Library preparation of tagged nucleic acids using a single-tube addition protocol

Abstract

The present application relates to the preparation of a library of tagged nucleic acids using a single-tube addition scheme. The present application provides a method for preparing a library of tagged nucleic acid fragments, the method comprising: directly contacting a cell population with a cleavage reagent having one or more proteases to generate a cell lysate; inactivating the protease to generate an inactivated cell lysate, and applying a transposase and a transposon end component containing a transferred strand to the inactivated cell lysate under conditions in which the target nucleic acid and the transposon end component undergo a transposition reaction. The present application also provides a kit for preparing a library of tagged nucleic acid fragments, the kit comprising: (a) a cleavage reagent having one or more proteases, and (b) a transposition reaction composition having at least one transposase and at least one transposon end component containing a transferred strand.

Description

[0001] The present application is a divisional application of an application filed on June 25, 2015, with application number 201580046283.6 and invention name “Library preparation of labeled nucleic acids using a single-tube addition protocol”. Technical Field

[0002] The present disclosure relates generally to methods for preparing libraries of nucleic acid fragments, and more particularly to methods for preparing libraries of nucleic acid fragments using proteases in a single tube for a variety of applications including, for example, next generation DNA sequencing. Background Art

[0003] There are a variety of methods and applications for which it is desirable to generate libraries of fragmented and tagged nucleic acids, for example for use as templates for DNA sequencing and / or for analysis of copy number variation.

[0004] Recently developed "next generation" DNA sequencing technologies, such as those developed by Illumina, Inc. (San Diego, CA), utilize large-scale parallel or multiple formats to enable the generation of sequence data from millions of sequencing templates in a single sequence run. The large-scale parallel nature of "next generation" sequencing requires the generation of a nucleic acid fragment library containing a set or a population of nucleic acid fragments from a target nucleic acid sample, such as genomic DNA. More importantly, it requires that the combination of these nucleic acid fragments be presented as a sequence that is qualitatively and / or quantitatively represented by the sequence from the target nucleic acid sample. When the nucleic acid sample is from a cell, the current method for generating a library of nucleic acid fragments generally requires a separation step for separating the target nucleic acid from the cell before nucleic acid fragmentation. The nucleic acid extraction step generally wastes the target nucleic acid sample, and generally causes the prepared nucleic acid to be unable to qualitatively represent the target nucleic acid from the sample. When the amount of the sample is limited or difficult to obtain, this becomes a particularly serious problem. In order to address this problem, some current methods use nucleic acid amplification before fragmentation. However, amplification cannot ensure the representativeness of the target nucleic acid because the target nucleic acid is still partially lost during the extraction period before amplification.

[0005] Therefore, there is a need for new methods that can quickly and efficiently prepare nucleic acid fragment libraries. The present disclosure addresses this need by providing methods for preparing libraries of nucleic acid fragments using proteases in a single reaction mixture, such as in a single tube. Related advantages are also provided. Summary of the invention

[0006] In one aspect, provided herein is a method for preparing a library of tagged nucleic acid fragments, the method comprising: (a) contacting a cell population directly with a lysis reagent to generate a cell lysate, wherein the lysis reagent has one or more proteases, and wherein the cell lysate comprises a target nucleic acid; (b) inactivating the one or more proteases to form an inactivated cell lysate, and (c) applying at least one transposase and at least one transposon end component comprising a transferred strand directly to the inactivated cell lysate under conditions wherein the target nucleic acid and the transposon end component undergo a transposition reaction to generate a mixture, wherein (i) the target nucleic acid is fragmented to generate a plurality of target nucleic acid fragments, and (ii) the transferred strand of the transposon end component is ligated to the 5′ end of each of the plurality of target nucleic acid fragments to generate a plurality of 5′-tagged target nucleic acid fragments.

[0007] In some embodiments, steps (a), (b) and (c) provided herein are performed in a single reaction mixture, for example, in one tube. In some embodiments, the cell colony is a minimum colony of cells. In some embodiments, the minimum colony of cells comprises one, two, three, four or five cells.

[0008] In some embodiments, one or more proteases are selected from the group consisting of: serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, glutamic proteases and metalloproteases. In some embodiments, one or more proteases are subtilisin and variants thereof. In some embodiments, the concentration of one or more proteases in cell lysates is 0.1mg / ml to 10mg / ml. In some embodiments, the concentration of one or more proteases in cell lysates is 0.1mg / ml to 2.5mg / ml. In some embodiments, the concentration of one or more proteases in cell lysates is 0.5mg / ml. In some embodiments, the concentration of one or more proteases in cell lysates is 4.5mAU / ml to 500mAU / ml. In some embodiments, the concentration of one or more proteases in cell lysates is 22.5mAU / ml.

[0009] In some embodiments, the cell population is contacted with a lysis reagent at pH 7.0 to pH 10.0 in step (a). In some embodiments, the cell population is contacted with a lysis reagent at pH 7.0 to pH 9.0.

[0010] In some embodiments, in step (b), the one or more proteases are inactivated by increasing the temperature. In some embodiments, the one or more proteases are inactivated by increasing the temperature to 50° C.-80° C. In some embodiments, the one or more proteases are inactivated by increasing the temperature to 70° C. In some embodiments, the one or more proteases are inactivated by adding one or more inhibitors of the one or more proteases.

[0011] In some embodiments, the lysis reagent comprises one or more detergents. In some embodiments, the one or more detergents are non-ionic detergents. In some embodiments, the one or more detergents include Triton.

[0012] In some embodiments, the target nucleic acid is double-stranded DNA, and wherein the target nucleic acid maintains double-stranded DNA before applying the transposase and the transposon end components in step (c). In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid comprises chromosomal DNA or a fragment thereof. In some embodiments, the target nucleic acid comprises a genome or a partial genome.

[0013] In some embodiments, the at least one transposase is a Tn5 transposase. In some embodiments, the at least one transposon end component comprises a Tn5 transposon end.

[0014] In some embodiments, the transferred strand comprises a tag domain comprising one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, and an addressing tag domain.

[0015] In some embodiments, the methods provided herein further comprise: (d) incubating the mixture from step (c) directly with at least one nucleic acid modification enzyme under conditions wherein the 3' tag is ligated to the 5'-tagged target nucleic acid fragment to generate a plurality of di-tagged target nucleic acid fragments. In some embodiments, steps (a), (b), (c), and (d) are performed in a single reaction tube.

[0016] In some embodiments, the nucleic acid modifying enzyme is a polymerase, and wherein the 3' tag is formed by extension of the 3' end of the 5'-tagged target nucleic acid fragment. In some embodiments, the nucleic acid modifying enzyme is a ligase, and wherein the 3' tag is formed by ligating an oligonucleotide to the 3' end of the 5'-tagged target nucleic acid fragment.

[0017] In some embodiments, the methods provided herein further include (e) amplifying one or more di-tagged target nucleic acid fragments to generate a library of tagged nucleic acid fragments having additional sequences at the 5' end and / or 3' end of the di-tagged nucleic acid fragments. In some embodiments, steps (a), (b), (c), (d), and (e) are performed in a single reaction tube.

[0018] In some embodiments, amplification includes utilizing one or more of a polymerase chain reaction (PCR), a strand displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, or a loop-mediated amplification reaction. In some embodiments, amplification includes PCR using a single primer complementary to the 3' tag of the di-tagged target DNA fragment. In some embodiments, amplification includes PCR using a first primer and a second primer, wherein at least the 3' terminal portion of the first primer is complementary to at least a portion of the 3' tag of the di-tagged target nucleic acid fragment, and wherein at least the 3' terminal portion of the second primer exhibits the sequence of at least a portion of the 5' tag of the di-tagged target nucleic acid fragment. In some embodiments, the 5' terminal portion of the first primer is not complementary to the 3' tag of the di-tagged target nucleic acid fragment, and the 5' terminal portion of the second primer does not exhibit the sequence of at least a portion of the 5' tag of the di-tagged target nucleic acid fragment. In some embodiments, the first primer comprises a first universal sequence, and / or wherein the second primer comprises a second universal sequence.

[0019] In some embodiments, the methods provided herein further comprise sequencing the tagged nucleic acid fragments. In some embodiments, sequencing of the tagged nucleic acid fragments comprises using one or more of synthesis sequencing, bridge PCR, chain termination sequencing, hybridization sequencing, nanopore sequencing, and ligation sequencing. In some embodiments, sequencing of the tagged nucleic acid fragments comprises using next generation sequencing.

[0020] In some embodiments, the methods provided herein further comprise analyzing copy number variation.In some embodiments, the methods provided herein further comprise analyzing single nucleotide variation.

[0021] In another aspect, the present disclosure provides a kit for preparing a library of tagged nucleic acid fragments, the kit comprising: (a) a cleavage reagent having one or more proteases, and (b) a transposition reaction composition having at least one protease and at least one transposon end component, the at least one transposon end component comprising a transferred strand.

[0022] In some embodiments, one or more proteases are selected from the group consisting of: serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, glutamic proteases and metalloproteases. In some embodiments, one or more proteases are subtilisin and variants thereof. In some embodiments, the lysing agent comprises one or more detergents. In some embodiments, one or more detergents include Triton.

[0023] In some embodiments, at least one transposon end component comprises: a tag domain and a 3' portion comprising a transferred strand. In some embodiments, the tag domain comprises one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, and an addressing tag domain. In some embodiments, the transposition reaction composition comprises two or more transposon end components, each of the two or more transposon end components comprising a transferred strand that differs by at least one nucleotide. In some embodiments, the transposase is a Tn5 transposase. In some embodiments, the transposon end component comprises a Tn5 transposon end.

[0024] In some embodiments, the kits provided herein further include a polymerase. In some embodiments, the kits provided herein further include a ligase.

[0025] In some embodiments, the kit provided herein also includes reagents for amplification reactions. In some embodiments, the reagents for amplification reactions are reagents for PCR. In some embodiments, the reagents for amplification reactions include at least one primer. In some embodiments, at least one primer includes a 3' portion that exhibits a sequence of at least a portion of the transferred strand. In some embodiments, at least one primer includes a 5' portion that includes a universal sequence.

[0026] In some embodiments, the kits provided herein further include a size selection reagent. In some embodiments, the size selection reagent includes AMPure XP beads. In some embodiments, the kits provided herein further include a library normalization reagent.

[0027] In some embodiments, the kits provided herein further comprise a device having a solid surface. In some embodiments, the device is a flow cytometry device. In some embodiments, the solid surface comprises a patterned surface suitable for immobilizing molecules in an ordered pattern.

[0028] Specifically, this application provides the following:

[0029] 1. A method for preparing a library of tagged nucleic acid fragments, the method comprising:

[0030] (a) directly contacting a cell population with a lysis reagent to generate a cell lysate, wherein the lysis reagent has one or more proteases, and wherein the cell lysate contains a target nucleic acid;

[0031] (b) inactivating the one or more proteases to form an inactivated cell lysate, and

[0032] (c) applying at least one transposase and at least one transposon end component comprising the transferred strand directly to the inactivated cell lysate under conditions wherein the target nucleic acid and the transposon end component undergo a transposition reaction to generate a hybrid, wherein:

[0033] (i) the target nucleic acid is fragmented to generate a plurality of target nucleic acid fragments, and

[0034] (ii) the transferred strand of the transposon end element is ligated to the 5′ end of each of the plurality of target nucleic acid fragments to generate a plurality of 5′-tagged target nucleic acid fragments.

[0035] 2. The method according to item 1, wherein steps (a), (b) and (c) are carried out in a single reaction tube.

[0036] 3. The method according to item 1, wherein the cell population is a minimal cell population, and wherein the minimal cell population comprises one, two, three, four or five cells.

[0037] 4. The method according to item 1, wherein the one or more proteases are subtilisin and variants thereof.

[0038] 5. The method according to item 1, wherein the concentration of the one or more proteases in the cell lysate is 4.5 mAU / ml to 500 mAU / ml.

[0039] 6. The method according to item 5, wherein the concentration of the one or more proteases in the cell lysate is 22.5 mAU / ml.

[0040] 7. The method according to item 1, wherein in step (a) the cell population is contacted with the lysis reagent at pH 7.0 to pH 10.0.

[0041] 8. The method according to item 7, wherein the cell population is contacted with the lysis reagent at pH 7.0 to pH 9.0.

[0042] 9. The method according to item 1, wherein in step (b) the one or more proteases are inactivated by increasing the temperature.

[0043] 10. The method according to item 9, wherein the one or more proteases are inactivated by increasing the temperature to 50°C-80°C.

[0044] 11. The method according to item 10, wherein the one or more proteases are inactivated by increasing the temperature to 70°C.

[0045] 12. The method according to item 1, wherein the one or more proteases are inactivated by adding one or more inhibitors of the one or more proteases.

[0046] 13. A method according to item 1, wherein the lysis reagent comprises one or more detergents.

[0047] 14. The method according to item 13, wherein the one or more detergents are non-ionic detergents.

[0048] 15. The method of claim 13, wherein the one or more detergents comprises Triton.

[0049] 16. The method according to item 1, wherein the target nucleic acid is a double-stranded DNA, and wherein the target nucleic acid maintains the double-stranded DNA before applying the transposase and the transposon end components in step (c).

[0050] 17. The method according to item 16, wherein the target nucleic acid is genomic DNA.

[0051] 18. The method according to item 16, wherein the target nucleic acid comprises chromosomal DNA or a fragment thereof.

[0052] 19. The method according to item 16, wherein the target nucleic acid comprises a genome or a portion of a genome.

[0053] 20. The method according to item 1, wherein the at least one transposase is Tn5 transposase.

[0054] 21. The method according to item 1, wherein the at least one transposon end component comprises a Tn5 transposon end.

[0055] 22. The method according to item 1, wherein the transferred strand comprises a tag domain, and the tag domain comprises one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain and an addressing tag domain.

[0056] 23. The method according to item 1, further comprising:

[0057] (d) incubating the mixture from step (c) directly with at least one nucleic acid modifying enzyme under conditions wherein the 3′ tag is ligated to the 5′-tagged target nucleic acid fragment to generate a plurality of di-tagged target nucleic acid fragments.

[0058] 24. The method according to item 23, wherein steps (a), (b), (c) and (d) are carried out in a single reaction tube.

[0059] 25. The method according to item 23, wherein the at least one nucleic acid modifying enzyme is a polymerase, and wherein the 3' tag is formed by extension of the 3' end of the 5'-tagged target nucleic acid fragment.

[0060] 26. The method according to item 23, wherein the at least one nucleic acid modifying enzyme is a ligase, and wherein the 3′ tag is formed by ligating an oligonucleotide to the 3′ end of the 5′-tagged target nucleic acid fragment.

[0061] 27. The method according to item 23, further comprising:

[0062] (e) amplifying one or more di-tagged target nucleic acid fragments to generate a library of tagged nucleic acid fragments having additional sequences at the 5′ end and / or the 3′ end of the di-tagged target nucleic acid fragments.

[0063] 28. The method according to item 27, wherein steps (a), (b), (c), (d) and (e) are carried out in a single reaction tube.

[0064] 29. The method according to item 27, wherein the amplification includes utilizing one or more of polymerase chain reaction (PCR), strand displacement amplification reaction, rolling circle amplification reaction, ligase chain reaction, transcription-mediated amplification reaction or loop-mediated amplification reaction.

[0065] 30. The method according to item 27, wherein the amplification comprises PCR using a single primer that is complementary to the 3' tag of the di-tagged target nucleic acid fragment.

[0066] 31. A method according to item 27, wherein the amplification comprises PCR using a first primer and a second primer, wherein at least the 3' terminal portion of the first primer is complementary to at least a portion of the 3' tag of the double-tagged target nucleic acid fragment, and wherein at least the 3' terminal portion of the second primer exhibits the sequence of at least a portion of the 5' tag of the double-tagged target nucleic acid fragment.

[0067] 32. A method according to item 31, wherein the 5' terminal portion of the first primer is not complementary to the 3' tag of the double-tagged target nucleic acid fragment, and wherein the 5' terminal portion of the second primer does not exhibit the sequence of at least a portion of the 5' tag of the double-tagged target nucleic acid fragment.

[0068] 33. A method according to any one of items 31-32, wherein the first primer comprises a first universal sequence, and / or wherein the second primer comprises a second universal sequence.

[0069] 34. The method according to item 1, further comprising sequencing the tagged nucleic acid fragments.

[0070] 35. A method according to item 34, wherein the sequencing of the tagged nucleic acid fragments includes using one or more of synthesis sequencing, bridge PCR, chain termination sequencing, hybridization sequencing, nanopore sequencing and ligation sequencing.

[0071] 36. The method of item 34, wherein the sequencing of the tagged nucleic acid fragments comprises using next generation sequencing.

[0072] 37. The method according to item 34 further comprises analyzing copy number variation.

[0073] 38. The method according to item 34, further comprising analyzing single nucleotide variations.

[0074] 39. A kit for preparing a library of tagged nucleic acid fragments, the kit comprising:

[0075] (a) a cleavage reagent having one or more proteases, and

[0076] (b) a transposition reaction composition comprising at least one transposase and at least one transposon end component comprising a transferred strand.

[0077] 40. The kit according to item 39, wherein the one or more proteases are subtilisin and variants thereof.

[0078] 41. A kit according to item 39, wherein the lysis reagent comprises one or more detergents.

[0079] 42. A kit according to item 41, wherein the one or more detergents comprise Triton.

[0080] 43. The kit according to item 39, wherein the at least one transposon end component comprises: a tag domain and a 3' portion comprising the transferred strand.

[0081] 44. A kit according to item 43, wherein the tag domain comprises one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain and an addressing tag domain.

[0082] 45. The kit according to item 39, wherein the transposition reaction composition comprises two or more transposon end components, each of the two or more transposon end components comprises a transferred strand that differs in at least one nucleotide.

[0083] 46. ​​The kit according to item 39, wherein the transposase is Tn5 transposase.

[0084] 47. The kit according to item 39, wherein the transposon end components comprise Tn5 transposon ends.

[0085] 48. The kit according to item 39, further comprising a polymerase.

[0086] 49. The kit according to item 39, further comprising a ligase.

[0087] 50. The kit according to item 39, further comprising reagents for an amplification reaction.

[0088] 51. The kit according to item 50, wherein the reagent for amplification reaction is a reagent for PCR.

[0089] 52. The kit according to item 51, wherein the reagents for the amplification reaction include at least one primer.

[0090] 53. Kit according to item 52, wherein the at least one primer comprises a 3' portion which exhibits the sequence of at least a part of the transferred strand.

[0091] 54. A kit according to item 52, wherein at least one of the primers comprises a 5' portion comprising a universal sequence.

[0092] 55. The kit according to item 39, further comprising a size selection reagent.

[0093] 56. A kit according to item 55, wherein the size selection reagent comprises AMPure XP beads.

[0094] 57. The kit according to item 39, further comprising a library normalization reagent.

[0095] 58. The kit according to item 39, further comprising a device having a solid surface.

[0096] 59. The kit according to item 58, wherein the device is a flow cytometric device.

[0097] 60. The kit according to item 58, wherein the solid surface comprises a patterned surface suitable for immobilizing molecules in an ordered pattern. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 is a bar graph showing the percentage of unique mapped reads in sequencing of whole cells or nuclei treated with 0 mg / ml, 0.1 mg / ml, 0.5 mg / ml or 2.5 mg / ml of protease.

[0099] Figure 2 Shown are bar graphs of copy number analysis results using total DNA, single cells treated with sufficient protease activity, and single cells treated with insufficient protease activity.

[0100] Figure 3A Shown are bar graphs of copy number analysis results in single cells treated with 0.5 mg / ml active protease, 2 mg / ml active protease, or 2 mg / ml previously heat-inactivated protease. Figure 3B Shown are bar graphs of the percentage of uniquely mapped reads in sequencing of single cells treated with 0.5 mg / ml active protease, 1 mg / ml active protease, 2 mg / ml protease at reaction temperature, or 2 mg / ml pre-heat-inactivated protease and cell-free control samples. Figure 3C Shown are histograms of read count differences between neighboring bins (interquartile range of read count differences between neighboring bins) in sequencing of single cells treated with 0.5 mg / ml active protease, 1 mg / ml active protease, 2 mg / ml active protease, or 2 mg / ml pre-heat-inactivated protease and cell-free control samples.

[0101] Figure 4A is a bar graph showing the relative activity of the protease at pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0 or pH 10.0. Figure 4B Shown are bar graphs of the percentage of uniquely mapped reads in sequencing experiments of single cells treated with proteases at pH 7.0, pH 8.0, pH 9.0, or pH 10.0. Figure 4C Shown are histograms of read count differences between neighboring bins (interquartile range of read count differences between neighboring bins) in sequencing experiments of single cells treated with proteases at pH 7.0, pH 8.0, pH 9.0, or pH 10.0.

[0102] Figure 5A is a bar graph showing relative protease activity at room temperature, 50°C, 60°C or 70°C. Figure 5B Shown are bar graphs of the percentage of uniquely mapped reads in sequencing experiments of single cells, triplicate cells, or 15 pg of genomic DNA treated with proteases preheated at room temperature, 50°C, 60°C, or 70°C. Figure 5C Shown are histograms of read count differences between adjacent bins (interquartile range of read count differences between adjacent bins) in sequencing experiments of single cells, three cells, or 15 pg of genomic DNA treated with proteases preheated at room temperature, 50°C, 60°C, or 70°C.

[0103] Fig. 6A Insert sizes of libraries generated by treatment with 1 μl Tn5 or 2 μl Tn5 are shown. Figure 6B Insert sizes of libraries generated by treatment with 1 μl Tn5 or 2 μl Tn5 are shown. Figure 6C Shown are the diversity of libraries generated by treatment with 1 μl Tn5 or 2 μl Tn5.

[0104] Figure 7 Shown are bar graphs of the results of count and copy number analysis in sequencing experiments of single cells using PCR with 16 cycles, 18 cycles, or 20 cycles according to the methods provided herein.

[0105] Fig. 8A Shown are the read distributions of three single-cell sequencing experiments. Figure 8B The read distribution of single-cell sequencing, three-cell sequencing, or five-cell sequencing is shown. Figure 8C Histograms showing average library diversity and estimated genome coverage using a single cell, three cells, or five cells are shown. Fig.8D The overall program success rate is shown.

[0106] Fig. 9A Shown are copy number analysis using REPLIg Single Cell (MDA) and Nexteral XT library preparation. Fig. 9B Copy number analysis using SurePlex versus Nexteral XT library preparation is shown. Fig. 9C Shown is copy number analysis using the Nextera Single Cell provided herein.

[0107] Fig. 10AShown are data from chromosome 18 copy number analysis using triplicate individual GM50121 cells. Fig. 10B Shown are count number data of individual GM20916 cells using triplicates. Fig. 10C Shown are copy number analysis data for chromosome 15, chromosome X, and chromosome 10 using triplicate individual GM20916 cells. Fig. 10D Shown are copy number analysis data for chromosome 1 and chromosome 11 using triplicate individual GM1021 cells. DETAILED DESCRIPTION

[0108] The present disclosure relates generally to methods for preparing libraries of nucleic acid fragments, and more particularly to methods for preparing libraries of nucleic acid fragments using proteases in a single reaction mixture, such as a single tube, for use in a variety of applications including, for example, next generation DNA sequencing.

[0109] definition

[0110] As used herein, the terms "includes," "including," "includes," "including," "contains," "containing," "have," "having" and any variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, product defined by a method, or composition of matter that includes, includes, or contains an element or a list of elements includes not only those elements but may include other elements not expressly listed or inherent to such process, method, product defined by a method, or composition of matter.

[0111] As used herein, the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0112] As used herein, the term "about" or "approximately" means within 5% of a given value or range.

[0113] As used herein, the term "minimal cell population" means a cell population comprising a DNA copy amount lower than the nucleic acid sequencing capability in the absence of a separation step such as DNA extraction before labeling. Exemplary separation steps include extracting DNA content and / or DNA amplification from a cell lysate. The minimal cell population may comprise one, two, three, four or five cells. The minimal cell population may be a single cell. As used herein, "nucleic acid sequencing capability" means the sequencing capability that can produce clear genome copy number variation data.

[0114] As used herein, the term "nucleic acid" means a single-stranded polymer and a double-stranded polymer of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA) connected by phosphodiester bonds between nucleotides or nucleotide analogs, and associated counterions, such as H+, NH4+, trialkylammonium, tetraalkylammonium, Mg2+, Na+, etc. Nucleic acids include polynucleotides and oligonucleotides. Nucleic acids can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or include mixtures inlaid therein. Nucleotide monomer units may include any nucleotide described herein, including but not limited to naturally occurring nucleotides and nucleotide analogs. The size range of nucleic acids is generally from several monomer units, such as 5-40 to several thousand monomer nucleotide units. Nucleic acids include but are not limited to genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA, fragmented nucleic acids, nucleic acids obtained from subcellular organelles such as mitochondria or chloroplasts, and nucleic acids obtained from microorganisms or DNA or RNA viruses that may be present on or in biological samples.

[0115] As used herein, the term "target nucleic acid" is intended to mean a nucleic acid of a target object for analysis or action. Analysis or action includes subjecting the nucleic acid to copying, amplification, sequencing and / or other procedures for nucleic acid interrogation. The target nucleic acid may include a nucleotide sequence other than the target sequence to be analyzed. For example, the target nucleic acid may include one or more adapters, which are included in the adapters that function as primer binding sites on the flanks of the target nucleic acid sequence to be analyzed. The target nucleic acid hybridized with a capture oligonucleotide or a capture primer may include nucleotides that extend beyond the 5' or 3' ends of the capture oligonucleotide, so that not all target nucleic acids are suitable for extension in this way.

[0116] As used herein, the terms "isolate" and "purify" as used herein refer to the reduction of the amount of at least one contaminant, such as a protein and / or nucleic acid sequence, from a sample or from a source from which a substance is isolated or purified.

[0117] As used herein, the term "size selection" means a procedure during which a subpopulation of nucleic acid fragments is selected from a population of nucleic acid fragments, the majority of which have a number of nucleotides falling within a defined range, and thus the percentage of nucleic acid fragments having a number of nucleotides falling within a defined range is increased.

[0118] As used herein, the term "protease" refers to a protein, polypeptide or peptide that exhibits the ability to hydrolyze a polypeptide or a substrate having a polypeptide portion. The protease provided in the present method can be a single protease with wide specificity. The present method can use a mixture of multiple proteases. The protease provided herein can be heat-labile and therefore inactivated by heat. In certain embodiments, the protease provided herein can be inactivated at a temperature higher than about 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or higher than about 85°C. The protease provided herein can digest chromatin proteins and other DNA binding proteins to release naked genomic DNA, and can also digest endogenous DNA enzymes to protect DNA from degradation. The protease provided herein includes but is not limited to serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, glutamic proteases and metalloproteases. Typically, aspartic proteases, glutamic proteases and metalloproteases activate water molecules, which carry out nucleophilic attacks on peptide bonds to hydrolyze them. Serine proteases, threonine proteases and cysteine ​​proteases usually use affinity residues to carry out nucleophilic attack, to covalently connect the protease to the substrate protein, releasing the first half of the product. The covalent acyl-enzyme intermediate is then hydrolyzed by activated water, to complete catalysis by releasing the second half of the product and regenerating the free enzyme. Exemplary proteases used herein include serine proteases separated from recombinant Bacillus strains. Exemplary proteases used herein include subtilisin and variants thereof, including subtilisin Carlsberg, alcalase and subtilisin S41.Subtilisin and its variants are known to those skilled in the art and include, for example, alcalase, alcalase 0.6L, alcalase 2.5L, ALK enzyme, bacitracin A, bacitracin B, subtilisin bioprase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, genenase I, esperase, maxatase, thermostable protease PC 10, protease XXVII, thermostable enzyme, superase, subtilisin Carlsberg, subtilisin DY, subtilisin, SP 266, savinase 8.0L, savinase 4.0T, kazusase, protease VIII, opticlean, protin A 3L, savinase, savinase 16.0L, savinase 32.0L EX, orientase 10B, proteinase S, serine endopeptidase. In certain embodiments of the methods and compositions presented herein, thermolabile proteases such as subtilisin and thermolabile variants of subtilisin can be used, as shown by the exemplary disclosure of Davail et al., 1994, J. Biol. Chem., 26: 17448-17453, which is incorporated herein by reference in its entirety.

[0119] As used herein, the term "protease inhibitor" refers to a substance, such as a compound, that is capable of at least partially reducing the ability of a protease to hydrolyze a peptide.

[0120] As used herein, the term "ligase" refers to a nucleic acid modification enzyme that catalyzes the intramolecular and intermolecular formation of a phosphodiester bond between the 5'-phosphate terminus and the 3'-hydroxy terminus of a nucleic acid strand. Ligases include, for example, template-independent ligases such as CIRCLIGASE TMssDNA ligase, which can connect the ends of single-stranded RNA and DNA; and template-dependent, which closes the nick in double-stranded DNA. As used herein, "template-dependent ligase" means a DNA ligase that catalyzes the intramolecular and intermolecular formation of phosphodiester bonds between 5'-phosphate ends and 3'-hydroxyl ends of DNA chains adjacent to each other when annealed to complementary polynucleotides. The polynucleotide to which the two DNA ends to be connected are adjacently annealed is referred to as a "ligation template" herein, and the connection is referred to as "template-dependent connection". The ligation template can be a complementary DNA sequence in a genome or other DNA in a biological sample, or the ligation template can be a "bridge oligodeoxyribonucleotide" or "ligation splint oligodeoxyribonucleotide (ligation splintoligodeoxyribonucleotide)" (or "ligation splint") that is synthesized and / or specially provided for a specific assay or method. Examples of template-dependent DNA ligases include: NAD-type DNA ligases, such as Escherichia coli (E. coli) DNA ligase, Tth DNA ligase, TfI DNA ligase and DNA ligase (EPICENTRE Biotechnologies, Madison, WI, USA), NAD-type DNA ligases that catalyze intramolecular ligation of ssDNA molecules only in the presence of a ligation template; and ATP-type DNA ligases, such as T4 DNA ligase or FASTLINK TM DNA ligase (EPICENTRE Biotechnologies).

[0121] As used herein, the term "tagmentation" refers to the modification of DNA by a transposome complex comprising a transposase complexed with an adapter comprising a transposon end sequence. Tagging results in simultaneous DNA fragmentation and connection of the adapter to the 5' ends of the two chains of the duplex fragment. Additional sequences can be added to the ends of the suitable fragments, for example, by PCR, connection, or any other suitable method known to those skilled in the art. As used herein, the term "transposome complex" refers to a transposase that is non-covalently bound to a double-stranded nucleic acid. For example, the complex can be a transposase pre-incubated with a double-stranded transposon DNA under conditions that support the formation of a non-covalent complex. Double-stranded transposon DNA may include, but is not limited to, Tn5 DNA, a portion of Tn5 DNA, a transposon end component, a mixture of transposon end components, or other double-stranded DNAs that can interact with a transposase such as a hyperactive Tn5 transposase.

[0122] As used herein, the term "transposition reaction" refers to a reaction in which one or more transposons are inserted into a target nucleic acid, for example, at a random site or a nearly random site. The important components in the transposition reaction are a transposase and a DNA oligonucleotide that exhibits the nucleotide sequence of the transposon, the DNA oligonucleotide comprising the transposon sequence to be transferred and its complement (non-transferred transposon end sequence) and other components required to form a functional transposition or transposome complex. The DNA oligonucleotide may also comprise additional sequences (e.g., adapter or primer sequences) as needed or desired. In some embodiments, the methods provided herein are exemplified by employing a transposition complex formed by a hyperactive Tn5 transposase and a Tn5-type transposon end (Goryshin and Reznikoff, 1998, J. Biol. Chem., 273: 7367) or by a MuA transposase and a Mu transposon end comprising R1 and R2 terminal sequences (Mizuuchi, 1983, Cell, 35: 785; Savilahti et al., 1995, EMBO J., 14: 4893). However, any transposition system capable of inserting transposon ends in a random or nearly random manner with sufficient efficiency to 5' tag and fragment target DNA for its intended purpose can be used in the present invention.Examples of transposition systems known in the art that can be used in the present method include, but are not limited to, Staphylococcus aureus Tn552 (Colegio et al., 2001, J Bacterid., 183:2384-8; Kirby et al., 2002, Mol Microbiol, 43:173-86), TyI (Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72 and International Patent Application No. WO 95 / 23875), transposon Tn7 (Craig, 1996, Science. 271:1512; Craig, 1996, Review in: Curr Top Microbiol Immunol, 204:27-48), Tn10 and IS10 (Kleckner et al., 1996, Curr Top Microbiol Immunol, 204:49-82), Mariner transposase (Lampe et al., 1996, EMBO J., 15:5470-9), Tci (Plasterk, 1996, Curr Top Microbiol Immunol, 204:125-43), P factor (Gloor, 2004, Methods Mol Biol, 260:97-114), TnJ (Ichikawa and Ohtsubo, 1990, J Biol Chem. 265:18829-32), bacterial insertion sequences (Ohtsubo and Sekine, 1996, Curr. Top. Microbiol. Immunol. 204:1-26), retroviruses (Brown et al., 1989, Proc Natl Acad Sci USA, 86:2525-9) and yeast retrotransposons (Boeke and Corces, 1989, Annu Rev Microbiol. 43:403-34). The method for inserting the transposon end into the target sequence can be performed in vitro using any suitable transposon system, for which a suitable in vitro transposition system is available or can be developed based on knowledge in the art. Generally, a suitable in vitro transposition system for the methods provided herein requires at least: a transposase of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity; a transposon end with which the transposase forms a functional complex, wherein the functional complex has a respective transposase capable of catalyzing a transposition reaction. Suitable transposase transposon end sequences that can be used in the present invention include, but are not limited to, a wild-type transposon end sequence, a derived transposon end sequence, or a mutated transposon end sequence that forms a complex with a transposase, wherein the transposase is selected from a wild-type transposase, a derived form of a transposase, or a mutated form of a transposase.

[0123] As used herein, the term "transposase" refers to an enzyme that can form a functional complex with a component containing a transposon end (e.g., a transposon, a transposon end, a transposon end component) and can catalyze the insertion or transposition of a component containing a transposon end into a double-stranded target nucleic acid incubated with the component containing the transposon end, for example, in an in vitro transposition reaction. Transposases as shown herein can also include integrases from retrotransposons and retroviruses. Transposases, transpososomes, and transpososome complexes are generally known to those skilled in the art, as exemplified by the disclosure of US 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety. Although many embodiments described herein relate to Tn5 transposases and / or hyperactive Tn5 transposases, it will be appreciated that any transposition system capable of inserting transposon ends with sufficient efficiency for the purpose of its intent to add 5'-tags and fragments to target nucleic acids can be used in the present invention. In certain embodiments, the transposition system is capable of inserting transposon ends in a random or nearly random manner to 5'-tag and fragment target nucleic acids.

[0124] As used herein, the term "transposon end" refers to a double-stranded nucleic acid DNA that exhibits only the nucleotide sequence ("transposon end sequence") necessary to form a complex with a transposase or integrase that is functional in an in vitro transposition reaction. The transposon end forms a "complex" or "synaptic complex" or "transposome complex" or "transposome composition" together with a transposase or integrase that recognizes and binds to the transposon end, and the complex is capable of inserting the transposon end into the target DNA incubated with the complex in an in vitro transposition reaction. The transposon end exhibits two complementary sequences consisting of a "transferred strand" and a "non-transferred strand." For example, with a highly active Tn5 transposase (e.g., EZ-Tn5 TM Transposase, EPICENTRE Biotechnologies, Madison, WI, USA) together form a complex with one transposon end comprising a transferred strand exhibiting a "transferred transposon end sequence" as follows: 5'AGATGTGTATAAGAGACAG 3' (SEQ ID NO: 1) and a non-transferred strand exhibiting a "non-transferred transposon end sequence" as follows: 5'CTGTCT CTTATACACATCT 3' (SEQ ID NO: 2). The 3' end of the transferred strand is ligated or transferred to a target nucleic acid in an in vitro transposition reaction. The non-transferred strand exhibiting a transposon end sequence complementary to the transferred transposon end sequence is not ligated or transferred to a target nucleic acid in an in vitro transposition reaction.

[0125] As used herein, the term "transposon end element" refers to an element comprising a transposon end (a minimal double-stranded DNA segment capable of acting with a transposase to undergo a transposition reaction), optionally plus one or more additional sequences 5'- to the transferred transposon end sequence and / or 3' to the non-transferred transposon end sequence. For example, a transposon end attached to a tag is a "transposon end element".

[0126] As used herein, the term "transferred strand" refers to the transferred portion of both the "transposon end" and the "transposon end element" (regardless of whether the transposon end is attached to a tag or other moiety). Similarly, the term "non-transferred strand" refers to the non-transferred portion of both the "transposon end" and the "transposon end element".

[0127] As used herein, the term "tag" refers to a non-target nucleic acid component, typically DNA, which provides a means for searching for (addressing) a nucleic acid fragment to which the tag is attached. For example, in some embodiments, the tag includes a nucleotide sequence that allows identification, recognition, and / or molecular or biochemical manipulation of the DNA to which the tag is attached (e.g., by providing a site for annealing an oligonucleotide, such as a primer for extension by a DNA polymerase, or an oligonucleotide for capture or for a ligation reaction). The method of connecting a tag to a nucleic acid molecule is sometimes referred to herein as "tagging" and a nucleic acid that undergoes tagging or contains a tag is referred to as "tagged" (e.g., "tagged DNA").

[0128] As used herein, the term "tag domain" refers to a portion or domain of a tag that exhibits a sequence for a desired intended purpose or application. One tag domain is a "transposon end domain," which exhibits the transferred transposon end sequence. In some embodiments, the transferred strand also exhibits one or more other nucleotide sequences at the 5'-end of the transferred transposon end sequence, and the tag also has one or more other "tag domains" in the 5' portion, each of which is provided for any desired purpose. For example, some embodiments comprise a transposon end component, the transposon end component comprising a tag domain, the tag domain being selected from one or more of a restriction site tag domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, an addressing tag domain, and a transcription promoter domain.

[0129] As used herein, the term "restriction site domain" refers to a tag domain that exhibits a sequence for the purpose of facilitating cleavage using a restriction endonuclease. For example, a restriction site domain can be used to generate di-tagged linear ssDNA fragments. A restriction site domain can also be used to generate compatible double-stranded 5' ends in the tag domain so that the ends can be ligated to another DNA molecule using a template-dependent DNA ligase.

[0130] As used herein, the term "capture tag domain" refers to a tag domain that exhibits a sequence for the purpose of facilitating capture of nucleic acid fragments to which the tag domain is attached (e.g., providing an annealing site or affinity tag for capturing di-tagged linear ssDNA fragments on a bead or other surface, e.g., where the annealing site of the tag domain sequence allows capture by annealing to a specific sequence on a surface, such as a probe on a bead, or on a microchip or microarray, or on a sequencing bead). In some embodiments, the capture tag domain comprises the 5' portion of the transferred strand, which is linked to a chemical group or moiety, which includes an affinity binding molecule (e.g., biotin, streptavidin, an antigen, or an antibody that binds to an antigen, which allows capture of di-tagged linear ssDNA fragments on a surface to which a second affinity binding molecule is attached to form a specific binding pair with the first affinity binding molecule).

[0131] As used herein, the term "sequencing tag domain" refers to a tag domain that exhibits a sequence for the purpose of facilitating sequencing of the nucleic acid fragment to which the tag is attached (e.g., providing a priming site for synthesis sequencing, or providing an annealing site for ligation sequencing, or providing an annealing site for hybridization sequencing).

[0132] As used herein, the term "amplification tag domain" refers to a tag domain that exhibits a sequence for the purpose of facilitating amplification of the nucleic acid to which the tag is attached. For example, in some embodiments, the amplification tag domain provides a priming site for a nucleic acid amplification reaction (e.g., a PCR amplification reaction or a strand displacement amplification reaction or a rolling circle amplification reaction) using a DNA polymerase, or a ligation template for ligating probes using a template-dependent ligase in a nucleic acid amplification reaction (e.g., a ligation chain reaction).

[0133] As used herein, the term "detection tag domain" refers to a tag domain that exhibits a sequence or a detectable chemical or biochemical moiety (e.g., a visible dye, a fluorescent dye, a chemiluminescent dye, or other detectable dye; an enzyme detectable in the presence of a substrate, such as alkaline phosphatase with NBT plus BCIP, or a peroxidase with a suitable substrate; a detectable protein, such as green fluorescent protein; and an affinity binding molecule that is bound to a detectable moiety or can form an affinity binding pair or specific binding pair with another detectable affinity binding molecule; or any of the many other detectable molecules or systems known in the art) for the purpose of facilitating detection of the tagged nucleic acid fragments.

[0134] As used herein, the term "addressable tag domain" means a tag domain that exhibits a sequence that allows identification of a specific sample (eg, where the transferred strands have different addressable tag domains that exhibit different sequences for each sample).

[0135] As used herein, the terms "amplify" or "amplified" as used in relation to nucleic acids or nucleic acid reactions "amplifying" refers to an in vitro method of making copies of a specific nucleic acid, such as a target nucleic acid or a tagged nucleic acid. Many methods of amplifying nucleic acids are known in the art, and amplification reactions include, but are not limited to, polymerase chain reaction, ligase chain reaction, strand displacement amplification reaction, rolling circle amplification reaction. The amplified nucleic acid can be DNA. Regardless of whether the starting nucleic acid is DNA, RNA, or both, the products resulting from the amplification of one or more nucleic acid molecules ("amplification products") can be DNA or RNA, or a mixture of both DNA and RNA nucleosides or nucleotides, or they can include modified DNA or RNA nucleosides or nucleotides. "Copy" does not necessarily mean a sequence that is perfectly complementary to the target sequence or identical to the target sequence. For example, a copy can include: nucleotide analogs, such as deoxyinosine or deoxyuridine; intentional sequence changes (such as sequence changes introduced by primers that include sequences that are hybridizable but not complementary to the target sequence); and / or sequence errors that occur during amplification.

[0136] As used herein, the term "library of tagged nucleic acid fragments" refers to a collection or population of tagged nucleic acid fragments (e.g., di-tagged nucleic acid fragments) generated from a source, such as a whole genome, wherein the combination of tagged nucleic acid fragments in the collection or population exhibits sequences that are qualitatively and / or quantitatively representative of the sequences of the source, such as the whole genome, from which the tagged nucleic acid fragments were generated. It is possible that the library of tagged nucleic acid fragments does not contain tagged nucleic acid fragments that represent every sequence exhibited by the source.

[0137] As used herein, the term "nucleic acid modifying enzyme" refers to an enzyme that acts on nucleic acid, such as DNA, to affect modification, such as cleavage, ligation, polymerization, phosphorylation, etc. Nucleic acid modifying enzymes include, for example, polymerases, nucleases, transferases, ligases, phosphorylases, phosphatases, methylases, transposases, etc. "DNA modifying enzymes" include any enzyme that acts on DNA, including enzymes that also act on other substrates such as RNA.

[0138] As used herein, the term "DNA polymerase" refers to a modification enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA chain. DNA polymerases include "template-dependent DNA polymerases" that require a template nucleic acid to determine the smoothness of the addition of deoxyribonucleotides in the polymer, or they can be "template-independent" so that they catalyze polymerization without reference to the template sequence. In addition to synthesizing DNA polymers, DNA polymerases may include other features or activities. For example, DNA polymerases may be characterized as having or lacking 5' to 3' exonuclease activity (also referred to as 5' exonuclease or 5' nuclease activity), 3' to 5' exonuclease activity, and strand displacement activity.

[0139] As used herein, the term "primer" is an oligonucleotide ("oligo"), typically with a free 3'-OH group that can be extended by a nucleic acid polymerase. For template-dependent polymerases, typically at least the 3' portion of the primer oligonucleotide is complementary to a portion of the template nucleic acid, the oligonucleotide "binds" (or "complexes", "anneals" or "hybridizes") to the portion of the template nucleic acid and the template to give a primer / template complex for initiating synthesis by a DNA polymerase, and at least the 3' portion of the primer oligonucleotide is extended during DNA synthesis by adding a covalently bound base complementary to the template at its 3' end. As a result, a primer extension product is obtained.

[0140] As used herein, the term "universal sequence" refers to a region of nucleotide sequence that is common or shared by two or more nucleic acid molecules. Optionally, the two or more nucleic acid molecules also have regions that differ in sequence. Thus, for example, a 5' tag may comprise the same or universal nucleic acid sequence and a 3' tag may comprise the same or universal sequence. A universal sequence that may be present in different members of a plurality of nucleic acid molecules may allow a single universal primer that is complementary to the universal sequence to be used to replicate or amplify a plurality of different sequences.

[0141] As used herein, the terms "solid surface", "solid support" and other grammatical equivalents herein refer to any material that is suitable or can be modified to be suitable for the attachment of polynucleotides. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylic resins, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, TMIn some embodiments, the solid support and the solid surface are located in a flow cell device. In some embodiments, the solid support includes a patterned surface suitable for fixing the transposome complex in an ordered pattern. "Patterned surface" refers to the arrangement of different regions in or on the exposed layer of the solid support. In some embodiments, the solid support includes an array of holes or recesses in the surface. The composition and geometry of the solid support may vary with its use. In some embodiments, the solid support is a planar structure, such as a glass slide, a chip, a microchip and / or an array. Therefore, the surface of the substrate can be in the form of a planar layer. In some embodiments, the solid support includes one or more surfaces of a flow cell. The term "flow cell" as used herein refers to a chamber comprising a solid surface through which one or more fluid reagents can flow. Examples of flow cells and related fluid systems and detection platforms that can be easily used in the methods of the present disclosure are described in, for example, Bentley et al., Nature 456:53-59 (2008); WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US2008 / 0108082, each of which is incorporated herein by reference. In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the solid support includes microspheres or beads. In this article, "microspheres", "beads", "particles" or grammatical equivalents are intended to mean small discrete particles made of a variety of materials including but not limited to plastics, ceramics, glass and polystyrene. In certain embodiments, the microspheres are magnetic microspheres or beads. Alternatively or additionally, the beads may be porous. The size of the beads ranges from nanometers, such as 100 nm, to millimeters, such as 1 mm.

[0142] Methods for preparing a library of tagged nucleic acid fragments

[0143] The present disclosure relates generally to methods for preparing libraries of nucleic acid fragments, and more particularly to methods for preparing libraries of nucleic acid fragments using proteases in a single reaction mixture, such as a single reaction tube or other container, for a variety of applications, including, for example, next generation DNA sequencing, analysis of copy number variation, and analysis of single nucleotide variation.

[0144] There are various methods and applications, for which it is desirable to prepare a library of nucleic acid fragments from a minimum cell population, such as a single cell, for various applications such as genome sequencing. The current method for preparing a library of nucleic acid fragments requires separation of nucleic acid extracts and / or amplification steps before DNA fragmentation. Typically, cells are first treated to generate cell lysates, from which target nucleic acid contents are extracted and purified. Then in the separation step, the purified target nucleic acid is subjected to fragmentation using, for example, Nextera transposases available from Illumina, Inc (San Diego, CA). The separation of nucleic acid extracts and the transfer of samples between reaction tubes or containers usually waste target nucleic acid samples, and therefore make it less likely that the prepared nucleic acid fragments will successfully fully represent the entire target nucleic acid from the sample. When the amount of a cell sample is limited or difficult to obtain, this inadequate representation becomes particularly challenging. In the case of a single or a few cells being input by a pre-amplification step, some methods have been developed to solve this problem. However, these methods cannot effectively solve the problem of inadequate representation and usually introduce high noise. The present disclosure provides a solution to this problem by using a single reaction mixture in, for example, a single tube with an addition scheme to generate a library of nucleic acid fragments. The methods provided herein integrate multiple steps, including generating cell lysates, labeling, etc. in a single reaction tube, optionally using one or more addition schemes. In such a single tube addition method, the amount of the starting nucleic acid material from the cell is maintained, and the library generated therefrom can therefore better represent the target nucleic acid, such as a genome.

[0145] In one aspect, the present disclosure provides a method for preparing a library of tagged nucleic acid fragments, the method comprising: (a) contacting a cell population directly with a lysis reagent to generate a cell lysate, wherein the lysis reagent has one or more proteases, and wherein the cell lysate comprises a target nucleic acid; (b) inactivating the one or more proteases to form an inactivated cell lysate, and (c) applying at least one transposase and at least one transposon end component comprising a transferred strand directly to the inactivated cell lysate under conditions wherein the target nucleic acid and the transposon end component undergo a transposition reaction to generate a hybrid, wherein (i) the target nucleic acid is fragmented to generate a plurality of target nucleic acid fragments, and (ii) the transferred strand of the transposon end component is ligated to the 5′ end of each of the plurality of target nucleic acid fragments to generate a plurality of 5′-tagged target nucleic acid fragments.

[0146] In some embodiments, the cell sample is directly contacted with a combined cleavage reagent comprising one or more proteases, and therefore the protease provided herein can be directly contacted with intact cells. In some embodiments, the cell sample is contacted with a first cleavage reagent comprising a detergent to generate a first cell lysate, and then a second cleavage reagent comprising one or more proteases is added to the reaction tube comprising the first cell lysate. In this alternative, the protease provided herein is contacted with the cell lysate. The following embodiment 1 provided illustrates the method for generating a cell lysate comprising a target nucleic acid. An exemplary total mixture (master mixture) of cleavage comprising a detergent and QIAGEN (San Diego, CA) protease (product number 19155) is illustrated in Example 1 and Table 1-3.

[0147] The starting material according to the method provided herein can be a minimum cell population, and using the minimum cell population, traditional sequencing schemes may generally only produce noise sequencing data and copy number variation data due to insufficient representation of the entire target nucleic acid, such as a genome. In some embodiments, the minimum cell population can include one, two, three, four or five cells. In some embodiments, the minimum cell population can be less than 10 cells, less than 15 cells, less than 20 cells, less than 25 cells, less than 30 cells, less than 35 cells, less than 40 cells, less than 45 cells, less than 50 cells, less than 60 cells, less than 70 cells, less than 80 cells, less than 90 cells or less than 100 cells. In some embodiments, the starting material used in this method only includes a single cell. In some embodiments, the target nucleic acid is a genomic DNA. In some embodiments, the target nucleic acid includes chromosomal DNA or a fragment thereof. In some embodiments, the target nucleic acid includes a genome or a partial genome.

[0148] The protease used herein can digest chromatin proteins, such as histones and other DNA binding proteins, to release naked genomic DNA. In addition, the protease provided herein can digest endogenous DNA enzymes to protect the genome from degradation. In some embodiments, the method herein only uses a protease with a broad spectrum of specificity, and therefore the protease can digest a variety of different proteins and polypeptides, including some or many proteins in the cell. In some other embodiments, the broad spectrum of specificity can be achieved by using a mixture of multiple proteases, and the combination of multiple proteases can digest a variety of different proteins and polypeptides, including some or many proteins in the cell. Exemplary proteases include subtilisin, such as alcalase, subtilisin carlsberg, subtilisin S41, heat-labile proteinase K and Qiagen protease. Example 4 illustrates that protease activity is useful for uniformly approaching genomic DNA. It should be appreciated that, depending on different conditions, such as cell type and sample size, different proteases and / or mixtures of proteases can be used.

[0149] The amount and concentration of the protease used in each reaction provided herein can vary according to the amount of chromosomal DNA and / or the number of cells used and the activity of the protease. In some embodiments, the concentration of one or more proteases in the cell lysate is 0.1mg / ml to 10mg / ml. In some embodiments, the concentration of one or more proteases in the cell lysate is 0.1mg / ml to 2.5mg / ml. In some embodiments, the concentration of one or more proteases in the cell lysate is 2mAU / ml to 500mAU / ml. In some embodiments, the concentration of one or more proteases in the cell lysate is 4.5mAU / ml to 500mAU / ml. In some embodiments, the concentration of one or more proteases in the cell lysate is 10mAU / ml to 100mAU / ml. The present disclosure illustrates the use of protease such as QIAGEN protease (product number 19155) to test and optimize protease concentration, as shown in Example 5. As shown in this example, when single cells were treated with 0.5 mg / ml (equal to 22.5 mAU / ml) or 2 mg / ml (equal to 90 mAU / ml) of protease at normal reaction temperature (e.g., room temperature), similarly clear copy number analysis results were obtained, as shown in Figure 3AIn some embodiments, the concentration of protease in the cell lysate is 0.5 mg / ml to 2 mg / ml. The exemplary concentration of protease in the cell lysate includes 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml and 2.0 mg / ml. In some embodiments, the concentration of one or more protease in the cell lysate is 20 mAU / ml to 90 mAU / ml. Exemplary concentrations of one or more proteases in the cell lysate include 20 mAU / ml, 30 mAU / ml, 40 mAU / ml, 50 mAU / ml, 60 mAU / ml, 70 mAU / ml, 80 mAU / ml, 90 mAU / ml.

[0150] A variety of conditions including pH values ​​can affect both the digestion of the protease in the reaction tube and the activity of other enzymes, and therefore these conditions such as pH values ​​can be optimized. Example 6 illustrates the optimization of the pH conditions of the protease digestion reaction, balancing the protease activity and sequencing results. As shown, the QIAGEN protease activity is analyzed under different pH conditions, and the activity of the protease increases with the increase of pH value in the range of pH 7.0 to pH 10.0, and the protease has the lowest activity at pH 7.0 and the highest activity at pH 10.0. Then, the percentage of uniquely mapped reads and noise in the copy number data is also analyzed under different pH conditions. As shown, when the pH is 7, 8 or 9, about 70% of clear uniquely mapped reads can be obtained. However, when the pH is 10, a smaller percentage of uniquely mapped reads can be obtained, and the data variation is significantly increased. Similarly, when the pH is 7, 8 or 9, the count difference between adjacent bins is relatively small (about 20%), with small variation; while at pH 10.0, the count difference between adjacent bins increases significantly, with huge variation. Thus, in some embodiments, the cell population is contacted with a lysis reagent at pH 7.0 to pH 10.0. In some embodiments, the cell population is contacted with a lysis reagent at pH 7.0 to pH 9.0. Exemplary pH conditions include pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0, and pH 9.5.

[0151] Because nucleic acid preparation and fragmentation steps are carried out in the same reaction tube, it can be beneficial that the protease according to the present method can be effectively inactivated without interfering with the next labeling step that usually requires double-stranded DNA. In some embodiments, the protease can be inactivated by increasing the temperature before the labeling step. High temperature can denature the double-stranded DNA conformation. Therefore, in some embodiments, the protease provided herein can be inactivated at a relatively low temperature without denaturing the double-stranded DNA. Example 7 illustrates the thermal inactivation of the test protease. As shown, the protease activity is tested at different temperatures, and the protease activity gradually decreases as the temperature increases, and is completely inactivated at 70°C. Therefore, in some embodiments, one or more proteases are inactivated by increasing the temperature to 50°C-80°C. In some embodiments, one or more proteases are inactivated by increasing the temperature to 70°C.

[0152] In some embodiments, the protease provided herein can also be inactivated by adding a protease inhibitor to the reaction tube. The protease inhibitor provided herein does not interfere with the labeling and amplification steps to be carried out later in the same reaction tube. Exemplary protease inhibitors include, for example, AEBSF, benstatin, E-64, pepstatin A, phosphamidone, leupeptin, aprotinin, benstatin hydrochloride, leupeptin, phosphamidone disodium salt, elastatin, aprotinin, nafamostat mesylate, antipain, PMSF (phenylmethanesulfonylfluoride), PefaBloc, diisopropyl fluorophosphate and Streptomyces subtilisin inhibitor.

[0153] As discussed above, one or more detergents may also be added to the cells. In some embodiments, detergents are added to the cells along with proteases. In other embodiments, detergents are added to the cells first and then proteases are added to the reaction tubes. The functions of the detergents used herein include disrupting cell membranes and releasing intracellular substances in soluble form. In some embodiments, the detergents used herein do not interfere with downstream enzyme activity. Therefore, in some embodiments, nonionic detergents are used. These detergents disrupt protein-lipid associations and lipid-lipid associations, but do not disrupt protein-protein interactions, and are therefore less likely to interfere with other downstream enzymes. Typically, nonionic detergents contain uncharged, hydrophilic head groups. Typically nonionic detergents are based on polyoxyethylene or glycosides. Exemplary nonionic detergents include 80. 20Tween, X-100, X-100-R, X-114, NP-40, C-100, X-100, CA 630, 200, 96 / 97 Triton, 98. 58. 35Brij series, L64, P84, non-detergent sulfobetaine (NDSB 201), amphipols (PMAL-C8), CHAPS, octyl β-D-pyranoglucoside, saponin, monododecyl nonaglycone ether (C12E9, polidocanol), sodium lauryl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, cetyltrimethylammonium bromide, SB3-10, SB3-12, amidino sulfobetaine-14, octyl thioglucoside, maltoside, HEGA and MEGA series.

[0154] After the protease is inactivated, an in vitro transposition reaction can be performed in the same reaction mixture, such as in the same reaction tube, by adding a transpososome composition comprising a stable complex formed between the transposase and the transposon end component or using separate transposase and transposon end components. The in vitro transposition reaction catalyzed by the transposase results in the simultaneous destruction of the target nucleic acid into fragments and the attachment of a tag to the 5' end of each fragment. It should be understood that any method describing the use of a transposase and a transposon end component may also use a transposome composition prepared from the transposase and the transposon end component, and any method describing the use of a transposome composition may also use separate transposase and transposon end components contained in the transposome composition.

[0155] In some embodiments, the methods provided herein include incubating an inactivated cell lysate comprising a target nucleic acid with at least one transposase and a transposon end component with which the transposase forms a transposition complex in an in vitro transposition reaction, under conditions in which multiple insertions into the target nucleic acid occur, and for a time sufficient for multiple insertions into the target nucleic acid to occur, the transposon end component comprising: (i) a transferred strand exhibiting a transferred transposon end sequence and optionally an additional sequence 5′-to-1 of the transferred transposon end sequence, and (ii) a non-transferred strand exhibiting a sequence complementary to the transferred transposon end sequence, each of the multiple insertions resulting in attachment of a first tag comprising the transferred strand to the 5′ end of a nucleotide in the target nucleic acid, thereby fragmenting the target nucleic acid and generating a population of annealed 5′-tagged DNA fragments, each of which has the first tag at the 5′ end of the target nucleic acid fragment.

[0156] In some embodiments, the methods described above are performed using separate transposases and transposon end components. In other embodiments, the methods described above are performed using a transposome composition comprising a complex formed between a transposase and a transposon end component.

[0157] In some specific embodiments, the methods provided herein are performed using Nextera transposomes available from Illumina Inc (San Diego, CA), as generally described in the disclosure of US 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety.

[0158] Transposases and transposome compositions are generally known to those skilled in the art, as exemplified by the disclosure of US2010 / 0120098, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the methods provided herein employ transposome compositions formed by hyperactive Tn5 transposases and Tn5-type transposon ends (Goryshin and Reznikoff, 1998, J. Biol. Chem., 273: 7367). In some embodiments, the methods provided herein employ transposome compositions formed by MuA transposases and Mu transposon ends comprising R1 and R2 terminal sequences (Mizuuchi, 1983, Cell, 35: 785; Savilahti et al., 1995, EMBO J., 14: 4893). Any transposition system capable of inserting transposon ends in a random or nearly random manner with sufficient efficiency to add 5' tags and fragmentation to target nucleic acids for their intended purposes can be used in the present disclosure.Exemplary transposome composition systems include, but are not limited to, Staphylococcus aureus Tn552 (Colegio et al., 2001, J Bacterid., 183:2384-8; Kirby et al., 2002, Mol Microbiol, 43:173-86), TyI (Devine and Boeke, 1994, Nucleic Acids Res., 22:3765-72 and International Patent Application No. WO95 / 23875), transposon Tn7 (Craig, 1996, Science. 271:1512; Craig, 1996, Review in: Curr Top Microbiol Immunol, 204:27-48), Tn10 and IS10 (Kleckner et al., 1996, Curr Top Microbiol Immunol, 204:49-82), Mariner transposase (Lampe et al., 1996, EMBO J., 15:5470-9), Tci (Plasterk, 1996, Curr Top Microbiol Immunol, 204:125-43), P factor (Gloor, 2004, Methods Mol Biol, 260:97-114), TnJ (Ichikawa and Ohtsubo, 1990, J Biol Chem. 265:18829-32), bacterial insertion sequences (Ohtsubo and Sekine, 1996, Curr. Top. Microbiol. Immunol. 204:1-26), retroviruses (Brown et al., 1989, Proc Natl Acad Sci USA, 86:2525-9) and yeast retrotransposons (Boeke and Corces, 1989, Annu Rev Microbiol. 43:403-34).

[0159] As non-limiting examples, the transposon end may include a 19-bp outer end ("OE") transposon end, an inner end ("IE") transposon end, or a "mosaic end" ("ME") transposon end recognized by a wild-type or mutant Tn5 transposase, or R1 and R2 transposon ends as described in the disclosure of US 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety. The transposon end may include a nucleic acid or nucleic acid analog suitable for forming a functional complex with a transposase or integrase in an in vitro transposition reaction. For example, the transposon end may include DNA, RNA, modified bases, non-natural bases, modified backbones, and may contain gaps in one or both strands.

[0160] In some embodiments, wherein the transferred strand comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the transferred transposon end sequence, and the 5' portion of the transferred strand exhibits a sequence comprising one or more tag domains for a specific purpose (e.g., a sequencing tag domain or an amplification tag domain for next generation sequencing or amplification, and optionally an addressing tag domain). Exemplary tag domains include restriction site tag domains, capture tag domains, sequencing tag domains, amplification tag domains, detection tag domains, addressing tag domains, and transcription promoter domains.

[0161] In some embodiments, two different transposomes are used in an in vitro transposition reaction, and each of the two transposomes contains the same transposase but different transposon end components. In some embodiments, two different transposomes are used, and each of the two different transposomes contains the same transposase and the transposon end components contain different transferred strands. In some embodiments, two different transposomes are used, and each of the two transposomes contains a different transposase and a different transposon end component, each of the different transposon end components forming a functional complex with the respective transposase.

[0162] In some embodiments, the amount of transposase and transposon end components or the amount of transposome composition used in the in vitro transposition reaction is between about 1 picomoles and about 25 picomoles per 50 nanograms of target nucleic acid per 50 microliters reaction. In some embodiments, the amount of transposase and transposon end components or the amount of transposome composition used in the in vitro transposition reaction is between about 5 picomoles and about 50 picomoles per 50 nanograms of target nucleic acid per 50 microliters reaction. In some embodiments, the concentration of the transposase is 0.5-1 nM. In some embodiments, the concentration of the transposase is 0.01-0.02 picomoles / 20 μl reaction.

[0163] Example 2 illustrates a protocol for the tagging step using the methods provided herein. In embodiments where a single cell is used to prepare a library for sequencing, there are only two copies of the genome, and therefore a smaller insert size tends to increase library diversity. As shown in Example 8, counts increase as the insert size decreases, and therefore the diversity represented by the library increases as the insert size decreases. Therefore, in some embodiments, the methods herein use a higher amount of transposase in the tagging step to increase fragmentation and reduce the insert size of the tagged nucleic acid fragments. As shown, when 1 μl Tn5 was used in the tagging reaction, the average fragment size was about 550 bp; and when 2 μl Tn5 was used in the tagging reaction, the average fragment size was about 400 bp. Consistent with the smaller insert size, the diversity of the library when treated with 2 μl Tn5 increased compared to the diversity of the library treated with 1 μl Tn5. Tn5 is used to illustrate the adjustment of transposase. It should be appreciated that other transposases may also be used in the present method, and their amounts may be adjusted and optimized using the methods provided herein and methods known to those skilled in the art.

[0164] In some embodiments, the reaction time for the in vitro transposition reaction is 2 hours or less, 1 hour or less, 30 minutes or less, 15 minutes or less, or 10 minutes or less. In some embodiments, the reaction time for the in vitro transposition reaction is 5 minutes or less.

[0165] In some embodiments, the reaction temperature for the in vitro transposition reaction is from about 40° C. to about 70° C., from about 45° C. to about 65° C., or from about 50° C. to about 60° C. In some embodiments, the reaction temperature for the in vitro transposition reaction is about 55° C.

[0166] In some embodiments, the in vitro transposition reaction can be terminated by keeping the sample in the tube, for example, at 4° C. In some embodiments, the tagmentation buffer is neutralized with the tagmentation product, and the sample is incubated at room temperature for 5 minutes.

[0167] The target nucleic acid fragments are tagged at the 5' end by an in vitro transposition reaction. In some embodiments, the methods provided herein further comprise the step of incorporating a 3' end tag into the 5' tagged nucleic acid fragments to prepare a library of di-tagged nucleic acid fragments. In some embodiments, the library of di-tagged nucleic acid fragments is generated from the 5' tagged target nucleic acid in a single tube without any intermediate purification steps. Adding the 3' end tag can be performed by a variety of methods, for example, by using a DNA polymerase, a terminal transferase and / or a ligase as described in WO2010 / 048605, the contents of which are incorporated by reference in their entirety.

[0168] Thus, in some embodiments, the methods provided herein further comprise: (d) incubating the mixture from step (c) directly with at least one nucleic acid modification enzyme under conditions wherein the 3' tag is attached to the 5' tagged target nucleic acid fragment to generate a plurality of di-tagged target nucleic acid fragments. In some embodiments, steps (a), (b), (c), and (d) are performed in a single reaction tube. Embodiments exemplifying the generation of a library of di-tagged nucleic acid fragments are discussed below.

[0169] In some embodiments, the di-tagged nucleic acid fragments are generated by using a polymerase, such as a DNA polymerase, having strand displacement or 5' nuclease activity. In some embodiments, the methods provided herein comprise: incubating a population of annealed 5'-tagged nucleic acid fragments with a DNA polymerase having strand displacement or 5' nuclease activity under conditions without thermal cycling and wherein the annealed 5'-tagged nucleic acid fragments are not denatured, wherein the DNA polymerase extends the 3' end of each strand of the annealed 5'-tagged nucleic acid fragments using the complementary strand as a template and displaces or digests the non-transferred strand, thereby generating a library of di-tagged double-stranded DNA fragments. In one embodiment, the extension step is performed at 72° C. using the 5' tag on the opposite strand as a template.

[0170] In some embodiments, the di-tagged double-stranded DNA fragments generated by the methods provided above are denatured to generate a library of tagged DNA fragments comprising di-tagged single-stranded DNA fragments (eg, by heating to 95° C. and rapid cooling).

[0171] In other embodiments, the di-tagged nucleic acid fragments are generated by using a terminal transferase. In some embodiments, the 5'-tagged double-stranded nucleic acid fragments are denatured to generate 5'-tagged single-stranded nucleic acid fragments. The 5'-tagged single-stranded nucleic acid fragments are incubated with a DNA polymerase consisting of a terminal transferase and at least one substrate for the terminal transferase, during which the terminal transferase attaches a second tag to the 3' end of the 5'-tagged nucleic acid fragments, thereby generating a library of tagged nucleic acid fragments comprising di-tagged nucleic acid fragments. In some embodiments, the 3' end of the non-transferred transposon end comprising the transposon end component is blocked (e.g., by using a non-transferred transposon end having a dideoxynucleotide or a 3'-O-methyl nucleotide as the 3' terminal nucleotide), which blocks the 3' nucleotide and prevents addition by the terminal transferase, thereby preventing background tagging of the non-transferred transposon end.

[0172] In other embodiments, the 5'-tagged double-stranded nucleic acid fragments are not denatured to generate the 5'-tagged single-stranded nucleic acid fragments. Instead, the 5'-tagged nucleic acid fragments are incubated with a DNA polymerase consisting of a terminal transferase and at least one substrate for the terminal transferase, under conditions in which the terminal transferase attaches the second tag to the 3' end of the 5'-tagged nucleic acid fragments, for a time sufficient for the terminal transferase to attach the second tag to the 3' end of the 5'-tagged nucleic acid fragments, without a prior denaturation step, thereby generating a library of di-tagged nucleic acid fragments. In some embodiments, the 3' end of the non-transferred transposon end comprising the transposon end component is blocked (e.g., by using a non-transferred transposon end having a dideoxynucleotide or a 3'-O-methyl nucleotide as the 3' terminal nucleotide).

[0173] In other embodiments, the di-tagged nucleic acid fragments are generated by using a DNA polymerase and a terminal tagging oligonucleotide. In some embodiments, the 5′-tagged double-stranded nucleic acid fragments are denatured to generate 5′-tagged single-stranded nucleic acid fragments (e.g., by heating to 95° C. and rapidly cooling), and a second tag is attached to the 3′ end of the 5′-tagged single-stranded nucleic acid fragments using a DNA polymerase and a terminal tagging oligonucleotide, thereby generating a library of di-tagged nucleic acid fragments. In some embodiments, the step of attaching a second tag to the 3′ end of the 5′-tagged nucleic acid fragment using a DNA polymerase and a terminal tagging oligonucleotide comprises: (1) providing a terminal tagging oligonucleotide having a 5′ portion and a 3′ portion, the 5′ portion exhibiting a sequence complementary to the sequence of the second tag desired to be attached to the 3′ end of the 5′-tagged single-stranded nucleic acid fragment, and the 3′ portion exhibiting a random sequence comprising between 3 random nucleotides and 8 random nucleotides, the 3′ terminal nucleotide of the terminal tagging oligonucleotide being blocked so that it cannot be extended by the DNA polymerase; (2) annealing the 5′-tagged single-stranded nucleic acid fragment to the 5′-tagged single-stranded nucleic acid fragment under conditions wherein the terminal tagging oligonucleotide anneals to the 5′-tagged single-stranded nucleic acid fragment; contacting the tagged single-stranded nucleic acid fragments with the terminal tagging oligonucleotide for a time sufficient to allow the terminal tagging oligonucleotide to anneal to the 5'-tagged single-stranded nucleic acid fragments; and (3) contacting the 5'-tagged single-stranded nucleic acid fragments to which the terminal tagging oligonucleotide has annealed with a DNA polymerase in a reaction mixture and under DNA polymerization conditions in which the 3' ends of the 5'-tagged single-stranded nucleic acid fragments are extended using the terminal tagging oligonucleotide as a template, and for a time sufficient to allow the 3' ends of the 5'-tagged single-stranded nucleic acid fragments to be extended using the terminal tagging oligonucleotide as a template, thereby attaching a second tag to their 3' ends and generating 5'- and 3'-tagged single-stranded nucleic acid fragments.

[0174] In still other embodiments, the di-tagged nucleic acid fragments are generated by using a template-dependent ligase and a ligation tagging oligonucleotide. In some embodiments, the 5′-tagged nucleic acid fragments are incubated with a template-dependent DNA ligase and a ligation tagging oligodeoxynucleotide having a 3′ portion and a 5′ portion under conditions in which a second tag is ligated to the annealed 5′-tagged DNA fragments, and for a time sufficient for the second tag to be ligated to the annealed 5′-tagged DNA fragments, wherein the 3′ portion exhibits a second tag that exhibits any sequence desired to be ligated to the 3′ end of the 5′-tagged DNA fragments, and the 5′ portion has a 5′ monophosphate group and exhibits a random sequence, thereby generating a library of DNA fragments comprising the annealed di-tagged DNA fragments. In some embodiments, the method further comprises a step of denaturing the library of DNA fragments comprising the annealed di-tagged DNA fragments (e.g., by heating to 95° C. and rapidly cooling), thereby generating a library of di-tagged single-stranded DNA fragments.

[0175] After the library of tagged nucleic acid fragments is generated, the tagged nucleic acid fragments can be amplified using, for example, limited cycle polymerase chain reaction (PCR) to introduce additional end sequences or adapters, such as indexes, universal primers, and other sequences required for cluster formation and sequencing. In some embodiments, such amplification is performed on the library of 5' tagged nucleic acid fragments. In some embodiments, such amplification is performed on the library of di-tagged nucleic acid fragments. In some embodiments, amplification is performed in the same reaction tube in which the library of tagged nucleic acid fragments is generated, and the agents used for amplification are added directly to the same reaction tube.

[0176] Thus, the methods provided herein further comprise (e) amplifying one or more di-tagged target nucleic acid fragments to generate a library of tagged nucleic acid fragments having additional sequences at the 5' end and / or the 3' end of the di-tagged nucleic acid fragments. In some embodiments, steps (a), (b), (c), (d), and (e) are performed in a single reaction tube. Exemplary amplification methods include polymerase chain reaction (PCR), strand displacement amplification reaction, rolling circle amplification reaction, ligase chain reaction, transcription-mediated amplification reaction, or loop-mediated amplification reaction.

[0177] In some embodiments, the methods provided herein include amplifying a library of double-tagged single-stranded nucleic acid fragments using PCR. In some embodiments, the methods provided herein utilize single-primer PCR amplification of a library of tagged DNA fragments. In some embodiments, the step of amplifying the double-tagged DNA fragments includes using a DNA polymerase and at least one primer complementary to the second tag. In some embodiments, the step of amplifying the library of double-tagged DNA fragments includes amplifying the library of double-tagged DNA fragments by PCR using only one oligodeoxyribonucleotide that exhibits at least a portion of the sequence of the transferred strand as a PCR primer and using the double-tagged DNA fragments as a template. In some embodiments, the primer comprises a 5' portion, and the 5' portion comprises an additional sequence, such as an adapter sequence.

[0178] In some embodiments, two different PCR primers are used, each of which exhibits the sequence of at least a portion of the transferred transposon end comprising the transposon end component. In some embodiments, each PCR primer comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the respective transferred transposon end sequence, and the 5' portion exhibits the sequence of the respective tag domain or adapter for a specific purpose (e.g., a sequencing tag domain / adapter or an amplification tag domain / adapter for next-generation sequencing or amplification, and optionally an addressing tag domain / adapter). For example, when a single transposon end component is used in an in vitro transposition reaction to generate a library of double-tagged DNA fragments using a DNA polymerase with strand displacement or 5' nuclease activity, the double-tagged DNA fragments can be amplified by PCR using two different PCR primers. Each PCR primer comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the respective transferred transposon end sequence, and the 5' portion exhibits the sequence of the respective tag domain / adapter for a specific purpose (e.g., a sequencing tag domain / adapter or an amplification tag domain / adapter for next generation sequencing or amplification, and optionally an addressing tag domain / adapter). In some embodiments, the 5' portion of each PCR primer is different from the 5' portion of another primer, and thus the sequences of the two ends of the PCR product are different. For example, one end comprises an index and / or universal primer sequence, and the other end comprises a different index and / or universal primer sequence.

[0179] In some embodiments, the two ends of the double-tagged nucleic acid fragment are derived from two different transfer strand sequences. For example, in some embodiments, two different transpososomes can be used in an in vitro transposition reaction, and each of the two transpososomes contains the same transposase but different transposon end components. In some embodiments, two different transpososomes are used, and each of the two different transpososomes contains the same transposase and the transposon end components contain different transfer strands. In some embodiments, two different transpososomes are used, and each of the two transpososomes contains different transposases and different transposon end components, and each of the different transposon end components forms a functional complex with each transposase. In some embodiments, wherein two different transposon end components are used in an in vitro transposition reaction, and a library of double-tagged single-stranded nucleic acid fragments is generated using a DNA polymerase with chain displacement or 5' nucleic acid activity, the first tag shows the sequence of the transfer strand of a transposon end component and the second tag shows the sequence of the non-transferred strand of another transposon end component.

[0180] In the above-mentioned embodiments and other embodiments in which two different transferred strands are attached to the 5' end of each opposite strand of the double-stranded nucleic acid, the methods provided herein may further include the step of amplifying the di-tagged nucleic acid fragments by PCR using two different PCR primers. One of the PCR primers exhibits the sequence of at least a portion of one transferred strand comprising one transposon end component, and the other of the PCR primers exhibits the sequence of at least a portion of another transferred strand comprising another transposon end component.

[0181] In some embodiments where two primers are used, each PCR primer comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the respective transferred transposon end sequence, and the 5' portion exhibits the sequence of the respective tag domain / adapter (e.g., a sequencing tag domain or amplification tag domain for next-generation sequencing or amplification, and optionally an addressing tag domain) for a specific purpose. In some embodiments, the 5' portion of each PCR primer is different from the 5' portion of another primer, and thus different sequences are introduced into the two ends of the PCR product. In some embodiments, the 5' portion of the first PCR primer or the 5' portion of the second PCR primer, or the 5' portion of both the first PCR primer and the second PCR primer respectively comprises the first sequencing tag / adapter or the second sequencing tag / adapter, for generating a template for next-generation sequencing for a specific sequencing platform (e.g., a sequencing tag for an Illumina Nextera sequencing platform). In some embodiments, the 5' portion of the first PCR primer or the 5' portion of the second PCR primer additionally comprises an addressing tag domain / adapter or another tag domain / adapter for a specific purpose.

[0182] Example 3 illustrates limited cycle PCR amplification that can add other sequences at both ends of the tagged nucleic acid fragments, such as index 1 (i7) and index 2 (i5) (from Illumina, Inc, San Diego, CA) and sequences required for other purposes such as cluster formation. In single-cell sequencing, the input DNA is relatively small, and therefore the number of cycles of PCR can be adjusted to achieve better sequencing results. In Example 9, the number of cycles of PCR was tested and optimized using a single cell as the starting material. As shown, when PCR with 16 cycles was used in copy number analysis, the noise was large, and when PCR with 18 cycles or 20 cycles was used, the noise was significantly reduced. Therefore, in some embodiments, the number of PCR cycles is 18, 19 or 20.

[0183] For amplification reactions by PCR known to those skilled in the art, a wide variety of enzymes and kits are available. For example, in some embodiments, FAILSAFE from EPICENTRE Biotechnologies, Madison, WI is used. TM PCR system or MASTERAMP TM PCR amplification was performed using the Extra-Long PCR System as described by the manufacturer. However, the present disclosure is not limited to the use of those products or conditions for the amplification reaction, and any suitable thermostable DNA polymerase and reaction mixture that allows amplification of the sequence between the primer annealed to the target sequence and the primer annealed to the transposon may be used.

[0184] The methods provided herein are not limited to using PCR to amplify libraries of tagged nucleic acid fragments. Any suitable amplification method (e.g., rolling circle amplification, riboprimer amplification (e.g., U.S. Patent No. 7,413,857), ICAN, UCAN, ribospia, terminal tagging (U.S. Patent Application No. 20050153333), Eberwine-type aRNA amplification or strand displacement amplification) that amplifies the same sequence and generates a suitable composition and amount of amplified product for the intended purpose can be used in embodiments of the present invention. For example, some strand displacement methods that can be used are described in PCT Patent Publication Nos. WO 02 / 1661, WO 00 / 56877, and AU00 / 29742 to Takara Shuzo Company, Kyoto, Japan; U.S. Pat. Nos. 5,523,204, 5,536,649, 5,624,825, 5,631,147, 5,648,211, 5,733,752, 5,744,311, 5,756,702, and 5,916,779 to Becton Dickinson and Company; U.S. Pat. Nos. 6,238,868, 6,309,833, and 6,326,173 to Nanogen / Becton Dickinson Partnership; Nos. 5,849,547, 5,874,260, and 6,218,151 to Merieux; 5,786,183, 6,087,133, and 6,214,587 to Gen-Probe, Inc.; 6,063,604 to Wick et al.; 6,251,61 to Kurn; 6,410,278 to Eiken Kagaku Kabushiki Kaishi, Tokyo, Japan, and PCT Publication No. WO No. 00 / 28082; U.S. Patent Nos. 5,591,609, 5,614,389, 5,773,733, 5,834,202, and 6,448,017 to Auerbach; and U.S. Patent Nos. 6,124,120 and 6,280,949 to Lizardi.

[0185] In some embodiments, the library of tagged nucleic acid fragments prepared by any of the methods of the present disclosure can then be subjected to steps for purifying the library nucleic acids and, optionally, for providing size selection. These steps can help clean up PCR products and remove nucleic acids of undesirable sizes. Many methods in the art can be used to clean up the nucleic acid fragments generated in the present methods, including but not limited to: using columns to clean up the fragments, such as using the Qiagen QIAquick PCR purification kit; and using gel size selection, such as using the Pippin Prep electrophoresis platform. Other methods known in the art for cleaning up nucleic acid fragments and / or for selecting nucleic acid size can also be used in the methods provided herein.

[0186] For example, in some implementation methods, AMPure XP beads (from Beckman Coulter Genomics) are used to purify tagged nucleic acid fragments. Nucleic acid fragments can be bound to solid phase reversible immobilization (SPRI) beads, and the affinity of nucleic acid fragments with different lengths to the beads can be controlled by changing the PEG / NaCl concentration. Therefore, by changing the PEG / NaCl concentration, nucleic acids with different sizes can be selectively purified. In some embodiments, the methods provided herein use a single AMPure XP treatment to remove nucleic acid fragments less than a certain size (e.g., 150-200bp). In some embodiments, double (upper and lower) size selection can be performed by two consecutive AMPure XP steps. In the first selection step, a low concentration of AMPure XP beads is added to the sample to bind larger DNA fragments. In this step, beads containing larger fragments are discarded. Then, in the second selection step, more beads are then added to the supernatant. In this second step, the amount of PEG and NaCl is increased so that the smaller fragment size will be bound. Next, the supernatant containing very short library fragments is discarded, the beads are washed, and the intermediate fragments are eluted. One skilled in the art will appreciate that depending on the concentration of PEG and NaCl in the first and final SPRI steps, different size ranges can be generated, as exemplified in Bronner et al., 2009, Curr Protoc Hum Genet. 18:10.

[0187] A general procedure for cleaning up a library of nucleic acid fragments using AMPure XP beads includes: (1) vortexing the AMPure XP beads to ensure that the beads are evenly dispersed; (2) adding a certain amount of AMPure XP beads to each PCR product generated and incubating at room temperature; (3) placing the tube in a tube holder on a magnetic stand until the supernatant is clear; (5) removing and discarding the supernatant; (6) washing the beads one or more times without removing the tube from the magnetic stand; (7) allowing the beads to air dry with the tube still on the magnetic stand; (8) removing the tube from the magnetic stand and adding resuspension buffer and incubating at room temperature; and (9) transferring the supernatant to a new tube.

[0188] After the library of nucleic acid fragment is cleaned and size selected, it can be further subjected to library standardization step, so that the amount of each library is standardized and ensures roughly equal library representatives in each mixed sample. In some embodiments, in the method provided herein, the library standardization method based on pearl is used. In the library standardization method based on pearl, the pearl of roughly equal amount is added to each hole of the sample comprising nucleic acid fragment. Because the amount of the pearl added to each hole is roughly equal, the amount of the nucleic acid fragment attached to the pearl in each hole is also roughly equal. Therefore, after removing the supernatant, the amount of the nucleic acid fragment eluted from the pearl in each hole can be roughly equal.

[0189] A typical bead-based library normalization method includes: (1) adding a roughly equal amount of beads (e.g., in bead buffer) to each well containing nucleic acid fragments generated in the methods provided above; (2) incubating and / or shaking to allow the beads to bind to the nucleic acid fragments; (3) placing the wells (which can be on a plate) on a magnetic stand and allowing the supernatant to become clear; (4) with the wells on the magnetic stand, carefully remove and discard the supernatant; (5) washing the beads one or more times; and (6) eluting the nucleic acid fragments attached to the beads.

[0190] In some embodiments, the libraries of tagged nucleic acid fragments generated by the methods provided herein can be used as templates for nucleic acid sequencing.

[0191] In some embodiments, prior to sequencing, the tagged nucleic acid fragments in the library are amplified to enhance the signal relative to noise during sequencing, e.g., in synthetic sequencing. In some embodiments, the library of tagged nucleic acid fragments is used as a template for an amplification reaction (e.g., a PCR amplification reaction using PCR primers that are complementary to the end sequences of the tagged nucleic acid fragments). In some embodiments, the library of amplified tagged nucleic acid fragments comprises most or approximately all of the sequence exhibited by the target nucleic acid. In some embodiments, where the target nucleic acid comprises genomic DNA of an organism, the amplification reaction is a whole genome amplification reaction.

[0192] In some embodiments, the tagged nucleic acid fragments can be fixed to a solid surface. For example, the solid surface can be attached to a polynucleotide complementary to the terminal sequence of the tagged nucleic acid fragment, and thus the tagged nucleic acid fragment can be fixed on the solid surface. Then, the fixed nucleic acid fragments are amplified on the surface. For example, in some embodiments, the fixed nucleic acid fragments are amplified using a cluster amplification method, as exemplified by the disclosure of U.S. Patents Nos. 7,985,565 and 7,115,400, each of which is incorporated herein by reference in its entirety. The incorporated materials of U.S. Patents Nos. 7,985,565 and 7,115,400 describe a method of solid phase nucleic acid amplification, which allows the amplification product to be fixed on a solid support to form an array of clusters or "clusters" of fixed nucleic acid molecules. Each cluster or cluster on such an array is formed by a plurality of identical fixed polynucleotide chains and a plurality of identical fixed complementary polynucleotide chains. The array so formed is generally referred to as a "clustered array" herein. The product of a solid phase amplification reaction, such as those described in U.S. Pat. Nos. 7,985,565 and 7,115,400, is a so-called "bridge" structure formed by annealing of an immobilized pair of polynucleotide strands and an immobilized pair of complementary strands, both strands being immobilized on a solid support at the 5' end via, for example, covalent attachment. Cluster amplification methods are examples of methods in which an immobilized nucleic acid template is used to produce an immobilized amplicon. Other suitable methods known in the art can also be used to produce an immobilized amplicon from immobilized tagged nucleic acid fragments produced according to the methods provided herein.

[0193] Libraries of tagged nucleic acid fragments prepared according to the methods provided herein can be sequenced according to any suitable sequencing method, such as direct sequencing, including synthesis sequencing, ligation sequencing, hybridization sequencing, nanopore sequencing, etc. In some embodiments, the immobilized DNA fragments are sequenced on a solid support. In some embodiments, the solid support used for sequencing is the same solid support on which amplification occurs.

[0194] In some embodiments, the sequencing method used in the methods provided herein is sequencing by synthesis (SBS). In SBS, the extension of a nucleic acid primer along a nucleic acid template (e.g., a target nucleic acid or its amplicon) is monitored to determine the sequence of the nucleotides in the template. Potential chemical methods can be polymerization (e.g., as catalyzed by a polymerase). In specific polymerase-based SBS embodiments, fluorescently labeled nucleotides are added to primers (thus extending primers) in a template-dependent manner so that the order and type of the nucleotides added to the primers can be used to determine the sequence of the template.

[0195] Other sequencing programs using cyclic reactions can be used, such as pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) as specific nucleotides are incorporated into nascent nucleic acid chains (Ronaghi, et al., 1996, Analytical Biochemistry 242 (1), 84-9; Ronaghi, 2001, Genome Res. 11 (1), 3-11; Ronaghi et al., 1998, Science 281 (5375), 363; US 6,210,891; US ​​6,258,568 and US. 6,274,320, each of which is incorporated herein by reference). In pyrophosphate sequencing, the released PPi can be detected by being immediately converted into adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP produced can be detected via protons produced by luciferase. Therefore, the sequencing reaction can be monitored via a luminescent detection system. The excitation radiation source for the fluorescence-based detection system is not necessary for the pyrophosphate sequencing program. Useful fluidic systems, detectors, and procedures applicable to applying pyrosequencing to amplicons produced according to the present disclosure are described, for example, in WIPO Patent Application Serial Nos. PCT / US11 / 57111, US2005 / 0191698 A1, US 7,595,883, and US 7,244,559, each of which is incorporated herein by reference.

[0196] Some embodiments may utilize methods including real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET) interactions between a fluorophore-loaded polymerase and a γ-phosphate-labeled nucleotide, or by using a zero-mode waveguide (ZMW). Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. 2003, Science 299, 682–686; Lundquist et al., 2008, Opt. Lett. 33, 1026–1028; Korlach et al., 2008, Proc. Natl. Acad. Sci. USA 105, 1176–1181, the disclosures of which are incorporated herein by reference.

[0197] Some SBS embodiments include detection of the proton that Nucleotide is incorporated into the extension product after release.For example, the sequencing based on the proton that detects release can use electronic detector and the commercially available correlation technology from Ion Torrent (Guilford, CT, a Life Technologies subsidiary), or the sequencing method and system described in US 2009 / 0026082 A1, US 2009 / 0127589 A1, US 2010 / 0137143 A1 or US 2010 / 0282617 A1, each of which is incorporated herein by reference.The method for utilizing kinetics to exclude amplification of target nucleic acid described herein can be easily applied to the substrate that is used to detect proton.More specifically, the method described herein can be used to produce the clonal colony of the amplicon that is used to detect proton.

[0198] Another useful sequencing technology is nanopore sequencing (see, e.g., Deamer et al., 2000, Trends Biotechnol., 18, 147-151; Deamer et al., 2002, Acc. Chem. Res. 35: 817-825; Li et al., 2003, Nat. Mater. 2: 611-615), the disclosures of which are incorporated herein by reference. In some nanopore embodiments, the target nucleic acid or individual nucleotides removed from the target nucleic acid pass through a nanopore. As the nucleic acid or nucleotide passes through the nanopore, each nucleotide type can be identified by measuring the conductivity fluctuations of the pore. (U.S. Patent No. 7,001,792; Soni et al., 2007, Clin. Chem., 53, 1996-200; Healy, 2007, Nanomed. 2, 459-481; Cockroft et al., 2008, J. Am. Chem. Soc., 130, 818-820, the disclosures of which are incorporated herein by reference).

[0199] In some embodiments, the method provided herein also includes analyzing the copy number variation of cells. Copy number analysis tests DNA copy number variation in samples. Such analysis helps detect chromosome copy number variation, which may lead to or increase the risk of multiple serious disorders. For example, autism has been reported to be associated with copy number mutations (Sebat et al., 2007, Strong association of de novo copy number mutations with autism, Science 316 (5823): 445–9). It has also been reported that schizophrenia is associated with copy number variation (St Clair, 2008, Copy number variation and schizophrenia, Schizophr Bull 35 (1): 9–12). A variety of methods have been developed for detecting copy number variation. However, when the starting material is limited and comes from a minimal cell population, the noise is significant and the results are discounted. The present method provides a method for detecting copy number variation in such cases. The following examples provide copy number variation analysis using the present method, and for copy number variation analysis, several parameters are optimized. In some embodiments, the minimum cell population used in the copy number variation analysis comprises one, two, three, four or five cells. Typically, as the number of cells increases, a more complete read distribution can be achieved, and therefore there is less noise in the data, as shown in Example 10. In this example, the read distribution using one, three or five cells is analyzed in this example. As shown, the genome coverage increases with the increase in the number of cells, and it is evaluated that one cell can cover about 40% of the genome, and three cells can cover more than 50% of the genome, and five cells can cover about 60% of the genome. The average library counts using one cell, three cells and five cells are about 5 million, 15 million and 20 million, respectively. It is also shown in this example that when a single cell is used, the overall success rate is relatively high 94% (N=187). The failure of a cell assay may be caused by the quality of the cell itself, such as selecting a cell that is replicating or apoptotic / necrotic cells.

[0200] Example 11 compares the present method with some current single cell preparation methods. When the REPLI-g single cell kit developed by QIAGEN (San Diego, CA) is used to prepare nucleic acids, the copy number variation data is noisy when derived from a single cell, three cells or five cells. When the SurePlex (PicoPlex) developed by Illumina, Inc (San Diego, CA) is used to prepare nucleic acids, it reduces noise compared to the REPLI-g single cell kit. As shown, the present method (Nextera SC) further reduces noise compared to using the SurePlex amplification system. Therefore, the present method provides an advanced method for analyzing copy number variation.

[0201] One aspect of copy number variation analysis is to detect mosaicism. Mosaicism or mosaicism indicates the presence of two or more genotypes in an individual. There are two major categories of mosaicism: somatic mosaicism and germline mosaicism. Somatic mosaicism occurs when somatic cells contain more than one genotype, for example due to mitotic errors during the first division or subsequent divisions. Researchers have shown that the presence of somatic mutations increases throughout life and is the cause of many leukemias, lymphomas, and solid tumors (Jacobs et al., 2012, Detectable Clonal Mosaicism and Its Relationship to Aging and Cancer, Nature Genetics 44(6):651–1268). In germline mosaicism, some gametes (sperm or oocytes) carry mutations but other gametes are normal, which also leads to many diseases. Therefore, detecting mosaicism can provide valuable diagnostic information. The present disclosure provides methods for detecting mosaicism. In Example 12, the detection of mosaicism using the method provided herein is illustrated. As shown, in the copy number analysis of chromosome 18 of a single GM50121 cell, a population representing 15.4MB of DNA was detected in each single cell sequencing. Similarly, additional populations representing other chromosomes were detected using the copy number analysis data of chromosome 15, chromosome X, and chromosome 10 of a single GM20916 cell, and the copy number analysis data of chromosome 1 and chromosome 11 of a single GM1021 cell.

[0202] The present method can also be used for other applications, such as pre-transplantation gene screening, single cell research, analysis of circulating tumor cells, fine needle aspiration biopsy, buffy coat and amniocyte analysis. In these applications, the starting nucleic acid material is usually limited, and therefore the present method can improve the analysis of these applications. Except copy number variation analysis, the present method can also be used to detect single nucleotide variants (single nucleotide variants) present in the smallest cell population in the above-mentioned application. Single nucleotide variants include single nucleotide polymorphisms (SNPs) and point mutations. Single nucleotide polymorphisms (SNPs) are common types of genetic variation, which include polymorphisms in DNA positions, where two or more alternative bases exist (usually greater than or equal to 1%) at a considerable frequency in a population at the DNA position. Point mutations are base variations with a frequency less than 1%. Single nucleotide polymorphisms (SNPs) and point mutations represent the largest source of diversity in the human genome. These single nucleotide polymorphisms (SNPs) and point mutations can serve as biomarkers for disease localization on the human genome atlas, because they are usually located near the gene place relevant to a certain disease. Therefore, detection of single nucleotide polymorphisms (SNPs), point mutations and similar mutations is very important for clinical activity, human health and control of genetic diseases. The present method provides the advantage of uniformly approaching genomic DNA and helps to maintain the target nucleic acid material. Therefore, it can improve the detection of single nucleotide variations using a minimal cell population.

[0203] In the description of some embodiments of the various methods above, a "reaction tube" or "tube" is used. It should be appreciated that other reaction media and / or containers may also be used in the present methods.

[0204] Kit for preparing a library of tagged nucleic acid fragments

[0205] In another aspect, the present disclosure provides a kit for preparing a library of tagged nucleic acid fragments, the kit comprising: (a) a cleavage reagent having one or more proteases, and (b) a transposition reaction composition having at least one transposase and at least one transposon end component comprising a transferred strand.

[0206] In some embodiments, the cleavage reagent provided comprises only one protease with a broad spectrum of specificity, and thus the protease can digest a variety of proteins and polypeptides. In some other embodiments, the cleavage reagent provided herein comprises a mixture of a variety of proteases, and the combination of a variety of proteases can digest a variety of proteins and polypeptides. Exemplary proteases provided herein include serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, glutamic proteases, and metalloproteases. Exemplary proteases used herein include serine proteases separated from recombinant Bacillus strains. Exemplary proteases used herein include subtilisin and variants thereof, including subtilisin Carlsberg, alcalase, and subtilisin S41. Subtilisin and variants thereof are known to those skilled in the art and include, for example, alcalase, alcalase 0.6L, alcalase 2.5L, ALK enzyme, bacitracin A, bacitracin B, subtilisin alkaline protease bioprase, bioprase AL 15, bioprase APL 30, colistinase, subtilisin J, subtilisin S41, subtilisin Sendai, subtilisin GX, subtilisin E, subtilisin BL, genenase I, esperase, maxatase, thermostable protease PC 10, protease XXVII, thermostable enzyme, superase, subtilisin Carlsberg, subtilisin DY, subtilisin, SP 266, savinase 8.0L, savinase 4.0T, kazusase, protease VIII, opticlean, myosadsorption protein A 3L, savinase, savinase 16.0L, savinase 32.0L EX, orientase 10B, proteinase S, serine endopeptidase. In certain embodiments of the methods and compositions presented herein, thermolabile proteases such as subtilisin and thermolabile variants of subtilisin can be used, as shown by the exemplary disclosure of Davail et al., 1994, J. Biol. Chem., 26: 17448-17453, which is incorporated herein by reference in its entirety.

[0207] In some embodiments, the lysis reagent comprises one or more detergents. In some embodiments, the detergents provided herein do not interfere with downstream enzyme activity. Thus, in some embodiments, the lysis reagent comprises a non-ionic detergent. Typically, the non-ionic detergent comprises an uncharged, hydrophilic head group. Typically, the non-ionic detergent is based on polyoxyethylene or glycosides. Exemplary non-ionic detergents include 80. 20Tween, X-100, X-100-R, X-114, NP-40, C-100, X-100, CA 630, 200, 96 / 97 Triton, 98. 58. 35Brij series, L64, P84, non-detergent sulfobetaine (NDSB 201), amphipols (PMAL-C8), CHAPS, octyl β-D-pyranoglucoside, saponin, monododecyl nonaethylene glycol (C12E9, polydocanol) ether, sodium lauryl sulfate, N-lauryl sarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidinosulfobetaine-14, octylthioglucoside, maltoside, HEGA and MEGA series. In one embodiment, the lysis reagent includes the components provided in Tables 1-3.

[0208] In some embodiments, the transposition composition comprises at least one transposase and at least one transposon end component, wherein the at least one transposon end component comprises: (i) a transferred strand, the transferred strand having a 3' portion and a 5' portion, the 3' portion exhibiting a transferred transposon end sequence, the 5' portion exhibiting a sequence of a tag domain for a next generation sequencing or amplification reaction; and (ii) a non-transferred strand comprising a 5' phosphate, the non-transferred strand comprising a 5' phosphate exhibiting only a non-transferred transposon end sequence, wherein the transposase forms a complex with the transposon end component active in an in vitro transposition reaction. In some embodiments, the kit further comprises a reaction buffer, the reaction buffer comprising dimethylformamide in an amount that results in the presence of dimethylformamide in an in vitro transposition reaction at a final concentration of 10%. In some embodiments, the tag domain comprises one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, and an addressing tag domain.

[0209] In some embodiments, the transposition reaction composition comprises two or more transposon end components, each of the two or more transposon end components comprising a transferred strand that differs by at least one nucleotide.

[0210] In some embodiments, the transposase is a Tn5 transposase. In some embodiments, the transposon end component comprises a Tn5 transposon end. In one embodiment of the kit, the transposome comprises a wild-type Tn5 transposase or a hyperactive Tn5 transposase or a MuA transposase provided at a concentration of at least 250 nM in the in vitro transposition reaction. In some other embodiments, the wild-type Tn5 transposase or the hyperactive Tn5 transposase or the MuA transposase has a final concentration of at least 500 nM.

[0211] In one embodiment, the transposase in the kit is a wild-type or mutant form of Tn5 transposase (e.g., EZ-Tn5) at a concentration greater than or equal to about 5 units / μl, about 10-20 units / μl, about 20-40 units / μl, about 40-60 units / μl, about 60-80 units / μl, or about 80-100 units / μl. TM In some embodiments, the kits provided herein comprise the components provided in Table 6.

[0212] In some embodiments, the kit further comprises a modifying enzyme. In some embodiments, the modifying enzyme is a polymerase or a ligase. In some embodiments, the kit comprises at least one other enzyme component selected from the following: a DNA polymerase having 5' nuclease or strand displacement activity, a DNA polymerase lacking 5' nuclease activity, a template-dependent NAD ligase, and a template-independent ligase. In some embodiments, at least one other enzyme component is selected from the following: FAILSAFE TM DNA polymerase mix, Taq DNA polymerase, TfI DNA polymerase, T4 DNA polymerase, E. coli DNA ligase, bacteriophage TS2126 thermostable RNA ligase, Mth Rn 1 thermostable RNA ligase, and CIRCLIGASE TM Thermostable ssDNA ligase.

[0213] In some embodiments where at least one enzyme in the kit is a template-dependent ligase (e.g., E. coli DNA ligase), a high proportion of the ligase molecules are adenylated and ATP is not provided in the kit. In some embodiments where at least one enzyme in the kit is a template-dependent ligase (e.g., E. coli DNA ligase), the kit further comprises a ligation tagging oligonucleotide comprising a 3' portion and a 5' portion, wherein the 3' portion exhibits the sequence of the tag domain and the 5' portion exhibits a random sequence consisting of about 3 nucleotides to about 8 nucleotides. In some embodiments, the ligation tagging oligonucleotide comprises a 5' portion that exhibits a random sequence consisting of 4 nucleotides.

[0214] In some embodiments wherein at least one enzyme in the kit is a non-template-dependent ligase, the non-template-dependent ligase is selected from bacteriophage TS2126 thermostable RNA ligase, Mth Rn 1 thermostable RNA ligase, and CIRCLIGASE TM Thermostable ssDNA ligase, non-template-dependent ligase is provided in a highly adenylated form and no ATP is provided in the kit. In one embodiment, the kit comprises EZ-Tn5 TM Transposase and template-independent nucleic acid ligase, the EZ-Tn5 pMEDS transposon end component contains both an EZ-Tn5METS transferred strand having a 5' monophosphate group and an EZ-Tn5 pMENTS non-transferred strand having a 5' monophosphate group.

[0215] In some embodiments, the kit further comprises reagents for amplification reaction. In some embodiments, the reagents for amplification reaction are reagents for PCR. In some embodiments, the reagents for amplification reaction comprise at least one primer. In some embodiments, at least one primer comprises a 3' portion, the 3' portion exhibiting a sequence of at least a portion of the transferred strand. In some embodiments, at least one primer comprises a 5' portion, the 5' portion comprising a universal sequence.

[0216] In some embodiments, the kit includes two primers, each PCR primer comprises a 3' portion and a 5' portion, wherein the 3' portion exhibits the respective transferred transposon end sequence, and the 5' portion exhibits the sequence of the respective tag domain / adapter for a specific purpose (e.g., a sequencing tag domain or an amplification tag domain for next-generation sequencing or amplification, and optionally an addressing tag domain). In some embodiments, the 5' portion of each PCR primer is different from the 5' portion of another primer. In some embodiments, the 5' portion of the first PCR primer or the 5' portion of the second PCR primer, or the 5' portion of both the first PCR primer and the second PCR primer comprises a first sequencing tag / adapter or a second sequencing tag / adapter, respectively. In one embodiment, the kit provided herein comprises the components provided in Table 7.

[0217] In some embodiments, the kit further comprises a size selection agent. In some embodiments, the size selection agent comprises AMPure XP beads (from Beckman Coulter Genomics). The nucleic acid fragments can be bound to solid phase reversible immobilization (SPRI) beads. In some embodiments, the size selection agent further comprises PEG and NaCl.

[0218] In some embodiments, the kit provided herein also includes a library standardization reagent. In some embodiments, the library standardization reagent includes a library standardization additive (Library Normalization Additives) provided by Illumina, Inc (San Diego, CA, product number 1502511) and a library standardization bead (Library Normalization Beads) provided by Illumina, Inc (product number 15022566). In some embodiments, the library standardization reagent also includes a library standardization wash (Library Normalization Wash) provided by Illumina, Inc (product number 15022565). In some embodiments, the library standardization reagent also includes a library standardization storage buffer provided by Illumina, Inc (San Diego, CA, product number 1502511).

[0219] In some embodiments, the kit also includes a device with a solid surface. In some embodiments, the solid surface is attached to a colony of oligonucleotides. In some embodiments, the device is a flow cell device. In some embodiments, the solid surface includes a patterned surface suitable for fixing molecules in an orderly pattern.

[0220] From the foregoing description it will be apparent that the invention described herein may be modified and adapted to various usages and conditions.Such embodiments are also within the scope of the following claims.

[0221] The reference to a list of elements in the definition of any variable herein includes the definition of that variable as any single element or combination (or subcombination) of the listed elements. The reference to an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0222] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

[0223] The following examples are offered by way of illustration and not by way of limitation.

[0224] Example

[0225] Example 1 Generation of cell lysate containing target nucleic acid

[0226] In some embodiments, during the step of generating cell lysate, the cell membrane is destroyed by a detergent, during which protein-lipid association and lipid-lipid association are destroyed, and thereby releases intracellular substances in soluble form. The main function of a broad-spectrum specific protease is to remove DNA binding proteins, such as histones from DNA, to allow transposase to uniformly approach DNA. In some embodiments, as illustrated in this embodiment, a detergent and a protease are provided in a single lysis reagent mixture. The mixture is directly applied to cells for generating a cell lysate comprising a target nucleic acid. As discussed above, in some embodiments, when heat is used to inactivate the protease, it is important that the heat does not denature the double-stranded nucleic acid and ensure that the labeling step is not disturbed.

[0227] In this example, the protease can be heat inactivated at 70°C, and at this temperature, the double-stranded conformation of the DNA is maintained. The protocol for generating a cell lysate containing a target nucleic acid is exemplified in Example 1 as follows:

[0228] (1) Mix the reagents thoroughly by gently inverting and flicking the tube 3-5 times, then spin briefly in a microcentrifuge.

[0229] (2) In a clean microcentrifuge tube, mix the components in Table 1 to prepare a 5X lysis master mix. The lysis master mix can be scaled up proportionally based on the number of samples, for example, an additional 10% can be included to compensate for losses during pipetting.

[0230] Table 1 Components of the total pyrolysis mixture

[0231] Components of the total lysis mixture Volume (μl) 5X Lysis Buffer 1.1 5X Protease Stock Solution 1.1 total 2.2

[0232] The 5X lysis buffer in Table 1 above can be prepared according to Table 2 below:

[0233] Table 2 Components of 5X Lysis Buffer

[0234]

[0235] All reagents can be mixed thoroughly by gently vortexing the tube several times, followed by a brief spin in a microcentrifuge. This step can be repeated 3-5 times. The 5X Lysis Buffer can be stored at room temperature to prevent detergent precipitation.

[0236] 5X protease stock solution can be prepared as follows: (1) prepare a single-use stock aliquot by resuspending a protease, such as QIAGEN protease, directly in a glass vial by adding 2.38 ml Super Q H2O to a final concentration of 3150 mAU / ml. Ensure that the protease is fully dissolved by gently vortexing the vial several times. Aliquot the solution into 25 μl aliquots and immediately freeze at -80°C, and (ii) remove the single-use stock aliquot from the freezer and thaw, and prepare a 5X protease stock solution according to Table 3 below:

[0237] Table 3 Composition of 5X protease storage solution

[0238]

[0239] Accordingly, the final concentration of the 5X protease stock solution was 450 mAU / ml.

[0240] (3) Add 2 μl of the total lysis mixture prepared above to each tube containing cells, positive control genomic DNA, or negative control. Incubate the samples in a thermal cycler according to the following program: 50°C for 30 min, 70°C for 20 min, and 4°C hold.

[0241] In some embodiments, positive control genomic DNA (about 30 pg) is included in each experiment. Positive control genomic DNA can be prepared in two-step serial dilutions from a 10 ng / μl stock solution as prepared in Tables 4 and 5 below:

[0242] Table 4 Composition of intermediate genomic DNA dilutions

[0243] Components Storage concentration Intermediate concentration Volume (μl) DNA 10ng / μl 100pg / μl 2 1x RS1 198 total 200

[0244] The intermediate DNA dilutions prepared according to the table above can then be subsequently diluted according to the following Table 5:

[0245] Table 5 Composition of the final genomic DNA dilution

[0246] Components Storage concentration Intermediate concentration Volume (μl) DNA 100pg / μl 10pg / μl 10 1X PBS 90 total 100

[0247] 3 μl of the final dilution prepared in the above table can be used as input for the positive control genomic DNA. This corresponds to 30 pg or the genome equivalent of 5 cells. More or less genomic DNA can also be used according to the methods provided herein.

[0248] Example 2 Direct fragmentation of target nucleic acid in cell lysate

[0249] In some embodiments, genomic DNA in a cell lysate (e.g., as prepared in Example 1) can be tagged (labeled and fragmented) by Nextera transposomes (available from Illumina, Inc, San Diego, CA). Nextera transposomes can simultaneously fragment the input DNA and add tag / adapter sequences to the ends. The tagging master mix can be added directly to the cell lysate prepared in Example 1 without any prior DNA purification or amplification steps. The tagging master mix can be prepared as shown in Table 6 below, and the master mix can be expanded according to the number of samples, for example, by an additional 10% to compensate for losses during pipetting.

[0250] Table 6 Components of the labelled total mixture

[0251] Components Volume (μl) DNA labeling buffer 11 Nextera Amplicon Tagmentation Mix 2.2 <![CDATA[Super Q H2O]]> 3.3 total 16.5

[0252] The tagging DNA buffer and Nextera amplicon tagging mixture are available from Illumina, Inc (San Diego, CA; Product Nos. 15027866 and 15031561). The tagging DNA buffer contains Tris(hydroxymethyl)aminomethane, MgCl2, and dimethylformamide. The Nextera amplicon tagging mixture contains transposase. 15 μl of the total tagging mixture can then be added to each cell lysate generated from, for example, Example 1, and incubated with the cell lysate at 55°C for 5 min, and then the reaction is terminated at 4°C. A neutralizing tagging buffer containing SDS (available from Illumina, Inc, San Diego, CA) can then be added to the tube and incubated at room temperature for 5 minutes.

[0253] Example 3 Limited-Cycle PCR Amplification

[0254] The tagged DNA fragments (e.g., as prepared in Example 2) can be amplified by a limited cycle PCR procedure. This PCR step can also add other sequences at both ends of the tagged nucleic acid fragments, such as index 1 (i7) and index 2 (i5) (available from Illumina, Inc, San Diego, CA) and sequences required for other purposes such as cluster formation. For example, the components in Table 7 below (available from Illumina, Inc, San Diego, CA) can be added to the neutralized tagging product generated from Example 2.

[0255] Table 7 Components used for limited cycle PCR

[0256] Components Volume (μl) PCR master mix 15 Index 1 Primer (P5 Primer) 5 Index 2 Primer (P7 Primer) 5

[0257] The PCR master mix in Table 7 can be prepared as shown in Table 8 below:

[0258] Table 8 Components of the PCR Master Mixture

[0259] Components Storage concentration Total mixture concentration Volume (μl) KAPA HiFi Fidelity Buffer 5X 3.33X 999 dNTP Mix 25mM each 1.00mM each 59.94 KAPA HiFi DNA Polymerase 1U / μl 0.033U / μl 49.95 <![CDATA[Super Q H2O]]> 11.11 total 1500

[0260] An exemplary PCR program is as follows: 72°C for 3 min, 98°C for 30 sec, and then 20 cycles of 98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec, and finally holding the sample at 4°C.

[0261] Example 4 Protease activity is useful for uniform access to DNA

[0262] In this example, the effect of protease activity on uniform access to DNA was analyzed. In particular, whole cells and nuclei were treated with 0 mg / ml, 0.1 mg / ml (4.5 mAU / ml), 0.5 mg / ml (22.5 mAU / ml) or 2.5 mg / ml (112.5 mAU / ml) protease. The percentage of uniquely mapped reads was analyzed for each sequencing. Figure 1 It is a histogram showing the percentage of uniquely mapped reads in sequencing of whole cells or nuclei treated with 0 mg / ml, 0.1 mg / ml, 0.5 mg / ml or 2.5 mg / ml protease. As shown, the uniquely mapped reads increase with increasing protease concentrations, and this is true using both whole cells and nuclei as starting materials. It is also noted that the percentage of uniquely mapped reads using 0.5 mg / ml protease is similar to the percentage of uniquely mapped reads using 2.5 mg / ml.

[0263] The effect of protease activity on uniform access to DNA was also analyzed by comparing count and copy number analysis results between using a total genomic DNA control with Nextera XT library preparations, using single cells with sufficient protease activity, and using single cells with insufficient protease activity. Figure 2 Bar graphs showing the results of count and copy number analysis using total DNA, single cells treated with sufficient protease activity, and single cells treated with insufficient protease activity are shown. As shown, when relatively large amounts of genomic DNA are used with the current Nextera XT library preparation method, Figure 2 As shown in the figure above, relatively clear copy number analysis results can be obtained with insignificant noise. When only a single cell is used for sequencing, the noise is significant and the copy number analysis data show a discrete distribution pattern, such as Figure 2Unexpectedly, when single cells were treated with sufficient protease (0.5 mg / ml), the copy number analysis results were restored to be comparable to those using total genomic DNA, showing clear data with insignificant noise, as shown in Figure 2. Figure 2 This suggests that the protease increases the accessibility of the genomic DNA to the transposase, as the DNA-binding proteins are uniformly removed.

[0264] These results show that protease activity is useful for uniform access to DNA during sequencing.

[0265] Example 5 Optimizing protease concentration

[0266] In this example, the concentration of the protease used in the present method was analyzed. Figure 3A A bar graph showing the results of copy number analysis in single cells treated with 0.5 mg / ml active protease, 2 mg / ml active protease or 2 mg / ml HI protease is shown. As shown, when single cells were treated with 0.5 mg / ml or 2 mg / ml active protease, similarly clear copy number analysis results were obtained, such as Figure 3A In contrast, when the reaction was performed with the protease pre-heat-inactivated at 70°C, no clear copy number results were obtained, as shown in the upper two bar graphs of . Figure 3A The results show that both 0.5 mg / ml and 2 mg / ml protease concentrations are effective and sufficient.

[0267] The percentage of uniquely mapped reads was also analyzed in sequencing of single cells treated with 0.5 mg / ml active protease, 1 mg / ml active protease, 2 mg / ml active protease, or 2 mg / ml pre-heat-inactivated (70°C) protease. Figure 3B Shown in the sequencing of single cells treated with 0.5mg / ml active protease, 1mg / ml active protease, 2mg / ml active protease, or 2mg / ml pre-heat-inactivated protease and a control sample without cells, a bar graph of the percentage of uniquely mapped reads. As shown, the percentage of uniquely mapped reads in the sequencing of single cells treated with 0.5mg / ml active protease, 1mg / ml active protease, and 2mg / ml active protease is all about 65%, with small variations. In contrast, when the protease is inactivated at 70°C, even with a higher amount of protease, the percentage of uniquely mapped reads is much lower, with huge variations.

[0268] Additionally, noise in the copy number data was analyzed by analyzing the count differences between neighboring bin counts. Figure 3CA histogram of read count differences between neighboring bins (interquartile range of read count differences between neighboring bins) in sequencing of single cells treated with 0.5 mg / ml active protease, 1 mg / ml active protease, 2 mg / ml active protease, or 2 mg / ml pre-heat-inactivated protease and a cell-free control sample is shown. As shown, in sequencing using single cells treated with 0.5 mg / ml active protease, 1 mg / ml active protease, and 2 mg / ml active protease, the count differences between neighboring bin counts were all relatively small (about 20%) with small variations. In contrast, when the protease was inactivated at 70°C, the count differences between neighboring bin counts were much larger with huge variations, even with a higher amount of protease (2 mg / ml).

[0269] Taken together, these results show that proteases having a concentration range from 0.5 mg / ml to 2.0 mg / ml (22.5 mAU / ml to 90 mAU / ml) are sufficient and effective in the methods provided herein.

[0270] Example 6 Optimizing pH conditions for protease digestion reactions

[0271] In this example, the pH conditions of the protease digestion reaction were optimized to balance protease activity and sequencing results.

[0272] The protease activity was analyzed under different pH conditions. The results are shown in Figure 4A middle. Figure 4A Is a bar graph showing the relative activity of the protease at pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0 or pH 10.0 (relative to the protease activity at pH 8.0). As shown, the activity of the protease increases with increasing pH, with the protease having the lowest activity at pH 7.0 and the highest activity at pH 10.0.

[0273] Then, the percentage of uniquely mapped reads was analyzed under different pH conditions. Figure 4B A bar graph showing the percentage of uniquely mapped reads in sequencing of single cells treated with proteases at pH 7.0, pH 8.0, pH 9.0, or pH 10.0 is shown. As shown, approximately 70% of clear uniquely mapped reads were obtained when the pH was 7, 8, or 9. However, when the pH was 10, a smaller percentage of uniquely mapped reads was obtained, and the data variation increased significantly.

[0274] Noise in the copy number data was also analyzed by comparing count differences between adjacent bins. Figure 4CShown are histograms of read count differences between adjacent bins (interquartile range of read count differences between adjacent bins) in sequencing of single cells treated with 0.5 mg / ml protease at pH 7.0, pH 8.0, pH 9.0, or pH 10.0. As shown, consistent with the uniquely mapped read results, the count differences between adjacent bins are relatively small (about 20%) with small variations, whereas at pH 10.0, the count differences between adjacent bins increase significantly with large variations.

[0275] In some embodiments, the pH of the digestion reaction is between pH 7.0 and pH 9.0.

[0276] Example 7 Testing for thermal inactivation of proteases

[0277] In some embodiments, the protease provided herein can be heat-inactivated. As discussed above, in preparative embodiments, the protease can be inactivated at a relatively low temperature (e.g., 70° C.) so that the double-stranded DNA conformation can be maintained for labeling reactions. In this embodiment, the protease (from QIAGEN) is analyzed for heat inactivation and its impact on sequencing results.

[0278] The protease was preheated at different temperatures and the activity of the protease was tested. The results are shown in Figure 5A , which shows a bar graph of relative protease activity when preheated at room temperature, 50° C., 60° C. or 70° C. As shown, the protease activity gradually decreases with increasing temperature and is completely inactivated at 70° C. This result is consistent with the results shown in Example 5 above.

[0279] The percentage of uniquely mapped reads in sequencing of single cells, triplicate cells, and 15 pg of genomic DNA at different temperatures was analyzed. Figure 5B Shown is a bar graph of the percentage of uniquely mapped reads in sequencing of single cells, three cells, or 15 pg of genomic DNA treated with 2.0 mg / ml protease at room temperature, 50°C, 60°C, or 70°C. As shown, the percentage of uniquely mapped reads decreases as temperature increases. However, because a relatively high protease concentration (2.0 mg / ml) is used in this embodiment, the protease activity reduced at 70°C is more tolerable. Therefore, the percentage of uniquely mapped reads at 70°C is still relatively high, although it is lower than the percentage of uniquely mapped reads processed at lower temperatures.

[0280] The count differences between adjacent bins in the sequencing of single cells, three cells, and 15 pg of genomic DNA at different temperatures were also analyzed. Figure 5CShown are histograms of read count differences between adjacent bins (interquartile range of read count differences between adjacent bins) in sequencing of single cells, three cells, or 15 pg of genomic DNA treated with 2 mg / ml protease at room temperature, 50° C., 60° C., or 70° C. As shown, at lower temperatures (e.g., at room temperature and 50-60° C.), the count differences between adjacent bins are relatively small with small variations, whereas at 70° C., the count differences between adjacent bins increase significantly with larger variations.

[0281] Example 8 Library diversity increases with smaller insert size

[0282] In single-cell sequencing, there are only two copies of the genome, and therefore smaller insert sizes tend to increase library diversity. Fig. 6A As shown in , counts increase as insert size decreases, and thus the diversity represented by the library increases as insert size decreases. Thus, in some embodiments, the methods herein use a higher amount of transposase in the tagmentation step to increase fragmentation and reduce the insert size of the tagged nucleic acid fragments. Figure 6B The insert sizes of libraries treated with 1 μl Tn5 or 2 μl Tn5 are shown. As shown, when 1 μl Tn5 was used in the tagmentation reaction, the average fragment size was about 550 bp; while when 2 μl Tn5 was used in the tagmentation reaction, the average fragment size was about 400 bp. Consistent with the smaller insert size, the diversity of the library when treated with 2 μl Tn5 increased compared to the diversity of the library treated with 1 μl Tn5, as shown in FIG. Figure 6C Displayed in.

[0283] Example 9 Optimization of PCR Cycles

[0284] In sequencing using a minimal cell population, the input DNA is relatively small, and therefore the number of cycles of PCR can be adjusted to achieve better sequencing results. In this example, the number of cycles of PCR was tested and optimized using a single cell as the starting material. Figure 7 Shown are histograms of count and copy number analysis results in single cell sequencing using PCR with 16 cycles, 18 cycles, or 20 cycles according to the methods provided herein. As shown, when PCR with 16 cycles is used, the noise is large, and when PCR with 18 cycles or 20 cycles is used, the noise is significantly reduced.

[0285] Example 10 Read distribution using one, three or five cells

[0286] In this example, the distribution of reads using one, three, or five cells was analyzed. Fig. 8AThe distribution of reads from three single-cell sequencing runs is shown. As shown, the read regions do not completely overlap between the three single-cell sequencing runs. Therefore, increasing the number of cells can help broaden the coverage. Figure 8B The read distribution of single-cell sequencing, three-cell sequencing, or five-cell sequencing is shown. As shown, genome coverage increases with the number of cells. Figure 8C A histogram of the average library diversity and estimated genome coverage using a single cell, three cells, or five cells is shown. As shown, it is estimated that one cell can cover about 40% of the genome, and three cells can cover more than 50% of the genome, and five cells can cover about 60% of the genome. The average library counts using one cell, three cells, and five cells are about 5 million, 15 million, and 20 million, respectively.

[0287] Fig.8D The overall success rate is shown. As shown, when more than one cell was used, the overall success rate was 99% (N=81). When a single cell was used, the overall success rate was still relatively high at 94% (N=187).

[0288] Example 11 Comparison of count and copy number data between different library preparation methods

[0289] In this example, the methods provided herein are compared with some current single cell preparation methods.

[0290] Fig. 9A Copy number analysis using REPLIg Single Cell (MDA) and Nexteral XT library preparation is shown. The REPLI-g single cell kit developed by QIAGEN is specifically designed to amplify genomic DNA from single cells (1 to <1000 cells) or purified genomic DNA with genome coverage. The REPLI-g single cell kit developed by QIAGEN utilizes multiple displacement amplification (MDA) technology. See Spits et al., 2006, Whole-genome multiple displacement amplification from single cells, Nature protocols 1(4): 1965-70. However, due to the over-amplification bias introduced by MDA, the copy number variation data is noisy when derived from a single cell, three cells, or five cells, such as Fig. 9A Displayed in.

[0291] Fig. 9BCopy number analysis using SurePlex (PicoPlex) and Nexteral XT library preparation is shown. The SurePlex amplification system developed by Illumina, Inc (San Diego, CA) is a solution for extracting and amplifying DNA from a single cell or very few single cells. As shown, the SurePlex amplification system significantly reduces noise compared to MDA.

[0292] Fig. 9C Shown is a copy number analysis using the methods provided herein (Nextera SC). As shown, the noise is further reduced compared to using the SurePlex amplification system.

[0293] Example 12 Detection of mosaic phenomenon

[0294] In this embodiment, the detection of mosaicism using the method provided herein is demonstrated. Fig. 10A Data from a single GM50121 cell copy number analysis of chromosome 18 are shown. Copy number data from three single-cell sequencing runs are shown. In each single-cell sequencing run, a population representing 15.4 MB of DNA was detected. Fig. 10B Shown are count number data using single GM20916 cells. As shown, arrows indicate counts resulting from mosaicism. Fig. 10C Using a single GM20916 cell, the copy number analysis data for chromosome 15, chromosome X, and chromosome 10 are shown. The copy number data for each chromosome analyzed detected an additional population representing another chromosome. Similarly, Fig. 10D Using a single GM1021 cell, data for chromosome 1 and chromosome 11 copy number analysis are shown. As shown in these figures, Fig. 10D The copy number data for each chromosome analyzed in also detected an additional population representing another chromosome.

[0295] A number of embodiments have been described. However, it will be appreciated that various changes may be made. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method for preparing a library of tagged nucleic acid fragments, the method comprising: (a) directly contacting a cell population with a lysis reagent to generate a cell lysate, the cell population comprising at least two cells and less than 15 cells, wherein the lysis reagent has one or more proteases, and wherein the cell lysate comprises a target nucleic acid; (b) inactivating the one or more proteases to form an inactivated cell lysate, and (c) applying at least one transposase and at least one transposon end component comprising the transferred strand directly to the inactivated cell lysate under conditions wherein the target nucleic acid and the transposon end component undergo a transposition reaction to generate a hybrid, wherein: (i) the target nucleic acid is fragmented to generate a plurality of target nucleic acid fragments, (ii) the transferred strand of the transposon end element is ligated to the 5′ end of each of the plurality of target nucleic acid fragments to generate a plurality of 5′-tagged target nucleic acid fragments, (iii) the target nucleic acid is double-stranded DNA, and (iv) the target nucleic acid is still double-stranded DNA before applying the transposase and the transposon end components in step (c), wherein steps (a), (b) and (c) are performed in a single reaction tube and there is no DNA purification and amplification step between steps (a) and (c).

2. The method of claim 1, wherein the cell population comprises two, three, four or five cells.

3. The method of claim 1, wherein the one or more proteases are subtilisin and variants thereof. The method according to claim 1 , wherein the concentration of the one or more proteases in the cell lysate is 4.5 mAU / ml to 500 mAU / ml. The method according to claim 4 , wherein the concentration of the one or more proteases in the cell lysate is 22.5 mAU / ml.

6. The method of claim 1, wherein in step (a) the cell population is contacted with the lysis reagent at pH 7.0 to pH 10.

0.

7. The method of claim 6, wherein the cell population is contacted with the lysis reagent at pH 7.0 to pH 9.

0.

8. The method of claim 1, wherein in step (b) the one or more proteases are inactivated by increasing the temperature.

9. The method of claim 8, wherein the one or more proteases are inactivated by increasing the temperature to 50°C-80°C.

10. The method of claim 9, wherein the one or more proteases are inactivated by increasing the temperature to 70°C.

11. The method of claim 1, wherein the one or more proteases are inactivated by adding one or more inhibitors of the one or more proteases.

12. The method of claim 1, wherein the lysis reagent comprises one or more detergents.

13. The method of claim 12, wherein the one or more detergents are non-ionic detergents.

14. The method of claim 12, wherein the one or more detergents comprises Triton.

15. The method of claim 1, wherein the target nucleic acid is genomic DNA.

16. The method of claim 1, wherein the target nucleic acid comprises chromosomal DNA or a fragment thereof.

17. The method of claim 1, wherein the target nucleic acid comprises a genome or a portion of a genome.

18. The method of claim 1, wherein the at least one transposase is Tn5 transposase.

19. The method of claim 1, wherein the at least one transposon end element comprises a Tn5 transposon end.

20. The method of claim 1, wherein the transferred strand comprises a tag domain comprising one or more of a restriction site domain, a capture tag domain, a sequencing tag domain, an amplification tag domain, a detection tag domain, and an addressing tag domain.

21. The method according to claim 1, further comprising: (d) incubating the mixture from step (c) directly with at least one nucleic acid modifying enzyme under conditions wherein the 3′ tag is ligated to the 5′-tagged target nucleic acid fragment to generate a plurality of di-tagged target nucleic acid fragments.

22. The method of claim 21, wherein steps (a), (b), (c) and (d) are performed in a single reaction tube.

23. The method of claim 21, wherein the at least one nucleic acid modification enzyme is a polymerase, and wherein the 3' tag is formed by extension of the 3' end of the 5'-tagged target nucleic acid fragment. 24 . The method of claim 21 , wherein the at least one nucleic acid modification enzyme is a ligase, and wherein the 3′ tag is formed by ligating an oligonucleotide to the 3′ end of the 5′-tagged target nucleic acid fragment.

25. The method according to claim 21, further comprising: (e) amplifying one or more di-tagged target nucleic acid fragments to generate a library of tagged nucleic acid fragments having additional sequences at the 5′ end and / or the 3′ end of the di-tagged target nucleic acid fragments.

26. The method of claim 25, wherein steps (a), (b), (c), (d) and (e) are performed in a single reaction tube.

27. The method of claim 25, wherein the amplifying comprises utilizing one or more of polymerase chain reaction (PCR), strand displacement amplification reaction, rolling circle amplification reaction, ligase chain reaction, transcription-mediated amplification reaction, or loop-mediated amplification reaction.

28. The method of claim 25, wherein the amplifying comprises polymerase chain reaction using a single primer that is complementary to the 3' tag of the di-tagged target nucleic acid fragments.

29. The method of claim 25, wherein the amplifying comprises a polymerase chain reaction using a first primer and a second primer, wherein at least the 3′ terminal portion of the first primer is complementary to at least a portion of the 3′ tag of the di-tagged target nucleic acid fragments, and wherein at least the 3′ terminal portion of the second primer exhibits the sequence of at least a portion of the 5′ tag of the di-tagged target nucleic acid fragments.

30. The method of claim 29, wherein the 5′ terminal portion of the first primer is not complementary to the 3′ tag of the di-tagged target nucleic acid fragments, and wherein the 5′ terminal portion of the second primer does not exhibit the sequence of at least a portion of the 5′ tag of the di-tagged target nucleic acid fragments.

31. The method of any one of claims 29-30, wherein the first primer comprises a first universal sequence, and / or wherein the second primer comprises a second universal sequence.

32. The method of claim 1, further comprising sequencing the tagged nucleic acid fragments.

33. The method of claim 32, wherein the sequencing of the tagged nucleic acid fragments comprises using one or more of sequencing by synthesis, bridge polymerase chain reaction, chain termination sequencing, sequencing by hybridization, nanopore sequencing, and sequencing by ligation.

34. The method of claim 32, wherein the sequencing of the tagged nucleic acid fragments comprises using next generation sequencing.

35. The method of claim 32, further comprising analyzing copy number variation.

36. The method of claim 32, further comprising analyzing single nucleotide variations.

37. The method of claim 35, wherein the analyzing copy number variation is used to detect mosaicism.

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