Method for preparation of directional tagmentation sequencing libraries using transposon-based technology with unique molecular identifiers for error correction - Patent Application 20070236333

Abstract

Materials and methods for preparing nucleic acid libraries for next generation sequencing are described herein.Various approaches are described for the use of unique molecular identifiers using transposon-based technology in the preparation of sequencing libraries.Sequencing materials and methods for identifying and correcting amplification and sequencing errors are also described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 168,802, filed March 31, 2021, which is incorporated herein 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 in a file entitled "2022-03-29_01243-0024-00PCT_Sequence_Listing_ST25.txt" (created March 29, 2022, size 4 kilobytes). The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] This application relates to the preparation of DNA and RNA sequencing libraries using transposon-based technology to incorporate unique molecular identifiers (UMIs) that increase the sequencing sensitivity of low frequency variants. [Background technology]

[0004] Next-generation sequencing (NGS) has enabled cancer researchers to evaluate a large number of genes in a single assay using highly accurate sequencing data. However, all synthesis-based methods contain inherent errors. Although error rates are low enough (<0.5%) to successfully accomplish many NGS-based applications, new methods using non-invasive or other methods for sample collection that yield lower concentrations of target nucleic acid may require lower error rates. For example, analysis of cell-free DNA (cfDNA) can be used to detect somatic mutations in blood without the need for biopsy, but the low proportion of circulating tumor DNA (ctDNA) in total cfDNA results in mutant allele frequencies near the detection limit of existing methods. Artifacts that may arise from library preparation methods may be mistaken for low-frequency variants, thereby reducing the sensitivity and reliability of the method.

[0005] Transposon-based technologies can be used to prepare whole genome sequencing libraries. For example, Illumina DNA Prep (RUO), formerly known as Nextera DNA Flex Library Prep, supports a wide nucleic acid input range (1-500 ng), multiple sample types, and both small and large genomes. In less than 4 hours, libraries of 350 base pair fragments can be generated by treating the target nucleic acid with the transposome complex, which allows the nucleic acid to be simultaneously fragmented and tagged ("tagmented") for sequencing.

[0006] Libraries prepared according to transposon-based technology can be improved by incorporating unique molecular identifiers (UMIs) to reduce the inherent error rate in NGS data. The incorporation of UMIs into sequencing libraries allows the UMI Error Correction App to recognize multiple reads from the same target molecule and collapse them into a single read, reducing errors in the final variant call. UMIs combined with stranded (i.e., forked) libraries can resolve individual stranded molecules in sequencing data. The present disclosure provides materials and methods for preparing UMI libraries using transposon-based technology. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates to materials, compositions, and methods for preparing nucleic acid sequencing libraries containing UMIs using transposon-based technology.

[0008] Embodiment 1 is a method of generating a double-stranded nucleic acid library, each fragment in the library comprising a unique molecular identifier (UMI), the method comprising: (a) applying a sample comprising a double-stranded target nucleic acid to a first transposome complex comprising: (i) a first transposase; (ii) a first transposon comprising a first 3'-end transposon end sequence, a first adapter sequence, and a first UMI; and (iii) a second transposon comprising a sequence that is fully or partially complementary to the first 3'-end transposon end sequence; and (b) tagging the double-stranded target nucleic acid with the first transposome complex to generate tagmented double-stranded target nucleic acid fragments. (c) releasing the tagmented double-stranded target nucleic acid fragments from the first transposome complex; (d) optionally extending the tagmented double-stranded target nucleic acid fragments; (e) optionally ligating a first transposon to the tagmented double-stranded target nucleic acid fragments or the extended tagmented double-stranded target nucleic acid fragments; (f) generating the tagmented double-stranded target nucleic acid fragments; and (g) amplifying the tagmented double-stranded target nucleic acid fragments.

[0009] Embodiment 2 is the method of embodiment 1, wherein the first UMI in the first transposon is located between the first adapter sequence and the first 3' transposon end sequence.

[0010] Embodiment 3 is the method of embodiment 1 or 2, wherein the first adapter sequence in the first transposon is located between the first UMI and the first 3' transposon end sequence.

[0011] Embodiment 4 is the method of any one of embodiments 1 to 3, further comprising a second transposome complex comprising: (a) a second transposase; (b) a third transposon comprising a second adapter sequence and a second 3' transposon end sequence; and (c) a fourth transposon comprising a sequence that is fully or partially complementary to the second 3' end transposon end sequence.

[0012] Embodiment 5 is the method of embodiment 4, wherein the tagmentation step generates a tagmented double-stranded target nucleic acid fragment comprising: (a) a first strand comprising a first adapter sequence and a first UMI; and (b) a second strand comprising a second adapter sequence.

[0013] Embodiment 6 is the method of embodiment 4 or 5, wherein (a) the third transposon further comprises a second UMI, and (b) the second adapter sequence is located between the second UMI and the second 3' transposon end sequence.

[0014] Embodiment 7 is the method of embodiment 6, wherein the tagmentation step generates a double-stranded target nucleic acid fragment comprising (a) a first strand comprising a first adapter sequence and a first UMI, and (b) a second strand comprising a second adapter sequence and a second UMI.

[0015]

[0023] Embodiment 8 is a method of making a double-stranded nucleic acid library, each fragment in the library comprising a UMI, the method comprising: (a) applying a sample comprising a double-stranded target nucleic acid to a transposome complex comprising: (i) a transposase; (ii) a first transposon comprising a first 3'-end transposon end sequence and a first adaptor sequence; and (iii) a second transposon comprising a sequence that is fully or partially complementary to the first 3'-end transposon end sequence; (b) tagmenting the first strand of the double-stranded target nucleic acid with the transposome complex to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising a first adaptor sequence; and (c) tagging the first strand of the double-stranded target nucleic acid with the transposome complex to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising a first adaptor sequence; (d) releasing the acid fragment from the transposome complex; (e) hybridizing a polynucleotide comprising a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3'-terminal transposon sequence; (f) optionally extending a second strand of the tagmented double-stranded target nucleic acid fragment; (g) optionally ligating a polynucleotide to the tagmented double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment; (g) generating a tagmented double-stranded target nucleic acid fragment comprising a UMI, wherein the UMI is located immediately adjacent to the 3'-end of the insert DNA; and (h) amplifying the tagmented double-stranded target nucleic acid fragment comprising a UMI.

[0016]

[0023] Embodiment 9 is a method of generating a double-stranded nucleic acid library, each fragment in the library comprising a UMI, the method comprising: (a) applying a sample comprising a double-stranded target nucleic acid to a transposome complex comprising: (i) a transposase; (ii) a first transposon comprising a first 3'-end transposon end sequence and a first adaptor sequence; and (iii) a second transposon comprising a sequence that is fully or partially complementary to the first 3'-end transposon end sequence; (b) tagmenting the first strand of the double-stranded target nucleic acid with the transposome complex to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising a first adaptor sequence; and (c) subjecting the tagmented double-stranded target nucleic acid fragments to transposome amplification. (d) hybridizing a first polynucleotide comprising a UMI and a second adapter sequence; (e) optionally adding a second polynucleotide comprising a region complementary to the first polynucleotide to generate a double-stranded adapter; (f) optionally extending the second strand of the tagmented double-stranded target nucleic acid fragment; (g) optionally ligating the second polynucleotide to the second strand of the extended tagmented double-stranded target nucleic acid fragment; (h) generating a tagmented double-stranded target nucleic acid fragment comprising a UMI, wherein the UMI is located between the double-stranded target nucleic acid fragment and the second adapter sequence; and (i) amplifying the tagmented double-stranded target nucleic acid fragment comprising a UMI.

[0017] Embodiment 10 is the method of embodiment 9, wherein after the hybridizing step, the method further comprises: (a) extending the second strand of the double-stranded target nucleic acid fragment; and (b) copying the first polynucleotide.

[0018]

[0023] Embodiment 11 is a method of making a double-stranded nucleic acid library, wherein each fragment in the library comprises two different UMIs, the method comprising: (a) subjecting a sample comprising a double-stranded target nucleic acid to (i) a first transposome complex comprising (1) a first transposase and (2) a first forked adapter comprising (a) a first transposon on a first strand of the double-stranded target nucleic acid fragment and (b) a second transposon, the first transposon comprising a first 3' end transposon end sequence, a first copy of the first adapter sequence, and a first UMI, and the second transposon comprising a first 3' end transposon end sequence, a first copy of the first adapter sequence, and a first UMI. (ii) a first transposome complex, the first copy of the first adapter sequence being single-stranded and the first copy of the second adapter sequence comprising a double-stranded portion; and (iii) a second transposome complex, the second transposome complex comprising (1) a second transposase and (2) (a) a third transposon on the second strand of the double-stranded target nucleic acid fragment and (b) a fourth transposon. a forked adaptor; and a third transposon comprising a second 3'-end transposon end sequence, a second copy of the first adaptor sequence, and a second UMI, wherein the third transposon comprises a second copy of the second adaptor sequence and a sequence that is fully or partially complementary to the second 3'-end transposon end sequence and the second UMI, further wherein the second copy of the first adaptor sequence is single-stranded and the second copy of the second adaptor sequence comprises a double-stranded portion; and (b) a second transposome complex comprising a double-stranded target nucleic acid. with a forked adapter to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising a first and a second copy of a first adapter sequence, a first UMI, a first and a second copy of a second adapter sequence, and a second UMI; (c) releasing the tagmented double-stranded target nucleic acid fragments from the transposome complex; (d) optionally extending the tagmented double-stranded target nucleic acid fragments; and (e) combining a second and a fourth transposon with(f) ligating the double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment to generate a tagmented double-stranded target nucleic acid fragment; and (g) amplifying the tagmented double-stranded target nucleic acid fragment.

[0019]

[0023] Embodiment 12 is a method of making a double-stranded nucleic acid library, each fragment in the library comprising four different UMIs, the method comprising: (a) subjecting a sample comprising a double-stranded target nucleic acid to (i) a first transposome complex comprising (1) a first transposase and (2) a first forked adapter comprising (a) a first transposon on a first strand of the double-stranded target nucleic acid fragment and (b) a second transposon, the first transposon comprising a first 3' end transposon end sequence, a first copy of the first adapter sequence, a first copy of the first UMI, and a first copy of a second adaptor sequence, the second transposon comprising a sequence that is fully or partially complementary to the first 3' end transposon end sequence, a first copy of a third adaptor sequence, a first copy of a second UMI, and a fourth adaptor sequence, further wherein the first, second, and third adaptor sequence first copies are single-stranded and the fourth adaptor sequence comprises a double-stranded portion; and (i) a second transposome complex comprising (1) a second transposase and (2)(a) a double-stranded target nucleic acid fragment. and (b) a second forked adapter comprising a fourth transposon, wherein the third transposon comprises a second 3'-end transposon end sequence, a first copy of a fifth adapter sequence, a first copy of a third UMI, and a first copy of a sixth adapter sequence, the fourth transposon comprises a sequence that is fully or partially complementary to the second 3'-end transposon end sequence, a first copy of a seventh adapter sequence, a first copy of a fourth UMI, and an eighth adapter sequence, and further comprising the fifth, sixth, and eighth adapter sequences. (b) tagmenting the double-stranded target nucleic acid with the forked adapter to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising first copies of the first, second, third, fifth, sixth, and seventh adapter sequences, first copies of the first, second, third, and fourth UMIs, the sixth adapter sequence, and the eighth adapter sequence;(c) releasing the tagmented double-stranded target nucleic acid fragment from the transposome complex; (d) optionally extending the tagmented double-stranded target nucleic acid fragment; (e) ligating a second and a fourth transposon to the double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment; (f) generating a tagmented double-stranded target nucleic acid fragment; and (g) amplifying the tagmented double-stranded target nucleic acid fragment.

[0020] Embodiment 13 is the method of any one of embodiments 6, 7, 11, or 12, wherein the first, second, third, and fourth UMI can be complementary or different sequences.

[0021] Embodiment 14 is the method according to any one of embodiments 1 to 13, wherein the double-stranded target nucleic acid is double-stranded DNA.

[0022] Embodiment 15 is the method according to any one of embodiments 1 to 13, wherein the double-stranded target nucleic acid is ctDNA.

[0023] Embodiment 16 is the method of any one of embodiments 1 to 13, wherein the double-stranded target nucleic acid is cfDNA.

[0024] Embodiment 17 is the method according to any one of embodiments 1 to 13, wherein the double-stranded target nucleic acid is RNA.

[0025] Embodiment 18 is the method according to any one of embodiments 1 to 13, wherein the double-stranded target nucleic acid is cDNA or the DNA:RNA duplex is generated from RNA.

[0026] Embodiment 19 is the method according to any one of embodiments 1 to 18, wherein the first adaptor sequence is a 5' first read sequencing adaptor sequence.

[0027] Embodiment 20 is the method according to any one of embodiments 1 to 19, wherein the second adaptor sequence is a 5' second read sequencing adaptor sequence.

[0028] Embodiment 21 is the method according to any one of embodiments 1 to 20, wherein the first and second adaptor sequences are 5' first read and 5' second read sequencing adaptor sequences.

[0029] Embodiment 22 is the method of any one of embodiments 1 to 21, wherein the 5' first read and 5' second read sequencing adapter sequences comprise a unique primer binding site.

[0030] Embodiment 23 is the method of any one of embodiments 1, 2, 4-8, or 13-22, wherein the first UMI is on the first strand of the tagmented double-stranded target nucleic acid fragment.

[0031] Embodiment 24 is a method according to any one of embodiments 1, 3, 5-7, 13-22, wherein a first copy of the first UMI is on a first strand of the tagmented double-stranded target nucleic acid fragment and a second copy of the first UMI is on a second strand.

[0032] Embodiment 25 is a method according to any one of embodiments 1 to 7, 13 to 22, wherein the first UMI is on a first strand of the tagmented double-stranded target nucleic acid fragment and the second UMI is on a second strand of the tagmented double-stranded target nucleic acid fragment.

[0033] Embodiment 26 is the method of any one of embodiments 1 to 25, wherein the first, second, third, or fourth transposon further comprises a biotin tag.

[0034] Embodiment 27 is the method of any one of embodiments 1 to 26, wherein the first, second, third, or fourth transposon further comprises a first unique primer binding sequence.

[0035] Embodiment 28 is the method of embodiment 27, wherein the first, second, third, or fourth transposon further comprises a second unique primer binding sequence.

[0036] Embodiment 29 is the method of embodiment 27 or 28, wherein the unique primer binding sequence comprises A2, A14, and / or B15.

[0037] Embodiment 30 is the method according to any one of embodiments 8 to 10 or 14 to 22, wherein the hybridizing step produces a forked adapter.

[0038] Embodiment 31 is the method of any one of embodiments 1 to 30, further comprising extending from the 3' end of the double-stranded target nucleic acid fragment to the 5' end of the transposon.

[0039] Embodiment 32 is a method according to any one of embodiments 1 to 7 or 11 to 31, wherein the ligation step comprises ligating the 3' end of the tagmented double-stranded target nucleic acid fragment or the 3' end of the extended tagmented double-stranded target nucleic acid fragment to the 5' end of the first, second, or fourth transposon.

[0040] Embodiment 33 is the method according to any one of embodiments 1 to 32, wherein the extension and / or ligation steps are optionally carried out in an extension-ligation mixture.

[0041] Embodiment 34 is the method of any one of embodiments 8, 15-22, and 26-33, wherein the polynucleotide comprises (a) a hairpin UMI, (b) a hairpin UMI and a universal hybridizing tail, (c) a splint ligation adaptor, or (d) a 3' adaptor comprising a 3' template switch oligonucleotide.

[0042] Embodiment 35 is the method of embodiment 34, wherein the hairpin UMI is stable during the extension and / or ligation steps but not during the amplification step.

[0043] Embodiment 36 is the method of embodiment 34 or 35, wherein the hairpin UMI comprises a 3 or 4 base pair stem.

[0044] Embodiment 37 is the method of any one of embodiments 34 to 36, wherein the universal hybridizing tail comprises a nucleotide that can bind to any DNA nucleotide.

[0045] Embodiment 38 is the method of any one of embodiments 34 to 37, wherein the ligation step comprises ligating the 3' end of the second strand of the tagmented double-stranded target nucleic acid fragment to the 5' end of the universal hybridizing tail.

[0046] Embodiment 39 is the method of embodiment 34, wherein (a) the polynucleotide comprises a 3' adaptor comprising a hairpin UMI, and (b) the extension step comprises extending from the 3' end of the second strand of the tagmented double-stranded target nucleic acid fragment to the 5' end of the hairpin UMI.

[0047] Embodiment 40 is the method of embodiment 39, wherein the ligation step comprises ligating the 3' end of the second strand of the extended tagmented double-stranded target nucleic acid fragment to the 5' end of the hairpin UMI.

[0048] Embodiment 41 is the method of embodiment 34, wherein (a) the polynucleotide comprises a splint ligation adaptor, and (b) the extending step comprises extending from the 3'-end of the second strand of the tagmented double-stranded target nucleic acid fragment to the 5'-end of the splint ligation adaptor.

[0049] Embodiment 42 is the method of embodiment 41, wherein the extending step comprises extending 9 bases.

[0050] Embodiment 43 is the method of embodiment 41 or 42, wherein the ligation step comprises ligating the 3' end of the second strand of the extended tagmented double-stranded target nucleic acid fragment to the 5' end of the first strand of the splint ligation adaptor.

[0051] Embodiment 44 is the method of any one of embodiments 34, wherein (a) the polynucleotide comprises a template switch oligonucleotide; (b) the extending step comprises extending from a 3' end of a second strand of the tagmented double-stranded target nucleic acid fragment to a junction in the template switch oligonucleotide by copying a first strand of the tagmented double-stranded target nucleic acid fragment; (c) switching the template from the first strand to the unpaired region of the 3' template switch oligonucleotide; and (d) copying the unpaired region of the 3' template switch oligonucleotide from the junction to the 5' end of the unpaired region of the 3' template switch oligonucleotide.

[0052] Embodiment 45 is the method of embodiment 44, wherein the extending, switching and copying are performed by a polymerase capable of DNA-directed template switching.

[0053] Embodiment 46 is the method of embodiment 44 or 45, wherein the polymerase capable of DNA-directed template switching comprises MMLV reverse transcriptase.

[0054] Embodiment 47 is a method according to any one of embodiments 1 to 33, wherein the ligation step comprises ligating the 3' end of the tagmented double-stranded target nucleic acid fragment to the 5' end of the first, second, or fourth transposon.

[0055] Embodiment 48 is the method of any one of embodiments 1 to 33 or 47, further comprising selecting amplified nucleic acid fragments within a size range after the amplification step.

[0056] Embodiment 49 is the method of any one of embodiments 1 to 48, wherein the amplifying step comprises adding oligonucleotides to one or both ends of the tagmented double-stranded target nucleic acid fragments to attach the library to a solid support.

[0057] Embodiment 50 is the method according to any one of embodiments 1 to 49, wherein the amplifying step comprises adding at least a first lead sequencing oligonucleotide and / or a second lead sequencing oligonucleotide.

[0058] Embodiment 51 is the method according to any one of embodiments 1 to 50, wherein the amplifying step comprises adding at least a P5 oligonucleotide and a P7 oligonucleotide.

[0059] Embodiment 52 is the method according to any one of embodiments 1 to 51, wherein the amplifying step comprises adding at least a plurality of i5 oligonucleotides and a plurality of i7 oligonucleotides.

[0060] Embodiment 53 is the method of any one of embodiments 1 to 52, wherein the transposome complex, the first transposome complex and / or the second transposome complex are on a solid support.

[0061] Embodiment 54 is the method of any one of embodiments 1 to 53, wherein the transposome complex, the first transposome complex and / or the second transposome complex are in solution.

[0062] Embodiment 55 is a method for sequencing a double-stranded nucleic acid library generated by the method according to any one of embodiments 1 to 54, wherein UMIs are sequenced to increase sensitivity in DNA sequencing.

[0063] Embodiment 56 is the method of embodiment 55, comprising combining sequencing primers having similar melting temperatures.

[0064] Embodiment 57 is the method of embodiment 55 or 56, comprising binding a sequencing primer comprising a sequence that is fully or partially complementary to the unique primer binding sequence.

[0065] Embodiment 58 is the method according to any one of embodiments 55 to 57, comprising sequencing a primer having at least the A2 sequence.

[0066] Embodiment 59 is the method according to any one of embodiments 55 to 57, comprising sequencing a primer having at least the A14 sequence and the B15 sequence.

[0067] Embodiment 60 is the method according to any one of embodiments 55 to 59, comprising sequencing the primers with at least the bridging primer.

[0068] Embodiment 61 is the method of any one of embodiments 55 to 60, further comprising a dark cycle, wherein data is not recorded for a portion of the sequencing method.

[0069] Embodiment 62 is the method according to any one of embodiments 55 to 60, wherein the unrecorded data is sequence data relating to the 3' transposon end sequence.

[0070] Embodiment 63 is the method of any one of embodiments 55 to 60, wherein the method eliminates the need for a dark cycle.

[0071] Embodiment 64 is the method of embodiment 1 or 9, wherein the extension step comprises a polymerase that copies the UMI or the first UMI to generate a double-stranded UMI.

[0072] Embodiment 65 is a transposome complex comprising: (a) a transposase; (b) a first transposon comprising a 3' transposon end sequence and a 5' adapter sequence; and (c) a second transposon comprising a sequence that is fully or partially complementary to the first 3' end transposon end sequence.

[0073] Embodiment 66 is a transposome complex described in embodiment 65, wherein the 5' adapter sequence of the first transposon comprises the A14 sequence (sequence number 4), the A2 sequence (sequence number 7), and / or the B15 sequence (sequence number 5).

[0074] Embodiment 67 is a transposome complex according to embodiment 65 or 66, wherein the first transposon further comprises a UMI sequence.

[0075] Embodiment 68 is a transposome complex according to any one of embodiments 65 to 67, wherein the first or second transposon comprises A14-ME (SEQ ID NO: 1).

[0076] Embodiment 69 is a transposome complex according to any one of embodiments 65 to 67, wherein the first or second transposon comprises B15-ME (SEQ ID NO: 2).

[0077] Embodiment 70 is the transposome complex according to any one of embodiments 65 to 67, wherein the 3′ transposon end sequence of the first transposon comprises ME (SEQ ID NO: 6) or ME′ (SEQ ID NO: 3).

[0078] Embodiment 71 is the transposome complex according to any one of embodiments 65 to 67, wherein the 3′ transposon end sequence of the second transposon comprises ME (SEQ ID NO: 6) or ME′ (SEQ ID NO: 3).

[0079] Embodiment 72 is the transposome complex of embodiment 67, wherein the second transposon further comprises a 3' adapter sequence, and the 3' adapter sequence of the second transposon is partially or completely complementary to the 5' adapter sequence of the first transposon.

[0080] Embodiment 73 is the transposome complex of embodiment 67, wherein the second transposon further comprises a 3' adapter sequence, and no portion of the 3' adapter sequence of the second transposon is complementary to the 5' adapter sequence of the first transposon.

[0081] Embodiment 74 is a transposome complex according to embodiment 72 or 73, wherein the 3' adapter sequence of the second transposon comprises the A14 sequence (SEQ ID NO: 4), the A2 sequence (SEQ ID NO: 7), the B15 sequence (SEQ ID NO: 5), the X sequence, the Y' sequence, the A sequence, and / or the B sequence.

[0082] Embodiment 75 is a transposome complex according to embodiment 72 or 74, wherein the second transposon further comprises a sequence that is complementary to the UMI sequence of the first transposon.

[0083] Embodiment 76 is the transposome complex of embodiment 73 or 74, wherein the second transposon further comprises a UMI, and the UMI of the second transposon comprises a sequence that differs from the UMI of the first transposon.

[0084] Embodiment 77 is a transposome complex according to embodiment 75 or 76, further comprising an oligonucleotide complementary to the B15 sequence or the A14 sequence.

[0085] Embodiment 78 is a transposome complex according to embodiment 76, further comprising: (a) an A adaptor sequence adjacent to the A14 sequence; (b) a B adaptor sequence adjacent to the B15 sequence; (c) an X adaptor sequence adjacent to the ME sequence; and / or (d) a Y' adaptor sequence adjacent to the ME' sequence.

[0086] Embodiment 79 is the transposome complex according to any one of embodiments 65 to 78, wherein the transposome complex is immobilized on a solid support via the first or second transposon.

[0087] Embodiment 80 is the transposome complex of embodiment 77, wherein the transposome complex is immobilized to a solid support via complementary oligonucleotides.

[0088] Embodiment 81 is a transposome complex according to embodiment 79 or 80, wherein the solid support is a bead.

[0089] Embodiment 82 is a kit comprising the transposome complex according to any one of embodiments 65 to 81.

[0090] Embodiment 83 is a kit for producing the transposome complex according to any one of embodiments 65 to 81.

[0091] 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.

[0092] 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.

[0093] 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]

[0094] [Figure 1]1 illustrates an embodiment in which a capture oligonucleotide is used to tagment a DNA fragment using a bead-linked transposome (BLT). [Diagram 2] We demonstrate the incorporation of unique molecular identifiers (UMIs) using A2 adapters. This method combines BLT with the Hyb2Y workflow to generate tagmented DNA libraries suitable for sequencing with the advantage of double-stranded UMI error correction. UMIs can contain randomized sequences. [Figure 3A] Figure 3 shows the sequencing of a duplex UMI DNA library prepared as described in Example 1. Figure 3A shows the standard sequence of Illumina DNA Prep with enrichment of Illumina DNA Prep and primers standard read 1, standard read 2, standard i5, and standard i7. Figure 3B shows the Nextera sequencing method with four custom primers and 19 dark cycles. The gray arrows indicate where the custom primers anneal. Figure 3C shows the quality of each cycle in an exemplary sequencing run, expressed as the percentage probability of Q30 or higher. Figure 3D shows the sequencing signal intensity using i7 and i5 primers in an exemplary sequencing run. Figure 3E compares the percentage of duplex families of the BLT duplex UMI design (described in Figure 2) and the TruSight UMI (TruSight Duplex) method. [Figure 3B]Figure 3 shows the sequencing of a duplex UMI DNA library prepared as described in Example 1. Figure 3A shows the standard sequence of Illumina DNA Prep with enrichment of Illumina DNA Prep and primers standard read 1, standard read 2, standard i5, and standard i7. Figure 3B shows the Nextera sequencing method with four custom primers and 19 dark cycles. The gray arrows indicate where the custom primers anneal. Figure 3C shows the quality of each cycle in an exemplary sequencing run, expressed as the percentage probability of Q30 or higher. Figure 3D shows the sequencing signal intensity using i7 and i5 primers in an exemplary sequencing run. Figure 3E compares the percentage of duplex families of the BLT duplex UMI design (described in Figure 2) and the TruSight UMI (TruSight Duplex) method. [Figure 3C] Figure 3 shows the sequencing of a duplex UMI DNA library prepared as described in Example 1. Figure 3A shows the standard sequence of Illumina DNA Prep with enrichment of Illumina DNA Prep and primers standard read 1, standard read 2, standard i5, and standard i7. Figure 3B shows the Nextera sequencing method with four custom primers and 19 dark cycles. The gray arrows indicate where the custom primers anneal. Figure 3C shows the quality of each cycle in an exemplary sequencing run, expressed as the percentage probability of Q30 or higher. Figure 3D shows the sequencing signal intensity using i7 and i5 primers in an exemplary sequencing run. Figure 3E compares the percentage of duplex families of the BLT duplex UMI design (described in Figure 2) and the TruSight UMI (TruSight Duplex) method. [Figure 3D]Figure 3 shows the sequencing of a duplex UMI DNA library prepared as described in Example 1. Figure 3A shows the standard sequence of Illumina DNA Prep with enrichment of Illumina DNA Prep and primers standard read 1, standard read 2, standard i5, and standard i7. Figure 3B shows the Nextera sequencing method with four custom primers and 19 dark cycles. The gray arrows indicate where the custom primers anneal. Figure 3C shows the quality of each cycle in an exemplary sequencing run, expressed as the percentage probability of Q30 or higher. Figure 3D shows the sequencing signal intensity using i7 and i5 primers in an exemplary sequencing run. Figure 3E compares the percentage of duplex families of the BLT duplex UMI design (described in Figure 2) and the TruSight UMI (TruSight Duplex) method. [Figure 3E] Figure 3 shows the sequencing of a duplex UMI DNA library prepared as described in Example 1. Figure 3A shows the standard sequence of Illumina DNA Prep with enrichment of Illumina DNA Prep and primers standard read 1, standard read 2, standard i5, and standard i7. Figure 3B shows the Nextera sequencing method with four custom primers and 19 dark cycles. The gray arrows indicate where the custom primers anneal. Figure 3C shows the quality of each cycle in an exemplary sequencing run, expressed as the percentage probability of Q30 or higher. Figure 3D shows the sequencing signal intensity using i7 and i5 primers in an exemplary sequencing run. Figure 3E compares the percentage of duplex families of the BLT duplex UMI design (described in Figure 2) and the TruSight UMI (TruSight Duplex) method. [Figure 4] 1 shows sequencing of a double-stranded UMI DNA library by bridging primer rehybridization. [Figure 5A] The transposome structure (FIG. 5A) and workflow (FIG. 5B) of UMI-BLT are shown. TsTn5=transposase. [Figure 5B]The transposome structure (FIG. 5A) and workflow (FIG. 5B) of UMI-BLT are shown. TsTn5=transposase. [Figure 6A] Sequencing of a double-stranded UMI library with (Figure 6A) and without (Figure 6B) a dark cycle is shown. [Figure 6B] Sequencing of a double-stranded UMI library with (Figure 6A) and without (Figure 6B) a dark cycle is shown. [Figure 7] 4 shows Q30% scores for sequencing runs using IDPE, TruSeq™, unforked UMI-BLT with dark cycle, and unforked UMI-BLT with bridging primer rehybridization. Q30% scores are shown for read 1 and read 2. [Figure 8] 1 shows the BLT and enrichment workflow used to prepare a DNA library with a single UMI from cfDNA. In some embodiments, cfDNA was extracted using the Circulating Nucleic Acid Kit (Qiagen, Cat #55114). [Figure 9] The incorporation of a single UMI using a classical Nextera adapter is shown. Although this method does not allow indexing of the sample, standard sequencing methods can capture the incorporated UMI from the index read. In some embodiments, the UMI is read using a standard sequencing primer. [Figure 10] Shown is the % of total reads indicating that UMIs were successfully incorpo- rated into tagmented DNA fragments and were evenly distributed across the tagmented library. [Figure 11A] The single UMI-BLT library shows greater average target coverage and higher conversion of cfDNA to the library than the TruSeq™ library (shown as "No UMI" in FIG. 11A). FIG. 11A shows the deduplicated average target coverage provided by Read Collapsing analysis. FIG. 11B compares the TruSeq™ method with the single UMI-BLT method (shown as "eBBN" in FIG. 11B). [Figure 11B] The single UMI-BLT library shows greater average target coverage and higher conversion of cfDNA to the library than the TruSeq™ library (shown as "No UMI" in FIG. 11A). FIG. 11A shows the deduplicated average target coverage provided by Read Collapsing analysis. FIG. 11B compares the TruSeq™ method with the single UMI-BLT method (shown as "eBBN" in FIG. 11B). [Figure 12] 1 shows the incorporation of a duplex UMI using a forked adapter capture oligonucleotide in BLT to generate a unique double index (UDI)-matched DNA library for sequencing. [Figure 13] 1 shows the incorporation of a duplex UMI using a forked adapter capture oligonucleotide in a BLT to generate a sequencing-ready DNA library compatible with the UDI. [Figure 14] Shown is ligation with Hyb2Y and a 3' adaptor containing a hairpin-UMI and a universal hybridization 5' tail (universal hybridization tail). This method utilizes Tn5 at A14 only. The ligation step is performed after Hyb2Y and no extension step is required. In some embodiments, the universal hybridization tail contains inosine bases capable of universal Watson-Crick base pairing. In some embodiments, the universal hybridization tail can hybridize to A14 and / or B15. * indicates the ligation junction. In some embodiments, the universal hybridization 5' can hybridize to A14 and B15. [Figure 15] Shown is Hyb2Y, extension, and ligation to a 3' adaptor containing a hairpin UMI. After Hyb2Y, an extension step occurs, followed by a ligation step. In some embodiments, the hairpin stem contains 3-4 base pairs for stability. In some embodiments, the hairpin loop contains about 4 bases. * indicates the ligation junction. [Figure 16] Hyb2Y, extension, and ligation with the 3' adaptor complex are shown. This method utilizes A14-only Tn5. In some embodiments, the splint ligation adaptor comprises two portions, a splint portion and a tail portion. Each portion is about 50 nucleotides in length. In some embodiments, A14', ME, and / or X can be cleaved or removed. * indicates the ligation junction. [Figure 17] 1 shows a template switch-off ME sequence method utilizing Tn5 on A14 only. A template switch extension step is performed after the hybridization step. In some embodiments, a template switch as long as about 70 nucleotides can be used. In some embodiments, the switch oligonucleotide can form a secondary structure (i.e., fold) on itself, thereby preventing it from functioning as intended in certain embodiments. Folding of the switch oligonucleotide can be avoided by using a TruSeq™ adapter sequence in place of the ME on the P7 side (indicated with ***). In some embodiments, A14' can be truncated or omitted. ** indicates the template switch junction. [Figure 18A] Addition of 3'UMI and adapter sequences using polymerase template switching is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 18A). Single stranded polymerase template switch adapters are added using Hyb2Y (Figure 18B). Insert DNA is extended using a polymerase that can switch templates from insert DNA to polymerase template switch adapters (Figure 18C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 18D). [Figure 18B]Addition of 3'UMI and adapter sequences using polymerase template switching is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 18A). Single stranded polymerase template switch adapters are added using Hyb2Y (Figure 18B). Insert DNA is extended using a polymerase that can switch templates from insert DNA to polymerase template switch adapters (Figure 18C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 18D). [Figure 18C] Addition of 3'UMI and adapter sequences using polymerase template switching is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 18A). Single stranded polymerase template switch adapters are added using Hyb2Y (Figure 18B). Insert DNA is extended using a polymerase that can switch templates from insert DNA to polymerase template switch adapters (Figure 18C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 18D). [Figure 18D] Addition of 3'UMI and adapter sequences using polymerase template switching is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 18A). Single stranded polymerase template switch adapters are added using Hyb2Y (Figure 18B). Insert DNA is extended using a polymerase that can switch templates from insert DNA to polymerase template switch adapters (Figure 18C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 18D). [Figure 19A]Addition of a 5' adapter sequence and a 3' UMI using polymerase extension and proximity is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 19A). A 5' double stranded adapter is added using Hyb2Y (Figure 19B). A UMI is added to the insert DNA using polymerase extension and proximity 5' ligation (Figure 19C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 19D). [Figure 19B] Addition of a 5' adapter sequence and a 3' UMI using polymerase extension and proximity is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 19A). A 5' double stranded adapter is added using Hyb2Y (Figure 19B). A UMI is added to the insert DNA using polymerase extension and proximity 5' ligation (Figure 19C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 19D). [Figure 19C] Addition of a 5' adapter sequence and a 3' UMI using polymerase extension and proximity is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 19A). A 5' double stranded adapter is added using Hyb2Y (Figure 19B). A UMI is added to the insert DNA using polymerase extension and proximity 5' ligation (Figure 19C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 19D). [Figure 19D] Addition of a 5' adapter sequence and a 3' UMI using polymerase extension and proximity is shown. Tagmentation of target DNA was performed using A14 transposomes (Figure 19A). A 5' double stranded adapter is added using Hyb2Y (Figure 19B). A UMI is added to the insert DNA using polymerase extension and proximity 5' ligation (Figure 19C). PCR is used to amplify libraries from A14 and B15 using sample index and flow cell primers (Figure 19D). [Figure 20]Compare certain embodiments that add a 3'UMI that is in-line with the insert DNA, i.e. adjacent to it. In certain embodiments, template switch extension is used. In certain embodiments, extension and ligation is used. [Figure 21A] Specific embodiments are shown for attaching transposome complex oligonucleotides to a solid support surface. These embodiments provide an option to aid the utility of BLT as a target enrichment method that may be compromised by the presence of 5' biotinylated library fragments. Figure 21A shows indirect 3' biotinylated binding of Tsm adaptors through complementary base pairing in the adaptors. Figure 21B shows direct 3' biotinylated binding. Figure 21C shows direct 5' biotinylated binding. [Figure 21B] Specific embodiments are shown for attaching transposome complex oligonucleotides to a solid support surface. These embodiments provide an option to aid the utility of BLT as a target enrichment method that may be compromised by the presence of 5' biotinylated library fragments. Figure 21A shows indirect 3' biotinylated binding of Tsm adaptors through complementary base pairing in the adaptors. Figure 21B shows direct 3' biotinylated binding. Figure 21C shows direct 5' biotinylated binding. [Figure 21C] Specific embodiments are shown for attaching transposome complex oligonucleotides to a solid support surface. These embodiments provide an option to aid the utility of BLT as a target enrichment method that may be compromised by the presence of 5' biotinylated library fragments. Figure 21A shows indirect 3' biotinylated binding of Tsm adaptors through complementary base pairing in the adaptors. Figure 21B shows direct 3' biotinylated binding. Figure 21C shows direct 5' biotinylated binding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0095] Array Description Table 1 provides a list of specific sequences referenced herein. All sequences are shown either N-terminus to C-terminus or 5' to 3' for protein and nucleic acid sequences, respectively. The specific sequences in Table 1 represent exemplary sequences from a library of sequences. For example, as described in Section II.A below, "UMI" represents a library of UMI sequences. In another example, an ME sequence may contain sequence mutations when compared to the exemplary ME of SEQ ID NO:6. Similarly, an A14-ME sequence may contain sequence mutations when compared to the exemplary A14-ME of SEQ ID NO:1. Sequence mutations may include, for example, nucleic acid mutations, nucleic acid substitutions, nucleic acid deletions, nucleic acid additions, nucleic acid insertions, sequence truncations, longer sequences, shorter sequences, UMI sequences, primer sequences, index tag sequences, capture sequences, barcode sequences, cleavage sequences, anchor sequences, universal sequences, spacer sequences, transposon end sequences, sequencing-related sequences, and any combination thereof. In another example, sequencing-related primers and adapters may refer to a library of primers and adapters. The library of i5 and i7 sequences is provided by Illumina Adapter Sequences Document#1000000002694 v15, which is incorporated herein by reference in its entirety. In exemplary custom primers such as SEQ ID NOs: 10 and 11, the i5 and i7 portions may contain sequence mutations as provided by Illumina Adapter Sequences Document#1000000002694 v15.

[0096] [Table 1]

[0097] I. Definition As used herein, "hybridization sequence" or "HYB" refers to a sequence that can hybridize to a complementary hybridization sequence. Hybridization of HYB in one library product to HYB' in another library product can result in a hybridization adduct, where the two library products anneal to each other via HYB / HYB' hybridization.

[0098] As used herein, "Hyb2Y" or "Hyb2Y workflow" refers to the use of HYB / HYB' to generate forked adaptor structures (also known as Y adaptor structures). In some, but not all, cases, this process also involves the replacement of one oligonucleotide with another.

[0099] In the context of bead-linked transposomes (BLT), generating a forked adapter structure using "Hyb2Y", i.e., HYB / HYB', results in removing the non-transferred strand from the Tn5 transposome product complex and replacing it with another oligonucleotide that may contain additional sequence relative to the replacing oligonucleotide, thereby creating a new forked structure or maintaining an existing forked structure of the adapter being used.

[0100] As used herein, "insert sequence" refers to a region of a target nucleic acid that is included in a polynucleotide. A polynucleotide may contain multiple insert sequences.

[0101] As used herein, "stacked read" refers to a sequencing read of multiple insert sequences generated from a single polynucleotide. These sequencing reads can be consecutive. For example, a polynucleotide containing two or more insert sequences and two or more primer sequences can be used to generate stacked reads. As used herein, "stacked read library" refers to a library of polynucleotides containing multiple insert sequences that can be used to generate stacked reads.

[0102] As used herein, "sequencing by synthesis" or "SBS" refers to sequences incorporated into a polynucleotide to improve binding of a lead primer. In embodiments where the polynucleotides are generated from library products produced by tagmentation, the SBS may be a mosaic end sequence and the SBS' may be the complement of a mosaic end sequence such as ME and ME'. When the library products are produced using the TruSeq™ method (Illumina), the SBS and SBS' sequences may also be included in the adapters.

[0103] II. Preparation of UMI libraries using transposon-based technology Unique molecular identifiers (UMIs) are nucleic acid sequences that are incorporated into double-stranded nucleic acid libraries for identification and correction of sequencing errors and PCR replication. UMIs are used to distinguish one source DNA molecule from another when many DNA molecules are sequenced together. UMIs can help identify sequencing and PCR artifacts, as well as errors from strand-specific DNA damage, such as those typically found in formalin-fixed, paraffin-embedded, FFPE tissues. UMIs allow for the reduction of noise from errors that occur during PCR amplification and sequencing, allowing the detection of single-base variants (SNVs) (e.g., in cell-free DNA, cfDNA) at allele frequencies below 1%.

[0104] The materials and methods described herein can be used in conjunction with transposon-based technology to incorporate UMIs into double-stranded nucleic acid libraries. As used herein, a "UMI library" is a library of double-stranded nucleic acid fragments, each fragment containing at least one UMI. In certain embodiments described herein, each fragment can contain one, two, or more UMIs.

[0105] Disclosed herein are methods for generating sequencing libraries that are combined with transposon-based technology. In some embodiments, the transposon-based technology includes a workflow for a series of DNA Prep products by Illumina® to generate a population of double-stranded nucleic acid fragments tagged with unique adapter sequences at the ends of the fragments. Disclosed are various HYB or HYB' sequences for use in transposition reactions. In some embodiments, the method is carried out in a solution mixture. In some embodiments, a solid support such as BLT is used.

[0106] In many embodiments, the method of preparing a UMI library includes a first step of applying a sample having a double-stranded target nucleic acid to one, two, or more transposome complexes.

[0107] In some embodiments, after the first step, the method of preparing a UMI library further comprises: (1) tagmenting the nucleic acid to generate a nucleic acid fragment comprising a UMI and an adapter sequence; (2) releasing the nucleic acid fragment from the transposome complex; (3) ligating the transposon or an extended transposon to the nucleic acid fragment; and (4) generating a nucleic acid fragment comprising a UMI. In some embodiments, the method further comprises an optional extension step after the release step, in which the double-stranded target nucleic acid fragment is extended. This extension step is also known as gap filling.

[0108] In some embodiments, after the first step, the method of preparing a UMI library further comprises: (1) tagmenting the nucleic acid to generate a nucleic acid fragment comprising an adaptor sequence; (2) releasing the nucleic acid fragment from the transposome complex; and (3) hybridizing a polynucleotide comprising the adaptor sequence and the UMI for incorporation of the UMI. The polynucleotide further comprises a sequence that is fully or partially complementary to the 3'-terminal transposon sequence. The method may further comprise an optional step of extending the second strand of the double-stranded target nucleic acid fragment. The method may further comprise an optional step of ligating the polynucleotide or the extended polynucleotide. In some embodiments, the method further comprises generating a double-stranded target nucleic acid fragment with a UMI, where the UMI is located immediately adjacent to the 3'-end of the insert DNA.

[0109] In some embodiments, after the first step, the method of preparing a UMI library further comprises (1) tagmenting the first strand of the double-stranded target nucleic acid with a transposon to generate double-stranded target nucleic acid fragments comprising a first adapter sequence, (2) releasing the double-stranded target nucleic acid fragments from the transposome complex, and (3) hybridizing a first polynucleotide comprising the UMI and a second adapter sequence. In some embodiments, the method may further comprise the optional steps of (1) adding a second polynucleotide comprising a region complementary to the first polynucleotide to generate double-stranded adapters, (2) extending the second strand of the double-stranded target nucleic acid fragments, and / or (3) optionally ligating the double-stranded adapters to the double-stranded target nucleic acid fragments.

[0110] In some embodiments, after the first step, the method of preparing a UMI library further comprises: (1) tagmenting the double-stranded target nucleic acid with a forked adapter transposon to generate a double-stranded target nucleic acid fragment comprising a first and second copy of a first adapter sequence, a first UMI, a first and second copy of a second adapter sequence, and a second UMI; (2) releasing the double-stranded target nucleic acid fragment from the transposome complex; and (3) ligating the forked adapter transposon to the double-stranded target nucleic acid fragment. In some embodiments, after the releasing step, the double-stranded target nucleic acid fragment is extended, in which case a subsequent ligation step ligates the extended forked adapter transposon to the double-stranded target nucleic acid fragment.

[0111] In many embodiments, after the UMI library is generated, the method further comprises amplifying the UMI library.

[0112] In some embodiments, the UMI is incorporated during tagmentation using a transposon adaptor. In some embodiments, the UMI is incorporated after tagmentation using a polynucleotide adaptor. In some embodiments, the UMI is incorporated by extending and / or ligating a polynucleotide adaptor. In some embodiments, the UMI is incorporated prior to library amplification.

[0113] Aspects of each of these steps are described in the following sections.

[0114] A. Unique Molecular Identifier (UMI) Unique molecular identifiers (UMIs) are sequences of nucleotides applied to or identified in nucleic acid molecules that can be used to distinguish individual nucleic acid molecules from one another. UMIs can be sequenced along with the nucleic acid molecules to which they are associated to determine whether the read sequences are one source nucleic acid molecule or another. The term "UMI" can be used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide itself. UMIs are similar to barcodes that are commonly used to distinguish reads of one sample from reads of other samples, but UMIs are instead used to distinguish a nucleic acid template fragment from another fragment when many fragments from individual samples are sequenced together. UMIs can be defined in many ways, as described in WO2019 / 108972 and WO2018 / 136248, which are incorporated herein by reference.

[0115] UMIs may be single-stranded or double-stranded and may be at least 5 bases, at least 6 bases, at least 7 bases, at least 8 bases, or more. In certain embodiments, UMIs are 5-8 bases, 5-10 bases, 5-15 bases, 5-25 bases, 8-10 bases, 8-12 bases, 8-15 bases, or 8-25 bases, etc., in length. Further, in certain embodiments, UMIs are 30 bases or less, 25 bases or less, 20 bases or less, 15 bases or less in length. It should be understood that the length of the UMI sequences provided herein may refer to the unique / distinguishable portions of the sequence and may exclude adjacent common or adapter sequences (e.g., p5, p7) that may function as sequencing primers and are common among multiple UMIs with different identifier sequences.

[0116] UMIs can be defined in many ways, as described in WO 2018 / 136248, which is incorporated herein by reference. UMIs can be random, pseudorandom or partially random, or non-random nucleotide sequences inserted into adapters or otherwise incorporated into source DNA molecules to be sequenced. In some embodiments, UMIs are unique, and each UMI can provide a unique identification for any given source DNA molecule present in a sample. As described herein, transposon adapters and polynucleotide adapters can be used to incorporate UMIs into the target nucleic acid to be sequenced, and each individual sequenced molecule has a UMI that helps distinguish it from all other fragments. In some embodiments, multiple different physical UMIs can be used to uniquely identify DNA fragments in a sample. In some embodiments, the UMI is long enough to ensure uniqueness for each and every source DNA molecule.

[0117] In some embodiments, the library of UMIs includes nonrandom sequences. In some embodiments, nonrandom UMIs (nrUMIs) are predefined for a particular experiment or application. In certain embodiments, rules are used to generate a set of sequences or to select samples from a set to obtain nrUMIs. For example, the set of sequences can be generated such that the sequences have a particular pattern. In some embodiments, each sequence differs from all other sequences in the set by a particular number of nucleotides (e.g., 2, 3, or 4). That is, an nrUMI sequence cannot be converted to any other available nrUMI sequence by replacing fewer than a particular number of nucleotides. In some implementations, the set of UMIs used in the sequencing process includes fewer UMIs than all possible UMIs given a particular sequence length. For example, a set of nrUMIs with 6 nucleotides will have a total of 4 UMIs. AInstead of 6=4096 possible different sequences, a total of 96 different sequences may be included, in some embodiments, the library of UMIs includes 120 non-random sequences.

[0118] In some implementations where the nrUMIs are selected from a set having fewer than all possible distinct sequences, the number of nrUMIs is less than the number of source DNA molecules, sometimes significantly less. In such implementations, the nrUMI information can be combined with other information, such as virtual UMIs, read positions on a reference sequence, and / or sequence information of the reads, to identify sequence reads that originate from the same source DNA molecule.

[0119] A "virtual unique molecular index" or "virtual UMI" is a unique subsequence in a source DNA molecule. In some embodiments, the virtual UMI is located at or near the end of the source DNA molecule. One or more such unique end positions can uniquely identify a source DNA molecule, alone or in combination with other information. Depending on the number of different source DNA molecules and the number of nucleotides in the virtual UMI, one or more virtual UMIs can uniquely identify a source DNA molecule in a sample. In some cases, a combination of two virtual unique molecular identifiers is required to identify a source DNA molecule. Such combinations are extremely rare and can possibly only be found once in a sample. In some cases, one or more virtual UMIs in combination with one or more physical UMIs can together uniquely identify a source DNA molecule. In some embodiments, the virtual UMI is present at a fragmentation end point resulting from the Nextera fragmentation process.

[0120] In some embodiments, a library of rUMIs may include random UMIs (rUMIs) selected as random samples, with or without replacement, from a set of UMIs consisting of all possible different oligonucleotide sequences given one or more sequence lengths. For example, if each UMI in a set of UMIs has n nucleotides, the set may include 4 UMIs with sequences that differ from each other. AContains n UMIs. 4 A A random sample selected from the n UMIs constitutes the rUMI.

[0121] In some embodiments, the library of UMIs is pseudo-random or partially random and may include a mixture of nrUMIs and rUMIs.

[0122] In many embodiments, the UMI is added to the target double-stranded nucleic acid using an oligonucleotide or polynucleotide during or after tagmentation of the nucleic acid. In many embodiments, the UMI is added to the target double-stranded nucleic acid prior to the library amplification step.

[0123] In some embodiments, UMI reagents from the TruSight® Oncology workflow (Illumina catalog number 20024586) may be utilized in accordance with the present disclosure.

[0124] In some embodiments, each double-stranded nucleic acid molecule in a UMI library contains one unique UMI sequence, or a single UMI. In many embodiments, the UMI may be located on either side of the insert DNA. In some embodiments, an adapter sequence or other nucleotide sequence may be present between the UMI and the insert DNA.

[0125] In some embodiments, the UMI library includes a double-stranded UMI, which may reduce the error detection limit compared to the use of a single UMI. The double-stranded UMI allows one of skill in the art to pair the plus strand with its minus strand despite errors that may occur in the sequencing reaction. Such sequencing mismatches are identified during sequencing, and the sequence of the nucleic acid fragment can still be accurately reconstructed despite having a mismatch. In some embodiments, the method of generating a UMI library including a double-stranded UMI includes a forked adaptor, as discussed in detail in Section II.C below. In some embodiments, the forked adaptor is a BLT fork adaptor.

[0126] In some embodiments, each double-stranded nucleic acid fragment in a UMI library contains two, three, or four UMI sequences. The UMI sequences may have sequences that are complementary to each other or may each have a different sequence.

[0127] In some embodiments, an adapter sequence or other nucleotide sequence may be present between each UMI and the insert DNA.

[0128] In some embodiments, the UMI is located 5' of the insert DNA. In some embodiments, the UMI is located 3' of the insert DNA. In some embodiments, a sequence of nucleic acid representing one or more adapter sequences may be located between the UMI and the insert DNA. In some embodiments, the UMI is located between the adapter sequence and the transposon end sequence.

[0129] In many embodiments, the UMI may be on the first strand, the second strand, or both strands of the double-stranded target nucleic acid fragment. In some embodiments, the UMI is on the first strand. In some embodiments, the first copy of the UMI is on the first strand of the double-stranded target nucleic acid fragment, and the second copy of the UMI is on the second strand. In some embodiments, the first UMI is on the first strand, and the second UMI is on the second strand.

[0130] 1. Inline UMI The UMI may be located anywhere on the double-stranded nucleic acid molecule. In many embodiments, the location of the UMI on the double-stranded nucleic acid molecule varies. In some embodiments, the UMI is located directly adjacent to the insert DNA, i.e., the UMI is an "in-line UMI." In some embodiments, the in-line UMI is adjacent to the 3' of the insert DNA. In some embodiments, the in-line UMI is adjacent to the 5' of the insert DNA. Current BLT methods contain MEs adjacent to the target insert, precluding the use of Illumina ligation adapters with UMIs. UMIs are useful for removing PCR duplicates and detecting low frequency variants in double-stranded nucleic acids, while UDIs are useful for mitigating sample inaccuracies due to index hopping in library sequencing and demultiplexing. UDIs are unique i5 and i7 index sequences that are added to the ends of the target nucleic acid such that both ends contain a UDI. UDIs are used with patterned flow cells such as Illumina's NovaSeq 6000 system (see, e.g., International Publication Nos. WO 2018 / 204423, WO 2018 / 208699, WO 201 / 9055715, and WO 2016 / 176091, which are incorporated by reference in their entireties). One of skill in the art will appreciate that in-line UMIs allow compatibility of UMI libraries with standard downstream library preparations that utilize UDIs, such as sample multiplexed PCR and sequencing reagent recipes in Illumina's TruSeq™ and AmpliSeq™ workflows. In some embodiments, the sequencing methods used with in-line UMIs do not require custom primers or custom reads.

[0131] In some embodiments, standard sequencing methods are used to sequence the UMI library with in-line UMIs. In these embodiments, the UMI is adjacent to the 3' end of the insert nucleic acid (Figure 20). Thus, each UMI and insert nucleic acid sequence is captured using read 2 without the need to sequence the ME sequence between them. In these embodiments, the sequencing method does not include a dark cycle. The dark cycle is described below in Section III.A.

[0132] In some embodiments, the "in-line UMI" is located between the insert DNA and the adapter sequence. In some embodiments, the adapter sequence is a second adapter sequence.

[0133] B. Transposome Complex In general, the transposon complexes of the invention comprise a transposase and a first and a second transposon, along with one or more elements that mediate targeting to one or more nucleic acid sequences of interest.

[0134] As used herein, a "transposome complex" is composed of at least one transposase (or other enzymes described herein) 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 aspects, the transposon recognition sequence is a double-stranded transposon end sequence. The transposase binds to a transposase recognition site in a 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 transcribed into the target nucleic acid and a cleavage event occurs. Exemplary transposition procedures and systems can be readily adapted for use with transposases.

[0135] In some embodiments, the methods include one, two, or more transposome complexes, each of which can include a different transposase and transposon than other transposome complexes that can also be used in the same methods.

[0136] In some embodiments, the transposome complex comprises a transposase and one, two, or more transposons.

[0137] In some embodiments, the transposome complex comprises a transposase and a first transposon comprising a 3' transposon end sequence and a 5' adaptor sequence. The 5' adaptor sequence of the first transposon can comprise an A14 sequence (SEQ ID NO:4), an A2 sequence (SEQ ID NO:7), and / or a B15 sequence (SEQ ID NO:5). In some embodiments, the first transposon also comprises a UMI sequence.

[0138] In some embodiments, the transposome complex also comprises a first and a second transposon. The second transposon comprises a 5' transposon end sequence. The 5' transposon end sequence of the second transposon can be complementary to the 3' transposon end sequence of the first transposon.

[0139] In some embodiments, the second transposon also comprises a 3' adapter sequence. The 3' adapter sequence of the second transposon can be partially or completely complementary to the 5' adapter sequence of the first transposon.

[0140] In some embodiments, the 3' adapter sequence of the second transposon does not contain a portion that is complementary to the 5' adapter sequence of the first transposon.

[0141] In some embodiments, the 3' adapter sequence of the second transposon comprises an A14 sequence (SEQ ID NO:4), an A2 sequence (SEQ ID NO:7), a B15 sequence (SEQ ID NO:5), and / or a sequence complementary to the UMI sequence of the first transposon.

[0142] In some embodiments, the second transposon further comprises a UMI. The UMI of the second transposon may be the same sequence as the UMI of the first transposon or a different sequence.

[0143] In some embodiments, the transposome complex comprises one, two, or more transposons, each having a sequence comprising A14-ME (SEQ ID NO:1) and / or B15-ME (SEQ ID NO:2).

[0144] In some embodiments, the transposon complex comprises a first transposon having a 3' transposon end sequence comprising ME (SEQ ID NO:6) or ME' (SEQ ID NO:3). In some embodiments, the transposon complex comprises a second transposon having a 3' transposon end sequence comprising ME (SEQ ID NO:6) or ME' (SEQ ID NO:3).

[0145] In some embodiments, the transposome complex comprises an additional adapter sequence adjacent to the A14 sequence (SEQ ID NO:4), the A2 sequence (SEQ ID NO:7), the B15 sequence (SEQ ID NO:5), the ME sequence (SEQ ID NO:6), and / or the ME' sequence (SEQ ID NO:3). Many sequences can be used as additional adapter sequences, such as those disclosed in Illumina Adapter Sequences Document#1000000002694 v15, which is incorporated herein by reference. In some embodiments, the additional adapter sequence is an A adapter sequence, a B adapter sequence, an X adapter sequence, or a Y' adapter sequence.

[0146] In some embodiments, the transposome complex comprises oligonucleotides complementary to the B15 and / or A14 sequences.

[0147] In some embodiments, the transposome complex is immobilized on a solid support, such as a bead or other material. In some embodiments, the transposome complex is immobilized via the first or second transposon. In some embodiments, the transposome complex is immobilized via an oligonucleotide that is complementary to an adapter sequence (such as the B15 sequence or the A14 sequence) of the first or second transposon.

[0148] 1. Transposase "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.

[0149] 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. More 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.

[0150] 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.

[0151] 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.

[0152] 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 (see, 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; International Patent Application No. 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, which are incorporated by reference in their entireties.

[0153] 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 of the transposase.

[0154] In some embodiments, the transposase comprises a Tn5 transposase. In some embodiments, the Tn5 transposase is a hyperactive Tn5 transposase.

[0155] 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 transposons in each population are 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.

[0156] The term "transposon end" refers to a double-stranded nucleic acid molecule that exhibits only the nucleotide sequences ("transposon end sequences") necessary to form a complex with a transposase or integrase enzyme that functions in an in vitro transposition reaction. In some embodiments, the double-stranded nucleic acid molecule is DNA. In some embodiments, the transposon end is capable of forming a functional complex with a transposase in a transposition reaction. As a non-limiting example, the transposon end may include a 19-bp outer end ("outer end, OE") transposon end, an inner end ("inner end, IE") transposon end, or a "mosaic end ("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 herein by reference in its entirety. Transposon ends can comprise any nucleic acid or nucleic acid analogue 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, unnatural bases, modified backbones, and can include 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.

[0157] 2. Transferred and non-transferred strands Similarly, the term "transferred strand" refers to the transferred portions of both transposon ends. Similarly, the term "non-transferred strand" refers to the non-transferred portions of both "transposon ends." The 3' end of the transferred strand is bound or transferred to target DNA in an in vitro transposition reaction. The non-transferred strand, which exhibits a transposon end sequence complementary to the transferred transposon end sequence, is not bound or transferred to target DNA in an in vitro transposition reaction.

[0158] 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.

[0159] In some embodiments, the transposome complex comprises a first transposon comprising a 3' transposon end sequence and a 5' adaptor sequence. In some embodiments, the transposome complex comprises a second transposon comprising a 5' transposon end sequence, wherein the 5' transposon end sequence is complementary to the 3' transposon end sequence.

[0160] Thus, in some embodiments, the tagmentation process generates a double-stranded target nucleic acid fragment that includes (1) a first strand that includes a first adaptor sequence and a first UMI, and (2) a second strand that includes a second adaptor sequence. In some embodiments, the second strand may further include a second UMI.

[0161] 3. Tagmentation "Tagmentation" refers to the use of a transposase to fragment and tag nucleic acids. Tagmentation involves modification of nucleic acids with a transposome complex that includes a transposase enzyme complexed with one or more adapter sequences that include transposon end sequences (referred to herein as transposons). Tagmentation can result in simultaneous fragmentation of DNA and ligation of adapters to the 5' ends of both strands of a double-stranded fragment.

[0162] In many embodiments, tagmentation may include multiple transposome complexes, each of which includes a transposase complexed with a transposon that includes a transposon end sequence and an adapter sequence. In some embodiments, tagmentation is symmetric tagmentation, where all adapter sequences in the multiple transposome complexes are identical. In some embodiments, tagmentation is standard or asymmetric tagmentation, where the multiple transposome complexes include two different sets of adapter sequences. Adapter sequences are described below in Section II.C. Symmetric and asymmetric tagmentation are described in WO 2015 / 168161 and WO 2017 / 040306, which are incorporated by reference in their entireties.

[0163] In some embodiments, the method includes a first transposase, a first transposon, and a second transposon. In some embodiments, the method further includes a second transposase, a third transposon, and a fourth transposon.

[0164] In many embodiments, the tagmentation process produces double-stranded target nucleic acid fragments with adaptor sequences and / or UMIs that can be arranged in a variety of ways. The positions of the adaptor sequences and UMIs (or the order of the adaptor sequences and UMIs from 5' to 3') depend on the transposon adaptor used in tagmentation. In some embodiments, the tagmentation process produces double-stranded target nucleic acid fragments that include a first adaptor sequence and a first UMI. In some embodiments, the first adaptor sequence and the first UMI are on the first strand of the nucleic acid fragment.

[0165] In some embodiments, the tagmentation step generates a double-stranded target nucleic acid fragment comprising a first adapter sequence, a first UMI, and a second adapter sequence, in some embodiments, the first adapter sequence and the first UMI are on a first strand of the nucleic acid fragment, while the second adapter sequence is on a second strand of the nucleic acid fragment.

[0166] In some embodiments, the tagmentation step generates a duplex comprising a first adaptor sequence, a first UMI, a second adaptor sequence, and a second UMI, in some embodiments, the first adaptor sequence and the first UMI are on a first strand of the nucleic acid fragment, while the second adaptor sequence and the second UMI are on a second strand of the nucleic acid fragment.

[0167] In some embodiments, the tagmentation process generates a double-stranded target nucleic acid having a forked adapter transposon to generate a double-stranded target nucleic acid fragment comprising a first and second copy of a first adapter sequence, a first UMI, a first and second copy of a second adapter sequence, and a second UMI.

[0168] In some embodiments, the tagmentation process generates double-stranded target nucleic acid fragments that further comprise a third UMI and / or a fourth UMI.

[0169] In some embodiments, the tagmentation step generates a double-stranded target nucleic acid that includes one or more adapter sequences and is UMI-free. In some embodiments, the one or more adapter sequences are on a first strand of the nucleic acid fragment.

[0170] 4. Immobilized Transposome Complexes As described in U.S. Pat. No. 9,683,230, which is incorporated herein in its entirety, several different types of immobilized transposomes can be used in these methods. In the methods and compositions presented herein, the transposome complexes are immobilized to a solid support. In some embodiments, the transposome complexes and / or capture oligonucleotides are immobilized to the support via one or more polynucleotides, such as a polynucleotide that includes a transposon end sequence. In some embodiments, the transposome complexes can 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 to the 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., nucleic acid) remain immobilized or associated with 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.

[0171] In some embodiments, the transposome is immobilized using a transposon that contains a biotin tag.

[0172] In some embodiments, the transposome complex is 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.

[0173] In some embodiments, the length of the double-stranded fragments in the immobilized library is adjusted by increasing or decreasing the density of transposome complexes on the solid support.

[0174] a) Capture Oligonucleotides In some embodiments, the capture oligonucleotide is immobilized on a solid support.

[0175] In some embodiments, the 3' end of the target DNA is attached to a capture oligonucleotide.

[0176] In some embodiments, the 3' end of the target RNA is attached to a capture oligonucleotide. In some embodiments, the capture oligonucleotide may function to immobilize the target RNA on a solid support.

[0177] In some embodiments, the capture probe comprises a poly-T sequence.

[0178] In some embodiments, the target RNA is mRNA, and the mRNA binds to a capture oligonucleotide that includes a poly-T sequence.

[0179] In some embodiments, the capture oligonucleotide does not include a poly-T sequence.

[0180] In some embodiments, the capture oligonucleotide is immobilized to the bead via the P5 or P7 sequence.

[0181] In some embodiments, the capture oligonucleotide comprises a tag that is also present in a first tag contained in a first polynucleotide of the immobilized transposome.

[0182] b) solid support 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 (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.

[0183] 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.

[0184] 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 described herein are described in U.S. Patent Application Serial No. 13 / 661,524 or U.S. Patent Application Publication No. 2012 / 0316086(A1), each of which is incorporated herein by reference.

[0185] In some embodiments, the solid support comprises an array of wells or depressions on its surface, which can be fabricated as commonly 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.

[0186] 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 2004 / 018497, U.S. Pat. No. 7,057,026, WO 1991 / 06678, WO 2007 / 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] The binding of nucleic acid to the support, whether rigid or semi-rigid, can occur via covalent or non-covalent bonds. Exemplary bindings are described in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, and U.S. Patent Application Publication No. 2011 / 0059865(A1), 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.

[0191] 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.

[0192] In some embodiments, a solid support has a library of prepared tagged DNA fragments immobilized thereon.

[0193] 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.

[0194] In some embodiments, the solid support further comprises a transposase bound to the first polynucleotide to form a transposome complex.

[0195] 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.

[0196] In some embodiments, the solid support further comprises a transposase bound to a second polynucleotide to form a transposome complex.

[0197] 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.

[0198] 5. Fluid-phase Transposome Complex The transposome complex may be a liquid-phase transposome complex. These liquid-phase transposome complexes may be mobile and may not be immobilized on a solid support. In some embodiments, the liquid-phase transposome complex is used to generate tagged fragments in solution.

[0199] Furthermore, the method may include a step involving a liquid-phase transposome complex. For example, the method presented herein may further include providing a transposome complex in a solution and contacting the liquid-phase transposome complex with an immobilized fragment under conditions in which DNA is fragmented by the transposome complex solution, thereby obtaining an immobilized nucleic acid fragment with one end in the solution. In some embodiments, the transposome complex in the solution may include a second tag, such that the method produces an immobilized nucleic acid fragment with a second tag, the second tag being in the solution. The first and second tags may be different or the same.

[0200] In some embodiments, the method may further include contacting the liquid-phase transposome complex with the double-stranded nucleic acid under conditions where the DNA fragments are further fragmented by the liquid-phase transposome complex, thereby obtaining immobilized nucleic acid fragments having one end in solution.

[0201] In some embodiments, the liquid phase transposome complexes comprise a second tag, thereby generating immobilized nucleic acid fragments in solution with the second tag. In some embodiments, the first and second tags are different. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the liquid phase transposome complexes comprise the second tag.

[0202] In some embodiments, one form of surface-bound transposomes is present primarily on a solid support. For example, in some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the tags present on the solid support contain the same tag domain. In such embodiments, after a first tagmentation reaction with surface-bound transposomes, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the bridge structures contain the same tag domain at each end of the bridge. A second tagmentation reaction can be performed by adding transposomes from solution, which further fragments the bridge. In some embodiments, most or all of the solution-phase transposomes contain a tag domain that is different from the tag domain present on the bridge structure generated in the first tagmentation reaction. For example, in some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the tags present in the liquid phase transposomes comprise a tag domain that is different from the tag domain present on the bridge structure generated in the first tagmentation reaction.

[0203] In some embodiments, the length of the template is longer than can be suitably amplified using standard cluster chemistry. For example, in some embodiments, the length of the template is at least 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp, 1900bp, 2000bp, 2100bp, 2200bp, 2300bp, 2400bp, 2500bp, 2600bp, , 2700bp, 2800bp, 2900bp, 3000bp, 3100bp, 3200bp, 3300bp, 3400bp, 3500bp, 3600bp, 3700bp, 3800bp, 3900bp, 4000bp, 4100bp, 4200bp, 4300bp, 4400bp, 4500bp, 4600bp, 4700bp, 4800bp, 4900bp, 5000bp, 10000bp, 30000bp, or 100,000bp. In such embodiments, a second tagmentation reaction can then be performed by adding transposomes from solution to further fragment the bridge, as described in U.S. Patent No. 9,683,230, which is incorporated herein in its entirety. Thus, a second tagmentation reaction can remove the internal span of the bridge, leaving short truncations immobilized on the surface that can be converted into clusters ready for further sequencing steps. In certain embodiments, the length of the template can be within a range defined by upper and lower limits selected from those exemplified above.

[0204] C. Adapter As used herein, "adapter" refers to a transposon or polynucleotide that exhibits one or more "adapter sequences" for one or more desired intended purposes or uses. An adapter can include any sequence that provides for any desired purpose.

[0205] The adaptor may be a 5' adaptor or a 3' adaptor. A 5' adaptor is used that is intended to be linked to the 5' end of a target nucleic acid molecule. A 3' adaptor is intended to be linked to the 3' end of a target nucleic acid molecule.

[0206] In some embodiments, the adapter sequence comprises one or more regions suitable for hybridization with a primer for an amplification reaction. In some embodiments, the adapter sequence comprises one or more regions suitable for hybridization with a primer for a sequencing reaction. In some embodiments, the adapter sequence comprises one or more regions suitable for hybridization with a polynucleotide for incorporation of a UMI. In such embodiments, the UMI can be incorporated using the HYB / HYB' or Hyb2Y workflow.

[0207] In some embodiments, the adapter sequence comprises a UMI, a primer sequence, an index tag sequence, a capture sequence, a barcode sequence, a cleavage sequence, an anchor sequence, a universal sequence, a spacer region, a transposon end sequence, or a sequencing-related sequence, or a combination thereof. As used herein, a sequencing-related sequence may be any sequence associated with a subsequent sequencing step. A sequencing-related sequence may act to simplify a downstream sequencing step. For example, a sequencing-related sequence may 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. It is understood that any other suitable features may be incorporated into the adapter, and that the adapter sequence may be used in any combination and may be arranged in any order from 5' to 3'. In some embodiments, the transposon end sequence is a mosaic end sequence (ME).

[0208] The adapter may comprise one, two, or more lead sequencing adapter sequences. In some embodiments, the adapter sequence is a 5' first lead sequencing adapter sequence. In some embodiments, the adapter sequence is a 5' second lead sequencing adapter sequence. In some embodiments, the first lead and / or second lead sequencing adapter sequence comprises a unique primer binding site.

[0209] In some embodiments, the adapter sequence comprises a sequence having a length of 5 bp to 200 bp. In some embodiments, the adapter sequence comprises a sequence having a length of 10 bp to 100 bp. In some embodiments, the adapter sequence comprises a sequence having a length of 20 bp to 50 bp. In some embodiments, the adapter sequence 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.

[0210] Although a variety of sequences can be used in the adapters, provided below are specific sequences that can be used in the adapter sequence, unique primer binding site, polynucleotide, or transposon end sequence (ME). The sequences may be used in any combination and may be arranged in 5' to 3' order. Exemplary sequences for A14-ME, ME, B15-ME, ME', A14, B15, and ME are shown below. A14-ME: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1) B15-ME: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 2) ME': 5'-phoS-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 3) A14: 5'-TCGTCGGCAGCGTC-3' (SEQ ID NO: 4) B15: 5'-GTCTCGTGGGCTCGG-3' (SEQ ID NO: 5) ME: AGATGTGTATAAGAGACAG (SEQ ID NO: 6) A2: TCACTCAAGAACAGC (SEQ ID NO: 7)

[0211] In some embodiments, the adapter sequence is incorporated during tagmentation. In these embodiments, a transposon carrying the adapter sequence is used in the tagmentation step.

[0212] In some embodiments, the adaptor sequence is incorporated during the adaptor ligation step. In these embodiments, a polynucleotide having an adaptor sequence is used in the ligation step. In some embodiments, one, two, or more polynucleotides may be used.

[0213] 1. Fork-type adapter In some embodiments, the adapter may be a forked adapter, also known as a Y-shaped adapter. Forked adapter-based technology can be utilized to generate polynucleotides, for example, as exemplified in the workflow of the TruSeq™ Sample Preparation Kit (Illumina, Inc.). Reagents in the workflow of the TruSight® Oncology Kit (Illumina, Inc.) can also be used to assemble the forked adapter. In many embodiments, the HYB / HYB' workflow is used to generate the forked adapter.

[0214] As used herein, a "forked adapter" refers to an adapter that includes two strands of a 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 adapter 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 adapter is ligated to both strands at the ends of the double-stranded DNA fragment. In some embodiments, the forked adapter is ligated to one end of the double-stranded DNA fragment. In some embodiments, the forked adapter is ligated to both ends of the double-stranded DNA fragment. In some embodiments, the forked adapters on the opposing ends of the fragment are different. In some embodiments, one strand of the forked adapter is phosphorylated at its 5' to facilitate ligation to the fragment. In some embodiments, one strand of the forked adapter 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, the phosphorothioate bond blocks 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.

[0215] In some embodiments, the transposome complex has the following structure:

[0216] [ka]

[0217] In some embodiments, the transposome complex has the following structure:

[0218] [ka] 2. Transposon Adapters In some embodiments, the UMI is incorporated during the tagmentation step. In these embodiments, the adapter used to incorporate the UMI is a transposon. In some embodiments, the UMI is located between the adapter sequence and the 3' transposon end sequence. In some embodiments, the adapter sequence is located between the UMI and the 3' end transposon end sequence. In some embodiments, the adapter sequence may comprise a sequence that is fully or partially complementary to the 3' end transposon end sequence.

[0219] In some embodiments, the transposon is a forked adapter transposon. The forked adapter may comprise two strands. In some embodiments, the first strand of the forked adapter transposon comprises a 3'-end transposon end sequence, an adapter sequence, and a UMI. In some embodiments, the second strand of the forked adapter transposon comprises an adapter sequence and a sequence that is fully or partially complementary to the first strand of the first forked adapter transposon. The sequences with full or partial complementarity in the first and second strands allow the two strands to hybridize and form a forked structure.

[0220] In some embodiments, two or more forked adapter transposons may be used to incorporate two or more UMIs and two or more adapter sequences into the library.

[0221] In some embodiments, two UMIs and four adapter sequences are incorporated into the library using two forked adapter transposons. In some embodiments, tagmentation of double-stranded nucleic acid with a forked adapter transposon generates double-stranded target nucleic acid fragments having two UMIs, a first and second copy of a first adapter sequence, and a first and second copy of a second adapter sequence.

[0222] In some embodiments, two forked adapter transposons are used to incorporate four UMIs and four adapter sequences into the library. In some embodiments, double-stranded target nucleic acid fragments having four UMIs and four adapter sequences are generated by tagmenting double-stranded nucleic acids with a forked adapter transposon.

[0223] In some embodiments, the transposon further comprises one, two, three, four or more unique primer binding sequences. In some embodiments, the unique primer binding sequences are used in the Hyb2Y workflow. In some embodiments, the unique primer binding sequences are used to anneal custom sequencing primers. In some embodiments, the unique primer binding sequences comprise A2, A14, and / or B15.

[0224] 3. Polynucleotide Adapters In some embodiments, the UMI is incorporated after tagmentation. In these embodiments, the adaptor used to incorporate the UMI is a polynucleotide. In some embodiments, the method includes one, two, or more polynucleotides. In some embodiments, the polynucleotide includes the UMI and one, two, or more adaptor sequences. In some embodiments, the polynucleotide includes a region for hybridizing to another polynucleotide or transposon via a complementary sequence. For example, the polynucleotide may include a sequence that is fully or partially complementary to the 3'-end transposon sequence. In some embodiments, the one or more polynucleotides are processed in a hybridization step to generate a forked adaptor.

[0225] In some embodiments, the portion of the polynucleotide may comprise a 3' adaptor. The 3' adaptor may comprise a hairpin UMI, a universal hybridizing tail, a splint ligation adaptor, and / or a template switch oligonucleotide.

[0226] In some embodiments, the polynucleotide comprises a hairpin UMI. In some of these embodiments, the polynucleotide further comprises a universal hybridizing tail. In some embodiments, the hairpin UMI is stable during the extension and / or ligation steps, but is not stable during the amplification step of the method. In some embodiments, the UMI comprises a 3 or 4 base pair stem. In some embodiments, the universal hybridizing tail comprises a nucleotide such as inosine that can bind to any DNA molecule.

[0227] In some embodiments, the polynucleotide comprises a splint ligation adaptor.

[0228] In some embodiments, the methods include a template switch oligonucleotide.

[0229] D. Post-tagmentation extension and ligation steps In some embodiments, gaps in the nucleic acid sequence left after the tagmentation event can be filled using an extension step. Generally, the extension step is followed by a ligation step. The extension and / or ligation is carried out using appropriate conditions. In some embodiments, the buffer used is an extension-ligation mix buffer (e.g., extension-ligation mix buffer 3, ELM3). Polymerases such as T4 DNA pol Exo- (New England BioLabs, Catalog No. M0203S) or Ttaq608 can be used in such extension and / or ligation steps. Taq polymerase, or any mutant, analog, or derivative of the aforementioned polymerases, can also be used instead in this step.

[0230] In some embodiments, the double-stranded target nucleic acid fragment is extended. In some embodiments, the second strand of the double-stranded target nucleic acid fragment is extended.

[0231] In some embodiments, the 3' end of the double-stranded target nucleic acid fragment is extended to the 5' end of the transposon.

[0232] In some embodiments, the extending step comprises extending from the 3'-end of the second strand of the double-stranded target nucleic acid fragment to the 5'-end of the hairpin UMI.

[0233] In some embodiments, the extension step is carried out using a strand displacement extension reaction, such as one that includes Bst DNA polymerase and a dNTP mix.

[0234] In some embodiments, the extension step is followed by ligation. In these embodiments, the method may include treating with a polymerase and a ligase to extend and ligate the nucleic acid strands to generate the complete double-stranded tagged fragment.

[0235] In some embodiments, the extending step comprises extending by 9 bases.

[0236] In some embodiments, the extending step comprises extending from the 3' end of the second strand of the double-stranded target nucleic acid fragment to the 5' end of the splint ligation adaptor.

[0237] In some embodiments, the extending step comprises copying a first strand of the double-stranded target nucleic acid fragment to extend from the 3' end of a second strand of the double-stranded target nucleic acid fragment to a junction within the template switch oligonucleotide.

[0238] In some embodiments, there are no gaps in the nucleic acid sequence left after the transposition event, in these embodiments, the method comprises ligating the transposon or polynucleotide to the double-stranded target nucleic acid fragment using a ligase and no extension step is used.

[0239] A wide variety of library preparation methods that include a step of adapter ligation 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). Exemplary ligated forked adapters are described in International Publication No. WO 2007 / 052006, US Patent Application Publication No. 2020 / 0080145, US Patent No. 9,868,982, and International Publication No. WO 2020 / 144373, which are incorporated by reference in their entirety. Adapters used with other ligation methods may also be used in this method (see, e.g., Illumina Adapter Sequences, Illumina, 2021). In particular, adapter ligation may allow for more flexible incorporation of adapters (such as longer adapters) compared to methods of tagging fragments by tagmentation (wherein adapter sequences are incorporated into the fragments during the transposition reaction). In some methods involving tagmentation, additional adapter sequences may be incorporated by PCR reactions, and this method may eliminate the need for an additional PCR step to incorporate additional adapter sequences.

[0240] 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 the DNA fragments. In some embodiments, A bases are added to the blunt ends of each strand to prepare them for ligation to the 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.

[0241] 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 index reads. In some embodiments, adapter ligation eliminates the need for an additional PCR step to add index tags and index primer sites.

[0242] In some embodiments, the ligation step comprises ligating the 3' end of the double-stranded target nucleic acid fragment to the 5' end of the transposon.

[0243] In some embodiments, the ligation step comprises ligating the 3' end of the double-stranded target nucleic acid fragment to the 5' ends of multiple transposons.

[0244] In some embodiments, the ligation step comprises ligating the 3' end of the second strand of the double-stranded target nucleic acid fragment to the 5' end of the universal hybridizing tail.

[0245] In some embodiments, the ligation step comprises ligating the 3' end of the second strand of the extended double-stranded target nucleic acid fragment to the 5' end of the first strand of the splint ligation adaptor.

[0246] E. Mold switching In some embodiments, a template switching or strand exchange step can be performed after release of the nucleic acid fragment from the transposome complex. In some embodiments, this template switching step is followed by gap filling and ligation. In some embodiments, this method can be performed in a tube or in a flow cell.

[0247] Template switching refers to the ability of a polymerase to pause extension while still bound to a newly synthesized strand and resume synthesis on another nucleic acid strand. In some embodiments, the steps of (1) extension, (2) template switching, and (3) resumption of synthesis after tagmentation are performed by a polymerase capable of DNA template switching. In some embodiments, the polymerase is Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.

[0248] In some embodiments, the template is switched from a first strand of a double-stranded target nucleic acid fragment to an unpaired region of the 3' template switch oligonucleotide. In some embodiments, the template switching step is followed by a copying step, which copies the unpaired region of the 3' switch oligonucleotide from the junction in the template switch oligonucleotide to the 5' end of such unpaired region.

[0249] F. Amplification The UMI library can be optionally amplified according to any suitable amplification method known in the art and sequenced using one or more sequencing primers. In some embodiments, the UMI library is amplified on a solid support. In some embodiments, the solid support is the same solid support on which BLT tagmentation 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.

[0250] For example, in some embodiments, the UMI library is amplified using cluster amplification methods, as exemplified by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are incorporated herein by reference in their entirety. The incorporated materials of U.S. Patent Nos. 7,985,565 and 7,115,400 describe methods of solid-phase nucleic acid amplification that allow 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 those 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 of which are 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 immobilized nucleic acid templates. Other suitable methods can also be used to generate immobilized amplicons from the UMI library generated 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.

[0251] In other embodiments, the UMI library is amplified in solution. For example, in some embodiments, the nucleic acid 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 nucleic acid fragments for one or more initial amplification steps, followed by subsequent amplification steps in solution. Thus, in some embodiments, immobilized nucleic acid templates can be used to produce solution-phase amplicons.

[0252] It should be understood that any of the amplification methods described herein or generally known in the art can be utilized with universal or target-specific primers to amplify the UMI library. 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 utilized to amplify the UMI library. In some embodiments, a primer specifically directed to the nucleic acid of interest is included in the amplification reaction.

[0253] Other suitable methods for amplifying polynucleotides can 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). It should be understood that these amplification methodologies can be designed to amplify UMI libraries. 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.

[0254] 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 is described in detail in the disclosure of U.S. Patent No. 7,670,810, which is incorporated herein by reference in its entirety.

[0255] 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 that are complementary to the constant 5' region.

[0256] In some embodiments, the amplification step involves adding oligonucleotides to one or both ends of the nucleic acid fragments to attach the library to a solid support.

[0257] In some embodiments, the amplifying step comprises adding at least a first lead sequencing oligonucleotide and / or a second lead sequencing oligonucleotide. In some embodiments, the amplifying step comprises adding at least a P5 oligonucleotide and a P7 oligonucleotide. In some embodiments, the amplifying step comprises adding at least a plurality of i5 oligonucleotides and a plurality of i7 oligonucleotides.

[0258] In some embodiments, after the amplification step, the method may include selecting amplified nucleic acid fragments within a size range after the amplification step.

[0259] G. Method for creating a UMI library Although the adapter may include two or more adapter sequences in any combination or order from 5' to 3', the present disclosure provides adapters that may be used in various embodiments. The present disclosure also provides a number of methods that may be used with the adapters described herein. The methods of the present disclosure may include one or more of the following adapters and methods.

[0260] 1. Method for creating a UMI library using a single UMI As shown in FIG. 1, an exemplary adapter includes, from 5' to 3' on its first strand, the adapter sequences of B15, A2, UMI, and ME. In the adapter, the UMI is located between A2 and ME. The UMI can include nrUMI and / or rUMI. On its second strand, the adapter includes a sequence that is complementary to ME. The adapter also includes a biotin tag, such that the adapter can be used with a solid support. In other embodiments, a solid support is not used, and researchers can use a liquid phase transposome complex.

[0261] As shown in FIG. 2 and described in Example 1, an exemplary method of generating a UMI library includes: (1) generating a double-stranded nucleic acid library, each fragment in the library including a UMI, the method including: (a) applying a sample including a double-stranded target nucleic acid to a first transposome complex including: (i) a first transposase; (ii) a first transposon including a first 3′-end transposon end sequence, a first adapter sequence, and a first UMI; and (iii) a second transposon including a sequence that is fully or partially complementary to the first 3′-end transposon end sequence; (2) tagmenting the double-stranded target nucleic acid with a first and a second transposon to generate a double-stranded target nucleic acid fragment comprising a first adapter sequence and a first UMI; (3) releasing the double-stranded target nucleic acid fragment from the first transposome complex; (4) optionally extending the double-stranded target nucleic acid fragment, thereby copying the single UMI to generate a double-stranded UMI; (5) ligating the transposon or the extended transposon to the double-stranded target nucleic acid fragment; (6) generating a double-stranded target nucleic acid fragment comprising the UMI; and (7) amplifying the double-stranded target nucleic acid fragment.

[0262] In this exemplary method, a first UMI in a first transposon is located between a first adapter sequence and a first 3' transposon end sequence.

[0263] As shown in Figure 3B and described in Example 2, an exemplary method for sequencing a UMI library involves 19 dark cycles (discussed in Section III.A below). In this method, 19 bases of the ME sequence are not imaged during the 19 dark cycles. This method uses four primers: Custom Primer 1 UMI + Read 1, Custom Primer i5, Custom Primer i7, and Custom Primer 4 UMI + Read 2.

[0264] Using this exemplary adaptor and method, a UMI library is created in which a first UMI is located on a first strand of a double-stranded target nucleic acid fragment and a second UMI is located on the second strand of the double-stranded target nucleic acid fragment.

[0265] Another exemplary method of sequencing a UMI library may be used. As shown in Figure 4 and described in Example 3, the exemplary method includes six custom primers: custom UMI 1 read (SEQ ID NO: 8), custom bridge primer for insert 1 read (SEQ ID NO: 9), custom i7 read (SEQ ID NO: 10), custom i5 read (SEQ ID NO: 11), custom UMI 2 read (SEQ ID NO: 12), and custom bridge primer for insert 2 read (SEQ ID NO: 13). In this sequencing method, primers with SEQ ID NO: 1 and 5 are combined, primers with SEQ ID NO: 3 and 4 are combined, and primers with SEQ ID NO: 2 and 6 are combined.

[0266] 2. How to create a UMI library using UMI-BLT Two exemplary adapters are shown in Figure 5A. The first adapter contains, from 5' to 3' on its first strand, the sequences of A15 and ME. The first adapter also contains a sequence on its second strand that is complementary to ME.

[0267] The second adaptor comprises, from 5' to 3' on its first strand, the sequences B15, A2, UMI, and ME. UMI is located between A2 and ME. The second adaptor also comprises a sequence on its second strand that is complementary to ME. The first and second adaptors comprise biotin tags.

[0268] As shown in FIG. 5B and described in Example 4, an exemplary method of generating a UMI library includes (1) generating a double-stranded nucleic acid library, wherein each fragment in the library includes a UMI, the method including (a) subjecting a sample including a double-stranded target nucleic acid to (i) a first transposase, (ii) a first transposon including a first 3′-end transposon end sequence, a first adapter sequence, and a first UMI, and (iii) a second transposon including a sequence that is fully or partially complementary to the first 3′-end transposon end sequence. (2) tagmenting the double-stranded target nucleic acid with the first and second transposons to generate a double-stranded target nucleic acid fragment comprising a first adapter sequence and a first UMI; (3) liberating the double-stranded target nucleic acid fragment from the first transposome complex; (4) optionally extending the double-stranded target nucleic acid fragment; (5) generating a double-stranded target nucleic acid fragment comprising a UMI; and (7) amplifying the double-stranded target nucleic acid fragment.

[0269] In this exemplary method, a first UMI in a first transposon is located between a first adapter sequence and a first 3' transposon end sequence.

[0270] This exemplary method further includes a second transposome complex comprising: (1) a second transposase; (2) a third transposon comprising a second adapter sequence and a second 3' transposon end sequence; and (3) a fourth transposon comprising a sequence that is fully or partially complementary to the second 3' end transposon end sequence.

[0271] Using the exemplary adaptors and methods described herein, a UMI library is generated in which the first UMI is located on the first strand of a double-stranded target nucleic acid fragment.

[0272] As shown in FIG. 6A and described in Example 5, an exemplary method for sequencing a UMI library includes a dark cycle and the following four primers: standard insert read 1, custom i7, standard i5, and UMI+insert read 2.

[0273] Another exemplary method of sequencing a UMI library may be used. As shown in Figure 6B and described in Example 6, the exemplary method includes four primers: standard insert read 1, custom i7, standard i5, UMI primer, and insert read 2 bridge primer. This method uses a bridge primer rehybridization step in which the UMI primer is replaced by the insert read 2 bridge primer.

[0274] 3. Methods for generating UMI libraries prepared from cell-free DNA (cfDNA) Two exemplary adaptors are shown in Figure 9. The first adaptor contains, from 5' to 3' on its first strand, the sequences P5, UMI, A14, and ME. The first adaptor also contains a sequence on its second strand that is complementary to ME. The UMI is located between P5 and A14.

[0275] The second adaptor comprises, from 5' to 3' on its first strand, the sequences P7, UMI, B15, and ME. UMI is located between P7 and B15. The second adaptor also comprises a sequence on its second strand that is complementary to ME. The first and second adaptors comprise a biotin tag.

[0276] As shown in FIG. 9 and described in Example 7, an exemplary method of generating a UMI library includes (1) generating a double-stranded nucleic acid library, wherein each fragment in the library includes a UMI, the method including (a) subjecting a sample including a double-stranded target nucleic acid to (i) a first transposase, (ii) a first transposon including a first 3′-end transposon end sequence, a first adapter sequence, and a first UMI, and (iii) a second transposon including a sequence that is fully or partially complementary to the first 3′-end transposon end sequence. (2) tagmenting the double-stranded target nucleic acid with the first and second transposons to generate a double-stranded target nucleic acid fragment comprising a first adapter sequence and a first UMI, (3) liberating the double-stranded target nucleic acid fragment from the first transposome complex, (4) optionally extending the double-stranded target nucleic acid fragment, (5) generating a double-stranded target nucleic acid fragment comprising a UMI, and (7) amplifying the double-stranded target nucleic acid fragment. The first adapter sequence in the first transposon is located between the first UMI and the first 3' transposon end sequence.

[0277] This exemplary method further includes a second transposome complex comprising: (1) a second transposase; (2) a third transposon comprising a second adapter sequence and a second 3' transposon end sequence; and (3) a fourth transposon comprising a sequence that is fully or partially complementary to the second 3' end transposon end sequence.

[0278] The method further includes: (1) the third transposon further comprises a second UMI; and (2) the second adaptor sequence is located between the second UMI and the second 3' transposon end sequence. In this method, the tagmentation step generates a double-stranded target nucleic acid fragment comprising: (1) a first strand comprising the first adaptor sequence and the first UMI; and (2) a second strand comprising the second adaptor sequence and the second UMI.

[0279] Using the exemplary adaptors and methods described herein, a UMI library is created in which a first copy of a first UMI is on a first strand of a double-stranded target nucleic acid fragment and a second copy of the first UMI is on the second strand.

[0280] As shown in FIG. 9 and described in Example 8, an exemplary method for sequencing a UMI library includes four primers: Read 1 (standard primer), UMI Read (standard i7 primer), UMI Read (standard i5 primer), and Read 2 (standard primer).

[0281] Another exemplary method of sequencing a UMI library may be used. As shown in Figure 6B and described in Example 6, the exemplary method includes four primers: standard insert read 1, custom i7, standard i5, UMI primer, and insert read 2 bridge primer. This method uses a bridge primer rehybridization step in which the UMI primer is replaced by the insert read 2 bridge primer.

[0282] 4. First method for generating a UMI library using UDIs and double-stranded UMIs Two exemplary adaptors are shown in Figure 12. The first and second adaptors are forked adaptors.

[0283] The first adaptor comprises, from 5' to 3' on its first strand, the sequences of A14, UMI-A, and ME. The first adaptor also comprises, from 5' to 3' on its second strand, the sequences of ME', UMI-A', and a B15 duplex in which B15 hybridizes to B15'. UMI-A is located between A14 and ME. UMI-A' is located between ME' and the B15 duplex.

[0284] The second adaptor contains, from 5' to 3' on its first strand, the sequences A14, UMI-B, and ME. The second adaptor also contains, from 5' to 3' on its second strand, the sequences ME', UMI-B', and B15 duplex. UMI-B is located between A14 and ME.

[0285] The first and second adaptors each contain a biotin tag.

[0286] As shown in FIG. 12 and described in Example 9, an exemplary method for generating a UMI library includes: (1) applying a sample containing a double-stranded target nucleic acid to a first transposome complex and a second transposome complex; (2) tagmenting the double-stranded target nucleic acid with a forked adapter transposon to generate double-stranded target nucleic acid fragments containing a first and second copy of a first adapter sequence, a first UMI, a first and second copy of a second adapter sequence, and a second UMI; (3) releasing the double-stranded target nucleic acid fragments from the transposome complex; (4) optionally extending the double-stranded target nucleic acid fragments; (5) ligating the forked adapter transposon or the extended forked adapter transposon to the double-stranded target nucleic acid fragments; (6) generating double-stranded target nucleic acid fragments containing a UMI; and (7) amplifying the double-stranded target nucleic acid fragments.

[0287] In this method, the first transposome complex comprises (1) a first transposase and (2) a first forked adapter transposon on a first strand of a double-stranded target nucleic acid fragment, where (i) the first strand of the first forked adapter transposon comprises a first 3' terminal transposon end sequence, a first copy of the first adapter sequence, and a first UMI, and (ii) the second strand of the first forked adapter transposon comprises a first copy of the second adapter sequence, and a sequence that is fully or partially complementary to the first strand of the first forked adapter transposon.

[0288] Additionally, the second transposome complex comprises a second transposome complex comprising: (1) (i) a second transposase; and (ii) a second forked adapter transposon on a second strand of the double-stranded target nucleic acid fragment, wherein (a) the first strand of the second forked adapter transposon comprises a second 3' terminal transposon end sequence, a second copy of the first adapter sequence, and a second UMI; and (b) the second strand of the second forked adapter transposon comprises a second copy of the second adapter, and a sequence that is fully or partially complementary to the first strand of the second forked adapter transposon.

[0289] As shown in FIG. 6A and described in Example 5, an exemplary method for sequencing a UMI library includes a dark cycle and the following four primers: standard insert read 1, custom i7, standard i5, and UMI+insert read 2.

[0290] Another exemplary method of sequencing a UMI library may be used. As shown in Figure 6B and described in Example 6, the exemplary method includes four primers: standard insert read 1, custom i7, standard i5, UMI primer, and insert read 2 bridge primer. This method uses a bridge primer rehybridization step in which the UMI primer is replaced by the insert read 2 bridge primer.

[0291] As shown in FIG. 12 and described in Example 10, an exemplary method for sequencing a UMI library includes a dark cycle and the following primers: A14 read, B15 read, i7 read, and i5 read.

[0292] 5. Second method for generating UMI libraries using UDIs and double-stranded UMIs Two exemplary adaptors are shown in Figure 13. The first and second adaptors are forked adaptors. Due to the use of double-stranded sequencing in this method of generating a UMI library, the annealed pair of UMIs within each forked adaptor are not complementary. (See Figure 12 for comparison).

[0293] Each adapter in this method is double-stranded and contains two UMIs, with one UMI on each strand (Figure 13). The two strands are annealed at the ME region to generate a forked adapter with a non-complementary double-stranded UMI. Because the double-stranded UMI does not contain a complementary sequence, each adapter is annealed separately to each other.

[0294] The first adaptor contains, from 5' to 3' on its first strand, the sequences A14, A, UMI-1, X, and ME. The first adaptor also contains, from 5' to 3' on its second strand, the sequences ME', Y, UMI-2', B, and a B15 duplex where B15 hybridizes to B15'. UMI-1 is located between A and UMI-1. UMI-2' is located between ME' and B.

[0295] The second adaptor comprises, from 5' to 3' on its first strand, the sequences A14, A, UMI-4', X, and ME. The second adaptor also comprises, from 5' to 3' on its second strand, the sequences ME', Y', UMI-3, B, and B15 duplex. UMI-4' is located between A and X. UMI-3 is located between B and Y'.

[0296] The first and second adaptors each contain a biotin tag.

[0297] As shown in FIG. 13 and described in Example 11, an exemplary method for generating a UMI library includes: (1) applying a sample containing a double-stranded target nucleic acid to a first transposome complex and a second transposome complex; (2) tagmenting the double-stranded target nucleic acid with a forked adapter transposon to generate double-stranded target nucleic acid fragments containing a first and second copy of a first adapter sequence, a first UMI, a first and second copy of a second adapter sequence, and a second UMI; (3) releasing the double-stranded target nucleic acid fragments from the transposome complex; (4) optionally extending the double-stranded target nucleic acid fragments; (5) ligating the forked adapter transposon or the extended forked adapter transposon to the double-stranded target nucleic acid fragments; (6) generating double-stranded target nucleic acid fragments containing a UMI; and (7) amplifying the double-stranded target nucleic acid fragments.

[0298] In this method, the first transposome complex comprises (1) a first transposase and (2) a first forked adapter transposon on a first strand of a double-stranded target nucleic acid fragment, where (i) the first strand of the first forked adapter transposon comprises a first 3' terminal transposon end sequence, a first copy of the first adapter sequence, and a first UMI, and (ii) the second strand of the first forked adapter transposon comprises a first copy of the second adapter sequence, and a sequence that is fully or partially complementary to the first strand of the first forked adapter transposon.

[0299] Additionally, the second transposome complex comprises a second transposome complex comprising: (1) (i) a second transposase; and (ii) a second forked adapter transposon on a second strand of the double-stranded target nucleic acid fragment, wherein (a) the first strand of the second forked adapter transposon comprises a second 3' terminal transposon end sequence, a second copy of the first adapter sequence, and a second UMI; and (b) the second strand of the second forked adapter transposon comprises a second copy of the second adapter, and a sequence that is fully or partially complementary to the first strand of the second forked adapter transposon.

[0300] Further, (1) the first strand of the first forked adapter transposon further comprises a third adapter sequence, (2) the second strand of the first forked adapter transposon further comprises a fourth adapter sequence and a third UMI, (3) the first strand of the second forked adapter transposon further comprises a sequence that is fully or partially complementary to the third adapter sequence, (4) the second strand of the second forked adapter transposon further comprises a sequence that is fully or partially complementary to the fourth adapter sequence and the fourth UMI, and (5) the tagmentation step generates a double-stranded target nucleic acid fragment that further comprises a third UMI and a fourth UMI.

[0301] As shown in FIG. 13 and described in Example 12, an exemplary method for sequencing a UMI library includes a dark cycle and six custom primers: Custom 1, Custom UMI i7, Custom i7, Custom 2, Custom UMI i5, and Custom i5.

[0302] 6. How to Generate an In-line UMI Using a Hairpin UMI and an Adapter Containing a Universal Hybridizing Tail An exemplary 3' adapter is shown in Figure 14 and described in Example 13. The adapter includes, from 5' to 3', a universal hybridizing tail, a hairpin UMI, ME', and B15. The hairpin UMI includes a 3 or 4 base pair stem structure that forms a bulge. The universal hybridizing tail includes an inosine that can bind to any DNA molecule, allowing hybridization to the exposed 5' base of the transferred strand.

[0303] As described in Example 13, an exemplary method for generating a UMI library using an in-line UMI includes: (1) applying a sample containing double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3'-terminal transposon end sequence and a first adapter sequence; (2) tagmenting a first strand of the double-stranded target nucleic acid with the transposon to generate a double-stranded target nucleic acid fragment containing the first adapter sequence; (3) releasing the double-stranded target nucleic acid fragment from the transposome complex; (4) hybridizing a polynucleotide containing a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3'-terminal transposon sequence; (5) ligating the polynucleotide to the double-stranded target nucleic acid fragment; (6) generating a double-stranded target nucleic acid fragment containing a UMI, where the UMI is located immediately adjacent to the 3'-end of the insert DNA; and (7) amplifying the double-stranded target nucleic acid fragment.

[0304] Additionally, the ligation step includes ligating the 3' end of the second strand of the double-stranded target nucleic acid fragment to the 5' end of the universal hybridizing tail.

[0305] Furthermore, the hairpin UMI is stable during the extension and / or ligation steps, but not during the amplification step.

[0306] According to this method, the UMI is on the first strand of a double-stranded target nucleic acid fragment.

[0307] The exemplary adapters and methods described herein generate a UMI library in which an in-line UMI is adjacent to the 3' end of the insert DNA (Figure 20). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0308] 7. Manufacturing method of in-line UMI including hairpin UMI An exemplary 3' adapter is shown in Figure 15 and described in Example 14. The adapter is a polynucleotide that includes, from 5' to 3', a hairpin UMI, ME', and B15. The hairpin UMI includes a 3 or 4 base pair stem structure that forms a bulge.

[0309] As described in Example 14, an exemplary method for generating a UMI library using in-line UMIs includes: (1) applying a sample containing a double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3'-terminal transposon end sequence and a first adapter sequence; (2) tagmenting the first strand of the double-stranded target nucleic acid with the transposon to generate double-stranded target nucleic acid fragments that contain the first adapter sequence; and (3) releasing the double-stranded target nucleic acid fragments from the transposome complex. (4) hybridizing a polynucleotide comprising a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3' terminal transposon sequence; (5) extending the second strand of the double-stranded target nucleic acid fragment; (6) ligating the extended polynucleotide to the double-stranded target nucleic acid fragment; (7) generating a double-stranded target nucleic acid fragment comprising a UMI, wherein the UMI is located immediately adjacent to the 3' end of the insert DNA; and (8) amplifying the double-stranded target nucleic acid fragment.

[0310] Additionally, the extension step includes extending from the 3'-end of the second strand of the double-stranded target nucleic acid fragment to the 5'-end of the hairpin UMI.

[0311] Additionally, the ligation step includes ligating the 3' end of the second strand of the double-stranded target nucleic acid fragment to the 5' end of the pin UMI.

[0312] Furthermore, the hairpin UMI is stable during the extension and / or ligation steps, but not during the amplification step.

[0313] According to this method, the UMI is on the first strand of a double-stranded target nucleic acid fragment.

[0314] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 20). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0315] 8. First Method for Fabricating In-line UMI Containing Splint Ligation Adapters An exemplary 3' adapter is shown in Figure 16 and described in Example 15a. The adapter is a polynucleotide that includes a 3' splint ligation adapter complex that includes a partial duplex. The two parts of the adapter are the splint (see Figure 16, 3' splint ligation adapter, bottom strand) and the tail (see Figure 16, 3' splint ligation adapter, top strand). The splint portion includes, 5' to 3', ME, UMI', ME', truncated A14'. The tail portion includes, 5' to 3', UMI, ME', and B15. The complex is formed by hybridization of the UMI and ME sequences.

[0316] As described in Example 15a, an exemplary method for generating a UMI library using an in-line UMI includes: (1) applying a sample containing double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3'-terminal transposon end sequence and a first adapter sequence; (2) tagmenting a first strand of the double-stranded target nucleic acid with the transposon to generate a double-stranded target nucleic acid fragment containing the first adapter sequence; (3) releasing the double-stranded target nucleic acid fragment from the transposome complex; (4) hybridizing a polynucleotide containing a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3'-terminal transposon sequence; (5) ligating the polynucleotide to the double-stranded target nucleic acid fragment; (6) generating a double-stranded target nucleic acid fragment containing a UMI, where the UMI is located immediately adjacent to the 3'-end of the insert DNA; and (7) amplifying the double-stranded target nucleic acid fragment.

[0317] Additionally, the extension step includes extending 9 bases from the 3' end of the second strand of the double-stranded target nucleic acid fragment to the 5' end of the splint ligation adaptor.

[0318] Additionally, the ligation step includes ligating the 3' end of the second strand of the extended double-stranded target nucleic acid fragment to the 5' end of the first strand of the splint ligation adaptor.

[0319] According to this method, the UMI is on the first strand of a double-stranded target nucleic acid fragment.

[0320] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 20). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0321] 9. Second Method for Fabricating In-line UMI Containing Splint Ligation Adapters An exemplary 3' adapter is shown in Figure 16 and described in Example 15b. The adapter is a polynucleotide that includes a 3' splint ligation adapter complex that includes a partial duplex. The two parts of the adapter are the splint (see Figure 16, 3' splint ligation adapter, bottom strand) and the tail (see Figure 16, 3' splint ligation adapter, top strand). The splint portion includes, from 5' to 3', X, UMI', ME', truncated A14', where X is the 3' TruSeq™ adapter sequence, which can be full-length or truncated. The tail portion includes, from 5' to 3', UMI, X', and B15. The complex is formed by hybridization of the UMI and X sequences.

[0322] As described in Example 15b, an exemplary method for generating a UMI library using an in-line UMI includes: (1) applying a sample containing double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3'-end transposon end sequence and a first adapter sequence; (2) tagmenting a first strand of the double-stranded target nucleic acid with the transposon to generate double-stranded target nucleic acid fragments containing the first adapter sequence; (3) releasing the double-stranded target nucleic acid fragments from the transposome complex; (4) hybridizing a polynucleotide containing a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3'-end transposon sequence; (5) ligating the polynucleotide to the double-stranded target nucleic acid fragments; (6) generating double-stranded target nucleic acid fragments containing a UMI, where the UMI is located immediately adjacent to the 3'-end of the insert DNA; and (7) amplifying the double-stranded target nucleic acid fragments.

[0323] Additionally, the extension step includes extending 9 bases from the 3' end of the second strand of the double-stranded target nucleic acid fragment to the 5' end of the splint ligation adaptor.

[0324] Additionally, the ligation step includes ligating the 3' end of the second strand of the extended double-stranded target nucleic acid fragment to the 5' end of the first strand of the splint ligation adaptor.

[0325] According to this method, the UMI is on the first strand of a double-stranded target nucleic acid fragment.

[0326] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 20). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0327] 10. First Method for Producing an In-Line UMI Containing a 3' Template Switch Oligonucleotide An exemplary 3' adapter is shown in Figure 17 and described in Example 16a. The adapter is a polynucleotide that comprises a template switch oligonucleotide about 70 nucleotides in length and contains, from 5' to 3', B15', ME or X, UMI', ME', and A14'.

[0328] As described in Example 16a, an exemplary method for generating a UMI library using an in-line UMI includes: (1) applying a sample containing a double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3'-end transposon end sequence and a first adapter sequence; (2) tagmenting a first strand of the double-stranded target nucleic acid with the transposon to generate a double-stranded target nucleic acid fragment containing the first adapter sequence; (3) releasing the double-stranded target nucleic acid fragment from the transposome complex; (4) hybridizing a polynucleotide containing a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3'-end transposon sequence; (5) ligating the polynucleotide to the double-stranded target nucleic acid fragment; (6) generating a double-stranded target nucleic acid fragment containing a UMI, where the UMI is located immediately adjacent to the 3'-end of the insert DNA; and (7) amplifying the double-stranded target nucleic acid fragment.

[0329] Furthermore, the extension step includes (1) extending the second strand of the double-stranded target nucleic acid fragment from the 3' end to a junction in the template switch oligonucleotide by copying the first strand of the double-stranded target nucleic acid fragment; (2) template switching from the first strand to the unpaired region of the 3' template switch oligonucleotide; and (3) copying the unpaired region of the 3' template switch oligonucleotide from the junction to the 5' end of the unpaired region of the 3' template switch oligonucleotide.

[0330] According to this method, the UMI is on the first strand of a double-stranded target nucleic acid fragment.

[0331] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 20). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0332] 11. A second method for generating an in-line UMI containing a template switch oligonucleotide, wherein the oligonucleotide contains a modification in A14' An exemplary 3' adapter is shown in Figure 17 and described in Example 16b. The adapter is a polynucleotide comprising a template switch oligonucleotide about 70 nucleotides in length and contains, from 5' to 3', B15', ME or X, UMI', ME', and optionally a portion of A14'. The A14' sequence has been truncated or removed. Thus, this adapter is the same as the adapter described in II.G.10 above, except that the adapter in II.G.10 above has the A14' sequence, but in this embodiment, the A14' sequence has been truncated or removed.

[0333] As described in Example 16b, this exemplary method includes the steps disclosed in II.G.10 above.

[0334] According to this method, the UMI is on the first strand of a double-stranded target nucleic acid fragment.

[0335] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 20). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0336] 12. A method for producing an in-line UMI comprising a 5' double-stranded adapter, a polymerase extension step, and a proximity ligation step An exemplary adapter is shown in Figure 19B. The adapter comprises a 5' duplex comprising two oligonucleotides. The first oligonucleotide comprises, from 5' to 3', B15, X, and UMI. The second oligonucleotide comprises, from 5' to 3', UMI', X', and B15'. The first and second oligonucleotides are hybridized to form a double-stranded adapter.

[0337] As described in Example 16d and shown in Figures 19A-19C, an exemplary method for generating a UMI library using in-line UMIs includes: (1) applying a sample containing a double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3'-terminal transposon end sequence and a first adapter sequence; (2) tagmenting the first strand of the double-stranded target nucleic acid with the transposon to generate double-stranded target nucleic acid fragments containing the first adapter sequence; and (3) releasing the double-stranded target nucleic acid fragments from the transposome complex. (4) hybridizing a first polynucleotide comprising a UMI and a second adaptor sequence, (5) adding a second polynucleotide comprising a region complementary to the first polynucleotide to generate a double-stranded adaptor, (6) extending the second strand of the double-stranded target nucleic acid fragment, (7) ligating the double-stranded adaptor to the double-stranded target nucleic acid fragment, (8) generating a double-stranded target nucleic acid fragment comprising a UMI, the UMI being located between the double-stranded target nucleic acid fragment and the second adaptor sequence, and (9) amplifying the double-stranded target nucleic acid fragment. The above ligation step is referred to as "proximity ligation" because the 5' phosphate and 3' OH being ligated are not hybridized to the same template strand (as shown in FIG. 19B).

[0338] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 19d). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0339] 13. Methods for Producing an In-Line UMI Containing a 5' Single-Stranded Polymerase Template Switch Oligonucleotide An exemplary adaptor is shown in Figure 18B. The adaptor comprises, from 5' to 3', a 5' polymerase template switch oligonucleotide having B15, X, and a UMI.

[0340] As described in Example 16c and shown in Figures 18A-18C, an exemplary method for generating a UMI library using in-line UMIs includes: (1) applying a sample containing a double-stranded target nucleic acid to a transposome complex containing (i) a transposase and (ii) a transposon containing a first 3' terminal transposon end sequence and a first adapter sequence; and (2) tagmenting the first strand of the double-stranded target nucleic acid with the transposon to generate double-stranded target nucleic acid fragments containing the first adapter sequence. (3) releasing the double-stranded target nucleic acid fragment from the transposome complex, (4) hybridizing a first polynucleotide comprising a UMI and a second adapter sequence, (5) extending the second strand of the double-stranded target nucleic acid fragment, (6) copying the first polynucleotide, (7) generating a double-stranded target nucleic acid fragment comprising a UMI, the UMI being located between the double-stranded target nucleic acid fragment and the second adapter sequence, and (9) amplifying the double-stranded target nucleic acid fragment. The extension step includes template switching from the target nucleic acid strand to the adapter strand.

[0341] The exemplary adapters and methods described herein generate a UMI library in which the UMI is adjacent to the 3' end of the insert DNA (Figure 18d). Using standard sequencing methods, each UMI and insert DNA sequence is captured using read 2 without sequencing the ME sequence. The use of this exemplary adapter and method to generate a UMI library eliminates the need for a dark cycle when the UMI library is sequenced.

[0342] H. Samples and Target Nucleic Acids The biological sample used according to the present disclosure can be of any type that contains target nucleic acid. However, the sample does not need to 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, the 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, the 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 tagmentation 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., which are incorporated herein by reference.

[0343] In some embodiments, the sample applied to the solid support has a 260 / 280 absorbance ratio that is 1.7 or less.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] 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.

[0348] 1.DNA In some embodiments, the sample comprises target double-stranded DNA. In some embodiments, the DNA is genomic DNA. In some embodiments, the DNA is cell-free DNA (cfDNA). In some embodiments, the DNA is circulating tumor DNA (ctDNA). In some embodiments, the DNA is a DNA:RNA duplex, as described in detail in section II.H.3 below.

[0349] 2.RNA In some embodiments, the sample comprises target RNA. In some embodiments, the sample comprises RNA and DNA. In some embodiments, the target RNA is mRNA. In some embodiments, the target RNA comprises coding, untranslated region (UTR) sequences, intron sequences, and / or intergenic sequences.

[0350] In some embodiments, the target RNA comprises a sequence that is complementary to at least a portion of one or more of the capture oligonucleotides.

[0351] In some embodiments, the target RNA is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA). Appropriate capture oligonucleotides can be designed based on the type of target RNA.

[0352] In some embodiments, the 3' end of the target RNA is attached to a capture oligonucleotide.

[0353] In some embodiments, the target RNA is an mRNA. In some embodiments, the target RNA is polyadenylated (i.e., comprises a stretch of RNA that contains only adenine bases). In some embodiments, the mRNA comprises a polyA tail. In some embodiments, the 3' end of the mRNA comprises a polyA tail.

[0354] In some embodiments, the target mRNA contains a polyA sequence and binds to a capture oligonucleotide that contains a polyT sequence.

[0355] 3.DNA:RNA double strand In some embodiments, cDNA is synthesized from a sample containing RNA as the first step of library preparation. In other words, DNA:RNA duplexes can be generated in solution before tagmentation by BLT. In some embodiments, DNA:RNA duplexes are then captured on BLT by capture oligonucleotides. In some embodiments, DNA:RNA duplexes are directly bound to BLT based on their affinity for transposase contained in transposome complex.

[0356] In some embodiments, cDNA synthesis is performed by reverse transcriptase. In some embodiments, this cDNA synthesis results in a DNA:RNA duplex, producing a strand of DNA that can hybridize to a strand of RNA. In some embodiments, reverse transcriptase polymerase is added to a sample containing RNA under conditions to synthesize cDNA. In some embodiments, the conditions for synthesizing cDNA include the presence of nucleotides and / or primers that can bind to RNA (such as poly-T primers and / or randomer primers).

[0357] In some embodiments, the reverse transcriptase only prepares DNA from RNA (without producing additional copies of the DNA to obtain double-stranded DNA).

[0358] In some embodiments, the DNA:RNA duplexes generated in solution can then be bound to a BLT and tagmented. As described above for RNA in section II.H.2, the target RNA can contain a polyA tail that binds to a capture oligonucleotide that contains a polyT sequence.

[0359] In some embodiments, fragments of the DNA:RNA duplex can be used to generate coding, untranslated regions (UTRs), introns, and / or intergenic sequences of a target RNA.

[0360] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA includes adding a reverse transcriptase polymerase to a sample containing the target RNA under conditions to synthesize cDNA and generate DNA:RNA duplexes, immobilizing the DNA:RNA duplexes to a solid support on which a transposome complex is immobilized, the transposome complex comprising a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion comprising a transposon end sequence and a first tag, the sample being applied to the solid support under conditions in which the DNA:RNA duplexes bind directly to a capture oligonucleotide or to the transposase, and performing fragmentation on the DNA:RNA duplexes with the transposome complexes under conditions in which the DNA:RNA duplexes are tagged at the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, at least one strand being 5' tagged with the first tag. In some embodiments, the 5' end of one strand is the 5' end of the RNA strand. In some embodiments, the 5' end of one strand is the 5' end of a DNA strand.

[0361] III. Methods for sequencing UMI libraries The present disclosure further relates to sequencing the UMI library produced according to the methods provided herein. The UMI library 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, and the like. In some embodiments, the library is sequenced on a solid support. In some embodiments, the solid support for sequencing is the same solid support on which surface-bound tagmentation occurs. In some embodiments, the solid support for sequencing is the same solid support on which amplification occurs.

[0362] 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 polymer-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.

[0363] 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 produced 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.

[0364] Other sequencing procedures using cyclic reactions can be used, such as pyrosequencing, which detects the release of inorganic pyrophosphate (PPi) when a particular 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 Nos. 6,210,891, 6,258,568, and 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. Thus, the sequencing reaction can be monitored via a luminescence detection system. The excitation radiation source used in fluorescence-based detection systems is not required for pyrosequencing procedures. Useful fluidic systems, detectors, and procedures that can be adapted for application of pyrosequencing to amplicons produced by the present disclosure are described, for example, in International Application PCT / US11 / 57111, U.S. Patent Application Publication No. 2005 / 0191698(A1), U.S. Patent Nos. 7,595,883, and 7,244,559, each of which is incorporated herein by reference.

[0365] 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.

[0366] 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.

[0367] 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, each nucleotide type 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).

[0368] 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 Application Publication Nos. 2005 / 0053980(A1), 2009 / 0186349(A1), or 2005 / 0181440(A1), each of which is incorporated herein by reference.

[0369] 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 nucleic acid 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 No. 2010 / 0111768(A1) and U.S. Patent Application No. 13 / 273,666, 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, Calif.) and the devices described in U.S. Patent Application No. 13 / 273,666, which is incorporated herein by reference.

[0370] In some embodiments, the method of sequencing a UMI library of the present disclosure sequences the UMIs to provide increased sensitivity in DNA sequencing. In some embodiments, the sequencing method comprises NextSeq 500 / 550 (Illumina).

[0371] A. Dark Cycle In some embodiments, a custom sequencing recipe is prepared and selected using NextSeq software to include a dark cycle that is used to skip recording of a particular sequence. The sequencing chemistry of that sequence is still performed, but the sequence is not imaged by the instrument. The dark cycle is used to mitigate the phasing / pre-phasing problem associated with repeatedly sequencing low diversity sequences, such as ME sequence libraries, which can lead to overall poor sequencing results. After the dark cycle, imaging of the sequence is resumed so that the insert sequence of the target nucleic acid is recorded.

[0372] The custom sequencing recipe includes a variation of the standard recipe to include an appropriate number of dark cycles that span the length of the sequence to be skipped. In other words, the number of dark cycles is equal to the number of bases that are intended to be skipped. For example, if the sequence to be skipped is a ME sequence that is 19 bases long, 19 dark cycles are used. In some embodiments, the sequence to be skipped is an ME sequence. In an embodiment with a ME that is 19 nucleotides long, the number of dark cycles is 19. For MEs with a different number of nucleotides, the dark cycle is generally the number of nucleotides. To obtain the maximum benefit from the dark cycle, the user can skip the entire ME, but it is also possible to skip the ME domain and most of its sequence portion and ignore those nucleotides in the results.

[0373] In some embodiments, the sequencing method includes a dark cycle in which data is not recorded for a portion of the sequencing method. In some embodiments, the unrecorded data is sequence data related to 3' transposon end sequence. In some embodiments, the unrecorded sequence data is ME sequence. In some embodiments, the dark cycle includes 19 cycles.

[0374] In some embodiments, the sequencing method does not include a dark cycle. In these embodiments, the method of preparing a UMI library eliminates the need for a dark cycle because each UMI is adjacent to the 3' end of the insert nucleic acid and there is no ME sequence between them (Figure 20).

[0375] In some embodiments, custom primers are used to eliminate the need for a dark cycle. In these embodiments, the custom primers are bridging primers that contain sequences that align with the ME (Figures 4 and 6B). In these embodiments, the ME sequence is not imaged.

[0376] B. Sequencing primers Sequencing primers and adapter sequences that may be used to sequence UMI libraries using Illumina library preparation kits and sequencing platforms, such as Nextera, Illumina Prep, Illumina PCR, AmpliSeq™, TruSight®, and TruSeq™, are as disclosed in Illumina Adapter Sequences Document#1000000002694 v15, which is incorporated by reference in its entirety. These sequencing primers and adapters may be modified according to the present disclosure. Examples of such primers and adapters include Read 1, Read 2, Index 1 Read, Index 2 Read, Index 1 (i7) adapter, Index 2 (i5) adapter, Index adapters 1-27, TruSeq Universal Adapter, Index PCR Primer, Multiplexing Adapter, Multiplexing Read Sequencing Primer, Multiplexing Index Read Sequencing Primer, and PCR Primer Index Sequences 1-12.

[0377] In some embodiments, the sequencing method comprises combining sequencing primers that have similar melting temperatures.

[0378] 1. Custom primer Custom primers can be used in the sequencing reaction to serve different functions.

[0379] In some embodiments, the UMI sequence is included in a custom primer to enable primer binding to the UMI.

[0380] In some embodiments, custom primers may contain sequences that function to lengthen the primer and / or affect the melting temperature of the primer. In some embodiments, custom and standard sequencing primers that may be used in the same reaction may have similar melting temperatures.

[0381] In some embodiments, the custom primer is a bridge primer that includes one or more spacers that allow the bridge primer to align with any nucleic acid sequence.

[0382] In some embodiments, the spacer is capable of binding to a target nucleic acid sequence, hi some embodiments, the spacer comprises a universal hybridization sequence, such as inosine.

[0383] In some embodiments, the spacer may align with the target nucleic acid sequence without binding to it, hi some embodiments, the spacer comprises a non-nucleic acid linker.

[0384] In some embodiments, the spacer aligns with a variable sequence. In some embodiments, the spacer aligns with a UMI sequence. In some embodiments, the spacer aligns with a UDI sequence.

[0385] In some embodiments, the sequencing primer comprises a sequence that is fully or partially complementary to one or more unique primer binding sequences, hi some embodiments, the sequencing primer comprises at least the A2 sequence, at least the A14 sequence, or at least the B15 sequence.

[0386] In some embodiments, the unique primer binding sequences are A2, A14, and / or B15.

[0387] a) Spacer As used herein, the spacer region in a sequence refers to a nucleic acid sequence that does not carry any structural or classification information for known gene function. The spacer region on a polynucleotide or oligonucleotide can be aligned with a variety of sequences. In some embodiments, the spacer region can be aligned with a range of i5 sequences, which are disclosed in Illumina Adapter Sequences Document#1000000002694 v15, which is incorporated herein by reference. In some embodiments, the spacer region is aligned with a UMI sequence. In some embodiments, the spacer region is aligned with a ME sequence.

[0388] In some embodiments, the spacer region is a universal sequence. In some embodiments, the spacer region is a non-DNA spacer. In some embodiments, the spacer region includes a universal base such as inosine or nitroindole. Alternatively, the spacer may include a synthetic linker. Examples of synthetic linkers include C3 spacer, hexanediol, 1',2'-dideoxyribose (dSpacer), photocleavable spacer (PC spacer), spacer 9, and spacer 18. The C3 spacer is a C3 spacer phosphoramidite that can be incorporated internally or at the 5' end of the oligonucleotide. Multiple C3 spacers can be added to either end of the oligonucleotide to introduce long hydrophilic spacer arms for the addition of fluorophores or other pendant groups. Hexanediol is a 6-carbon glycol spacer that can block extension by DNA polymerase. This 3' modification can support the synthesis of longer oligonucleotides. The dSpacer modification can be used to introduce stable abasic sites within the oligonucleotide. The PC spacer can be placed between DNA bases or between the oligonucleotide and the 5'-modification group. The PC spacer provides a 10 atom spacer arm that can be cleaved by exposure to UV light in the 300-350 nm spectral range. Cleavage liberates an oligonucleotide bearing a 5'-phosphate group. Spacer 9 is a triethylene glycol spacer that can be incorporated at the 5' or 3' end of the oligonucleotide, or internally. Multiple insertions can be used to create longer spacer arms. Spacer 18 (iSp18) is an 18 atom hexaethylene glycol spacer and can be considered the longest spacer arm that can be added as a single modification.

[0389] In some embodiments, the spacer comprises an iSp18 linker. As used herein, an iSp18 linker is a standard modified linker with a C18 spacer (18 atom hexa-ethylene glycol spacer) and is equal to 4 base pairs in length. Thus, a 2x sp18 linker is equal to 8 base pairs in length. In some embodiments, the spacer region comprises a 2x iSp18 synthetic linker. In some embodiments, the spacer region comprises one or more C18 spacers, such as 1, 2, 3, 4, 5, 6 or more C18 spacers. In some embodiments, the spacer region comprises two C18 spacers (equivalent to 8 nucleotides in length). In some embodiments, the spacer is a C9 spacer equal to 2 base pairs in length. In some embodiments, the spacer region comprises one or more C9 spacers (triethylene glycol spacers), such as 1, 2, 3, 4, 5, 6 or more C9 spacers. In some embodiments, the spacer is a conventional spacer used with existing indexes, such as a 10 base pair spacer. In some embodiments, the spacer region is a combination of spacers, for example, a combination of one or more C18 spacers and one or more C9 spacers, or any combination of any spacers described herein. In some embodiments, the spacer region is equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, or 30 base pairs in length. In some embodiments, the spacer region is approximately equal to 8 or 10 base pairs or nucleotides in length. In some embodiments, the spacer region is specifically selected to be the same length as the index region. In some embodiments, the index region is 8 nucleotides in length and the spacer region comprises two C18 spacers. In some embodiments, the index region is 10 nucleotides in length and the spacer region comprises two C18 spacers and one C9 spacer.

[0390] In some embodiments, the spacer comprises an abasic nucleotide. The abasic nucleotide can be introduced at any position of the spacer. Examples of spacers having an abasic nucleotide include dSpacer (1',2'-dideoxyribose, DNA abasic), rSpacer (i.e., RNA abasic), and Abasic II. In some embodiments, the dSpacer is an abasic furan, tetrahydrofuran (THF), a THF derivative, or an apurine / apyrimidine (AP) nucleotide.

[0391] In some embodiments, the spacer includes a wobble base. The wobble base can be introduced at any position of the spacer. A wobble base pair is a pairing between two nucleotides that does not follow the Watson-Crick base pairing rules, such as guanine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine.

[0392] IV. Kits Containing Transposome Complexes In some embodiments, the kits include components of the transposome complexes disclosed herein, hi some embodiments, the kits include components for generating such transposome complexes, including transposases and oligonucleotides including transposons, 5' and 3' transposon end sequences, adapter sequences, UMI sequences, and / or other HYB / HYB' sequences.

[0393] The kits may include any of a variety of adaptors, in many embodiments, the adaptors may be selected from 3' adaptors, polynucleotide adaptors, forked adaptors, hairpin UMI adaptors, hairpin UMI and universal hybridizing tail adaptors, splint ligation adaptors, template switch oligonucleotide adaptors, and any suitable oligonucleotides.

[0394] In some embodiments, the kit may include components for Hyb2Y, such as adaptors and buffers.

[0395] In some embodiments, the kit may include a solid support, such as beads.

[0396] In some embodiments, the kit may include a reverse transcriptase polymerase.

[0397] In some embodiments, the kit may include a sequencing primer. EXAMPLES

[0398] The following examples describe methods related to preparing DNA sequencing libraries with UMIs. The generation of sequencing libraries using BLT methods (e.g., Illumina DNA Prep (for research use only, RUO), formerly known as Nextera DNA Flex Library Prep and Nextera XT DNA Library Preparation Kits) is a convenient and efficient method that is compatible with NGS library preparation workflows. For many of these, it is desirable to be able to track the relative orientation and identity of sequenced DNA molecules (i.e., the strandedness or orientation of the target DNA) and bioinformatically resolve them. The methods described in the examples relate to the use of UMIs to provide strandedness or orientation, a feature not available with current generation BLT methods. UMIs are incorporated without the use of Illumina TruSeq™ methods. The following examples disclose different methods of incorporating UMIs.

[0399] Example 1. Preparation of DNA libraries for sequencing using UMI-BLT to enable double-stranded UMI error correction This example describes the asymmetric tagmentation BLT method used to prepare a DNA sequencing library with a unique double index (UDI) and a double-stranded UMI. This example describes a method to combine UDI and UMI for error correction. A single UMI is used to tag a DNA library, and then the single UMI is copied to generate a double-stranded UMI.

[0400] The method of this example combined the BLT method with the Hyb2Y workflow. In the tagmentation step, a first UMI was added to the first strand of the target DNA, and a second UMI was added to the second strand of the target DNA.

[0401] In this method, an additional A2 adapter sequence was added to the transposon arm in the BLT and the UMI was copied using the Hyb2Y workflow. The addition of the A2 sequence to the BLT adapter serves two purposes. First, it allows for the annealing of a Hyb2Y oligonucleotide that can be extended to have a paired UMI on the opposite strand. Hybridization of the Hyb2Y oligonucleotide to A2 allows for a longer extension that can copy the UMI and adapter sequence, rather than relying on other methods where extension is minimal. Second, the A2 sequence allows for the development of custom sequencing recipes and custom primers for sequencing that have the same annealing temperature (Tm) as standard sequencing primers. Furthermore, libraries prepared according to this method reduce the amount of adapter dimers that are occasionally observed when a forked adapter BLT design is used. By avoiding adapter dimers, this method also increases library yields.

[0402] A. Material The following materials were used in this example: (1) Genomic DNA (gDNA) Horizon Tru-Q 7 Standard (Horizon catalog no. HD734), (2) Illumina DNA Prep with Enrichment (IDPE, Illumina catalog no. 20025523 and 20025524, formerly Nextera Flex for Enrichment), (3) TruSight Oncology UMI Reagents (Illumina catalog no. 20024586), (4) TruSight Tumor 170 Reagents (Illumina catalog no. 20028821), (5) New Enrichment Blocker NHB2 (Illumina reference no. 20031771), (6) Extension Ligation Mix ELM3 (Illumina catalog no. 20019117), (7) NextSeq 500 / 550 v2.5 Kit (Illumina catalog no. 20024906), and (8) custom primers.

[0403] B. BLT library with double-stranded UMI In this method, the BLT for tagmentation of the target DNA fragment was first prepared in the reaction mixture using a capture oligonucleotide with UMI-BLT (Figure 1). The target DNA for tagmentation was added to the reaction mixture with UMI-BLT (Figure 2). 10 ng and 50 ng of gDNA Horizon Tru-Q 7 standard samples were used as target DNA.

[0404] A tagmented library containing an AB-long single UMI was prepared using a BLT made at a density similar to the eBLT used in IDPE. The library was prepared using a TruSight™ Tumor (TST170, Illumina) probe according to the IDPE protocol guidelines. The tagmentation process was stopped by adding tagmentation stop buffer ST2.

[0405] The resulting tagmented library was heated at 55°C for 5 minutes to release the tagmented library into solution. The 3'-biotinylated ME remained bound to the beads and was not transferred. The reaction mixture was incubated at room temperature for 5 minutes, and the reaction mixture was washed twice with Tagmentation Wash Buffer (TWB).

[0406] Hyb2Y oligonucleotide (5'P-A2'A14'-3' in Figure 2) was then added and annealed at 65° C. for 10 minutes. The reaction mixture was allowed to slowly cool to 37° C. The supernatant of the reaction mixture was then removed and mixed with extension-ligation mix ELM3 for gap filling.

[0407] The 34 bases are gap-filled by extension and ligation in ELM3 for 30 min at 37°C. The UMI sequence is copied during this step, which allows the UMI to be used to identify and group the top and bottom strands, thereby allowing UMI double-strand error correction. The reaction mixture was then washed using solid-phase reversible immobilization beads (SPRI) to generate a solution with tagmented DNA. Nine cycles of PCR were performed using UDI primers to amplify the tagmented DNA. The PCR product was then purified using SPRI to capture the tagmented DNA that fell within the correct size range. Finally, the library (approximately 500 ng of DNA) was enriched using IDPE and TST170 probes. An additional blocker was added for hybridization of the AB-long BLT probe.

[0408] These steps generated a standard structure BLT library with a double-stranded UMI. The library contained the A14 and B15 oligonucleotide sequences that could be used for PCR amplification with Illumina UDI (Figure 2).

[0409] C. BLT library with a single UMI A second BLT library was prepared, this one containing a single UMI and was made with an AB-short single UMI. The library was prepared using the steps described above for the AB-long single UMI, except that no additional blocker was used for the BLT hybridization.

[0410] D. Control Library For comparison, a separate tagmented library was prepared using TruSight Oncology UMI reagents following the TruSight Tumor 170 protocol guidelines.

[0411] For further comparison, a UMI-free library was prepared using NFE.

[0412] Example 2. Sequencing of DNA libraries containing double-stranded UMIs with dark cycles This example describes a method for sequencing the DNA library of Example 1.

[0413] A. Material The following systems and materials were used in this example: (1) A NextSeq 500 sequencing system was used (Illumina Document #15046563), and (2) sequencing primers specific for the libraries in Example 1 and custom primers, as required (Illumina Document #15057456).

[0414] B. Method The libraries from Example 1 were pooled, denatured, and added to the NextSeq 500 sequencing cartridge following the protocol guidelines. Custom primers were diluted and added to the relevant positions in the cartridge according to the NextSeq 500 and NextSeq 550 Sequencing Systems Custom Primers Guide.

[0415] A custom sequencing recipe was loaded onto the sequencing instrument and selected using the NextSeq software. The recipe involved modifying the standard recipe to include 19 dark cycles across the ME region. A dark cycle is a sequencing cycle without imaging, which corrected for phasing / prephasing issues that can lead to poor overall sequencing results. The dark cycle is described in detail above in section III.A. During the dark cycle, the 19 bases of the ME region were not imaged. After the dark cycle, imaging was resumed and the insertion sequence was imaged.

[0416] The sample sheet contained the settings found in the TruSight Oncology UMI Reagents Guide.

[0417] Data analysis was performed in Basespace Sequence Hub using an internal UMI folding APP and the Dragen Enrichment App.

[0418] 1. Primer The custom sequencing primers used are as shown in Figure 3B. The four custom primers contain melting temperatures (Tm) compatible with the standard sequencing primers and therefore can be mixed and used in the same sequencing reaction. The custom primers were (1) Custom Primer 1 UMI+Read 1, (2) Custom Primer i5, (3) Custom Primer i7, and (4) Custom Primer 4 UMI+Read 2, as shown in Figure 3B. The custom primers were designed to anneal to their respective regions as indicated by the blue arrows in Figure 3B. Custom Primer 1 UMI+Read 1 annealed to the A14-A2 sequence. Custom Primer i5 annealed to the A14'-A2' sequence. Custom Primer i7 annealed to the A2'-B15' sequence. Custom Primer 4 UMI+Read 2 annealed to the B15-A2 sequence. The sequence of the insert DNA was read using Custom Primer 1 UMI+Read 1 and Custom Primer 4 UMI+Read 2.

[0419] Three custom primer ports containing a total of six primers were used in this sequencing method. i7 and i5 custom primers were added to one custom primer port according to the standard operating procedure for sequencing. The primers used and prepared according to this example can be useful for those skilled in the art who have a limited number of available primer ports on a sequencing cartridge. For example, some sequencing platforms only have three available primer ports. This method allows the mixing of different custom sequencing primers in a single reaction that are used at different times during the sequencing process, thereby allowing those skilled in the art to minimize the number of custom primer ports required on a sequencing cartridge.

[0420] Optionally, this method may instead include only two primers: Custom Primer 1 UMI + Read 1 and Custom Primer 2 UMI + Read 2. These two primers can be pre-mixed, requiring only two custom primer ports.

[0421] C. Results FIG. 3C shows the quality score for each cycle in a sequencing run. Briefly, the quality score is a prediction of the probability of an error in a base call. A high quality score means that the base call is more reliable and less likely to be incorrect. For a base call with a quality score of Q30, 1 in 1,000 base calls are predicted to be incorrect. When sequencing quality reaches Q30, virtually all reads are complete and free of errors and ambiguities. Q30 is considered the benchmark for quality in next-generation sequencing.

[0422] Figure 3C shows the % > Q30, while Figure 3D shows the intensity of the sequencing cycles per cycle in the sequencing run of this example. Dark cycles were used to speed up sequencing and to avoid recording uninformative images of the reaction across the adapter sequence. Dark cycles (and light cycles) reduce the quality of subsequent sequencing compared to starting a new read at the insert (Figures 3C and 3D).

[0423] In sequencing reactions with 50 ng template input, the TruSight UMI method showed excellent performance. The Hyb2Y workflow in Example 1 may have required optimization to allow for improved sequencing performance.

[0424] As shown in FIG. 3E, the TruSight UMI method (TruSight-Duplex) demonstrated superior performance in reactions with 50 ng template input. This may have been caused by UMI reads being discarded in the first step of the analysis due to errors introduced into the UMI sequence by the polymerase used during the extension and ligation steps in Example 1. In FIG. 3E, designs without duplex UMIs were called 0. Adapter blocking of the fork-duplex library was also suboptimal. Nevertheless, the fork-duplex data set was called 20% duplex families. This number should be improved by optimization of the biochemical reactions in the Hyb2Y workflow in Example 1. Examples of parameters that can be optimized include oligonucleotide concentration, hybridization time, hybridization temperature, and the choice of sequences used for hybridization.

[0425] Example 3. Sequencing of DNA libraries containing double-stranded UMIs by bridging primer rehybridization This example describes a method for sequencing the DNA library of Example 1.

[0426] A. Material Materials are as described in Example 2 above.

[0427] B. Method The method is as described in Example 2 above, with the following modifications.

[0428] Here, a custom sequencing recipe is used that does not include a dark cycle. The recipe further includes an additional primer rehybridization between read 1 and read 4 (Figure 4).

[0429] 1. Primer The custom primers in this example are as provided in Table 2 and Figure 4. Primers for Lead 1 and Lead 6 are bridging primers.

[0430] [Table 2]

[0431] Each bridge primer contains a sequence that anneals to the A14-A2 sequence, two spacers that span but do not anneal to the UMI sequence, and a sequence that anneals to the ME sequence. In the tagmented library, the A14-A2 and ME sequences are constant sequences, but the UMI sequence varies. In this example, two copies of iSp18 are used, two spacers in each of primers 2 and 6.

[0432] In the sequencing method of this example, primer 1 is annealed first and then removed to allow primer 2 to anneal. Similarly, primer 5 is annealed and then removed to allow primer 6 to anneal. The sequence of the insert DNA was read using a custom bridging primer for the Insert 1 read and a custom bridging primer for the Insert 2 read.

[0433] Example 4. Preparation of DNA libraries for sequencing using UMI-BLT to enable double-stranded UMI error correction This example describes the asymmetric tagmentation BLT method used to prepare DNA sequencing libraries with UDIs and double-stranded UMIs for error correction. Materials are as described in Example 1. In the tagmentation step, UMIs are added to the first strand of target DNA, and the second strand of target DNA is not tagmented by UMIs.

[0434] In this method, the transposome structure containing UMI-BLT for tagmenting the target DNA is as shown in Figure 5A. The tagmented DNA is treated as shown in Figure 5B. The tagmented DNA is washed with sodium dodecyl sulfate (SDS) to remove the transposase TsTn5 (shown in Figures 5A and 5B). The tagmented DNA library is amplified by PCR using UDI primers.

[0435] Example 5. Sequencing of DNA libraries containing double-stranded UMIs with dark cycles This example describes a method for sequencing the DNA library of Example 4 that includes a dark cycle (FIG. 6A).

[0436] A. Material Materials are as described in Example 2 above.

[0437] B. Method The method is as described in Example 2 above, with the following modifications.

[0438] 1. Primer Four primers were used in this method: (1) Standard Insert Read 1, (2) Custom i7, (3) Standard i5, and (4) UMI+Insert Read 2. Primers were designed to anneal to their respective regions as indicated by the black arrows in Figure 6A. Standard Insert Read 1 annealed to the A14-ME sequence. Custom i7 annealed to the A2'-B15' sequence. Standard i5 annealed to the ME'-A14' sequence. UMI+Insert Read 2 annealed to the B15-A2 sequence.

[0439] C. Results The sequencing method of this example (FIG. 6A) was compared to a sequencing run using the TruSeq™ method or the IDPE standard method (FIG. 3A). The Q30% of standard sequencing read 1 and R4 UMI+insert read 2 for this method shown in FIG. 7 ("Dark") indicates that the method was successful, although it did not perform as well as the IDPE ("IDPE Standard") and TruSeq™ ("TruSeq Standard") methods. A decrease in Q30% score was also observed after the dark cycle. This sequencing method uses only three primers and may be the preferred method when used with a sequencing instrument with a cartridge that can support three or fewer primers.

[0440] Example 6. Sequencing of DNA libraries containing double-stranded UMIs by bridging primer rehybridization This example describes a method for sequencing the DNA library of Example 4 that includes bridging primer rehybridization in place of the dark cycle (Figure 6B).

[0441] A. Material Materials are as described in Example 5 above.

[0442] B. Method The method is as described in Example 5 above, with the following modifications.

[0443] 1. Primer This method uses five primers: (1) Standard Insert Read 1, (2) Custom i7, (3) Standard i5, (4) UMI, and (5) Insert Read 2 Bridge Primer. The primers were designed to anneal to their respective regions as indicated by the black arrows in Figure 6B. Primers (1)-(4) anneal to the regions described in the previous paragraph. Primer 5 contains a sequence that anneals to the A2-B13 sequence, a spacer that spans but does not anneal to the UMI sequence, and a sequence that anneals to the ME sequence. Primer 5 eliminates the need for dark cycles in the sequencing method. In this method, primer 4 is annealed first and then removed to allow primer 5 to anneal. The sequence of the insert DNA is read using the Standard Insert Read 1 and Insert Read 2 Bridge Primers.

[0444] C. Results The sequencing method of this example (FIG. 6B) was compared to sequencing runs using the TruSeq™ method or the IDPE standard method (FIG. 3A). The Q30% of standard sequencing Read 1 and R5-inserted Read 2 bridge primers for this method shown in FIG. 7 ("Re-hyb") indicates that the method performed similarly to the TruSeq™ ("TruSeq Standard") and IDPE ("IDPE Standard") methods, and sequenced better than the method using a dark cycle ("Dark" see also Example 5).

[0445] Example 7. Preparation of DNA libraries from cell-free DNA (cfDNA) using UMI BLT for sequencing This example describes the asymmetric tagmentation BLT method used to prepare DNA sequencing libraries with UDIs and double-stranded UMIs for error correction. Materials are as described in Example 1. In the tagmentation step, a first UMI was added to the first strand of the target DNA, and a second UMI was added to the second strand of the target DNA.

[0446] cfDNA was extracted from 5 mL of plasma from one patient. 2+cfDNA was extracted using BLT Tn5 without Tn5. As shown in Figure 8, cfDNA was processed using the TruSeq™ workflow as a control or the method described in this example ("eBBN" in Figure 8).

[0447] First, cfDNA was processed using the TruSeq™ workflow as follows: (1) end repair for 30 min, (2) A-tailing for 30 min, (3) UMI ligation for 30 min, (4) adapter ligation for 30 min, (5) SPRI clean, and (6) PCR amplification.

[0448] As shown in Figure 9, another sample of cfDNA was processed according to the tagmentation workflow of the existing method with the following steps: (1) cfDNA was tagmented with a capture oligonucleotide containing a single UMI adapter for 5 min, (2) tagmentation was stopped, (3) the tagmented cfDNA, i.e., the UMI library, was washed using a 5-10 min wash, and (4) the generated UMI library was amplified by PCR.

[0449] In this method, a UMI was added to the BLT capture oligonucleotide instead of a UDI, thus precluding further indexing using a UDI: the UMI is not on the same strand as the strand bearing the BLT capture moiety, the UMI is on the transferred strand while the BLT capture moiety is on the non-transferred strand.

[0450] Ten UMI sequences were used in the i7 position and ten UMI sequences were used in the i5 position. The tagmented DNA fragments were gap-filled and amplified by PCR using P5 and P7 primers. This method generated a standard structure BLT library with A14 and B15 oligonucleotide sequences ready for sequencing using standard sequencing primers.

[0451] Example 8. Sequencing of a DNA library containing a single UMI This example describes a method for sequencing the DNA library of Example 7.

[0452] A. Material Materials are as described in Example 2 above.

[0453] B. Method The method is as described in Example 2 above, with the following modifications.

[0454] 1. Primer This example included a standard sequencing run and standard sequencing primers Nextera Read primer 1 (NR1 read), i7 read, i5 read, and Nextera Read primer 2 (NR2 read). The primers were designed to anneal to their respective regions as indicated by the black arrows in Figure 9. The i7 and i5 regions are compromised by UMIs, so UMIs were captured from the index read.

[0455] C. Results The uniform distribution of UMI reads across the DNA library indicates successful incorporation of a single UMI into tagmented DNA fragments (Figure 10). A Read Collapsing analysis step was performed on the sequencing reads to group overlapping reads and collapse them into a single consensus aligned read. The resulting reads (deduplicated reads) have higher per-base quality and lower noise from various sources. Read Collapsing is a useful metric for quality control when UMIs are involved.

[0456] As shown in Figures 11A and 11B, the single UMI-BLT library (shown as "eBBN" in Figure 11B) has greater deduplicated average target coverage and higher conversion of cfDNA to the library than the TruSeq™ library (shown as "No UMI" in Figure 11A).

[0457] Example 9. Preparation of DNA libraries using double-stranded UMI-BLT for sequencing and double-stranded sequence error correction by UDI This example describes a symmetric tagmentation BLT method used to prepare DNA sequencing libraries with UDIs and double-stranded UMIs for error correction. Materials are as described in Example 1. This method includes a double-stranded UMI in the forked adapter capture oligonucleotide of the BLT (FIG. 12). In the tagmentation step, the UMI is added to both strands of the target DNA.

[0458] First, a pool of UMIs containing 120 different UMI duplexes is formed. Each UMI duplex is prepared separately and then mixed together to form a pool of UMIs. This pool is used to prepare forked adapter capture oligonucleotides, which are then used to prepare a universal UMI BLT (Universal UMI Tsm). Target DNA fragments are tagmented using Universal UMI Tsm. Gap filling and ligation are performed using ELM. The tagmented DNA is amplified by PCR using Nextera Index primers and is ready for sequencing.

[0459] Example 10. Sequencing of DNA libraries containing double-stranded UMIs and UDIs This example describes a method to sequence the DNA library of Example 9 containing double-stranded UMIs and UDIs. The method involves the use of four standard primers and a dark cycle to avoid imaging of the ME region.

[0460] A. Material Materials are as described in Example 2 above.

[0461] B. Method The method is as described in Example 2 above, with the following modifications.

[0462] 1. Primer This example includes a sequencing run with 19 dark cycles and sequencing primers (1) A14read, (2) i7read, (3) B15read, and (4) i5read. The primers were designed to anneal to their respective regions as indicated by the gray arrows in FIG.

[0463] The standard A14 and B15 read primers anneal to the A14 and B15 regions. These regions contain short nucleotide sequences (i.e., 14 base pairs), which results in the design of a low Tm in the A14 and B15 read primers. The primers benefit from modifications such as an additional 10 base pairs to increase the respective Tm so that they can read the UMI sequence.

[0464] Example 11. Preparation of DNA libraries for sequencing allowing indexing and double stranded sequence error correction This example describes a symmetric tagmentation BLT method used to prepare DNA sequencing libraries with UDIs and double-stranded UMIs for error correction. Materials are as described in Example 1. This method includes a UMI in the forked adapter capture oligonucleotide of the BLT (FIG. 13). In the tagmentation step, the UMI is added to both strands of the target DNA.

[0465] The steps for preparing UMI, BLT, and tagmented DNA are as described in Example 9 above.

[0466] Example 12. Sequencing of DNA libraries This example describes a method for sequencing the DNA library of Example 11.

[0467] A. Material Materials are as described in Example 2 above.

[0468] B. Method The method is as described in Example 2 above, with the following modifications.

[0469] 1. Primer This example includes six custom sequencing primers: (1) Custom 1, (2) Custom UMIi7, (3) Custom i7, (4) Custom 2, (5) Custom UMIi5, and (6) Custom i5. The primers were designed to anneal to their respective regions as indicated by the black arrows in FIG.

[0470] Example 13. Preparation of DNA libraries for sequencing using 3' adapters containing hairpin UMIs and universal hybridizing tails This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with a UMI, where the UMI is incorporated after tagmentation (FIG. 14). A hairpin-UMI and a 3' adaptor containing a universal hybridizing tail are used to incorporate the UMI.

[0471] Materials were as described in Example 1.

[0472] The method involves tagmenting the target DNA with a 5' sequencing adaptor (5' adaptor), and then hybridizing the 3' sequencing adaptor (3' adaptor) to the 5' adaptor ME sequence such that the UMI is positioned immediately adjacent to the 3' end of the insert DNA. This generates an in-line UMI that ensures compatibility with standard downstream library preparation steps (i.e., sample multiplexed PCR) and sequencing reagent recipes.

[0473] The double-stranded DNA is tagmented using a transposome containing only the 5' adapter sequence A14, and ME, a non-transferred Tn5-mosaic end sequence, is modified. The 3' adapter is an oligonucleotide containing a 3' universal hybridizing tail, which may contain an inosine base capable of general Watson-Crick base pairing. The 3' universal hybridizing tail further contains a UMI hairpin, and ME' sequence, and 3' adapter sequence B15.

[0474] The 3' adapter is hybridized to the 5' adapter ME using Hyb2Y. The universal hybridizing tail is hybridized to the exposed 5' bases of the transferred strand (adjacent to the 5' adapter). With a 9 nucleotide universal hybridizing tail, the 9 exposed nucleotides of the transferred strand are fully hybridized and the 5' of the universal hybridizing tail is ligated to the 3' of the non-transferred strand by E. coli DNA ligase. If a universal hybridizing tail of less than 9 nucleotides is used, an additional extension step of the non-transferred strand may be required prior to ligation.

[0475] Using standard sequencing methods (described in Example 2 and shown in Figures 3B and 20), the libraries in this example can be sequenced at the beginning of read 2 or the end of read 1, preceding and following the insert DNA, respectively. A read is more likely to be captured at the beginning of read 2 due to the quality of the insert and the variable insert length.

[0476] The universal hybridizing tail oligonucleotide provides the possibility to track and resolve the unique copy (Unique Copy Index, UCI) of each (original) DNA molecule. Different copies of the original insert molecule can have 9 nucleotide universal hybridizing tail sequences that differ by the same UMI. Like the UMI, the UCI is inline with a given position in the sequencing read. It can therefore be identified bioinformatically.

[0477] Example 14. Preparation of DNA libraries for sequencing using 3' adapters containing hairpin-UMIs This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with an in-line UMI, where the UMI is incorporated after tagmentation (FIG. 15). A 3' adapter containing a hairpin-UMI is used to incorporate the UMI.

[0478] Materials were as described in Example 1.

[0479] The 3' adaptor contains a hairpin UMI as described in Example 13, but does not contain a universal hybridizing tail.

[0480] The 5' adapter tagmentation and 3' adapter hybridization steps are carried out as described in Example 13. After 3' adapter hybridization, the 3' of the non-transferred strand is extended by DNA polymerase until it reaches the 5' end of the hybridized 3' adapter. (The DNA polymerase does not involve strand displacement and does not contain 5' to 3' exonuclease activity.) This positions the 5' end of the UMI-hairpin close to the 3' end of the 3' adapter.

[0481] Using standard sequencing methods (described in Example 2 and shown in Figures 3B and 20), the libraries in this example can be sequenced at the beginning of read 2 or the end of read 1, preceding and following the insert DNA, respectively. A read is more likely to be captured at the beginning of read 2 due to the quality of the insert and the variable insert length.

[0482] Example 15a. Preparation of DNA libraries for sequencing using 3' splint ligation adapters. This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with an in-line UMI, where the UMI is incorporated after tagmentation (Figure 16). A 3' splint ligation adapter is used to incorporate the UMI.

[0483] Materials were as described in Example 1.

[0484] The 5' adapter tagmentation and 3' adapter hybridization steps are carried out as described in Example 13.

[0485] The 3' splint ligation adapter is a partially double-stranded complex that creates a splint for ligation between the UMI-ME'-B15 and the non-transferred strand (Figure 16). Each strand of the 3' splint ligation adapter forms one of the two parts of the adapter, with each strand being approximately 50 nucleotides long. The two parts of the adapter are the splint (see Figure 16, 3' splint ligation adapter, bottom strand) and the tail (see Figure 16, 3' splint ligation adapter, top strand). The adapter splint portion includes, from 5' to 3', a region of ME, UMI', ME', and truncated A14'. Both the ME and A14' sequences can be truncated to improve the desired hybridization specificity and to reduce adapter oligonucleotide costs. For example, the ME is truncated to prevent intramolecular hybridization with the complete ME' sequence required for 5' to 3' adapter binding. The adaptor tail portion hybridizes to the adaptor splint portion via the UMI and ME sequences, which may improve efficiency by stabilizing hybridization between the 5' and 3' adaptors. The adaptor tail portion contains, from 5' to 3', the UMI, ME', and B15 regions. The adaptor tail portion is not cleaved. The non-transferred strand of the target DNA is extended to the 5' end of the adaptor tail and ligated as specified according to the ligation steps described in Example 14.

[0486] Using standard sequencing methods (described in Example 2 and shown in Figures 3B and 20), the libraries in this example can be sequenced at the beginning of read 2 or the end of read 1, preceding and following the insert DNA, respectively.

[0487] Example 15b. Preparation of DNA libraries for sequencing using 3' splint ligation adapters. This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with an in-line UMI, where the UMI is incorporated after tagmentation (Figure 16). A 3' splint ligation adapter is used to incorporate the UMI. This example describes the method provided by Example 15a with the following modifications.

[0488] The 3' splint ligation adapter is as described in Example 15a above with the following modifications: The adapter splint portion contains, from 5' to 3', the regions X, UMI', ME'. Compared to the splint portion of Example 15a, the splint portion in this example does not contain A14' so that the 3' splint adapter can facilitate 3' adapter addition on the bead. The X sequence is part of the 3' TruSeq™ adapter sequence and can be truncated to improve the desired hybridization specificity and reduce adapter oligonucleotide costs. The adapter tail portion contains, from 5' to 3', the regions UMI, X', and B15.

[0489] The library in this example is sequenced using standard sequencing methods (described in Example 2 and shown in Figure 3B and Figure 20) with the following modifications: a custom Read 2 primer is required.

[0490] Example 16a. Preparation of DNA libraries for sequencing using 3' template switch oligonucleotides This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with an in-line UMI, where the UMI is incorporated after tagmentation (FIG. 17). The UMI is incorporated using a 3' template switch oligonucleotide.

[0491] Materials were as described in Example 1.

[0492] The 3' template switch oligonucleotide is approximately 70 nucleotides in length and contains, from 5' to 3', the following regions: B15', ME or X, UMI', ME', and A14'.

[0493] The 5' adapter tagmentation and 3' adapter hybridization steps are carried out as described in Example 13. After hybridization, extension is performed using a polymerase capable of DNA-directed template switching, such as murine leukemia virus (MMLV) reverse transcriptase. The non-transferred strand is extended to copy the 5' end of the transferred strand up to 9 nucleotides. The template switch junction (in FIG. 17) is ** ), the polymerase can switch from using the non-transferred DNA strand as a template to the 3' template switch oligonucleotide. In this way, the UMI, ME' / X', and B15 sequences are copied from the 3' template switch oligonucleotide.

[0494] Using standard sequencing methods (described in Example 2 and shown in Figures 3B and 20), the libraries in this example can be sequenced at the beginning of read 2 or the end of read 1, preceding and following the insert DNA, respectively.

[0495] Example 16b. Preparation of DNA libraries for sequencing using 3' template switch oligonucleotides This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with an in-line UMI, where the UMI is incorporated after tagmentation (FIG. 17). A 3' template switch oligonucleotide is used to incorporate the UMI. This example describes the method provided by Example 16a with the following changes to the 3' template switch oligonucleotide:

[0496] The A14' sequence of the 3' template switch oligonucleotide is cleaved or removed to facilitate on-bead attachment of the 3' template switch oligonucleotide.

[0497] Using standard sequencing methods (described in Example 2 and shown in Figures 3B and 20), the libraries in this example can be sequenced at the beginning of read 2 or the end of read 1, preceding and following the insert DNA, respectively.

[0498] Example 16c. Preparation of DNA libraries for sequencing using 5' single stranded polymerase template switch oligonucleotides This example describes an asymmetric tagmentation BLT method used to prepare a DNA sequencing library with an in-line UMI, where the UMI is incorporated after tagmentation (Figures 18A-D). The UMI is incorporated using a 5' polymerase template switch oligonucleotide.

[0499] The materials are as described in Example 1. Circulating tumor DNA (ctDNA) is used as the target DNA.

[0500] The 5' single stranded polymerase template switch oligonucleotide is a 5' adaptor that has from 5' to 3' regions B15, X, and UMI (Figure 18B).

[0501] The tagmentation and adapter hybridization steps are performed (FIGS. 18A-B) as described in Example 13. In this example, a 5' adapter is added to the 5' of the ME' (FIG. 18B).

[0502] A polymerase template switch is then used to add a 5' adaptor to the DNA insert. The polymerase switches from using the insert DNA as a template to using the added 5' adaptor as a template (Figure 18C). Once extension is complete, the B15, X, and UMI sequences can be fused to the 3' end of the insert DNA and used as a template in a PCR reaction to add additional flow cell and sample index adaptor elements (Figure 18D).

[0503] The library of this example is sequenced using standard sequencing methods (described in Example 2). The X region serves to extend the B15 region to reach an appropriate Tm for sequencing from B15 in the absence of ME.

[0504] Example 16d. Preparation of DNA libraries for sequencing using 5' double-stranded adapters, polymerase extension and proximity ligation This example describes an asymmetric tagmentation BLT method used to prepare DNA sequencing libraries with in-line UMIs, where the UMI is incorporated after tagmentation (Figures 19A-D). A 5' double-stranded adapter is used to incorporate the UMI.

[0505] The materials are as described in Example 1. Circulating tumor DNA (ctDNA) is used as the target DNA.

[0506] In this example, the 5' double stranded adapter contains, from 5' to 3', the regions B15, X, and UMI on its first strand. The second strand contains, from 5' to 3', the complementary sequences UMI', X', and B15' listed herein. A 5'-phosphate is present on the second strand of the 5' adapter, but the ME' on the tagmentation adapter is dephosphorylated to prevent ligation of the ME' to the 5' adapter (Figure 19B).

[0507] The tagmentation and adapter hybridization steps are performed as described in Example 13 (FIG. 19A-B). A 5' adapter is added to the 5' of the ME' (FIG. 19B). During adapter hybridization, the first and second strands of the 5' adapter are mixed to form a duplex. Additionally, the ME' on the tagmentation adapter is dephosphorylated to prevent ligation with the 5' adapter (FIG. 19B).

[0508] The gap from the initial transposition reaction is then extended across it using a polymerase such as T4 DNA pol Exo (New England BioLabs, Cat. No. M0203S) or Ttaq608 (FIG. 19C). Taq polymerase, or a mutant, analog, or derivative of any of the aforementioned polymerases, may also be used instead in this step. The polymerase used lacks strand displacement or exonuclease activity. The gap extension terminates at the junction with ME'.

[0509] A proximity ligation step then occurs between the 3' extension product and the second strand of the 5' adapter (Figure 19C).

[0510] The library of this example (Figure 19D) is sequenced using standard sequencing methods (described in Example 2). The X region serves to extend the B15 region to reach a suitable Tm for sequencing from B15 in the absence of ME. Reads are more likely to be captured at the start of read 2 due to the quality of the inserts and the variable insert length.

[0511] Example 17. Preparation of DNA libraries for detection of low frequency mutants This example describes the asymmetric tagmentation-BLT method used to prepare DNA sequencing libraries for the detection of low-frequency single nucleotide variants (SNVs) and structural variants (SVs).

[0512] A first DNA library is prepared using the method described above in Example 7. A second DNA library is prepared using the TruSeq™ method.

[0513] DNA containing specific amounts of SNV and SV are used, namely 2%, 0.5% and 0.2%.

[0514] 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.

[0515] As used herein, the term about refers to numerical values, including, for example, integers, ratios, and percentages, whether or not explicitly stated. The term 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 of generating a double-stranded nucleic acid library, each fragment in the library comprising a unique molecular identifier (UMI), the method comprising: a. a sample containing a double-stranded target nucleic acid, i. a first transposase; ii. a first transposon comprising a first 3' end transposon end sequence, a first adapter sequence, and a first UMI; iii. a second transposon comprising a sequence that is fully or partially complementary to the first 3' end transposon end sequence; b. tagmenting the double-stranded target nucleic acid with the first transposome complex to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising the first adapter sequence and the first UMI; c. Releasing the tagmented double-stranded target nucleic acid fragment from the first transposome complex; d. Optionally, extending the tagmented double-stranded target nucleic acid fragments; e. Optionally, ligating the first transposon to the tagmented double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment; f. generating tagmented double-stranded target nucleic acid fragments; g. amplifying the tagmented double-stranded target nucleic acid fragments.

2. 2. The method of claim 1, wherein the first UMI in the first transposon is located between the first adapter sequence and the first 3' transposon end sequence.

3. The method of claim 1 or 2, wherein the first adapter sequence in the first transposon is located between the first UMI and the first 3' transposon end sequence.

4. a. a second transposase; b. a third transposon comprising a second adapter sequence and a second 3' transposon end sequence; and c. a fourth transposon comprising a sequence that is fully or partially complementary to the second 3' end transposon end sequence.

5. The tagmentation step comprises: a. a first strand comprising the first adapter sequence and the first UMI; and a second strand comprising the second adapter sequence.

6. a. the third transposon further comprises a second UMI; b) The method of claim 4 or 5, wherein the second adapter sequence is located between the second UMI and the second 3' transposon end sequence.

7. The tagmentation step comprises: a. a first strand comprising the first adapter sequence and the first UMI; and a second strand comprising said second adaptor sequence and said second UMI.

8. 1. A method of generating a double-stranded nucleic acid library, each fragment in the library comprising a UMI, the method comprising: a. a sample containing a double-stranded target nucleic acid, i. a transposase; ii. a first transposon comprising a first 3' end transposon end sequence and a first adapter sequence; a second transposon comprising a sequence that is fully or partially complementary to the first 3' end transposon end sequence; b. tagmenting a first strand of the double-stranded target nucleic acid with the transposome complex to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising the first adapter sequence; c. Releasing the tagmented double-stranded target nucleic acid fragment from the transposome complex; d. hybridizing a polynucleotide comprising a second adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3' end transposon sequence; e. optionally extending a second strand of said tagmented double-stranded target nucleic acid fragments; f. Optionally, ligating the polynucleotide to the tagmented double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment; g. generating a tagmented double-stranded target nucleic acid fragment containing the UMI, the UMI being located immediately adjacent to the 3' end of the insert DNA; h. amplifying the tagmented double-stranded target nucleic acid fragment containing the UMI.

9. 1. A method of generating a double-stranded nucleic acid library, each fragment in the library comprising a UMI, the method comprising: a. a sample containing a double-stranded target nucleic acid, i. a transposase; ii. a first transposon comprising a first 3' end transposon end sequence and a first adapter sequence; a second transposon comprising a sequence that is fully or partially complementary to the first 3' end transposon end sequence; b. tagmenting a first strand of the double-stranded target nucleic acid with the transposome complex to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising the first adapter sequence; c. Releasing the tagmented double-stranded target nucleic acid fragment from the transposome complex; d. hybridizing a first polynucleotide comprising a UMI and a second adapter sequence; e. optionally, adding a second polynucleotide that comprises a region complementary to the first polynucleotide to generate a double stranded adapter; f. Optionally, extending a second strand of said tagmented double-stranded target nucleic acid fragments; g. Optionally, ligating the second polynucleotide to the second strand of the extended tagmented double-stranded target nucleic acid fragment; h. generating a tagmented double-stranded target nucleic acid fragment comprising the UMI, the UMI being located between the double-stranded target nucleic acid fragment and the second adaptor sequence; i. amplifying the tagmented double-stranded target nucleic acid fragment containing the UMI.

10. 1. A method for generating a double-stranded nucleic acid library, each fragment in the library comprising two different UMIs, the method comprising: a. a sample containing a double-stranded target nucleic acid, i. a first transposome complex, 1. A first transposase; 2. (a) a first transposon on the first strand of the double-stranded target nucleic acid fragment, and (b) a first forked adapter comprising a second transposon; the first transposon comprises a first 3' end transposon end sequence, a first copy of a first adapter sequence, and a first UMI; the second transposon comprises a first copy of a second adapter sequence, and a sequence that is fully or partially complementary to the first 3' end transposon end sequence and the first UMI; a first transposome complex, wherein the first copy of the first adapter sequence is single-stranded and the first copy of the second adapter sequence comprises a double-stranded portion; and ii. a second transposome complex, 1. A second transposase; 2. (a) a third transposon on the second strand of the double-stranded target nucleic acid fragment, and (b) a second forked adapter comprising a fourth transposon; the third transposon comprises a second 3' end transposon end sequence, a second copy of the first adapter sequence, and a second UMI; the third transposon comprises a second copy of the second adapter sequence, and a sequence that is fully or partially complementary to the second 3' end transposon end sequence and the second UMI; a second transposome complex, wherein the second copy of the first adaptor sequence is single-stranded and the second copy of the second adaptor sequence comprises a double-stranded portion; b. tagmenting the double-stranded target nucleic acid with the forked adapter to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising the first and second copies of the first adapter sequence, the first UMI, the first and second copies of the second adapter sequence, and the second UMI; c. Releasing the tagmented double-stranded target nucleic acid fragment from the transposome complex; d. Optionally, extending the tagmented double-stranded target nucleic acid fragments; e. ligating the second and fourth transposons to the double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment; f. generating tagmented double-stranded target nucleic acid fragments; g. amplifying the tagmented double-stranded target nucleic acid fragments.

11. 1. A method of generating a double-stranded nucleic acid library, each fragment in the library comprising four different UMIs, the method comprising: a. a sample containing a double-stranded target nucleic acid, i. a first transposome complex, 1. A first transposase; 2. (a) a first transposon on the first strand of the double-stranded target nucleic acid fragment, and (b) a first forked adapter comprising a second transposon; the first transposon comprises a first 3' end transposon end sequence, a first copy of a first adapter sequence, a first copy of a first UMI, and a first copy of a second adapter sequence; the second transposon comprises a sequence that is fully or partially complementary to the first 3' end transposon end sequence, a first copy of a third adapter sequence, a first copy of a second UMI, and a fourth adapter sequence; a first transposome complex, wherein the first copies of the first, second, and third adaptor sequences are single-stranded and the fourth adaptor sequence comprises a double-stranded portion; and ii. a second transposome complex, 1. A second transposase; 2. (a) a third transposon on the second strand of the double-stranded target nucleic acid fragment, and (b) a second forked adapter comprising a fourth transposon; the third transposon comprises a second 3' end transposon end sequence, a first copy of a fifth adapter sequence, a first copy of a third UMI, and a first copy of a sixth adapter sequence; the fourth transposon comprises a sequence that is fully or partially complementary to the second 3' end transposon end sequence, a first copy of a seventh adapter sequence, a first copy of a fourth UMI, and an eighth adapter sequence; a second transposome complex, wherein the first copies of the fifth, sixth, and seventh adaptor sequences are single-stranded and the eighth adaptor sequence comprises a double-stranded portion; b. tagmenting the double-stranded target nucleic acid with the forked adapter to generate tagmented double-stranded target nucleic acid fragments, each tagmented double-stranded target nucleic acid fragment comprising the first copy of the first, second, third, fifth, sixth, and seventh adapter sequence, the first copy of the first, second, third, and fourth UMI, the sixth adapter sequence, and the eighth adapter sequence; c. Releasing the tagmented double-stranded target nucleic acid fragment from the transposome complex; d. Optionally, extending the tagmented double-stranded target nucleic acid fragments; e. ligating the second and fourth transposons to the double-stranded target nucleic acid fragment or the extended tagmented double-stranded target nucleic acid fragment; f. generating tagmented double-stranded target nucleic acid fragments; g. amplifying the tagmented double-stranded target nucleic acid fragments.

12. 12. The method of any one of claims 1 to 11, wherein the double-stranded target nucleic acid is double-stranded DNA, ctDNA, cfDNA, RNA, cDNA, or a DNA:RNA duplex.

13. 13. The method of any one of claims 1, 2, 4-8, or 12, wherein the first UMI is on the first strand of the tagmented double-stranded target nucleic acid fragment.

14. 13. The method of any one of claims 1, 3, 5-7, or 12, wherein a first copy of the first UMI is on the first strand of the tagmented double-stranded target nucleic acid fragment and a second copy of the first UMI is on the second strand.

15. 15. The method of any one of claims 1-7 or 12-14, wherein the ligation step comprises ligating the 3' end of the tagmented double-stranded target nucleic acid fragment or the 3' end of the extended tagmented double-stranded target nucleic acid fragment to the 5' end of the first, second, or fourth transposon.

16. The polynucleotide comprises: a. Hairpin UMI, b. A hairpin UMI and a universal hybridizing tail; c. splint ligation adaptors, or d. a 3' template switch oligonucleotide.

17. a. the polynucleotide comprises a 3' adaptor that comprises a hairpin UMI; 17. The method of claim 16, wherein the extending step comprises extending from the 3'-end of the second strand of the tagmented double-stranded target nucleic acid fragment to the 5'-end of the hairpin UMI.

18. a. the polynucleotide comprises a splint ligation adaptor; 17. The method of claim 16, wherein the extending step comprises extending from the 3' end of the second strand of the tagmented double-stranded target nucleic acid fragment to the 5' end of the splint ligation adaptor.

19. a. the polynucleotide comprises a template switch oligonucleotide; b. the extending step extends the first strand of the tagmented double-stranded target nucleic acid fragment from the 3' end of the second strand of the tagmented double-stranded target nucleic acid fragment to a junction within the template switch oligonucleotide by copying the first strand of the tagmented double-stranded target nucleic acid fragment; c. template switching from the first strand to the unpaired region of the 3′ template switch oligonucleotide; d. copying the unpaired region of the 3' template switch oligonucleotide from the junction to the 5' end of the unpaired region of the 3' template switch oligonucleotide.

20. 20. A method for sequencing a double-stranded nucleic acid library generated by the method of any one of claims 1 to 19, wherein the UMIs are sequenced to increase sensitivity in DNA sequencing.

21. A transposome complex comprising: a. a transposase; b. a first transposon comprising a 3' transposon end sequence and a 5' adapter sequence; c) a second transposon comprising a sequence that is fully or partially complementary to the first 3' end transposon end sequence.

22. A kit comprising the transposome complex of claim 21.

23. A kit for producing the transposome complex of claim 21.

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