Improved library preparation methods

Abstract

Disclosed herein are modified transposon end sequences, including mosaic end sequences, where the mosaic end sequences include one or more mutations compared to a wild-type mosaic end sequence, where the mutations include substitutions with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, or modified pyrimidine. Also disclosed are transposome complexes that include these modified transposon end sequences, and library preparation methods that use these modified transposon end sequences.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 167,150, filed March 29, 2021, and U.S. Provisional Patent Application No. 63 / 224,201, filed July 21, 2021, each of which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application has been filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled "2022-03-25_01243-0027-00PCT_Sequence_Listing_ST25.txt" (created March 25, 2022, size 5,634 bytes). The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] explanation The present disclosure relates to modified transposon end sequences, including mosaic end sequences, where the mosaic end sequences include one or more mutations compared to a wild-type mosaic end sequence, where the mutations include substitutions with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, or modified pyrimidine. The present disclosure also relates to transposome complexes that include these modified transposon end sequences, and to library preparation methods that use these modified transposon end sequences. [Background technology]

[0004] NGS requires fragmentation of DNA samples, but current methods are limited to (A) mechanical approaches that require expensive capital equipment, (B) enzymatic strategies whose performance varies based on sample concentration and time, and (C) tagmentation-based approaches that impose restrictions on library adapter structure.

[0005] The first step in preparing libraries for NGS is DNA fragmentation, which produces DNA fragments with a size distribution centered around an optimal length, typically in the range of several hundred base pairs. There are various methods for DNA fragmentation that can be classified as either mechanical or enzymatic. Mechanical methods include sonication, acoustic shearing, and nebulization (see Maria S. Poptsova et al., Scientific Reports 4 (2014)). All of these mechanical methods require specialized capital equipment and have the potential to introduce DNA damage. In contrast, enzymes do not require specialized equipment, reducing the user's initial costs. For this reason, users may prefer library preparation products that rely on enzymatic fragmentation.

[0006] Beyond transposases, such as those included in some Illumina library preparation products, alternative classes of enzymes that can be used for DNA fragmentation include restriction enzymes and nicking enzymes. Restriction enzymes recognize and cleave specific sites, which results in fragmentation bias and are therefore not commonly used for NGS applications. In contrast, nicking enzymes introduce random single-strand breaks into DNA substrates. An example of a product that allows enzymatic fragmentation based on nicking enzymes is NEBNext Fragmentase. In this product, one enzyme generates random nicks in the substrate DNA and a separate enzyme cleaves the complementary strand, resulting in DNA fragmentation. An exemplary protocol that uses this method is NEBNext dsDNA Fragmentase (see NEBNext® for DNA Sample Prep for the Illumina Platform, NEB, 2019).

[0007] Because NEB Fragmentase fragments DNA without adding adapter sequences, this workflow is compatible with a variety of existing ligation-based library preparation workflows, including PCR-free approaches. However, these fragmentation enzymes can turn over several times, making fragmentation time- and concentration-dependent, and therefore optimizing the reaction for the user's specific sample type is often necessary to achieve an appropriate fragment size distribution (see Joseph P. Dunham and Maren L. Friesen, Cold Spring Harbor Protocols 9:820-34 (2013)). In contrast, transposase-mediated fragmentation is limited to one turnover based on its dependency on preloaded transposon substrates, but transposase-mediated fragmentation requires the introduction of mosaic end sequences into DNA fragments.

[0008] In summary, enzymatic fragmentation methods are preferred by many users because they do not require specialized equipment and are more suitable for high-throughput applications. However, current enzymatic fragmentation methods do not have the advantages of BLT, such as DNA quantification and library normalization by BLT, and therefore BLT-based methods are distinguished from methods that use fragmentation enzymes.

[0009] A key requirement for transposition of the transposon Tn5 are the "mosaic ends" (MEs) that are specifically recognized by the Tn5 transposase and are required for its transposition activity. Tn5 transposase naturally recognizes "outside end" (OE) and "inside end" (IE) sequences, which have been shown to be highly intolerant of mutations, with most mutations resulting in reduced activity. Later studies demonstrated that chimeric sequences derived from IEs and OEs, termed "mosaic ends" (MEs) along with mutant Tn5 enzymes, increase transposition activity by approximately 100-fold compared to the native system. This highly active system forms the basis of the Illumina DNA Flex PCR-Free (for research use only, RUO) technology, formerly known as Illumina's Nextera technology. The crystal structure of Tn5 transposase in complex with a DNA substrate shows that 13 of the 19 base pairs have nucleobase-specific crystal contacts, although other bases have been shown to play a role in catalysis.

[0010] Tn5 transposase and bead-linked transposomes (BLT) are powerful tools that mediate simultaneous enzymatic DNA fragmentation and adapter ligation, or tagmentation, for NGS library preparation. The tagmentation process eliminates the need for mechanical or enzymatic fragmentation of sample DNA, enzymatic end repair, and adapter ligation, resulting in a facile library preparation method. However, a limitation of these systems is the requirement that single-stranded 19-nucleotide mosaic end sequences be incorporated adjacent to the 5' end of the library insert. While this can be easily exploited for standard library preparation, the formation of libraries with additional features, such as forked adapters, barcodes, and unique molecular identifiers (UMIs), while retaining compatibility with standard sequencing methods, is challenging.

[0011] The fragmentase BLT (fBLT) technology described herein overcomes these technical challenges by leveraging the inherent advantages of BLT, while also eliminating the constraints of previous tagmentation approaches that require defined 19-base pair sequences flanking the library insert. Decoupling the enzymatic fragmentation and adapter tagmentation steps can allow for the addition of features such as forked adapters, barcodes, and UMIs while retaining compatibility with standard sequencing methods. Based on these inherent advantages, fBLT can be used for a variety of applications, such as UMI library preparation and PCR-free library preparation. The modified transposon end sequences disclosed herein can eliminate the constraint of requiring 19-bp mosaic end sequences flanking the library insert, enabling a hybrid Tn5 ligation library preparation approach, thus allowing the utilization of BLT in library preparation workflows developed based on ligation chemistry. The present disclosure describes that Tn5 can tolerate several mutations and nucleobase modifications within the mosaic end substrate. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Maria S. Poptsova et al.,Scientific Reports 4(2014) [Non-Patent Document 2] Joseph P. Dunham and Maren L. Friesen,Cold Spring Harbor Protocols 9:820-34(2013) Summary of the Invention [Means for solving the problem]

[0013] According to the present specification, the library preparation method can include transposition by bead-linked transposomes (BLT), cleavage of modified mosaic end sequences contained in the transposon ends, and adapter ligation.

[0014] Embodiment 1. A modified transposon end sequence comprising a mosaic end sequence, the mosaic end sequence comprising one or more mutations compared to a wild-type mosaic end sequence, the mutations being: Uracil, B. Inosine, C. ribose, d. 8-oxoguanine, e. Thymine glycol, f. modified purine, or g. Modified transposon end sequences, including modified pyrimidine substitutions.

[0015] Embodiment 2. The modified transposon end sequence of embodiment 1, wherein the wild-type mosaic end sequence comprises SEQ ID NO:1, and further wherein the one or more mutations comprise substitutions at A16, C17, A18, and / or G19.

[0016] Embodiment 3. The modified transposon end sequence of embodiments 1 and 2, wherein the mosaic end sequence comprises eight or fewer mutations compared to the wild-type sequence.

[0017] Embodiment 4. The modified transposon end sequence of embodiment 2, wherein the mosaic end sequence comprises one or more mutations compared to SEQ ID NO:1, in addition to one or more mutations at A16, C17, A18, and / or G19.

[0018] Embodiment 5. The modified transposon end sequence of embodiment 2, wherein the mosaic end sequence comprises one or more mutations at A16, C17, A18, and / or G19, as well as one to four substitution mutations compared to SEQ ID NO:1.

[0019] Embodiment 6. The modified transposon end of embodiment 2, wherein the mosaic end sequence has one or more mutations at A16, C17, A18, and / or G19, as well as one substitution mutation compared to SEQ ID NO:1.

[0020] Embodiment 7. The modified transposon end of embodiment 2, wherein the mosaic end sequence has two substitution mutations compared to SEQ ID NO:1, in addition to one or more mutations at A16, C17, A18, and / or G19.

[0021] Embodiment 8. The modified transposon end of embodiment 2, wherein the mosaic end sequence has three substitution mutations compared to SEQ ID NO:1, in addition to one or more mutations at A16, C17, A18, and / or G19.

[0022] Embodiment 9. The modified transposon end of embodiment 2, wherein the mosaic end sequence has four substitution mutations compared to SEQ ID NO:1, in addition to one or more mutations at A16, C17, A18, and / or G19.

[0023] Embodiment 10. the substitution at A16 is A16T, A16C, A16G, A16U, A16 inosine, A16 ribose, A16-8-oxoguanine, A16 thymine glycol, A16 modified purine, or A16 modified pyrimidine; b. the substitution at C17 is C17T, C17A, C17G, C17U, C17 inosine, C17 ribose, C17-8-oxoguanine, C17 thymine glycol, C17 modified purine, or C17 modified pyrimidine; c. the substitution at A18 is A18G, A18T, A18C, A18U, A18 inosine, A18 ribose, A18-8-oxoguanine, A18 thymine glycol, A18 modified purine, or A18 modified pyrimidine; and / or d. The modified transposon end sequence of any one of embodiments 2-9, wherein the substitution at G19 is G19T, G19C, G19A, G19U, G19 inosine, G19 ribose, G19-8-oxoguanine, G19 thymine glycol, G19 modified purine, or G19 modified pyrimidine.

[0024] Embodiment 11. The mutation is: Uracil, B. Inosine, C. ribose, d. 8-oxoguanine, e. Thymine glycol, f. modified purines, and / or g. The modified transposon end sequence of any one of embodiments 2-9, comprising a substitution with a modified pyrimidine.

[0025] Embodiment 12. The modified transposon end sequence of any one of embodiments 2 to 11, comprising a mutation in A16, C17, A18, or G19.

[0026] Embodiment 13. The modified transposon end sequence of any one of embodiments 2 to 11, comprising two mutations selected from mutations in A16, C17, A18, or G19.

[0027] Embodiment 14. The modified transposon end sequence of any one of embodiments 2 to 11, comprising three mutations selected from mutations in A16, C17, A18, or G19.

[0028] Embodiment 15. The modified transposon end sequence of any one of embodiments 2 to 11, comprising four mutations at A16, C17, A18, and G19.

[0029] Embodiment 16. The modified transposon end of any one of embodiments 2 to 11, wherein the modified transposon end sequence has one to four substitution mutations at A16, C17, A18, and / or G19 compared to SEQ ID NO:1.

[0030] Embodiment 17. The modified transposon end of any one of embodiments 1 to 11, wherein the modified transposon end sequence has one substitution mutation compared to the wild-type sequence.

[0031] Embodiment 18. The modified transposon end of any one of embodiments 1 to 11, wherein the modified transposon end sequence has two substitution mutations compared to the wild-type sequence.

[0032] Embodiment 19. The modified transposon end of any one of embodiments 1 to 11, wherein the modified transposon end sequence has three substitution mutations compared to the wild-type sequence.

[0033] Embodiment 20. The modified transposon end of any one of embodiments 1 to 11, wherein the modified transposon end sequence has four substitution mutations compared to the wild-type sequence.

[0034] Embodiment 21. The modified transposon end of any one of embodiments 1 to 20, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0035] Embodiment 22. The modified transposon end of any one of embodiments 1 to 20, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0036] Embodiment 23. A transposome complex comprising: A transposase; b. a first transposon comprising modified transposon end sequences comprising uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines; c. a second transposon comprising a second transposon end sequence that is complementary to at least a portion of the first transposon end sequence.

[0037] Embodiment 24. The transposome complex of embodiment 23, wherein the first transposon comprises ribose, uracil, inosine, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines, and the transposome complex is in solution.

[0038] Embodiment 25. The transposome complex of embodiment 23, wherein the first transposon comprises uracil, inosine, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines, and the transposome complex is immobilized on a solid support.

[0039] Embodiment 26. The transposome complex of any one of embodiments 23 to 25, wherein the first transposon comprises a modified transposon end sequence of any one of embodiments 1 to 22.

[0040] Embodiment 27 The transposome complex of any one of embodiments 23 to 26, wherein the transposase is Tn5.

[0041] Embodiment 28 The transposome complex of any one of embodiments 23 to 27, wherein the first transposon is the transferred strand.

[0042] Embodiment 29. The transposome complex of any one of embodiments 23 to 28, wherein the second transposon is the non-transferred strand.

[0043] Embodiment 30 The transposome complex of any one of embodiments 23 to 29, wherein a uracil in the first transposon is base-paired with an A in the second transposon.

[0044] Embodiment 31 The transposome complex of any one of embodiments 23 to 30, wherein an inosine in the first transposon is base-paired with a C in the second transposon.

[0045] Embodiment 32. The transposome complex of any one of embodiments 23 to 31, wherein a ribose in the first transposon is base-paired with an A, C, T, or G in the second transposon.

[0046] Embodiment 33 The transposome complex of any one of embodiments 23 to 32, wherein a thymine glycol in the first transposon is base-paired with an A in the second transposon.

[0047] Embodiment 34 The transposome complex of any one of embodiments 23 to 33, wherein the modified purine is a 3-methyladenine in the first transposon that is base-paired with a T in the second transposon.

[0048] Embodiment 35. The transposome complex of any one of embodiments 23 to 34, wherein the modified purine is a 7-methylguanine in the first transposon that is base-paired with a C in the second transposon.

[0049] Embodiment 36. The transposome complex of any one of embodiments 23 to 34, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine in the first transposon base-paired with G in the second transposon.

[0050] Embodiment 37. The transposome complex of any one of embodiments 23 to 36, wherein the first or second transposon comprises an affinity element.

[0051] Embodiment 38 The transposome complex of embodiment 37, wherein the first transposon comprises an affinity element.

[0052] Embodiment 39 The transposome complex of embodiment 38, wherein the affinity element is attached to the 5' end of the first transposon.

[0053] Embodiment 40 The transposome complex of embodiment 38 or 39, wherein the first transposon contained in the targeted transposome complex comprises a linker.

[0054] Embodiment 41. The transposome complex of embodiment 40, wherein the linker has a first end bound to the 5' end of the first transposon and a second end bound to the affinity element.

[0055] Embodiment 42 The transposome complex of embodiment 37, wherein the second transposon comprises an affinity element.

[0056] Embodiment 43 The transposome complex of embodiment 42, wherein the affinity element is attached to the 3' end of the second transposon.

[0057] Embodiment 44 The transposome complex of embodiment 43, wherein the second transposon comprises SEQ ID NO:13.

[0058] Embodiment 45 The transposome complex of embodiment 44, wherein the second transposon comprises a linker.

[0059] Embodiment 46 The transposome complex of embodiment 45, wherein the linker has a first end bound to the 3' end of the second transposon and a second end bound to the affinity element.

[0060] Embodiment 47. The transposome complex of any one of embodiments 37 to 46, wherein the affinity element comprises biotin, avidin, streptavidin, an antibody, or an oligonucleotide.

[0061] Embodiment 48. The second transposon comprises: a. a second transposon end sequence complementary to SEQ ID NO:1, or b. The transposome complex of any one of embodiments 23 to 47, comprising a second transposon end that is perfectly complementary to the first transposon end.

[0062] Embodiment 49. The transposome complex of embodiment 48, wherein the first transposon comprises a modified transposon end sequence comprising an A16U, A16-8-oxoguanine, or A16 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0063] Embodiment 50. The transposome complex of embodiment 48, wherein the first transposon comprises a modified transposon end sequence comprising a C17-8-oxoguanine or a C17 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end fully complementary to the first transposon end.

[0064] Embodiment 51. The transposome complex of embodiment 48, wherein the first transposon comprises a modified transposon end sequence comprising an A18-8-oxoguanine or A18 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end fully complementary to the first transposon end.

[0065] Embodiment 52. The transposome complex of embodiment 48, wherein the first transposon comprises a modified transposon end sequence comprising a G19-8-oxoguanine or G19 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end fully complementary to the first transposon end.

[0066] Embodiment 53. The transposome complex of any one of embodiments 23 to 52, which is in solution.

[0067] Embodiment 54. A solid support on which the transposome complex of any one of embodiments 23 to 52 is immobilized.

[0068] Embodiment 55. A method for fragmenting a double-stranded nucleic acid, comprising combining a sample containing double-stranded nucleic acid with a transposome complex of any one of embodiments 23 to 53 or a solid support of embodiment 54, and preparing fragments.

[0069] Embodiment 56. A method for preparing a double-stranded nucleic acid fragment lacking all or part of a first transposon end, comprising: a. combining a sample containing nucleic acid with a transposome complex according to any one of embodiments 23 to 53 or a solid support according to embodiment 54, and preparing a fragment; b. combining the sample with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites, and cleaving the first transposon end at a uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within the mosaic sequence to remove all or a portion of the first transposon end from the fragment.

[0070] Embodiment 57 The method of embodiment 56, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0071] Embodiment 58 The method of embodiment 56, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0072] Embodiment 59 The method of embodiment 57 or 58, further comprising sequencing the fragment after removing all or part of the first transposon end from the fragment.

[0073] Embodiment 60 The method of embodiment 59, which does not require amplification of the fragment prior to sequencing.

[0074] Embodiment 61 The method of embodiment 59, wherein the fragment is amplified prior to sequencing.

[0075] Embodiment 62 The method of any one of embodiments 59 to 61, further comprising concentrating the fragments of interest after ligating the adaptors and prior to sequencing.

[0076] Embodiment 63. A method for preparing a double-stranded nucleic acid fragment comprising an adaptor, comprising: a. combining a sample containing nucleic acid with a transposome complex according to any one of embodiments 23 to 53 or a solid support according to embodiment 54, and preparing a fragment; b. combining the sample with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites, and cleaving the first transposon end at a uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within the mosaic end sequence to remove all or a portion of the first transposon end from the fragment; c. ligating adaptors onto the 5' and / or 3' ends of the fragments.

[0077] Embodiment 64 The method of embodiment 63, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0078] Embodiment 65 The method of embodiment 63, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0079] Embodiment 66 The method of any one of embodiments 56 to 65, wherein the nucleic acid is double-stranded DNA.

[0080] Embodiment 67. The method of any one of embodiments 56 to 65, wherein the nucleic acid is RNA and a double-stranded cDNA or a DNA:RNA duplex is generated prior to combining with the transposome complex.

[0081] Embodiment 68 The method of any one of embodiments 56 to 67, wherein all or part of the first transposon end that is cleaved is split off from the remainder of the sample.

[0082] Embodiment 69. The method of any one of embodiments 63 to 68, further comprising filling in the 3' ends of the fragments and phosphorylating the 3' ends of the fragments with a kinase prior to ligation.

[0083] Embodiment 70. The method of embodiment 69, wherein the filling is carried out using T4 DNA polymerase.

[0084] Embodiment 71 The method of embodiment 70, further comprising adding a single A overhang to the 3' end of the fragment.

[0085] Embodiment 72 The method of embodiment 71, wherein the polymerase adds a single A overhang.

[0086] Embodiment 73. The method of embodiment 72, wherein the polymerase is (i) Taq or (ii) Klenow fragment, exo-.

[0087] Embodiment 74. The method of any one of embodiments 56 to 73, wherein the fragment comprises 0 to 3 bases of a mosaic end sequence.

[0088] Embodiment 75. The method of any one of embodiments 56 to 74, wherein preparing the fragments results in preparation of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the number of fragments compared to preparing the fragments using a transposome complex comprising a first transposon comprising a transposon end sequence comprising a wild-type mosaic end sequence comprising SEQ ID NO:1.

[0089] Embodiment 76 The method of any one of embodiments 63 to 75, further comprising sequencing the fragments after ligating the adaptors.

[0090] Embodiment 77 The method of embodiment 76, which does not require amplification of the fragment prior to sequencing.

[0091] Embodiment 78 The method of embodiment 77, wherein the fragment is amplified prior to sequencing.

[0092] Embodiment 79. The method of any one of embodiments 76 to 78, further comprising concentrating the fragments of interest after ligating the adaptors and prior to sequencing.

[0093] Embodiment 80. The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises uracil, and the combination of the DNA glycosylase and the abasic site-recognizing endonuclease / lyase is a uracil-specific excision reagent (USER).

[0094] Embodiment 81 The method of embodiment 80, wherein the USER is a mixture of uracil DNA glycosylase and endonuclease VIII or endonuclease III.

[0095] Embodiment 82 The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises inosine and the endonuclease is endonuclease V.

[0096] Embodiment 83 The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises a ribose and the endonuclease is RNAse HII.

[0097] Embodiment 84. The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises 8-oxoguanine and the endonuclease is formamidopyrimidine-DNA glycosylase (FPG) or oxoguanine glycosylase (OGG).

[0098] Embodiment 85. The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises thymine glycol and the DNA glycosylase is endonuclease EndoIII (Nth) or Endo VIII.

[0099] Embodiment 86 The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises a modified purine, the DNA glycosylase is human 3-alkyladenine DNA glycosylase, and the endonuclease is endonuclease III or VIII.

[0100] Embodiment 87 The method of embodiment 86, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0101] Embodiment 88. The method of any one of embodiments 56 to 79, wherein the modified transposon end sequence comprises a modified pyrimidine and (1) the DNA glycosylase is thymine-DNA glycosylase (TDG) or mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4) and the endonuclease / lyase that recognizes the abasic site is an endonuclease that is endonuclease III or VIII, or (2) the endonuclease is DNA glycosylase / lyase ROS1 (ROS1).

[0102] Embodiment 89. The method of embodiment 88, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0103] Embodiment 90. The method of any one of embodiments 56 to 89, wherein the first transposon comprises a modified transposon end sequence comprising two or more mutations selected from uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, or modified pyrimidine, and wherein (1) the endonuclease, or (2) a combination of a DNA glycosylase and an endonuclease / lyase that recognizes heat, basic conditions, or abasic sites, is an enzyme mixture.

[0104] Embodiment 91 The method of embodiment 90, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0105] Embodiment 92 The method of embodiment 90, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0106] Embodiment 93 The method of any one of embodiments 63 to 92, wherein cleaving the first transposon end generates a sticky end for ligating an adaptor.

[0107] Embodiment 94 The method of embodiment 93, wherein the sticky end is longer than one base.

[0108] Embodiment 95 The method of any one of embodiments 63 to 94, wherein the adaptor comprises a double-stranded adaptor.

[0109] Embodiment 96 The method of any one of embodiments 63 to 95, wherein adaptors are added to the 5' and 3' ends of the fragments.

[0110] Embodiment 97. The method of embodiment 96, wherein the adaptors added to the 5' and 3' ends of the fragment are different.

[0111] Embodiment 98. The method of any one of embodiments 63 to 97, wherein the adapter comprises a unique molecular identifier (UMI), a primer sequence, an anchor sequence, a universal sequence, a spacer region, an index sequence, a capture sequence, a barcode sequence, a cleavage sequence, a sequencing-related sequence, and combinations thereof.

[0112] Embodiment 99. The method of any one of embodiments 98, wherein the adapter includes a UMI.

[0113] Embodiment 100. The method of embodiment 99, wherein an adaptor containing a UMI is ligated to both the 3' and 5' ends of the fragment.

[0114] Embodiment 101. The method of any one of embodiments 63 to 100, wherein the adapter is a fork-type adapter.

[0115] Embodiment 102. The method of any one of embodiments 63 to 101, wherein the ligating is carried out using a DNA ligase.

[0116] Embodiment 103. The method of any one of embodiments 63 to 102, carried out in a single reaction vessel.

[0117] Embodiment 104. The method of any one of embodiments 56 to 103, wherein the density of the transposomes immobilized on the solid surface is selected to regulate the fragment size and library yield of the immobilized fragments.

[0118] Embodiment 105. The method of any one of embodiments 56 to 104, which enables bead-based normalization.

[0119] Embodiment 106 The method of any one of embodiments 56 to 105, wherein the sample contains partially fragmented DNA.

[0120] Embodiment 107. The method of any one of embodiments 56 to 106, wherein the sample is formalin-fixed paraffin-embedded tissue or cell-free DNA.

[0121] Embodiment 108. The method of any one of embodiments 56 to 107, wherein the library comprises fragments prepared by a single tagmentation event.

[0122] Embodiment 109. A transposon pair having a first transposon and a second transposon, wherein the first transposon comprises a modified transposon end sequence of any one of embodiments 1 to 22, and the second transposon comprises: a. a transposon end sequence that contains a mosaic end sequence that is complementary to a wild-type mosaic end sequence; or b. A transposon pair, the transposon comprising a transposon end sequence that is perfectly complementary to the end of a first transposon.

[0123] Embodiment 110. The transposon pair of embodiment 109, wherein the first transposon comprises a modified transposon end sequence comprising an A16U, A16-8-oxoguanine, or A16 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0124] Embodiment 111. The transposon pair of embodiment 109, wherein the first transposon comprises a modified transposon end sequence comprising a C17-8-oxoguanine or C17 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0125] Embodiment 112. The transposon pair of embodiment 109, wherein the first transposon comprises a modified transposon end sequence comprising an A18-8-oxoguanine or A18 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0126] Embodiment 113. The transposon pair of embodiment 109, wherein the first transposon comprises a modified transposon end sequence comprising a G19-8-oxoguanine or G19 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0127] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0128] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0129] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0130] [Figure 1A] An overview of the fragmentation method is shown. (A) The present fragmentation-Tn5 approach uses modification of the Tn5-mosaic end substrate to allow selective cleavage of mosaic ends and subsequent adapter ligation. (B) Standard competitive workflow, where input DNA is mechanically sheared or enzymatically fragmented, followed by end repair and adapter ligation. In both Figures 1A and 1B, the binding of Y-shaped adapters containing all standard adapter sequences for Illumina sequencing (P5-i5-A14-ME and ME'-B15'-i7'-P7') is shown. In an alternative configuration, a short Y-shaped adapter containing only A14-ME and ME'-B15' can be used, and additional adapter sequences can be added by PCR, such as the method described in Figure 2 of US Patent Application Publication No. 2018 / 0201992(A1), which is incorporated by reference in its entirety. [Figure 1B]An overview of the fragmentation method is shown. (A) The present fragmentation-Tn5 approach uses modification of the Tn5-mosaic end substrate to allow selective cleavage of mosaic ends and subsequent adapter ligation. (B) Standard competitive workflow, where input DNA is mechanically sheared or enzymatically fragmented, followed by end repair and adapter ligation. In both Figures 1A and 1B, the binding of Y-shaped adapters containing all standard adapter sequences for Illumina sequencing (P5-i5-A14-ME and ME'-B15'-i7'-P7') is shown. In an alternative configuration, a short Y-shaped adapter containing only A14-ME and ME'-B15' can be used, and additional adapter sequences can be added by PCR, such as the method described in Figure 2 of US Patent Application Publication No. 2018 / 0201992(A1), which is incorporated by reference in its entirety. [Diagram 2] The mechanism of Tn5 transposase in standard tagmentation library preparation is outlined. The Tn5 transposase enzyme is preloaded with a transposon DNA substrate consisting of cognate "mosaic ends" and additional adapter sequences (such as A14 and B15 for Illumina methods). During tagmentation, these transpososomes act on genomic DNA, resulting in simultaneous fragmentation and tagging with the adapter sequences. The A14 and B15 sequences are SEQ ID NOs: 11 and 12, respectively. The ME sequence and its complement (ME') are SEQ ID NOs: 1 and 4, respectively. [Diagram 3] We outline how Bead-Linked Transposomes (BLT) enable a normalization-free workflow. By conjugating transposomes to magnetic beads, the amount of DNA converted into the library is normalized. Furthermore, some control of library fragment size is achieved through the selection of transposome density. Libraries can also be subjected to size selection based on Solid Phase Reversible Immobilization (SPRI) to gain further control of fragment size. gDNA = genomic DNA. [Figure 4]FIG. 1 shows an overview of enzymatic fragmentation with a fragmentation enzyme. In the method shown, enzyme 1 introduces a random nick into one strand, and enzyme 2 introduces a cut opposite the nick, generating a dsDNA break. The resulting DNA fragments typically have 1-4 base overhangs at the 5' end. An exemplary protocol using this method is NEBNext dsDNA Fragmentase (see NEBNext® for DNA Sample Prep for the Illumina Platform, NEB, 2019, and details of the NEBNext dsDNA Fragmentase Product available at www.nebj.jp / products / detail / 1020.com (accessed March 17, 2021)). [Diagram 5] 1 shows potential mechanisms for the removal of mosaic end sequences. Possible enzymatic strategies include the use of restriction enzymes, single-stranded DNAses, or DNA repair enzymes. In some embodiments, DNA repair enzymes are attractive due to their specificity. [Figure 6A] Analysis of Tn5v3 activity with mutant mosaic end sequences. (A) Canonical substitutions at various positions are reported based on the transferred strand sequence (SEQ ID NO:1) with the corresponding substitutions in the non-transferred strand (SEQ ID NO:4), except for the bases marked with *, which indicate that the substitutions were made only in the transferred strand and the wild-type non-transferred strand was annealed. At position 16A, substitutions were made to T, C, and G. At position 17C, substitutions were made to T, A, and G. At position 18A, substitutions were made to G, T, and C. At position 19G, substitutions were made to T, C, and A. Other substitutions to SEQ ID NO:1 were made as marked. (B) Activity of Tn5v3 transposomes prepared with DNA modifications at TS. Uracil base-paired with A and inosine base-paired with C. Sequences shown are the transferred strand TS (SEQ ID NO:1) and the non-transferred strand NTS (SEQ ID NO:4). AU = arbitrary units. [Figure 6B]Analysis of Tn5v3 activity with mutant mosaic end sequences. (A) Canonical substitutions at various positions are reported based on the transferred strand sequence (SEQ ID NO:1) with the corresponding substitutions in the non-transferred strand (SEQ ID NO:4), except for the bases marked with *, which indicate that the substitutions were made only in the transferred strand and the wild-type non-transferred strand was annealed. At position 16A, substitutions were made to T, C, and G. At position 17C, substitutions were made to T, A, and G. At position 18A, substitutions were made to G, T, and C. At position 19G, substitutions were made to T, C, and A. Other substitutions to SEQ ID NO:1 were made as marked. (B) Activity of Tn5v3 transposomes prepared with DNA modifications at TS. Uracil base-paired with A and inosine base-paired with C. Sequences shown are the transferred strand TS (SEQ ID NO:1) and the non-transferred strand NTS (SEQ ID NO:4). AU = arbitrary units. [Figure 7A] Library preparation using the Tn5-fragmentase approach. (A) Diagram of the workflow used to prepare libraries. (B) Diagram of how a modification-specific endonuclease can cleave the modified base in a one-step reaction or a modification-specific glycosylase followed by AP lyase / endonuclease or heat can cleave the modified base in a two-step reaction. (C) Electropherograms of libraries prepared with DNA modifications. Libraries were treated with either USER, Endonuclease V, or RNAse HII according to the manufacturer's protocol (NEB). In this experiment, a large amount of adapter dimers (peak at approximately 160 bp) was observed, which is likely due to suboptimal ligation adapter concentration. ATL=A-tailing. LIG=ligation. [Figure 7B]Library preparation using the Tn5-fragmentase approach. (A) Diagram of the workflow used to prepare libraries. (B) Diagram of how a modification-specific endonuclease can cleave the modified base in a one-step reaction or a modification-specific glycosylase followed by AP lyase / endonuclease or heat can cleave the modified base in a two-step reaction. (C) Electropherograms of libraries prepared with DNA modifications. Libraries were treated with either USER, Endonuclease V, or RNAse HII according to the manufacturer's protocol (NEB). In this experiment, a large amount of adapter dimers (peak at approximately 160 bp) was observed, which is likely due to suboptimal ligation adapter concentration. ATL=A-tailing. LIG=ligation. [Figure 7C] Library preparation using the Tn5-fragmentase approach. (A) Diagram of the workflow used to prepare libraries. (B) Diagram of how a modification-specific endonuclease can cleave the modified base in a one-step reaction or a modification-specific glycosylase followed by AP lyase / endonuclease or heat can cleave the modified base in a two-step reaction. (C) Electropherograms of libraries prepared with DNA modifications. Libraries were treated with either USER, Endonuclease V, or RNAse HII according to the manufacturer's protocol (NEB). In this experiment, a large amount of adapter dimers (peak at approximately 160 bp) was observed, which is likely due to suboptimal ligation adapter concentration. ATL=A-tailing. LIG=ligation. [Figure 8A] Comparison of uracil modification sites within MEs. (A) Electropherograms of libraries generated with alternative mosaic ends. (B) Qubit yields of libraries with alternative MEs. USER incubation times of 20 and 60 minutes were tested. [Figure 8B]Comparison of uracil modification sites within MEs. (A) Electropherograms of libraries generated with alternative mosaic ends. (B) Qubit yields of libraries with alternative MEs. USER incubation times of 20 and 60 minutes were tested. [Figure 9] Summarizing, the fragmentase libraries show the expected ME "scars" flanking the library insert. Due to variable UMI lengths, some libraries are shifted by 1 bp. The ME scars for each modification site are present as expected. The A16U transferred strand and T16A non-transferred strand sequences are SEQ ID NOs: 5 and 6, respectively. The C17U transferred strand and G17A non-transferred strand sequences are SEQ ID NOs: 7 and 8, respectively. The A18U transferred strand and T18A non-transferred strand sequences are SEQ ID NOs: 9 and 10, respectively. [Figure 10A] A representative fBLT library preparation is shown. (A) Workflow used in this study. (B) Library yields of enriched BLT (eBLT) and fragmentase (fBLT) library preparations. (C) Representative Bioanalyzer traces of eBLT and fBLT libraries. Additional workflows involving fBLT are disclosed herein. [Figure 10B] A representative fBLT library preparation is shown. (A) Workflow used in this study. (B) Library yields of enriched BLT (eBLT) and fragmentase (fBLT) library preparations. (C) Representative Bioanalyzer traces of eBLT and fBLT libraries. Additional workflows involving fBLT are disclosed herein. [Figure 10C] A representative fBLT library preparation is shown. (A) Workflow used in this study. (B) Library yields of enriched BLT (eBLT) and fragmentase (fBLT) library preparations. (C) Representative Bioanalyzer traces of eBLT and fBLT libraries. Additional workflows involving fBLT are disclosed herein. [Figure 11]Schematic of a fBLT with representative modified transposon ends including a transferred strand with a G19I mutation (SEQ ID NO: 14) and a biotinylated non-transferred strand (SEQ ID NO: 13) that can be used to immobilize transposomes. B=biotin. [Figure 12] Results are shown using fBLT with different modified bases at the mosaic end (ME sequence) of the first transposon. 16-19 represent the position of the modification from SEQ ID NO: 1. OxoG = oxoguanine; AU = activity units. [Figure 13A] Results for different types of fBLTs are shown. (A) Results for percentage conversion with A18 inosine (I18), C17-8-oxoguanine (O17), and G19U (U19) mutations. (B) Results for variant calling performance with I18, O17, and U19. (C) Results for percentage conversion with I18, G19I (I19), O17, A18O (O18), and G19O (O19). Results showed that the performance of BLTs with mosaic ends substituted with inosines was generally high, with G19I (I19) performing the best. [Figure 13B] Results for different types of fBLTs are shown. (A) Results for percentage conversion with A18 inosine (I18), C17-8-oxoguanine (O17), and G19U (U19) mutations. (B) Results for variant calling performance with I18, O17, and U19. (C) Results for percentage conversion with I18, G19I (I19), O17, A18O (O18), and G19O (O19). Results showed that the performance of BLTs with mosaic ends substituted with inosines was generally high, with G19I (I19) performing the best. [Figure 13C]Results for different types of fBLTs are shown. (A) Results for percentage conversion with A18 inosine (I18), C17-8-oxoguanine (O17), and G19U (U19) mutations. (B) Results for variant calling performance with I18, O17, and U19. (C) Results for percentage conversion with I18, G19I (I19), O17, A18O (O18), and G19O (O19). Results showed that the performance of BLTs with mosaic ends substituted with inosines was generally high, with G19I (I19) performing the best. [Figure 14] Data regarding chimeric reads are presented: the use of modified transposon ends containing uracil can result in a higher percentage of chimeric reads compared to modified transposon ends containing inosine or oxoguanine. [Figure 15A] Comparison of fBLT versus other library preparation fragmentation methods (i.e., NEBNext® dsDNA Fragmentase® or sonication performed with a Covaris Ultrasonicator following standard procedures). (A) Overview of the workflow. (B) Overview of the sensitivity and specificity of variant calling performance by the different methods measuring a 50 ng input gDNA 1% mixture of NA12877 into a NA12878 background (with 84 heterozygous variants and 0.5% variant allele frequency (VAF)). [Figure 15B] Comparison of fBLT versus other library preparation fragmentation methods (i.e., NEBNext® dsDNA Fragmentase® or sonication performed with a Covaris Ultrasonicator following standard procedures). (A) Overview of the workflow. (B) Overview of the sensitivity and specificity of variant calling performance by the different methods measuring a 50 ng input gDNA 1% mixture of NA12877 into a NA12878 background (with 84 heterozygous variants and 0.5% variant allele frequency (VAF)). [Figure 16] Error rates for different methods of library preparation by fragmentation are shown, including a substantially higher error rate for samples prepared by sonication. [Figure 17] Library conversion efficiency of different fragmentation methods is shown. Overall, fBLT outperformed the other library conversion methods. Sample 1 is a 1% mixture of genomic DNA of NA12877 in a NA12878 background. Samples 2-6 are formalin fixed paraffin embedded (FFPE) tissues. dCq is a measure of DNA quality, with higher values ​​corresponding to lower quality samples. Thus, the higher conversion efficiency of sample 1 relative to the other samples highlights the fact that library conversion from FFPE tissues is generally reduced due to the lower DNA quality of FFPE tissues. [Figure 18A] We summarize the method involving a single tagmentation event using fBLT to generate fragments from samples of FFPE tissue. (A) Overview of the workflow. (B) Percentage of fragments rescued from different tissues. Sample numbers are the same as those outlined for Figure 17. dCq is a measure of DNA quality, with higher values ​​corresponding to samples of lower quality. Thus, the lower percentage of fragments rescued for sample 1 indicates a higher quality in this genomic DNA sample compared to samples 2-6 with FFPE tissue (i.e., there were fewer fragments derived from a single tagmentation event, with most fragments derived from two tagmentation events). In contrast to genomic DNA, a higher percentage of samples was rescued from FFPE tissue. The reason is that in FFPE tissue, there may be more single tagmentation events due to its lower quality. [Figure 18B]We summarize the method involving a single tagmentation event using fBLT to generate fragments from samples of FFPE tissue. (A) Overview of the workflow. (B) Percentage of fragments rescued from different tissues. Sample numbers are the same as those outlined for Figure 17. dCq is a measure of DNA quality, with higher values ​​corresponding to samples of lower quality. Thus, the lower percentage of fragments rescued for sample 1 indicates a higher quality in this genomic DNA sample compared to samples 2-6 with FFPE tissue (i.e., there were fewer fragments derived from a single tagmentation event, with most fragments derived from two tagmentation events). In contrast to genomic DNA, a higher percentage of samples was rescued from FFPE tissue. The reason is that in FFPE tissue, there may be more single tagmentation events due to its lower quality. [Figure 19] We summarize some advantages and flexibility of tagmentation protocol using fBLT. In particular, the method allows ligation of adapters that allow different workflows that users may want to pursue. As shown, the adapters can include unique molecular identifiers (UMIs) to determine different unique fragments from the same fragment amplicon. Alternatively, forked adapters can be used in workflows involving PCR to incorporate indexes, or indexed forked adapters can be used in PCR-free workflows. [Figure 20] A standard workflow for library preparation using fBLT and optional enrichment is outlined. Boxes and triangles indicate steps where the user should handle the reaction samples. The overall library preparation time of about 5.5 hours is similar to other ligation-based library preparation methods. Optional enrichment can be used, for example, to enrich for cancer-related panels when preparing libraries from FFPE tissue samples from cancer patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0131] Array Description Table 1 provides a list of specific sequences referenced herein, in which / 3BiotinN / and / 5Phos / refer to the 3' biotin and 5' phosphate, respectively, / i8oxodG / refers to an internal 8-oxoG nucleotide, and / 38oxodG / refers to an 8-oxoG nucleotide at the 3' position.

[0132] [Table 1] [Mode for carrying out the invention]

[0133] I. Modified transposon ends with mutations in the mosaic end sequences Described herein are modified transposon end sequences that contain mosaic end sequences. In some embodiments, these modified transposon end sequences contain mosaic end sequences that allow for the cleavage and removal of the mosaic end sequences after transposition. A key requirement for transposition is the "mosaic end" (ME) that is specifically recognized by Tn5 and is required for its transposition activity. Tn5 naturally recognizes "outer end" (OE) and "inner end" (IE) sequences (as shown in Table 2) that have been shown to be highly intolerant of mutations, with most mutations resulting in reduced activity (see JC Makris et al. PNAS 85(7): 2224-28 (1988)). Subsequent studies demonstrated that chimeric sequences derived from IE and OE, called "mosaic ends" (Table 2), together with mutant Tn5 enzymes, increase transposition activity by approximately 100-fold compared to the native system (see Maggie Zhou et al., Journal of Molecular Biology 276(5):913-25 (1998)). This highly active system is used in Illumina's Illumina DNA Flex PCR-Free (RUO) product. The crystal structure of Tn5 complexed with a DNA substrate shows that 13 of the 19 base pairs have nucleobase-specific crystal contacts (see Douglas R. Davies et al., Science 289 5476:77-85 (2000)), while other bases have been shown to play a role in catalysis (see Mindy Steiniger-White et al., Journal of Molecular Biology 322(5):971-82 (2002)). Typically, the activity of Tn5 is assessed by an in vivo reporter system (the papillation assay described in Zhou et al. J. Mol. Biol.276:913-925 (1998)).

[0134] [Table 2]

[0135] In Table 2, sequences in normal font indicate the shared sequences, double underlined italicized sequences are derived from natural OE substrates, and bold italicized sequences are derived from natural IE substrates.

[0136] A representative wild-type mosaic end sequence (transferred strand) is SEQ ID NO: 1. Various mutant Tn5 and transposon ends are described in WO 2015 / 160895 and U.S. Pat. No. 9,080,211, each of which is incorporated by reference in its entirety and may be suitable for use in the methods described herein.

[0137] Several DNA enzymes or combinations of enzymes can mediate the selective removal of modified bases, such as uracil, inosine, ribose bases, 8-oxoG, thymine glycol, modified purines, and modified pyrimidines, among others (see Table 3 and Properties of DNA Repair Enzymes and Structure-specific Endonucleases, New England Biolabs, and Jacobs and Schar Chromosoma 121:1-20 (2012), downloaded on January 20, 2022 from www.international.neb.com / tools-and-resources / selection-charts). Such enzymes include modification-specific endonucleases or modification-specific glycosylases. Modified purines for use with modification-specific glycosylases include 3-methyladenine (3mA) and 7-methylguanine (7mG). Modified pyrimidines for use with modification-specific glycosylases can include 5-methylcytosine (5mC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). Selective removal of uracil and 8-oxoG using DNA repair enzymes is already used in certain sequencing platforms.

[0138] Since only one strand of the mosaic end, called the "transferred strand," is covalently added to the library insert during transposition, incorporation of such modified bases, specifically into the mosaic end transferred strand, may allow for selective cleavage and removal of the mosaic end transferred strand. However, this type of mosaic end cleavage and removal requires mutation of the mosaic end sequence from its reference sequence (SEQ ID NO:1).

[0139] [Table 3] * N-glycosylases can be paired with AP lyases / endonucleases (e.g., EndoIII or EndoVIII). Alternatively, abasic sites are chemically labile and can be cleaved under heat and / or basic conditions.

[0140] In Table 3, Endo = endonuclease, FPG = formamidopyrimidine-DNA glycosylase, OGG = oxoguanine glycosylase (OGG), hAAG = human 3-alkyladenine DNA glycosylase, UNG = uracil-N-glycosylase, Nth = cloned nth gene, TDG = thymine-DNA glycosylase, MBD4 = mammalian DNA glycosylase-methyl-CpG binding domain protein 4, and ROS1 = endonuclease ROS1 (with bifunctional DNA glycosylase / lyase activity).

[0141] Disclosed herein are modified transposon end sequences, including mosaic end sequences, which include one or more mutations compared to a wild-type mosaic end sequence, the mutations including substitutions with inosine, ribose, 8-oxoguanine, thymine glycol, modified purines (e.g., 3mA or 7mG), or modified pyrimidines. In some embodiments, these substitutions are used in methods of truncating transposon ends after transposition, as described below.

[0142] In some embodiments, the mosaic end sequence can be a mosaic end sequence for use with a Tn5 transposase. In some embodiments, the modified transposon end sequence has a mutation in the mosaic end sequence compared to SEQ ID NO:1.

[0143] In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising one or more mutations compared to SEQ ID NO:1, the one or more mutations comprising a substitution at A16, C17, A18, and / or G19. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at A16. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at C17. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at A18. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at G19. In some embodiments, the modified transposon end sequence comprises SEQ ID NO:5, 7, 9, or 14-22. Data with representative modified transposon end sequences are shown in FIG. 6A (involving transposition in solution) and FIG. 12 (involving transposition mediated by fBLT).

[0144] In some embodiments, the mosaic end sequence comprises two or more mutations, hi some embodiments, the mosaic end sequence comprises eight or fewer mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO: 1).

[0145] In addition to one or more mutations at A16, C17, A18, and / or G19, further mutations may also be present in the mosaic end sequence. In some embodiments, the mosaic end sequence comprises one or more mutations compared to SEQ ID NO:1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence comprises one to four substitution mutations compared to SEQ ID NO:1 in addition to one or more mutations at A16, C17, A18, and / or G19.

[0146] In some embodiments, the mosaic end sequence has one substitution mutation compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence has two substitution mutations compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence has three substitution mutations compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence has four substitution mutations compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19.

[0147] In some embodiments, the substitution at A16 is A16T, A16C, A16G, A16U, A16 inosine, A16 ribose, A16-8-oxoguanine, A16 thymine glycol, A16 modified purine, or A16 modified pyrimidine, and the substitution at C17 is C17T, C17A, C17G, C17U, C17 inosine, C17 ribose, C17-8-oxoguanine, C17 thymine glycol, C17 modified purine, or C17 modified pyrimidine. and the substitution at A18 is A18G, A18T, A18C, A18U, A18 inosine, A18 ribose, A18-8-oxoguanine, A18 thymine glycol, A18 modified purine, or A18 modified pyrimidine, and / or the substitution at G19 is G19T, G19C, G19A, G19U, G19 inosine, G19 ribose, G19-8-oxoguanine, G19 thymine glycol, G19 modified purine, or G19 modified pyrimidine. In some embodiments, the modified purine is 3mA or 7mG. In some embodiments, the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0148] In some embodiments, the mutations include substitutions with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines. In some embodiments, these mutations allow for a method of cleavage of the mosaic terminal sequence following transposition.

[0149] In some embodiments, the modified transposon end sequence comprises a mutation at A16, C17, A18, or G19.

[0150] In some embodiments, the modified transposon end sequence comprises two mutations selected from mutations at A16, C17, A18, or G19. In some embodiments, the modified transposon end sequence comprises three mutations selected from mutations at A16, C17, A18, or G19. In some embodiments, the modified transposon end sequence comprises four mutations at A16, C17, A18, and G19.

[0151] In some embodiments, the modified transposon end sequence has one to four substitution mutations at A16, C17, A18, and / or G19 compared to SEQ ID NO:1. In some embodiments, the modified transposon end sequence has one substitution mutation compared to the wild-type sequence (in some embodiments, SEQ ID NO:1). In some embodiments, the modified transposon end sequence has two substitution mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO:1). In some embodiments, the modified transposon end sequence has three substitution mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO:1). In some embodiments, the modified transposon end sequence has four substitution mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO:1).

[0152] II. Transposition-ligation library preparation method Disclosed herein are library preparation methods that combine transposition and adapter ligation. These library preparation methods may therefore be referred to as "hybrid transposition-ligation library preparation." Such methods may use modified Tn5-mosaic end sequences that allow for cleavage of the transposed transposon ends after transposition (as shown in FIG. 1A). As used herein, "hybrid Tn5-ligation approach" refers to a method involving transposition, cleavage of mosaic end sequences, and adapter ligation.

[0153] In some embodiments, cleavage of the mosaic end sequence allows for its removal from the library fragments. The method uses ligation after cleavage of the mosaic end sequence to incorporate adaptors for potential downstream sequencing, although the method is not limited to embodiments requiring ligation of adaptor sequences.

[0154] A BLT designed for fragmentation of mosaic end sequences is sometimes called a "fragmentase BLT" (fBLT). Although an fBLT does not itself contain a fragmentase, the fBLT is designed to prepare fragments similar to those prepared with a fragmentase, in that the resulting fragments lack all or part of the mosaic end sequence. The fBLT is designed for cleavage (post-transposition) to remove all or part of the mosaic end sequence after fragment generation by transposition.

[0155] The method can uncouple the enzymatic fragmentation and adaptor ligation activities of a transposase, such as Tn5 transposase, by programmed cleavage of mosaic end sequences from library fragments. As described herein, a transposase, in some embodiments Tn5, can tolerate several mutations and nucleobase modifications in the mosaic end substrate. By incorporating modified bases in the transferred strand of the mosaic end, the enzyme can enable selective cleavage and removal. This technology eliminates the constraint of needing 19-bp mosaic end sequences flanking the library insert, enabling a hybrid transposase-ligation library preparation approach, which in turn allows fBLT to be utilized in library preparation workflows developed based on ligation chemistry. Thus, the method improves upon conventional workflows for mechanically shearing or enzymatically fragmenting dsDNA followed by end repair and adaptor ligation (Figure 1B).

[0156] Described herein is a method for preparing double-stranded nucleic acid fragments containing adaptors, comprising combining a sample containing nucleic acid with a transposome complex and preparing fragments, combining the sample with an enzyme or enzyme mixture, and cleaving a first transposon end at a uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within a mosaic end sequence to remove all or part of the first transposon end from the fragment, and ligating an adaptor onto the 5' and / or 3' end of the fragment. In some embodiments, the modified purine is 3-methyladenine or 7-methylguanine. In some embodiments, the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine. In some embodiments, the enzyme or enzyme mixture is a modification-specific endonuclease or a modification-specific DNA glycosylase. In some embodiments, a modification-specific DNA glycosylase is used with an endonuclease / lyase that does not need to be modification specific. Instead, the endonuclease / lyase recognizes an abasic site.

[0157] In some embodiments, the enzyme or enzyme mixture is (1) an endonuclease, or (2) a combination of a DNA glycosylase and a heat, basic, or abasic site recognizing endonuclease / lyase.

[0158] In some embodiments, the method is carried out in a single reaction vessel, in other words, the method may be carried out without the need to separate reaction products from one another.

[0159] A. Transposome Complex As used herein, a "transposome complex" or "transposome" is composed of at least one transposase (or other enzymes described herein) and a transposon recognition sequence. The present invention is not limited to a particular transposase.

[0160] A "transposome complex" is composed of at least one transposase enzyme and a transposon recognition sequence. In some such systems, the transposase binds to the transposon recognition sequence to form a functional complex capable of catalyzing a transposition reaction. In some embodiments, the transposon recognition sequence is a double-stranded transposon end sequence. The transposase or integrase binds to the transposase recognition site in the target nucleic acid and inserts the transposase recognition sequence into the target nucleic acid. In some such insertion events, one strand of the transposase recognition sequence (or end sequence) is transferred to the target nucleic acid, which also results in a cleavage event. Exemplary transposition procedures and systems that can be readily adapted for use with the transposases of the present disclosure are described, for example, in WO 10 / 048605, U.S. Patent Application Publication Nos. 2012 / 0301925, 2012 / 13470087, or 2013 / 0143774, each of which is incorporated by reference in its entirety.

[0161] In some such systems, the transposase binds to the transposon recognition sequence to form a functional complex capable of catalyzing a transposition reaction. In some embodiments, the transposon recognition sequence is a double-stranded transposon end sequence. The transposase binds to the transposase recognition site in the target nucleic acid and inserts the transposon recognition sequence into the target nucleic acid. In some such insertion events, one strand of the transposon recognition sequence (or end sequence) is transferred to the target nucleic acid, resulting in a cleavage event. Exemplary transposition procedures and systems can be readily adapted for use with transposases.

[0162] "Transposase" refers to an enzyme that can form a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and catalyze the insertion or transposition of the transposon end-containing composition into a double-stranded target nucleic acid. Transposases as provided herein can also include integrases from retrotransposons and retroviruses.

[0163] Exemplary transposases that may be used in certain embodiments provided herein include (or are encoded by) Tn5 transposase, Sleeping Beauty (SB) transposase, Vibrio harveyi, MuA transposase and Mu transposase recognition sites including R1 and R2 end sequences, Staphylococcus aureus Tn552, Ty1, Tn7 transposase, Tn / O and IS10, mariner transposase, Tc1, P Element, Tn3, bacterial insertion sequences, retroviruses, and yeast retrotransposons. Further examples include IS5, Tn10, Tn903, IS911, and engineered versions of transposase family enzymes. The methods described herein also include combinations of transposases, not just a single transposase.

[0164] In some embodiments, the transposase is a Tn5, Tn7, MuA, or Vibrio harveyi transposase, or an active variant thereof. In other embodiments, the transposase is a Tn5 transposase or a variant thereof. In other embodiments, the transposase is a Tn5 transposase or a variant thereof. In other embodiments, the transposase is a Tn5 transposase or an active variant thereof. In some embodiments, the Tn5 transposase is a hyperactive Tn5 transposase or an active variant thereof. In some aspects, the Tn5 transposase is a Tn5 transposase described in PCT Publication WO 2015 / 160895, which is incorporated herein by reference. In some aspects, the Tn5 transposase is a hyperactive Tn5 having mutations at positions 54, 56, 372, 212, 214, 251, and 338 relative to wild-type Tn5 transposase. In some aspects, the Tn5 transposase is a hyperactive Tn5 with the following mutations relative to the wild-type Tn5 transposase: E54K, M56A, L372P, K212R, P214R, G251R, and A338V. In some embodiments, the Tn5 transposase is a fusion protein. In some embodiments, the Tn5 transposase fusion protein comprises a fusion elongation factor Ts (Tsf) tag. In some embodiments, the Tn5 transposase is a hyperactive Tn5 transposase comprising mutations at amino acids 54, 56, and 372 relative to the wild-type sequence. In some embodiments, the hyperactive Tn5 transposase is a fusion protein, and optionally the fusion protein is an elongation factor Ts (Tsf). In some embodiments, the recognition site is a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367, 1998). In one embodiment, a transposase recognition site that forms a complex with a hyperactive Tn5 transposase is used (e.g., EZ-Tn5TM transposase, Epicentre Biotechnologies, Madison, Wis.). In some embodiments, the Tn5 transposase is a wild-type Tn5 transposase.

[0165] As used throughout, the term "transposase" refers to an enzyme that can form a functional complex with a transposon-containing composition (e.g., a transposon, a transposon composition) and catalyze the insertion or transposition of the transposon-containing composition into a double-stranded target nucleic acid with which it is incubated in an in vitro transposition reaction. Transposases of the provided methods also include integrases from retrotransposons and retroviruses. Exemplary transposases that can be used in the provided methods include wild-type or mutant forms of Tn5 transposase and MuA transposase.

[0166] A "transposition reaction" is a reaction in which one or more transposons are inserted into a target nucleic acid at random or near-random sites. The essential components of a transposition reaction are a transposase and a DNA oligonucleotide that represents the nucleotide sequence of the transposon, including the transferred transposon sequence and its complement (i.e., the non-transferred transposon end sequence), as well as other components necessary to form a functional transposition or transposome complex. The methods of the present disclosure are exemplified by using a transposition complex formed by hyperactive Tn5 transposase and Tn5-type transposon ends, or by MuA or HYPERMu transposase and Mu transposon ends containing R1 and R2 end sequences (see, e.g., Goryshin, I. and Reznikoff, WS, J. Biol. Chem., 273:7367, 1998, and Mizuuchi, Cell, 35:785, 1983; Savilahti, H, et al., EMBO J., 14:4893, 1995, which are incorporated herein by reference in their entirety). However, any transposition system capable of inserting transposon ends in a random or near-random manner with sufficient efficiency to tag target nucleic acids for the intended purpose may be used in the methods provided.Other examples of known transposition systems that can be used in the provided methods include Staphylococcus aureus Tn552, Tyl, transposons Tn7, Tn / O and IS10, mariner transposase, Tel, P elements, Tn3, bacterial insertion sequences, retroviruses, and yeast retrotransposons (e.g., Colegio OR et al, J. Bacteriol., 183:2384-8, 2001; Kirby C et al, Mol. Microbiol., 43:173-86, 2002; Devine SE, and Boeke J D., Nucleic Acids Res., 22:3765- 72, 1994; WO 95 / 23875; Craig, NL, Science. 271:1512, 1996; Craig, NL, Review in: Curr Top Microbiol. Immunol.,204:27-48,1996, Kleckner N,et al.,Curr Top Microbiol Immunol.,204:49-82,1996,Lampe DJ,et al.,EMBO J.,15:5470-9,1996,Plasterk RH,Curr Top Microbiol Immunol,204:125-43,1996, Gloor,GB,Methods Mol.Biol,260:97-1 14,2004, Ichikawa H,and Ohtsubo E.,J Biol.Chem.265:18829-32,1990, Ohtsubo,F and Sekine, Y, Curr. Top. Microbiol. Immunol. 204:1-26, 1996, Brown PO, et al, Proc Natl Acad. Sci USA, 86:2525-9, 1989; Boeke JD and Corces VG, Annu Rev Microbiol. 43:403-34, 1989, all of which are incorporated herein by reference in their entireties).

[0167] The method for inserting a transposon into a target sequence can be carried out in vitro using any suitable transposon system that is available or can be developed based on knowledge in the art. In general, a suitable in vitro transposition system for use in the disclosed method requires at least a transposase enzyme of sufficient purity, sufficient concentration, and sufficient in vitro transposition activity, and a transposon that forms a functional complex with the respective transposase that can catalyze the transposition reaction. Suitable transposase transposon end sequences that can be used include, but are not limited to, wild-type, derivative, or mutant transposon end sequences that form a complex with a transposase selected from wild-type, derivative, or mutant forms of the transposase.

[0168] In some embodiments, the transposase comprises a Tn5 transposase. In some embodiments, the Tn5 transposase is a hyperactive Tn5 transposase.

[0169] In some embodiments, the transposome complex comprises a dimer of two molecules of transposase. In some embodiments, the transposome complex is a homodimer, where the two molecules of transposase are each bound to a first and a second transposon of the same type (e.g., the sequences of the two transposons bound to each monomer are the same, forming a "homodimer"). In some embodiments, the compositions and methods described herein employ two populations of transposome complexes. In some embodiments, the transposase in each population is the same. In some embodiments, the transposome complexes in each population are homodimers, where the first population has a first adaptor sequence in each monomer and the second population has a different adaptor sequence in each monomer.

[0170] The term "transposon end" refers to a double-stranded nucleic acid DNA that exhibits only the necessary nucleotide sequences ("transposon end sequences") to form a complex with a transposase or integrase enzyme that functions in an in vitro transposition reaction. In some embodiments, the transposon end can form a functional complex with a transposase in a transposition reaction. As a non-limiting example, the transposon end can include a 19-bp outer end ("outer end, OE") transposon end, an inner end ("inner end, IE") transposon end, or a "mosaic end ("ME") transposon end recognized by a wild-type or mutant Tn5 transposase, or an R1 and R2 transposon end, as described in the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, which is incorporated by reference in its entirety. The transposon end can include any nucleic acid or nucleic acid analogue that is suitable for forming a functional complex with a transposase or integrase enzyme in an in vitro transposition reaction. For example, transposon ends can comprise DNA, RNA, modified bases, non-natural bases, modified backbones, and can contain nicks in one or both strands. Although the term "DNA" is used throughout this disclosure in reference to transposon end compositions, it should be understood that any suitable nucleic acid or nucleic acid analogue can be utilized in the transposon ends.

[0171] The term "transferred strand" refers to the portion of a transposon pair that is transferred to a fragment of nucleic acid from a sample during a transposition reaction. Similarly, the term "non-transferred strand" refers to the portion of a transposon pair that is not transferred to a fragment of nucleic acid from a sample during a transposition reaction. Within a transposon pair, the transferred strand and the non-transferred strand may be fully or partially complementary. The 3' end of the transferred strand is bound or transferred to the target DNA in an in vitro transposition reaction. The non-transferred strand that exhibits a transposon end sequence that is fully or partially complementary to the transferred transposon end sequence is not bound or transferred to the target DNA in an in vitro transposition reaction.

[0172] In some embodiments, the transferred strand and the non-transferred strand are covalently linked. For example, in some embodiments, the transferred and non-transferred strand sequences are provided on a single oligonucleotide, for example in a hairpin configuration. Thus, the free end of the non-transferred strand is not directly attached to the target DNA by the translocation reaction, but is indirectly attached to the DNA fragment because the non-transferred strand is linked to the transferred strand by the loop of the hairpin structure. Further examples of transposome structures and methods of preparing and using transposomes can be found in the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety.

[0173] As used herein, "transposome complex" and "transposome" are equivalent.

[0174] In some embodiments, the first transposon comprises the transferred strand in the transposition reaction. In some embodiments, the second transposon comprises the non-transferred strand in the transposition reaction.

[0175] In some embodiments, the transposome comprises modified transposon ends having mutations in the mosaic end sequences.

[0176] In some embodiments, the transposome complex comprises a transposase, a first transposon comprising a modified transposon end sequence comprising uracil, inosine, ribose, 8-oxoguanine, thymine glycol, a modified purine, and / or a modified pyrimidine, and a second transposon comprising a second transposon end sequence that is complementary to at least a portion of the first transposon end sequence.

[0177] In some embodiments, the first transposon comprises ribose and the transposome complex is in solution. In some embodiments, the first transposon comprises uracil, inosine, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines, and the transposome complex is immobilized on a solid support.

[0178] In some embodiments, the first transposon comprises a modified transposon end sequence. In some embodiments, the transposase is Tn5. In some aspects, the first transposon is a transferred strand. In some embodiments, the second transposon is a non-transferred strand.

[0179] In some embodiments, the uracil in the first transposon is base-paired with an A in the second transposon. In some embodiments, the inosine in the first transposon is base-paired with a C in the second transposon. In some embodiments, the ribose in the first transposon is base-paired with an A, a C, a T, or a G in the second transposon. In some embodiments, the thymine glycol in the first transposon is base-paired with an A in the second transposon. In some embodiments, the modified purine is a 3-methyladenine in the first transposon that is base-paired with a T in the second transposon. In some embodiments, the modified purine is a 7-methylguanine in the first transposon that is base-paired with a C in the second transposon. In some embodiments, the modified pyrimidine is a 5-methylcytosine, a 5-formylcytosine, or a 5-carboxycytosine in the first transposon that is base-paired with a G in the second transposon.

[0180] In some embodiments, the second transposon comprises a sequence complementary to SEQ ID NO: 1. In some embodiments, the second transposon comprises a second transposon end sequence complementary to SEQ ID NO: 1. In some embodiments, the second transposon comprises SEQ ID NO: 4.

[0181] In some circumstances, the second transposon end may be fully complementary to the first transposon end, while in other circumstances it may be partially complementary. Without being bound by theory, a transposase may have higher activity when a pair of transposon ends (i.e., the first transposon and the second transposon) contains fewer mutations. For example, a transpososome complex containing a second transposon containing a sequence complementary to SEQ ID NO:1 (i.e., the complement of the wild-type mosaic end sequence) may mediate higher activity than a transpososome complex containing a second transposon end complementary to a first transposon end containing a modified transposon end sequence as described herein. In other circumstances, the second transposon may be fully complementary to the first transposon to promote closer annealing of the transposon pair.

[0182] In some embodiments, the first transposon comprises a modified transposon end sequence comprising an A16U, A16-8-oxoguanine, or A16 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0183] In some embodiments, the first transposon comprises a modified transposon end sequence comprising a C17-8-oxoguanine, or a C17 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0184] In some embodiments, the first transposon comprises a modified transposon end sequence comprising an A18-8-oxoguanine, or an A18 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0185] In some embodiments, the first transposon comprises a modified transposon end sequence comprising a G19-8-oxoguanine, or a G19 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0186] In some embodiments, the transposome complex comprises a transposon pair comprising a first transposon and a second transposon, the first transposon comprising a modified transposon end sequence as described herein, and the second transposon comprising a transposon end sequence comprising a mosaic end sequence that is complementary to a wild-type mosaic end sequence. The transposon pair may comprise any of the modified transposon end sequences described herein.

[0187] In some embodiments, the first transposon comprises a modified transposon end sequence comprising an A16U, A16-8-oxoguanine, or A16 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0188] In some embodiments, the first transposon comprises a modified transposon end sequence comprising a C17-8-oxoguanine, or a C17 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0189] In some embodiments, the first transposon comprises a modified transposon end sequence comprising an A18-8-oxoguanine, or an A18 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0190] In some embodiments, the first transposon comprises a modified transposon end sequence comprising a G19-8-oxoguanine, or a G19 inosine substitution compared to SEQ ID NO:1, and the second transposon comprises a second transposon end sequence that is complementary to SEQ ID NO:1 or a second transposon end that is fully complementary to the first transposon end.

[0191] In some embodiments, there is a mismatch between the first transposon and the second transposon at the position where the first transposon contains a mutation compared to the wild-type mosaic end sequence. In other words, the first transposon and the second transposon do not have to be perfectly complementary (i.e., a U in the first transposon does not have to be base-matched with an A in the second transposon).

[0192] 1. Transposome Complexes in Solution In some embodiments, the transposome complex is in solution, which may be referred to as a solution-phase transposome complex or a soluble transposome complex. In some embodiments, double-stranded nucleic acid (such as DNA) bound to the solution-phase transposome complex undergoes tagmentation to generate nucleic acid fragments that are free in solution. The process referred to herein as "tagmentation" often involves modification of DNA by a transposome complex that includes a transposase enzyme complexed with an adaptor that includes a transposon end sequence. In some embodiments, the solution is a tagmentation buffer.

[0193] Protocols available for soluble tagmentation are well known in the art, such as those described for the Illumina DNA Nextera® XT DNA Library Preparation Kit (see Nextera XT Reference Guide, Document 770-2012-011). Representative data for soluble tagmentation are shown in Figures 7C-9.

[0194] In some embodiments, certain modified transposon ends may function better when the transposition reaction is carried out in solution, for example, modified transposon ends that include ribose may function better when included in a transposome complex in solution compared to when the transposome complex is immobilized on a solid support.

[0195] In some embodiments, the modified transposon ends included in the solution phase transposomes include uracil, inosine, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines. In some embodiments, the modified transposon ends included in the solution phase transposome complexes include ribose.

[0196] In another example, modified transposon ends containing a modification at position 16 of SEQ ID NO:1 may perform better when contained in a transposome complex in solution compared to when contained in a transposome complex immobilized on a solid support, This difference may be due to several factors, including the affinity of the different modified transposon ends for the transposase and the procedure used to prepare bead-linked transposomes.

[0197] 2. Immobilized Transposome Complexes and Bead-linked Transposomes In some embodiments, the transposome complex is immobilized on a solid support. In some embodiments, the solid support is included in a tagmentation buffer. In some embodiments, double-stranded nucleic acid (such as DNA) bound to the immobilized transposome complex undergoes tagmentation to generate immobilized nucleic acid fragments. Such bead-immobilized transposome complexes that can be used for fragmentation can be referred to as "fBLTs". A representative protocol for performing library preparation using fBLTs is shown in FIG. 20.

[0198] In some embodiments, the first transposon comprises uracil, inosine, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines, and the transposome complex is immobilized on a solid support.

[0199] In some embodiments, the density of transposomes immobilized on a solid surface is selected to control the fragment size and library yield of the immobilized fragments. 2 At least 10 per 3 , 10 4 , 10 5 , or 10 6 The complexes are present on the solid support at a density of 100 .mu.m.

[0200] In some embodiments, the length of the double-stranded nucleic acid fragments in the immobilized library is adjusted by increasing or decreasing the density of transposome complexes on the solid support.

[0201] As described in US Pat. No. 9,683,230, which is incorporated herein in its entirety, several different types of immobilized transposomes can be used in these methods.

[0202] In the methods and compositions presented herein, the transposome complexes are immobilized on a solid support. In some embodiments, the transposome complexes and / or capture oligonucleotides are immobilized on a support via one or more polynucleotides, such as a polynucleotide that includes a transposon end sequence. In some embodiments, the transposome complexes may be immobilized via a linker that connects the transposase enzyme to the solid support. In some embodiments, both the transposase enzyme and the polynucleotide are immobilized on a solid support. When referring to the immobilization of a molecule (e.g., a nucleic acid) to a solid support, the terms "immobilized" and "attached" are used interchangeably herein, and both terms are intended to encompass direct or indirect, covalent or non-covalent attachment, unless otherwise indicated, either explicitly or by context. In some embodiments, covalent attachment may be used, but generally, what is required is that the molecule (e.g., a nucleic acid) remains immobilized or attached to the support under conditions under which the support is intended to be used, for example, in applications requiring nucleic acid amplification and / or nucleic acid sequencing.

[0203] Certain embodiments may utilize solid supports comprised of an inert substrate or matrix (e.g., glass slides, polymeric beads, etc.) that have been functionalized by application of a layer or coating of an intermediate material that contains reactive groups that allow for covalent attachment to biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly those described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecule (e.g., polynucleotide) may be covalently attached directly to the intermediate material (e.g., hydrogel), although the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g., glass substrate). The term "covalent attachment to a solid support" should be interpreted accordingly to encompass this type of sequence.

[0204] 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 binding transposome complexes. As will be appreciated by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (e.g., acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, plastics, fiber optic bundles, and various other polymers. In some embodiments, particularly useful solid supports and solid surfaces are placed in a flow cell device. Exemplary flow cells are shown in more detail below.

[0205] In some embodiments, the solid support comprises a patterned surface suitable for immobilization of transposome complexes in a regular pattern. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more of the regions can be features in which one or more transposome complexes are present. The features can be separated by interstitial regions in which no transposome complexes are present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some implementations, the pattern can be a random arrangement of features and / or interstitial regions. In some embodiments, the transposome complexes are randomly distributed on the solid support. In some embodiments, the transposome complexes are distributed on the patterned surface. Exemplary patterned surfaces that can be used in the methods and compositions provided herein are described in U.S. Application Serial No. 13 / 661,524 or U.S. Patent Application Publication No. 2012 / 0316086(A1), each of which is incorporated herein by reference.

[0206] In some embodiments, the solid support comprises an array of wells or depressions on its surface, which can be fabricated as generally known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As will be understood in the art, the technique used will depend on the composition and shape of the array substrate.

[0207] The composition and geometry of the solid support may vary depending on its use. In some embodiments, the solid support is a planar structure, such as a slide, a chip, a microchip, and / or an array. Thus, the surface of the substrate may be in the form of a planar layer. In some embodiments, the solid support comprises one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber that comprises a solid surface through which one or more fluidic reagents can flow. Examples of flow cells and associated fluidic systems and detection platforms that can readily be used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. Nos. 7,329,492, 7,211,414, 7,315,019, 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0208] In some embodiments, the solid support or its surface is non-planar, such as the interior or exterior surface of a tube or container. In some embodiments, the solid support comprises a microsphere or bead. By "microsphere" or "bead" or "particle" or grammatical equivalents herein is meant a small discrete particle. Suitable bead compositions include, but are not limited to, plastic, ceramic, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran such as sepharose, cellulose, nylon, cross-linked micelles, and Teflon, as well as any other material outlined herein for solid supports can all be used. The "Microsphere Selection Guide" from Bangs Laboratories, Fishers, Ind. is a useful guide. In certain embodiments, the microsphere is a magnetic microsphere or bead.

[0209] The beads need not be spherical. Irregular particles may be used. Alternatively or additionally, the beads may be porous. Bead sizes range from nanometers, i.e., 100 nm, to millimeters, i.e., 1 mm, with beads being about 0.2 microns to about 200 microns, or about 0.5 to about 5 microns, although in some embodiments smaller or larger beads may be used.

[0210] In some embodiments, on-bead tagmentation allows for a more homogeneous tagmentation reaction compared to an in-solution tagmentation reaction.

[0211] The density of these surface-bound transposomes can be adjusted by varying the density of the first polynucleotide or by the amount of transposase added to the solid support. For example, in some embodiments, the transposome complexes are present on the solid support at a density of at least 10, 10, 10, or 10 complexes per mm.

[0212] The binding of nucleic acid to the support, whether rigid or semi-rigid, can occur through covalent or non-covalent bonds. Exemplary linkages are described in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, and 7,427,678, and U.S. Patent Publication No. 2011 / 0059865, each of which is incorporated herein by reference. In some embodiments, nucleic acid or other reaction components can be bound to a gel or other semi-solid support, which is then bound or attached to a solid support. In such embodiments, nucleic acid or other reaction components are considered to be solid phase.

[0213] In some embodiments, the solid support comprises a microparticle, a bead, a planar support, a patterned surface, or a well. In some embodiments, the planar support is the interior or exterior surface of a tube.

[0214] In some embodiments, a solid support has a library of prepared tagged DNA fragments immobilized thereon.

[0215] In some embodiments, the solid support comprises a capture oligonucleotide and a first polynucleotide immobilized thereon, the first polynucleotide comprising a 3' portion comprising a transposon end sequence and a first tag.

[0216] In some embodiments, the solid support further comprises a transposase bound to the first polynucleotide to form a transposome complex.

[0217] In some embodiments, the solid support comprises a capture oligonucleotide and a second polynucleotide immobilized thereon, the second polynucleotide comprising a 3' portion comprising a transposon end sequence and a second tag.

[0218] In some embodiments, the solid support further comprises a transposase bound to a second polynucleotide to form a transposome complex.

[0219] In some embodiments, the kit comprises a solid support as described herein. In some embodiments, the kit further comprises a transposase. In some embodiments, the kit further comprises a reverse transcriptase polymerase. In some embodiments, the kit further comprises a second solid support for immobilizing DNA.

[0220] A wide variety of different means of immobilizing transposome complexes are described, for example, in International Publication No. WO 2018 / 156519, which is incorporated herein in its entirety. In some embodiments, the first transposon contained in the transposome complex comprises an affinity element. In some embodiments, the affinity element is attached to the 5' end of the first transposon. In some embodiments, the first transposon comprises a linker. In some embodiments, the linker has a first end attached to the 5' end of the first transposon and a second end attached to the affinity element.

[0221] In some embodiments, the transposome complex further comprises a second transposon that is complementary to at least a portion of the first transposon end sequence. In some embodiments, the second transposon comprises an affinity element. In some embodiments, the affinity element is attached to the 3' end of the second transposon. In some embodiments, the second transposon comprises SEQ ID NO: 13. In some embodiments, the second transposon comprises a linker. In some embodiments, the linker has a first end attached to the 3' end of the second transposon and a second end attached to the affinity element.

[0222] In some embodiments, the affinity element comprises biotin, avidin, streptavidin, an antibody, or an oligonucleotide. In some embodiments, the affinity element is biotin. In some embodiments, the affinity element comprises an oligonucleotide capable of binding to a capture oligonucleotide contained on the surface of the solid support. In some embodiments, the affinity element comprises an antibody capable of binding to a ligand contained on the surface of the solid support.

[0223] As used herein, "bead-linked transposomes" in "BLT" refers to transposomes immobilized on beads. Bead-linked transposomes (BLT) are a key technology in certain NGS library preparation methods, such as Illumina's library preparation products. Bead-linked transposomes leverage the inherent advantages of enzymatic Tn5-mediated tagmentation, with the added advantage of providing library normalization and obviating the need for input DNA quantification (see FIG. 3 and Stephen Bruinsma et al., BMC Genomics 19(1): 1-16 (2018)). A drawback of solution-based tagmentation protocols is that control of the ratio of genomic DNA substrate to Tn5 enzyme directly impacts library fragment size, creating a source of variability in performance. By conjugating a predetermined amount of transposomes to a solid support, BLT allows for greater control of library fragment size. Furthermore, the known amount of transposomes bound to beads provides an upper limit to the amount of DNA substrate that can be converted into a library, resulting in library normalization.

[0224] In some embodiments, the solid support comprises a transposome complex as described herein immobilized thereon. In some embodiments, the solid support comprises beads (i.e., fBLT). Representative data generated using fBLT are shown in Figures 10A-18B.

[0225] B. Rearrangement reaction for fragmentation Transposition is an enzyme-mediated process by which DNA sequences are inserted, deleted, and replicated within a genome. This process has been adapted for widespread use on fragmented double-stranded nucleic acids (such as double-stranded DNA and DNA:RNA duplexes). Transposition can generate DNA fragments without the use of standard fragmentation enzyme protocols as outlined in FIG. 4. In some embodiments, the method of preparing library fragments using modified transposon end sequences is carried out using transposomes (such as fBLT as shown in FIG. 19) immobilized on a solid support. The library preparation method using fBLT can take approximately 5.5 hours (as shown in FIG. 20), which is similar to other ligation-based library preparation methods.

[0226] In some embodiments, the generation of fragments by this method using modified transposon ends (such as fBLT) avoids the DNA damage associated with oxidation during sonication. Such oxidative DNA damage generated from sonication is well known in the art (see, for example, Costello Nucleic Acids Research 41(6): e67(2013)). For example, the use of fBLT reduces false positive G>T transversions by about 50-fold. This is because these transversions are likely driven by oxidative damage to guanine during sonication.

[0227] This transposition reaction is illustrated using Tn5, although other transposases (described below) can mediate a similar reaction.

[0228] The well-tested E. coli Tn5 transposon is mobilized by a "cut and paste" transposition mechanism. First, the Tn5 transposase Tnp (hereafter referred to as Tn5) recognizes conserved substrate sequences on either side of the transposon DNA, which is then excised or "cut" from the genome. Tn5 then inserts or "pastes" this transposon DNA into the target DNA.

[0229] Tn5 has been exploited in many library preparation reagents (such as those from Illumina) for its ability to "tagment", i.e., simultaneously "tag" and "fragment", genomic DNA, thus significantly reducing the time and complexity involved in traditional sonication / ligation-based library preparation protocols. To support its use in library preparation, Tn5 is preloaded with a transposon consisting of conserved substrate sequences, called "mosaic ends" or "end sequences", appended to an adapter sequence (e.g., Illumina's A14 and B15 adapter sequences). This transposome complex, containing Tn5 transposase and the transposon sequence with the adapter, is then mixed with the genomic DNA sample. The resulting library preparation transposon has only the short adapter sequence, thus resulting in simultaneous fragmentation of genomic DNA and tagging with the short adapter sequence (Figure 2).

[0230] In some embodiments, transposition with the modified transposon ends described herein provides results equivalent to transposition with wild-type (i.e., transposon ends that do not contain a mutation described herein). In some embodiments, preparing fragments with a transposome complex described herein results in preparation of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the number of fragments compared to preparing fragments with a transposome complex comprising a first transposon that comprises a transposon end sequence that comprises a wild-type mosaic end sequence comprising SEQ ID NO:1.

[0231] In some embodiments, the transposition reaction is carried out with a transposome complex that contains a modified transposon end at the 3' end of the transferred strand.

[0232] 1. Library fragments generated by a single tagmentation event Typically, tagmentation methods for library preparation require tagging both ends of a fragment to incorporate an adapter sequence used in sequencing. However, the present fragmentation method (e.g., using fBLT) allows the possibility of preparing fragments by a single tagmentation event, where a mosaic end sequence is added to only one end of the fragment. As shown in Figure 18A, after tagmentation / cleavage by a single tagmentation event, both ends of the fragment can undergo end repair and subsequent adapter ligation. Since a single tagmentation event usually results in fragments that are unsequenceable by standard methods (because the adapter sequence is incorporated only at one end), being able to sequence fragments after a single tagmentation event can be called the "rescue" of the single tagmentation event (as shown in the data in Figure 18B).

[0233] In some embodiments, the transposition reaction for fragmentation improves library preparation from samples containing partially fragmented nucleic acids. In some embodiments, the transposition reaction for fragmentation can be used to fragment one end of a DNA molecule, followed by end-repair and ligation of adapters at both the fragmented and unfragmented ends of the molecule. In such a workflow, as shown in FIG. 18A, cleavage of the ME sequence is performed only at one end of the fragment, but the other end of the fragment can also be end-repaired and subsequently adapter-ligated. In this way, adapters are ligated to both ends of the fragment.

[0234] In some embodiments, the fBLT workflow allows the rescue of library fragments prepared using a single tagmentation event. The rescue of library fragments prepared using a single tagmentation event may particularly improve results for samples containing partially fragmented DNA. This is because fragments prepared by two tagmentation events from partially fragmented DNA may be shorter than the preferred length for sequencing, resulting in loss of successful sequencing. This effect may be partially offset by the ability to rescue a single tagmentation event using the methods described herein.

[0235] In some embodiments, the sample comprises partially fragmented DNA. In some embodiments, the sample comprising partially fragmented DNA is formalin-fixed paraffin-embedded (FFPE) tissue or cell-free DNA. In some embodiments, the library comprises fragments prepared by a single tagmentation event.

[0236] 2. Normalization with fBLT The fBLT described herein can be used for library normalization. "Normalization" or "library normalization" as used herein refers to the process of diluting a library of variable 441 concentrations to the same or similar concentrations prior to volume pooling.

[0237] In some embodiments, normalization helps ensure uniform read distribution for all samples during sequencing. In other words, normalizing libraries can help ensure uniform representation in the final sequencing data. In some embodiments, using fBLT for library normalization avoids downstream steps of manual normalization protocols.

[0238] The requirements for manually normalizing library concentrations are well known in the art (see, e.g., Best Practices for Manually Normalizing Library Concentrations, Illumina, April 22, 2021). In some embodiments, the method of normalizing using fBLT does not require calculation of library concentrations. In this way, users can avoid time-consuming and tedious calculations and dilutions during normalization.

[0239] Some bead-linked transposome (BLT) methodologies allow for bead-based normalization, such as Illumina® DNA Prep, (M) Tagmentation (previously known as Nextera DNA Flex). In some embodiments, fBLT also allows for bead-based normalization. The ability to normalize using a bead-based method avoids the time and potential sample loss associated with performing a separate normalization protocol after library preparation.

[0240] Tunable library fragment size determination using C.fBLT In some embodiments, fBLT (instead of solution-phase transposomes) produces uniform fragment sizes and library yields. U.S. Patent No. 9,683,230 and U.S. Patent Application Publication No. 2018-0155709, each of which is incorporated herein by reference in its entirety, describe the use of BLT to control library fragment size.

[0241] Fragment size is a function of the ratio of transposome to the amount and size of DNA and to the duration of reaction.However, even if these parameters are controlled in solution phase tagmentation reaction, size selection fragmentation is generally required as an additional step to remove excess small fragments.In other words, fragment size control can be better managed with BLT compared to solution phase tagmentation.

[0242] In some embodiments, the fBLT allows for the selection of final fragment size as a function of the spatial separation of bound transposomes, independent of the amount of transposome beads added to the tagmentation reaction. A further limitation of solution-based fragmentation is that some form of purification of the products of the tagmentation reaction must typically be performed both before and after PCR amplification. This typically requires some movement of the reaction from tube to tube. In contrast, the tagmentation products on the fBLT can be washed and later released for amplification or other downstream processing, thus avoiding the need for sample transfer. For example, in embodiments in which the transposomes are assembled on paramagnetic beads, purification of the tagmentation reaction products can be easily achieved by immobilizing the beads with a magnet and washing them. Thus, in some embodiments, tagmentation and other downstream processing, such as PCR amplification, can all be performed in a single tube, vessel, droplet, or other vessel.

[0243] In some embodiments, the density of transposomes immobilized on a solid surface is selected to control the fragment size and library yield of immobilized fragments. In some embodiments, the spacing of active transposomes on the bead surface of fBLT can be used to control the size distribution of inserts. For example, gaps on the bead surface can be filled with inactive transposomes (e.g., transposomes with inactive transposons).

[0244] D. Mosaic end removal To allow transformation of Tn5 into a fragmentase system, a mechanism is needed for selective removal of mosaic end sequences after transposition. Such potential mechanisms could include: (1) a restriction enzyme that recognizes a sequence within the mosaic end, (2) a single-stranded DNAse that exploits the 9-nucleotide gap present on either side of the transposition insert, and (3) either (a) an endonuclease, or (b) a combination of a DNA glycosylase with heat, basic conditions, or an endonuclease / lyase that recognizes an abasic site (see FIG. 5). Restriction enzymes are disadvantageous because they cut at other cognate sites in the genomic DNA, resulting in bias. Single-stranded nucleases could also potentially be used to remove mosaic end sequences. However, double-stranded DNA is known to "breathe" at its ends, which often results in off-target digestion of double-stranded DNA and is difficult to control (see Neelam A. Desai and Vepatu Shankar, FEMS Microbiology Reviews 26(5): 457-91 (2003)).

[0245] This method of selectively cleaving mosaic ends using an enzyme is a very attractive mechanism for transforming Tn5 into a fragmentation enzyme system (i.e., generating fragments lacking mosaic ends). As used herein, "base modification" or "DNA base modification" refers to the position of a modified base in a double-stranded nucleic acid (e.g., a modified base described in Table 3) that is recognized by an enzyme (e.g., (a) an endonuclease, or (b) a combination of a DNA glycosylase with heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites) and triggers cleavage at the modified base. In some embodiments, the endonuclease or DNA glycosylase is modification specific.

[0246] In some embodiments, the base modification is cleaved using (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an abasic site-recognizing endonuclease / lyase. For example, a DNA glycosylase can generate an abasic site that is then acted upon by heat, basic conditions, or an abasic site-recognizing endonuclease / lyase. The USER Reagent is an exemplary enzyme mix that includes a DNA glycosylase and an abasic site-recognizing endonuclease / lyase. Users can choose how to cleave at the abasic site depending on their preferred workflow. Figure 7B outlines how a modification-specific endonuclease can cleave the modified base in a one-step reaction, or a modification-specific glycosylase followed by an AP lyase / endonuclease or heat can cleave the modified base in a two-step reaction.

[0247] Fragments prepared from such a rearrangement reaction followed by cleavage at the modified bases contain a 5' overhang having a 5' phosphate and a 3'-OH, and an insert having 0 to 3 bases of the ME sequence, depending on the site of the modification at one or more of positions 16 to 19 of SEQ ID NO:1.

[0248] In some embodiments, cleavage of the modified mosaic end sequence is mediated by (a) an endonuclease, or (b) a combination of a DNA glycosylase and heat, basic conditions, or an abasic site-recognizing endonuclease / lyase. In some embodiments, (a) an endonuclease, or (b) a combination of a DNA glycosylase and heat, basic conditions, or an abasic site-recognizing endonuclease / lyase can mediate cleavage at uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines.

[0249] In some embodiments, the (a) endonuclease or (b) combination of DNA glycosylase and heat, basic condition, or abasic site recognizing endonuclease / lyase is USER, endonuclease V, RNAse HII, formamidopyrimidine-DNA glycosylase (FPG), oxoguanine glycosylase (OGG), endonuclease III (Nth), endonuclease VIII, a mixture of human alkyladenine DNA glycosylase + endonuclease VIII or endonuclease III, a mixture of either thymine-DNA glycosylase (TDG) or mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4) + endonuclease VIII or endonuclease III, or DNA glycosylase / lyase ROS1 (ROS1). In some embodiments, ROS1 can function as a modified endonuclease based on its bifunctional glycosylase / lyase activity.

[0250] In some embodiments, the modified transposon end sequence comprises uracil, the mixture is an N-glycosylase, and the apurinic or apyrimidinic site (AP) lyase / endonuclease is a uracil-specific excision reagent (USER). In some embodiments, the USER is a mixture of uracil DNA glycosylase and endonuclease VIII or endonuclease III.

[0251] In some embodiments, the modified transposon end sequence comprises inosine and the endonuclease is endonuclease V. In some embodiments, the modified transposon end sequence comprises ribose and the endonuclease is RNAse HII.

[0252] In some embodiments, the modified transposon end sequence comprises 8-oxoguanine and the endonuclease is formamidopyrimidine-DNA glycosylase (FPG) or oxoguanine glycosylase (OGG).

[0253] In some embodiments, the modified transposon end sequence comprises thymine glycol and the DNA endonuclease is endonuclease III (Nth) or endonuclease VIII.

[0254] In some embodiments, the modified transposon end sequences include modified purines and the DNA glycosylase and abasic site-recognizing endonuclease / lyase are a mixture of human alkyladenine DNA glycosylase (hAAG) plus endonuclease VIII or endonuclease III.

[0255] In some embodiments, the modified transposon end sequence comprises a modified pyrimidine, the DNA glycosylase is TDG or MBD4, and the abasic site-recognizing endonuclease / lyase is endonuclease VIII or endonuclease III. An alternative modification-specific endonuclease for use with modified transposon ends that contain modified pyrimidines is ROS1.

[0256] In some embodiments, the first transposon comprises a modified transposon end sequence comprising two or more mutations selected from uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine, and the mixture comprises an endonuclease or DNA glycosylase and an endonuclease / lyase that recognizes abasic sites. In some embodiments, the mixture of endonuclease or DNA glycosylase and an endonuclease / lyase that recognizes abasic sites comprises a plurality of enzymes selected from USER, endonuclease V, RNAse HII, formamidopyrimidine-DNA glycosylase (FPG), oxoguanine glycosylase (OGG), endonuclease III (Nth), endonuclease VIII, a mixture of hAAG+endonuclease VIII / endonuclease III, or a mixture of TDG or MBD4 and endonuclease VIII / endonuclease III, or ROS1. In some embodiments, methods using modified transposon end sequences containing two or more mutations and a mixture of endonucleases and / or DNA glycosylases in combination with abasic endonucleases / lyases improve the cleavage efficiency of mosaic end sequences compared to methods using modified transposon end sequences containing a single mutation and a single endonuclease or DNA glycosylase in combination with abasic endonucleases / lyases. For ROS1, a single endonuclease has both glycosylase and lyase functions.

[0257] In some embodiments, a method of fragmenting double-stranded nucleic acid includes combining a sample containing double-stranded nucleic acid with a transposome complex and preparing fragments.

[0258] In some embodiments, a method of preparing double-stranded nucleic acid fragments lacking all or a portion of a first transposon end includes combining a sample containing nucleic acid with a transposome complex and preparing the fragments, combining the sample with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites, and cleaving the first transposon end at a uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine in the mosaic sequence to remove all or a portion of the first transposon end from the fragment. In some embodiments, the modified purine is 3-methyladenine or 7-methylguanine. In some embodiments, the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine. In some embodiments, the method cleaves all or a portion of the first transposon end (transferred strand) from the fragment.

[0259] In some embodiments, the first transposon end is cut to generate a sticky end for adapter ligation. As used herein, a "sticky end" is an end of a double-stranded fragment, where one strand is longer than the other (i.e., there is an overhang), which allows for ligation of an adapter that contains a complementary overhang.

[0260] In some embodiments, the adaptor is added after removing all or part of the first transposon end from the fragment. In some embodiments, the adaptor is added by ligation. In some embodiments, an end repair and A-tailing mix allows for ligation of the adaptor. Those skilled in the art will be aware of other means for adding adaptors, such as PCR amplification or click chemistry.

[0261] E. Adapter Ligation In some embodiments, a method for preparing double-stranded nucleic acid fragments containing adaptors includes combining a sample containing nucleic acid with a transposome complex described herein and preparing the fragments, combining the sample with (1) an endonuclease or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites, and cleaving the first transposon end at a uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within the mosaic end sequence to remove all or a portion of the first transposon end from the fragments, and ligating adaptors onto the 5' and / or 3' ends of the fragments. A representative overview of the steps is shown in FIG. 7A.

[0262] In some embodiments, adaptors containing sequence sequences are ligated onto the library fragments after removal of all or part of the mosaic end sequences. Fragments that have been subjected to adaptor ligation to the 5' and / or 3' ends of the fragments may be referred to as "tagged fragments."

[0263] In some embodiments, the ligating is carried out with a DNA ligase.

[0264] In some embodiments, the adaptor comprises a double-stranded adaptor.

[0265] In some embodiments, adaptors are added to the 5' and 3' ends of the fragments. In some embodiments, the adaptors added to the 5' and 3' ends of the fragments are different.

[0266] A wide variety of library preparation methods that include an adapter ligation step are known in the art, such as TruSeq and TruSight Oncology 500 (see, e.g., TruSeq® RNA Sample Preparation v2 Guide, 15026495 Rev. F, Illumina, 2014). Adapters used in other ligation methods can be used in the present method (see, e.g., Illumina Adapter Sequences, Illumina, 2021). Adapters for use in the present invention also include those described in WO 2008 / 093098, WO 2008 / 096146, WO 2018 / 208699, and WO 2019 / 055715, each of which is incorporated herein by reference in its entirety.

[0267] In some embodiments, adapter ligation may allow for more flexible incorporation of adapters (such as adapters with longer lengths) compared to methods that tag fragments by tagmentation (wherein adapter sequences are incorporated into the fragments during the transposition reaction). In some methods involving tagmentation, additional adapter sequences can be incorporated by PCR reactions (such as those described in U.S. Patent Application Publication No. 2018 / 0201992(A1)), and the method may eliminate the need for an additional PCR step to incorporate additional adapter sequences.

[0268] Ligation techniques are commonly used to prepare NGS libraries for sequencing. In some embodiments, the ligation step uses enzymes to attach specialized adapters to both ends of DNA fragments. In some embodiments, A bases are added to the blunt ends of each strand to prepare them for ligation to sequencing adapters. In some embodiments, each adapter contains a T base overhang, providing a complementary overhang for ligating the adapter to the A-tailed fragmented DNA.

[0269] Adapter ligation protocols are known to have advantages over other methods. For example, adapter ligation can be used to generate the perfect complement of sequencing primer hybridization sites for single reads, paired-end reads, and indexed reads. In some embodiments, adapter ligation eliminates the need for an additional PCR step to add index tags and index primer sites.

[0270] In some embodiments, the adapter comprises a unique molecular identifier (UMI), a primer sequence, an anchor sequence, a universal sequence, a spacer region, an index sequence, a capture sequence, a barcode sequence, a cleavage sequence, a sequencing-related sequence, and combinations thereof. As used herein, a "barcode sequence" refers to a sequence that can be used to distinguish samples. As used herein, a sequencing-related sequence can be any sequence that is associated with a subsequent sequencing step. A sequencing-related sequence can act to simplify downstream sequencing steps. For example, a sequencing-related sequence can be a sequence that is otherwise incorporated via a step of ligating an adapter to a nucleic acid fragment. In some embodiments, the adapter sequence comprises a P5 or P7 sequence (or their complements) to facilitate binding to a flow cell in a particular sequencing method.

[0271] In some embodiments, the adaptor comprises a UMI. In some embodiments, an adaptor comprising a UMI is ligated to both the 3' and 5' ends of the fragment.

[0272] In some embodiments, the adaptor may be a forked adaptor. As used herein, a "forked adaptor" refers to an adaptor that includes two strands of nucleic acid, each of which includes a region that is complementary to the other strand and a region that is not complementary to the other strand. In some embodiments, the two strands of nucleic acid in the forked adaptor are annealed together prior to ligation, and the annealing is based on the complementary regions. In some embodiments, the complementary regions each include 12 nucleotides. In some embodiments, the forked adaptor is ligated to both strands at the ends of the double-stranded DNA fragment. In some embodiments, the forked adaptor is ligated to one end of the double-stranded DNA fragment. In some embodiments, the forked adaptor is ligated to both ends of the double-stranded DNA fragment. In some embodiments, the forked adaptors on both ends of the fragment are different. In some embodiments, one strand of the forked adaptor is phosphorylated at its 5' to facilitate ligation to the fragment. In some embodiments, one strand of the forked adaptor has a phosphorothioate bond immediately preceding the 3'T. In some embodiments, the 3'T is an overhang (i.e., not paired with a nucleotide in the other strand of the forked adaptor). In some embodiments, the 3'T overhang can base pair with an A-tail present on the library fragment. In some embodiments, phosphorothioate bonds block exonuclease digestion of the 3'T overhang. In some embodiments, PCR with partially complementary primers is used after adaptor ligation to extend the ends and resolve the forks.

[0273] In some embodiments, the adaptor may include a tag. As used herein, the term "tag" refers to a portion or domain of a polynucleotide that exhibits a sequence for a desired intended purpose or use. A tag domain may include any sequence provided for any desired purpose. For example, in some embodiments, the tag domain includes one or more restriction endonuclease recognition sites. In some embodiments, the tag domain includes one or more regions suitable for hybridization with primers for a cluster amplification reaction. In some embodiments, the tag domain includes one or more regions suitable for hybridization with primers for a sequencing reaction. It will be understood that any other suitable features may be incorporated into the tag domain. In some embodiments, the tag domain includes a sequence having a length of 5 bp to 200 bp. In some embodiments, the tag domain includes a sequence having a length of 10 bp to 100 bp. In some embodiments, the tag domain includes a sequence having a length of 20 bp to 50 bp. In some embodiments, the tag domain comprises a sequence having a length of 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 bp.

[0274] A tag can include one or more functional sequences or components (eg, a primer sequence, an anchor sequence, a universal sequence, a spacer region, or an index tag sequence) as necessary or desired.

[0275] In some embodiments, the tag comprises a region for cluster amplification, hi some embodiments, the tag comprises a region for priming a sequencing reaction.

[0276] In some embodiments, the method further comprises amplifying the fragment on a solid support by reacting a polymerase with an amplification primer corresponding to a portion of the tag. In some embodiments, the portion of the adaptor ligated onto the fragment after removal of all or a portion of the mosaic end sequence comprises an amplification primer. In some embodiments, the tag of the first transposon comprises an amplification primer.

[0277] In some embodiments, the tag comprises an A14 primer sequence. In some embodiments, the tag comprises a B15 primer sequence.

[0278] In some embodiments, transposomes on individual beads carry a unique index, and when large numbers of such indexed beads are used, phased transcripts result.

[0279] Adapters ligated onto library fragments may have advantages over adapters incorporated during tagmentation. For example, unique molecular identifiers (UMIs) can be used to enable sensitive variant detection by labeling single fragments with unique sequence tags prior to PCR (see Jesse J. Salk, et al., Nature Reviews Genetics 19(5):269-85(2018)). Some library preparation products, such as TSO 500 (Illumina), include a ligation-based UMI offering in which the UMI sequence is incorporated adjacent to the library insert, allowing for simultaneous sequencing as part of the insert read. Thus, the development of fBLT allows existing ligation-based products to be leveraged (such as the use of existing adapters and protocols), while at the same time allowing compatibility with existing enrichment workflows and on-board sequencing primers.

[0280] FIG. 19 presents some representative different adapter workflows that users may wish to use with fBLT. For example, a high-sensitivity UMI workflow can be used, in which the adapter has a UMI built into it. Alternatively, a PCR workflow that adds UMIs during PCR amplification may be used with standard forked adapters. In addition, a PCR-free workflow can be used with indexed forked adapters that avoid the need for PCR. Thus, an advantage of fBLT is that it allows users to select the most targeted adapters for a particular library preparation. Other library preparation methods (such as tagmentation) have higher stringency in the composition of the adapter sequences that can be used.

[0281] F. Samples and Target Nucleic Acids In some embodiments, the sample comprises a nucleic acid. The nucleic acid contained in the sample may be referred to as a "target nucleic acid." In some embodiments, the sample comprises DNA. In some embodiments, the DNA is genomic DNA. In some embodiments, the target nucleic acid is double-stranded DNA.

[0282] In some embodiments, the sample comprises RNA, hi some embodiments, the RNA may be converted to double-stranded cDNA or a DNA:RNA duplex (i.e., RNA hybridized to a single strand of cDNA).

[0283] In some embodiments, the nucleic acid is double-stranded DNA. In some embodiments, the nucleic acid is RNA and a double-stranded cDNA or DNA:RNA duplex is generated prior to combining with the transposome complex.

[0284] A biological sample can be of any type that contains nucleic acid. For example, a sample can contain nucleic acid in various states of purification, including purified nucleic acid. However, a sample need not be completely purified and can contain, for example, nucleic acid mixed with protein, other nucleic acid species, other cellular components, and / or any other contaminants. In some embodiments, a biological sample contains a mixture of nucleic acid, protein, other nucleic acid species, other cellular components, and / or any other contaminants that are present in approximately the same proportions as found in vivo. For example, in some embodiments, the components are found in the same proportions as found in intact cells. In some embodiments, a biological sample has a 260 / 280 absorbance ratio of 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or 0.60 or less. In some embodiments, the biological sample has a 260 / 280 absorbance ratio of at least 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, or 0.60. The methods provided herein allow nucleic acids to be bound to a solid support, so that other contaminants can be removed by simply washing the solid support after surface-bound transfer has occurred. The biological sample can include, for example, a crude cell lysate or whole cells. For example, the crude cell lysate applied to the solid support in the methods described herein does not need to undergo one or more of the separation steps traditionally used to isolate nucleic acids from other cellular components. Exemplary separation steps are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., incorporated herein by reference.

[0285] In some embodiments, the sample applied to the solid support has a 260 / 280 absorbance ratio that is 1.7 or less.

[0286] Thus, in some embodiments, a biological sample may include, for example, blood, plasma, serum, lymph, mucus, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, feces, and macerated tissues thereof, or lysates thereof, or any other biological specimen containing nucleic acid.

[0287] In some embodiments, the sample is blood. In some embodiments, the sample is a cell lysate. In some embodiments, the cell lysate is a crude cell lysate. In some embodiments, the method further comprises lysing cells in the sample after applying the sample to the solid support to produce a cell lysate.

[0288] In some embodiments, the sample is a biopsy sample. In some embodiments, the biopsy sample is a liquid or solid sample. In some embodiments, a biopsy sample from a cancer patient is used to evaluate a sequence of interest to determine if the subject has a particular mutation or variant in a predictor gene.

[0289] One advantage of the methods and compositions presented herein is that a biological sample can be added to a flow cell and subsequent lysis and purification steps can occur all within the flow cell without further movement or handling steps, simply by flowing the necessary reagents through the flow cell.

[0290] G. Gap-filling ligation, phosphorylation, and A-tailing In some embodiments, gaps in the DNA sequence remaining after the translocation event can also be filled in using a strand displacement extension reaction, such as one that includes Bst DNA polymerase and a dNTP mix. In some embodiments, the gap filling ligation is performed using an extension-ligation mix buffer.

[0291] In some embodiments, the method comprises treating multiple 5' fragments with a polymerase and a ligase to extend and ligate the strands to produce a complete double-stranded fragment.

[0292] The library of double-stranded DNA fragments can then optionally be amplified (such as by cluster amplification) and sequenced using a sequencing primer.

[0293] In some embodiments, all or a portion of the first transposon end that is cleaved is split off from the remainder of the sample.

[0294] In some embodiments, the method further comprises filling in the 3' ends of the fragments and phosphorylating the 3' ends of the fragments with a kinase prior to ligation. In some embodiments, the ends generated by cleavage of the mosaic end sequence are not blunt (i.e., one strand of the double-stranded fragment has a sticky overhang compared to the other end). In some embodiments, the sticky overhang is not filled in and an adaptor is ligated onto the sticky overhang, where the adaptor has a complementary sticky end.

[0295] In some embodiments, the fragment comprises 0-3 bases of the mosaic end sequence. In some embodiments, a strand of the double-stranded fragment has a different number of bases from the mosaic end sequence compared to the other strand (i.e., the end of the fragment has an overhang and is not blunt ended). In some embodiments, the overhang created by cleavage of the mosaic end sequence is filled in. In some embodiments, filling of the end created by cleavage of the mosaic end sequence is performed using T4 DNA polymerase.

[0296] In some embodiments, the method further comprises adding a single A overhang to the 3' end of the fragment. In some embodiments, adding a single A overhang may be referred to as "A-tailing." In some embodiments, A-tailing improves ligation of an adaptor, such as a forked adaptor. In some embodiments, one strand of the forked adaptor comprises a T overhang that can base pair with an A tail on the fragment.

[0297] In some embodiments, the polymerase adds a single A overhang, hi some embodiments, the polymerase is (i) Taq or (ii) Klenow fragment, exo-.

[0298] H. Amplification The present disclosure further relates to the amplification of fragments generated according to the methods provided herein. In some embodiments, the fragments are tagged by adapter ligation or at one or both ends of the fragment. In some embodiments, the immobilized fragments are amplified on a solid support. In some embodiments, the solid support is the same solid support on which the surface-bound translocation occurs. In such embodiments, the methods and compositions provided herein allow sample preparation to proceed on the same solid support from the initial sample introduction step, through amplification, and optionally through the sequencing step.

[0299] In some embodiments, the fragments are amplified prior to sequencing.

[0300] For example, in some embodiments, the immobilized fragments are amplified using cluster amplification methodology, as exemplified by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, each of which is incorporated herein by reference in its entirety. The incorporated materials in U.S. Patent Nos. 7,985,565 and 7,115,400 describe a method of solid-phase nucleic acid amplification that allows the amplification products to be immobilized on a solid support to form an array consisting of clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. Arrays so formed are generally referred to herein as "clustered arrays." The product of a solid-phase amplification reaction such as described in U.S. Patent Nos. 7,985,565 and 7,115,400 is a so-called "bridged" structure formed by annealing a pair of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on a solid support at their 5' ends, in some embodiments, via covalent bonds. The cluster amplification methodology is an example of a method for producing immobilized amplicons using an immobilized nucleic acid template. Other suitable methodologies can also be used to produce immobilized amplicons from the immobilized DNA fragments produced according to the methods provided herein. For example, one or more clusters or colonies can be formed by solid-phase PCR, regardless of whether one or both primers of each pair of amplification primers are immobilized.

[0301] In other embodiments, the fragments are amplified in solution. For example, in some embodiments, the fragments are cleaved or otherwise released from the solid support, and then the amplification primers are hybridized to the released molecules in solution. In other embodiments, the amplification primers are hybridized to the tagged fragments for one or more initial amplification steps, followed by subsequent amplification steps in solution. In some embodiments, immobilized nucleic acid templates can be used to generate solution-phase amplicons.

[0302] It will be understood that any of the amplification methods described herein or generally known in the art can be used with universal or target-specific primers to amplify tagged fragments. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA), as described in U.S. Pat. No. 8,003,354, the entirety of which is incorporated herein by reference. The above amplification methods can be used to amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR, SDA, TMA, NASBA, etc., can be used to amplify immobilized DNA fragments. In some embodiments, primers specifically directed to the nucleic acid of interest are included in the amplification reaction.

[0303] Other suitable methods for amplifying nucleic acids include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), incorporated herein by reference), and oligonucleotide ligation assay (OLA) (see generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907, EP 0320308(B1), EP 0336731(B1), EP 0439182(B1), WO 90 / 01069, WO 89 / 12696, and WO 89 / 09835, all of which are incorporated by reference) techniques. It should be understood that these amplification methodologies can be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method can include a ligation probe amplification or oligonucleotide ligation assay (OLA) reaction that includes a primer specifically directed to the nucleic acid of interest. In some embodiments, the amplification method can include a primer extension ligation reaction that includes a primer specifically directed to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify the nucleic acid of interest, the amplification can include primers used in the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified in U.S. Patent Nos. 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.

[0304] Exemplary isothermal amplification methods that can be used in the methods of the present disclosure include, but are not limited to, Multiple Displacement Amplification (MDA), as exemplified, for example, by Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification, as exemplified, for example, by U.S. Pat. No. 6,214,587, each of which is incorporated herein by reference in its entirety. Other non-PCR-based methods that can be used in the present disclosure include strand displacement amplification (SDA), as described, for example, in Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, U.S. Patent Nos. 5,455,166 and 5,130,238, and Walker et al., Nucl.Acids Res.20:1691-96 (1992), or hyperbranched strand displacement amplification, as described, for example, in Lage et al., Genome Research 13:294-307 (2003), each of which is incorporated herein by reference in its entirety. Isothermal amplification methods can be used with strand-displacing Phi 29 polymerase or large fragment, 5'→3' exo- of Bst DNA polymerase for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacement activity. High processivity allows the polymerase to produce fragments of 10-20 kb in length. As noted above, polymerases with low processivity and strand displacement activity, such as Klenow polymerase, can be used to produce smaller fragments under isothermal conditions. Further description of the amplification reaction, conditions, and components are described in detail in the disclosure of U.S. Patent No. 7,670,810, which is incorporated herein by reference in its entirety.

[0305] Another nucleic acid amplification method useful in the present disclosure is tagged PCR, which uses a population of two-domain primers with a constant 5' region followed by a random 3' region, as described, for example, in Grothues, et al. Nucleic Acids Res. 21(5): 1321-2 (1993), which is incorporated herein by reference in its entirety. The first round of amplification is carried out to allow multiple initiations on heat-denatured DNA based on individual hybridization from the randomly synthesized 3' region. Due to the nature of the 3' region, the initiation sites are considered to be random throughout the genome. Unbound primers can then be removed, and further replication can be carried out using primers complementary to the constant 5' region.

[0306] I. Sequencing In some embodiments, the method further comprises sequencing the fragment after removing all or part of the first transposon end from the fragment.

[0307] In some embodiments, the method further comprises sequencing the fragments after adaptor ligation. In some embodiments, the method does not require amplification of the fragments prior to sequencing. In some embodiments, the fragments are amplified prior to sequencing.

[0308] In some embodiments, the method further comprises enriching the fragments of interest after adaptor ligation and prior to sequencing. Enrichment can be performed using a variety of commercially available reagents, such as RNA Prep with Enrichment Reference Guide (Illumina Document No: 1000000124435).

[0309] The present disclosure further relates to sequencing the tagged fragments generated according to the methods provided herein. In some embodiments, the method comprises sequencing one or more of the 5'-tagged fragments and / or the 3'-tagged fragments or the complete double-stranded tagged fragments after ligation of an adaptor at one or both ends of the fragment. In some embodiments, the adaptor comprises a sequence primer binding sequence to facilitate sequencing.

[0310] The tagged fragments can be sequenced according to any suitable sequencing method, such as direct sequencing, including sequencing by synthesis, sequencing by ligation, sequencing by hybridization, nanopore sequencing, etc. In some embodiments, the tagged fragments are sequenced on a solid support. In some embodiments, the solid support for sequencing is the same solid support on which adapter ligation is performed. In some embodiments, the solid support for sequencing is the same solid support on which amplification occurs.

[0311] One exemplary sequencing methodology 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 an amplicon thereof) is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme). In certain polymerase-based SBS embodiments, fluorescently labeled nucleotides are added to the primer (thereby extending the primer) in a template-dependent manner such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template.

[0312] The flow cell provides a convenient solid support for housing the amplified DNA fragments produced by the disclosed method. One or more amplified DNA fragments in such a format can be subjected to SBS or other detection techniques that involve repeated delivery of reagents in cycles. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc. can be flowed / passed through the flow cell housing one or more amplified nucleic acid molecules. The site at which the primer extension incorporates the labeled nucleotide can be detected. Optionally, the nucleotide can further include a reversible termination property that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator portion can be added to the primer such that no further extension occurs until a deblocking agent is delivered to remove the portion. Thus, in embodiments using a reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between the various delivery steps. The cycle can then be repeated n times to extend the primer with n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidics systems and detection platforms that can be readily adapted for use with amplicons generated by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. Nos. 7,329,492, 7,211,414, 7,315,019, 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0313] Other sequencing procedures using cyclic reactions can be used, such as pyrosequencing, which detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); U.S. Patent No. 6,210,891; U.S. Patent No. 6,258,568; and U.S. Patent No. 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, the released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP produced can be detected via luciferase-generated photons. Therefore, the sequencing reaction can be monitored through a luminescence detection system. The excitation radiation source used in the fluorescence-based detection system is not necessary for the pyrosequencing procedure. Useful fluid systems, detectors and procedures that can be adapted to the application of pyrosequencing to the amplicons generated according to the present disclosure are described, for example, in International Publication No. 2012058096, US Patent Application Publication No. 2005 / 0191698(A1), US Patent No. 7,595,883 and US Patent No. 7,244,559, each of which is incorporated herein by reference.

[0314] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation may be detected via fluorescence resonance energy transfer (FRET) interactions between fluorophore-bearing polymerases and γ-phosphate-labeled nucleotides or using zeromode waveguides (ZMW). Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference.

[0315] Some SBS embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use electrical detectors and related technology available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082(A1), 2009 / 0127589(A1), 2010 / 0137143(A1), or 2010 / 0282617(A1), each of which is incorporated herein by reference. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be easily adapted to substrates used to detect protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons used to detect protons.

[0316] Another useful sequencing technique is nanopore sequencing (see, e.g., Deamer et al. Trends Biotechnol. 18, 147-151 (2000); Deamer et al. Acc. Chem. Res. 35:817-825 (2002); Li et al. Nat. Mater. 2:611-615 (2003), 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 the nanopore. As the nucleic acid or nucleotide passes through the nanopore, the type of each nucleotide can be identified by measuring the fluctuation in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni et al. Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference).

[0317] Exemplary methods for array-based expression and genotyping that can be applied for detection according to the present disclosure are described in U.S. Pat. Nos. 7,582,420, 6,890,741, 6,913,884, or 6,355,431, or U.S. Patent Publication Nos. 2005 / 0053980(A1), 2009 / 0186349(A1), or 2005 / 0181440(A1), each of which is incorporated herein by reference.

[0318] An advantage of the methods described herein is that they provide rapid and efficient detection of multiple target nucleic acids in parallel. Thus, the present disclosure provides an integrated system that can prepare and detect nucleic acids using techniques known in the art, such as those exemplified above. Thus, the integrated system of the present disclosure can include fluidic components that can deliver amplification and / or sequencing reagents to one or more immobilized DNA fragments, the system including components such as pumps, valves, reservoirs, fluid lines, etc. A flow cell can be configured and / or used in the integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0111768 (A1) and 2012 / 0270305 (A1), each of which is incorporated herein by reference. As exemplified for the flow cell, one or more of the fluidic components of the integrated system can be used for amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more of the fluidic components of the integrated system can be used for delivery of sequencing reagents in the amplification methods described herein and in the sequencing methods as exemplified above. Alternatively, an integrated system may include separate fluidic systems for performing the amplification method and for performing the detection method. Examples of integrated sequencing systems that can generate amplified nucleic acids and sequence the nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, CA) and the devices described in U.S. Patent Application Publication No. 2012 / 0270305 (hereby incorporated by reference), which is hereby incorporated by reference. EXAMPLES

[0319] Example 1. Representative library preparation method using fBLT A method of using Tn5 with fBLT may include the following steps. 1. Tn5 enzyme is complexed with a mutant mosaic end (ME) transposon that contains an encoded DNA base modification (e.g., uracil, 8-oxoG, etc.) near the 3' end of the transferred strand. If desired, the transposon DNA can be biotinylated to facilitate the formation of a bead-linked transposome (BLT, in this case fBLT). 2. The resulting transposomes are used to fragment input DNA, such as genomic DNA. 3. The resulting fragmented DNA is treated with an appropriate enzyme, which can be (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes an abasic site (e.g., USER or Fpg), resulting in cleavage of the transferred strand. Depending on the site of the modified base and the identity of the enzyme for cleavage, some bases of the mosaic end may remain attached to the library fragment. 4. Use a DNA polymerase to fill in the 3' ends of the library fragments. Depending on the enzyme used, a kinase may also be required to ensure proper phosphorylation for ligation. 5. Use an A-tailing polymerase (e.g., Taq, Klenow Exo-) to add single A overhangs to the 3' ends of the library fragments. 6. Appropriate library adaptors are ligated to the library inserts using DNA ligase.

[0320] In this way, transposition can be used to generate library fragments, while adapters are added to the library fragments by ligation. This method allows for removal of all or part of the first transposon end from the fragments prior to adapter ligation. In some embodiments, all or part of the first transposon end is split off from the remainder of the sample prior to ligation.

[0321] Other modifications of this protocol are possible. For example, alternative strategies such as chemical approaches can be used to selectively cleave the mosaic ends. Furthermore, the short sequences of the remaining "mosaic ends" could potentially be used as an alternative to A-tailing the sample DNA and to facilitate robust "sticky end" ligations of >1 bp, relying on weaker hybridization of single-base overhangs between the sample and the adaptor prior to ligation.

[0322] Example 2. Analysis of mosaic end mutations caused by Tn5v3 Mutation screening experiments were performed focusing on four base pairs at the 3' end of the transferred strand. A FRET activity assay was used to measure the activity of Tn5v3 with modified transposon ends containing mutant mosaic end sequences.

[0323] As shown in Figure 6A, Tn5v3 was able to recognize a variety of canonical mutations within the mosaic end sequence with only a small decrease in activity. Even mosaic ends with multiple mutations were tolerated, but with an approximately two-fold loss of activity. Interestingly, although the A18C mutation resulted in low activity, annealing the mutant transferred strand to the wild-type non-transferred strand rescued activity.

[0324] Having demonstrated that Tn5v3 can tolerate canonical mutations within the mosaic termini, specifically at positions proximal to the 3' end of the transferred strand, we investigated whether it could also tolerate modified bases such as those listed in Table 3. Transposons were prepared with single uracil, inosine, or ribose and assayed using the FRET activity assay. All of the modified base-containing MEs tested were tolerated by Tn5, although with slightly reduced activity (Figure 6B).

[0325] Example 3. Library preparation and sequencing using transposition ligation Based on the finding that uracil, inosine, and ribose modifications of the ME transferred strand were well tolerated by Tn5v3, library preparation using the fragmentase-Tn5 approach was attempted using soluble transposomes (also known as solution-phase transposomes, such as the method outlined in FIG. 7A). Transposomes with modified bases were incubated with 1 ng lambda DNA (NEB N3011S) based on available protocols for soluble tagmentation, such as those described for the Illumina DNA Nextera® XT DNA Library Preparation Kit (see Nextera XT Reference Guide, Document 770-2012-011). The DNA library was then treated with the appropriate enzyme (i.e., (1) an endonuclease, or (2) a combination of DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites), followed by treatment with T4 DNA polymerase to fill in the 3' ends of the library fragments. The libraries were then treated with Illumina A-tailing and ligation mix to ligate UMI-containing forked adapters. After PCR amplification, the resulting libraries were analyzed by Bioanalyzer. All three modified base-enzyme pairs led to the formation of libraries, with USER showing the highest conversion, evident from the 300-400 bp fragment peak (Figure 7C).

[0326] Example 4: Comparison of USER-Fragmentase Library with Alternative Modification Sites The uracil-USER pair (i.e., USER as the enzyme for uracil substitution in the mosaic end sequence and cleavage of the mosaic end sequence after transposition) was selected for further characterization of the fragmentation enzyme library preparation workflow. Transposomes with U16, U17, U18, and U19 modifications at the mosaic end were tested in parallel with the wild-type (WT) mosaic end (SEQ ID NO:1). Electrophoretic analysis of the resulting libraries showed that the fragment size distribution differed based on the modification site (Figure 8A). Furthermore, Qubit quantification of library yields showed that the yield was highest for the U16 ME, and the yield decreased as the uracil modification approached the 3' end (Figure 8B). Since transposomes were normalized by FRET activity for use in library preparation, these differences may be due to variability in USER recognition of these alternative ME substrates. Importantly, wild-type transposomes did not result in library formation, suggesting that cleavage of the mosaic end-transferred strand was required to generate library fragments compatible for ligation, presumably by a gap-filling step with T4 DNA polymerase. In other words, adapters will not ligate onto library fragments unless the mosaic end sequence is cleaved.

[0327] The USER enzyme mix acts by excising uracil bases, and therefore for these modification sites, 0-3 bases of the mosaic end are expected to remain in the resulting libraries. After sequencing the U16, U17, and U18 libraries, evidence of this ME "scar" flanking the library inserts was assessed. The UMI ligation adapters used in this study contained variable 6-7 base pair UMI sequences flanking "T" overhangs, and thus a distribution of library fragments shifted by 1 base pair was expected. Sampling 100,000 sequences from each library type showed the expected sequence signature for each ME modification site (Figure 9).

[0328] Example 5. Fragmentase bead-linked transposome (fBLT) Bead-linked transposomes are typically prepared by biotinylation of transposon DNA, which allows the binding of the resulting transposomes to streptavidin beads. Initial attempts to immobilize U16 transposomes resulted in significantly lower BLT activity than expected (data not shown). Based on this finding, mixed transposons consisting of U16 transferred strands and wild-type non-transferred strands were used in pilot studies for improved performance against BLT. These fBLTs were loaded at a transposome density of 66 activity units / μL (AU / μL) to achieve a library fragment distribution similar to the enriched BLT (eBLT) used in Illumina DNA Prep for Enrichment.

[0329] Preliminary studies were performed to assess the feasibility of fBLT-based library preparation: input DNA consisted of an equimolar mixture of NA12877 and NA12878 human gDNA and SspI-linearized phiX DNA (approximately 15,000 genomic copies each, equivalent to 50 ng human gDNA).

[0330] Fragmentase-BLT libraries were prepared using a streamlined workflow in which USER cleavage, end repair, and A-tailing steps are combined (Figure 10A). For comparison, libraries were also prepared using eBLT according to the protocol described in the DNA Prep with Enrichment Reference Guide (1000000048041). The resulting fBLT libraries had a median library yield of approximately 300 ng and a similar fragment size distribution compared to the eBLT libraries (Figures 10B and 10C). The slightly larger fragment size of the fBLT libraries may be due to the 0.8X SPRI used after adapter ligation.

[0331] After library preparation, libraries were enriched using a composite panel consisting of TruSight Cancer and a custom panel targeting the whole genome of PhiX, following the enrichment protocol described in the RNA Prep with Enrichment Reference Guide (Illumina Document No: 1000000124435). Libraries were sequenced and FASTQ files were trimmed to remove UMI sequences from fBLT samples. Comparative analysis of fBLT and eBLT was performed without UMIs using Dragen Enrichment v3.7.5 to characterize performance with the TruSight Cancer panel. Replicate samples consisting of 10% NA12877 in a background of NA12878 were analyzed for each library type. fBLT libraries performed similarly to eBLT libraries, despite a lower average target coverage depth (Table 4). fBLT performance could be improved by optimizing the workflow and BLT.

[0332] [Table 4]

[0333] Data are reported as the average of two replicates in Table 4. Samples contained a 10% spike of NA12877 gDNA into a background of NA12878 gDNA and were enriched using the TruSight Cancer panel.

[0334] This method enables an enzymatic approach for fragmentation of DNA samples for NGS library preparation, where the resulting fragments are available for adapter ligation. The benefit to the user is that they eliminate the need to purchase expensive sonication equipment as capital equipment and gain the ease and speed of use of a high-throughput enzymatic method for fragmentation of sample nucleic acids. The fBLT technique leverages the inherent advantages of the BLT technique and extends its compatibility to include and repurpose a variety of ligation-based approaches. The key innovation enabling this advance is the incorporation of mutations into the mosaic end sequence to generate modified bases that allow site-specific cleavage of the transferred first transposon end while maintaining recognition by Tn5. By uncoupling the enzymatic fragmentation and adapter tagging steps in the library preparation protocol, the addition of features such as forked adapters, barcodes, and UMIs may be possible while retaining compatibility with standard sequencing methods. Based on these inherent advantages, fBLT can be used for a variety of applications such as UMI library preparation and PCR-free library preparation.

[0335] Example 6. Optimization of conditions for methods using fBLT A variety of fBLTs were examined that contained different modified transposon ends, which contained substitutions at positions A16, C17, A18, or G19 within the mosaic end sequence compared to SEQ ID NO:1.

[0336] Based on available protocols for BLT tagmentation, such as those described for the Illumina DNA Prep with Enrichment Library Preparation kit (see Illumina DNA Prep with Enrichment reference guide, document 1000000048041), bead-linked transposomes with modified mosaic ends (fBLT) were incubated with 10–50 ng of human sample DNA. The DNA library was subsequently treated with an appropriate DNA endonuclease to cleave the mosaic ends. The samples were then subjected to a ligation-based library preparation workflow. The fragmented sample DNA was treated with ligation reagents that enable Illumina end repair, A-tailing, and adapter ligation.

[0337] The results of library conversion using different fBLTs are shown in Figure 12. Experiments were performed to directly evaluate BLT activity without the need for downstream sequencing. Inosine, oxo-guanine, and uracil mutations at position A16 all resulted in lower BLT activity compared to the same mutations at positions C17, A18, and G19. Thus, the modification at position A16 of SEQ ID NO:1 was well tolerated in soluble transposomes, while modifications at other positions resulted in higher BLT activity. Thus, transposon ends with modifications at position A16 may be of higher value for methods using soluble transposomes.

[0338] A variety of different fBLTs with inosine, oxo-guanine, or uracil mutations at positions C17, A18, and G19 were then evaluated, as shown in Figures 13A-13C. The data showed that the inosine modification had the best performance, as measured by library conversion efficiency and variant calling metrics. In particular, the G19I (I19) modification had high performance and could be used with the non-transferred strand biotinylated to enable immobilization onto the fBLT (as outlined in Figure 11).

[0339] While G19I showed a superior profile, G19U (U19) resulted in a relatively large number of chimeric reads, with some of the reads mapping to different chromosomes, in association with the A18I (I18) and C17O (O17) modifications (Figure 14). These chimeric reads may be due to several potential factors, such as the performance of different endonucleases (e.g., cleavage of uracil by the USER Reagents may be less robust than cleavage of other modified nucleotides by their respective endonucleases). Chimeric reads are undesirable sequencing artifacts, and therefore users may prefer to avoid uracil modifications (and subsequent cleavage of mosaic end sequences by the USER Reagents) for certain methods to reduce the risk of chimeric reads.

[0340] Taken together, these data suggest that A18 and G19 modifications, such as G19I and A18I, may exhibit high activity in fBLT.

[0341] Example 7. Comparison of fBLT with other fragmentation methods Fragmentation by A18I(I18)fBLT was compared to fragmentation by NEBNext® dsDNA Fragmentase® or sonication using the workflow shown in Figure 15A.

[0342] Sonicated samples were sheared by a Covaris sonicator (LE220 model) according to the manufacturer's recommended protocol designed for 175 bp fragments (see, e.g., Quick Guide to DNA Shearing with LE220, Covaris, May 2020).

[0343] Sample fragmentation with NEBNExt dsDNA Fragmentase was performed according to the manufacturer's protocol and reagents. To predetermine optimal sample incubation conditions, a time course study was performed using various samples of interest. A 10 minute incubation at room temperature (approximately 20° C.) was the best single condition available for all samples of interest.

[0344] Fragmentation by fBLT was carried out as described in Example 6.

[0345] End repair and A-tailing were performed in the same manner for all fragments using Illumina reagents. The same pool of UMI-containing forked adaptors was ligated to fragments prepared by each method, outlining the advantage of the fBLT method, which can use existing adaptors developed for other types of ligation-based library preparation.

[0346] After PCR amplification, the resulting libraries were enriched using the TruSight Cancer panel following the enrichment protocol described in the RNA Prep with Enrichment Reference Guide (Illumina Document No: 1000000124435).

[0347] The sample used for evaluation was a 50ng input genomic DNA (gDNA) 1% mixture of NA12877 in a NA12878 background (50ng input). Based on this mixture, there are 84 expected heterozygous variants (resulting in a variant allele frequency (VAF) of 0.5%). The results are shown in Figure 15B, and the fBLT method shows higher sensitivity and specificity compared to either the NEBNext® dsDNA Fragmentase® or sonication protocols.

[0348] Error rates were also evaluated using different fragmentation methods. As shown in Figure 16, substantially higher duplex and simplex forward error rates were observed for sonicated samples compared to samples prepared using fBLT or NEBNext® dsDNA Fragmentase®. Such increased error rates generally indicate that there is more noise in the data, i.e., there is more variability in the sequenced library fragments.

[0349] The strand G>T error rate for samples prepared using fBLT was 1.4 × 10 -5 This error rate was 70 x 10 for samples prepared via sonication, whereas -5 These data indicate that there was an approximately 50-fold reduction in false-positive G>T transversions for the fBLT method compared with the sonication method. The improved error rate with fBLT is likely due to the fBLT method avoiding oxidative damage to guanine that can be induced by sonication.

[0350] FIG. 17 shows that across a range of samples, the fBLT method outperformed the enzymatic NEBNext® dsDNA Fragmentase® method, giving similar library conversion efficiencies as the sonication protocol.

[0351] Thus, fBLT is a means of preparing libraries that has the advantages of improved sensitivity / specificity and reduced error rate compared to other currently used library preparation methods involving fragmentation. Sample 1 in FIG. 17 represents a genomic DNA sample, while samples 2-6 represent formalin-fixed paraffin-embedded (FFPE) samples. The higher conversion efficiency of sample 1 compared to the other samples is a function of the higher quality of DNA in the genomic DNA sample compared to the FFPE sample, as is well known in the art. The increase in dCq from samples 2-6 indicates that sample quality was poorer for the higher numbered samples and higher quality for sample 1 with genomic DNA.

[0352] Figure 19 summarizes some advantages of the fBLT method, including the ability of users to select a variety of different adapters to ligate onto fragments prepared with fBLT based on their preferred downstream workflow. For example, users desiring a streamlined workflow can use indexed forked adapters, which can avoid downstream PCR to incorporate index sequences. Similarly, users desiring high sensitivity for calling different fragments can use adapters containing UMIs (UMI adapters), allowing amplicons of the same fragment to be identified from sequencing results after PCR amplification. Thus, fBLT can combine the advantages of tagmentation for library preparation with the flexibility of ligation-based protocols, allowing a wide range of different adapters to be incorporated into library fragments.

[0353] Example 8. fBLT for use with formalin-fixed paraffin-embedded samples We developed a protocol for preparing library fragments from formalin-fixed paraffin-embedded (FFPE) samples using fBLT. FFPE samples can contain important information, such as profiles from tumor samples, but FFPE material is often highly fragmented, which can interfere with standard library preparation protocols.

[0354] As shown in Figure 18A, DNA is often partially fragmented in FFPE tissue. Standard tagmentation protocols require two tagmentation events per library fragment (i.e., a tagmentation event at each end of the fragment). However, two tagmentation events with DNA from FFPE tissue may result in a high proportion of very small fragments that are not suitable for sequencing due to this partial fragmentation of the starting DNA in FFPE samples.

[0355] In contrast, fBLT can be used to prepare single tagmented fragments (i.e., fragments where fBLT has tagged only one end of the fragment), which can be rescued by adaptor ligation. After mosaic end-cleavage, both ends of the fragment can be repaired and adaptors ligated. In this way, library fragments from FFPE tissues can be generated by a single tagmentation event followed by ligation at both ends of the fragment, resulting in the rescue of these fragments.

[0356] As shown in Figure 18B, fragments prepared from DNA in FFPE can be rescued when prepared by a single tagmentation event with fBLT. Thus, the fBLT workflow can improve library preparation from FFPE tissues and other samples that may contain partially fragmented DNA.

[0357] Example 9. Workflow for fBLT library preparation and optional enrichment Based on optimization experiments, a preliminary workflow for fBLT library preparation and subsequent enrichment was developed. This workflow is shown in FIG. 20. In summary, after tagmentation with BLT, the tagmentation products are cleaned and then the mosaic ends (MEs) are cleaved. After end repair and A-tailing, adapters are ligated (users can choose adapters such as those containing UMIs). If desired, users can then perform solid-phase reversible immobilization (SPRI) bead purification, followed by indexing PCR and another SPRI bead purification. Such a workflow can take approximately 5.5 hours. The time for this workflow is similar to other ligation-based library preparation protocols.

[0358] If the user wishes to enrich the library, this can be done by hybridization followed by capture, etc. Such a method can take approximately 5 hours.

[0359] Equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples set forth certain embodiments in detail and explain the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in the text, it will be understood that the embodiments can be embodied in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0360] As used herein, the term about refers to numerical values, including, for example, integers, ratios, and percentages, whether or not expressly indicated. The term about generally refers to a range of numerical values ​​(e.g., ±5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When a term such as at least and about precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges provided in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. 1. A method for preparing an adaptor-containing double-stranded nucleic acid fragment, comprising: a. A sample containing nucleic acid, i. a transposase; ii. a first transposon comprising modified transposon end sequences comprising uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines; a second transposon comprising a second transposon end sequence complementary to at least a portion of the first transposon end sequence; b. Preparing a fragment; c. combining the sample with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites, and cleaving the first transposon end with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within a mosaic end sequence to remove all or a portion of the first transposon end from the fragment; d. ligating adaptors onto the 5' and / or 3' ends of the fragments.

2. 2. The method of claim 1, wherein the modified purine is 3-methyladenine or 7-methylguanine, and / or the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

3. The method of claim 1 or 2, wherein (1) the nucleic acid is double-stranded DNA, or (2) the nucleic acid is RNA, and a double-stranded cDNA or a DNA:RNA duplex is generated prior to combination with the transposome complex.

4. 4. The method according to any one of claims 1 to 3, wherein all or part of the first transposon end that is cleaved is split off from the remainder of the sample, or the method further comprises filling in the 3' end of the fragment and phosphorylating the 3' end of the fragment with a kinase prior to ligation, optionally wherein the filling is carried out using T4 DNA polymerase.

5. 5. The method of claim 4, further comprising adding a single A overhang to the 3' end of said fragment, optionally wherein a polymerase adds said single A overhang, and optionally wherein said polymerase is (i) Taq or (ii) Klenow fragment, exo-.

6. The method of any one of claims 1 to 5, wherein the fragment comprises 0 to 3 bases of the mosaic end sequence.

7. The method further comprising sequencing the fragments after ligating the adapters, and optionally: a. the method does not require amplification of the fragment prior to sequencing, or b. The fragment is amplified prior to sequencing; The method according to any one of claims 1 to 6.

8. 8. The method of claim 7, further comprising enriching the fragments of interest after ligating the adaptors and prior to sequencing.

9. a. The modified transposon end sequence contains uracil, and the combination of DNA glycosylase and abasic site-recognizing endonuclease / lyase is a uracil-specific excision reagent (USER); b. the modified transposon end sequence comprises an inosine and the endonuclease is endonuclease V; c. the modified transposon end sequence comprises a ribose and the endonuclease is RNAse HII; d. the modified transposon end sequence comprises 8-oxoguanine and the endonuclease is formamidopyrimidine-DNA glycosylase (FPG) or oxoguanine glycosylase (OGG); e. the modified transposon end sequence comprises thymine glycol and the DNA glycosylase is endonuclease EndoIII (Nth) or Endo VIII; f. the modified transposon end sequence comprises a modified purine, and the DNA glycosylase is human 3-alkyladenine DNA glycosylase, and the endonuclease is endonuclease III or VIII; or g. the modified transposon end sequence comprises a modified pyrimidine; i. the DNA glycosylase is thymine-DNA glycosylase (TDG) or mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4), and the endonuclease / lyase that recognizes abasic sites is an endonuclease that is endonuclease III or VIII; or ii. The endonuclease is DNA glycosylase / lyase ROS1 (ROS1); The method according to any one of claims 1 to 8.

10. a. The USER is a mixture of uracil DNA glycosylase and endonuclease VIII or endonuclease III; b. the modified purine is 3-methyladenine or 7-methylguanine, or c. the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine; 10. The method of claim 9.

11. 11. The method of any one of claims 1 to 10, wherein the first transposon comprises a modified transposon end sequence comprising two or more mutations selected from uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, or modified pyrimidine, and the (1) endonuclease or (2) a combination of a DNA glycosylase and a heat, basic condition, or abasic site recognizing endonuclease / lyase is an enzyme mixture, and optionally the modified purine is 3-methyladenine or 7-methylguanine, and / or the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

12. 12. The method of any one of claims 1 to 11, wherein cleaving the first transposon end generates a sticky end for ligating the adapter, optionally the sticky end being more than one base long.

13. a. The adaptor comprises a double-stranded adaptor; b. Adapters are added to the 5' and 3' ends of the fragments; c. The adapter comprises a unique molecular identifier (UMI), a primer sequence, an anchor sequence, a universal sequence, a spacer region, an index sequence, a capture sequence, a barcode sequence, a cleavage sequence, a sequencing-related sequence, and combinations thereof; d. the adapter comprises a UMI, and / or e. The adapter is a fork-type adapter; The method according to any one of claims 1 to 12.

14. 14. The method of claim 13, wherein the adaptors added to the 5' and 3' ends of the fragment are different, and adaptors optionally containing UMIs are ligated to both the 3' and 5' ends of the fragment.

15. The method of claim 1, wherein the ligation is carried out using a DNA ligase. b. the method is carried out in a single reaction vessel; c. The density of transposomes immobilized on a solid surface is selected to control the fragment size and library yield of the immobilized fragments; d. The method allows for bead-based normalization. e. the sample contains partially fragmented DNA; f. the sample is formalin-fixed, paraffin-embedded tissue or cell-free DNA; and / or g. The library comprises fragments prepared by a single tagmentation event. The method according to any one of claims 1 to 14.

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