cannot be sequenced. Therefore, although these MPS sequencing platforms are cost-effective and efficient, the length of sequence reads obtained from these platforms is limited. There are additional challenges in sequencing DNA molecules with highly repetitive nucleotide sequences; for example, it is often difficult to discern whether two identical sequence reads are associated with different locations in the genome or whether they are simply duplicate sequence reads at the same location in the genome. Therefore, there is still a need to prepare sequencing libraries so that sequence information of long DNA molecules can be collected from short sequence reads in an accurate and efficient manner. Summary of the invention

[0004] In one aspect, disclosed herein is a method of preparing a single-stranded adapter-bearing construct for sequencing, optionally without the use of nanodroplets, comprising: preparing a plurality of nested sets of single-stranded nucleic acid constructs, optionally in a single mixture, wherein each single-stranded nucleic acid construct in each nested set comprises a target sequence portion flanked at the 5' end by a first adapter sequence and at the 3' end by a second adapter sequence, wherein the first adapter sequence comprises, from 5' to 3', a primer binding sequence, a barcode sequence, and a first hybridization sequence and the second adapter sequence comprises a second hybridization sequence, wherein the first and second hybridization sequences are complementary to each other, wherein each target sequence portion has a first end and a second end, wherein a distance between the first end and the barcode sequence is shorter than a distance between the second end and the barcode sequence, wherein each single-stranded nucleic acid construct in each nested group shares the same barcode sequence and single-stranded nucleic acid constructs in different nested groups have different barcode sequences, and wherein for each nested group of single-stranded nucleic acid constructs, the target sequence portions in the nested group have the same nucleotide sequence near the first end and differ from each other near the second end due to truncation, such that each nested group of single-stranded nucleic acid constructs comprises a plurality of target sequence portions having different lengths.

[0005] In another aspect, disclosed herein is a method of generating a single-stranded DNA circle comprising a single-stranded adapter-bearing construct for sequencing, optionally without the use of nanodroplets, comprising: preparing a plurality of nested sets of single-stranded nucleic acid constructs in a single mixture, wherein each single-stranded nucleic acid construct in each nested set comprises a target sequence portion flanked by a first adapter sequence and a second adapter sequence, wherein the first adapter sequence comprises a barcode sequence and a primer binding sequence, wherein each target sequence portion has a first end and a second end, wherein a distance between the first end and the barcode sequence is shorter than a distance between the second end and the barcode sequence, and wherein The single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences, wherein for each nested group of single-stranded nucleic acid constructs, (a) the target sequence portions in the nested group have the same nucleotide sequence near the first end and differ from each other due to truncation near the second end, so that each nested group of single-stranded nucleic acid constructs includes multiple target sequence portions with different lengths, and (b) the single-stranded nucleic acid constructs in each nested group are circularized to produce a single-stranded DNA loop, wherein the first adapter sequence is connected to the second adapter sequence.

[0006] In another aspect, disclosed herein is a method of generating a single-stranded DNA circle comprising a single-stranded adapter-bearing construct for sequencing, optionally without the use of nanodroplets, comprising: preparing, in a single mixture, a plurality of nested sets of single-stranded nucleic acid constructs, wherein each single-stranded nucleic acid construct in each nested set comprises a target sequence portion flanked by a first adapter sequence and a second adapter sequence, wherein the first adapter sequence comprises a barcode sequence and a primer binding sequence, wherein each target sequence portion has a first end and a second end, wherein a distance between the first end and the barcode sequence is shorter than a distance between the second end and the barcode sequence, The single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences, wherein for each nested group of single-stranded nucleic acid constructs, (a) the target sequence portions in the nested group have the same nucleotide sequence near the first end and differ from each other by truncation near the second end, so that each nested group of single-stranded nucleic acid constructs contains multiple target sequence portions with different lengths, and (b) the single-stranded nucleic acid constructs in each nested group are circularized to produce a single-stranded DNA circle, wherein the first adapter sequence and the second adapter sequence are connected.

[0007] In another aspect, disclosed herein is a method for preparing a double-stranded adapter-bearing construct for sequencing, optionally without the use of nanodroplets, wherein the method comprises: (i) amplifying a plurality of genomic fragments, each genomic fragment comprising a target sequence, to produce a plurality of groups of amplified nucleic acid fragments in a mixture, wherein the amplified nucleic acid fragments in each group share the same target sequence, optionally amplified using target-specific primers, and for each group, the method further comprises (ii) contacting the amplified nucleic acid fragments with an enzyme, wherein the enzyme introduces breaks in the amplified nucleic acid fragments, (iii) partitioning the mixture of fragments into a plurality of aliquots, (iv) performing nick translation on the aliquots of the fragments to synthesize DNA chains under conditions such that the DNA chains synthesized in different aliquots have different lengths, wherein each of the DNA chains comprises a portion of the target sequence having a first end and a second end, and wherein the DNA chains in the different aliquots share the same sequence near the first end and have different sequences near the second end, and (v) for each aliquot, ligating a second adapter to the second end of the DNA chain synthesized in (iv) via branched ligation, wherein each second adapter is a first adapter oligonucleotide and a second adapter oligonucleotide. Partially double-stranded adapters, wherein both the first adapter oligonucleotide and the second adapter oligonucleotide are complementary and hybridize to each other, wherein each of the second adapters comprises a positional barcode sequence, wherein each ligation comprises ligating the 5-primer end of the first adapter oligonucleotide of the second adapter to the second end of the synthetic DNA strand, wherein the first adapter oligonucleotides ligated to the second ends of the synthetic DNA strands in different aliquots comprise different positional barcode sequences and the first adapter oligonucleotides ligated to the second ends of the synthetic DNA strands in the same aliquot share the same positional barcode sequence, (vi) ligating the first adapter oligonucleotides from (v) combining different aliquots of the synthesized DNA chains connected to the second adapter into a single mixture, (vii) extending the primer hybridized to the first adapter oligonucleotide connected to the synthetic DNA chain to produce a double-stranded fragment with a blunt end, and (viii) optionally selecting the double-stranded fragment of (vii) from the single mixture, whose size range is 200bp-1.5kb, such as 300bp-1.2kb, 300bp-1kb or 500-1000bp, and (ix) connecting the third adapter to the blunt end of the double-stranded fragment to produce a double-stranded construct with an adapter.

[0008] In another aspect, disclosed herein is a method for preparing multiple nested sets of adapter-attached fragments, optionally without the use of nanodroplets, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer sequence, and a barcode sequence, wherein in each nested set of adapter-attached fragments, the target sequence fragments have the same nucleotide sequence at the first end and differ from each other at the second end due to truncation, such that each nested set of adapter-attached fragments comprises multiple target sequence fragments of different lengths, wherein the first end is closer to the barcode sequence than the second end, wherein the method comprises: (a) providing a population of single-stranded DNA concatemers in a reaction of a single mixture, wherein each concatemer comprises a plurality of identical monomers, and each monomer comprises a complement of the target sequence, a complement of the barcode sequence that identifies the concatemer, and a primer binding sequence shared by the population of single-stranded concatemers, wherein the primer binding sequence comprises a sequence complementary to the primer sequence, wherein both the primer binding sequence and the barcode sequence complement are at the target sequence. (a) at the complement of the 3-primer of the plurality of concatemers; (b) annealing a primer comprising a primer sequence to the primer binding sequence of the plurality of monomers of each concatemer in the plurality of concatemers; (c) extending at least some of the primers hybridized to the primer binding sequence using a DNA polymerase having 5'-->3' exonuclease activity and not having strand displacement activity, wherein the extension produces a plurality of extension primers, each of the extension primers comprising a target sequence fragment having a barcode sequence and a primer sequence, wherein the extension primers hybridize to the concatemers; wherein the extension primers are separated by a gap, and ( d) contacting the multiple extension primers with a 5-primer adapter comprising a 5-primer adapter sequence, a 3-primer adapter comprising a 3-primer adapter sequence, a DNA ligase, and an exonuclease having single-stranded DNA exonuclease activity under conditions where the exonuclease degrades a portion of the target sequence fragments in the extension primers to produce shortened extension primers, the 5-primer adapter being connected to the 5' end of the shortened extension primer, and the 3-primer adapter being connected to the 3' end of the shortened extension primer, thereby producing multiple nested groups of fragments with adapters.

[0009] In another aspect, disclosed herein is a method for preparing multiple nested sets of adapter-attached fragments, optionally without the use of nanodroplets, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer binding sequence, and a complement of a barcode sequence, wherein in each nested set of adapter-attached fragments, the target sequence fragments have the same nucleotide sequence at the first end and are different from each other at the second end, such that each nested set of adapter-attached fragments comprises a plurality of target sequence fragments having different lengths, wherein the first end is closer to the barcode sequence than the second end, wherein the method comprises (a) providing a barcoded fragment comprising a barcode sequence, a target sequence, and a primer binding sequence, wherein the barcoded fragment is immobilized on a bead at one end, (b) annealing a primer comprising the 5-primer adapter sequence to the bead, The invention relates to a method for preparing a primer binding sequence in a barcoded fragment, wherein the 5-primer adapter sequence comprises i) a complement of the barcode sequence, and ii) a primer sequence complementary to the primer binding sequence in the barcoded fragment, (c) extending the primer to produce an extended primer comprising a target sequence fragment and the complement of the barcode sequence, (d) contacting the extended primer with a branched adapter comprising a 3-primer adapter sequence to produce adapter-attached fragments, (e) separating the adapter-attached fragments from the barcoded fragments still immobilized on the beads, and (f) repeating one or more cycles of steps (b)-(e) under extension control conditions to produce one or more adapter-attached fragments, wherein the adapter-attached fragments produced from step (e) and the adapter-attached fragments produced from step (f) constitute nested sets of adapter-attached fragments, and wherein the adapter-attached fragments in each nested set comprise target sequence fragments having different lengths.

[0010] In another aspect, disclosed herein is a method for preparing multiple sets of adapter-attached fragments, optionally without the use of nanodroplets, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer binding sequence, and a complement of a barcode sequence, wherein the method comprises: (a) providing a barcoded fragment comprising a barcode sequence, a target sequence, and a primer binding sequence, wherein the barcoded fragment is immobilized on a bead at one end, (b) annealing a primer comprising a 5-primer adapter sequence to the primer binding sequence in the barcoded fragment, wherein the 5-primer adapter sequence comprises i) a barcode sequence complement, and ii) a primer sequence complementary to a primer binding sequence in a barcoded fragment, (c) extending the primer to produce an extended primer comprising a target sequence fragment and the barcode sequence complement, (d) contacting the extended primer with a first branched adapter comprising a 3-primer portion containing a degenerate sequence region, thereby forming a first extension product comprising a degenerate sequence region at the 3-primer portion, wherein the 3-primer portion hybridizes to the barcoded fragment through the degenerate sequence region, (e) extending the 3-primer portion of the first extension product to produce a second extension product, and (f) contacting the second extension product with a second branched adapter to produce a fragment with an adapter.

[0011] In another aspect, disclosed herein is a DNA complex comprising (a) a barcoded fragment immobilized on a solid support, wherein the barcoded fragment comprises a barcode sequence and a target sequence, and (b) a polynucleotide hybridized to the barcoded fragment, wherein the polynucleotide comprises a 5-primer portion comprising a complement of the barcode sequence, a 3-primer portion comprising a fragment of the target sequence, wherein the 5-primer portion and the 3-primer portion anneal to the barcoded fragment, and the middle portion does not anneal to the barcoded fragment, thereby forming a bubble.

[0012] In another aspect, disclosed herein is a composition comprising nested sets of adapter-attached fragments, each comprising a barcode sequence and a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, and a 3-primer adapter sequence, wherein the target sequence fragments have the same nucleotide sequence at the first end and differ from each other at the second end due to truncation, such that the nested sets of adapter-attached fragments comprise multiple target sequence fragments having different lengths, and wherein the nested sets of adapter-attached fragments share the same barcode sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings and their descriptions illustrate exemplary embodiments of the present disclosure. The methods and compositions provided in the present disclosure are not limited to the embodiments shown in these drawings.

[0014] Figure 1An embodiment of the method of the present disclosure is shown. The top panel represents a double-stranded genomic fragment with adapters, which comprises a target sequence having a first end and a second end. The target sequence is flanked by adapter 1 at 3-primer and adapter 3 at 5-primer. The first adapter comprises a primer binding site and a barcode sequence, the primer binding site being located at 3-primer relative to the barcode sequence (not shown).

[0015] Figure 2 An exemplary embodiment of the method of the present disclosure is shown. Various method steps are shown, including adding barcodes to genomic fragments and amplifying barcoded genomic fragments.

[0016] Figure 3A An embodiment of a DNA ring-based approach is shown for Figure 2 The amplified genomic fragments in the produce single-stranded DNA circles.

[0017] Figure 3B Another embodiment of a DNA ring-based approach is shown for Figure 2 The amplified genomic fragments in the produce single-stranded DNA circles.

[0018] Figure 3C Another embodiment of a DNA ring-based approach is shown for Figure 2 The amplified genomic fragments in the produce single-stranded DNA circles.

[0019] Figure 4A An embodiment of a DNA ring-based approach is shown for Figure 3A or Figure 3B The single-stranded DNA circle formed as shown generates a double-stranded adaptor-bearing construct.

[0020] Figure 4B Another embodiment of a DNA ring-based approach is shown for Figure 3A or Figure 3B The single-stranded DNA circle formed as shown generates a double-stranded adaptor-bearing construct.

[0021] Figure 5 One embodiment of a linear DNA-based protocol for generating double-stranded adaptor-attached constructs for sequencing is shown.

[0022] Fig. 6A and 6B A concatemer-based approach of the present invention is shown. Figure 1A shows that a double-stranded DNA molecule comprising a barcode 110 and a target sequence 120 is denatured into a single-stranded nucleic acid. The single-stranded nucleic acid is circularized and amplified by rolling circle replication to form a concatemer comprising a plurality of monomers, each monomer comprising a complement of the target nucleic acid sequence, a complement of the barcode sequence 121, and a primer binding sequence 131. In each monomer, a primer 130 is annealed to the primer binding sequence 131 at the 3-primer relative to the complement of the barcode sequence 111, and is extended using a polymerase that does not have strand displacement activity but has 5-primer to 3-primer exonuclease activity. The extended primers 150 are separated by a gap 160. Optionally, the gap 160 is widened by a gapping enzyme, thereby generating a gap 170. If the primer 130 is an RNA primer, the gapping enzyme can be RNase H. The L-adapter is then attached to the 5-primer of the extension primer, and then the branch adaptor is attached to the 3-primer of the extension primer in the presence of a 3-primer to 5-primer exonuclease. Note that although extension, gapping and connection are shown in separate steps, in some embodiments, reagents for one or more of these reactions can be added to a single reaction mixture at the same time. After denaturation, a nested group of single-stranded adapter fragments 191 with different target sequence fragment (122-125) lengths is generated, each having an L-adapter sequence at the 5-primer and a branch adaptor sequence at the 3-primer. The barcode is located at the 5-primer portion of the adapter fragment.

[0023] Figure 7A-7B Another concatemer-based method of the present invention is shown. Similar to FIG. 6 , a polymerase is used to extend a primer 230 annealed to a primer binding sequence 231. The DNA polymerase does not have a strand displacement activity, but has a 5-primer to 3-primer exonuclease activity. 210 is a barcode and 220 is a target sequence. Unlike FIG. 6 , the primer binding sequence 231 is at the 5-primer relative to the complement of the barcode sequence 211 (the primer binding sequence 131 in FIG. 6 is at the 3-primer relative to the complement of the barcode sequence 111). In addition, unlike FIG. 6 , the connection of the L-adapter 280 and the branch adapter 290 is performed in the presence of a 5-primer to 3-primer exonuclease (rather than a 3-primer to 5-primer exonuclease in FIG. 6 ). After denaturation, a nested group of adapter-bearing fragments 291 is formed, each fragment having an L-adapter sequence at the 5-primer and a branch adapter sequence at the 3-primer. The adapter-bearing fragments include target sequence fragments 222, 223, 224, and 225. These target sequence fragments are generated with different lengths. The barcode 210 is located at the 3-primer portion of the adapter-bearing fragments.

[0024] Figure 8An embodiment of the method based on the combinatorial approach of the present invention is shown. Genomic DNA is first fragmented to produce staggered fragments with single-strand breaks, as described in U.S. Provisional Application No. 63 / 224,731. Figure 2 StLFR is performed to generate a common barcoded fragment comprising a branched adapter sequence at one end and an L-adapter sequence at the other end. These common barcoded fragments are then released from the beads and then Figure 6A-6B or Figure 7A-7B Cyclization and workup were performed as described in .

[0025] Fig. 9 Another exemplary embodiment of the method based on the combinatorial scheme of the present invention is shown. The barcoded fragments comprising a barcode sequence 410, a primer binding sequence 433 and a target sequence 420 are fixed on beads, and the 3-primer ends of the barcoded fragments are also fixed on beads. The tailed primer comprises a tail 431 (which is optional) that does not hybridize with the barcoded fragments. The tailed primer comprises a primer sequence 432 that hybridizes with the primer binding sequence 433, and the complement of the barcode sequence 411 that hybridizes with the barcode sequence in the barcoded fragment. The tailed primer is extended to produce an extended tailed primer 435, which comprises a target sequence fragment 436 and the complement of the barcode sequence 411. A branch adapter 440 is connected to the 3-primer end of the target sequence fragment 436 to produce an adapter-attached fragment 450. The adapter fragment 450 is then separated from the barcoded fragment 460, which is still immobilized on the bead. The barcoded fragment is then used as a template for subsequent extension cycles of the tailed primer 430 to generate a plurality of extended tailed primers comprising target sequence fragments 451-453. The extension is controlled so that the target sequence fragments 451-453 have different lengths.

[0026] Figure 10A-10C Another exemplary embodiment of a method based on a combinatorial approach is shown. The barcoded fragments and tailed primers are as follows Fig. 9 shown. Fig. 10A It is shown that after an initial extension period with normal deoxynucleotides, uracil is added to the reaction mixture to generate an extended tailed primer containing uracil 550, and then normal deoxynucleotides (e.g., deoxynucleotides without uracil) and a reversible terminator are added. Different concentrations of terminators can be added in different cycles to generate extended tailed primers containing target sequence fragments with different lengths. Fig. 10BThe extended tailed primer 550 is then ligated to the branch adapter 540 to produce an adapter fragment 551. If a reversible terminator is used, it must be reversed before ligation. The adapter fragment 551 is then USER digested to remove uracil, leaving a gap 560 flanked by an exposed 3-primer end 570 and an exposed 5-primer end 580. The internal branch adapter 551 is then ligated to the exposed 3-primer end 570, and the L-adapter 552 is then ligated to the exposed 5-primer end 580 in the gap. The splint oligonucleotide 590 is then hybridized to the 3-primer portion of the internal branch adapter 551 and the 5-primer portion of the L adapter 552 to allow ligation between the two. The ligation produces a shortened adapter fragment 600 and a loop 591 (still fixed) in the barcoded fragment. The shortened adapter fragment 600 can be separated from the barcoded fragment after denaturation.

[0027] Fig. 10C Shows duplicates Fig. 10B The fragments generated after multiple cycles of the process shown. Each shortened adapter-bearing fragment comprises a shortened target sequence fragment generated by a different cycle (eg, 610 or 620 or 630).

[0028] Fig.11 Shortened target sequence fragments are shown, such as 610, 620 and 630, whose sequences correspond to Figure 10A-10C Different regions of the target sequence generated by the process shown in . Since all shortened adapter fragments contain the same complement of barcode sequence 511, sequencing reads from these adapter fragments can be assembled based on the shared barcode sequence to achieve complete coverage along the target sequence.

[0029] Fig.12 Another exemplary embodiment of a method based on a combinatorial approach is shown. Primers annealed to primer binding sequences in barcoded fragments are extended with a first extension under extension control conditions. The extension primer is connected to a branch adapter 720 having a degenerate sequence region 730 at the 3-primer portion. The first branch adapter can hybridize to a random position in the barcoded fragment through the degenerate sequence region, which forms a loop 740 and causes replication of certain random portions of the barcoded fragment to be skipped. Then, a second extension is performed by extending the 3-primer end of the first branch adapter 750 to form a second extension product under extension control conditions. The second branch adapter is connected to the 3-primer end of the second extension product to produce a fragment with an adapter. 710 is a barcode. 711 is the complement of the barcode.

[0030] Fig.13A and Fig. 13BAnother embodiment of the invention is shown, namely preparing nested sets of target sequence fragments for loop-mediated complete stLFR.

[0031] Fig.14 An embodiment of the loop-mediated complete stLFR is shown.

[0032] Fig.15 Shows the preparation Fig.14 An embodiment of loop-mediated intact stLFR-generated molecules for sequencing is shown. DETAILED DESCRIPTION

[0033] 1. Overview

[0034] The method disclosed herein involves preparing a library using large-scale parallel short-read sequencing to fully sequence long molecules. The length of these long DNA molecules is generally in the range of 1-20kb, for example, more than 1000bp, or more than 1500bp, or more than 2000bp, or more than 3000bp. These strategies disclosed herein do not require cloning barcoded beads and can be performed completely in solution, i.e., during the entire library preparation process, genomic fragments and adapters are in solution. Therefore, compared with the strategy requiring barcoded beads, they can be easily used to add barcodes to a large number of molecules in a library (e.g., 1 million to 10 million to 100 million to 1 billion molecules), and the cost is lower.

[0035] The method disclosed herein generates nested groups of nucleic acid constructs for each genomic fragment, and generates multiple nested groups for multiple genomic fragments. The nucleic acid construct can be single-stranded or double-stranded. Each nucleic acid construct in each nested group comprises a barcode and a target sequence portion, and the nucleic acid constructs in each nested group have different lengths. The nucleic acid constructs in each nested group share a unique barcode sequence. The target sequence portion has a first end and a second end. The nucleic acid constructs in each nested group share the same nucleotide sequence near the first end, but have different nucleotide sequences near the second end. These methods can sequence the first end and the second end of all nucleic acid constructs in the nested group, and the sequence reads are assembled to generate the sequence information of the entire long genomic DNA fragment. Various approaches to achieve this goal are described below.

[0036] In some approaches, the method provides a way to retain information that can be used to identify the position of each nucleic acid sequence (corresponding to each sequence read) in the original long DNA genomic DNA molecule. This position information helps to interpret the sequence information of long DNA molecules with repetitive sequences.

[0037] 2. Definition

[0038] The components or reactions in a "single reaction mixture" means that the reactions occur in a single mixture and do not need to be separated into separate tubes, containers, aliquots, wells, chambers or droplets during the labeling step. The components can be added simultaneously or in any order to make a single reaction mixture.

[0039] As used herein, "first end" and "second end" are used to define the two ends of each nucleic acid molecule in a nested group of nucleic acid molecules. The target sequence near the first end of the nucleic acid molecule shares the same nucleotide sequence, but the nucleotide sequence near the second end is different. In a double-stranded DNA molecule, the first end can be the 5-primer end or the 3-primer end. Similarly, in a double-stranded DNA molecule, the second end can be the 5-primer end or the 3-primer end. Relative to the second end in the same molecule, the first end is closer to the barcode sequence.

[0040] As used herein, a "unique molecular identifier" (UMI) refers to a nucleotide sequence present in a DNA molecule that can be used to distinguish individual DNA molecules. See, for example, Kivioja, Nature Methods 9, 72-74 (2012). UMIs and DNA sequences associated with them can be sequenced to identify sequencing reads from the same source nucleic acid. The term "UMI" is used herein to refer to both the nucleotide sequence and the physical nucleotides of the UMI, as can be seen from the context. A UMI can be a random, pseudo-random, or partially random, or non-random nucleotide sequence inserted into an adapter or otherwise incorporated into a source nucleic acid molecule to be sequenced. In some embodiments, each UMI is expected to uniquely identify any given source DNA molecule present in a sample. For the purposes of this disclosure, the term "UMI" is used interchangeably with the term "barcode".

[0041] As used herein, the term "single tube LFR" or "stLFR" refers to, for example, the process described in U.S. Patent Publication 2014 / 0323316 and Wang et al., Genome Research, 29: 798-808 (2019), the entire contents of each of which are incorporated herein by reference in their entirety. In stLFR, multiple copies of the same unique barcode sequence (or "tag") are associated with a single long nucleic acid fragment. In one embodiment of a single tube LFR, the long nucleic acid fragments are labeled with barcodes at regular intervals. In one embodiment, barcodes are introduced into long nucleic acid molecules using one or more enzymes (e.g., transposases, nickases, and ligases). The barcode sequence in the nucleic acid fragment can be conveniently carried out, for example, in a single container without separation. This process allows the analysis of a large number of single DNA fragments without separating the fragments into separate tubes, containers, aliquots, holes, or droplets in the labeling step.

[0042] As used herein, a "unique" barcode refers to a nucleotide sequence that is used to identify a single group of polynucleotides and distinguish it from other groups of polynucleotides in a multi-group mixture. For example, a unique barcode for a nested group of nucleic acid constructs means that the barcode sequence associated with one nested group is different from the barcode sequence associated with at least 90% of the total nested groups (more commonly at least 99% of the total nested groups, even more commonly at least 99.5% of the total nested groups, and most commonly at least 99.9% of the total nested groups). In some embodiments, a unique barcode is used to identify the position of a group of nucleic acid fragments relative to the genomic DNA from which the group of nucleic acid fragments is derived. This type of barcode is also referred to as a positional barcode in the present disclosure. In some cases, there are different groups of nucleic acid fragments in a single mixture, each group carrying a unique positional barcode. See, for example, Figure 3C

[316] In some cases, different groups of nucleic acid fragments (each with a unique positional barcode) are present in separate aliquots, which can then be combined into a mixture. See, e.g. Figure 5

[504] and

[505] in.

[0043] The term "in solution", when used in conjunction with an adaptor (or any other nucleic acid construct or polynucleotide complex) used in a method or composition disclosed herein, means that the adaptor (or any other polynucleotide or polynucleotide complex) is not fixed to a substrate, but is free to move in solution. When used to describe a reaction, such as "a reaction performed in solution", it means a reaction that occurs between nucleic acids, all of which are in solution.

[0044] The terms "nucleic acid fragment with adapters" and "fragment with adapters" are used interchangeably to refer to a polynucleotide comprising a target nucleic acid fragment and one or more adapter sequences.

[0045] The term "adapter sequence" refers to a sequence on either strand of the adaptor, as is clear from the context. That is, an "adapter sequence" may refer to either or both of an adaptor sequence on one strand and a complement sequence on a second strand. Similarly, the term "barcode sequence" refers to a barcode sequence on one strand or its complement sequence.

[0046] The terms "reversible terminator nucleotide" and "reversible terminator" are used interchangeably and refer to a nucleotide having a 3-primer reversible blocking group. A "reversible blocking group" refers to a group that can be cleaved to provide a hydroxyl group at the 3'-position of a nucleotide that can be linked to the 5-primer phosphate group of another nucleotide. The reversible blocking group can be cleaved by an enzyme, a chemical reaction, heat and / or light. Exemplary nucleotides having a 3-primer reversible blocking group are known in the art and are also disclosed in U.S. Patent No. 10,988,501; the entire disclosure of which is incorporated herein by reference.

[0047] The term "target sequence" refers to the sequence information of a DNA molecule, such as a genomic DNA fragment. The methods and compositions provided herein can be used to determine the target sequence.

[0048] The term "target sequence portion" refers to a portion of the entire target sequence or the complement of the target sequence. Multiple nucleic acid fragments may include sequences corresponding to different portions of the same target sequence.

[0049] The term "extended primer" refers to a DNA strand generated by extending a primer annealed to a template.

[0050] The term "copy" refers to the complementary nucleotide strand of the template generated by primer extension.

[0051] The term "corresponding to" refers to a DNA sequence having the same or complementary sequence as another DNA sequence.

[0052] The term "near" refers to a nucleotide sequence within a specified length from the reference point when referring to a sequence near a reference point (e.g., a nucleotide sequence near the first end). The specified length is typically less than 200 bases, less than 100 bases, less than 50 bases, less than 20 bases, or less than 10 bases. In some embodiments, the specified length is in the range of 1-50 bases (e.g., 1-30 bases, or 1-20 bases).

[0053] The term "exposed 5-primer" refers to the 5-primer end of a DNA fragment formed after the bond between two nucleotides in an otherwise continuous DNA strand is broken. Similarly, the term "exposed 3-primer" refers to the 3-primer end of a DNA fragment formed only after the bond between two nucleotides in an otherwise continuous DNA strand is broken.

[0054] As used herein, the term "length suitable for sequencing" refers to a DNA chain length equal to the length of a sequence read generated by MPS sequencing. The length may be determined by the sequencing method, but in general, the length of a single DNA chain suitable for sequencing falls within the range of 200 bases to 1.5 bases (e.g., 300-1000 bases, 300-500 bases, or 400-600 bases or 500-1000 bases), and the length of a DNA double strand suitable for sequencing falls within the range of 200-1.5 base pairs (e.g., 300-1000 base pairs, 300-500 base pairs, or 400-600 base pairs or 500-1000 base pairs).

[0055] When used in relation to a polynucleotide and a substrate (e.g., a bead), the term "attached" means that the polynucleotide (or one end of the polynucleotide) is directly in contact with or covalently attached to the substrate. For example, the surface may have reactive functionalities that react with functionalities on the polynucleotide molecule to form a covalent bond. As an illustrative example, a barcoded fragment is attached to Fig. 9 The term "linked" may also be used to describe the joining of one polynucleotide to another polynucleotide to form a single continuous polypeptide, e.g. Fig. 10B 551 and 552 in are connected to form a single continuous fragment with a linker.

[0056] As used in this context, a "fragment" is single-stranded, although as discussed above and elsewhere herein, a fragment may hybridize to a complementary strand, e.g., to form a nucleic acid complex. The term "fragment" is often used interchangeably with the term "polynucleotide."

[0057] In this context, "barcode region" refers to the region of a DNA molecule where the barcode or the complement of the barcode is located.

[0058] The term "barcoded fragment" refers to a fragment comprising a barcode sequence or the complement of a barcode sequence.

[0059] The term "branch adapter" refers to a partially double-stranded adapter. The partially double-stranded adapter includes (i) a double-stranded blunt end comprising the 5' end of one strand and the 3' end of the complementary strand, and (ii) a single-stranded region comprising a barcode sequence. The 5' end of the double-stranded region of the branch adapter can be connected to the 3' end of the nucleic acid fragment via a branch connection, as further described below.

[0060] The term "nested group" refers to a plurality of nucleic acid fragments that (i) have different lengths, (ii) share the same nucleotide sequence at one end, and (iii) have different nucleotide sequences at the other end due to truncation. An example of a nested group is Figure 6B As shown in 191.

[0061] The term "5-primer portion" of a polynucleotide refers to a contiguous nucleotide sequence region of a polynucleotide including the 5-primer end. The 5-primer portion may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the total length of the polynucleotide. The "5-primer portion" of a polynucleotide does not include the 3-primer end of the polynucleotide.

[0062] The term "3-primer portion" of a polynucleotide refers to a contiguous nucleotide sequence region of a polynucleotide including the 3-primer end. The 5-primer portion may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the total length of the polynucleotide. The "3-primer portion" of a polynucleotide does not include the 5-primer end of the polynucleotide.

[0063] The term "middle portion" of a polynucleotide refers to the portion between the 3-primer portion and the 5-primer portion.

[0064] The term "bubble" refers to a DNA structural configuration consisting of two DNA strands that contains a non-hybridizing region flanked by two double-stranded regions. The non-hybridizing region contains two single-stranded loops that lack sufficient complementarity so that they do not anneal to each other. Fig. 10C An illustration of the bubble region is shown.

[0065] The term "gap" refers to the space between two single-stranded nucleic acid fragments.

[0066] The term "gap" refers to a space that has been widened (used interchangeably with the term "lengthened"). A space is widened to form a gap. However, this does not necessarily mean that the length of a space is always less than the length of a gap. For example, the length of one particular space may be greater than the length of a gap formed by widening a different space.

[0067] The process of long fragment library preparation for sequencing can be carried out according to a variety of schemes. An exemplary embodiment of the method is described below. Under the guidance of the present disclosure, practitioners with skills in molecular biology and sequencing will recognize that a variety of changes in the various steps and reagents can be incorporated into the following scheme.

[0068] 3. Methods

[0069] 3.1 Adding adapters to the ends of nucleic acid molecules

[0070] Various approaches can be used to add adapter sequences to one or both ends of a nucleic acid molecule (e.g., a genomic fragment). This can be accomplished by, for example, adapter ligation, PCR amplification, and other methods known in the art.

[0071] In some approaches, each of the multiple genomic fragments is connected to an adapter that contains a unique barcode sequence for each genomic fragment. The unique barcode sequence can then be used to identify all reads from a specific genomic fragment. Methods for labeling each genomic fragment with a unique barcode are well known and will also be described further below, see the section entitled "Barcodes".

[0072] In various approaches, each genomic fragment is connected to a first adapter at one end and to a third adapter at the other end, and amplified by extending a primer that hybridizes to the two adapters. The terms "first", "second" or "third" are arbitrary and are used to refer to individual adapters. Unless specifically defined in the context of the present disclosure, they do not imply any particular physical relationship between the positions in which they appear in the genomic fragments, nor any particular order of the adapters used in the method.

[0073] In some approaches, the first adapter comprises a barcode sequence and a primer binding sequence as described above, configured such that when a primer bound to the primer binding sequence is extended, the extension product will comprise the primer sequence, the barcode sequence, and the target sequence in the order from 5-primer to 3-primer.

[0074] 3.2 Cyclization

[0075] Methods for producing DNA rings are known. In an exemplary embodiment, for example, splint oligonucleotides of 8-40 bases are annealed to the two ends of the single-stranded molecule. These annealed oligonucleotides allow 1-10 bases to overlap between the two ends of the product. The connection can then be performed with T4 DNA ligase to create a single-stranded ring with a small area of ​​double-stranded DNA at the connection site.

[0076] The cyclization of single-stranded DNA molecules can be carried out using methods well known in the art. In some approaches, for each of some single-stranded nucleic acid molecules, a splint oligonucleotide is then added, which is hybridized with the adapter sequence added to both ends of the target nucleic acid fragment. The single-stranded nucleic acid is then cyclized in the presence of a ligase (e.g., T4 or Taq ligase). The DNA polymerase used for RCR can be any DNA polymerase with strand displacement activity, such as Klenow fragments of Phi29, Bst DNA polymerase, DNA polymerase I, and Deep-VentR NDA polymerase (NEB#MO258). It is known that these DNA polymerases have strand displacement activity of varying strengths. One of ordinary skill in the art can select one or more DNA polymerases suitable for method and composition disclosed herein.

[0077] 3.3 Aliquots

[0078] Some approaches disclosed herein involve aliquoting the reaction mixture. Aliquots are used interchangeably with "pools" to refer to the partitioning of the whole. When forming aliquots, the volumes and components of different aliquots of the whole are similar. As used in the present application, different aliquots can be subjected to different processing procedures, so they can obtain different components. For example, in some approaches disclosed herein, adapters with different position barcodes are added to different aliquots, which causes the aliquots to have different components. Preferably, the DNA fragments in each aliquot have similar lengths. For aliquots with long and short DNA, specific methods can be used to minimize the over-coverage of shorter fragments. The product is amplified by PCR, then divided into 10-20 pools, followed by controlled extension or ExoIII digestion or controlled nick translation, and the duration of this process in different pools is different. Short DNA fragments will be fully extended to form blunt ends, and methods known in the art can be used, for example, DNA tailing or 3' blocking by terminal transferase to prevent these fragments with blunt ends from branching and connecting. Exemplary methods for blocking short fragment ligation are disclosed in WO2023001262, for example, Section 7.2, entitled “Remove excel adapters”, the entire disclosure of which is incorporated herein by reference in its entirety.

[0079] 3.4 Controlled Extension

[0080] In some approaches, the method includes extending primers hybridized to the DNA fragment under conditions that allow control of the extent of the extension reaction. These extension control conditions include, but are not limited to, selecting a polymerase with a suitable polymerization rate or other properties, and using various reaction parameters, including, but not limited to, reaction temperature, extension duration, primer composition, DNA polymerase, primer and nucleotide concentrations, additives, and buffer composition. In some cases, the extension can be controlled by a mixture of reversible terminators and normal nucleotides to extend. The ratio of the amount of reversible terminators to the amount of normal nucleotides can be adjusted to achieve the desired extent of extension. In general, the higher the ratio of the amount of reversible terminators to the amount of normal nucleotides, the more incomplete the extension will result.

[0081] In some approaches, the amplified genomic fragments are distributed into a plurality of aliquots, and each aliquot of the amplified genomic fragments is subjected to different extension control conditions, so that the extension products in different aliquots have different lengths. Each aliquot can be located in a different container or a different well. A single aliquot can also be located in different partitions (e.g., droplets) in the same container.

[0082] The number of required aliquots depends on the length of the target sequence and the length of the sequence reading generated from the sequencing platform. Generally, the larger the amplicon size, the more the number of required aliquots. In an illustrative example, for a 5kb amplicon and 500 bases per reading (double-ended read length is 250 bases or single-ended length is 500 bases), the method generally uses 10-20 aliquots. In some approaches, there are at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 aliquots. In some approaches, the number of aliquots can fall within the scope of 3-100 (e.g., 5-50, 6-40 or 10-20).

[0083] In order to generate extension products in each aliquot, the primer is annealed to the primer binding sequence in the genomic fragment with the adapter, and the primer is extended to replicate the barcode sequence and the region outside, that is, to extend to the target sequence portion of the genomic fragment with the adapter. The extension reaction in each aliquot is controlled as described above, resulting in the extension product having different sequences near both ends of the extension product. In some approaches, the extension of different aliquots is terminated at different times for different aliquots. In some approaches, the amount of extension time for each aliquot is gradually increased; for example, the first aliquot is extended for 2 minutes, the second aliquot is extended for 4 minutes, and so on. Figure 3B The length of extension time for the aliquot can range from 10 seconds to 20 minutes. Extension can also be controlled by limiting the concentration of nucleotides so that extension stops after 100 to 1,000 bases due to exhaustion of the nucleotide supply. In some approaches, extension is performed using a polymerase with 5' to 3' exolytic activity, which may be suitable for nick translation. Non-limiting examples of DNA polymerases that can be used in this method include Escherichia coli DNA polymerase 1.

[0084] 3.5 Controlled digestion

[0085] In some approaches, the amplified genomic fragments are distributed into multiple aliquots and then digested with nucleases. In some embodiments, the nuclease is a double-stranded DNA nuclease with 3'→5' nuclease activity, such as ExoIII and Klenow. In some approaches, digestion is controlled so that the lengths of the polynucleotides remaining after digestion in different aliquots are different. The extent of digestion can be controlled by parameters such as reaction temperature, digestion duration, and nuclease concentration. In an exemplary approach, the digestion time of each aliquot is different, so that the polynucleotides remaining after digestion have different lengths. In one approach, the digestion of each aliquot occurs at gradually increasing time intervals, so that the digested fragments in different aliquots have gradually decreasing lengths, for example, the lengths of the fragments in different aliquots are separated by 500 bases. Then the second adapter in each aliquot is connected to the two ends newly formed after digestion via branch connection, thereby producing a single-stranded nucleic acid construct, each of which includes a target sequence portion with two wings being a first adapter sequence and a second adapter sequence.

[0086] 3.6 Size selection

[0087] In some approaches, adapter fragments or amplified adapter fragments with lengths within a suitable range for sequencing are selected. Methods for selecting DNA fragments of a desired length are well known. An exemplary approach is to use AMPure XP beads (e.g., beads available from Pacific Biosciences (Menlo Park, Calif.), part number 100-265-900) to select fragments of a desired length.

[0088] 3.7 Amplification

[0089] Various methods are all related to amplification, such as amplification of genomic fragments or DNA fragments with adapters. Such amplification methods include, but are not limited to: multiple displacement amplification (MDA), polymerase chain reaction (PCR), ligation chain reaction (sometimes referred to as oligonucleotide ligase amplification OLA), cycle probe technology (CPT), strand displacement analysis (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCR) (for circularized fragments) and invasive cutting techniques. Amplification can be performed before or after any step of fragmentation or as outlined herein.

[0090] In some approaches, genomic fragments with adapters are amplified by extending primers that anneal to the adapter sequence. In some approaches, genomic fragments with different target sequences are connected to adapters at both ends, and the adapters share the same sequence. The genomic fragments are then amplified using primers that hybridize to the adapters at both ends. In some approaches, at least one of the adapters includes a barcode.

[0091] In some approaches, amplification is performed using target-specific primers (i.e., primers that hybridize to target sequences in genomic DNA). In some approaches, the target-specific primers contain a common adapter tag with a random barcode to amplify a specific region.

[0092] In some approaches, the amplification can be multiplex PCR, i.e., using multiple primer pairs targeting different target sequences in genomic DNA. In some approaches, the amplification is multiplex PCR, where 2-1000 different target regions are amplified in one reaction using target-specific primers, so that the reaction mixture contains amplified genomic fragments with different target sequences.

[0093] In some approaches, rolling circle amplification (RCR) can be used to amplify fragments or genomic fragments with adapters. The genomic fragment is first denatured into a single-stranded nucleic acid molecule. Add a splint oligonucleotide and hybridize it with the adapter sequence of the target sequence on both wings, and then in the presence of a ligase (e.g., T4 or Taq ligase), the single-stranded nucleic acid is cyclized. The DNA polymerase for RCR can be any DNA polymerase with strand displacement activity, and exemplary DNA polymerases include Phi29, BstDNA polymerase, Klenow fragment of DNA polymerase I and Deep-VentR NDA polymerase (NEB#MO258). It is known that these DNA polymerases have strand displacement activities of different intensities. One of ordinary skill in the art can select one or more DNA polymerases suitable for the present invention.

[0094] 3.8 Incision

[0095] In various embodiments of the present disclosure, genomic fragments or amplified genomic fragments (including fragments incorporating one or more adapter sequences) are combined with one or more nicking agents to produce nicks in genomic DNA fragments. In some approaches, the nicking agent is an enzyme (commonly referred to as a "nicking enzyme"). The nicking enzyme can be an endonuclease that cuts the phosphodiester bond in a polynucleotide or removes one nucleotide from a polynucleotide. In some cases, the nicking enzyme is a non-sequence-specific endonuclease that cuts the DNA chain at a random position. Non-limiting examples of nicking agents include Vibrio vulnificus nuclease (Vvn), Shrimps DNA-specific endonuclease, and DNAse I. In some approaches, the nicking agent is a site or sequence-specific nuclease, for example, a restriction endonuclease that cuts DNA at its recognition sequence. Non-limiting examples of site-specific nickases include Nt.CviPII (CCD), Nt.BspQI, and Nt.BbvCI, as described in Shuang-yong Xu, BioMol Concepts 2015; 6(4):253-267, the entire disclosure of which is incorporated herein by reference.

[0096] In some approaches, the nicking agent disclosed herein is a chemical nicking agent. Non-limiting examples of chemical nicking agents include the dipeptide seryl histidine (Ser-His), Fe2+ / H2O2 or Cu(II) complex / H2O2.

[0097] In some approaches, the method uses two or more nicking agents. In some approaches, the method uses two or more nicking agents from the same category of nicking agents (e.g., any class of non-specific nicking enzymes, site-specific nicking enzymes or chemical nicking agents). In some approaches, the method uses nicking agents from different categories.

[0098] After treatment, the length of the genomic fragments separated by the nick can be different. Generally, the higher the concentration of the nicking agent, the more nicks will be produced, thereby producing shorter fragments. Similarly, the longer the treatment time, the more nicks will be produced, thereby producing shorter fragments. By adjusting one or more of these parameters, the length of the fragment can be controlled within the desired range. In some approaches, the average length of the nucleic acid fragments produced by the nick is between 200 and 10000 nucleotides, for example, 200-500 nucleotides or 400-1000 nucleotides, or 1000-10000 nucleotides. Figure 3A An exemplary embodiment of using a nicking agent to create nicks in a genomic fragment is shown.

[0099] In some embodiments, the nick produced by the nickase is extended (widened) by the exonuclease to form a gap. This process may be referred to as "gapification", and the exonuclease used in the process may be referred to as "gapification enzyme". Examples of enzymes with 3' exonuclease activity include DNA polymerase I, Klenow fragment (no nucleotides present), exonuclease III, and other enzymes known in the art. Examples of enzymes with 5' exonuclease activity include Bst DNA polymerase, T7 exonuclease, truncated exonuclease VIII, lambda exonuclease, T5 exonuclease, and other exonucleases known in the art. Low processability exonucleases (i.e., exonucleases that remove nucleotides from one end of a polynucleotide at a relatively low rate) preferably open short gaps (e.g., 2-7 bases, 3-10 bases, or 3-20 bases) and separate from DNA to allow adapters to connect. If the use of exonucleases is desired, protection of the DNA adaptors from exonuclease digestion can be achieved by introducing phosphorothioate bonds between the bases (or modified bases) at the 5' and 3' ends of the adaptors.

[0100] Nicking and gapping of the amplified double-stranded genomic fragments produce DNA fragments of different lengths that end with exposed 3-primer ends, and a second adapter comprising a second adapter sequence can be connected to the 3-primer end via branched ligation. At least some of the ligation products produced by this process are flanked by a first adapter sequence and a second adapter sequence comprising a barcode sequence. The ligation products are separated from their hybridized complementary strands by denaturation, thereby forming a nested set of single-stranded nucleic acid constructs comprising portions of the target sequence.

[0101] 3.9 Cut Translation

[0102] Nick translation is performed on a nick in a DNA strand by a DNA polymerase (e.g., Escherichia coli DNA polymerase I). DNA polymerases suitable for nick translation generally have three activities: (1) 5' to 3' polymerase activity, which requires a single-stranded template and a primer with a 3' hydroxyl group to synthesize a new nucleotide chain complementary to the template; (2) 5' to 3' exonuclease activity, which degrades double-stranded DNA from the free 5' end; and (3) 3' to 5' exonuclease activity, which degrades double-stranded or single-stranded DNA from the free 3' hydroxyl end. The latter activity is a proofreading or editing function. On double-stranded DNA, the 3' to 5' exonuclease activity is blocked by the 5' to 3' polymerase activity. During nick translation, the 5' to 3' polymerase activity of the DNA polymerase adds nucleotides to the 3'-OH produced by the nick, while the 5' to 3' exonuclease activity removes nucleotides from the 5' side of the nick. The result of these synergistic activities is that nucleotides are removed from the 5' side of the nick, while nucleotides are added to the 3' side of the nick. This causes the nick to move or translate along the DNA. See Susan J. Karcher, Molecular Biology, A Project Approach, 1995, pp. 135-192, the relevant parts of which are incorporated herein by reference. Nick translation can be used in various embodiments of these methods, for example in Fig.13A middle.

[0103] 3.10 Branch Connection

[0104] Branched ligation, also known as "3-primer ligation" or "3-primer branched ligation", relies on the properties of T4 ligase to ligate double-stranded DNA adapters to DNA 3-primer ends in a space or gap. See, Wang et al., DNA Research, Feb. 1, 2019, 16(1):45-53, the entire disclosure of which is incorporated herein by reference. Branched ligation is very efficient in ligating adapters because it does not require the degenerate single-stranded bases at the adapter ends to hybridize in the gap.

[0105] Adapters suitable for branch ligation generally include: (i) a double-stranded blunt end comprising a 5-primer end of one chain and a 3-primer end of a complementary chain; (ii) a single-stranded region comprising a barcode sequence. The double-stranded blunt end provides a 5-primer phosphate group, which can be connected to the 3-primer of the target nucleic acid fragment via a 3-primer branch connection. In some embodiments, the double-stranded blunt end provides a 3-primer, which is blocked from connection by a dideoxynucleotide, a 3' phosphate group, a 3' overhang, or the like. The 3-primer branch connection involves covalently connecting the 5-primer phosphate group from the blunt-ended adapter (donor DNA) to the 3-primer hydroxyl end of the double-stranded DNA acceptor at the 3-primer recessed chain, gap, or interval. Compared to traditional DNA ligation, the 3-primer branch connection does not require complementary base pairing. 3-primer branch ligation is described in Wang et al., DNA Res. 26(1):45-53, doi:10.1093 / dnares / dsy037; PCT Publication No. WO 2019 / 217452; U.S. Patent Publication No. US2018 / 0044668 and International Application No. WO 2016 / 037418, U.S. Patent Publication No. 2018 / 0044667, all of which are incorporated by reference for all purposes.

[0106] In various embodiments, the adapter is connected to the genomic fragment using a branch connection. In some approaches, a nick is introduced into the amplified genomic fragment, resulting in exposed 3-primer ends and 5-primer ends, and then the second adapter is connected to the nick via a branch connection to form a fragment with an adapter. In some approaches, a controlled extension or digestion is performed using the genomic fragment as a template, and then the second adapter is connected to the newly formed 3-primer end of the extension product. Therefore, the connection produces a fragment with an adapter having a barcode sequence at the first end and a second adapter sequence at the second end.

[0107] In some cases, the adapter used in the branch connection contains additional information useful for the assembly of sequence reads. In some approaches, the second adapter includes a position barcode specific to each aliquot. The fragments into which the second adapter is incorporated in different aliquots contain different position barcode sequences, while the fragments into which the second adapter is incorporated in the same aliquot share the same position barcode sequence. The aliquots in which the DNA fragments incorporating the position barcode are now located can be combined and sequenced. The presence of position barcodes can be used to determine long genomic fragments with highly repetitive sequences. For example, the same sequence read from two aliquots will be assigned as a repeat of two different genomic locations, rather than being mistakenly regarded as a sequence read of one genomic location. The methods and compositions disclosed herein can accurately determine the sequence information of highly repetitive sequences and can therefore be used to sequence target sequences (e.g., DNA fragments near telomeres) located in genomic loci where highly repetitive sequences are found. The methods and compositions using these position barcodes can also be valuable for repeated sequencing of target genes associated with disease conditions.

[0108] In some approaches, genomic fragments are ligated to a first adapter having a barcode and then ligated to a second adapter via a branch ligation. In some approaches, the second adapter comprises a positional barcode, and the branch ligation to the second adapter produces a genomic fragment flanked by a first adapter sequence comprising a unique barcode sequence for each genomic fragment and a second adapter sequence comprising a unique positional barcode for each aliquot. Dual barcoding allows all aliquots of all nested groups to be combined in one reaction for sequencing, greatly improving sequencing efficiency.

[0109] 3.11 Sequencing

[0110] Libraries of adapter-attached fragments can be sequenced using methods known in the art, including, for example, but not limited to, polymerase-based sequencing-by-synthesis (e.g., HiSeq 2500 system by Illumina, San Diego, California), ligation-based sequencing (e.g., SOLiD 5500 by Life Technologies Corporation, Carlsbad, California), ion semiconductor sequencing (e.g., Ion PGM or Ion proton sequencer by Life Technologies Corporation, Carlsbad, California), zero-mode waveguide (e.g., PacBio RS sequencer by Pacific Biosciences, Menlo Park, California), nanopore sequencing (e.g., Oxford Nanopore Technologies Ltd., Oxford, England), pyrosequencing (e.g., 454 Life Sciences, Branford, Connecticut), or other sequencing techniques. Some of these sequencing technologies are short-read technologies, but others produce longer reads (e.g., GSFLX+ (454 Life Sciences; up to 1000 bp), PacBio RS (Pacific Biosciences; approximately 1000 bp), and nanopore sequencing (Oxford Nanopore Technologies Ltd.; 100 kb). For haplotype phasing, longer reads are advantageous and require less computation, although they tend to have higher error rates, and errors in such long reads may need to be identified and corrected according to the methods presented in this article prior to haplotype phasing.

[0111] In some approaches, sequencing is performed using combined probe anchor ligation (cPAL), such as described in US20140051588, US20130124100, both of which are herein incorporated by reference in their entirety for all purposes.

[0112] In some approaches, sequencing is performed using a DNBseq sequencer. The adapter-bearing fragments or their amplified products are denatured to produce single-stranded molecules. These rings are then used to make DNA nanoballs (DNBs) for the DNBseq sequencer.

[0113] In some approaches, the adapter-attached fragments or their amplification products are sequenced on an Illumina or other system that does not require circularization.

[0114] In some approaches, sequencing is double-end sequencing, including sequencing from either end of the same DNA fragment. In some approaches, the first read reading is generated by extending the sequencing primer annealed to the adapter sequence, which is closer to the first end of the target sequence fragment than the second end ("first read sequencing"), and the second sequence reading is generated by extending the sequencing primer annealed to the adapter sequence, which is closer to the second end of the target sequence fragment than the first end ("second read sequencing"). The first read sequencing will generate a barcode sequence. The second read sequencing will generate overlapping reads to substantially or completely cover molecules up to 500bp or 700bp or 1000bp in length. These overlapping sequencing reads will be clustered according to the barcode sequence determined by the first read sequencing in the de novo assembly.

[0115] In some approaches, sequencing is single-end sequencing, and the sequence information of the genomic fragment is determined based on the first read sequencing only.

[0116] 3.12 Assembly sequence information

[0117] Sequence reads from the same nested set of nucleic acid constructs (derived from one genomic fragment to be sequenced that has been ligated to adapters with unique barcode sequences) can be aligned based on the presence of the same barcode sequence. Sequence reads, each containing sequence information near a first end (identical) and a second end (variable), are assembled to provide the full-length sequence of the long genomic DNA fragment.

[0118] 4. Composition

[0119] 4.1 Sample

[0120] The sample containing the target nucleic acid can be obtained from any suitable source. For example, the sample can be obtained or provided from any target organism. Such organisms include, for example, plants; animals (e.g., mammals, including humans and non-human primates); or pathogens, such as bacteria and viruses. In some cases, the sample can be a cell, tissue or polynucleotide of such a target organism population or can be obtained therefrom. As another example, the sample can be a microbiome or a microbial population. Optionally, the sample is an environmental sample, such as a sample of water, air or soil.

[0121] Samples from a target organism or such a population of target organisms may include, but are not limited to, body fluid samples (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen); cells; tissues; biopsy samples, research samples (e.g., products of nucleic acid amplification reactions such as PCR amplification reactions); purified samples, such as purified genomic DNA; RNA preparations; and original samples (bacteria, viruses, genomic DNA, etc.). Methods for obtaining target polynucleotides (e.g., genomic DNA) from organisms are well known in the art.

[0122] 4.2 Target nucleic acid

[0123] As used herein, the term "target nucleic acid" (or polynucleotide) or "target nucleic acid" refers to any nucleic acid (or polynucleotide) suitable for processing and sequencing by the methods described herein. In some approaches, the target nucleic acid is a genomic fragment generated by fragmenting genomic DNA extracted from a sample. Note that although genomic fragments are used to illustrate the methods and compositions disclosed herein, these methods and compositions can also be used to prepare sequencing libraries to sequence any target nucleic acid or fragment thereof, including those containing nucleotide modifications (e.g., nucleotide analogs).

[0124] Nucleic acids can be single-stranded or double-stranded and can include DNA, RNA or other known nucleic acids. Target nucleic acids can be nucleic acids of any organism, including but not limited to viruses, bacteria, yeast, plants, fish, reptiles, amphibians, birds and mammals (including but not limited to mice, rats, dogs, cats, goats, sheep, cattle, horses, pigs, rabbits, monkeys and other non-human primates, and humans). Target nucleic acids can be obtained from one individual or multiple individuals (i.e., groups). Samples for obtaining nucleic acids can contain nucleic acids from cell mixtures or even organisms, for example: human saliva samples containing human cells and bacterial cells; mouse xenografts including mouse cells and cells from transplanted human tumors; and the like. Target nucleic acids can be unamplified or can be amplified by any suitable nucleic acid amplification method known in the art. Target nucleic acids can be purified according to methods known in the art to remove cell and subcellular contaminants (lipids, proteins, carbohydrates, nucleic acids other than nucleic acids to be sequenced, etc.), or they can be unpurified, i.e., include at least some cell and subcellular contaminants, including but not limited to intact cells that are destroyed to release their nucleic acids for processing and sequencing. Target nucleic acids can be obtained from any suitable sample using methods known in the art. Such samples include, but are not limited to, biological samples, such as tissues, isolated cells or cell cultures, body fluids (including but not limited to blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat, and semen); and environmental samples, such as air, agricultural, water, and soil samples, etc.

[0125] The target nucleic acid can be genomic DNA (e.g., from a single individual), cDNA, and / or can be a complex nucleic acid, including nucleic acids from multiple individuals or genomes. Examples of complex nucleic acids include microbial groups, circulating fetal cells in the blood of pregnant women (e.g., see Kavanagh et al., J. Chromatol. B 878: 1905-1911, 2010), circulating tumor cells (CTCs) from the blood of cancer patients. In one embodiment, this complex nucleic acid has a complete sequence comprising at least one gigabase (Gb) (the diploid human genome comprises approximately 6Gb of sequence).

[0126] In some cases, the target nucleic acid is a genomic fragment. In some approaches, the genomic fragment is longer than 10kb, for example, 10-100kb, 10-500kb, 20-300kb, 50-200kb, 100-400kb or longer than 500kb. In some cases, the target nucleic acid is 5,000 to 100,000Kb. In some approaches, the length of the target nucleic acid is 500 bases to 50,000 bases, for example, 1000 bases to 20,000 bases, or 5000 bases to 10,000 bases. The amount of the DNA (for example, human genomic DNA) used in a single mixture can be <10ng, <3ng, <1ng, <0.3ng or <0.1ng of DNA. In some approaches, the amount of DNA used in a single mixture can be less than 3,000x the amount of haploid DNA, such as less than 900x, less than 300x, less than 100x, or less than 30x. In some approaches, the amount of DNA used in a single mixture can be at least 1x the amount of haploid DNA, such as at least 2x or at least 10x the amount of haploid DNA.

[0127] The target nucleic acid can be isolated using conventional techniques, such as those described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, cited above. In certain circumstances, particularly if small amounts of nucleic acid are used in a particular step, it is advantageous to provide carrier DNA (e.g., an unrelated circular synthetic double-stranded DNA) which is mixed with the sample nucleic acid and used when only small amounts of sample nucleic acid are available and which runs the risk of being lost through nonspecific binding (e.g., to container walls, etc.).

[0128] According to some embodiments of the present invention, genomic DNA or other complex target nucleic acids can be obtained from a single cell or a small number of cells with or without purification by any known method.

[0129] As described above, the method disclosed herein can be used to sequence long nucleic acid fragments. Long fragments of genomic DNA can be isolated from cells by any known method. For example, Peters et al., Nature 487: 190–195 (2012) describes a scheme for isolating long genomic DNA fragments from human cells. In one embodiment, cells are lysed, and intact nuclei are precipitated by a gentle centrifugation step. Then, genomic DNA is released after several hours of proteinase K and RNase digestion. The material can be treated to reduce the concentration of remaining cell waste, for example, by dialysis for a period of time (i.e., 2-16 hours) and / or dilution. Since such methods do not require the use of many destructive processes (e.g., ethanol precipitation, centrifugation, and vortexing), genomic nucleic acids remain substantially intact, and most of the fragments produced are more than 150 kilobases in length. In some approaches, the length of the fragment is about 5 to about 750 kilobases. In other embodiments, the fragment length is about 150 to about 600, about 200 to about 500, about 250 to about 400, and about 300 to about 350 kilobases. The smallest fragment useful for haplotype analysis is approximately 2-5 kb; although fragment length may be limited by shearing resulting from manipulation of the starting nucleic acid preparation, there is no maximum theoretical size.

[0130] In other embodiments, long DNA fragments are isolated and manipulated in a manner that minimizes shearing of the DNA or its absorption by the container, including, for example, separation of cells in agarose in agarose gel plugs or oil, or use of specially coated tubes and plates.

[0131] According to another embodiment, to obtain uniform genomic coverage when the number of sample cells is small (e.g., 1, 2, 3, 4, 5, 10, 10, 15, 20, 30, 40, 50, or 100 cells from a microbiopsy or circulating tumor or fetal cells), all long fragments obtained from the cells are barcoded using the methods disclosed herein.

[0132] 4.3 Barcode

[0133] According to one embodiment, a barcode sequence having two, three or more segments is used, one of which is, for example, a barcode sequence. For example, the introduced sequence may include one or more known sequence regions and one or more degenerate sequence regions used as barcodes or tags. The known sequence (B) may include, for example, PCR primer binding sites, transposon ends, restriction endonuclease recognition sequences (e.g., sites of rare cutters, such as NotI, Sac II, Mlu I, BssH II, etc.) or other sequences. The degenerate sequence (N) used as a tag is long enough to provide a population of different sequence tags, which is equal to or preferably greater than the number of target nucleic acid fragments to be analyzed. The higher the N value, the less likely it is that two molecules share the same barcode.

[0134] According to one embodiment, the barcode-containing sequence comprises a known sequence region of arbitrarily selected length. According to another embodiment, the barcode-containing sequence comprises two known sequence regions of selected length, which are located on both sides of the degenerate sequence region of selected length, i.e., B n N n B n , where N can have any length sufficient to label a long fragment of the target nucleic acid, including but not limited to N=10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, and B can have any length that accommodates the desired sequence (e.g., transposon ends, primer binding sites, etc.). For example, such an embodiment can be B 20 N 15 B 20 .

[0135] In one embodiment, the barcode used to mark the long fragment adopts a two-segment or three-segment design. This design allows a wider range of possible barcodes to be achieved by connecting different barcode segments together to form a complete barcode segment or by using segments as reagents in oligonucleotide synthesis. This combination design provides a larger possible barcode library while reducing the number of full-size barcodes that need to be generated. In other embodiments, a barcode of 8-12 base pairs (or longer) is used to achieve the unique identification of each long fragment.

[0136] In one embodiment, two different barcode segments are used. Segment A and segment B can be easily modified so that each of them contains a different half-barcode sequence, thereby generating thousands of combinations. In another embodiment, the barcode sequence is incorporated into the same adapter. This can be achieved by dividing the B adapter into two parts, each part having a half barcode sequence, separated by a common overlapping sequence for connection. The two tag components each have 4 to 6 bases. The 8-base (2x4 base) tag group can uniquely mark 65,000 sequences. Both 2x5 base tags and 2x6 base tags can use degenerate bases (i.e., "wildcards") to achieve optimal decoding efficiency.

[0137] In another embodiment, a unique identification of each sequence is achieved using an 8-12 base pair error correction barcode. The length of the barcode can be 5 to 20 information bases, typically 8 to 16 information bases, for exemplary but not limiting purposes.

[0138] 4.4UMI

[0139] In various embodiments, unique molecular identifiers (UMIs) are used to distinguish individual DNA molecules from one another. A set of adapters is generated, each with a UMI. These adapters are attached to the fragments to be sequenced or other source DNA molecules, and each of the sequenced molecules has a UMI that helps distinguish it from all other fragments. In such embodiments, a large number of different UMIs (e.g., thousands to millions) can be used to uniquely identify DNA fragments in a sample. An exemplary embodiment of a method using UMIs is described in Example 2.

[0140] The length of the UMI is sufficient to ensure the uniqueness of each source DNA molecule. In some approaches, the length of the unique molecular identifier is about 3-12 nucleotides, or 3-5 nucleotides in length. In some cases, the length of each unique molecular identifier is about 3-12 nucleotides, or 3-5 nucleotides in length. Therefore, the length of the unique molecular identifier can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides.

[0141] The process of long fragment library preparation for sequencing can be carried out according to a variety of schemes. These schemes can be used to generate nested groups of nucleic acid constructs for each genomic fragment, and the sequences near the two ends of each nucleic acid construct in each nested group can be determined. The nucleic acid constructs in each nested group can be single-stranded or double-stranded. These approaches allow the sequence information of long genomic fragments to be effectively generated. Some of these approaches involve making DNA rings. Other approaches use linear DNA molecules. Exemplary embodiments of these methods are described below. Practitioners with molecular biology skills and sequencing according to the disclosure will recognize the various changes of the various steps and reagents that can be incorporated into the following schemes.

[0142] 4.5 Barcoded beads

[0143] The beads are barcoded by the barcode oligonucleotides in the adapters immobilized thereon. Each bead comprises multiple adapters and therefore multiple barcode oligonucleotides. Each barcode oligonucleotide comprises at least one barcode. The barcode oligonucleotides on the same bead have the same barcode sequence and the barcode oligonucleotides on different beads have different barcode sequences. Thus, each bead carries many copies of a unique barcode sequence that can be transferred to a target nucleic acid fragment using the methods described above.

[0144] The diameter of the beads used can range from 1-20 μm, alternatively 2-8 μm, 3-6 μm or 1-3 μm (e.g., about 2.8 μm). For example, the spacing of the barcoded oligonucleotides on the beads can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 nm. In certain embodiments, the spacing is less than 10 nm (e.g., 5-10 nm), less than 15 nm, less than 20 nm, less than 30 nm, less than 40 nm, or less than 50 nm. In some embodiments, the number of different barcodes used for each mixture can be >1M, >10M, >30M, >100M, >300M, or >1B. As described below, a large number of barcodes can be generated for use in the present invention, for example, using the methods described herein. In some embodiments, the number of different barcodes used per mixture can be >1M, >10M, >30M, >100M, >300M, or >1B, and they are sampled from a pool of at least 10-fold greater diversity (e.g., from >10M, >0.1B, 0.3B, >0.5B, >1B, >3B, >10B different barcodes on a bead). In some embodiments, the number of barcodes per bead is between 100k and 10M (e.g., between 200k and 1M, between 300k and 800k, or about 400k).

[0145] In some embodiments, the length of the barcode region is about 3-15 nucleotides, for example, 5-12, 8-12 or 10 nucleotides in length. In some cases, the length of each barcode in the barcode region is about 3-12 nucleotides, or 3-5 nucleotides in length. Therefore, the length of the barcode (whether it is a sample barcode, a cell barcode or other barcode) can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. In one example, each barcode region contains three barcodes, each barcode consists of 10 bases, and the three barcodes are separated by 6 bases of a common sequence.

[0146] The barcode beads are transferred to the target nucleic acid sequence. In some embodiments, the transfer occurs at regular intervals by ligating the 3' end of an adapter oligonucleotide to the nucleic acid fragments generated by nicking and gapping, as disclosed.

[0147] In some embodiments, barcoded beads are constructed using three sets of double-stranded barcoded DNA molecules by a strategy based on partitioning and pooling ligation. In some embodiments, each set of double-stranded barcoded DNA molecules consists of 10 base pairs, and the nucleic acid sequences of the three sets are different. An exemplary method for generating barcoded beads by partitioning and pooling ligation is described in PCT Publication No. WO 2019 / 217452, the disclosure of which is incorporated herein by reference in its entirety. Fig.12The methodology of the split and pooling method is also illustrated in 13. In one approach, a common adapter sequence containing a PCR primer annealing site is attached to DynabeadsTMM-280 streptavidin (ThermoFisher, Waltham, Massachusetts) magnetic beads via a 5' double biotin linker. Three sets of 1,536 barcode oligonucleotides containing overlapping sequence regions were constructed by Integrated DNA Technologies (Coralville, Iowa). Ligation was performed in a 384-well plate with a reaction volume of 15 μL containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM ATP, 2.5% PEG-8000, 571 units of T4 ligase, 580 pmol barcode oligonucleotides and 65 million M-280 beads. The ligation reaction was incubated on a rotator at room temperature for 1 hour. Between ligations, the beads were pooled into a single container by centrifugation, collected to the side of the container with a magnet, and washed once with high salt wash buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.1 mM EDTA, and 0.05% Tween 20) and twice with low salt wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20). The beads were resuspended in 1X ligation buffer and distributed in a 384-well plate, and the ligation step was repeated.

[0148] In one aspect, the present invention provides a composition comprising beads, to which are attached adapter oligonucleotides comprising clonal barcodes, wherein the composition comprises more than 3 billion different barcodes, and wherein the barcode is a tripartite barcode having the structure 5'-CS1-BC1-CS2-BC2-CS3-BC3-CS4. In some embodiments, CS1 and CS4 are longer than CS2 and CS3. In some embodiments, CS2 and CS3 are 4-20 bases, CS1 and CS4 are 5 or 10 to 40 (e.g., 20-30) bases, and the length of the BC sequence is 4-20 bases (e.g., 10 bases). In some embodiments, CS4 is complementary to a splint oligonucleotide. In some embodiments, the composition comprises a bridging oligonucleotide. In some embodiments, the composition comprises a bridging oligonucleotide, a bead comprising a tripartite barcode as described above, and a genomic DNA comprising a hybridization sequence having a region complementary to the bridging oligonucleotide.

[0149] Another source of clonal barcodes, such as beads or other supports associated with multiple copies of a tag, can be prepared by emulsion PCR or CPG (controlled pore glass) or chemical synthesis to prepare other particles with copies of the barcode with adapters. A population of DNA sequences containing tags can be PCR amplified on beads in a water-in-oil (w / o) emulsion by known methods. See, for example, Tawfik and Griffiths, Nature Biotechnology 16:652–656 (1998); Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8820, 2003; and Shendure et al., Science 309:1728-1732 (2005). This results in multiple copies of a sequence containing a single tag on each bead.

[0150] Another way to make clonal barcode sources is to perform oligonucleotide synthesis on microbeads or CPGs through a “mix and split” combinatorial process. Using this process, one can create a population of beads that each have a population of barcode copies on them. For example, to make all B 20 N 15 B 20 (each of which is present in ~1000+ copies of each of the 100 beads), on average one could start with ~100 billion beads and synthesize B on all of them. 20 The common sequence (adapter) is then divided into 1024 synthesis columns, different 5-mers are made in each synthesis column, then they are mixed and divided into 1024 columns again and additional 5-mers are made, and then repeated once more to complete N15, and then they are mixed and synthesized in one large column for the final B 20 As a second adapter. Thus, in 3050 syntheses, one can create a molecule with ~1 trillion beads (1 12 beads) to identify the barcodes of the same set of “similar clones” because only one in ten beads will have the starting template (the other nine will not), preventing each bead from having two templates with different barcodes.

[0151] An exemplary process for barcode sequence assembly is described in PCT Publication No. WO 2019 / 217452, the disclosure of which is incorporated herein by reference.

[0152] 4.6 Reaction mixture

[0153] Provided herein is a reaction mixture that can be used to prepare a polynucleotide library. The reaction mixture includes: 1) a polymerase that lacks 5-3' exonuclease activity and does not have strand displacement activity; 2) a DNA complex that contains multiple fragments that are hybridized to one or more monomers of a DNA concatemer and separated by a nick or gap. In some embodiments, some or all of the fragments are generated by extending an RNA primer, so these fragments incorporate an RNA sequence at the 5-primer end. In some embodiments, the reaction mixture also includes one or more gap enzymes as described herein. In some embodiments, the gap enzyme has 5'→3' exonuclease activity. In some embodiments, the gap enzyme has 3'→5' exonuclease activity.

[0154] In some embodiments, each fragment is ligated to an L-adapter at the 5-primer end and a branched adaptor at the 3-primer end.

[0155] Also disclosed herein is a DNA complex comprising a barcoded fragment fixed on a solid support (e.g., a bead) and a fragment hybridized with the barcoded fragment. In some embodiments, the fragment comprises a plurality of uracils. In some embodiments, the fragment comprises a 5-primer portion, a 3-primer portion, and a middle portion therebetween, and the middle portion of the fragment does not hybridize with the barcoded fragment. In some embodiments, the 5-primer portion of the fragment is an adapter sequence, and the 3-primer portion of the fragment comprises a branch adapter sequence. Fig. 10A An illustrative embodiment is shown.

[0156] Also provided herein are compositions comprising groups of DNA fragments with overlapping target sequences. In some embodiments, the fragments have target sequences of different lengths but share the same barcode sequence. In some embodiments, the fragments share a common adapter sequence at the 5-primer end and a common adapter sequence at the 3-primer end. In some embodiments, the fragments in the series share a common sequence in the 5-primer portion, which sequence comprises a barcode sequence. Figure 6B An exemplary embodiment is shown in In some embodiments, the fragments in the series share a common sequence in the 3-primer portion, which sequence comprises a barcode sequence. Figure 7B An illustrative embodiment is shown.

[0157] A composition is also provided herein, comprising a plurality of nested groups of single-stranded DNA loops, wherein each loop comprises a target sequence portion with a first adapter sequence and a second adapter sequence on both sides. The first adapter sequence comprises a primer binding sequence, a barcode sequence and a first hybridization sequence from 5' to 3', and the second adapter sequence comprises a second hybridization sequence. The first hybridization sequence and the second hybridization sequence hybridize with each other to form a loop. Each target sequence portion has a first end and a second end, wherein the distance between the first end and the barcode sequence is shorter than the distance between the second end and the barcode sequence. The single-stranded nucleic acid constructs in each nested group have the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences. For each nested group of single-stranded nucleic acid constructs, the target sequence portions in the nested group have the same nucleotide sequence near the first end, and differ from each other due to truncation near the second end, so that the nested groups of single-stranded nucleic acid DNA loops comprise a plurality of target sequence portions with different lengths.

[0158] 5. DNA ring-based approach

[0159] In some approaches, the method uses a DNA circularization approach. The method includes circularizing the nucleic acid constructs in each nested group so that the two ends of each nucleic acid construct are connected together. See Figure 1 . Each nucleic acid construct comprises a target sequence flanked by a first adapter ("Adapter 1") sequence and a third adapter ("Adapter 3") sequence. Circularization of the nucleic acid construct allows sequences near both ends to be included in a single sequence read. Portions of target sequences from different nucleic acid constructs in the same nested group can be assembled to generate sequence information corresponding to the entire target sequence in the genomic fragment. This scheme is further elaborated below.

[0160] 5.1 Ligating adapters to both ends of the genomic fragment and amplifying the genomic fragment

[0161] Figure 2 An exemplary approach to adding adapters to both ends of a genomic fragment

[201] is shown. Step (i) shows connecting a double-stranded genomic fragment to two double-stranded adapters at both ends to produce a genomic fragment with adapters

[202] . Step (ii) shows amplifying the double-stranded genomic fragment with adapters. Amplification can be performed using primers that hybridize to the first and third adapter sequences (not shown). This step produces an amplified genomic fragment with blunt ends

[203] .

[0162] 5.2 Producing Nested Sets of Single-Stranded Nucleic Acid Constructs Containing Target Sequences and Circularizing

[0163] In the circularization pathway, amplified genomic fragments are processed to generate nested sets of single-stranded nucleic acid constructs (e.g., Figure 3A

[303] in), and the single-stranded nucleic acid construct is circularized. Each single-stranded DNA construct comprises a target sequence portion having a first end and a second end. The two wings of the target sequence portion are first adapter sequences adjacent to the first end. The second adapter sequence (adapter 2) is inserted near the second end. In some approaches, the first adapter sequence is located at the 5-primer of the single-stranded DNA construct, and the second adapter sequence is located at the 3-primer. The first adapter sequence comprises a unique barcode sequence for each nested group, that is, the single-stranded DNA constructs within the same nested group share the same barcode sequence, while the nested groups from different single-stranded DNA constructs have different barcode sequences. In some cases, the first end of the single-stranded DNA construct is closer to the barcode sequence than the second end.

[0164] A variety of approaches can be used to produce single-stranded nucleic acid constructs from amplified genomic fragments. In some approaches, a nested set of single-stranded DNA constructs is generated by contacting the amplified genomic fragments with a nicking agent to introduce a nick in the target sequence. Then, the second adapter is connected at the nick via a branch connection. Figure 3A An example of such an approach is shown. Figure 3A Step (iii) of Figure 2 (ii) generating) contacting with a nicking agent to generate nicks at random positions of the target sequence in the amplified genomic fragments. Each amplified genomic fragment can be nicked once or multiple times, and the nicks generated in the fragments can be extended to form gaps using one or more exonucleases. The process generates one or more fragments, but only one of the fragments contains a first adapter sequence (containing a barcode sequence

[311] ). Many parameters can affect the length of the nucleic acid fragments separated by the nicks and / or gaps. Generally, the higher the concentration of the nicking agent and the longer the treatment time of the nicking agent, the shorter the length of the fragments. By adjusting one or more of these parameters, the length of the fragments can be controlled within a desired range. In some embodiments, the average length of the nucleic acid fragments generated by the nicks is between 200 and 10,000 nucleotides, for example, 200-500 nucleotides or 400-1000 nucleotides or 1000-10,000 nucleotides. Step (iv) shows connecting a second adapter comprising a second adapter sequence at the nick via branch connection to form a connection product

[302] , each of some of the connection products comprising the first adapter sequence and the second adapter sequence. Step (v) shows denaturing the connection products to form a single-stranded nucleic acid construct

[303] , each of some of the connection products comprising the first adapter sequence and the second adapter sequence. The population of single-stranded nucleic acid constructs represents a nested group of constructs comprising the target sequence.

[0165] In some other approaches, generating a nested set of single-stranded DNA constructs involves annealing a primer to a primer binding sequence in a first adaptor that has been ligated to a genomic fragment, and extending the primer to produce a primer extension product. Figure 3B An example of this pathway is shown. Figure 3B Step (iii) shows that Figure 2 The amplified double-stranded genomic fragments with adapters shown in are distributed into multiple aliquots. Step (iv) shows that the amplified products in each sample are denatured to form single-stranded molecules

[304] , and then the primers are hybridized with the primer binding sequence. The primers are extended in the presence of a polymerase and dNTPs, and the extension is controlled so that the extension products in different aliquots have different lengths, thereby forming a nested group of extension products. The extension products each have a first end (primer start) and a second end (extension end), and the extension products have the same sequence near the first end and different sequences near the second end. Step (v) shows that a second adapter is added to the second end by branch connection. In some embodiments, these second adapters contain unique position barcode sequences of a single aliquot

[312] . Therefore, the single-stranded nucleic acid constructs

[305] formed by branch connections in different aliquots contain different position barcode sequences. The single-stranded nucleic acid constructs in the same aliquot share the same position barcode sequence. The aliquots are then combined into a single mixture

[306] . The dotted ellipse represents a single mixture. Subsequent steps are all completed in a single mixture. Step (vi) shows denaturing the product in a single mixture to form adapter-bearing fragments

[307] .

[0166] In some other approaches, generating nested sets of single-stranded DNA constructs involves ligating adapters after each specified period of time via branched ligation with positional barcode sequences. Figure 3C An exemplary pathway is shown. Figure 3B The pathways shown in Figure 3CThe reaction is carried out in a single tube throughout the process without the need for aliquoting. Step iii shows that after the primer is extended for a first period of time, the second adapter is connected to the extension primer, thereby producing a reaction mixture of fragments connected to the second adapter

[314] ; step iv shows that after the primer is extended for an additional period of time, the additional adapter is connected to the extension primer (in the same reaction mixture), thereby producing a mixture of fragments connected to the second adapter

[314] and fragments connected to the first additional adapter

[315] ; step v shows that after the primer is extended for another period of time, the second additional adapter is added, thereby producing a mixture of fragments connected to the second adapter

[314] , the first additional adapter

[315] and the second additional adapter

[316] , and so on. The process of adding adapters with unique positional barcodes can be repeated for 3-50 rounds, such as 10-40 rounds or 10-20 rounds. Each of the second adapter, the first additional adapter, the second additional adapter and the additional additional adapters contains a unique positional barcode sequence, and each of these adapters is connected to the extension primer via branch connection.

[0167] The molar amount of each adapter used in the branch ligation (e.g. Figure 3C In some embodiments, the amount of adapters used is 1-20%, such as 1-10% or 2-15%, of the total molar amount of amplified genomic fragments. In some embodiments, the amount of adapters (e.g., the second adapter, the first additional adapter, the second additional adapter) used in different rounds is the same. In some embodiments, the amount of adapters used in different rounds is different. Figure 3C Step (vi) of the method shows denaturing the reaction mixture

[316] to produce single-stranded adapter-bearing fragments

[317] . The single-stranded fragments are then circularized as described below.

[0168] 5.3 Cyclization

[0169] The single-stranded nucleic acid construct is then circularized to form a single-stranded ring. Methods for circularization of single-stranded nucleic acids are well known, see Section 3.2. At least some of these single-stranded DNA fragments contain a barcode sequence and a target sequence portion. In each nested group, the target sequence portion shares the same nucleotide sequence near the first end, but has a different nucleotide sequence near the second end. After circularization, the first adapter sequence and the second adapter sequence in each single-stranded nucleic acid construct are connected together, so that the first end and the second end of the target sequence portion are close to each other, so that a single sequence read can identify the sequence information near both ends. Figure 3A Step (vi) and Figure 3BAn exemplary pathway is shown in step (vii).

[0170] 5.4 Generation of linear double-stranded adapter constructs from single-stranded circles

[0171] Various approaches can be used to remove single-stranded DNA circles (e.g., using Figure 3A or Figure 3B or Figure 3C The invention relates to the random fragmentation of single-stranded loops to produce fragments, and the double-stranded constructs with adapters are generated from these fragments. The DNA loops can be fragmented using methods known in the art (e.g., ultrasonic treatment) to produce multiple single-stranded DNA fragments. Each single-stranded DNA fragment contains the barcode sequence in the DNA loop. Optionally, size selection is performed to select fragments with a length suitable for sequencing.

[0172] Figure 4A An example of such a pathway is shown. Complementary strands are synthesized using single-stranded DNA fragments as templates, thereby forming multiple double-stranded fragments

[401] . Step (viii) shows that adapters are connected to both ends of the double-stranded fragments to produce a double-stranded construct with adapters

[402] . Step (ix) shows that size selection is performed to generate a nucleic acid construct having a length suitable for sequencing

[403] .

[0173] In some other approaches, a linear, adapter-attached, double-stranded construct is generated by extending a primer that hybridizes to a loop under extension-controlled conditions to produce an extended primer of a length suitable for sequencing. Figure 4B An illustrative example is shown in steps (vii) and (viii) of . Typically, controlled extension does not result in copying the entire template sequence; rather, the extension primer remains hybridized to loops

[404] having exposed 3-primer ends (i.e., 3-primer recessed ends) that are ready for branch ligation. In some cases, the length of the extension primer ranges from 300-1000 bases, such as 300-500 bases or 400-600 bases, to achieve more efficient sequencing. Any short artifacts can be removed by exonuclease treatment or purification. Therefore, size selection is not required using this approach, and all extension products can be used to generate sequence reads.

[0174] The second adapter is then ligated to the recessed 3-primer end of the extension primers via branched ligation to form adapter-attached extension primers, each of which has a second adapter sequence at one end and a primer binding sequence and a barcode sequence at the other end. Figure 4B , step (ix)

[405] . Collect the extension primer with adapter

[406] ( Figure 4B, step (x)), and using the extension primer with the adapter as a template to perform primer extension to produce a complementary strand, and then form a double-stranded DNA fragment

[407] . Figure 4B , step (xi). The double-stranded DNA fragments can be amplified and sequenced.

[0175] The DNA ring-based scheme described above can be used to generate nested sets of linear double-stranded fragments of each genomic fragment to be sequenced. Each double-stranded DNA fragment in the nested set contains a different target sequence portion of the genomic fragment, and these different target sequence portions can be assembled together to decrypt the sequence of the original long DNA molecule. See the section entitled "Assembling Sequence Information" above.

[0176] 6. Linear DNA-based protocols

[0177] In some approaches, a linear DNA-based approach is used to generate a sequence library comprising double-stranded adaptor-containing constructs containing a target sequence, that is, DNA circles are not generated in the process.

[0178] 6.1 Add adapters to both ends of the genomic fragment and amplify the genomic fragment

[0179] First, adapters are added to both ends of the genomic fragment, such as Figure 2 In some approaches, amplification of genomic fragments is similar to that in Section 5.1 above, except that amplification is performed in a reaction mixture containing uracil by a polymerase or using primers containing uracil, thereby generating amplified nucleic acid fragments that incorporate uracil in the reaction mixture. Figure 5 An example is shown in step (i), where uracil is part of the amplification primer (not shown) and is incorporated into the amplified genomic fragment during the amplification process.

[0180] 6.2 Creating the cutout

[0181] Next, the nick is introduced into the amplified genomic fragment. In some approaches, the amplification is carried out in the presence of uracil as described above, and the nick can be introduced into the amplified genomic fragment by contacting the amplified genomic fragment containing uracil with uracil-DNA glycosylase. Uracil glycosylase can remove uracil to form abasic sites. An enzyme (e.g., APE1 or EndoIV) is also added in the reaction to remove sugar groups from abasic sites. Using the enzyme as described above, the genomic fragment containing uracil is treated in this way, resulting in a nick in the extension product in the region containing the uracil base, with the two wings of each nick being a 5-primer exposed end and a 3-primer exposed end.

[0182] Preferably, uracil is spiked into the amplification reaction after the extension of the amplification primers passes through the barcode region but before reaching an extension length of about the size of the desired read length (also referred to as a length suitable for sequencing). The length suitable for sequencing can range from 25 to 1000 bases, depending on the read length specified by the sequencing method. In some approaches, this is achieved by adding uracil to the reaction mixture after extension has begun, i.e., when all other components required for amplification have been added to the reaction mixture. In some approaches, uracil is spiked into the reaction mixture within about 10 seconds to 10 minutes after extension begins.

[0183] In other approaches, primers used to amplify genomic fragments contain uracils, which are incorporated into the amplified genomic fragments

[501] . In some embodiments, the forward primers contain one or more uracils. In some embodiments, each forward primer contains a single uracil, thereby generating a nick in each double-stranded nucleic acid fragment

[502] (after enzymatic treatment as described above to remove uracils).

[0184] 6.3 Aliquoting

[0185] The reaction mixture is then divided into aliquots. Figure 5 , step (iii).

[0186] 6.4 Nick Translation to Generate Nested Groups of Nucleic Acid Constructs

[0187] Next, the aliquots are subjected to nick translation using a DNA polymerase having 5'→3' exonuclease activity to synthesize a DNA chain having a newly formed end (second end). Non-limiting examples of DNA polymerases include DNA Pol1, Taq, Bst full length, and Pfu DNA polymerase. The end opposite to the second end is the first end. The extension is controlled so that the DNA chains synthesized in different aliquots have different lengths. Each synthesized DNA chain includes a first end and a second end, and the DNA chains in different aliquots share the same sequence near the first end, but have different sequences near the second end

[503] . Each synthesized DNA chain includes a target sequence portion having a first end and a second end, the second end being the end formed by nick translation and the first end being the end opposite to the second end. The DNA chains in different aliquots share the same sequence near the first end, but have different sequences near the second end. Figure 5 An illustrative example is shown in step (iv).

[0188] 6.5 Branch connections in individual aliquots

[0189] After the nick translation reaction is completed, an adapter (second adapter) is added to the aliquot. These second adapters are connected to the second end of the newly synthesized DNA chain. Each second adapter is partially double-stranded and comprises a first adapter oligonucleotide and a second adapter oligonucleotide. The first and second adapter oligonucleotides are complementary to each other and hybridize. In the branch connection process, the 5-primer end of the first adapter oligonucleotide is connected to the 3-primer end of the DNA chain synthesized by nick translation as described above (for example, Figure 5 in

[504] ).

[0190] In some approaches, the second adapter includes a unique position barcode for the aliquots. In some approaches, the aliquots, now containing unique position barcodes, are then combined into a single reaction mixture (e.g., Figure 5 in

[505] ).

[0191] In some approaches, the second adapter further comprises an anchoring component for separating the fragments connected to the second adapter from the fragments not connected to the second adapter. In some approaches, the anchoring component allows the fragments with the adapter to be captured by the solid support, and the captured fragments with the adapter can then be separated from other reagents in the solution. In some approaches, the anchoring component can be biotin, and the solid support is coated with streptavidin. In some approaches, the anchoring component is an oligonucleotide in the second adapter, and the solid support is a magnetic bead with the oligonucleotide immobilized thereon.

[0192] The synthesized DNA strands ligated to the second adapter from the different aliquots of (v) are then combined to form a single mixture (e.g., Figure 5 Step (vi)

[505] , where the dashed ellipse represents a single mixture). Subsequent steps are all completed in the single mixture.

[0193] 6.6 Extending the Second Adapter Oligonucleotide of the Second Adapter to Form a Double-Stranded Fragment

[0194] Branch connection causes the first adapter oligonucleotide to be connected to the nucleic acid construct, while the second adapter oligonucleotide is not connected but still hybridizes with the first adapter oligonucleotide that is now connected.Then the primer is hybridized with the first adapter oligonucleotide, and the hybridized primer is extended to generate a double-stranded fragment.In some approaches, the double-stranded fragment produced has a blunt end.In some approaches, the double-stranded fragment so produced comprises a unique position barcode of each aliquot. Figure 5 An illustrative example is shown in step (vii).

[0195] 6.7 Generation of linear double-stranded constructs with adapters

[0196] In some embodiments, double-stranded DNA molecules with a length suitable for sequencing are selected. In some approaches, double-stranded fragments with a length in the range of 200 bp-1.5 kb (e.g., 500-1000 bp) are selected. In some approaches, the selected double-stranded fragments are connected to an adapter ("third adapter") via blunt end connection or the like, thereby generating a double-stranded construct with an adapter. See Figure 5 , step (viii). The double-stranded adaptor-bearing construct can then be sequenced as disclosed herein.

[0197] The sequence near the positional barcode in the double-stranded fragments in each aliquot can be determined by sequencing, and sequence reads corresponding to different target sequence portions in each nucleic acid construct can be assembled to generate sequence information for the entire target sequence.

[0198] 7. Loop-mediated complete stLFR

[0199] A loop-mediated complete stLFR method is also provided for preparing libraries to sequence long DNA molecules. The loop-mediated complete stLFR of one embodiment of the method comprises preparing multiple nested groups of single-stranded nucleic acid constructs using any of the methods disclosed herein. Each single-stranded nucleic acid construct in each nested group comprises a target sequence portion of a long DNA molecule flanked by a first adapter sequence at the 5' end and a second adapter sequence at the 3' end (e.g., see Fig. 13B The first adapter sequence includes a primer binding sequence from 5' to 3' (e.g. Fig.13A 1311 in), barcode sequences (e.g. Fig.13A 1319) and the first hybridization sequence (e.g. Fig.13A 1432 in). The second adapter sequence includes a second hybridization sequence. The first and second hybridization sequences are complementary to each other. Each target sequence portion has a first end and a second end, and the distance between the first end and the barcode sequence is shorter than the distance between the second end and the barcode sequence. The single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences. For each nested group of single-stranded nucleic acid constructs, the target sequence portions in the nested group have the same nucleotide sequence near the first end, and are different from each other due to truncation near the second end, so that each nested group of single-stranded nucleic acid constructs contains multiple target sequence portions with different lengths. Various schemes can be used to generate nested groups of target sequence fragments, as further described below.

[0200] In some embodiments, the method further comprises subjecting the plurality of nested sets of single-stranded nucleic acid constructs to hybridization conditions in a reaction such that the first adapter sequence hybridizes to the second adapter sequence, thereby forming a loop (e.g., Fig.141431 in). The method also includes extending the second adapter sequence using a DNA polymerase to replicate the barcode sequence and primer binding sequence in the first adapter sequence. The method also includes denaturing the reaction to open the ring and form a linear single-stranded DNA construct. Each linear single-stranded DNA construct comprises a barcode sequence and a primer binding sequence at the 3' end. In some embodiments, the method also includes annealing a primer to the primer binding sequence at the 3' end of the linear single-stranded DNA construct, and extending the primer to generate an extension product having a length suitable for sequencing. The details of the complete stLFR method mediated by the ring will be further discussed below.

[0201] An exemplary embodiment of loop-mediated complete stLFR includes ligating two partially double-stranded blunt-end adapters (respectively comprising a first adapter sequence and a third adapter sequence) to an end-repaired DNA fragment with a 5'-phosphate group to prepare double-stranded genomic fragments with adapters, each comprising a target sequence flanked by a first adapter sequence and a third adapter sequence. In some embodiments, the third adapter sequence is added to the DNA fragment by nick translation, such as Fig.13A As described in Example 4.

[0202] In some embodiments, double-adapter, double-stranded genomic fragments are amplified. Random nicking and gapping are performed on the double-adapter, double-stranded genomic fragments. Random nicking generates nested groups of fragments that have target sequence portions of different lengths and share a common barcode sequence at the 5-primer. One such fragment is Fig. 13B .Displayed as 1341 in

[0152] .

[0203] In the case of using an exonuclease to open the gap as described above, protection of the DNA adaptors can be achieved, if desired, by phosphorothioate bonds between bases and / or modified bases at the 5' and 3' ends of the adaptors.

[0204] The second adapter (e.g., Fig. 13B AD153UMI_5R) shown in FIG is ligated to the 3'-side of the nick or ssDNA gap in the adapter-ligated DNA fragment. This generates a set of fragments with different lengths of target sequence portions, each flanked by two adapter sequences (e.g., Fig. 13B AD153UMI_5 and AD153UMI_5R in FIG. ... Fig. 13B The second adapter is a partially double-stranded DNA adapter molecule, comprising a longer strand (such as Fig. 13B 1320.1 in ) and a shorter chain (such as Fig. 13B 1320.2 in ). The longer chain has a 5'-phosphate group. The longer chain also includes a first hybridization sequence (e.g., Fig.14 1432 in the sequence), the first hybridization sequence being located at the 3' side of the barcode sequence (e.g., Fig.14 1319 in ). The longer strand of the second adapter includes a second hybridization sequence (e.g., Fig.14 The first and second hybridization sequences are complementary and can hybridize to each other under conditions suitable for hybridization, as further disclosed below.

[0205] The ligation of the first and / or second adapters to the DNA fragments via branched ligation can be performed in solution or on beads. In the case where the process is performed on the surface of beads, the DNA fragments with adapters are preloaded onto the beads with a high concentration of PEG (5%-15%) before adding other reaction components. In some embodiments, the branched ligation reaction is performed in the presence of an additive (e.g., polyethylene glycol or betaine) to increase the activity of the ligation and / or nicking enzyme. The reaction can be incubated at room temperature, 37°C, or cycled between different temperatures, such as 5-15°C and 37°C, with a pH range of 5.0 to 9.0. Incubation can last from 5 minutes to several hours. The amount of time and the synergy of the nicking enzyme vary depending on the number of nicks required for each DNA fragment. If performed in solution, the reaction can be stopped by a DNA purification method (e.g., Ampure XP beads), and if performed on beads, the reaction can be stopped simply by a washing step using a Tris NaCl buffer containing PEG (5%-15%).

[0206] In some embodiments, after branch ligation, the DNA fragments are denatured to generate single-stranded DNA molecules, each comprising a target sequence portion flanked by an adapter sequence having single-stranded hybridization sequences (e.g., Fig. 13B The molecule shown in the lower left). In some embodiments, after branch ligation, the branch-ligated DNA fragments can be heat denatured (90°C-95°C). Alternatively, the branch-ligated DNA fragments can be denatured with an alkaline agent (e.g., 0.05M-0.2M NaOH or KOH) and further neutralized with a neutralizing agent (e.g., HCl, Tris-HCl, MOPS). A single-stranded DNA molecule (e.g., Fig. 13B 1343 in ).

[0207] Alternatively, in some embodiments, rather than denaturing the branch-ligated DNA fragments, the branch-ligated DNA fragments are digested using one or more dsDNA-specific exonucleases (e.g., exonuclease III) with 3'-5' exonuclease activity to expose the 5' single-stranded first hybridization sequence in the first adapter (e.g., FIG. 13 or Fig.141432), which can be used to hybridize with the second hybridization sequence in the second adapter at the 3' end of the DNA fragment.

[0208] Hybridization between the first and second hybridization sequences can be carried out in a hybridization buffer containing a buffer (e.g., Tris-HCl, MOPS, sodium phosphate) and / or salt. In some embodiments, the hybridization buffer also includes auxiliary factors, such as MgCl and dNTP, for subsequent enzymatic reactions.

[0209] The DNA hybridization step is followed by extension of the hybridized 3'-ends of the branched adapters (e.g. Fig.14 AD153UMI_5R shown) to replicate the first adapter (e.g. Fig.14 The barcode and primer binding sequence on the AD153UMI_5 shown in FIG. 5A . In some embodiments, in order to increase specificity, one or more DNA polymerases lacking 3'-exonuclease activity are used for extension. Exemplary DNA polymerases that can be used include, but are not limited to, Taq DNA polymerase, Klenow fragment (3'→5' exonuclease) and BstDNA polymerase, large fragment. In some embodiments, extension is performed at a temperature suitable for the polymerase to perform polymerization. In some embodiments, the temperature range is 30°C to 75°C.

[0210] Linear extension products (e.g. Fig.14 The double-stranded or partially double-stranded DNA molecule comprises a double-stranded adapter containing a barcode sequence, and the barcode sequence is attached to the target sequence portion of the long DNA molecule. The double-stranded or partially double-stranded DNA molecule is then denatured to open the loop and form a single-stranded fragment (e.g., Fig.14 1441 in the fragment, which includes a target sequence portion flanked by a first and a second adapter sequence. By replicating the barcode, each end of the target sequence portions 1411, 1421, 1431, etc. of different lengths is brought to the vicinity of the barcode sequence 1319 on the same DNA chain, so that these ends (with different target sequences) can be sequenced and assembled based on the public barcode sequence 1319. The adapter sequence at the 3-primer of the target sequence portion now includes a copy of the barcode sequence and a copy of the primer binding sequence. In some embodiments, the primer binding site is recognized by a universal amplification primer. The primer can anneal to the primer binding sequence and extend to generate an extension product having a length suitable for sequencing. The extension product can be connected to a fourth adapter (e.g., Fig.15 The fragment thus produced (e.g. Fig.15 1510) was amplified by PCR and circularized for DNB sequencing.

[0211] Other exemplary schemes for generating nested groups of target sequence fragments are disclosed below in Section 8, "Concatemer-based methods" and Section 9, "Combined schemes"; the first scheme starts with ssDNA or dsDNA circles and the second scheme starts with linear ssDNA or dsDNA. Both schemes involve adding adapter sequences at both ends of the target molecule, which can be done by adapter ligation, amplicon PCR, and any number of other strategies. One of these adapters has a barcode sequence that can later be used to identify all reads from that particular DNA fragment / molecule.

[0212] 8. Concatemer-based methods

[0213] 8.1.1. Concatemer

[0214] In some embodiments, a method based on concatemers is provided herein, which produces nested groups of fragments with adapters of target sequence fragments of different lengths. In some embodiments, concatemers are produced by rolling circle replication of single-stranded circular templates. Single-stranded circular templates can be produced by cyclizing single-stranded DNA molecules using methods well known in the art. For example, cyclization can be performed by using a splint oligonucleotide with a sequence complementary to the adapter sequence at both ends of the molecule, so that the 5' end and the 3' end are connected together for connection. DNA concatemers can then be produced by extending the primers of the sequence annealed to the circular template by a DNA polymerase with strand displacement activity.

[0215] The circular DNA template disclosed in the present invention comprises a barcode, a primer sequence and a target sequence. After the ring is made, the next step is to form a DNA concatemer, such as a DNA nanoball (DNB). The incubation time for making concatemers can range from 20 minutes to several hours. Longer concatemer manufacturing times may result in very long concatemers (>100kb), which can be broken into separate concatemers. Since each ring contains a unique barcode, this break is not a problem because all reads from these separate concatemers can still be correctly identified using the barcode information. Each concatemer contains a plurality of identical monomers, each monomer comprising a complement of the target sequence, a complement of the barcode sequence that identifies the DNB, and a primer binding sequence. The primer binding sequence includes a sequence complementary to the primer sequence. In some embodiments, the primer binding sequence is shared by a population of single-stranded concatemers.

[0216] 8.1.2. Generating Multiple Extension Primers Separated by Spacers

[0217] After concatemers are formed, they are converted to dsDNA by extending primers that anneal to the primer binding sequences in the concatemers. In some embodiments, the primers are used at concentrations sufficient to ensure that almost all primer binding sites on the DNB are occupied by extension primers. Extension can then be performed using a polymerase that lacks 5-3' exonuclease activity and does not have strand displacement activity. This results in the formation of a DNA complex containing multiple extension primers that are complementary to one or more monomers of the DNA concatemer. These extension primers in the DNA complex hybridize to the DNA concatemer and are separated by spacers. For example, see Fig. 6A Each extension primer comprises a target sequence fragment. In some embodiments, the primer is a DNA primer. In some embodiments, the primer is an RNA primer. In some embodiments, the primer is a mixture of an RNA primer and a DNA primer.

[0218] Non-limiting examples of DNA polymerases known to lack 5-3' exonuclease activity and strand displacement activity include Klenow exo-, Q5, hemo klen Taq, T7 polymerase, T4 polymerase, which are readily available from commercial sources (e.g., New England BioLabs, Ipswich, Massachusetts).

[0219] 8.1.3. Creating Gaps for Adding Adapters (in Concatemer-Based Methods)

[0220] In some embodiments, the spacing between fragments in the DNA complex as described above is extended (widened) by an exonuclease to form a gap. See, e.g. Figure 6B . This process may be referred to as "gapification", and the exonuclease used in the process may be referred to as a "gap-forming enzyme". Examples of enzymes with 3' exonuclease activity include DNA polymerase I, Klenow fragment (in the absence of nucleotides), exonuclease III, and other enzymes known in the art. Examples of enzymes with 5' exonuclease activity include Bst DNA polymerase, T7 exonuclease, truncated exonuclease VIII, Lambda exonuclease, T5 exonuclease, and other exonucleases known in the art. Low-processing exonucleases (i.e., exonucleases that remove nucleotides from the ends of polynucleotides at a relatively low rate) preferably open short gaps (e.g., 2-7 bases, 3-10 bases, or 3-20 bases) to allow adapters to connect. Table 1 shows exemplary exonucleases that can be used.

[0221] Table 1. Exemplary exonucleases

[0222] For the above-mentioned case where fragments are generated by extending RNA primers, RNase H can be added to degrade the RNA primers, thereby extending the interval to form a gap. The gap will generally have the length of the RNA primer (e.g., 8-40 bases, 10-35 bases, or 10-25 bases). The 5' end and 3' end of the two wings of the interval can be connected to an L-adapter and a 3' branched ligation adapter, respectively.

[0223] Fig. 6A and Figure 6B The process of using one or more gapping enzymes (eg, exonucleases having 3'→5' exonuclease activity) to widen the gap (160) thereby creating a gap (170) is shown. Fig. 7A and Figure 7B The process of using one or more gapping enzymes (eg, exonucleases having 5'→3' exonuclease activity) to widen the gap (260) thereby creating a gap (270) is shown.

[0224] To maintain the integrity of the barcode sequence, the exonuclease to be used should only be used to digest the ends that are farther from the barcode sequence. For example, if the barcode sequence is closer to the 5-primer end of the target sequence fragment (e.g. Figure 6B If the barcode sequence is closer to the 3-primer end of the target sequence fragment (as shown in Figure 7B If the fragment is not cleaved, an exonuclease with 5' to 3' exonuclease activity is used to digest the fragment starting from the 5-primer end.

[0225] If the primer binding sequence is located at the 3-primer complement of the barcode sequence in each monomer (i.e., the position of the extension primer is at the 5' end relative to the barcode sequence in the extension primer), a 3-5' exonuclease can be added during the ligation step to create target sequence fragments that have different sequences at the 3-primer end due to truncation, but have the same sequence at the 5-primer end ( Figure 6B ). Importantly, this results in each adapter-bearing fragment containing the barcode and covering the entire length of the original molecule containing the target sequence. In addition, L-oligo adapters can be designed to recognize partial adapter sequences on concatemers to improve ligation efficiency.

[0226] If the primer is located at the 5-primer complement of the barcode sequence in each monomer (i.e., the position of the extension primer is at the 3-primer relative to the barcode sequence in the extension primer), then 5'-3' exonuclease can be used instead, which generates target sequence fragments that have different sequences at the 5-primer end due to truncation and have the same sequence at the 3-primer end ( Figure 7B ).

[0227] If the primers used are DNA extension primers, a low concentration of exonuclease can be added, followed by ligase, adapters, and more exonuclease. This will cause most gaps to become gaps before the ligase has a chance to reseal them. If the primer binding sequence is located at the 3-primer complement of the barcode sequence in each monomer (e.g. Fig. 6A ), an exonuclease with 3'→5' exonuclease activity ( Figure 6B If the primer is located at the 5-primer position that complements the barcode sequence in each monomer (e.g. Fig. 7A ), an exonuclease with 5'→3' exonuclease activity ( Figure 7B ).

[0228] 8.1.4. Simultaneous ligation and exonuclease treatment

[0229] In some embodiments, exonucleases are used to generate target sequence fragments of different sizes. Due to the randomness of exonucleases, exonuclease treatment results in the distribution of primers of different sizes, truncations, and extensions, which include target sequence fragments. These target sequence fragments have the same nucleotide sequence at the first end, but differ from each other at the second end due to truncation, so that each group of fragments with adapters contains target sequence fragments of different lengths. In some embodiments, the first end is the 5-primer end of the target sequence fragment, such as Figure 6B In some embodiments, the first end is the 3-primer end of the target sequence fragment, such as Figure 7B As shown in (291) in .

[0230] In some embodiments, exonuclease treatment of the extension primer and ligation of the extension primer to the adapter occur in the same reaction mixture. In some embodiments, ligation includes at least ligating the branch adapter to the nucleic acid fragment. In some embodiments, ligation includes ligating the branch adapter and the L-adapter in solution to the extension primer. The following are exemplary conditions under which exonuclease treatment and ligation can occur in the same reaction.

[0231] temperature

[0232] The reaction can be maintained at a temperature in the range of 5-65° C. (e.g., 5-42° C., 10-37° C., or 5-15° C.). In some embodiments, the reaction is maintained at room temperature, 37° C. In some embodiments, when a thermostable ligase and exonuclease are used, the reaction can be maintained at a temperature above 37° C.

[0233] pH

[0234] In some embodiments, the pH of the reaction mixture is maintained in a pH range of 5.0 to 9.0 (e.g., 7.0 to 9.0) to accommodate all enzymatic functions required for library preparation. The duration of the exonuclease treatment and ligation reaction may vary depending on the desired nucleic acid fragment size and other conditions (e.g., enzyme (including polymerase, exonuclease or both) concentration, time, temperature, amount of input DNA).

[0235] time

[0236] Typically, the duration of connection and exonuclease treatment reaction can last for 5 minutes to 5 hours, for example 15-90 minutes or 30-120 minutes. The reaction can be terminated using methods well known in the art. In some embodiments, exonuclease treatment and connection are carried out in solution, and the reaction can be terminated by DNA purification method (for example, AmpureXP beads from Beckman Coulter). In some embodiments, exonuclease treatment and connection are carried out on beads, and the reaction can be terminated by washing beads with buffer (for example, Tris NaCl buffer) to remove enzymes and components required for nick and ligation reaction.

[0237] 8.1.5. Connection of adapters

[0238] As discussed above and in Figure 6B As shown in , an extension primer (130) annealed to an adapter sequence in a DNA concatemer generates a plurality of extension primers (150), each primer having a 5' end and a 3' end. In some embodiments, each of at least some of the extension primers is connected to two adapters, one adapter at either end. In some embodiments, an L-adapter is connected to the 5' end of the extension primer, and a branch adapter is connected to the 3' end of the extension primer. The result is a plurality of adapter-attached fragments having two different adapter sequences; and all adapter-attached fragments generated in the reaction have the same defined arrangement (e.g., an L-adapter at 5' and a branch adapter at 3').

[0239] 9. Combination Solution

[0240] In some embodiments, the method disclosed herein can be combined with the stLFR method to sequence long genomic DNA (e.g., genomic fragments of 20kb to 200kb in length). In this case, we will perform stLFR as disclosed in, for example, U.S. Patent No. 9328382B2 and PCT Publication WO2023001262, and then perform the above method and adjust the size of the barcoded fragment from about 300bp to 1000bp or from 500bp to 1500bp or from 1kb to 3kb. The advantage of using a longer insert is that the deviation of the enzyme (e.g., transposase or DNA nickase) that realizes stLFR coordinated barcoding can be tolerated. It is also easier to remove stLFR adapter-adapter artifacts (artefact) by size selection (i.e., removing DNA less than 300bp in length). The barcode provided by the beads will become the barcode used for each ring. After performing the above process, each 2kb fragment will have a read coverage greater than 1x (X) and can be combined with other fragments sharing the same stLFR barcode to create long fragments (up to several megabase pairs) with a coverage of close to 1x or higher for the entire fragment (average read coverage is 5-15x). Figure 8 An exemplary method according to this embodiment is shown.

[0241] Overall, 10-fold or more coverage in long fragments of each haplotype (where each long fragment receives ~5-10-fold read coverage) will require 50-100-fold total read coverage per haplotype. As the cost of MPS (NGS) decreases further, such complete and accurate WGS will become more affordable.

[0242] In some embodiments, the method starts with any linear DNA molecule with an adapter at at least one end. In some embodiments, the method starts with a PCR amplicon, which can provide enough copies of each barcoded molecule. In some embodiments, the process can be performed in solution. In some embodiments, the method can be performed on one end (5-primer or 3-primer) bead to which the adapter of the linear DNA molecule is fixed ( Fig. 9 ), and the adapter comprises a unique barcode. In some embodiments, the barcoded fragment comprises a barcode sequence, a target sequence, and a primer binding sequence, wherein the 3-primer end of the barcoded fragment is immobilized on a bead. In some embodiments, the barcoded fragment comprises a barcode sequence, a target sequence, and a primer binding sequence, wherein the 5-primer end of the barcoded fragment is immobilized on a bead. In some embodiments, the primer binding sequence is at the 3 primer relative to the barcode sequence.

[0243] Polynucleotides (e.g., barcoded fragments) can be immobilized on beads in a variety of ways, including covalent and non-covalent attachment. In some embodiments, the 3' or 5' end of the polynucleotide adapter is attached to biotin, and the barcoded fragments are captured on beads coated with streptavidin. In some embodiments, the polynucleotide is connected to a substrate (e.g., a bead), that is, one end of the polynucleotide is directly in contact with or connected to the substrate. For example, the surface may have reactive functions that react with complementary functional groups on the polynucleotide molecules to form covalent bonds. Long DNA molecules (e.g., several nucleotides or larger) can also be effectively attached to hydrophobic surfaces, such as clean glass surfaces with low concentrations of various reactive functional groups (e.g., -OH groups). In another embodiment, the polynucleotide molecules can be adsorbed to the surface by non-specific interactions with the surface, or by non-covalent interactions (e.g., hydrogen bonds, van der Waals forces, etc.).

[0244] In some embodiments, polynucleotides (eg, barcoded fragments) are immobilized to a surface by hybridizing to a capture oligonucleotide on the surface and forming a complex (eg, a double-stranded duplex or a partially double-stranded duplex) with a component of the capture oligonucleotide.

[0245] In some embodiments, the method uses a primer comprising a primer sequence that is complementary to a primer sequence in the barcoded fragment. In some embodiments, the primer is a tailed primer comprising a tail that is not complementary to the barcoded fragment. In some embodiments, the tail comprises a common adapter sequence. Fig. 9 .

[0246] In some embodiments, extension is controlled so that the polymerase extends the primer beyond the barcode region on the barcoded fragment. In some embodiments, the polymerase extends the primer beyond the barcode region by a length that is approximately equal to a length suitable for sequencing (also known as a sequencing read length), such as in the range of 25-1000 bases. Fig. 9. The extension product, i.e., the extension primer, can be separated from the barcoded fragment and the barcoded fragment can be used as a template for subsequent extension reaction cycles. A variety of methods for controlling the extension reaction can be used, which will be further described in Section 3.4 ("Controlled Extension") below. In some embodiments, primer extension (one or more cycles) can be controlled in a manner such that subsequent extension cycles produce longer or shorter extension products than the previous extension cycle. In some embodiments, the controlled extension is performed in the presence of a reversible terminator. In some embodiments, controlled extension is performed by using different polymerases capable of longer or shorter extensions. One of the advantages of using a terminator is that the length of the addition polymerization can be controlled by the concentration of the terminator, which is easier to control than time. At the end of the polymerization, the terminator can be reversed and the beads washed. In some embodiments, the beads can be washed in a buffered NaCl solution. Branch adapters (e.g. Fig. 9 After the DNA is denatured using heat or alkaline conditions, the supernatant can be collected and purified.

[0247] This process (e.g., primer extension and denaturation) can be performed for multiple rounds to generate nested groups of adapter fragments that contain target sequence fragments of different lengths, thereby covering the entire original DNA molecule. These adapter fragments also share the same barcode sequence. The method will produce variable target sequence fragments ranging in size from about 100bp to 5000bp, 100bp to 3000bp, 100bp to 1000bp, 100bp to 750bp, or 100bp to 500bp.

[0248] The target sequence fragments generated above can be cyclized for DNA nanoball (DNB) preparation and sequencing. As described above, these target sequence fragments have the same nucleotide sequence at the first end (the end closer to the barcode sequence), and are different from each other at the second end due to truncation. In some embodiments, sequencing is double-end sequencing, including sequencing from either end of the same DNA fragment. In some embodiments, the first reading read is generated by extending the sequencing primer annealed to the adapter sequence, and the adapter sequence is closer to the first end of the target sequence fragment than the second end ("first reading sequencing"), and the second sequencing reading is generated by extending the sequencing primer annealed to the adapter sequence, and the adapter sequence is closer to the second end of the target sequence fragment than the first end ("second reading sequencing"). The first reading sequencing will generate a barcode sequence. The second reading sequencing will generate overlapping readings to substantially or completely cover molecules with a length of 500bp or 700bp or 1000bp. These overlapping sequencing reads will be clustered according to the barcode sequence determined by the first reading sequencing in the de novo assembly.

[0249] In some embodiments, to more efficiently cover the entire molecule but avoid generating overly long fragments (e.g., more than 700 bp, or more than 1000 bp, or more than 1500 bp, or more than 2000 bp, or more than 3,000 bp), uracil is incorporated into the middle part of the extension step. For example, uracil can be added to the reaction after the extension has passed the barcode region, but before reaching an extension length that is approximately the size of the desired read length (e.g., 25-1000 bases, depending on the read length specified by the sequencing method). This can be achieved by extending the primers for a first extension period, then adding uracil to the reaction mixture in bulk to allow the primers to continue to extend for a second extension period, then washing the beads, and adding a new uracil-free deoxynucleotide mixture (normal deoxynucleotides) to the reaction to allow the primers to further extend for a third extension period ( Figure 10A-10B In some embodiments, the third extension reaction (uracil-free extension) is performed in the presence of a reversible terminator, for example, using a mixture of normal nucleotides and a reversible terminator.

[0250] After the extension reaction is completed, the terminator (if used) can be reversed, the beads are washed, and the extension product is connected to the 3' branch connection adapter. Next, uracil glycosylase can be added to remove uracil to form an abasic site, and an enzyme that can remove glycosyl from the abasic site is added to the reaction. This will result in fragmentation of the extension product in the region containing the uracil base. Non-limiting examples of enzymes that can remove glycosyl from abasic sites include APE1 or EndoIV. Removal of these fragmented products will leave gaps with 5-primer exposed ends and 3-primer exposed ends on both wings. L-adapters and internal branch adapters are connected to the exposed 5-primer ends and the exposed 3-primer ends. In some embodiments, the L-adapter sequence, the internal branch adapter sequence, the adapter sequences at the 5' end and the 3' end of the extension fragment can all be distinguished from each other. The 5' and 3' ends of the two adapters can then be joined via the splint oligonucleotide and ligated by T4 ligase to rejoin the 5' and 3' sides of the extended fragment (the single-stranded portion of the template molecule will fold, bringing the two adapters into close proximity to allow hybridization of the splint oligonucleotide, Figure 5 B).

[0251] The product can now be denatured, separated from the beads and collected. In some cases, the beads can be reused for one or more cycles. Fig. 10C The products of each round of denaturation are collected and sequenced. This process reduces the total length of the target sequence fragment in each adapter-bearing fragment by removing a portion of the extension product. Fig. 10C . Fig.11Shown are shortened target sequence fragments having sequences corresponding to different regions of the target sequence of the original molecule. This enables molecular read coverage of up to 1500bp, 2000bp, 3000bp, 4000bp or 5000bp to be achieved by MPS. For a 3kb molecule, to obtain 10 times coverage (300 bases per read), 100 such reads are required, because of the loss in library preparation and DNB or clustering, so that library processing preferably starts with at least 300, at least 1000 or at least 10,000 copies of each DNA molecule. If beads are reused, the number of copies can be reduced by 2-10 times or even more. If there are longer sequencing reads (e.g., 600 bases) and 25-100 repetitions of shorter template molecules (e.g., 1500bp) are performed, the process can be run without initially amplifying molecules by linear or exponential PCR or other methods. However, the amplified molecules allow multiple reactions (2 or more, 3 or more, 4 or more, 2-6, or 4-8) in parallel with increasingly longer uracil regions to optimally cover all regions of DNA molecules ranging in length from 1 kb to 10 kb (e.g., from 1 kb to 5 kb, or from 1 kb to 3 kb).

[0252] Another exemplary method to solve this problem (i.e., the original molecule containing the target sequence is too long to be sequenced) is to use a first branch adapter having a degenerate sequence region in the 3-primer portion. The first branch adapter is connected to the extended tailed primer formed after the first extension as described above. The first extension is controlled so that the primer extends beyond the barcode region. In some embodiments, the degenerate sequence region contains 3-10, for example, 3-8, 5-10 or 6-10 degenerate nucleotides. The first branch adapter can hybridize to a random position in the barcoded fragment through the degenerate sequence region, thereby skipping the replication of certain random parts of the barcoded fragment. A second controlled extension is then performed by extending the 3-primer end of the first branch adapter. A second extension can be performed so that 100 to 300 bases are added to the 3-primer end to form a second extension product. The second branch adapter can then be connected to the 3-primer end of the second extension product to produce a fragment with an adapter. See. Fig.12 .

[0253] In some embodiments, the adapter fragments are denatured and released from the beads. The barcoded fragments can be used as extension templates for additional extension cycles to generate more adapter fragments. The first and second extensions in each cycle are controlled so that adapter fragments are generated from cycles with overlapping target sequence fragments. These adapter fragments can be sequenced, and sequencing reads of overlapping target sequence fragments can be assembled to generate sequence information for the entire target sequence.

[0254] In some embodiments, the barcoded fragments have been amplified so that multiple copies of the barcoded fragments are used as extension templates (e.g., for extending primers annealed to the barcoded fragments). In some embodiments, these multiple copies are immobilized on the same bead. In some embodiments, these multiple copies are immobilized on more than one bead. The copies can be identified by the same barcode they share. In this embodiment, one cycle (including a first extension, ligation to a first branch adapter, a second extension, and ligation to a second branch adapter) is generally sufficient to generate overlapping target sequence fragments. However, if desired, the extension products can be denatured and released from the beads, and the barcoded fragments can be reused for additional cycles of generating additional adapter-containing fragments as described above.

[0255] 10. Exemplary embodiments of the present disclosure

[0256] Embodiment 1 is a method for producing a single-stranded construct with an adapter for sequencing, comprising: preparing multiple nested groups of single-stranded nucleic acid constructs, wherein each single-stranded nucleic acid construct in each nested group comprises a target sequence portion, the target sequence portion is flanked by a first adapter sequence at the 5' end and a second adapter sequence at the 3' end, wherein the first adapter sequence comprises a primer binding sequence, a barcode sequence and a first hybridization sequence from 5' to 3', and the second adapter sequence comprises a second hybridization sequence, wherein the first and second hybridization sequences are complementary to each other, wherein each target sequence portion has a first end and a second end, wherein the distance between the first end and the barcode sequence is shorter than the distance between the second end and the barcode sequence, wherein the single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences, and wherein for each nested group of single-stranded nucleic acid constructs, the target sequence portions in the nested group have the same nucleotide sequence near the first end and differ from each other due to truncation near the second end, so that each nested group of single-stranded nucleic acid constructs comprises multiple target sequence portions with different lengths.

[0257] Embodiment 2 is the method of embodiment 1, wherein the method further comprises subjecting the plurality of nested sets of single-stranded nucleic acid constructs to hybridization conditions, whereby the first adapter sequence hybridizes to the second adapter sequence, thereby forming a loop.

[0258] Embodiment 3 is the method of embodiment 2, wherein the method further comprises extending the second adapter sequence using a DNA polymerase to replicate the barcode sequence and the primer binding sequence in the first adapter sequence to form an extension product.

[0259] Embodiment 4 is the method of embodiment 3, wherein the method further comprises denaturing the extension product to open the ring, thereby forming a linear single-stranded DNA construct, wherein each linear single-stranded DNA construct comprises a barcode sequence and a primer binding sequence, wherein the primer binding sequence is located at the 3' end relative to the barcode sequence. In some embodiments, the primer binding sequence is located at the 3' end of the linear single-stranded DNA construct.

[0260] Embodiment 5 is the method of embodiment 4, wherein the method further comprises annealing a primer to the primer binding sequence at the 3' end of the linear single-stranded DNA construct, and extending the primer to generate an extension product having a length suitable for sequencing.

[0261] Embodiment 6 is a method for generating a single-stranded DNA circle containing a single-stranded adapter-containing construct for sequencing, comprising: preparing multiple nested groups of single-stranded nucleic acid constructs, wherein each single-stranded nucleic acid construct in each nested group comprises a target sequence portion flanked by a first adapter sequence and a second adapter sequence, wherein the first adapter sequence comprises a barcode sequence and a primer binding sequence, wherein each target sequence portion has a first end and a second end, wherein the distance between the first end and the barcode sequence is shorter than the distance between the second end and the barcode sequence, wherein the single-stranded nucleic acid constructs in each nested group share the same barcode sequence and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences, wherein for each nested group of single-stranded nucleic acid constructs, (a) the target sequence portions in the nested group have the same nucleotide sequence near the first end and differ from each other due to truncation near the second end, so that each nested group of single-stranded nucleic acid constructs comprises multiple target sequence portions with different lengths, and (b) circularizing the single-stranded nucleic acid constructs in each nested group to generate a single-stranded DNA circle, wherein the first adapter sequence and the second adapter sequence are connected.

[0262] Embodiment 7 is a method as described in any one of embodiments 1-6, wherein each nested group of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each fragment comprising a target sequence flanked by a first adapter sequence and a third adapter sequence, (ii) amplifying the double-stranded genomic fragments with adapters by using primers that hybridize to the first and third adapter sequences to produce amplified genomic fragments, (iii) contacting the amplified genomic fragments from (ii) with a nicking agent to produce a nick in the target sequence in one strand of the amplified genomic fragments, (iv) ligating a second adapter comprising a second adapter sequence at the nick in (iii) via branch ligation to form a ligation product, and (v) denaturing the ligation product from (iv) to form single-stranded nucleic acid constructs, each single-stranded nucleic acid construct comprising a first adapter sequence and a second adapter sequence.

[0263] Embodiment 8 is a method described in any one of embodiments 1-6, wherein each nested group of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each fragment comprising a target sequence flanked by a first adapter sequence and a third adapter sequence, (ii) amplifying the double-stranded genomic fragments with adapters to produce amplified genomic fragments, (iii) distributing the amplified genomic fragments into a plurality of aliquots, (iv) denaturing the amplified genomic fragments in (iii) to prepare single-stranded genomic fragments, wherein at least some of the single-stranded genomic fragments each comprise a primer binding sequence, (iv) extending primers hybridized to the primer binding sequence under extension control conditions such that the lengths of extension products from different aliquots are different, thereby producing extension products having newly formed ends, and the extension products have different sequences near the newly formed ends in different aliquots, wherein each extension product comprises a portion of the target sequence, and (v) connecting a second adapter comprising a second adapter sequence at the newly formed end in each aliquot via branch connection, thereby producing single-stranded nucleic acid constructs, each of which comprises a first adapter sequence and a second adapter sequence.

[0264] Embodiment 9 is a method described in any one of embodiments 1-6, wherein each nested group of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each fragment comprising a target sequence flanked by a first adapter sequence and a third adapter sequence, (ii) amplifying the double-stranded genomic fragments with adapters to produce amplified genomic fragments, (iii) distributing the amplified genomic fragments into a plurality of aliquots, (iv) adding a double-stranded DNA nuclease having 3'5' nuclease activity to the plurality of aliquots under controlled conditions, such that the lengths of the products remaining after double-stranded DNA nuclease digestion in different aliquots are different, thereby producing digestion products having newly formed ends and having different sequences in different aliquots, wherein each digestion product comprises a portion of the target sequence, and (v) connecting a second adapter comprising a second adapter sequence at the newly formed end in each aliquot via branch connection, thereby producing single-stranded nucleic acid constructs, each of which comprises a first adapter sequence and a second adapter sequence.

[0265] The method of any one of embodiments 1-6, wherein each nested set of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each fragment comprising a target sequence flanked by a first adapter sequence and a third adapter sequence, (ii) amplifying the double-stranded genomic fragments with adapters to produce amplified genomic fragments, (iii) denaturing the amplified genomic fragments to prepare single-stranded genomic fragments, wherein at least some of the single-stranded genomic fragments each comprise a primer binding sequence, and (iv) for each single-stranded genomic fragment, extending a primer that hybridizes to the primer binding sequence for a first period of time to produce an extended primer, wherein the extension is incomplete such that the extension The length of the primer is a small fraction of the length of the single-stranded genomic fragment, wherein the extension primer comprises a portion of the target sequence, and a second adapter is connected to the end of the extension primer formed by the extension via branch connection, thereby producing single-stranded nucleic acid constructs in a reaction mixture, each single-stranded nucleic acid construct comprising a first adapter sequence and a second adapter sequence, (v) repeating step (iv) for multiple rounds, for each round, the primer is further extended for an additional period of time, and an additional adapter with a unique position barcode is connected to the further extended primer, wherein the molar amount of the additional adapter is a small fraction of the total molar amount of the amplified genomic fragment, thereby producing a mixture of nested groups of single-stranded nucleic acid constructs.

[0266] Embodiment 11 is the method described in embodiment 8, wherein the target sequence comprises a repetitive sequence, wherein the second adapter comprises a unique position barcode sequence for each aliquot, wherein the single-stranded nucleic acid constructs in different aliquots formed in (v) comprise different position barcode sequences, and the single-stranded nucleic acid constructs in the same aliquot share the same position barcode sequence.

[0267] Embodiment 12 is the method of embodiment 6, wherein the primer binding sequence is at 3-primer relative to the barcode sequence.

[0268] Embodiment 13 is the method described in embodiment 6, wherein the method further includes (vi) fragmenting the single-stranded DNA loop to produce multiple single-stranded DNA fragments, wherein at least some of the single-stranded DNA fragments contain barcode sequences, (vii) generating double-stranded DNA fragments from the single-stranded DNA fragments from step (vi), and (vii) connecting a second adapter to each double-stranded DNA fragment from step (vii) to thereby generate double-stranded fragments with adapters.

[0269] Embodiment 14 is the method of embodiment 13, further comprising (viii) amplifying double-stranded adapter-bearing fragments, and optionally (ix) selecting amplified double-stranded adapter-bearing fragments having a length ranging from 300 to 1000 bases.

[0270] Embodiment 15 is the method described in embodiment 6, wherein the method further comprises (vi) hybridizing a primer to a primer binding sequence in each single-stranded DNA loop, (vii) extending the primer under extension-controlled conditions using each single-stranded DNA loop as a template, wherein the extension produces an extension primer hybridized to the single-stranded DNA loop, thereby producing a plurality of extension primers having different lengths, wherein each of the extension primers comprises a barcode sequence and a primer binding sequence, and (viii) connecting a second adapter to the plurality of extension primers via branch connection to produce an extension primer with an adapter.

[0271] Embodiment 16 is the method described in any one of embodiments 6-15, wherein the method further comprises amplifying extension primers with adapters to produce amplified double-stranded fragments, selecting amplified double-stranded fragments with a length ranging from 300 bases to 1000 bases, and sequencing the selected amplified fragments with adapters.

[0272] Embodiment 17 is the method of embodiments 1-16, wherein the single-stranded DNA circle is prepared in solution without the need for a solid support.

[0273] Embodiment 18 is the method of embodiment 6, wherein the first end or the second end is attached to a solid support.

[0274] Embodiment 19 is a method for producing a double-stranded construct with adapters for sequencing, wherein the method comprises: (i) amplifying multiple genomic fragments, each genomic fragment comprising a target sequence, to produce multiple groups of amplified nucleic acid fragments in a mixture, wherein the amplified nucleic acid fragments in each group share the same target sequence, optionally, for each group, amplification is performed using target-specific primers, the method further comprising (ii) contacting the amplified nucleic acid fragments with an enzyme, wherein the enzyme introduces breaks in the amplified nucleic acid fragments, (iii) distributing the mixture of fragments into multiple aliquots, (iv) performing nick translation on the aliquots of the fragments to synthesize DNA chains under certain conditions so that the DNA chains synthesized in different aliquots have different lengths, wherein each of the DNA chains comprises a target sequence portion having a first end and a second end, and wherein the DNA chains in different aliquots share the same sequence near the first end and have different sequences near the second end, and (v) for each aliquot, connecting a second adapter to the second end of the DNA chain synthesized in (iv) via branch connection, wherein each second adapter is a partially double-stranded adapter comprising a first adapter oligonucleotide and a second adapter. oligonucleotides, wherein the first adapter oligonucleotide and the second adapter oligonucleotide are both complementary and hybridize to each other, wherein each of the second adapters comprises a positional barcode sequence, wherein each ligation comprises ligating the 5-primer end of the first adapter oligonucleotide of the second adapter to the second end of the synthesized DNA strand, wherein the first adapter oligonucleotides ligated to the second end of the synthesized DNA strand in different aliquots comprise different positional barcode sequences, and the first adapter oligonucleotides ligated to the second end of the synthesized DNA strand in the same aliquot share the same positional barcode sequence, and (vi) The synthesized DNA chains connected to the second adapter from different aliquots of (v) are combined into a single mixture, (vii) the primer hybridized to the first adapter oligonucleotide connected to the synthesized DNA chain is extended to produce a double-stranded fragment with a blunt end, and (viii) double-stranded fragments of (vii) with a size ranging from 200bp to 1.5kb (the optimal range of about 500-1000bp will be disclosed in the specification) are optionally selected from the single mixture, and (ix) the third adapter is connected to the blunt end of the double-stranded fragment to produce a double-stranded construct with an adapter.

[0275] Embodiment 20 is the method described in embodiment 19, wherein step (i) comprises amplifying multiple genomic fragments in a mixture comprising uracil, thereby producing amplified nucleic acid fragments doped with uracil, and wherein step (ii) comprises contacting the amplified nucleic acid fragments with uracil-DNA glycosylase, wherein the uracil-DNA glycosylase removes uracil from the amplified genomic fragments.

[0276] Embodiment 21 is the method of embodiment 19, wherein amplifying multiple genomic fragments in step (i) is performed using primers containing uracil, thereby generating multiple groups of amplified nucleic acid fragments containing uracil.

[0277] Embodiment 22 is the method of embodiment 21, wherein each of the plurality of genomic fragments is amplified using a forward primer and a reverse primer, and wherein each forward primer comprises one or more uracils.

[0278] Embodiment 23 is the method of embodiment 22, wherein each of the plurality of genomic fragments is amplified using a forward primer and a reverse primer, and wherein each reverse primer comprises a single uracil.

[0279] Embodiment 24 is the method of embodiment 19, wherein step (ii) comprises contacting the amplified genomic fragments with an endonuclease, wherein the endonuclease randomly cleaves the amplified genomic fragments.

[0280] Embodiment 25 is the method of embodiment 24, wherein the endonuclease is EndoIV or APE1.

[0281] Embodiment 26 is a reaction mixture comprising the single-stranded DNA circle produced in claim 6.

[0282] Embodiment 27 is a reaction mixture comprising the combinatorially synthesized DNA strands from step (vi) of claim 18.

[0283] Embodiment 28 is a method for preparing multiple nested groups of fragments with adapters, wherein each fragment with adapters is a single-stranded nucleic acid, which comprises a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer sequence and a barcode sequence, wherein in each nested group of fragments with adapters, the target sequence fragments have the same nucleotide sequence at the first end and differ from each other due to truncation at the second end, so that each nested group of fragments with adapters comprises multiple target sequence fragments with different lengths, wherein the first end is closer to the barcode sequence than the second end, wherein the method comprises: (a) providing a population of single-stranded DNA concatemers in a reaction, wherein each concatemer comprises a plurality of identical monomers, and each monomer comprises a complement of the target sequence, a complement of the barcode sequence that identifies the concatemer, and a primer binding sequence shared by the population of single-stranded concatemers, wherein the primer binding sequence comprises a sequence complementary to the primer sequence, wherein both the primer binding sequence and the barcode sequence complement are at the 3-primer position of the target sequence complement; (b) ) annealing a primer comprising a primer sequence to a primer binding sequence of a plurality of monomers of each concatemer in a plurality of concatemers; (c) extending at least some of the primers hybridized to the primer binding sequence with a DNA polymerase having 5'-->3' exonuclease activity and not having strand displacement activity, wherein the extension generates a plurality of extension primers, each of the extension primers comprising a target sequence fragment having a barcode sequence and a primer sequence, wherein the extension primers hybridize to the concatemers; wherein the extension primers are separated by a gap, and (d) extending the plurality of extension primers contacted with a 5-primer adapter comprising a 5-primer adapter sequence, a 3-primer adapter comprising a 3-primer adapter sequence, a DNA ligase, and an exonuclease having single-stranded DNA exonuclease activity under conditions where the exonuclease degrades a portion of the target sequence fragment in the extension primer to produce a shortened extension primer, the 5-primer adapter being connected to the 5' end of the shortened extension primer, and the 3-primer adapter being connected to the 3' end of the shortened extension primer, thereby producing groups of multiple nested groups of fragments with adapters.

[0284] Embodiment 29 is the method of embodiment 28, wherein the population of single-stranded DNA concatemers is generated by rolling circle replication of circular templates, wherein each circular template comprises a target sequence, a barcode sequence, and a primer sequence.

[0285] Embodiment 30 is the method of embodiment 28, wherein the 5-primer adapter is an L-adapter and the 3-primer adapter is a branched adapter.

[0286] Embodiment 31 is the method of embodiment 28, wherein the method further comprises adding a nuclease to extend the gap formed in step (c), wherein the nuclease has single-stranded exonuclease activity.

[0287] Embodiment 32 is the method of embodiment 31, wherein at least some of the primers are RNA primers, and wherein the nuclease is RNAse H, wherein the RNAse H digests the RNA primers, thereby extending the gap.

[0288] Embodiment 33 is a method described in embodiment 28, wherein the primer binding sequence is located at the 3-primer of the complement of the barcode sequence in step (a), wherein the exonuclease has 3'5' exonuclease activity, and wherein the barcode sequence in each of the group of fragments with adapters is located at the 5-primer relative to the target sequence fragment.

[0289] Embodiment 34 is a method described in embodiment 28, wherein the primer binding sequence is located at the 5-primer relative to the complement of the barcode sequence in step (a), wherein the exonuclease has 5'3' exonuclease activity, and wherein the barcode sequence is located at the 3-primer relative to the target sequence fragment in each fragment with an adapter.

[0290] Embodiment 35 is the method of any of the preceding claims, wherein the 5-primer adapter and the 3-primer adapter are both in solution.

[0291] Embodiment 36 is the method of embodiment 35, wherein the reaction does not contain a solid support.

[0292] Embodiment 37 is the method of any of the preceding claims, wherein the target sequence is between 500 bases and 50 kilobases in length.

[0293] Embodiment 38 is the method described in embodiment 30, wherein the branched adapter comprises a double-stranded blunt end, which comprises a 5' end of one chain and a 3' end of a complementary chain, and wherein the 5' end of the chain in the double-stranded blunt end is connected to the 3' end of at least one extension primer via a branch connection.

[0294] Embodiment 39 is the method of embodiment 30, wherein the L-adapter comprises 1-10 degenerate bases at the 3-primer.

[0295] Embodiment 40 is a method for preparing multiple nested groups of adapter-attached fragments, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer binding sequence, and a complement of a barcode sequence, wherein in each nested group of adapter-attached fragments, the target sequence fragments have the same nucleotide sequence at the first end and are different from each other at the second end, such that each nested group of adapter-attached fragments comprises a plurality of target sequence fragments having different lengths, wherein the first end is closer to the barcode sequence than the second end, wherein the method comprises (a) providing a barcoded fragment comprising a barcode sequence, a target sequence, and a primer binding sequence, wherein the barcoded fragment is immobilized on a bead at one end, (b) annealing a primer comprising a 5-primer adapter sequence to a primer in the barcoded fragment; The invention relates to a method of preparing a barcode fragment comprising a barcode bead, wherein the 5-primer adapter sequence comprises i) a barcode sequence complement, and ii) a primer sequence complementary to a primer binding sequence in the barcoded fragment, (c) extending the primer to produce an extended primer comprising a target sequence fragment and the barcode sequence complement, (d) contacting the extended primer with a branched adapter comprising a 3-primer adapter sequence to produce adapter-attached fragments, (e) separating the adapter-attached fragments from the barcoded fragments still immobilized on the beads, and (f) repeating one or more cycles of steps (b)-(e) under extension control conditions to produce one or more adapter-attached fragments, wherein the adapter-attached fragments produced from step (e) and the adapter-attached fragments produced from step (f) constitute nested groups of adapter-attached fragments, and wherein the adapter-attached fragments in each nested group include target sequence fragments having different lengths.

[0296] Embodiment 41 is a method described in embodiment 40, wherein the primer is extended with uracil in one or more extension cycles under extension-controlled conditions to produce an extended primer, thereby producing a fragment with an adapter having uracil incorporated at the 5-primer portion of the target sequence fragment, (g) the fragment with the adapter is contacted with an enzyme that removes the incorporated uracil, thereby producing at least one gap flanked by an exposed 3-primer end and an exposed 5-primer end of the fragment with the adapter, (h) an internal branch adapter is attached to the exposed 3-primer end within at least one interval, and an L-adapter is attached to the exposed 5-primer end within the gap, and (i) the internal branch adapter attached to the exposed 3-primer end in step (h) is attached to the L-adapter attached to the exposed 5-primer end, thereby producing shortened fragments with the adapter, thereby producing a group of shortened fragments with the adapter, wherein the shortened fragments with the adapter include shortened target sequence fragments having sequences corresponding to different regions of the target sequence, and the different regions are overlapping.

[0297] Embodiment 42 is the method described in embodiment 41, wherein connecting the internal branch adapter and the L-adapter includes contacting the internal branch adapter and the L-adapter with a splint oligonucleotide, wherein the splint oligonucleotide comprises a 5-primer portion complementary to a sequence in the internal branch adapter and a 3-primer portion complementary to the L-adapter, such that the splint oligonucleotide hybridizes to the internal branch adapter via the 5-primer portion and the splint oligonucleotide hybridizes to the L-adapter via the 3-primer portion, thereby connecting the internal branch adapter and the L-adapter.

[0298] Embodiment 43 is a method for preparing multiple sets of adapter-attached fragments, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer binding sequence, and a complement of a barcode sequence, wherein the method comprises (a) providing a barcoded fragment comprising a barcode sequence, a target sequence, and a primer binding sequence, wherein the barcoded fragment is immobilized on a bead at one end, (b) annealing a primer comprising a 5-primer adapter sequence to the primer binding sequence in the barcoded fragment, wherein the 5-primer adapter sequence comprises i) a complement of the barcode sequence, and ii) a primer sequence complementary to a primer binding sequence in the barcoded fragment, (c) extending the primer to produce an extended primer comprising the target sequence fragment and the complement of the barcode sequence, (d) contacting the extended primer with a first branched adapter comprising a 3-primer portion, the 3-primer portion comprising a degenerate sequence region, thereby forming a first extension product comprising the degenerate sequence region at the 3-primer portion, wherein the 3-primer portion hybridizes to the barcoded fragment through the degenerate sequence region, (e) extending the 3-primer portion of the first extension product to generate a second extension product, and (f) contacting the second extension product with a second branched adapter to produce a fragment with an adapter.

[0299] Embodiment 44 is the method of embodiment 43, wherein the method further comprises (g) denaturation to separate the adapter-bearing fragments from the barcoded fragments.

[0300] Embodiment 45 is the method of embodiment 44, wherein the method further comprises repeating one or more cycles of steps (b)-(g) under extension-controlled conditions to produce one or more fragments with adapters.

[0301] Embodiment 46 is a DNA complex comprising a plurality of fragments hybridized to one or more monomers of a DNA concatemer, wherein the plurality of fragments are separated by a spacer, wherein each of the plurality of fragments comprises a barcode sequence and a target sequence fragment having a first end and a second end, wherein the target sequence fragments in the plurality of fragments have the same nucleotide sequence at the first end and differ from each other at the second end due to truncation, such that the target sequence fragments in the plurality of fragments have different lengths.

[0302] Embodiment 47 is the DNA complex of embodiment 46, wherein each of the plurality of fragments is ligated to an L-adapter at the 5-primer end and a branched adaptor at the 3-primer end.

[0303] Embodiment 48 is a DNA complex comprising (a) a barcoded fragment immobilized on a solid support, wherein the barcoded fragment comprises a barcode sequence and a target sequence, and (b) a polynucleotide hybridized to the barcoded fragment, wherein the polynucleotide comprises a 5-primer portion comprising a complement of the barcode sequence, a 3-primer portion comprising a fragment of the target sequence, wherein the 5-primer portion and the 3-primer portion anneal to the barcoded fragment, leaving a middle portion unannealed to the barcoded fragment, thereby forming a bubble.

[0304] Embodiment 49 is a plurality of DNA complexes as described in any one of embodiments 46-48, wherein the DNA complexes share the same barcode sequence.

[0305] Embodiment 50 is a composition comprising nested groups of fragments with adapters, each comprising a barcode sequence and a target sequence fragment having a first end and a second end, a 5-primer adapter sequence and a 3-primer adapter sequence, wherein the target sequence fragments have the same nucleotide sequence at the first end and differ from each other due to truncation at the second end, so that the nested groups of fragments with adapters contain multiple target sequence fragments with different lengths, and wherein the nested groups of fragments with adapters share the same barcode sequence.

[0306] Embodiment 2.1. A method of generating a single-stranded DNA circle comprising a single-stranded adapter-containing construct for sequencing, comprising:

[0307] preparing a plurality of nested sets of single-stranded nucleic acid constructs, wherein each single-stranded nucleic acid construct in each nested set comprises a portion of a target sequence flanked by a first adapter sequence and a second adapter sequence, wherein the first adapter sequence comprises a barcode sequence and a primer binding sequence,

[0308] wherein each target sequence portion has a first end and a second end,

[0309] wherein the distance between the first end and the barcode sequence is shorter than the distance between the second end and the barcode sequence,

[0310] wherein the single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences,

[0311] Wherein for each nested group of single-stranded nucleic acid constructs,

[0312] (a) the target sequence portions in the nested groups have the same nucleotide sequence near the first end and differ from each other due to truncation near the second end, so that each nested group of the single-stranded nucleic acid construct contains a plurality of target sequence portions having different lengths, and

[0313] (b) circularizing the single-stranded nucleic acid construct in each nested group to generate a single-stranded DNA circle in which the first adapter sequence and the second adapter sequence are connected.

[0314] Embodiment 2.2 The method of embodiment 2.1, wherein each nested set of single-stranded nucleic acid constructs is prepared by:

[0315] (i) preparing double-stranded genomic fragments with adapters, each comprising a target sequence flanked by a first adapter sequence and a third adapter sequence,

[0316] (ii) amplifying the adapter-attached double-stranded genomic fragment by using primers that hybridize to the first and third adapter sequences to produce an amplified genomic fragment,

[0317] (iii) contacting the amplified genomic fragment from (ii) with a nicking agent to generate a nick in the target sequence in one strand of the amplified genomic fragment,

[0318] (iv) ligating a second adaptor comprising a second adaptor sequence at the nick in (iii) via branched ligation to form a ligation product, and

[0319] (v) denaturing the ligation products from (iv) to form single-stranded nucleic acid constructs, each comprising a first adapter sequence and a second adapter sequence.

[0320] Embodiment 2.3. The method of embodiment 2.1, wherein each nested set of single-stranded nucleic acid constructs is prepared by:

[0321] (i) preparing double-stranded genomic fragments with adapters, each comprising a target sequence flanked by a first adapter sequence and a third adapter sequence,

[0322] (ii) amplifying the double-stranded genomic fragment with the adapter to produce an amplified genomic fragment,

[0323] (iii) distributing the amplified genomic fragments into a plurality of aliquots,

[0324] (iv) denaturing the genomic fragments amplified in (iii) to prepare single-stranded genomic fragments, wherein at least some of the single-stranded genomic fragments each comprise a primer binding sequence,

[0325] (iv) extending a primer hybridized to the primer binding sequence under extension control conditions such that extension products from different aliquots have different lengths, thereby generating extension products having newly formed ends, and the extension products have different sequences near the newly formed ends in different aliquots,

[0326] wherein each extension product comprises a portion of the target sequence, and

[0327] (v) ligating a second adapter comprising a second adapter sequence at the newly formed end in each aliquot via branched ligation, thereby generating single-stranded nucleic acid constructs each comprising the first adapter sequence and the second adapter sequence.

[0328] Embodiment 2.4. The method of embodiment 2.1, wherein each nested set of single-stranded nucleic acid constructs is prepared by:

[0329] (i) preparing double-stranded genomic fragments with adapters, each comprising a target sequence flanked by a first adapter sequence and a third adapter sequence,

[0330] (ii) amplifying the double-stranded genomic fragment with the adapter to produce an amplified genomic fragment,

[0331] (iii) distributing the amplified genomic fragments into a plurality of aliquots,

[0332] (iv) adding a double-stranded DNA nuclease having 3'→5' nuclease activity to the plurality of aliquots under controlled conditions such that the lengths of the products remaining after digestion in different aliquots are different, thereby generating digestion products having newly formed ends and having different sequences in different aliquots,

[0333] wherein each digestion product contains a portion of the target sequence, and

[0334] (v) ligating a second adapter comprising a second adapter sequence at the newly formed end in each aliquot via branched ligation, thereby generating single-stranded nucleic acid constructs each comprising the first adapter sequence and the second adapter sequence.

[0335] Embodiment 2.5. The method of embodiment 2.1, wherein each nested set of single-stranded nucleic acid constructs is prepared by:

[0336] (i) preparing double-stranded genomic fragments with adapters, each comprising a target sequence flanked by a first adapter sequence and a third adapter sequence,

[0337] (ii) amplifying the double-stranded genomic fragment with the adapter to produce an amplified genomic fragment,

[0338] (iii) denaturing the amplified genomic fragments to prepare single-stranded genomic fragments, wherein at least some of the single-stranded genomic fragments each comprise a primer binding sequence,

[0339] (iv) For each single-stranded genomic fragment,

[0340] extending a primer hybridized to the primer binding sequence for a first period of time to produce an extended primer,

[0341] wherein the extension is incomplete, so that the length of the extension primer is a small fraction of the length of the single-stranded genomic fragment,

[0342] wherein the extension primer comprises a portion of the target sequence, and

[0343] ligating a second adaptor to the newly formed end of the extended primer via branched ligation, thereby generating single-stranded nucleic acid constructs in one reaction mixture, each comprising a first adaptor sequence and a second adaptor sequence,

[0344] (v) repeating step (iv) for multiple rounds, and for each round, further extending the primer for an additional period of time and ligating an additional adapter having a unique positional barcode to the further extended primer,

[0345] The molar amount of the additional adapters is a small fraction of the total molar amount of the amplified genomic fragments.

[0346] Thereby a mixture of nested sets of single-stranded nucleic acid constructs is generated.

[0347] Embodiment 2.6. The method of Embodiment 2.3, wherein the target sequence comprises a repetitive sequence, wherein the second adapter comprises a unique positional barcode sequence for each aliquot,

[0348] Wherein the single-stranded nucleic acid constructs formed in (v) in different aliquots contain different positional barcode sequences, and the single-stranded nucleic acid constructs in the same aliquot share the same positional barcode sequence.

[0349] Embodiment 2.7. The method of Embodiment 2.1, wherein the primer binding sequence is at 3-primer relative to the barcode sequence.

[0350] Embodiment 2.8. The method of Embodiment 2.1, wherein the method further comprises:

[0351] (vi) fragmenting the single-stranded DNA circle to generate a plurality of single-stranded DNA fragments, wherein at least some of the fragments comprise a barcode sequence,

[0352] (vii) generating double-stranded DNA fragments from the single-stranded DNA fragments in step (vi),

[0353] (vii) ligating a second adaptor to each double-stranded DNA fragment from step (vii), thereby generating double-stranded adaptor-bearing fragments.

[0354] Embodiment 2.9. The method of embodiment 2.8, further comprising (viii) amplifying the double-stranded adapter-bearing fragment, and

[0355] Optionally (ix) selecting amplified double-stranded adaptor-attached fragments ranging in length from 300 to 1000 bases.

[0356] Embodiment 2.10. The method of Embodiment 2.1, wherein the method further comprises:

[0357] (vi) hybridizing a primer to the primer binding sequence in each single-stranded DNA loop,

[0358] (vii) using each single-stranded DNA circle as a template, extending the primers under extension control conditions,

[0359] wherein extension produces an extension primer that hybridizes to the single-stranded DNA loop, thereby producing a plurality of extension primers having different lengths,

[0360] wherein each of the extension primers comprises a barcode sequence and a primer binding sequence,

[0361] (viii) ligating the second adaptor to the plurality of extension primers via branched ligation to generate adaptor-bearing extension primers.

[0362] Embodiment 2.11. The method of Embodiment 2.10, wherein the method further comprises:

[0363] Amplifying the extension primer with the adapter to generate an amplified double-stranded fragment,

[0364] Selecting amplified double-stranded fragments with a length ranging from 300 bases to 1000 bases (a nested range with 600 bases as the optimal length will be disclosed in the specification), and

[0365] The selected amplified double-stranded adapter-attached fragments are sequenced.

[0366] Embodiment 2.12. The method of Embodiments 2.1-2.11, wherein the single-stranded DNA circle is prepared in solution without the need for a solid support.

[0367] Embodiment 2.13. The method of Embodiment 2.1, wherein the first end or the second end is attached to a solid support.

[0368] Embodiment 2.14. A method of preparing a double-stranded adaptor-containing construct for sequencing, wherein the method comprises:

[0369] (i) amplifying a plurality of genomic fragments, each genomic fragment comprising a target sequence, to generate a plurality of sets of amplified nucleic acid fragments in a mixture, wherein the amplified nucleic acid fragments in each nested set share the same target sequence, optionally using target-specific primers for amplification,

[0370] For each nested group, the method further includes:

[0371] (ii) contacting the amplified nucleic acid fragments with an enzyme, wherein the enzyme introduces breaks in the amplified nucleic acid fragments,

[0372] (iii) distributing the fragment mixture into a plurality of aliquots,

[0373] (iv) nick translation is performed on aliquots of the fragments under conditions to synthesize DNA chains such that the DNA chains synthesized in different aliquots have different lengths, wherein each DNA chain comprises a portion of the target sequence having a first end and a second end, and wherein the DNA chains in the different aliquots share a common sequence near the first end and have different sequences near the second end,

[0374] (v) for each aliquot, ligating a second adapter to the second end of the DNA strand synthesized in (iv) via branched ligation, wherein each second adapter is a partially double-stranded adapter comprising a first adapter oligonucleotide and a second adapter oligonucleotide,

[0375] wherein the first adapter oligonucleotide and the second adapter oligonucleotide are complementary and hybridize to each other,

[0376] wherein each of the second adapters comprises a positional barcode sequence,

[0377] wherein each ligation comprises joining the 5-primer end of the first adapter oligonucleotide of the second adapter to the second end of the synthesized DNA strand,

[0378] wherein the first adapter oligonucleotides ligated to the second ends of the synthesized DNA strands in different aliquots comprise different positional barcode sequences, and the first adapter oligonucleotides ligated to the second ends of the synthesized DNA strands in the same aliquot share the same positional barcode sequence,

[0379] (vi) combining the synthesized DNA strands ligated to the second adapter from different aliquots of (v) into a single mixture,

[0380] (vii) extending the primer hybridized to the first adapter oligonucleotide ligated to the synthesized DNA strand to generate a double-stranded fragment with blunt ends, and

[0381] (viii) optionally selecting from the single mixture double-stranded fragments of (vii) in the size range of 200 bp-1.5 kb (the optimal range of about 500-1000 bp will be disclosed in the specification), and

[0382] (ix) Ligating a third adaptor to the blunt end of the double-stranded fragment, thereby generating a double-stranded adaptor construct.

[0383] Embodiment 2.15. The method of Embodiment 2.14, wherein step (i) comprises amplifying a plurality of genomic fragments in a mixture comprising uracil, thereby generating amplified nucleic acid fragments incorporating uracil, and

[0384] Wherein step (ii) comprises contacting the amplified nucleic acid fragments with uracil-DNA glycosylase, wherein the uracil-DNA glycosylase removes uracil from the amplified genomic fragments.

[0385] Embodiment 2.16. The method described in Embodiment 2.14, wherein the amplification of multiple genomic fragments in step (i) is performed using primers containing uracil, thereby generating multiple sets of amplified nucleic acid fragments containing uracil.

[0386] Embodiment 2.17. The method of Embodiment 2.16, wherein each of the plurality of genomic fragments is amplified using a forward primer and a reverse primer, and wherein each forward primer comprises one or more uracils.

[0387] Embodiment 2.18. The method of Embodiment 2.17, wherein each of the plurality of genomic fragments is amplified using a forward primer and a reverse primer, and wherein each reverse primer comprises a single uracil.

[0388] Embodiment 2.19. The method of Embodiment 2.14, wherein step (ii) comprises contacting the amplified genomic fragments with an endonuclease, wherein the endonuclease randomly cleaves the amplified genomic fragments.

[0389] Embodiment 2.20. The method of Embodiment 2.19, wherein the endonuclease is EndoIV or APE1.

[0390] Embodiment 2.21. A reaction mixture comprising the single-stranded DNA circle produced in Embodiment 2.7.

[0391] Embodiment 2.22. A reaction mixture comprising the combined synthetic DNA strands of step (vi) of Embodiment 1.19.

[0392] Example

[0393] 1. Example 1. Co-barcoding in solution using infrequent random nicking

[0394] The present embodiment describes the full coverage of generating 1-20kb DNA molecules. For most sequences, it can be a useful assembly method, especially using a sequencing platform (such as SE400-SE1000 or PE300+MPS reading). Only when the target nucleic acid comprises a highly repetitive sequence, the position described herein is required to be barcoded together. The method starts by connecting a barcode adapter at one end of the molecule and connecting a non-barcode adapter at the other end, which is achieved by connecting a Y adapter or other common methods. If a common adapter tag is added to each PCR primer, and an adapter tag in each PCR primer pair comprises a barcode, the method can also be used for the target sequence. After PCR, the molecule is treated with a non-specific nickase at low concentration, low temperature and / or a short period of time to introduce a nick in each template. If necessary, 5' or 3' exonucleases or polymerases can be used without using dNTP to widen this nick to a gap of several bases in the sequence. Then branch connection is performed to add another adapter. Then the splint oligonucleotide between the branch connection adapter and the adapter containing the barcode is used to cyclize the molecule. Figure 3A The circles are then fragmented into 500-1000 base pairs, followed by primer extension from the barcoded adapters to replicate the barcodes. After one or more rounds of adapter ligation, the molecules can now be sequenced directly or PCR amplified and sequenced.

[0395] Another embodiment of this process uses a controlled extension of -600 bases, followed by circularization, followed by 3' branch ligation, and then PCR. Figure 4B This has the benefit of generating products with a relatively narrow size range, compared to random fragmentation, which would produce a broad size distribution. Any short artifacts can be removed by exonuclease treatment or purification.

[0396] The end result of this process is a series of overlapping sequence reads from each original DNA molecule that all share the same barcode sequence. Random nicking provides similar coverage for short and long DNA molecules present in the same pool. This enables each original DNA molecule to be fully reassembled.

[0397] 2. Example 2. Co-barcoding of the positions of 1-20 kb fragments in solution

[0398] This embodiment is similar to Example 1, except that it contains a barcode that can be shared between all sub-fragments of the original molecule (common barcoding). The process is started by amplifying a specific region using a targeted PCR primer containing a common adapter tag with a random barcode, or by connecting an adapter with a common sequence and a random barcode to a dsDNA fragment of 1-20kb in length. Preferably, the pool has DNA fragments of similar length. For pools with long and short DNA, a specific method can be used to minimize the over-coverage of shorter fragments. The product is PCR amplified and then divided into 10-20 pools, followed by controlled extension or ExoIII digestion (as described above) or controlled nick translation in different amounts in each pool. Short DNA fragments will be fully extended to form flat ends, and these fragments with flat ends can be blocked for branch connection using methods known in the art (e.g., DNA tailing or 3' blocking by terminal transferase). After this step, a 3' branch connection is performed to add an adapter with a common sequence and a barcode sequence (position barcode) specific to each pool. Now, unlike above, the product is circularized, which connects the DNA molecular barcode to the positional barcode, with a common adapter sequence in between. Next, the circular is fragmented into 500-1000 base pairs, and the fragments are primer extended with a primer that will be 5' of the molecular and positional barcodes. After extension, a tertiary adapter is ligated, and now sequencing can be performed, or PCR and sequencing. Instead of primer extension, a blunt-ended tertiary adapter with a non-phosphorylated 5' end can be ligated, followed by PCR and sequencing. Figure 3B .

[0399] Another approach that can be taken based on this process is to have the above reaction performed in a single tube rather than in separate pools. This can be achieved by adding a limited amount of 3' branched ligation adapters with different sequences after each time interval. For example, the first 3' branched ligation adapter is added 10 minutes after the start of extension, the second 3' branched ligation adapter is added 20 minutes after the start of extension, and so on, and the first and second 3' branched ligation adapters have different sequences. The ideal amount of 3' branched ligation adapters is the amount that will result in 1-10% of the total number of molecules connected (depending on the length of the molecules used). This process of adding a limited amount of adapters will be repeated 10-20 times or more (equivalent to the number of total pools used in another approach). The advantage of this is that it is performed in a single tube, but multiple rounds of adapter pipetting will be required into the same tube.

[0400] 3. Example 3. 2-20 kb pools for target enrichment of non-related sequences

[0401] Long-distance PCR is used to carry out the method described in this embodiment to the genomic target region. In some cases, multiple PCR can be carried out so that 100s to 1000s different target regions can be amplified in one or more reactions. After amplification, the product is divided into different pools. According to the size of the amplicon and the sequencing read length expected to be used, the number of pools can be increased or decreased, but for a 5kb product (double-ended 250 or single-ended 500) with ~500 base readings, about 10-20 pools are good quantities. For each pool, ExoIII is used for timed digestion, or a polymerase (e.g., Escherichia coli DNA polymerase 1) with 5'-3' exo-active is used for controlled extension. Importantly, for each pool, the time of ExoIII processing is all step-by-step variation (e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, etc.), so that the amount of digestion between each pool in this embodiment is about 500 bases. Similarly, if controlled extension or nick translation is carried out, the time or ratio of dNTP can be changed to obtain similar results. It is worth noting that the amount of extension or digestion in each pool is different, and there is a range of products rather than products of specific lengths. This results in overlapping fragments between different pools, and after sequencing, the overlap will make the in silico assembly of each original molecule easier. After this step, a 3' branch connection is performed to add an adapter with a common sequence and a barcode sequence specific to each pool. The adapter can contain biotin at the 3' end to assist in the purification step. The pools are then merged and the products are fragmented into ~500-1000 base pairs, followed by primer extension from the adapter sequence and then ligation of a second common adapter. The products can then be PCR amplified or directly circularized for DNB formation and sequencing. Figure 5 .

[0402] 4. Example 4. Cyclic extension of complete stLFR

[0403] Loop-mediated complete stLFR involves ligation of two functionalized partially double-stranded blunt-end adapters to an end-repaired DNA fragment bearing a 5′-phosphate group.

[0404] Ligate blunt-ended adapters to end-repaired DNA fragments with 5'-phosphate groups

[0405] The first part is a double-stranded blunt-ended adapter (e.g., AD153UMI_5, Fig.13A) has a longer chain (1313) and a shorter chain (1314) that are annealed to form blunt ends and unpaired ends. The blunt ends can be connected. The longer chain (1313) includes a single-stranded 5'-overhang, which includes one or more barcodes (UID) (1319), such as a unique molecular identification sequence (UMI) or a multiple sample barcode (1319). The single-stranded 5'-overhang may also include a sequence complementary to a universal amplification primer. The barcode sequence can exist as a single sequence or as several separate sequences. Optionally, the single-stranded 5'-overhang includes a single T overhang at the 3' for connection to an A-tailed DNA fragment. The shorter chain (1314) is annealed to the longer chain (1313) and includes a 5' end that does not contain a 5'-phosphate group and therefore cannot be connected to a DNA fragment. The 3' end of the shorter chain (1314) is also unable to connect because it has been modified to prevent connection. These 3' modifications for preventing ligation include, but are not limited to, inverted nucleosides, dideoxynucleosides, 3'-amino groups, and 3'-phosphate groups. When the first portion of double-stranded blunt-end adapters contacts genomic DNA (1310) under ligation conditions, only the longer chain (1313) is ligated to the gDNA, while the shorter chain (1314) cannot be ligated to the genomic DNA fragment because both its 5' and 3' ends are not ligatable, thereby leaving a nick (1317) between the shorter chain and the gDNA chain. See Fig.13A .

[0406] The design of the second double-stranded blunt-end adapter (eg, Ad183 shown in FIG. 13 ) is similar to that of the first double-stranded blunt-end adapter, except that it does not contain a barcode sequence (UID).

[0407] The genomic fragments are then ligated to the above-mentioned adapters and purified using the SPRI bead purification method (Beckman Coulter Life Sciences, Indianapolis, Indiana). Using a DNA polymerase with strand displacement activity (e.g., BstDNA polymerase, large fragment; Phi29 DNA polymerase; Bsu DNA polymerase, large fragment; Bsm DNA polymerase, large fragment) or with 5'-3' exonuclease activity (e.g., rTaqDNA polymerase, Escherichia coli DNA polymerase I), the adapter-ligated DNA molecule is enzymatically extended from the 3' end of the genomic DNA fragment to the non-ligatable 5' end of the shorter strand (1314) of the partially double-stranded adapter. This forms a double-stranded adapter-ligated DNA molecule, wherein the double-stranded adapter is attached to the DNA fragment sequence (1318). The 3' end of the shorter strand (1320.2) of the branch adapter contains a 15-20 base sequence that is complementary to the 5' end of the longer strand of the branch adapter. The longer strand of the branched adapter contains the barcode sequence and has a melting temperature (Tm) of 50-70° C. The 3′ end of the short strand (1320.2) is blocked with a 3′ terminal modification that prevents ligation (eg, dideoxynucleoside, 3′-amino, 3′-phosphate). Fig. 13B The adapter sequence derived from the first portion of the double-stranded adapter contains a site for a universal amplification primer, so the double-stranded adapter-ligated DNA molecule (1318) can be amplified by PCR or other amplification methods that rely on two primer sequences. Fig.13A .

[0408] Random incision and gapping are performed. This can be achieved by using non-specific nicking nuclease, which catalyzes the DNA backbone that only destroys one chain at a time; such as Vvn and mutants, ShrimpdsDNA specific endonuclease, DNAseI. This can also be achieved by using a mixture of multiple nicking enzymes, such as several site-specific nicking enzymes (such as CCD). In some cases, it is also possible to add an additional enzyme with 3' exo-activity (such as DNA polymerase I, Klenow fragments without nucleotides, exonuclease III or analogs) or an additional enzyme with 5' exo-activity (Bst DNA polymerase full length without nucleotides, T7 exonuclease, truncated exonuclease VIII, Lambda exonuclease, T5 exonuclease or analogs), to increase the opening of the incision, so as to provide more space for the branch adapter connection. It is preferred that low processability exonuclease opens a short gap (such as 2-7 bases) and is separated from DNA to allow the adapter to connect. Fig.13A and 13B Random nicking generates a set of fragments with different lengths of target sequence fragments and sharing a common barcode sequence at the 5' end. One of such fragments is Fig. 13BAs shown in 1341.

[0409] The branched adapters (e.g., Fig. 13B The branched adapter (1320) is attached to the 3' side of the nick or ssDNA gap in the DNA fragment to which the adapter is attached. The branched adapter (1320) is a partially double-stranded DNA adapter molecule with a 5'-phosphate on the longer strand (1320.1). This produces a set of fragments with different lengths of target sequence fragments, each of which is flanked by the adapter sequences AD153UMI_5 and AD153UMI_5R, one of which is Fig. 13B It is displayed as 1342.

[0410] The longer chain of the first double-stranded blunt-end adapter and the longer chain of the branched adapter respectively comprise a first hybridization sequence (1432) and a second hybridization sequence (1433). The first hybridization sequence (1432) is located at the 3' end relative to the barcode sequence (1319).

[0411] In the case of using an exonuclease to open the gap as described above, protection of the DNA adaptors can be achieved, if desired, by phosphorothioate bonds between bases and / or modified bases at the 5' and 3' ends of the adaptors.

[0412] The ligation of branched adapters to adapter-bearing DNA fragments (e.g., adapter-bearing genomic DNA fragments) can be performed in solution or on beads. In the case where the process is performed on the surface of beads, the adapter-bearing DNA fragments are pre-loaded onto the beads with a high concentration of PEG (5%-15%) before adding other reaction components.

[0413] In some embodiments, the branch ligation reaction is carried out in the presence of an additive (e.g., polyethylene glycol or betaine) to increase the activity of the ligation and / or nicking enzyme. The reaction can be carried out at room temperature, 37°C, or cycled between different temperatures, such as 5-15°C and 37°C, with a pH range of 5.0 to 9.0. Over 5 minutes to several hours. The amount of time and the synergy of the nicking enzyme vary depending on the number of nicks required for each DNA fragment. If carried out in solution, the reaction can be stopped by a DNA purification method (e.g., Ampure XP beads), and if carried out on beads, the reaction can be stopped simply by a washing step using a Tris NaCl buffer containing PEG (5%-15%).

[0414] In some embodiments, after the branch connection, the DNA fragments are denatured. The reaction mixture is heated to a temperature between 90°C and 95°C (including endpoints). Alternatively, the branch-connected DNA fragments can be denatured with an alkaline agent (e.g., 0.05M-0.2M NaOH or KOH) and further neutralized with a neutralizing agent (e.g., HCl, Tris-HCl, MOPS). A single-stranded DNA molecule (1343) is formed containing genomic DNA and an adapter sequence set at both ends. Fig. 13B .

[0415] Alternatively, in some embodiments, rather than denaturing, the 5' single-stranded tails at the adapter-ligated DNA fragments required for hybridization of the 3' ends of the branched adapters are generated by digestion with one or more dsDNA-specific exonucleases having 3'-5' exonuclease activity (e.g., Exonuclease III) (e.g., FIG. 13 or Fig.14 1432 in ).

[0416] DNA loop formation and enzymatic extension at the 3' end of branched adapter hybridization

[0417] The longer chain of the branch adapter and the longer chain of the first part double-stranded blunt-end adapter contain complementary sequences (1432 and 1433) and can hybridize with each other. The hybridization is carried out in a hybridization buffer containing a buffer (e.g., Tris-HCl, MOPS, sodium phosphate), salts, and auxiliary factors (e.g., MgCl2, dNTP) that are essential for subsequent enzymatic reactions. Fig.14 .

[0418] The DNA hybridization step is followed by branching adapters (e.g. Fig.14 The linear extension step of the hybridized 3' end of the AD153UMI_5R shown in the figure is performed to replicate the barcode on the first adapter AD153UMI_5. In order to increase specificity, a DNA polymerase lacking 3'-exonuclease activity is used for linear extension, such as Taq DNA polymerase, Klenow fragment (3'→5' exonuclease), Bst DNA polymerase, large fragment. Depending on the DNA polymerase used, the extension can be performed at different temperatures from 30°C to 75°C.

[0419] The linearly extended product (1431) represents a partially double-stranded DNA molecule whose double-stranded adapter contains the UID sequence attached to the DNA fragment. The product is then denatured to form a single-stranded sequence (1441) with adapter sequences at both ends, so that each end of the target sequence fragments 1411, 1421, 1431, etc. is close to the barcode sequence 1319. The adapter sequence including the barcode sequence and the site for the universal amplification primer can therefore be used for controlled primer extension in the next step to generate fragments with a length suitable for sequencing.

[0420] ***

[0421] While the present invention has been disclosed with reference to certain aspects and embodiments, it is apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention.

[0422] Each publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not an admission that any such document is relevant prior art nor does it constitute an admission as to its contents or date.

Claims

1. A method for producing a single-stranded construct with an adapter for sequencing, the method comprising: preparing a plurality of nested sets of single-stranded nucleic acid constructs, wherein each single-stranded nucleic acid construct in each nested set comprises a target sequence portion flanked by a first adapter sequence at the 5' end and a second adapter sequence at the 3' end, wherein the first adapter sequence comprises, from 5' to 3', a primer binding sequence, a barcode sequence, and a first hybridization sequence, and the second adapter sequence comprises a second hybridization sequence, wherein the first hybridization sequence and the second hybridization sequence are complementary to each other, wherein each target sequence portion has a first end and a second end, wherein the distance between the first end and the barcode sequence is shorter than the distance between the second end and the barcode sequence, wherein the single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences, and Wherein for each nested group of single-stranded nucleic acid constructs, The target sequence portions in the nested groups have the same nucleotide sequence near the first end and differ from each other due to truncation near the second end, so that each nested group of single-stranded nucleic acid constructs contains multiple target sequence portions with different lengths.

2. The method of claim 1, further comprising subjecting the plurality of nested sets of single-stranded nucleic acid constructs to hybridization conditions, whereby the first adapter sequence hybridizes to the second adapter sequence, thereby forming a loop.

3. The method of claim 2, wherein the method further comprises extending the second adapter sequence using a DNA polymerase to replicate the barcode sequence and the primer binding sequence in the first adapter sequence to form an extension product.

4. The method of claim 3, wherein the method further comprises denaturing the extension product to open the loop, thereby forming linear single-stranded DNA constructs, wherein each linear single-stranded DNA construct comprises a barcode sequence and a primer binding sequence, wherein the primer binding sequence is located at the 3' side relative to the barcode sequence.

5. The method of claim 4, wherein the method further comprises annealing a primer to the primer binding sequence at the 3' position of the linear single-stranded DNA construct and extending the primer to generate an extension product having a length suitable for sequencing.

6. A method for generating a single-stranded DNA circle comprising a single-stranded adapter-containing construct for sequencing, the method comprising: preparing a plurality of nested sets of single-stranded nucleic acid constructs, wherein each single-stranded nucleic acid construct in each nested set comprises a target sequence portion flanked by a first adapter sequence and a second adapter sequence, wherein the first adapter sequence comprises a barcode sequence and a primer binding sequence, wherein each target sequence portion has a first end and a second end, wherein a distance between the first end and the barcode sequence is shorter than a distance between the second end and the barcode sequence, wherein the single-stranded nucleic acid constructs in each nested group share the same barcode sequence, and the single-stranded nucleic acid constructs in different nested groups have different barcode sequences, Wherein for each nested group of single-stranded nucleic acid constructs, (a) the target sequence portions in the nested groups have the same nucleotide sequence near the first end and differ from each other due to truncation near the second end, so that each nested group of single-stranded nucleic acid constructs contains a plurality of target sequence portions having different lengths, and (b) circularizing the single-stranded nucleic acid construct in each nested group to generate the single-stranded DNA circle, wherein the first adapter sequence and the second adapter sequence are connected.

7. The method of any one of claims 1 to 6, wherein each nested group of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each of which comprises a target sequence flanked by the first adapter sequence and the third adapter sequence, (ii) amplifying the adapter-attached double-stranded genomic fragment by using primers that hybridize to the first adapter sequence and the third adapter sequence to produce an amplified genomic fragment, (iii) contacting the amplified genomic fragment from (ii) with a nicking agent to generate a nick in the target sequence in one strand of the amplified genomic fragment, (iv) ligating a second adaptor comprising the second adaptor sequence at the nick in (iii) via branched ligation to form a ligation product, and (v) denaturing the ligation products from (iv) to form the single-stranded nucleic acid constructs, each of which comprises the first adaptor sequence and the second adaptor sequence.

8. The method of any one of claims 1 to 6, wherein each nested group of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each of which comprises a target sequence flanked by the first adapter sequence and the third adapter sequence, (ii) amplifying the double-stranded genomic fragment with adapters to produce an amplified genomic fragment, (iii) distributing the amplified genomic fragments into a plurality of aliquots, (iv) denaturing the amplified genomic fragments described in (iii) to prepare single-stranded genomic fragments, wherein at least some of the single-stranded genomic fragments each comprise the primer binding sequence, (iv) extending a primer hybridized to the primer binding sequence under extension control conditions such that the lengths of extension products from different aliquots are different, thereby generating extension products having newly formed ends, and wherein the extension products have different sequences near the newly formed ends in different aliquots, wherein each extension product comprises a portion of the target sequence, and (v) ligating a second adaptor comprising the second adaptor sequence to the newly formed end in each aliquot via branched ligation, thereby generating the single-stranded nucleic acid constructs, each of which comprises the first adaptor sequence and the second adaptor sequence.

9. The method of any one of claims 1 to 6, wherein each nested group of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each of which comprises a target sequence flanked by the first adapter sequence and the third adapter sequence, (ii) amplifying the double-stranded genomic fragment with adapters to produce an amplified genomic fragment, (iii) distributing the amplified genomic fragments into a plurality of aliquots, (iv) adding a double-stranded DNA nuclease having 3'→5' nuclease activity to the plurality of aliquots under controlled conditions, so that the lengths of the products remaining after digestion by the double-stranded DNA nuclease in different aliquots are different, thereby generating digestion products having newly formed ends and having different sequences in different aliquots, wherein each digestion product contains a portion of the target sequence, and (v) ligating a second adaptor comprising the second adaptor sequence to the newly formed end in each aliquot via branched ligation, thereby generating the single-stranded nucleic acid constructs, each of which comprises the first adaptor sequence and the second adaptor sequence.

10. The method of any one of claims 1 to 6, wherein each nested set of single-stranded nucleic acid constructs is prepared by: (i) preparing double-stranded genomic fragments with adapters, each of which comprises a target sequence flanked by the first adapter sequence and the third adapter sequence, (ii) amplifying the double-stranded genomic fragment with adapters to produce an amplified genomic fragment, (iii) denaturing the amplified genomic fragments to prepare single-stranded genomic fragments, wherein at least some of the single-stranded genomic fragments each comprise the primer binding sequence, (iv) For each single-stranded genomic fragment, extending a primer hybridized to the primer binding sequence for a first period of time to produce an extended primer, wherein the extension is incomplete such that the length of the extension primer is a fraction of the length of the single-stranded genomic fragment, wherein the extension primer comprises a target sequence portion, and ligating a second adaptor to the end of the extension primer formed by the extending via branched ligation, thereby generating single-stranded nucleic acid constructs in one reaction mixture, each single-stranded nucleic acid construct comprising the first adapter sequence and the second adapter sequence, (v) repeating step (iv) for multiple rounds, and for each round, further extending the primer for an additional period of time and ligating an additional adapter having a unique positional barcode to the further extended primer, The molar amount of the additional adapters is a small fraction of the total molar amount of the amplified genomic fragments, thereby generating a mixture of nested sets of single-stranded nucleic acid constructs.

11. The method of claim 8, wherein the target sequence comprises a repetitive sequence, wherein the second adapter comprises a unique positional barcode sequence for each aliquot, wherein the single-stranded nucleic acid constructs formed in (v) in different aliquots contain different positional barcode sequences, and the single-stranded nucleic acid constructs in the same aliquot share the same positional barcode sequence.

12. The method of claim 6, wherein the primer binding sequence is located 3-primer relative to the barcode sequence.

13. The method of claim 6, wherein the method further comprises: (vi) fragmenting the single-stranded DNA circle to generate a plurality of single-stranded DNA fragments, wherein at least some of the single-stranded DNA fragments comprise the barcode sequence, (vii) generating double-stranded DNA fragments from the single-stranded DNA fragments from step (vi), (vii) ligating a second adaptor to each of the double-stranded DNA fragments from step (vii), thereby generating double-stranded adaptor-bearing fragments.

14. The method of claim 13, further comprising (viii) amplifying the double-stranded adapter-bearing fragment, and Optionally (ix) selecting the amplified double-stranded adapter-attached fragments having a length ranging from 300 to 1000 bases.

15. The method of claim 6, wherein the method further comprises: (vi) hybridizing a primer to the primer binding sequence in each of the single-stranded DNA loops, (vii) extending the primers under extension control conditions using each of the single-stranded DNA circles as a template, wherein the extending generates an extended primer that hybridizes to the single-stranded DNA loop, thereby generating a plurality of extended primers having different lengths, wherein each of the extension primers comprises the barcode sequence and the primer binding sequence, (viii) ligating a second adaptor to the plurality of extension primers via branched ligation to generate adaptor-bearing extension primers.

16. The method of any one of claims 6 to 15, wherein the method further comprises: amplifying the adapter-bearing extension primer to generate an amplified double-stranded fragment, Selecting the amplified double-stranded fragments with a length ranging from 300 bases to 1000 bases (a nested range with 600 bases as the optimal length will be disclosed in the specification), and The selected amplified double-stranded adapter-bearing fragments are sequenced.

17. The method of claims 1-16, wherein the single-stranded DNA circle is prepared in solution without the need for a solid support.

18. The method of claim 6, wherein the first end or the second end is attached to a solid support.

19. A method for producing a double-stranded adaptor-containing construct for sequencing, wherein the method comprises: (i) amplifying a plurality of genomic fragments, each genomic fragment comprising a target sequence, to generate a plurality of sets of amplified nucleic acid fragments in a mixture, wherein the amplified nucleic acid fragments in each set share the same target sequence, optionally using target-specific primers for said amplification, For each group, the method further comprises: (ii) contacting the amplified nucleic acid fragments with an enzyme, wherein the enzyme introduces breaks in the amplified nucleic acid fragments, (iii) distributing said mixture of fragments into a plurality of aliquots, (iv) performing nick translation on the aliquots of the fragments under conditions to synthesize DNA chains such that the DNA chains synthesized in different aliquots have different lengths, wherein each of the DNA chains comprises a portion of the target sequence having a first end and a second end, and wherein the DNA chains in different aliquots share the same sequence near the first end and have different sequences near the second end, (v) for each aliquot, ligating a second adapter to the second end of the DNA strand synthesized in (iv) via branched ligation, wherein each second adapter is a partially double-stranded adapter comprising a first adapter oligonucleotide and a second adapter oligonucleotide, wherein the first adapter oligonucleotide and the second adapter oligonucleotide are complementary and hybridize to each other, wherein each of said second adapters comprises a positional barcode sequence, wherein each ligation comprises connecting the 5-primer end of the first adapter oligonucleotide of the second adapter to the second end of the synthesized DNA strand, wherein the first adapter oligonucleotides ligated to the second end of the synthesized DNA strand in different aliquots comprise different positional barcode sequences, and the first adapter oligonucleotides ligated to the second end of the synthesized DNA strand in the same aliquot share the same positional barcode sequence, (vi) combining the synthesized DNA strands ligated to the second adaptors from different aliquots of (v) into a single mixture, (vii) extending the primer hybridized to the first adapter oligonucleotide ligated to the synthesized DNA strand to generate a double-stranded fragment with blunt ends, and (viii) optionally selecting from said single mixture said double-stranded fragments of (vii) having a size ranging from 200 bp to 1.5 kb, and (ix) ligating a third adaptor to the blunt end of the double-stranded fragment, thereby generating a double-stranded adaptor construct.

20. The method of claim 19, wherein step (i) comprises amplifying the plurality of genomic fragments in a mixture containing uracil, thereby generating amplified nucleic acid fragments incorporating uracil, and Wherein step (ii) comprises contacting the amplified nucleic acid fragment with uracil-DNA glycosylase, wherein the uracil-DNA glycosylase removes the uracil from the amplified genomic fragment.

21. The method of claim 19, The amplification of the multiple genomic fragments in step (i) is performed using primers containing uracil, thereby generating multiple groups of amplified nucleic acid fragments containing uracil.

22. The method of claim 21, wherein each of the plurality of genomic fragments is amplified using a forward primer and a reverse primer, and wherein each forward primer comprises one or more uracils.

23. The method of claim 22, wherein each of the plurality of genomic fragments is amplified using a forward primer and a reverse primer, and wherein each reverse primer comprises a single uracil.

24. The method of claim 19, wherein step (ii) comprises contacting the amplified genomic fragments with an endonuclease, wherein the endonuclease randomly cleaves the amplified genomic fragments.

25. The method of claim 24, wherein the endonuclease is EndoIV or APE1.

26. A reaction mixture comprising the single-stranded DNA circle produced as claimed in claim 6.

27. A reaction mixture comprising the DNA strands resulting from the combinatorial synthesis of step (vi) of claim 19.

28. A method for preparing multiple nested sets of adapter-attached fragments, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer sequence, and a barcode sequence, wherein in each nested group of fragments with adapters, the target sequence fragments have the same nucleotide sequence at the first end and differ from each other due to truncation at the second end, such that each nested group of fragments with adapters comprises a plurality of target sequence fragments having different lengths, wherein the first end is closer to the barcode sequence than the second end, The method comprises: (a) providing a population of single-stranded DNA concatemers in a reaction, wherein each concatemer comprises a plurality of identical monomers, and each monomer comprises a complement of a target sequence, a complement of a barcode sequence that recognizes the concatemer, and a primer binding sequence shared by the population of single-stranded concatemers, wherein the primer binding sequence comprises a sequence complementary to the primer sequence, wherein the primer binding sequence and the complement of the barcode sequence are both at 3-primers of the complement of the target sequence; (b) annealing a primer comprising the primer sequence to the primer binding sequences of the plurality of monomers of each of the plurality of concatemers; (c) extending at least some of the primers hybridized to the primer binding sequence using a DNA polymerase having 5'-->3' exonuclease activity and not having strand displacement activity, wherein the extending generates a plurality of extension primers, each of the extension primers comprises a target sequence fragment having a barcode sequence and a primer sequence, wherein the extension primer hybridizes to the concatemer; wherein the extension primers are separated by a spacer, and (d) contacting the plurality of extension primers with the following under conditions where the exonuclease degrades a portion of the target sequence fragment in the extension primer: a 5-primer adapter comprising said 5-primer adapter sequence, a 3-primer adapter comprising said 3-primer adapter sequence, DNA ligase, and an exonuclease having single-stranded DNA exonuclease activity to generate a shortened extension primer, the 5-primer adapter being ligated to the 5' end of the shortened extension primer, and the 3-primer adapter being ligated to the 3' end of the shortened extension primer, This results in multiple nested groups of adapter-containing fragments.

29. The method of claim 28, wherein the population of single-stranded DNA concatemers is generated by rolling circle replication of circular templates, wherein each of the circular templates comprises the target sequence, the barcode sequence, and the primer sequence.

30. The method of claim 28, wherein the 5-primer adapter is an L-adapter and the 3-primer adapter is a branched adapter.

31. The method of claim 28, wherein the method further comprises adding a nuclease to extend the gap formed in step (c), wherein the nuclease has single-stranded exonuclease activity.

32. The method of claim 31, wherein at least some of the primers are RNA primers, and wherein the nuclease is RNAse H, wherein the RNAse H digests the RNA primers, thereby extending the gap.

33. The method of claim 28, wherein the primer binding sequence is located at the 3-primer of the complement of the barcode sequence in step (a), wherein the exonuclease has 3'→5' exonuclease activity, and wherein the barcode sequence in each of the groups of adapter-bearing fragments is located 5-primer relative to the target sequence fragment.

34. The method of claim 28, wherein the primer binding sequence is located at 5-primer relative to the complement of the barcode sequence in step (a), wherein the exonuclease has 5'→3' exonuclease activity, and Wherein in each of the adapter-bearing fragments, the barcode sequence is located at the 3-primer relative to the target sequence fragment.

35. The method of any of the preceding claims, wherein both the 5-primer adapter and the 3-primer adapter are in solution.

36. The method of claim 35, wherein the reaction does not contain a solid support.

37. The method of any of the preceding claims, wherein the target sequence is between 500 bases and 50 kilobases in length.

38. The method of claim 30, wherein the branched adapter comprises a double-stranded blunt end comprising a 5' end of one strand and a 3' end of a complementary strand, and The 5' end of the strand in the double-stranded blunt end is connected to the 3' end of at least one of the extension primers via a branched connection.

39. The method of claim 30, wherein the L-adapter comprises 1-10 degenerate bases at the 3-primer.

40. A method for preparing a nested set of multiple adapter-attached fragments, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer binding sequence, and a complement of a barcode sequence, wherein in each nested group of fragments with adapters, the target sequence fragments have the same nucleotide sequence at a first end and differ from each other at a second end, such that each nested group of fragments with adapters comprises a plurality of target sequence fragments having different lengths, wherein the first end is closer to the barcode sequence than the second end, The method comprises: (a) providing a barcoded fragment comprising a barcode sequence, a target sequence, and a primer binding sequence, wherein one end of the barcoded fragment is immobilized on a bead, (b) annealing a primer comprising the 5-primer adapter sequence to the primer binding sequence in the barcoded fragment, wherein the 5-primer adapter sequence comprises i) the complement of the barcode sequence, and ii) a primer sequence complementary to the primer binding sequence in the barcoded fragment, (c) extending the primer to generate an extended primer comprising the complement of the target sequence fragment and the barcode sequence, (d) contacting the extension primer with a branched adapter comprising the 3-primer adapter sequence to generate an adapter-bearing fragment, (e) separating the adapter-attached fragments from the barcoded fragments still immobilized on the beads, and (f) repeating steps (b)-(e) for one or more cycles under extension control conditions to generate one or more adapter-bearing fragments, wherein the adapter-attached fragments generated from step (e) and the adapter-attached fragments generated from step (f) constitute the nested group of adapter-attached fragments, and The adapter-attached fragments in each nested group contain target sequence fragments with different lengths.

41. The method of claim 40, wherein the primer is extended with uracil under extension control conditions for one or more extension cycles to generate the extended primer, thereby generating the adapter fragment incorporating the uracil in the 5 primer portion of the target sequence fragment, (g) contacting the adapter-attached fragment with an enzyme to remove the incorporated uracil, thereby generating at least one gap flanked by an exposed 3-primer end and an exposed 5-primer end of the adapter-attached fragment, (h) ligating an internal branching adaptor to the exposed 3-primer end in the at least one gap and ligating an L-adapter to the exposed 5-primer end in the gap, and (i) ligating the internal branched adaptor ligated to the exposed 3-primer end in step (h) to the L-adapter ligated to the exposed 5-primer end, thereby generating a shortened adaptor-bearing fragment, Thereby, a set of shortened adapter-attached fragments are generated, which include shortened target sequence fragments having sequences corresponding to different regions of the target sequence, and the different regions are overlapping.

42. The method of claim 41, wherein ligating the internal branch adapter and the L-adapter comprises contacting the internal branch adapter and the L-adapter with a splint oligonucleotide, wherein the splint oligonucleotide comprises a 5-primer portion complementary to a sequence in the internal branch adaptor and a 3-primer portion complementary to the L-adapter, Thereby, the splint oligonucleotide is hybridized to the internal branch adapter via the 5-primer portion, and the splint oligonucleotide is hybridized to the L-adapter via the 3-primer portion, thereby connecting the internal branch adapter and the L-adapter.

43. A method for preparing multiple sets of adapter-attached fragments, wherein each adapter-attached fragment is a single-stranded nucleic acid comprising a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, a 3-primer adapter sequence, a primer binding sequence, and a complement of a barcode sequence, The method comprises: (a) providing a barcoded fragment comprising a barcode sequence, a target sequence, and a primer binding sequence, wherein one end of the barcoded fragment is immobilized on a bead, (b) annealing a primer comprising the 5-primer adapter sequence to the primer binding sequence in the barcoded fragment, wherein the 5-primer adapter sequence comprises i) the complement of the barcode sequence, and ii) a primer sequence complementary to the primer binding sequence in the barcoded fragment, (c) extending the primer to generate an extended primer comprising the complement of the target sequence fragment and the barcode sequence, (d) contacting the extension primer with a first branched adapter comprising a 3-primer portion containing a degenerate sequence region, thereby forming a first extension product comprising the degenerate sequence region at the 3-primer portion, wherein the 3-primer portion hybridizes to the barcoded fragment through the degenerate sequence region, (e) extending the 3-primer portion of the first extension product to generate a second extension product, and (f) contacting the second extension product with a second branched adapter to generate the adapter-bearing fragment.

44. The method of claim 43, wherein the method further comprises: (g) denaturing to separate the adapter-bearing fragments from the barcoded fragments.

45. The method of claim 44, wherein the method further comprises: Steps (b)-(g) are repeated for one or more cycles under extension control conditions to generate one or more adapter-bearing fragments.

46. ​​A DNA complex comprising a plurality of fragments hybridized to one or more monomers of a DNA concatemer, wherein the plurality of fragments are separated by a spacer, wherein each of the plurality of fragments comprises a barcode sequence and a target sequence fragment having a first end and a second end, The target sequence fragments in the plurality of fragments have the same nucleotide sequence at the first end and are different from each other due to truncation at the second end, so that the target sequence fragments in the plurality of fragments have different lengths.

47. The DNA complex of claim 46, wherein each of the plurality of fragments is ligated to an L-adapter at the 5-primer end and a branched adaptor at the 3-primer end.

48. A DNA complex comprising: (a) barcoded fragments immobilized on a solid support, wherein the barcoded fragment comprises a barcode sequence and a target sequence, and (b) a polynucleotide that hybridizes to the barcoded fragment, wherein the polynucleotide comprises: a 5-primer portion comprising the complement of the barcode sequence, a 3-primer portion comprising a target sequence fragment; The 5-primer portion and the 3-primer portion anneal to the barcoded fragment, leaving the middle portion unannealed to the barcoded fragment, thereby forming a bubble.

49. A plurality of DNA complexes as described in any one of claims 46-48, wherein the DNA complexes share the same barcode sequence.

50. A composition comprising a nested set of adapter-attached fragments, each adapter-attached fragment comprising a barcode sequence and a target sequence fragment having a first end and a second end, a 5-primer adapter sequence, and a 3-primer adapter sequence, wherein the target sequence fragments have the same nucleotide sequence at the first end and differ from each other by truncation at the second end, so that the nested set of adapter-bearing fragments comprises a plurality of target sequence fragments having different lengths, and The nested groups of adapter-containing fragments share the same barcode sequence.

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