CN118308491A

CN118308491A - Single cell DNA methylation detection method

Info

Publication number: CN118308491A
Application number: CN202310018280.8A
Authority: CN
Inventors: 曹云龙; 谢晓亮; 袁天骄; 白雅丽
Original assignee: Beijing Changping Laboratory; Peking University
Current assignee: Beijing Changping Laboratory; Peking University
Priority date: 2023-01-06
Filing date: 2023-01-06
Publication date: 2024-07-09
Also published as: WO2024146540A1

Abstract

The present disclosure provides methods of single cell DNA methylation analysis that enable high throughput, applicable to whole genome DNA methylation sequencing of single cells within a variety of tissues.

Description

Single cell DNA methylation detection method

Technical Field

The present disclosure provides single cell DNA methylation detection methods.

Technical Field

DNA methylation, an important epigenetic modification, plays an important role in the genomic regulation process. The establishment and removal of DNA methylation is often closely related to cell fate decisions, embryonic development, disease development, and the like. Researchers explore the relationship between DNA methylation and cell type-specific gene expression, maintenance of cell status, and transformation by analyzing differences in DNA methylation modification distribution in the genomes of different tissues or different cell types. With the intensive research, researchers have found that the way in which genomic DNA of tissue is extracted for sequencing masks the heterogeneity between cells within the tissue. The high-throughput single-cell DNA methylation sequencing technology is particularly important for realizing high-precision DNA methylation analysis aiming at complex biological tissues such as brain, cancer and the like.

The principle of methylation sequencing is to distinguish cytosine carrying methylation modification from cytosine not carrying methylation modification by using chemical reaction difference, wherein the chemical reaction difference comprises a whole genome DNA methylation sequencing technology based on bisulfite conversion, and damage to DNA caused by the chemical reaction and DNA loss caused by purification in the TAPS flow (Liu et al.,Nature Biotechnology(2019)."Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution"),EM-seq flow (Williams et al.,New England Biolabs,Inc.(2019)."Enzymatic Methyl-seq:The Next Generation of Methylome Analysis"). limit the application of the methods in a single-cell initiation scene.

Single cell DNA methylation sequencing methods for DNA amplification following bisulfite conversion, which include scRRBS(Guo et al.,Genome Research(2013)."Single-Cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing"),scBS-seq(Smallwood et al.,Nature Methods(2014)."Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity"),scWGBS(Farlik et al.,Cell Reports(2015)."Single-Cell DNA Methylome Sequencing and Bioinformatic Inference of Epigenomic Cell-State Dynamics"). to prevent DNA damage and loss from bisulfite reaction, purification, have low levels of genome coverage for single cell DNA methylation sequencing.

In snmC-seq(Luo et al.,Science(2017)."Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex") flow published in 2017 in Science journal, random primers marked by tag sequences are utilized to amplify products after the bisulfite conversion, so that single-cell DNA carrying different tag sequences can be mixed for library construction, and flux improvement is realized. The method has the problems that before single cells are labeled, each cell needs to be subjected to bisulfite conversion and DNA purification separately, so that the reagent cost and the operation complexity are increased, the requirements on a laboratory platform are high, and the technical popularization is difficult to realize. According to the sci-MET(Mulqueen et al.,Nature Biotechnology(2018)."Highly scalable generation of DNA methylation profiles in single cells") method published in 2018 in Nature Biotechnology journal, labeled Tn5 is utilized to fragment DNA in cell nuclei, and cells marked with different labels can be converted into bisulfite in a reaction system in a mode of uniformly mixing and then sorting. However, the destructive nature of the bisulfite reaction on DNA causes the fragmented DNA to further break down and degrade, and the loss of substantial information ultimately severely affects detection sensitivity.

In patent application WO2021077415, a single-cell DNA methylation sequencing technology based on Tn5 transposition and enzymatic transformation is provided, which can be called Cabernet technology, wherein a second generation sequencing linker carrying methylation modification is connected to both ends of a DNA fragment through Tn5, and cytosine carrying methylation modification is distinguished from cytosine not carrying methylation modification in combination with enzymatic transformation reaction, so that single-cell whole genome DNA methylation detection is realized. The method replaces the bisulfite conversion, and the damage of chemical reaction to DNA is reduced as much as possible by relying on an enzymatic conversion mode, however, each cell still needs to be independently operated, and the high flux requirement is difficult to meet.

The introduction of single cell independent tags using Tn5 followed by mixed amplification can achieve High throughput, including but not limited to the sci (single-cell combinatorial indexing) method (Mulqueen et al.,Nature Biotechnology(2018)."Highly scalable generation of DNA methylation profiles in single cells"),"Nature Biotechnology" journal published in 2021, s3 method (Mulqueen et al., nature Biotechnology (2021), "High-content single-cell combinatorial indexing"). In the s3 method, tn5 transposase is assembled with a DNA linker sequence containing uracil, so that incomplete extension can be realized during gap filling reaction, and further linker replacement can be realized. The method introduces a tag sequence by means of Tn5 multicellular transposition reaction to achieve flux enhancement, however, the method is not suitable for methods requiring sequence transformation such as methylation sequencing while limiting transposition reaction efficiency.

In view of the above, there is still a lack of single-cell DNA methylation detection methods with high throughput, low cost and high sensitivity in the field.

Summary of The Invention

The present disclosure provides methods for single cell methylation detection that achieve high throughput by introducing single cell tags followed by mixed enzymatic conversion and sequencing by library construction.

In one aspect, the present disclosure provides a method of analyzing methylation characteristics of single cell genomic DNA comprising:

Contacting genomic DNA from a single cell with a plurality of transposomes, wherein the transposomes comprise a transposase and a transposon DNA, wherein the transposon DNA comprises a double stranded transposase binding site and an overhang, wherein the overhang comprises a first primer binding sequence at the 5' end of the overhang and comprises uracil nucleotides downstream (e.g., 3' end) of the first primer binding sequence, upstream (e.g., 5' end) of the transposase binding site; to obtain double-stranded genomic DNA fragments comprising transposon DNA at each end;

Filling gaps between the transposon DNA and the genomic DNA fragment with uracil intolerant polymerase to form a first double stranded extension product of the genomic DNA fragment;

Contacting the first double-stranded extension product with a template switching oligonucleotide, wherein the template switching oligonucleotide comprises, from 5 'end to 3' end: a second primer binding sequence, a tag sequence, and a transposase binding site binding sequence; wherein the tag sequence has a unique nucleotide sequence corresponding to the cell;

Performing an extension reaction using uracil intolerant polymerase to obtain a second double-chain extension product; wherein the second double-stranded extension product comprises a library of tagged genomic DNA fragments of the cell;

Mixing libraries of tagged genomic DNA fragments obtained from different cells and processing the mixed libraries to convert cytosine to uracil;

The transformed mixed library is amplified using the first and second primers to generate amplicons.

In certain embodiments, the transposome has two transposases and two transposon DNA, wherein each transposon DNA comprises a transposase binding site and an overhang of the double strand.

In certain embodiments, the plurality of transposomes cleave the genomic DNA into a plurality of double-stranded genomic DNA fragments representing a library of genomic DNA fragments, wherein each double-stranded genomic DNA fragment comprises transposon DNA on each end of the genomic DNA fragment.

In certain embodiments, the first double-stranded extension product has a 5' overhang at each end, the overhang comprising a first primer binding sequence.

In certain embodiments, the second double-stranded extension product has a 5' overhang at one end, the overhang comprising the first primer binding sequence.

In certain embodiments, the template switching oligonucleotide comprises a transposase binding site binding sequence capable of hybridizing to a transposase binding site sequence 3' of the genomic fragment in the first double stranded extension product.

In certain embodiments, the methylation includes 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC).

In certain embodiments, the transposase binding site binding sequence included in the template switching oligonucleotide comprises a Locked Nucleotide (LNA) modification.

In certain embodiments, the transposase binding site binding sequence comprises a plurality of LNA modifications, e.g., 2,3, 4, or 5 LNA modifications.

In certain embodiments, the filling of the gap and/or extending step comprises using A, T, C, G nucleotides, wherein a cytosine nucleotide (e.g., dCTP) comprises a modified cytosine that is resistant to the transformation step. In certain embodiments, the tolerating transformation step refers to a reaction that is capable of tolerating the conversion of cytosine to uracil (e.g., apodec deamination) without sequence changes. In certain embodiments, the modified cytosine is a methylated cytosine, such as 5-methylcytosine (5-mdC) or 5-hydroxymethylcytosine (5-hmdC).

In certain embodiments, the first primer binding sequence is free of cytosine nucleotides.

In certain embodiments, the first primer binding sequence comprises a cytosine nucleotide comprising a modified cytosine, wherein the modified cytosine is resistant to the transformation step. In certain embodiments, the modified cytosine is a methylated cytosine, such as 5-methylcytosine (5-mdC) or 5-hydroxymethylcytosine (5-hmdC).

In certain embodiments, the transposon DNA comprises a plurality (e.g., 2, 3, or 4) consecutive uracil nucleotides downstream of the first primer binding sequence and upstream of the transposase binding site.

In certain embodiments, the 3' end of the first primer binding sequence is immediately adjacent to the uracil nucleotide. In certain embodiments, the 5' end of the transposase binding site is immediately adjacent to the uracil nucleotide. In certain exemplary embodiments, the first primer binding sequence and the transposase binding site are linked by 3 consecutive uracil nucleotides.

In certain embodiments, the combinatorial library is treated in the presence of vector DNA to convert cytosine to uracil. In certain embodiments, the vector DNA is selected from dsDNA fragments between 100bp and 4000bp (e.g., 100bp to 1000bp, 100bp to 800bp, 100bp to 600bp, 100bp to 500bp, 100bp to 400bp, 200bp to 400 bp) in length. In certain embodiments, the vector DNA is selected from about 200bp to 400bp, e.g., 300bp, in length. In certain embodiments, the vector DNA is sonicated lambda DNA.

In certain embodiments, the method further comprises sequencing the amplicon.

In certain embodiments, the transformed mixed library is amplified using the first and second primers to generate amplicons comprising sequencing adaptors at each end, thereby generating a sequencing library.

In certain embodiments, the sequencing linker is an Illumina sequencing linker.

In certain embodiments, the first primer is introduced into the P7 end and the second primer is introduced into the P5 end. In certain embodiments, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion 5' of the first portion comprising a P7 sequence, which may also comprise other functional sequences as desired, such as index sequences. In certain embodiments, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion 5' of the first portion comprising a P5 sequence, which may also comprise other functional sequences as desired, such as index sequences.

In certain embodiments, the first primer is introduced into the P5 end and the second primer is introduced into the P7 end. In certain embodiments, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion 5' of the first portion comprising a P5 sequence, which may also comprise other functional sequences as desired, such as index sequences. In certain embodiments, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion 5' of the first portion comprising a P7 sequence, which may also comprise other functional sequences as desired, such as index sequences.

In certain embodiments, the transposase IS a Tn5 transposase, mu transposase, tn7 transposase, or IS5 transposase.

In certain embodiments, the uracil intolerant polymerase is selected from the group consisting of Q5 polymerase, DEEP VENT DNA polymerase, phusion high fidelity polymerase, KAPA high fidelity polymerase, phanta polymerase, or any combination thereof.

In certain embodiments, the step of amplifying using the first and second primers is performed using uracil-tolerant polymerase. In certain embodiments, the uracil-resistant polymerase is selected from the group consisting of Q5U polymerase, KAPA u+ polymerase, phanta Uc polymerase, or any combination thereof.

In certain embodiments, the bound transposase is removed from the double stranded genomic DNA fragment prior to gap filling and extension of the double stranded fragment.

In certain embodiments, the method further comprises the step of, after the extending step but before the converting step: the purification step is performed by purifying the reaction medium comprising the second double-stranded extension product, for example by means of a DNA spin-column (DNA spin-column) or bead-based DNA purification (beads-based DNA purification).

In certain embodiments, the method further comprises the step, after the amplifying step, of: purification of the reaction medium containing the amplicon, such as by a DNA spin-column (DNA spin-column) or bead-based DNA purification (beads-based DNA purification).

In certain embodiments, the treatment that converts cytosine to uracil is an enzymatic conversion. In certain embodiments, the enzymatic conversion comprises an apodec deamination reaction. In certain embodiments, the enzymatic conversion comprises the use of T4-BGT enzyme and APOBEC3A enzyme. In certain embodiments, the enzymatic conversion comprises the use of a TET2 enzyme and an apodec 3A enzyme.

Drawings

The foregoing and other features and advantageous aspects of the invention will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings in which:

Fig. 1 shows a schematic diagram of the workflow of an exemplary embodiment. Wherein single cells are first sorted and lysed, then Tn5 transposes to form single cell genomic DNA fragments, and independent tag sequences are tagged to distinguish from other cells, then single cell DNA tagged with different tag sequences can be mixed for subsequent enzymatic transformation and pool-building sequencing procedures.

Fig. 2 shows a schematic diagram of the chemical reaction principle of an exemplary embodiment of the addition of a single cell tag.

FIG. 3 shows the data resolution results of the labeled single cells. The left panel shows the distribution of the amount of different single cell sequencing data split according to the tag, with the abscissa representing the number of sequencing reads split for multiple cells mixed in the same 96 well plate. The right panel shows the mixed pooling sequencing of human and mouse DNA in the same 96 well plate, with the split sequencing reads aligned in human and mouse reference genomes.

Fig. 4 shows quality control and correlation performance. A, lambda DNA fragment (C in the figure) without carrying any methylation modification, pUC19 DNA fragment (5 mC in the figure) with 5mC modification at CpG site, 5hmC DNA fragment (5 hmC in the figure), and 5mC and 5hmC detection are respectively carried out on three standard substances according to the TSO-Cabernet flow, and the obtained accuracy results are shown in a bar graph. B, heat maps show pearson correlation of genome 5mC modifications between single cell samples and multicellular samples detected by TSO-Cabernet technology (tso_1-tso_5) and Cabernet technology (Cabernet _1-Cabernet _3, bulk_cabernet_1, bulk_cabernet_2), with red indicating strong correlation.

Detailed Description

According to one aspect, a method of analyzing methylation characteristics of genomic DNA of a single cell is provided, the genomic DNA from the single cell being contacted with a plurality of transposomes. Each transposome has two transposases (e.g., tn5 transposase) and two transposon DNAs, each transposase being bound to a respective transposon DNA to form a transposase/transposon DNA complex dimer. Each transposon DNA includes a double stranded transposase binding site (e.g., a double stranded 19bp Tnp binding site) and an overhang (e.g., a 5' overhang). The overhang includes a first primer binding sequence at the 5' end of the overhang and comprises uracil nucleotides (e.g., one or more consecutive uracil nucleotides) downstream (e.g., 3' end) of the first primer binding sequence and upstream (e.g., 5' end) of the transposase binding site. The overhang may have any length suitable for including the first primer binding sequence or other functional sequence as desired.

The transposomes randomly bind to a target location along double-stranded genomic DNA and cleave the double-stranded genomic DNA into a plurality of double-stranded fragments, wherein each double-stranded fragment has a first complex linked to an upper strand through a transposase binding site and a second complex linked to a lower strand through a transposase binding site. Thus, the transposon DNA (i.e., the transposase binding site together with the overhang comprising the first primer binding sequence) is ligated to each 5' end of the double stranded fragment. According to one aspect, the transposase is removed from the complex.

The transposon DNA is ligated to the double stranded genomic DNA fragment, and a single stranded gap exists between one strand of the genomic DNA double stranded fragment and one strand of the transposon DNA. Filling the gap between the transposon DNA and the genomic DNA fragment with uracil intolerant polymerase, creating a double stranded ligation between the double stranded genomic DNA fragment and the double stranded transposon DNA to form a first double stranded extension product of the genomic DNA fragment, the first double stranded extension product having a 5' overhang at each end, the overhang comprising a first primer binding sequence. According to one aspect, uracil nucleotides are included between the downstream (e.g., 3 'end) of the first primer binding sequence and the upstream (e.g., 5' end) of the transposase binding site, thereby preventing the 3 'ends of both strands of the first double stranded extension product from continuing to extend toward the first primer binding sequence when gap-fill procedures are performed using uracil intolerant polymerases, thereby forming the first double stranded extension product with 5' overhangs at each end. According to one aspect, the first double stranded extension product comprises a top strand comprising, from the 5 'end to the 3' end: a first primer binding sequence, uracil nucleotides, a transposase binding site, a genomic DNA fragment upper strand, a filled gap, a transposase binding site, the lower strand comprising from 5 'to 3': a first primer binding sequence, uracil nucleotides, a transposase binding site, a genomic DNA fragment downlink, a filled gap, a transposase binding site.

Contacting the first double-stranded extension product with a template switching oligonucleotide, wherein the template switching oligonucleotide comprises, from 5 'end to 3' end: a second primer binding sequence, a tag sequence, and a transposase binding site binding sequence; wherein the tag sequence has a unique nucleotide sequence corresponding to the cell. According to one aspect, genomic DNA of different cells is tagged with different unique tag sequences to distinguish from each other by introducing tag sequences. According to one aspect, the transposase binding site binding sequence is capable of hybridizing to a transposase binding site sequence 3' of the genomic fragment in the first double stranded extension product.

Performing an extension reaction using uracil intolerant polymerase to obtain a second double-chain extension product; wherein the second double-stranded extension product has a 5' overhang at one end, the overhang comprising a first primer binding sequence; wherein the second double-stranded extension product constitutes a library of tagged genomic DNA fragments of the cell. According to one aspect, the template switch oligonucleotide anneals to one strand of the first double-stranded extension product, and the annealed template switch oligonucleotide serves as a template strand, allowing the strand of the first double-stranded extension product to undergo an extension reaction (e.g., PCR extension) to obtain the tag sequence and the second primer binding sequence, thereby allowing the genomic DNA fragment to be tagged. It will be appreciated by those skilled in the art that the tag sequence and the second primer binding sequence contained in the tagged genomic DNA strand obtained by the extension reaction described above are in fact complementary to the corresponding sequences in the template switch oligonucleotide, according to the base pairing rules. According to one aspect, one strand of the first double stranded extension product will also act as a template strand, extending the annealed template switch oligonucleotide to the 3' end, wherein a uracil nucleotide is included between the downstream (e.g., 3' end) of the first primer binding sequence and the upstream (e.g., 5' end) of the transposase binding site, thereby preventing the 3' end of the template switch oligonucleotide from continuing to extend toward the first primer binding sequence when extension reactions are performed using uracil intolerant polymerases, thereby forming the second double stranded extension product with a 5' overhang at one end. According to one aspect, the second double-chain extension product comprises a top strand comprising, from the 5 'end to the 3' end: a first primer binding sequence, uracil nucleotide, a transposase binding site, a genomic DNA fragment, a transposase binding site, a tag sequence, a second primer binding sequence, the lower strand comprising from 5 'to 3': a second primer binding sequence, a tag sequence, a transposase binding site, an extension strand with a genomic DNA fragment as a template, a transposase binding site.

Libraries of tagged genomic DNA fragments obtained from different cells are mixed and the mixed library is processed to convert cytosine to uracil. According to one aspect, the above steps are performed separately on genomic DNA from different individual cells, and separate second double-stranded extension products with unique tag sequences are obtained for each cell, followed by mixing of these second double-stranded extension products. According to one aspect, single cell genomic DNA carrying different tag sequences is mixed and transformed and pooled to significantly increase throughput.

The transformed mixed library is amplified using the first and second primers to generate amplicons. According to one aspect, the first and second primers specifically hybridize to a first primer binding sequence and a second primer binding sequence, respectively, at both ends of the tagged genomic DNA fragment. According to one aspect, the first and second primers are mixed with a mixed library and the tagged genomic DNA fragments are amplified. According to one aspect, sequencing adaptors are introduced at both ends of the tagged genomic DNA fragments from the first primer and the second primer, respectively, thereby generating a sequencing library. According to one aspect, amplicons that include a sequencing adapter at each end are sequenced using, for example, high throughput sequencing methods known to those skilled in the art.

Unless otherwise indicated, certain embodiments or implementations of features of certain embodiments may employ conventional techniques of molecular biology, microbiology, recombinant DNA, etc., which are within the ability of one of ordinary skill in the art. Such techniques are well explained in the literature. See, sambrook, fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2 nd edition (1989), OLIGONUCLEOTIDE SYNTHESIS (m.j.gai edit, 1984), ANIMAL CELL CULTURE (r.i.freshney, edit ,1987),the series METHODS IN ENZYMOLOGY(Academic Press,Inc.);GENE TRANSFER VECTORS FOR MAMMALIAN CELLS(J.M.Miller and m.p.calos edit 1987), HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, (d.m.weir and c.blackwell, edit ),CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(F.M.Ausubel,R.Brent,R.E.Kingston,D.D.Moore,J.G.Siedman,J.A.Smith,and K.Struhl,eds.,1987),CURRENT PROTOCOLS IN IMMUNOLOGY(J.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach and w.strober, edit 1991); ANNUAL REVIEW OF IMMUNOLOGY; and journal articles such as ADVANCES IN IMMUNOLOGY. All patents, patent applications, and publications mentioned herein above and below are hereby incorporated by reference.

The terms and symbols of nucleic acid chemistry, biochemistry, genetics and molecular biology as used herein follow the terms and symbols of the art standard papers and texts (e.g., kornberg and Baker, DNA Replication, 2 nd edition (W.H. Freeman, new York, 1992), lehninger, biochemistry, 2 nd edition (Worth Publishers, new York, 1975), strachan and Read, human Molecular Genetics, 2 nd edition (Wiley-List, new York, 1999), eckstein, editor, oligonucleotides and Analogs: A PRACTICAL Aproch (Oxford University Press, new York, 1991), gait, editor, oligonucleotide Synthesis: A PRACTICAL Aproch (IRL Press, oxford, 1984), and the like).

Definition of terms

As used herein, "DNA methylation" typically includes modification of cytosine bases in a DNA molecule or DNA fragment to 5-methylcytosine (5 mC). Furthermore, although the incidence is less than 5mC, few cytosine bases are modified to 5-hydroxymethylcytosine (5 hmC), 5-aldehyde cytosine (5 fC), 5-carboxyl cytosine (5 caC), etc. References herein to methylation generally refer to any instance in which a cytosine base is modified, and may refer to modification to 5mC, and may refer to modification to 5hmC, 5fC, 5caC, etc., unless the context indicates otherwise. In certain embodiments, the methylation described herein comprises 5-methylcytosine (5 mC) and/or 5-hydroxymethylcytosine (5 hmC).

As used herein, "methylation signature" refers to information about the methylation status in a DNA molecule or DNA fragment, including, but not limited to, methylation site, methylation level, methylation pattern (5 mC or 5 hmC), and the like. Herein, "methylation level", which may also be referred to as "degree of methylation", refers to the proportion (or frequency) of methylation modification of a particular methylation site in a sample. Methylation detection is generally based on the following principle: converting one of methylated cytosine and unmethylated cytosine to uracil (U) or a base substantially identical to uracil in base pairing (e.g., dihydrouracil, DHU); in the subsequent amplification process, the corresponding uracil is paired with adenine (A) as thymine (T), with the end result that the cytosine or methylated cytosine at the methylation site is represented as thymine in the detection result (e.g., sequencing result); by comparison with the reference sequence, it is possible to determine whether or not the cytosine in the DNA molecule or DNA fragment is methylated. The reference sequence may be a sequence from the same sample but not transformed as described above, or a corresponding sequence in a healthy population. In addition, as described below, different methylation patterns (e.g., 5mC and 5 hmC) can also be distinguished by some means.

As used herein, "single cell" refers to one cell. Individual cells useful in the methods described herein may be obtained from a tissue of interest or from a biopsy, blood sample, or cell culture. In addition, cells from specific organs, tissues, tumors, neoplasms (neoplasm), etc., may be obtained and used in the methods described herein. In addition, generally, cells from any population can be used in the method, such as a population of prokaryotic or eukaryotic single-cell organisms (including bacteria or yeast). Single cell suspensions may be obtained using standard methods known in the art, including, for example, enzymatic digestion of cell-linked proteins in tissue samples using trypsin or papain, or release of adherent cells in culture, or mechanical separation of cells in samples. The single cells may be placed in any suitable reaction vessel that allows the single cells to be treated individually. For example, a 96-well plate such that each individual cell is placed in an individual well.

As used herein, the term "genome" is defined as a collection of genes carried by an individual, cell, or organelle. As used herein, the term "genomic DNA" is defined as DNA material comprising a collection of part or all of the genes carried by an individual, cell, or organelle.

As used herein, the term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to a sugar moiety through an ester linkage. Exemplary nucleotides include nucleoside monophosphates, diphosphate and triphosphate. The terms "polynucleotide," "oligonucleotide," and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides of any length (deoxyribonucleotides or ribonucleotides) joined together by phosphodiester linkages between 5 'and 3' carbon atoms. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The term also refers to double-stranded and single-stranded molecules. Unless otherwise indicated or required, any embodiment of the invention comprising a polynucleotide includes both the double stranded form and each of the two complementary single stranded forms known or predicted to make up the double stranded form. Polynucleotides consist of a specific sequence of four nucleotide bases: adenine (a); cytosine (C); guanine (G); thymine (T); and uracil (U) corresponding to thymine when the polynucleotide is RNA. Thus, the term polynucleotide sequence is a alphabetical representation of a polynucleotide molecule. The alphabetical representation may be entered into a database in a computer with a central processing unit and used for bioinformatic applications such as functional genomics and homology searches.

The nucleotides described herein include natural nucleotides as well as nucleotide analogs or modified nucleotides. The terms "nucleotide analog", "modified nucleotide" refer to non-standard nucleotides, including non-naturally occurring ribonucleotides or deoxyribonucleotides. In certain exemplary embodiments, nucleotide analogs can be modified at any position to alter certain chemical properties of the nucleotide, but retain the ability of the nucleotide analog to perform its intended function. Examples of the positions of nucleotides that can be derivatized include the 5-position, e.g., 5- (2-amino) propyluridine, 5-bromouridine, 5-propynyluridine, 5-propenyl uridine, etc.; etc.; position 6, e.g., 6- (2-amino) propyluridine; adenosine and/or guanosine at the 8-position, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine; o-and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or other modifications known in the art) nucleotides; other heterocycle-modified nucleotide analogs, such as those described in Herdewijn, ANTISENSE NUCLEIC ACID DRUG dev, 2000aug.10 (4): 297-310.

Nucleotide analogs may also include modifications to the sugar portion of a nucleotide. For example, the 2' OH-group may be replaced by a group selected from the group consisting of: H. OR, R, F, cl, br, I, SH, SR, NH ₂、NHR、NR₂, COOR, OR OR, wherein R is substituted OR unsubstituted C ₁-C₆ alkyl, alkenyl, alkynyl, aryl, OR the like. Other possible modifications include those described in U.S. patent nos. 5,858,988 and 6,291,438.

The phosphate group of a nucleotide may also be modified, for example, by replacing one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioate), or by making other substitutions that allow the nucleotide to perform its intended function, such as described, for example, in Eckstein, ANTISENSE NUCLEIC ACID DRUG Dev.2000Apr.10 (2): 117-21, rusckowski et al Antisense Nucleic Acid Drug Dev.2000Oct.10(5):333-45,Stein,Antisense Nucleic Acid Drug Dev.2001Oct.11(5):317-25,Vorobjev et al ANTISENSE NUCLEIC ACID DRUG Dev.2001Apr.11 (2): 77-85 and U.S. Pat. No. 5,684,143). Some of the above modifications (e.g., phosphate group modifications) reduce the rate of hydrolysis of, for example, polynucleotides comprising the analogs in vivo or in vitro.

As used herein, the terms "complementary" and "complementarity" are used in reference to nucleotide sequences related to the base pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 5 '-ACT-3'. The complementarity may be partial or complete. Partial complementarity occurs when one or more nucleobases do not match according to the base pairing rules. Full or complete complementarity between nucleic acids occurs when each nucleic acid base matches another base under the base pairing rules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, the term "hybridization" refers to pairing of complementary nucleic acids. Hybridization and hybridization intensity (i.e., the intensity of association between nucleic acids) are affected by factors such as: the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the T _m of the hybrids formed, and the G to C ratio within the nucleic acids. The single molecule that contains within its structure a pairing of complementary nucleic acids is called "self-hybridizing" (self-hybridized).

As used herein, the term "T _m" refers to the melting temperature of a nucleic acid. The melting temperature is the temperature at which half of the population of double-stranded nucleic acid molecules dissociate into single strands. Equations for calculating T _m for nucleic acids are well known in the art. As shown in the standard reference, a simple estimate of the T _m value can be calculated by the following formula when the nucleic acid is in an aqueous solution of 1M NaCl: t _m =81.5+0.41 (% g+c) (see, e.g., anderson and Young, quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include more complex calculations that take structural as well as sequence features into account in the calculation of T _m.

As used herein, the term "stringency" refers to conditions of temperature, ionic strength, and the presence of other compounds, such as organic solvents, under which nucleic acid hybridization occurs.

When used in nucleic acid hybridization, "low stringency conditions" include conditions equivalent to: when using probes of about 500 nucleotides in length, binding or hybridization was performed at 42℃in a solution consisting of 5 XSSPE comprising 43.8g/l NaCl,6.9g/l NaH ₂PO₄(H₂ O) and 1.85g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5 XDenhardt's reagent (50 XDenhardt per 500 ml: 5g Ficoll (Type 400, pharmacia), 5g BSA (Fraction V; sigma)); then washed in a solution containing 5 XSSPE, 0.1% SDS at 42 ℃.

When used in nucleic acid hybridization, "moderately stringent conditions" include conditions equivalent to those described below: when probes of about 500 nucleotides in length are used, binding or hybridization is performed at 42℃in a solution consisting of 5 XSSPE and 100mg/ml denatured salmon sperm DNA, the 5 XSSPE comprising 43.8g/l NaCl,6.9g/l NaH ₂PO₄(H₂ O) and 1.85g/l EDTA, the pH being adjusted to 7.4 with NaOH), 0.5% SDS, 5 XDenhardt's reagent; then washed in a solution containing 1.0 XSSPE, 1.0% SDS at 42 ℃.

When used in nucleic acid hybridization, "high stringency conditions" include conditions equivalent to: when probes of about 500 nucleotides in length are used, binding or hybridization is performed at 42℃in a solution consisting of 5 XSSPE and 100mg/ml denatured salmon sperm DNA, the 5 XSSPE comprising 43.8g/l NaCl,6.9g/l NaH ₂PO₄(H₂ O) and 1.85g/l EDTA, the pH being adjusted to 7.4 with NaOH), 0.5% SDS, 5 XDenhardt's reagent; then washed in a solution containing 0.1 XSSPE, 1.0% SDS at 42 ℃.

When hybridization occurs between two single stranded polynucleotides in an antiparallel configuration, the reaction is referred to as "annealing" and those polynucleotides are described as "complementary". If hybridization can occur between one of the strands of a first polynucleotide and one of the strands of a second polynucleotide, then the double-stranded polynucleotide is complementary or homologous to the other polynucleotide. Complementarity or homology (the degree of complementarity of one polynucleotide with another polynucleotide) may be quantified according to the proportion of bases in the opposite strand expected to form hydrogen bonds with each other according to the commonly accepted base pairing rules.

As used herein, the term "amplification" refers to the process of forming additional or multiple copies of a particular polynucleotide. Amplification methods include PCR methods known to those of skill in the art, and also include rolling circle amplification (Blanco et al, j. Biol. Chem.,264,8935-8940,1989), hyperbranched rolling circle amplification (Lizard et al, nat. Genetics,19,225-232,1998), and loop-mediated isothermal amplification (Notomi et al, nuc. Acids res.,28, e63, 2000), each of which is hereby incorporated by reference in its entirety. Such methods are known in the art and widely practiced. The term "amplification reagents" may include primers, nucleic acid templates, and amplification enzymes (e.g., polymerases), but may also include other reagents required for amplification, such as nucleotides (e.g., deoxyribonucleotide triphosphates), buffers, and the like. Typically, the amplification reagents are placed and contained in reaction vessels (test tubes, microwells, etc.) along with the other reaction components.

As used herein, the term "primer" generally includes natural or synthetic oligonucleotides that are capable of acting as a point of initiation of nucleic acid synthesis (such as a sequencing primer) upon formation of a duplex with a polynucleotide template, and extending along the template from its 3' end, thereby forming an extended duplex. The sequence of the nucleotides added during extension is determined by the sequence of the template polynucleotide. Typically, the primer is extended by a DNA polymerase. The primers are typically 3 to 36 nucleotides, 5 to 24 nucleotides, 14 to 36 nucleotides or 17 to 30 nucleotides in length. A "primer" can be considered a short polynucleotide, typically having a free 3' -OH group that binds to a target or template that may be present in a sample of interest by hybridizing to the target, and subsequently facilitates polymerization of a polynucleotide complementary to the target.

Obtaining single cell genomic DNA

According to one aspect, the present disclosure provides methods involving obtaining genomic DNA from a single cell.

In certain embodiments, the cells are identified and then individual cells are isolated. Cells within the scope of the present disclosure include any type of cell in which it is considered by those skilled in the art to be useful to understand the DNA content. Cells according to the present disclosure include any type of cancer cells, hepatocytes, oocytes, embryonic cells, stem cells, iPS cells, ES cells, neurons, erythrocytes, melanocytes, astrocytes, germ cells, oligodendrocytes, kidney cells, and the like.

In certain embodiments, individual cells useful in the methods described herein may be obtained from a tissue of interest or from a biopsy, blood sample, or cell culture. In addition, cells from specific organs, tissues, tumors, neoplasms (neoplasm), etc., may be obtained and used in the methods described herein. Single cell suspensions may be obtained using standard methods known in the art, including, for example, enzymatic digestion of cell-linked proteins in tissue samples using trypsin or papain, or release of adherent cells in culture, or mechanical separation of cells in samples. The single cells may be placed in any suitable reaction vessel that allows the single cells to be treated individually. For example, a 96-well plate such that each individual cell is placed in an individual well.

Methods of manipulating single cells are known in the art, including Fluorescence Activated Cell Sorting (FACS), flow cytometry (herzenberg., PNAS USA 76:1453-55 1979), micromanipulation and the use of semi-automatic pickers (e.g., quixell ^TM cell transfer system from Stoelting co.). For example, individual cells may be individually selected based on characteristics that can be detected by microscopic observation, such as location, morphology, or reporter gene expression. In addition, a combination of gradient centrifugation and flow cytometry may also be used to increase separation or sorting efficiency.

Once the desired cells are identified, the cells are lysed using methods known to those skilled in the art to release the cell contents, including DNA. The cell contents are contained within a container or collection space. In certain embodiments, cellular content, such as genomic DNA, may be released from cells by lysing the cells. Lysis may be achieved, for example, by heating the cells, or by using detergents or other chemical methods, or by a combination of these methods. Any suitable cleavage method known in the art may be used. In certain embodiments, the cells are heated in a cell lysate containing a detergent. In certain embodiments, cells are heated at 72℃for 2 minutes in the presence of Tween-20, sufficient to lyse the cells; alternatively, the cells may be heated in water to 65℃for 10 minutes (Esumi et al, neurosci Res60 (4): 439-51 (2008)); or heated to 70℃in a PCR buffer II (Applied Biosystems) supplemented with 0.5% NP-40 for 90 seconds (Kurimoto et al, nucleic Acids Res (5): e42 (2006)); cleavage may be achieved with proteases such as proteinase K or by using chaotropic salts such as guanidinium isothiocyanate (U.S. publication No. 2007/0281313). The cell lysate obtained may be used directly according to the methods described herein, e.g. the reaction mixture may be added to the cell lysate. Alternatively, the cell lysate may be divided into two or more volumes using methods known to those skilled in the art, such as into two or more containers, tubes, or areas, with a portion of the cell lysate contained in each volume of container, tube, or area. Genomic DNA contained in each container, tube or region may then be processed by the methods described herein.

Swivel base

According to one aspect, the present disclosure provides methods involving methods of genomic DNA fragmentation using a transposase. The method uses a transposase or transposome to fragment an original or starting nucleic acid sequence (such as genomic DNA) and ligates an overhang sequence comprising a first primer binding sequence to each end of a cleavage site or cleavage site, thereby generating a set of fragments (each member of the set having the same overhang sequence).

In certain embodiments, genomic DNA is cleaved into double-stranded fragments using multiple transposomes or a library of transposomes. Each transposome of the plurality of transposomes or libraries is a dimer of a transposase bound to a transposon DNA, i.e., each transposome comprises two separate transposon DNA. Each transposon DNA of the transposomes comprises a transposase binding site and an overhang sequence comprising a first primer binding sequence. Thus, a number of fragments from the original nucleic acid sequence are generated by a library of transposomes, wherein each fragment has the same overhang sequence comprising the first primer binding sequence at each end of the fragment.

In certain embodiments, the overhang may have any length suitable for including the first primer binding sequence or other functional sequence desired. In certain exemplary embodiments, the overhang is no more than 60bp in length, e.g., no more than 55bp, no more than 50bp. In certain exemplary embodiments, the overhang is at least 4bp in length, e.g., at least 5bp, at least 8bp, at least 10bp, at least 12bp, at least 15bp. In certain exemplary embodiments, the overhang is 10-50bp in length.

In certain embodiments, the first primer binding sequence is free of cytosine nucleotides. According to one aspect, the first primer binding sequence that does not comprise a cytosine nucleotide is capable of tolerating a reaction that converts a cytosine to a uracil (e.g., an apodec deamination reaction) without sequence changes.

In certain embodiments, the transposon DNA comprises a plurality (e.g., 2, 3, or 4) consecutive uracil nucleotides downstream of the first primer binding sequence and upstream of the transposase binding site. In certain embodiments, the 3' end of the first primer binding sequence is immediately adjacent to the uracil nucleotide. In certain embodiments, the 5' end of the transposase binding site is immediately adjacent to the uracil nucleotide. In certain embodiments, the 3 'end of the first primer binding sequence and the 5' end of the transposase binding site are linked by a plurality (e.g., 2, 3, or 4) consecutive uracil nucleotides.

In certain embodiments, exemplary transposon systems include Tn5 transposase, mu transposase, tn7 transposase, or IS5 transposase, among others. Other useful transposon systems are known to those skilled in the art, including the Tn3 transposon system (see Maekawa,T.,Yanagihara,K.,and Ohtsubo,E.(1996),A cell-free system of Tn3 transposition and transposition immunity,Genes Cells 1,1007-1016)、Tn7 transposon system (see Craig, N.L. (1991), tn7: A TARGET SITE-specific transposon, mol. Microbiol.5, 2569-2573), the Tn10 transposon system (see Chalmers,R.,Sewitz,S.,Lipkow,K.,and Crellin,P.(2000),Complete nucleotide sequence of Tn10,J.Bacteriol 182,2970-2972)、Piggybac transposon system (see Li,X.,Burnight,E.R.,Cooney,A.L.,Malani,N.,Brady,T.,Sander,J.D.,Staber,J.,Wheelan,S.J.,Joung,J.K.,McCray,P.B.,Jr. et al (2013), piggyBac transposase tools for Genome engineering, proc. Natl. Acad. Sci. USA 110, E2279-2287), the sleeping beauty transposon system (Sleeping beauty transposon system) (see Ivics, Z., hackett, P.B., plasterk, R.H. and Izsvak,Z.(1997),Molecular reconstruction of Sleeping Beauty,a Tc1-like transposon from fish,and its transposition in human cells,Cell 91,501-510)、Tol2 transposon systems (see Kawakami, K. (2007), tol2: A VERSATILE GENE TRANSFER vector in vertebrates, genome biol.8suppl.1, S7.)

Specific Tn5 transposition systems have been described and are known to those skilled in the art. See Goryshin, i.y. and w.s. reznikoff, tn5 in vitro transfer position.the Journal of biological chemistry,1998.273 (13): pages 7367-74; davies, d.r. ,Three-dimensional structure of the Tn5 synaptic complex transposition intermediate.Science,2000.289(5476)：, pages 77-85; goryshin, I.Y. et al ,Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes.Nature biotechnology,2000.18(1)： pages 97-100 and STEINIGER-White, M., pages 50-7 of I.Rayment and W.S.Reznikoff,Structure/function insights into Tn5 transposition.Current opinion in structural biology,2004.14(1)：, each of which is hereby incorporated by reference in its entirety for all purposes. Kits for using the Tn5 transposition system for DNA library preparation and other uses are known. See Adey, a. Et al ,Rapid,low-input,low-bias construction of shotgun fragment libraries by high-density in vitro transposition.Genome biology,2010.11(12)：, page R119; marine, R.et al ,Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.Applied and environmental microbiology,2011.77(22)：, pages 8071-9; p.125-33 of Parkinson, N.J. et al ,Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA.Genome research,2012.22(1)：; adey, A. And J.Shendure,Ultra-low-input,tagmentation-based whole-genome bisulfite sequencing.Genome research,2012.22(6)： pages 1139-43; picelli, S.et al, full-LENGTH RNA-seq from SINGLE CELLS using Smart-seq2.Nature protocols,2014.9 (1): each of the documents described on pages 171-81, buenrostro, j.d. et al ,Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin,DNA-binding proteins and nucleosome position.Nature methods,2013, are hereby incorporated by reference in their entirety for all purposes. See also WO 98/10077, EP 2527438 and EP 2376517, each of which is hereby incorporated by reference in its entirety. A commercially available transposon kit is sold under the trade name NEXTERA and is available from Illumina.

In certain exemplary embodiments, the transposase is a Tn5 transposase and the transposase binding site comprises a first strand as set forth in AGATGTGTATAAGAGACAG (SEQ ID NO: 11) and a second strand as set forth in its complement.

In certain embodiments, the bound transposase is removed from the double stranded genomic DNA fragment prior to gap filling and extension of the double stranded fragment. In certain embodiments, the transposase is inactivated by use of a protease. In certain embodiments, further comprising inactivating the protease by heat and/or a protease inhibitor. In certain embodiments, the residual transposase is inactivated by digestion with a protease (such as QIAGEN protease) at a final concentration of 1-500 μg/mL for 10-60 minutes at 37-55 ℃. The protease is then inactivated by heat and/or a protease inhibitor such as AEBSF.

Gap filling

The double-stranded fragments generated by the transposome methods described herein are then processed to fill the gaps. The gap filling described herein is performed using uracil intolerant polymerases. Uracil intolerant polymerases refer to DNA polymerases that are incapable of reading and amplifying uracil-containing nucleic acid templates. Such polymerases are known to those skilled in the art. According to one aspect, uracil nucleotides are included between the downstream (e.g., 3 'end) of the first primer binding sequence described herein and the upstream (e.g., 5' end) of the transposase binding site, thereby preventing the 3 'end from continuing to extend toward the first primer binding sequence when gap filling is performed using uracil intolerant polymerase, thereby forming a first double stranded extension product having a 5' overhang at each end.

In certain embodiments, the gap filling step comprises using A, T, C, G nucleotides, wherein a cytosine nucleotide (e.g., dCTP) comprises a modified cytosine that tolerates the transformation step. The tolerogenic transformation step described herein refers to the ability to tolerate reactions that convert cytosines to uracils (e.g., apodec deamination reactions) without sequence changes. In certain embodiments, the modified cytosine is a methylated cytosine, such as 5-methylcytosine (5-mdC) or 5-hydroxymethylcytosine (5-hmdC). In certain embodiments, the gap-filling step comprises using methylated dCTP in place of dCTP in the dNTP mix.

Labeling single cell genomic DNA

According to one aspect, the present disclosure provides methods that involve the introduction of tag sequences such that genomic DNA of different cells are tagged with different unique tag sequences to distinguish them from each other. The introduction of the tag sequence is achieved by the template switching oligonucleotides described herein and based on an extension reaction.

In certain embodiments, the tag sequence may have any length suitable for distinguishing between different cells. In certain exemplary embodiments, the tag sequence is 4-30bp in length, e.g., 4-25bp, 4-20bp. In certain exemplary embodiments, the tag sequence is 4bp to 16bp in length.

In certain embodiments, the transposase binding site binding sequence included in the template switching oligonucleotide comprises a Locked Nucleotide (LNA) modification. According to one aspect, there is a reverse complementary pairing of transposase binding site sequences at both ends of the genomic DNA fragment, and thus there is a possibility of single-stranded DNA looping during annealing. The transposase binding site binding sequence with the locked nucleotide modifications has a higher Tm value (e.g., each locked nucleotide modification can be raised by about 2 ℃) which allows for a higher annealing temperature to be set during annealing to achieve template switching oligonucleotide binding while avoiding single stranded DNA looping.

In certain embodiments, the transposase binding site binding sequence comprises a plurality of LNA modifications, e.g., 2, 3, 4, or 5 LNA modifications. In certain embodiments, the transposase binding site binding sequence comprises 5 LNA modifications.

In certain embodiments, the template switching oligonucleotide will anneal to a transposase binding site sequence 3' of the genomic fragment in the first double stranded extension product, followed by an extension reaction (e.g., PCR) to obtain the tag sequence and the second primer binding sequence for the first double stranded extension product, thereby tagging the genomic DNA fragment.

In certain embodiments, the extending step comprises using A, T, C, G nucleotides, wherein a cytosine nucleotide (e.g., dCTP) comprises a modified cytosine that is resistant to the converting step. The tolerogenic transformation step described herein refers to the ability to tolerate reactions that convert cytosines to uracils (e.g., apodec deamination reactions) without sequence changes. In certain embodiments, the modified cytosine is a methylated cytosine, such as 5-methylcytosine (5-mdC) or 5-hydroxymethylcytosine (5-hmdC). In certain embodiments, the extending step comprises using methylated dCTP in place of dCTP in the dNTP mix.

Vector DNA and optional purification

According to certain aspects, the transformation treatment is performed in the presence of vector DNA. According to certain aspects, a mixed library of tagged genomic DNA fragments of different cells is treated in the presence of vector DNA to convert cytosine to uracil. The vector DNA may be any dsDNA fragment between 100 base pairs (bp) and 4 kilobase pairs (e.g., 100bp to 1000bp, 100bp to 800bp, 100bp to 600bp, 100bp to 500bp, 100bp to 400bp, 200bp to 400 bp) in length. In certain embodiments, the vector DNA is selected from about 200bp to 400bp, e.g., 300bp, in length. In certain embodiments, the vector DNA may be a different DNA type than the target DNA. In certain embodiments, the vector DNA may be the same DNA type as the target DNA. In certain embodiments, the vector DNA is sonicated lambda DNA. In certain embodiments, the vector DNA does not include Illumina sequencing adaptors.

The vector DNA is used to reduce damage to the target DNA or loss of the target DNA by the transformation process. In certain embodiments, the vector DNA is added to the reaction medium in an amount of 100 to 1000 times (e.g., 100 to 1000 times) the amount of sample DNA.

According to certain aspects, the reaction medium, including the mixed library and vector DNA, may be purified by a DNA spin-column (DNA spin-column) or bead-based DNA purification (beads-based DNA purification) or other purification methods known to those skilled in the art prior to performing the transformation treatment required for methylation detection. Or the reaction medium may be directly converted.

Transformation

According to one aspect, the present disclosure provides methods involving mixing libraries of tagged genomic DNA fragments obtained from different cells and processing the mixed libraries to convert cytosine to uracil.

In certain embodiments, the transformation is performed in the presence of vector DNA.

In certain embodiments, the treatment that converts cytosine to uracil is enzymatic conversion. Reagents for converting cytosine to uracil are known to those skilled in the art. In certain embodiments, enzymatic reagents are used that convert cytosine to uracil, i.e., cytosine deaminase, including those of the ABOPEC family, such as apodec-seq or apodec 3A. An apodec family member is a cytidine deaminase that converts cytosine to uracil without altering the modified cytosine base. Other enzymatic reagents will become apparent to those skilled in the art based on the present disclosure.

In certain embodiments, the enzymatic conversion comprises the use of T4-BGT enzyme and APOBEC3A enzyme to detect 5hmC.

In certain embodiments, the enzymatic conversion comprises the use of a TET2 enzyme and an apodec 3A enzyme to detect 5mC or 5hmC.

In certain embodiments, the vector DNA may be removed after transformation, or the transformed fragment may be amplified without amplifying the vector DNA, thereby obtaining amplified fragmented DNA. The tagged genomic DNA fragments obtained by the methods as described herein have a first and a second primer binding sequence at each end such that the tagged genomic DNA fragments can be sufficiently distinguished from the vector DNA. In certain embodiments, the DNA linked by the first and second primer binding sequences is amplified while the vector DNA is not amplified. The vector DNA becomes single stranded DNA, ssDNA, and is removed from the mixture, resulting in a pure amplified target DNA fragment.

Optional purification

According to certain aspects, the reaction medium comprising the transformed fragments may be purified prior to amplification by a DNA spin column or bead-based DNA purification or other purification methods known to those of skill in the art. Or the reaction medium may be amplified directly. In certain embodiments, after the converting step but before the amplifying step, comprises: purifying a reaction medium comprising the transformed mixed library; preferably, the purification step is performed by a DNA spin-column (DNA spin-column) or bead-based DNA purification (beads-based DNA purification). In certain embodiments, the reaction medium comprising the converted fragments is directly subjected to the amplification step without purification.

Amplification of

According to one aspect, the present disclosure provides methods involving amplifying a mixed library that is transformed using first and second primers to generate amplicons.

In certain embodiments, the first primer introduces a first sequencing adapter sequence and the second primer introduces a second sequencing adapter sequence. In certain embodiments, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a first sequencing linker sequence. In certain embodiments, the second portion of the first primer may also comprise other functional sequences as desired, such as index sequences. In certain embodiments, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion 5' of the first portion comprising a second sequencing linker sequence. In certain embodiments, the second portion of the second primer may also comprise other functional sequences as desired, such as index sequences.

In certain embodiments, the sequencing linker is an Illumina sequencing linker.

In certain embodiments, the first primer is introduced into the P7 end and the second primer is introduced into the P5 end. In certain embodiments, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P7 sequence. In certain embodiments, the second portion of the first primer may also comprise other functional sequences as desired, such as an i7 index sequence. In certain embodiments, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P5 sequence. In certain embodiments, the second portion of the second primer may also comprise other functional sequences as desired, such as an i5 index sequence.

In certain embodiments, the first primer is introduced into the P5 end and the second primer is introduced into the P7 end. In certain embodiments, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P5 sequence. In certain embodiments, the second portion of the first primer may also comprise other functional sequences as desired, such as an i5 index sequence. In certain embodiments, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P7 sequence. In certain embodiments, the second portion of the second primer may also comprise other functional sequences as desired, such as an i7 index sequence.

In certain embodiments, the step of amplifying using the first and second primers is performed using uracil-tolerant polymerase. Uracil-tolerant polymerase refers to a DNA polymerase capable of reading and amplifying uracil-containing nucleic acid templates. Such polymerases are known to those skilled in the art. In certain embodiments, the uracil-resistant polymerase is selected from the group consisting of Q5U polymerase, KAPA u+ polymerase, phanta Uc polymerase, or any combination thereof.

In certain embodiments, amplification is achieved using PCR. Methods for PCR are well known in the art. PCR typically involves providing oligonucleotide primers and amplification reagents with the desired target sequence, followed by thermal cycling in the presence of a polymerase (e.g., a DNA polymerase). The primers are complementary to their respective strands of the double-stranded target sequence ("primer binding sequences"). To effect amplification, the double stranded target sequence is denatured and the primer is then annealed to its complementary sequence within the target molecule. After annealing, the primers are extended with a polymerase to form a pair of new complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated multiple times (i.e., denaturation, annealing, and extension constitute one "cycle; there can be multiple" cycles ") to obtain high concentrations of amplified segments of the desired target sequence.

Optional purification

According to certain aspects, the reaction medium comprising the amplified fragments may be purified by a DNA spin column or bead-based DNA purification or other purification methods known to those skilled in the art prior to sequencing. DNA purification following amplification (such as by PCR reactions) will remove most of the single stranded vector DNA, which will result in a pure amplified target DNA library ready for sequencing. In certain embodiments, after the expanding step but before the sequencing step, comprises: purifying a reaction medium comprising said amplicon; preferably, the purification step is performed by a DNA spin-column (DNA spin-column) or bead-based DNA purification (beads-based DNA purification).

Sequencing

The DNA amplified according to the methods described herein can be sequenced and analyzed using methods known to those of skill in the art. Sequencing of the target nucleic acid sequence can be determined using a variety of sequencing methods known in the art, including, but not limited to, sequencing by sequencing-by-synthesis (SBS), sequencing-by-hybridization (SBH), sequencing-by-ligation (SBL) (Shendure et al (2005) Science 309:1728), quantitative incremental fluorescent nucleotide addition sequencing (quantitative incremental fluorescent nucleotide addition sequencing) (QIFNAS), ligation and cleavage step by step (stepwise ligation AND CLEAVAGE), fluorescence Resonance Energy Transfer (FRET), molecular beacon, taqMan reporter probe digestion, pyrosequencing, fluorescent In Situ Sequencing (FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), swing sequencing (PCT/US 05/27695), multiplex sequencing (U.S. Ser. No. 12/027,039 submitted at 2/6 of 2008; pore et al (2007) Nat. Methods 4:931), polymeric colony (POLONY) sequencing (U.S. Pat. Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US 05/06425); nanomesh rolling circle sequencing (ROLONY) (U.S. serial No. 12/120,541 submitted on 5/14 of 2008), allele-specific oligonucleotide ligation assays (ole-specific oligo ligation assay) (e.g., oligonucleotide Ligation Assays (OLA), single template molecules OLA using ligated linear probes and Rolling Circle Amplification (RCA) reads, ligated padlock probes, and/or single template molecules OLA using ligated circular padlock probes and Rolling Circle Amplification (RCA) reads), and the like. High throughput sequencing methods may also be utilized, for example using platforms such as Roche 454, illumina, AB-SOLiD, helicos, polonator platforms. A variety of light-based sequencing techniques are known in the art (Landegren et al (1998) Genome Res.8:769-76; kwok (2000) Pharmacogenomics1:95-100 and Shi (2001) Clin.chem.47:164-172).

In certain embodiments, the amplified DNA may be sequenced using a high throughput sequencing method (such as an Illumina sequencing platform). In certain embodiments, the sequencing is sequencing-by-synthesis (SBS).

Amplification and sequencing methods are useful in the predictive medical field where diagnostic assays, prognostic assays, pharmacogenomics and monitoring clinical trials are used for prognostic (predictive) purposes to prophylactically treat individuals. Accordingly, one aspect of the invention relates to diagnostic assays for determining genomic DNA in order to determine whether an individual is at risk of developing a condition and/or disease. Such assays may be used for prognostic or predictive purposes, thereby prophylactically treating an individual prior to the onset of a disorder and/or disease. Thus, in certain exemplary embodiments, methods of diagnosing and/or prognosing one or more diseases and/or disorders using the methods of analyzing methylation characteristics of single cell genomic DNA described herein are provided.

It is to be understood that the embodiments of the application that have been described are merely illustrative of some of the applications of the principles of the application. Many modifications may be made by one of ordinary skill in the art based on the teachings herein without departing from the true spirit and scope of the application. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety for all purposes.

The application will now be described with reference to the following examples, which are intended to illustrate the application, but not to limit it. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the application by way of example and are not intended to limit the scope of the application as claimed.

Example 1

According to an exemplary embodiment of the present application, the present application provides a high throughput single cell 5mC/5hmC detection method, which may be referred to as TSO-Cabernet. The TSO-Cabernet method includes sorting individual cells by FACS; fragmenting single-cell genome DNA by using Tn5 transposition, and introducing sequencing primers and tag sequences at two ends of the fragments so as to mark the single cells with independent sequences; enzymatic transformation and pool sequencing were performed by single cell mix labeled with different tag sequences (FIG. 1).

According to an exemplary embodiment of the present application, a method for sequence tagging a genomic fragment of a single cell comprises: tn5 transposition, gap filling, linker replacement, and extension (FIG. 2). Wherein:

Tn5 transposition: genomic DNA was fragmented using Tn5 transposition while custom P7-terminal sequencing primer sequences (i.e., first primer binding sequences) were introduced at both ends of the genomic DNA. The cleavage of the genomic DNA and insertion of the transposon leaves gaps at both ends of the transposition/insertion site, resulting in a genomic DNA fragment having a transposon DNA Tnp binding site and a custom P7 end sequencing primer sequence attached to the 5 'position of the upper strand and a transposon DNA Tnp binding site and a custom P7 end sequencing primer sequence attached to the 5' position of the lower strand. Uracil bases are designed between the self-defined P7 end sequencing primer sequence and the ME sequence (namely, transposase binding site), so that the 3' -end of the DNA polymerase is prevented from continuing to extend to the P7 end sequence in the gap filling process of the DNA polymerase, and the subsequent joint replacement is facilitated. In addition, the P7 end sequencing primer sequence (the adaptor sequence upstream of the ME sequence) adopts a 'cytosine-free' design, and under the condition of uracil base blocking, the adaptor sequence exists in a single-chain form, so that the situation that BGT protein cannot be normally combined and glycosyl group protection (BGT is combined with double-chain DNA) cannot be provided for cytosine in the BGT protein in the enzymatic conversion reaction process, thus APOBEC deamination reaction cannot be tolerated is avoided, and therefore the 'cytosine-free' design is carried out on the P7 end sequence part, and the APOBEC deamination reaction can be tolerated, and no sequence change occurs.

Joint replacement: the adapter substitution is based on a PCR reaction, i.e. a custom oligonucleotide strand (i.e. a template switching oligonucleotide) carrying a tag sequence and a P5-end sequencing primer sequence (i.e. a second primer binding sequence) is bound to the ME sequence by base complementary pairing. In addition, the ME sequences of the reverse complementary pairing exist at the two ends of the sample DNA fragment, so that the possibility of single-stranded DNA looping in the cooling annealing process exists. To avoid this, 5 Locked Nucleotide (LNA) modifications were designed in the ME sequence portion of the custom oligonucleotide strand for linker substitution. The ME sequence with the locked nucleotide modifications has a higher Tm (each locked nucleotide modification can be raised by about 2 ℃), which allows for a higher annealing temperature to be set during annealing to achieve oligonucleotide strand binding while avoiding single-stranded DNA looping. Furthermore, it will be appreciated by those skilled in the art that the P5-terminal sequencing primer sequence may also be introduced by transposition, while the P7-terminal sequencing primer sequence is introduced at the time of linker substitution.

Annealing and extending: the annealed oligonucleotide chain is used as a template chain, so that the sample DNA can be subjected to PCR extension to obtain a P5 end sequencing primer sequence and a tag sequence, and the oligonucleotide chain can also extend to the 3' end to form a double-stranded DNA structure so as to ensure the efficiency of subsequent DNA purification and enzymatic conversion. In addition, the modified cytosine is adopted as a substrate in the PCR extension process, so that the sequence change of the part generated by extension can be prevented after enzymatic conversion, and the normal operation of amplification and library establishment can be further ensured.

The DNA duplex thus produced by the extension step already has tag sequence information. Single-cell DNA samples carrying different tag sequences can be mixed together for subsequent enzymatic conversion, and finally, sequences at two ends of the fragments are used as bridges to build a sequencing library which is suitable for an illuminea platform.

Example 2

Preparation of the reaction solution

2.1 Preparation of cell lysate

Table 2-1: cell lysate formula

* Sequence: TCAGGTTTTCCTGAA (SEQ ID NO: 1)

2.2 Preparation of transposition reaction solution (2X)

Table 2-2: transposition reaction liquid formula

2.3 Preparation of a transposition stop solution

Table 2-3: formula of swivel base stopping liquid

2.4 Preparation of transposon annealing buffer (10X)

Tables 2 to 4: transposon annealing buffer solution formula

2.5 Preparation of a stock solution of the transposable Complex

Tables 2-5: liquid storage formulation for transposition complex

2.6 Preparation of magnetic bead dilution solution

Tables 2-6: formula of magnetic bead diluted solution

2.7Lambda DNA fragmentation

The Lambda DNA (Thermo Scientific, SD 0021) without any modification was broken down to 300bp using a non-contact sonicator, purified and stored at-20℃for a long period of time.

Example 3

Preparation of a transposable Complex

3.1 Preparation of transposon

Table 3-1: transposon sequences

One strand of the transposon is Tn5_3U_oligo, which comprises a P7 terminal sequencing primer sequence, uracil nucleotides, and an ME sequence, exemplary sequences of which are shown in Table 3-1. The other strand is Tn5_comp, which contains the ME sequence, an exemplary sequence of which is shown in Table 3-1.

Table 3-2: transposon annealing reaction system

The ingredients shown in the above table were mixed, and the program (cooling to 25℃at 0.1℃every 3 seconds, 4℃hold) was run in a PCR apparatus at-20℃for a long period of time.

3.2 Assembly of the transposition Complex

The transposon was mixed with Tn5 transposase (Vazyme) at a ratio of 1.1:1, incubated at room temperature for 30min, diluted to 250nM with a stock solution of the transposable complex, and sub-packaged and stored at-80 ℃.

Example 4

Isolation of single cells and cell lysis

Single cell sorting is performed by means of an oral pipette or by means of a flow cytometer. Single cell sorting in a 0.2mL PCR tube containing 2.5. Mu.L of cell lysate, cell lysis PCR instrument program (50℃1h,65℃1h,70℃15min,4℃hold) was run. The single cell lysate was stored at-80 ℃.

Example 5

Labelling of single cells

5.1Tn5 transposition reaction

Table 5-1: transposon annealing reaction system

The reaction system was prepared in accordance with Table 5-1, and after incubation at 55℃for 10min, 1. Mu.L of 2mg/mL protease (QIAGEN, 19157) and 1. Mu.L of a transposition stop solution were added to the transposition reaction system, and PCR procedures (50℃40min,70℃15min,4℃hold) were run to inactivate the transposase.

5.2 Notch filling and joint conversion

To the DNA sample after the completion of the transposition reaction, 18. Mu.L of a adaptor-transformation premix (see Table 5-2) was added, and the procedure was run in a PCR apparatus (3 min at 50 ℃, 30s at 98 ℃,10 cycles (10 s at 98 ℃,20 s at 59 ℃, 1min at 72 ℃, 2min at 72 ℃,4 ℃ hold). Wherein the tagged TSO sequence (i.e., template switching oligonucleotide) comprises, from 5 'to 3': a P5-terminal sequencing primer sequence, a tag sequence, and an ME binding sequence, the ME binding sequence being bound to the ME sequence by base complementary pairing. Exemplary sequences of the tagged TSO sequences are shown in SEQ ID NO. 4: TCGTCGGCAGCGTC (P5-end sequencing primer sequence) TTACCGAC (tag sequence) AGATGTGTA +TA+AG+AG+AC+AG (ME binding sequence), note: "+" indicates that the nucleotide in its 3' adjacent position has LNA modification.

Table 5-2: premixed liquid for joint conversion reaction

* Methylated dCTP (NEB, N0356S) can be replaced by methylolated dCTP (Jena Bioscience, NU-932L)

Subsequently, 96-well plate samples were mixed, 40ng of fragmented Lambda DNA and 5mL of magnetic beads (Beckman, B23319) were added, DNA purification was performed with reference to the instructions, and finally eluted at 56. Mu.L of 1mM Tris-HCl (pH=8.0).

Example 6

Library preparation

6.1TET2 reaction (detection of 5 mC)

To 56. Mu.L of the eluted product, 44. Mu.L of TET2 reaction premix (see Table 6-1) was added and incubated for 1h at 37 ℃.

Table 6-1: TET2 reaction premix

TET2 reaction was terminated by adding 2. Mu.L of a termination reagent (NEB, E7125L) and incubating at 37℃for 30min. Subsequently, 183.6. Mu.L of magnetic beads were added for DNA purification by eluting with 8. Mu.L of 1mM Tris-HCl (pH=8.0).

6.2BGT reaction (detection of 5 hmC)

To 56. Mu.L of the eluted product, 44. Mu.L of BGT reaction premix (see Table 6-2) was added and incubated at 37℃for 2 hours.

Table 6-2: BGT reaction premix

End of the BGT reaction 5. Mu.L proteinase K (NEB, P8107S) was added and incubated at 37℃for 30min to terminate the BGT reaction. Then 189 μl magnetic beads were added for DNA purification as described above, eluting with 8 μl 1mM Tris-HCl (pH=8.0).

6.3APOBEC reaction

Table 6-3: APOBEC reaction premix

To 8. Mu.L of the eluted product obtained in 6.1 or 6.2, 2. Mu.L of 0.1M NaOH was added, and the mixture was incubated at 50℃for 10 minutes to denature DNA double strand, followed by rapid cooling with ice. The samples were kept on ice and 10. Mu.L of APOBEC reaction premix (see Table 6-3) was added, incubated at 37℃for 3h (5 mC. For detection) or 12h (5 hmC. For detection).

6.4 Library amplification

Mu.L of 2 XQ5U PCR premix (NEB, M0597L), 0.4. Mu.L of 100. Mu. M s3N501 primer and 0.4. Mu.L of 100. Mu. M s3N701 primer were added. The s3N501 primer refers to a primer referring to a P5-end sequencing adapter, which comprises a P5 sequence, an i5 index sequence, and a sequence that hybridizes to the P5-end sequencing primer sequence at one end of the introduced genomic fragment, an exemplary sequence of which is shown in XX. The s3N701 primer refers to a primer directed to a P7 end sequencing adapter comprising a P7 sequence, an i7 index sequence, and a sequence that hybridizes to the P7 end sequencing primer sequence at one end of the introduced genomic fragment, exemplary sequences of which are shown in tables 6-4.

Table 6-4: adaptor primer sequences

The procedure (98 ℃ C. 30s,11 cycles (98 ℃ C. 10s,60 ℃ C. 30s,65 ℃ C. 90 s), 65 ℃ C. 5min,4 ℃ C. Hold) was run in a PCR apparatus for whole genome amplification. The DNA purification kit was then used for purification and fragment selection as indicated.

Example 7

Library sequencing

The library obtained by purification and fragment selection was quantified using a Qubit fluorometer and the library fragment distribution was detected using a fragment analysis system. A20% base balance sequence was incorporated to increase base complexity, and the resulting library was subjected to 150bp read length double-ended sequencing at a concentration of 0.9pM using a illumina NovaSeq 6000 sequencer, with sequencing primer sequences shown in Table 7.

Table 7: sequencing primer sequences

Example 8

Sequencing results

The histology sequencing technology of mixed library construction after multicellular labelling generally causes the problems of uneven distribution of sequencing data and the like due to the fact that sequencing depth difference among single cells is too large and low-depth cell quality control is not closed. By counting the sequencing readings which can be split after the cells of the same 96-well plate are treated by the TSO-Cabernet method provided by the present disclosure, the method is found to be capable of obtaining uniform sequencing depth distribution (fig. 3, left diagram), and can effectively ensure that a plurality of cells in the same library can obtain sufficient sequencing data.

After a plurality of cells are mixed together for enzymatic conversion and library construction sequencing, the accurate splitting and tracing of sequencing data by using mutually independent tag sequences among each cell are key to ensuring the accuracy of each single cell analysis. To verify this problem, human and mouse genomic DNA was added separately to different wells in the same 96-well plate, the DNA in each well was labeled and mixed enzymatically transformed and sequenced according to the TSO-Cabernet procedure provided in the present disclosure, and finally the split data obtained was aligned according to human and mouse reference genomes. By observing the number of sequencing reads per sample alignment to human and mouse reference genomes, samples can be found that are substantially free of significant mixing of humans and mice (fig. 3, right panel), demonstrating that this method allows for accurate traceability of the vast majority (greater than 99%) of sequencing reads based on tag sequence information contained in the sequencing data, thereby ensuring accuracy of downstream analysis.

Three standards (C corresponding to Lambda DNA fragment; 5mC corresponding to pUC19 DNA fragment; 5hmC corresponding to 5hmC modified DNA fragment) were incorporated into the K562 sample for 5mC/5hmC detection according to TSO-Cabernet procedure, indicating the detection accuracy of C, 5mC, 5hmC, respectively (FIG. 4A). Statistics show that when 5hmC sequencing is performed, the ratio of C to 5mC incorrectly identified as 5hmC averages 0.576% and 1.54%, respectively, and the ratio of 5hmC correctly identified averages 99.6%; when 5mC sequencing was performed, the proportion of C that was incorrectly identified as 5mC averaged 0.818% and the proportion of 5mC that could be correctly identified averaged 98.3%. In addition, to verify the reliability of the TSO-Cabernet procedure to detect DNA methylation modification results, five single cell samples were drawn from them and subjected to a genome-wide DNA methylation modification correlation analysis (fig. 4B) with samples detected using the Cabernet technique disclosed in WO2021077415, including single cell and multicellular samples. From the figure, it can be seen that the TSO-Cabernet procedure and Cabernet procedure have high similarity in the results of detecting DNA methylation modifications in the genome. The above results may demonstrate that the TSO-Cabernet method can provide reliable 5mC and 5hmC detection results. The TSO-Cabernet method provided by the disclosure can realize simultaneous transformation and library construction treatment of multiple cells, and therefore, the method provided by the disclosure can remarkably improve flux while having detection accuracy.

Claims

1. A method of analyzing methylation characteristics of single cell genomic DNA, comprising:

Contacting genomic DNA from a single cell with a plurality of transposomes, wherein the transposomes comprise a transposase and a transposon DNA, wherein the transposon DNA comprises a double stranded transposase binding site and an overhang, wherein the overhang comprises a first primer binding sequence at the 5' end of the overhang and comprises uracil nucleotides downstream of the first primer binding sequence upstream of the transposase binding site; to obtain double-stranded genomic DNA fragments comprising transposon DNA at each end;

2. The method of claim 1, wherein the transposase binding site binding sequence comprised by the template switching oligonucleotide comprises a Locked Nucleotide (LNA) modification;

preferably, the transposase binding site binding sequence comprises a plurality of LNA modifications, e.g., 2,3, 4 or 5 LNA modifications.

3. The method of claim 1 or 2, wherein the filling of gaps and/or extending step comprises using A, T, C, G four nucleotides, wherein a cytosine nucleotide (e.g., dCTP) comprises a modified cytosine that is resistant to the converting step;

Preferably, the modified cytosine is a methylated cytosine, such as 5-methylcytosine (5-mdC) or 5-hydroxymethylcytosine (5-hmdC).

4. The method of any one of claims 1-3, wherein the first primer binding sequence is free of cytosine nucleotides.

5. The method of any one of claims 1-3, wherein the first primer binding sequence comprises a cytosine nucleotide comprising a modified cytosine, wherein the modified cytosine tolerates the transformation step;

6. The method of any one of claims 1-5, wherein the transposon DNA comprises a plurality (e.g., 2, 3, or 4) consecutive uracil nucleotides downstream of the first primer binding sequence and upstream of the transposase binding site.

7. The method of any one of claims 1-6, wherein the 3' end of the first primer binding sequence is immediately adjacent to the uracil nucleotide;

Preferably, the 5' end of the transposase binding site is immediately adjacent to the uracil nucleotide.

8. The method of any one of claims 1-7, wherein the mixed library is treated in the presence of vector DNA to convert cytosine to uracil;

Preferably, the vector DNA is selected from dsDNA fragments between 100bp and 4000bp in length;

Preferably, the vector DNA is sonicated lambda DNA.

9. The method of any one of claims 1-8, further comprising sequencing the amplicon.

10. The method of any one of claims 1-9, wherein the transformed mixed library is amplified using the first and second primers to produce amplicons comprising a sequencing adapter at each end, thereby generating a sequencing library.

11. The method of claim 10, wherein the sequencing linker is an Illumina sequencing linker.

12. The method of claim 11, wherein the first primer is introduced into a P7-end and the second primer is introduced into a P5-end;

preferably, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P7 sequence;

Preferably, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P5 sequence.

13. The method of claim 11, wherein the first primer is introduced into a P5 end and the second primer is introduced into a P7 end;

preferably, the first primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a first primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P5 sequence;

preferably, the second primer comprises (i) a first portion at the 3 'end comprising a sequence capable of hybridizing to a second primer binding sequence, and (ii) a second portion located 5' of the first portion comprising a P7 sequence.

14. The method of any one of claims 1-13, wherein the transposase IS a Tn5 transposase, mu transposase, tn7 transposase, or IS5 transposase.

15. The method of any one of claims 1-14, wherein the uracil intolerant polymerase is selected from the group consisting of Q5 polymerase, DEEP VENT DNA polymerase, phusion high-fidelity polymerase, KAPA high-fidelity polymerase, phanta polymerase, or any combination thereof.

16. The method of any one of claims 1-15, wherein the step of amplifying using the first and second primers is performed using uracil-tolerant polymerase;

preferably, the uracil-resistant polymerase is selected from the group consisting of Q5U polymerase, KAPA u+ polymerase, phanta Uc polymerase, or any combination thereof.

17. The method of any one of claims 1-16, wherein the bound transposase is removed from the double stranded genomic DNA fragment prior to gap filling and extension of the double stranded fragment.

18. The method of any one of claims 1-17, further comprising the step of, after the extending step but before the converting step: purifying a reaction medium comprising the second double chain extension product;

preferably, the purification step is performed by DNA spin columns or bead-based DNA purification.

19. The method of any one of claims 1-18, further comprising the step, after the amplifying step, of: purifying a reaction medium comprising said amplicon;

20. The method of any one of claims 1-19, wherein the treatment that converts cytosine to uracil is an enzymatic conversion.

21. The method of claim 20, wherein the enzymatic conversion comprises an apodec deamination reaction.

22. The method of claim 20, wherein the enzymatic conversion comprises the use of a T4-BGT enzyme and an apodec 3A enzyme, or a TET2 enzyme and an apodec 3A enzyme.