Preparation of RNA and DNA sequencing libraries using bead-linked transpososomes

Abstract

This application describes methods for preparing immobilized libraries of tagged RNA fragments. Also described herein are several methods for preparing DNA and RNA sequencing libraries from a single sample. These methods may include library preparation from a single cell. Also described herein are methods for preparing RNA libraries incorporating 3' unique molecular identifiers (UMIs) and methods for preparing strand-specific RNA libraries using tagmentation. Methods for preparing RNA and DNA libraries are described herein.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 061,885 filed on 6 August 2020, U.S. Provisional Patent Application No. 63 / 165,830 filed on 25 March 2021, U.S. Provisional Patent Application No. 63 / 168,802 filed on 31 March 2021, and U.S. Provisional Patent Application No. 63 / 219,014 filed on 7 July 2021, each of which is incorporated herein by reference in whole for all purposes.

[0002] (Field of Invention) This application relates to the preparation of RNA sequencing libraries using bead-linked transposomes. Methods for preparing RNA and DNA sequencing libraries from a single sample are also described. [Background technology]

[0003] RNA sequencing is important for several applications. For example, sequencing of complete RNA transcripts allows for the study of any mutations in the transcript, such as differences in splicing. RNA sequencing can also be used to perform transcript counts, counting the number of transcripts of various genes.

[0004] RNA sequencing (RNA-seq) is a method of profiling the transcriptome using next-generation sequencing. Common techniques involve converting single-stranded RNA into single-stranded or double-stranded cDNA fragments. Adapters are then added to the ends of each fragment during library preparation, which are required by many sequencing platforms. Depending on the approach used, information about which strand the RNA originates from may be preserved. This is known as strand-specific RNA-seq and improves upon standard approaches by precisely identifying antisense transcripts, determining the strands of non-coding RNAs (e.g., LncRNAs), and demarcating duplicate gene boundaries. Furthermore, strand-specific RNA-seq is a preferred approach because it provides a more accurate estimate of transcript expression.

[0005] Many current protocols for sequencing RNA samples employ sample preparation methods that convert the RNA in the sample into double-stranded cDNA format before sequencing. Methods are needed to improve the workflow and ease of use for RNA sequencing, such as methods that enable the preparation of strand-specific RNA libraries using tagmentation.

[0006] Furthermore, current single-cell RNA sequencing analysis relies on unique molecular identifier (UMI)-based methods to ensure quantitative measurement of expression. This approach is particularly relevant to single-cell RNA sequencing, which often relies on significant levels of cDNA pre-amplification to obtain sufficient input for sequencing. Amplification bias can be corrected by folding reads that have matching UMIs and mapping sites. However, UMI-based methods, such as CEL-seq2 (Hashimshony T et al. Genome Biol. 17:77 (2016)), Drop-seq (Macosko EZ, et al. Cell 161:1202-14 (2015)), and Smart-seq2 (Picelli S, et al. Nature Protocols 9:171-181 (2014)), provide reads derived only from the 3' end of the transcript, either because UMI is bound in modern protocols such as CEL-seq2 (Hashimshony T et al. Genome Biol. 17:77 (2016)), Drop-seq (Macosko EZ, et al. Cell 161:1202-14 (2015)), or because UMI can be bound at the 5' end in methods such as STRT-seq (Islam S, et al. Nat Methods 2013;11:163-6). Therefore, while alternative splicing studies have become a prominent application of RNA sequencing, single-cell level measurement of isoform expression remains limited due to the scarcity of available technologies that can enable single-cell isoform expression analysis. Therefore, methods for improving cDNA production from RNA can be used in a wide variety of different applications.

[0007] Transpososomes bound to a surface (such as beads) can tag long molecules of double-stranded (ds) DNA, allowing for the production of template libraries on beads or other surfaces (see U.S. Patent No. 9683230). Immobilizing transpososomes on beads can help control insertion size and yield during tagmentation and forms the basis of the Illumina DNA Flex PCR-Free (research use only, RUO) technology, formerly known as Illumina's Nextera technology. In these methods, the Tn5 enzyme catalyzes the rearrangement of adapters required for sequencing at the ends of double-stranded DNA or cDNA via a "cut-and-paste" mechanism called tagmentation. In library preparation, tagmentation is performed either in solution or on magnetic beads using bead-linked transpososomes (BLTs). Tagmentation-based methods require fewer steps, result in higher conversion efficiency, and are more accurate than ligation-based methods. However, there are no strand-specific RNA-seq methods available for use with tagmentation.

[0008] Furthermore, asymmetric tagmentation methods such as Illumina DNA Flex PCR-Free involve tagmentation using a BLT containing a mixture of A14 and B15 transpososomes, where A14 and B15 contain sequencing adapter sequences. Fragments tagged with only A14 or only B15 (i.e., fragments with A14 sequences at both ends or B15 sequences at both ends) do not produce a viable library product because standard Illumina SBS sequencing requires the presence of A14 at one end and B15 at the other. Consequently, approximately half of all tagged fragments are lost, leading to reduced library preparation efficiency in asymmetric tagmentation methods in the prior art. Methods to avoid such loss of tagmentation products, such as incorporating a symmetric tagmentation protocol in which all transpososome complexes contain identical transpososomes, are needed to improve yields in tagmentation-intensive methods.

[0009] Transposomals can also tagment "apparent" DNA:RNA double helix. For example, a poly-T capture oligo can bind to the surface of a flow cell, capture an mRNA transcript via its 3' poly-A tail, and subsequently be treated with reverse transcriptase (RT) enzyme to generate a double helix containing a "first strand synthesis" cDNA strand bound to the original RNA transcript. Transposomals from solution can then generate a clusterable and sequenceable template, as described in International Publication No. 2013 / 131962(A1). Transposomals were then added to a solution to generate a clusterable and sequenceable template without generating a second strand cDNA and therefore a double-stranded cDNA. This suggests that transposomals can tagment "apparent" RNA / DNA double helix. The hypothesis for this mechanism is that reverse transcriptase initiates second strand synthesis via a nick created in the RNA strand, and these dsDNA double helixes are substrates for transposomals. However, as a result of using transposomes from solution, reads are generated only from the 3' end of the transcript (as shown in Figures 1B and 1C). This application describes a method for tagging RNA to avoid the 3' bias. Furthermore, this application describes means for preparing RNA and DNA sequencing libraries from the same sample. Many sequencing-based assays benefit from the ability to characterize both the DNA and RNA content of a sample with “multi-omics” analysis. Current sample preparation / sequencing workflows for analyzing total nucleic acid (TNA, including both DNA and RNA) from a sample are limited to multi-omics because these workflows are either cumbersome and / or result in significant sample loss (e.g., requiring the splitting of a TNA sample into two biomolecule-specific library preparations) or do not distinguish between biomolecule types (e.g., next-generation sequencing (NGS) reads may originate from either RNA or DNA molecules). This disclosure describes various methodologies for efficient TNA sample preparation for NGS using methods for identifying the original biomolecule type (RNA or DNA). Thus, these methodologies can enable simplified multi-omics library preparation through tagmentation of double-stranded nucleic acids. Methods for preparing RNA libraries incorporating 3' unique molecular identifiers (UMIs), and methods for preparing strand-specific RNA libraries using tagmentation are also described herein. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] U.S. Patent No. 9683230 [Patent Document 2] International Publication No. 2013 / 131962(A1) [Non-patent literature]

[0011] [Non-Patent Document 1] Hashimshony T et al.Genome Biol.17:77(2016)

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0012] Methods for preparing RNA and DNA libraries are described herein.

[0013] Embodiment 1. A method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA, comprising: (a) applying a sample containing the target RNA to a solid support onto which a transpososome complex and a capture oligonucleotide are immobilized, wherein the transpososome complex comprises a transposase bound to a first polynucleotide, the first polynucleotide comprises a 3' portion containing a transposon end sequence and a first tag, and the sample is applied to the solid support under conditions such that the 3' end of the target RNA binds to the capture oligonucleotide; (b) adding a reverse transcriptase polymerase under conditions for synthesizing cDNA and generating a DNA:RNA duplex immobilized on the capture oligonucleotide; (c) performing tagmentation on the DNA:RNA duplex using the transpososome complex under conditions such that the DNA:RNA duplex is tagged at the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5'-tagged with the first tag.

[0014] Embodiment 2. The method according to Embodiment 1, wherein the transposome complex is reversibly inactivated before tagmentation, and tagmentation activates the transposome complex.

[0015] Embodiment 3. The method according to Embodiment 2, wherein the transposome complex is reversibly inactivated by a transposome inactivator bound to the transposome complex.

[0016] Embodiment 4. The method according to Embodiment 3, wherein the transposome inactivator is bound to the Tn5 binding site of the transposome complex.

[0017] Embodiment 5. The method according to Embodiment 3 or 4, wherein the transposome inactivator comprises dephosphorylated ME', an extra base, an inhibitory double helix, and / or a thermolabile antibody.

[0018] Embodiment 6. The method according to any one of Embodiments 2 to 5, wherein the transposome complex is activated in step (c) by removing the transposome inactivator.

[0019] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the captured oligonucleotide comprises a polyT sequence.

[0020] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the target RNA includes a sequence that is complementary to at least one portion of the captured oligonucleotide.

[0021] Embodiment 9. The method according to any one of Embodiments 1 to 8, wherein the transposome complex is immobilized on a solid support via a first polynucleotide.

[0022] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the transposome complex comprises a second polynucleotide containing a region complementary to the transposon terminal sequence.

[0023] Embodiment 11. The method according to Embodiment 10, wherein the transposome complex is immobilized on a solid support via a second polynucleotide.

[0024] Embodiment 12. The method according to any one of Embodiments 1 to 11, further comprising washing the solid support after step (a) to remove any unbound target RNA.

[0025] Embodiment 13. The transposome complex is 1 mm 2 At least 10 3 , 10 4 , 10 5 , or 10 6 The method according to any one of embodiments 1 to 12, wherein the composite is present on a solid support at a density of 1.

[0026] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein the transposase comprises Tn5 transposase.

[0027] Embodiment 15. The method according to Embodiment 14, wherein the Tn5 transposase is a highly active Tn5 transposase.

[0028] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the length of the double-stranded fragments in the immobilized library is adjusted by increasing or decreasing the density of transposome complexes on a solid support.

[0029] Embodiment 17. The method according to any one of Embodiments 1 to 16, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first tags include the same tag domain.

[0030] Embodiment 18. The method according to any one of Embodiments 1 to 17, wherein the tag includes a region for cluster amplification.

[0031] Embodiment 19. The method according to any one of Embodiments 1 to 18, wherein the tag includes a region for priming a sequencing reaction.

[0032] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the solid support comprises microparticles, beads, a planar support, a patterned surface, or wells.

[0033] Embodiment 21. The method according to Embodiment 20, wherein the planar support is the inner or outer surface of the tube.

[0034] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein tagmentation is performed to produce double-stranded DNA:RNA double helix cross-linked to two immobilized transposome complexes on a solid support, and optionally, the second strand of DNA is synthesized to prepare the double-stranded DNA before tagmentation.

[0035] Embodiment 23. The method according to Embodiment 22, wherein the length of the crosslinked double helix is ​​100 to 1500 base pairs.

[0036] Embodiment 24. The method according to any one of Embodiments 1 to 23, wherein the sample applied to the solid support is blood.

[0037] Embodiment 25. The method according to any one of Embodiments 1 to 24, wherein the sample applied to the solid support is a cell lysate.

[0038] Embodiment 26. The method according to Embodiment 25, wherein the cell lysate is crude cell lysate.

[0039] Embodiment 27. The method according to any one of Embodiments 1 to 26, wherein the sample applied to the solid support has an absorbance ratio of 260 / 280 of 1.7 or less.

[0040] Embodiment 28. The method according to any one of Embodiments 1 to 27, further comprising applying the sample to a solid support and then lysing the cells in the sample.

[0041] Embodiment 29.(d) The method according to any one of Embodiments 1 to 28, further comprising contacting a liquid-phase transposome complex with the immobilized DNA:RNA fragment under conditions in which the DNA:RNA fragment is further fragmented by the liquid-phase transposome complex, thereby obtaining an immobilized nucleic acid fragment having one end in solution.

[0042] Embodiment 30. The method according to Embodiment 29, wherein the liquid-phase transposome complex comprises a second tag, thereby generating an immobilized nucleic acid fragment having the second tag in solution.

[0043] Embodiment 31. The method according to Embodiment 30, wherein the first and second tags are different.

[0044] Embodiment 32. The method according to any one of Embodiments 29 to 31, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the liquid-phase transposome complex contains the second tag.

[0045] Embodiment 33. The method according to any one of Embodiments 29 to 32, further comprising amplifying a fragment on a solid support by reacting a polymerase with an amplification primer corresponding to a portion of a first polynucleotide.

[0046] Embodiment 34. A solid support on which a library of tagged RNA fragments, prepared according to the method of any one of Embodiments 1 to 33, is immobilized.

[0047] Embodiment 35. A method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA, comprising: a) applying a sample containing target RNA to a solid support on which a capture oligonucleotide and a first polynucleotide are immobilized thereon, wherein the first polynucleotide comprises a 3' portion containing a transposon terminal sequence and a first tag, and the sample is applied to the solid support under conditions that the 3' end of the target RNA binds to the capture oligonucleotide; (b) adding a transposase under conditions that the transposase binds to the first polynucleotide to form a transposomal complex; (c) adding a reverse transcriptase polymerase under conditions that cDNA is synthesized and an immobilized DNA:RNA double helix is ​​produced on a second capture oligonucleotide; and (d) performing tagmentation on the DNA:RNA double helix using a transposomal complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with the first tag.

[0048] Embodiment 36. A method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA, comprising: a) applying a sample containing target RNA to a solid support on which a capture oligonucleotide and a first polynucleotide are immobilized thereon, wherein the first polynucleotide comprises a 3' portion containing a transposon terminal sequence and a first tag, and the sample is applied to the solid support under conditions that the 3' end of the target RNA is bound to the capture oligonucleotide; (b) adding reverse transcriptase polymerase under conditions that cDNA is synthesized and a DNA:RNA double helix immobilized on the capture oligonucleotide is produced; (c) adding a transposase under conditions that the transposase binds to the first polynucleotide to form a transposomal complex; and (d) performing tagmentation on the DNA:RNA double helix using the transposomal complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with the first tag.

[0049] Embodiment 37. The method according to either Embodiment 35 or 36, wherein the application of the sample containing the target RNA to the solid support is performed in a droplet.

[0050] Embodiment 38. The method according to Embodiment 37, wherein applying a sample containing target RNA to a solid support comprises (a) providing a single cell in a droplet together with beads, (b) lysing the cell in the droplet, (c) releasing the target RNA from the single cell, and (d) capturing the target RNA on the beads.

[0051] Embodiment 39. The method according to Embodiment 37 or 38, wherein the droplet is removed before the cDNA is synthesized.

[0052] Embodiment 40. The method according to any one of Embodiments 35 to 39, further comprising delivering an immobilized library of DNA:RNA fragments on a solid support for sequencing.

[0053] Embodiment 41. The method of Embodiment 40, further comprising, after delivery, (a) capturing a solid support with an immobilized library of DNA:RNA fragments on the surface for sequencing, (b) releasing the immobilized fragments from the solid support, and (c) capturing the fragments on the surface for sequencing.

[0054] Embodiment 42. The method according to Embodiment 41, further comprising arranging fragments on a surface for arrangement determination.

[0055] Embodiment 43. The method according to Embodiment 42, wherein the surface for sequencing is a flow cell.

[0056] Embodiment 44. The method according to any one of Embodiments 35 to 43, wherein the application of a sample containing the target RNA to a solid support is performed in microwells on the solid support.

[0057] Embodiment 45. The method according to Embodiment 44, wherein applying a sample containing target RNA to a solid support comprises lysing cells and releasing target RNA from single cells in microwells.

[0058] Embodiment 46. The method according to any one of Embodiments 35 to 45, further comprising releasing an immobilized library of DNA:RNA fragments and sequencing the fragments in the same microwell.

[0059] Embodiment 47. The method according to any one of Embodiments 44 to 46, wherein the solid support is a flow cell containing microwells.

[0060] Embodiment 48. The method of Embodiment 47, wherein the sequencing data enables resolution of fragments that were immobilized on the same solid support based on the spatial proximity of the fragments on the surface for sequencing.

[0061] Embodiment 49. Tagmentation of DNA:RNA double helix using a transposome complex is performed. The method according to any one of Embodiments 1 to 48, wherein the method is carried out using two different transposomal complexes, the different transposomal complexes comprising a first transposon containing a different adapter sequence.

[0062] Embodiment 50. The method according to Embodiment 49, wherein at least some fragments are tagged with a first lead sequence adapter sequence at the 5' end of one strand and tagged with a second lead sequence adapter sequence at the 5' end of the other strand.

[0063] Embodiment 51. The method according to any one of Embodiments 1 to 48, wherein tagging of a DNA:RNA double helix using a transposome complex is performed using a transposome complex containing a first transposon containing the same adapter sequence.

[0064] Embodiment 52. The method according to Embodiment 51, wherein all transposome complexes are identical.

[0065] Embodiment 53. The method according to Embodiment 51 or 52, wherein the fragment is tagged with the same adapter sequence at the 5' ends of both strands of the double-stranded fragment.

[0066] Embodiment 54. The method according to any one of Embodiments 51 to 53, further comprising: (a) releasing a double-stranded target nucleic acid fragment from a transposome complex; (b) hybridizing a polynucleotide comprising an adapter sequence, a UMI, and a sequence that is fully or partially complementary to the first 3' terminal transposon sequence, wherein the adapter sequence in the polynucleotide is different from the adapter sequence in the transposome complex; (c) optionally extending the second strand of the double-stranded target nucleic acid fragment; (d) optionally ligating the polynucleotide or the extended polynucleotide with the double-stranded target nucleic acid fragment; and (e) producing a double-stranded target nucleic acid fragment comprising a UMI, wherein the UMI is located directly adjacent to the 3' end of the inserted DNA.

[0067] Embodiment 55. The method according to any one of Embodiments 51 to 53, further comprising: (a) releasing a double-stranded target nucleic acid fragment from a transposome complex; (b) hybridizing a first polynucleotide comprising a UMI and an adapter sequence, wherein the adapter in the first transposon is different from the adapter in the first polynucleotide; (c) optionally adding a second polynucleotide comprising a region complementary to the first polynucleotide to produce a double-stranded adapter; (d) optionally extending the second strand of the double-stranded target nucleic acid fragment; (e) optionally ligating the double-stranded adapter with the double-stranded target nucleic acid fragment; and (f) producing a double-stranded target nucleic acid fragment comprising a UMI, wherein the UMI is located between the double-stranded target nucleic acid fragment and the adapter sequence from the first polynucleotide.

[0068] Embodiment 56. The method according to Embodiment 54 or 55, wherein the fragment is tagged at the 5' end of one strand with a first read sequence adapter sequence from a first transposon and at the 5' end of the other strand with a second read sequence adapter sequence from a first polynucleotide.

[0069] Embodiment 57. A method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA, comprising: (a) applying a sample containing the target RNA to a solid support on which a capture oligonucleotide is immobilized; (b) Adding reverse transcriptase polymerase under conditions that synthesize cDNA and generate immobilized DNA:RNA double helixes on captured oligonucleotides, (c) A method comprising tagging a DNA:RNA double helix in solution using a transposome complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a first tag.

[0070] Embodiment 58. The method according to Embodiment 57, wherein the RNA is mRNA and the capture oligonucleotide comprises a polyT sequence.

[0071] Embodiment 59. The method according to Embodiment 58, wherein the fragment library comprises DNA:RNA fragments generated from the 3' ends of one or more RNAs.

[0072] Embodiment 60. The method according to any one of Embodiments 57 to 59, wherein the capture oligonucleotide further comprises a first read sequencing adapter sequence, a bead code, and / or one or more additional adapter sequences.

[0073] Embodiment 61. The method according to any one of Embodiments 57 to 60, wherein the transposome complex in solution comprises a first transposome comprising a second read sequence adapter sequence and / or one or more additional adapter sequences.

[0074] Embodiment 62. The method according to any one of Embodiments 57 to 61, wherein a library of DNA:RNA fragments is sequenced without amplifying the fragments before sequencing.

[0075] Embodiment 63. A solid support comprising a captured oligonucleotide and a first polynucleotide immobilized thereon, wherein the first polynucleotide comprises a 3' portion including a transposon terminal sequence and a first tag.

[0076] Embodiment 64. The solid support according to Embodiment 63, wherein the solid support is a bead.

[0077] Embodiment 65. The solid support according to Embodiment 64, wherein the first polynucleotide further comprises a bead cord.

[0078] Embodiment 66. The solid support according to Embodiment 65, wherein the beads are contained in a pool of beads, and each bead contains an immobilized first polynucleotide having a different bead cord compared to the bead cords contained in the other beads in the pool.

[0079] Embodiment 67. A solid support according to any one of Embodiments 63 to 66, further comprising a transposase bound to a first polynucleotide for forming a transposome complex.

[0080] Embodiment 68. The solid support according to Embodiment 67, wherein the transposome complex is reversibly inactivated.

[0081] Embodiment 69. The solid support according to Embodiment 68, wherein the transposome complex is reversibly inactivated by a transposome inactivator bound to the transposome complex.

[0082] Embodiment 70. The solid support according to Embodiment 69, wherein the transposome inactivator is bound to the Tn5 binding site of the transposome complex.

[0083] Embodiment 71. A solid support according to Embodiment 69 or 70, wherein the transposome inactivator comprises a dephosphorylated ME', an extra base, an inhibitory double helix, and / or a thermolabile antibody.

[0084] Embodiment 72. A solid support comprising a captured oligonucleotide and an immobilized oligonucleotide, wherein the immobilized oligonucleotide comprises a sequence for hybridizing to a hybridization sequence contained in a second transposon contained in a transposome complex.

[0085] Embodiment 73. The solid support according to Embodiment 72, wherein the solid support is a bead.

[0086] Embodiment 74. The solid support according to Embodiment 73, wherein the immobilized oligonucleotide further comprises a bead cord and / or one or more adapter sequences.

[0087] Embodiment 75. The solid support according to Embodiment 74, wherein the beads are contained in a pool of beads, and each bead contains an immobilized oligonucleotide that includes a different bead code compared to the bead code contained in the immobilized oligonucleotide that is contained in the other beads in the pool.

[0088] Embodiment 76. A solid support according to any one of Embodiments 63 to 75, wherein the captured oligonucleotide comprises a poly-T sequence.

[0089] Embodiment 77. A solid support according to any one of Embodiments 63 to 76, wherein the captured oligonucleotide comprises a sequence complementary to at least a portion of the target RNA.

[0090] Embodiment 78. A solid support according to any one of Embodiments 63 to 77, wherein the transposome complex is immobilized on the solid support via a first polynucleotide.

[0091] Embodiment 79. A solid support according to any one of Embodiments 63 to 78, wherein the transposome complex comprises a second polynucleotide containing a region complementary to the transposon terminal sequence.

[0092] Embodiment 80. The method according to embodiment 79, wherein the transposome complex is immobilized on a solid support via a second polynucleotide.

[0093] Embodiment 81. The transposome complex is at least 10 2 per mm 3 and at least 10 4 and at least 10 5 or at least 10 6 complexes per density of the solid support according to any one of embodiments 63 to 80.

[0094] Embodiment 82. The solid support according to any one of embodiments 63 to 81, wherein the transposase is Tn5 transposase.

[0095] Embodiment 83. The method according to embodiment 82, wherein the Tn5 transposase is a high-activity Tn5 transposase.

[0096] Embodiment 84. The solid support according to any one of embodiments 63 to 83, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first tags contain the same tag domain.

[0097] Embodiment 85. The solid support according to any one of embodiments 63 to 84, wherein the tag contains a region for cluster amplification.

[0098] Embodiment 86. The solid support according to any one of embodiments 63 to 85, wherein the tag contains a region for priming a sequencing reaction.

[0099] Embodiment 87. The solid support according to any one of embodiments 63 to 86, wherein the solid support includes microparticles, beads, a planar support, a patterned surface, or a well.

[0100] Embodiment 88. The solid support according to embodiment 87, wherein the planar support is the inner or outer surface of a tube.

[0101] Embodiment 89. A kit comprising a solid support as described in any one of Embodiments 63 to 88.

[0102] Embodiment 90. The kit according to Embodiment 89, further comprising a transposase.

[0103] Embodiment 91. The kit according to Embodiment 89 or 90, further comprising reverse transcriptase polymerase.

[0104] Embodiment 92. The kit according to Embodiment 90 or 91, further comprising a second solid support for immobilizing DNA, the second transpososome complex comprising a transposase, and a third polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a second tag.

[0105] Embodiment 93. A method for preparing a tagged DNA:RNA fragment immobilized library from a sample containing RNA and DNA, comprising: (a) applying the sample containing RNA and DNA to a first solid support for immobilizing DNA, comprising a first transposomal complex immobilized thereon, wherein the first transposomal complex comprises a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a first tag; and a second solid support on which a first capture oligonucleotide is immobilized thereon, wherein the sample is applied to a mixture of the first and second solid supports under conditions that the DNA is bound to the first transposomal complex on the first solid support, tagged, optionally tagged, and the RNA is bound to the first capture oligonucleotide on the second solid support; and (b) applying the RNA bound to the second solid support A method comprising: (c) transferring to a third solid support, the third solid support having a second capture oligonucleotide immobilized thereon and binding to the transferred RNA, and a second transpososome complex, wherein the second transpososome complex comprises a transposase and a second polynucleotide, the second polynucleotide comprising a 3' portion containing a transposon terminal sequence and a second tag; (c) adding reverse transcriptase polymerase under conditions that synthesize cDNA and produce an immobilized DNA:RNA double helix on the second capture oligonucleotide; and (d) performing tagmentation on the DNA:RNA double helix using the second transpososome complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a first tag.

[0106] Embodiment 94. The method according to Embodiment 93, wherein the first and / or second captured oligonucleotide comprises a polyT sequence.

[0107] Embodiment 95. The method according to Embodiment 93 or 94, wherein the RNA includes a sequence that is complementary to at least one portion of the first and / or second capture oligonucleotide.

[0108] Embodiment 96. The method according to any one of Embodiments 93 to 95, wherein the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotide.

[0109] Embodiment 97. The method according to any one of Embodiments 93 to 96, further comprising washing the solid support after step (a) to remove any unbound DNA or RNA.

[0110] Embodiment 98. A method for preparing a tagged DNA:RNA fragment immobilized library from a sample containing RNA and DNA, comprising: (a) applying a sample containing RNA and DNA to a first solid support for immobilizing DNA, comprising a first transposomal complex immobilized thereon, wherein the first transposomal complex comprises a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a first tag; and a second solid support on which a capture oligonucleotide and a second polynucleotide are immobilized thereon, wherein the second polynucleotide comprises a 3' portion containing a transposon terminal sequence and a second tag, wherein the sample is subjected to the first transposomal complex on the first solid support, fragmented, and optionally A method comprising: (b) applying a mixture of first and second solid supports under conditions that the RNA is tagged and bound to a capture oligonucleotide on a second solid support; (c) adding a reverse transcriptase polymerase under conditions that the transposase binds to a second polynucleotide to form a transposomal complex on the second solid support, synthesizing cDNA and producing an immobilized DNA:RNA double helix on the second capture oligonucleotide; and (d) tagging the DNA:RNA double helix using the second transposomal complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with the first tag.

[0111] Embodiment 99. The method according to Embodiment 98, wherein the captured oligonucleotide comprises a polyT sequence.

[0112] Embodiment 100. The method according to Embodiment 98 or 99, wherein the RNA includes a sequence that is complementary to at least one portion of the captured oligonucleotide.

[0113] Embodiment 101. The method according to any one of Embodiments 98 to 100, wherein the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotide.

[0114] Embodiment 102. The method according to any one of Embodiments 98 to 101, further comprising washing the solid support after step (a) to remove any unbound DNA or RNA.

[0115] Embodiment 103. A method for preparing a tagged DNA:RNA fragment immobilized library from a sample containing RNA and DNA, comprising: (a) a sample containing RNA and DNA, a first solid support for immobilizing DNA, comprising a first transposomal complex immobilized thereon, wherein the first transposomal complex comprises a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a first tag; and a second solid support for immobilizing RNA, comprising a reversibly inactivated capture oligonucleotide and a second transposomal complex immobilized thereon, wherein the second transposomal complex comprises a transposase bound to a second polynucleotide, the second polynucleotide comprising a 3' portion containing a transposon terminal sequence and a second tag; A method comprising: (b) applying a sample to a mixture of first and second solid supports under conditions that DNA is bound to a first transposome complex on a first solid support, fragmented, optionally tagged, and RNA is bound to a capture oligonucleotide on a second solid support; (b) adding reverse transcriptase polymerase under conditions that synthesize cDNA and produce a DNA:RNA double helix immobilized on a second capture oligonucleotide; (c) activating a second transposome complex; and (d) performing tagmentation on a DNA:RNA double helix using the activated second transposome complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with the first tag.

[0116] Embodiment 104. The method according to Embodiment 103, wherein the transposome complex is reversibly inactivated by a transposome inactivator bound to the transposome complex.

[0117] Embodiment 105. The method according to Embodiment 104, wherein the transposome inactivator is bound to the Tn5 binding site of the transposome complex.

[0118] Embodiment 106. The method according to Embodiment 104 or 105, wherein the transposome inactivator comprises a dephosphorylated ME', an extra base, an inhibitory double helix, and / or a thermolabile antibody.

[0119] Embodiment 107. The method according to any one of Embodiments 104 to 106, wherein the transposome complex is activated in step (c) by removal of the transposome inactivator.

[0120] Embodiment 108. The method according to any one of Embodiments 103 to 106, wherein the captured oligonucleotide comprises a polyT sequence.

[0121] Embodiment 109. The method according to any one of Embodiments 103 to 108, wherein the RNA includes a sequence that is complementary to at least one portion of the captured oligonucleotide.

[0122] Embodiment 110. The method according to any one of Embodiments 103 to 109, wherein the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotide.

[0123] Embodiment 111. The method according to any one of Embodiments 103 to 110, further comprising washing the solid support after step (a) to remove any unbound DNA or RNA.

[0124] Embodiment 112. A method for preparing a tagged DNA:RNA fragment immobilized library from a sample containing RNA and DNA, comprising: (a) applying the sample containing RNA and DNA to a first solid support for immobilizing DNA, the first solid support comprising a first transposomal complex immobilized thereon, the first transposomal complex comprising a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a first tag, the sample being applied under conditions that the DNA binds to the first transposomal complex on the first solid support, is fragmented, and optionally tagged; and (b) separating the first solid support having the bound DNA from the RNA; and (c) immobilizing the RNA immobilized thereon with a capture oligonucleotide and a second transposomal complex. A method comprising: (d) applying RNA to a second solid support, wherein the second transpososome complex comprises a transposase bound to a second polynucleotide, the second polynucleotide comprising a 3' portion containing a transposon terminal sequence and a second tag, and the RNA is bound to a capture oligonucleotide on the second solid support; (d) adding reverse transcriptase polymerase under conditions that synthesize cDNA and produce a DNA:RNA double helix immobilized on the second capture oligonucleotide; and (e) performing tagmentation on the DNA:RNA double helix using an activated second transpososome complex under conditions that the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a first tag.

[0125] Embodiment 113. The method according to Embodiment 112, wherein the captured oligonucleotide comprises a polyT sequence.

[0126] Embodiment 114. The method according to Embodiment 112 or 113, wherein the RNA includes a sequence that is complementary to at least one portion of the captured oligonucleotide.

[0127] Embodiment 115. The method according to any one of Embodiments 112 to 114, wherein the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotide.

[0128] Embodiment 116. The method according to any one of Embodiments 112 to 115, further comprising washing the solid support after step (c) to remove any unbound RNA.

[0129] Embodiment 117. The method according to any one of Embodiments 112 to 116, further comprising recombining a first solid support having bound DNA with a second solid support having an immobilized library of tagged DNA:RNA fragments.

[0130] Embodiment 118. A method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA, comprising: (a) adding reverse transcriptase polymerase to a sample containing target RNA under conditions for synthesizing cDNA and generating a DNA:RNA double helix; (b) immobilizing the DNA:RNA double helix on a solid support on which a transposomal complex is immobilized, wherein the transposomal complex comprises a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and a first tag, and the sample is applied to the solid support under conditions for the DNA:RNA double helix to directly bind to a capture oligonucleotide or transposase; and (b) performing tagmentation on the DNA:RNA double helix using the transposomal complex under conditions for the DNA:RNA double helix to be tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a first tag.

[0131] Embodiment 119. The method according to Embodiment 118, wherein the transposome complex is immobilized on a solid support via a first polynucleotide.

[0132] Embodiment 120. The method according to Embodiment 118 or 119, wherein the transposome complex comprises a second polynucleotide containing a region complementary to the transposon terminal sequence.

[0133] Embodiment 121. The method according to Embodiment 120, wherein the transposome complex is immobilized on a solid support via a second polynucleotide.

[0134] Embodiment 122. The transposome complex is 1 mm 2 At least 10 3 , 10 4 , 10 5 , or 10 6 The method according to any one of embodiments 118 to 121, wherein the composite is present on a solid support at a density of 1.

[0135] Embodiment 123. The method according to any one of Embodiments 118 to 122, wherein the transposase comprises Tn5 transposase.

[0136] Embodiment 124. The method according to Embodiment 123, wherein the Tn5 transposase is a highly active Tn5 transposase.

[0137] Embodiment 125. The method according to any one of Embodiments 118 to 124, wherein the length of the double-stranded fragments in the immobilized library is adjusted by increasing or decreasing the density of transposome complexes on a solid support.

[0138] Embodiment 126. The method according to any one of Embodiments 118 to 125, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first tags include the same tag domain.

[0139] Embodiment 127. The method according to any one of Embodiments 118 to 126, wherein the tag includes a region for cluster amplification.

[0140] Embodiment 128. The method according to any one of Embodiments 118 to 127, wherein the tag includes a region for priming a sequencing reaction.

[0141] Embodiment 129. The method according to any one of Embodiments 118 to 128, wherein the solid support comprises microparticles, beads, a planar support, a patterned surface, or wells.

[0142] Embodiment 130. The method according to Embodiment 129, wherein the planar support is the inner or outer surface of the tube.

[0143] Embodiment 131. The method according to any one of Embodiments 118 to 130, wherein tagmentation is performed to produce double-stranded DNA:RNA double helix crosslinked to two immobilized transposomal complexes on a solid support.

[0144] Embodiment 132. The method according to Embodiment 131, wherein the length of the crosslinked double helix is ​​100 to 1500 base pairs.

[0145] Embodiment 133. The method according to any one of Embodiments 118 to 132, wherein the sample applied to the solid support is blood.

[0146] Embodiment 134. The method according to any one of Embodiments 118 to 133, wherein the sample applied to the solid support is a cell lysate.

[0147] Embodiment 135. The method according to Embodiment 134, wherein the cell lysate is crude cell lysate.

[0148] Embodiment 136. The method according to any one of Embodiments 118 to 135, wherein the sample applied to the solid support has an absorbance ratio of 260 / 280 with an absorbance ratio of 1.7 or less.

[0149] Embodiment 137. The method according to any one of Embodiments 118 to 136, further comprising applying the sample to a solid support and then lysing the cells in the sample.

[0150] Embodiment 138.(d) The method according to any one of Embodiments 118 to 137, further comprising contacting a liquid-phase transposome complex with the immobilized DNA:RNA fragment under conditions in which the DNA:RNA fragment is further fragmented by the liquid-phase transposome complex, thereby obtaining an immobilized nucleic acid fragment having one end in solution.

[0151] Embodiment 139. The method according to Embodiment 138, wherein the liquid-phase transposome complex comprises a second tag, thereby generating an immobilized nucleic acid fragment having the second tag in solution.

[0152] Embodiment 140. The method according to Embodiment 139, wherein the first and second tags are different.

[0153] Embodiment 141. The method according to any one of Embodiments 138 to 140, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the liquid-phase transposome complex contains a second tag.

[0154] Embodiment 142. The method according to any one of Embodiments 138 to 141, further comprising amplifying a fragment on a solid support by reacting a polymerase with an amplification primer corresponding to a portion of a first polynucleotide.

[0155] Embodiment 143. The method according to any one of Embodiments 1 to 142, wherein the 5' end of one strand is the 5' end of an RNA strand.

[0156] Embodiment 144. The method according to any one of Embodiments 1 to 142, wherein the 5' end of one strand is the 5' end of a DNA strand.

[0157] Embodiment 145. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, and the method comprises: (a) combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support comprises a transposomal complex immobilized thereon, and the transposomal complex comprises a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; (b) immobilizing DNA; (c) performing tagmentation on the first solid support to prepare tagged fragments containing a DNA-specific barcode; and (d) RNA A method comprising: (e) preparing double-stranded cDNA from a sample; combining the sample with a second solid support to immobilize the cDNA, wherein the second solid support comprises a transposomal complex immobilized thereon, and the transposomal complex comprises a transposase and a transposon containing a transposon terminal sequence and an RNA-specific barcode; and (f) immobilizing the cDNA and performing tagmentation on the second solid support to prepare a tagged fragment containing an RNA-specific barcode, wherein optionally the transposomal complex is reversibly inactivated before tagmentation, and the tagmentation activates the transposomal complex.

[0158] Embodiment 146. The method according to Embodiment 145, further comprising combining the first and second solid supports after performing tagmentation on a second solid support, wherein each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0159] Embodiment 147. The method according to Embodiment 145 or 146, further comprising splitting the first solid support having immobilized tagged fragments containing DNA-specific barcodes from the remainder of the sample after tagmentation on the first solid support and before preparing double-stranded cDNA from RNA.

[0160] Embodiment 148. The method according to Embodiment 145 or 146, further comprising separating the first solid support having the immobilized DNA from the rest of the sample after immobilizing the DNA and before performing tagmentation on the first solid support, to prepare a tagged fragment containing a DNA-specific barcode.

[0161] Embodiment 149. The method according to any one of Embodiments 145 to 148, wherein double-stranded cDNA is prepared from RNA by template switching.

[0162] Embodiment 150. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, and the method comprises: (a) combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support comprises a transposomal complex immobilized thereon, and the transposomal complex comprises a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; (b) immobilizing the DNA; (c) performing tagmentation on the first solid support to prepare tagged fragments containing a DNA-specific barcode; and (d) preparing a single strand of cDNA from RNA to form a DNA:RNA A method comprising: (e) producing a double helix, combining a sample with a second solid support in order to immobilize the DNA:double helix, wherein the second solid support comprises a transposomal complex immobilized thereon, the transposomal complex comprising a transposase having activity for DNA:RNA double helix, and a transposon comprising a transposon terminal sequence and an RNA-specific barcode; and (f) immobilizing the DNA:RNA double helix and performing tagmentation on the second solid support to prepare a tagged fragment comprising an RNA-specific barcode, wherein the transposomal complex is reversibly inactivated before tagmentation, and the tagmentation activates the transposomal complex.

[0163] Embodiment 151. The method according to Embodiment 150, further comprising combining the first and second solid supports after performing tagmentation on a second solid support, wherein each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0164] Embodiment 152. The method according to Embodiment 150 or 151, further comprising splitting the first solid support having immobilized tagged fragments containing DNA-specific barcodes from the remainder of the sample after tagmentation on the first solid support and before preparing single strands of cDNA from RNA, to produce DNA:RNA double helix.

[0165] Embodiment 153. The method according to Embodiment 150 or 151, further comprising separating the first solid support having the immobilized DNA from the rest of the sample after immobilizing the DNA and before performing tagmentation on the first solid support, to prepare a tagged fragment containing a DNA-specific barcode.

[0166] Embodiment 154. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, and the method comprises: (a) combining the sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support comprises a transposomal complex immobilized thereon, and the transposomal complex comprises a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; (b) immobilizing the DNA; (c) performing tagmentation on the first solid support to prepare tagged fragments containing a DNA-specific barcode; (d) preparing double-stranded cDNA from RNA; and (e) in solution A method comprising: (f) performing tagmentation on double-stranded DNA to prepare a tagged fragment of double-stranded cDNA, wherein a transposomal complex in solution comprises a transposase and a transposon having a transposon terminal sequence, an RNA-specific barcode, and a sequence that hybridizes to a capture probe, and optionally the transposomal complex is reversibly inactivated before performing tagmentation, and performing tagmentation activates the transposomal complex, wherein the tagged fragment comprises an RNA-specific barcode and a sequence that hybridizes to a capture probe; (f) combining the sample with a second solid support having a surface containing a capture probe; and (g) immobilizing the tagged fragment of double-stranded cDNA on the second solid support.

[0167] Embodiment 155. The method according to Embodiment 154, further comprising immobilizing a tagged fragment of double-stranded cDNA on a second solid support, and then combining the first and second solid supports, wherein each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0168] Embodiment 156. The method according to Embodiment 154 or 155, further comprising splitting the first solid support having an immobilized tagged fragment containing a DNA-specific barcode from the remainder of the sample after tagmentation on the first solid support and before double-stranded cDNA from RNA.

[0169] Embodiment 157. The method according to Embodiment 154 or 155, further comprising separating the first solid support having the immobilized DNA from the rest of the sample after immobilizing the DNA and before performing tagmentation on the first solid support, to prepare a tagged fragment containing a DNA-specific barcode.

[0170] Embodiment 158. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, and the method comprises: (a) combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support comprises a transposomal complex immobilized thereon, and the transposomal complex comprises a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; (b) immobilizing the DNA; (c) performing tagmentation on the first solid support to prepare tagged fragments containing a DNA-specific barcode; (d) preparing a single strand of cDNA from RNA to produce a DNA:RNA double helix; and (e) A method for preparing a tagged fragment of a DNA:RNA double helix by performing tagmentation on the DNA:RNA double helix in solution, wherein the transposome complex in solution comprises a transposase and a transposon containing a transposon terminal sequence, an RNA-specific barcode, and a sequence that hybridizes to a capture probe, and optionally the transposome complex is reversibly inactivated before tagmentation, and tagmentation activates the transposome complex, wherein the tagged fragment contains an RNA-specific barcode and a sequence that hybridizes to a capture probe; (f) combining the sample with a second solid support having a surface containing a capture probe; and (g) immobilizing the tagged fragment of the DNA:RNA double helix on the second solid support.

[0171] Embodiment 159. The method according to Embodiment 158, further comprising immobilizing a DNA:RNA double-stranded tagged fragment onto a second solid support, and then combining the first and second solid supports, wherein each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0172] Embodiment 160. The method according to Embodiment 158 ​​or 159, further comprising splitting the first solid support having immobilized tagged fragments containing DNA-specific barcodes from the remainder of the sample after tagmentation on the first solid support and before preparing single strands of cDNA from RNA, to produce DNA:RNA double helix.

[0173] Embodiment 161. The method according to Embodiment 158 ​​or 160, further comprising separating the first solid support having the immobilized DNA from the rest of the sample after immobilizing the DNA and before performing tagmentation on the first solid support, to prepare a tagged fragment containing a DNA-specific barcode.

[0174] Embodiment 162. The method according to any one of Embodiments 154 to 161, wherein the capture probe comprises nucleic acid.

[0175] Embodiment 163. The method according to any one of Embodiments 145 to 162, further comprising adding synthetic double-stranded DNA to the first solid support after performing tagmentation on the first solid support.

[0176] Embodiment 164. The method according to Embodiment 163, wherein the synthetic double-stranded DNA contains uracil.

[0177] Embodiment 165. The method according to any one of Embodiments 145 to 164, wherein the DNA-specific barcode and the RNA-specific barcode include different primer-binding sequences.

[0178] Embodiment 166. The method according to Embodiment 165, further comprising amplifying a tagged fragment containing a DNA-specific barcode using a primer that binds to a primer-binding sequence contained in the DNA-specific barcode.

[0179] Embodiment 167. The method according to Embodiment 165, further comprising amplifying a tagged fragment containing an RNA-specific barcode using a primer that binds to a primer-binding sequence contained in the RNA-specific barcode.

[0180] Embodiment 168. The method of Embodiment 165, further comprising amplifying a tagged fragment containing a DNA-specific barcode and a tagged fragment containing an RNA-specific barcode using a primer mix comprising a primer that binds to a primer-binding sequence contained in a DNA-specific barcode and a primer that binds to a primer-binding sequence contained in an RNA-specific barcode.

[0181] Embodiment 169. The method according to any one of Embodiments 166 to 168, wherein amplification is performed using uracil-intolerant DNA polymerase.

[0182] Embodiment 170. The method according to Embodiment 168 or 169, wherein the amplification includes bridge amplification.

[0183] Embodiment 171. The method according to any one of Embodiments 145 to 170, further comprising sequencing a tagged fragment or an amplified tagged fragment.

[0184] Embodiment 172. The method according to any one of Embodiments 145 to 171, wherein the method is carried out in a single reaction vessel.

[0185] Embodiment 173. The transposome complex is 1 mm 2 At least 10 3 , 10 4 , 10 5 , or 10 6 The method according to any one of embodiments 145 to 172, wherein the composites are present on a solid support at a density of 1.

[0186] Embodiment 174. The method according to any one of Embodiments 145 to 173, wherein the transposase comprises Tn5 transposase.

[0187] Embodiment 175. The method according to Embodiment 174, wherein the Tn5 transposase is a highly active Tn5 transposase.

[0188] Embodiment 176. The method according to any one of Embodiments 145 to 175, wherein the length of the immobilized fragment is adjusted by increasing or decreasing the density of the transposome complex on the solid support.

[0189] Embodiment 177. The method according to any one of Embodiments 145 to 176, wherein the solid support comprises microparticles, beads, a planar support, a patterned surface, or wells.

[0190] Embodiment 178. The method according to Embodiment 177, wherein the solid support is a bead.

[0191] Embodiment 179. A solid support on which a library of tagged fragments, prepared according to the method of any one of Embodiments 145 to 178, is immobilized.

[0192] Embodiment 180. The solid support according to Embodiment 179, wherein the solid support is a bead.

[0193] Embodiment 181. A method for preparing a strand-specific library of single-stranded DNA from RNA, comprising: (a) preparing a first strand of cDNA from RNA contained in a sample using a nucleotide containing reverse transcriptase, a primer, and dTTP under conditions that inhibit DNA-dependent DNA synthesis; (b) preparing a second strand of cDNA from the first strand of cDNA using a nucleotide containing DNA polymerase, a primer, and dUTP to prepare double-stranded cDNA; and (c) applying the double-stranded cDNA to a solid support on which a transposome complex is immobilized, wherein each transposome complex comprises a transposase, a first transposon comprising a 3' portion containing a transposon terminal sequence and a first read sequencing adapter sequence, and a first transposon comprising a 5' affinity element for immobilizing the transposome complex to a solid support, and a second transposon sequence comprising a sequence that is completely or partially complementary to the transposon terminal sequence, and (b) using the transposome complex A method comprising: (c) performing tagmentation on double-stranded DNA to prepare a tagged double-stranded DNA fragment containing a first read sequencing adapter sequence, wherein optionally, the transposome complex is reversibly inactivated before tagmentation, and tagmentation activates the transposome complex; (d) removing a second transposon, filling the gap and extending the fragment; (e) hybridizing a primer containing the second read sequencing adapter sequence to a transposon terminal sequence or a sequence that is completely or partially complementary to the transposon terminal sequence, amplifying the fragment to prepare a DNA strand that is not bound to a solid support and contains the first and second read sequencing adapters; and (f) releasing the strand produced by amplification from the solid support, wherein the release releases a single-stranded DNA fragment containing the first and second read sequencing adapters.

[0194] Embodiment 182. The method according to Embodiment 181, wherein the condition for inhibiting DNA-dependent DNA synthesis is the presence of a buffer containing actinomycin D.

[0195] Embodiment 183. The method according to Embodiment 181 or 182, wherein the primer is one or more randomizer primers.

[0196] Embodiment 184. The method according to any one of Embodiments 181 to 183, wherein the primer is a mixture of a randomizer primer and a poly-T primer.

[0197] Embodiment 185. The method according to any one of Embodiments 181 to 184, wherein the primer for preparing the second strand of cDNA is the same as the primer for preparing the first strand of cDNA.

[0198] Embodiment 186. The method according to any one of Embodiments 181 to 185, wherein the RNA is a long non-coding RNA or an antisense transcript.

[0199] Embodiment 187. The method according to any one of Embodiments 181 to 186, wherein amplification is performed using uracil-intolerant polymerase.

[0200] Embodiment 188. The method according to Embodiment 187, wherein amplification is not performed from a DNA strand containing uracil.

[0201] Embodiment 189. The method according to any one of Embodiments 181 to 188, wherein a unique molecular identifier (UMI) is included in a primer containing a second read sequencing adapter sequence.

[0202] Embodiment 190. The method according to Embodiment 189, wherein UMI is located between a second read sequencing adapter sequence and a sequence that can bind to a transposon terminal sequence or a sequence that is completely or partially complementary to a transposon terminal sequence.

[0203] Embodiment 191. The method according to any one of Embodiments 181 to 188, wherein UMI is contained in the first transposon.

[0204] Embodiment 192. The method according to Embodiment 191, wherein the UMI is located between the transposon terminal sequence and the first read sequencing adapter sequence.

[0205] Embodiment 193. The method according to any one of Embodiments 189 to 192, wherein the RNA comprises a pool of different RNAs, and the single-stranded fragments comprising a first read sequencing adapter and a second read sequencing adapter comprise a pool of different fragments, and each fragment comprises a UMI different from other fragments in the pool of different fragments.

[0206] Embodiment 194. The method according to any one of Embodiments 181 to 193, wherein the affinity element is biotin or desthiobiotin, and the solid support comprises streptavidin or avidin on its surface.

[0207] Embodiment 195. The method according to Embodiment 194, wherein the affinity element is a double biotin.

[0208] Embodiment 196. The method according to any one of Embodiments 181 to 195, wherein the release is carried out by heat or sodium hydroxide treatment.

[0209] Embodiment 197. The method according to any one of Embodiments 181 to 196, wherein a single-stranded fragment including a first read sequencing adapter and a second read sequencing adapter is separated from a solid support after release.

[0210] Embodiment 198. The method according to any one of Embodiments 181 to 197, further comprising preparing an indexed fragment by performing index primer amplification using a single-stranded DNA fragment containing a first read sequencing adapter and a second read sequencing adapter after release.

[0211] Embodiment 199. The method according to Embodiment 198, wherein the index primer amplification is performed in a reaction vessel separate from the solid support.

[0212] Embodiment 200. The method according to Embodiment 198 or 199, wherein index primer amplification is performed using uracil-intolerant polymerase.

[0213] Embodiment 201. The method according to any one of Embodiments 181 to 200, further comprising sequencing a single-stranded DNA fragment or indexed fragment including a first read sequencing adapter and a second read sequencing adapter.

[0214] Embodiment 202. The method according to Embodiment 201, wherein the sequencing data is generated from the first strand of cDNA generated from RNA.

[0215] Embodiment 203. The method according to Embodiment 201, wherein the sequencing data is not generated from the second strand of cDNA generated from RNA.

[0216] Embodiment 204. The method according to any one of Embodiments 181 to 203, wherein the method does not require ligation.

[0217] Embodiment 205. The method according to any one of Embodiments 181 to 206, for demarcating the boundaries of duplicate sequences in RNA.

[0218] Embodiment 206. The method according to any one of Embodiments 181 to 207, wherein the method enables the estimation of transcript expression.

[0219] Embodiment 207. The method according to Embodiment 208, wherein the estimation of transcript expression is based on UMI analysis.

[0220] Embodiment 208. A method for preparing a library of double-stranded DNA fragments from RNA, comprising: (a) preparing a first strand of cDNA from full-length RNA in a sample using a poly-T primer comprising UMI and a first read sequencing adapter sequence; (b) preparing a second strand of cDNA to generate double-stranded cDNA; and (c) applying the double-stranded cDNA to beads on which a transposome complex is immobilized, wherein each transposome complex comprises a transposase, a first transposon comprising a 3' transposon terminal sequence, and a second transposon comprising a sequence and a hybridization sequence that are completely or partially complementary to the transposon terminal sequence, the transposome complex being immobilized by binding of the hybridization sequence to an oligonucleotide immobilized on the beads, the oligonucleotide comprising a 5' affinity element, a first read sequence A method comprising: (b) immobilizing double-stranded cDNA and performing tagmentation on beads to prepare a double-stranded DNA fragment, optionally including reversibly inactivating the transposomal complex before performing tagmentation and activating the transposomal complex by performing tagmentation; (c) removing a second transposon; (d) hybridizing a primer comprising a second read sequencing adapter sequence and a sequence fully or partially complementary to the transposon terminal sequence to the transposon terminal sequence; and (e) gap filling and extension to prepare a double-stranded DNA fragment comprising a first read sequencing adapter and a second read sequencing adapter.

[0221] Embodiment 209. A method for preparing a library of double-stranded DNA fragments from RNA, comprising: (a) preparing a first strand of cDNA from full-length RNA in a sample using a poly-T primer comprising a UMI and a first read sequencing adapter sequence; (b) preparing a second strand of cDNA to generate double-stranded cDNA; and (c) applying the double-stranded cDNA to beads on which a transposome complex is immobilized, wherein each transposome complex comprises a transposase, a first transposon comprising a 3' transposon terminal sequence, a bead code, and a second read sequencing adapter sequence, and further comprising a 5' affinity element for immobilizing the transposome complex on a solid support, and a sequence that is completely or partially complementary to the transposon terminal sequence. A method comprising: (b) immobilizing double-stranded cDNA and performing tagmentation on beads to prepare a double-stranded DNA fragment, wherein optionally the transposomal complex is reversibly inactivated before tagmentation and the tagmentation activates the transposomal complex; (c) removing the second transposon; (d) hybridizing a primer comprising a second read sequencing adapter sequence and a sequence that is fully or partially complementary to the transposon terminal sequence to the transposon terminal sequence; and (e) gap filling and extension to prepare a double-stranded DNA fragment comprising a first read sequencing adapter and a second read sequencing adapter.

[0222] Embodiment 210. The method according to Embodiment 208 or 209, wherein the sequence that is completely or partially complementary to the transposon terminal sequence is shorter than the transposon terminal sequence.

[0223] Embodiment 211. The method according to Embodiment 210, wherein fewer adapter dimers are produced when the sequence that is completely or partially complementary to the transposon terminal sequence is shorter than the transposon terminal sequence.

[0224] Embodiment 212. The method according to any one of Embodiments 208 to 211, wherein the primer comprises a 5' portion containing a second read sequence adapter and a 3' portion containing a sequence that is fully or partially complementary to the transposon terminal sequence.

[0225] Embodiment 213. The method according to any one of Embodiments 210 to 212, wherein when a sequence that is completely or partially complementary to the transposon terminal sequence is removed, the fragment remains bound to the transposome at one or both ends.

[0226] Embodiment 214. The method according to any one of Embodiments 210 to 213, wherein the full-length RNA comprises a pool of different full-length RNAs, and the poly-T primer comprises a pool of different poly-T primers containing different UMIs.

[0227] Embodiment 215. The method according to Embodiment 214, wherein each poly-T primer contained in a pool of different poly-T primers contains a different UMI.

[0228] Embodiment 216. The method according to Embodiments 210-215, wherein the full-length RNA comprises a pool of different full-length RNAs, and a 3' double-stranded DNA fragment prepared from a single full-length RNA contains a different UMI from a 3' double-stranded DNA fragment prepared from other full-length RNAs in the pool.

[0229] Embodiment 217. The method according to Embodiment 216, wherein the full-length RNA comprises a pool of different full-length RNAs, and the beads comprises a pool of beads.

[0230] Embodiment 218. The method of Embodiment 217, wherein each bead immobilizes a transposome complex containing a different bead code compared to the bead codes contained in the transposome complex immobilized on other beads in the pool.

[0231] Embodiment 219. The method according to any one of Embodiments 210 to 218, wherein all fragments prepared from double-stranded cDNA prepared from a single full-length RNA are tagged on the same beads.

[0232] Embodiment 220. The method according to any one of Embodiments 210 to 219, wherein all double-stranded fragments, including a first read sequencing adapter and a second read sequencing adapter prepared from double-stranded cDNA, are on the same solid support after gap filling and extension.

[0233] Embodiment 221. The method according to any one of Embodiments 210 to 220, wherein the full-length RNA comprises a pool of different full-length RNAs, and all double-stranded fragments, including a first read sequencing adapter and a second read sequencing adapter prepared from a single full-length RNA in the pool, are on the same solid support after gap filling and extension.

[0234] Embodiment 222. The method according to any one of Embodiments 210 to 221, further comprising amplifying a double-stranded fragment containing a first read sequencing adapter and a second read sequencing adapter to prepare an amplified fragment.

[0235] Embodiment 223. The method according to any one of Embodiments 210 to 222, further comprising sequencing an amplified fragment or double-stranded fragment including a first read sequencing adapter and a second read sequencing adapter.

[0236] Embodiment 224. The method according to Embodiment 223, wherein sequencing enables the detection of full-length RNA isoforms.

[0237] Embodiment 225. The method according to any one of Embodiments 210 to 224, wherein the preparation of double-stranded cDNA is by the strand method.

[0238] Embodiment 226. The method according to any one of Embodiments 223 to 225, wherein the presence of bead cords in the sequence obtained from a double-stranded fragment containing a first read sequencing adapter and a second read sequencing adapter or an amplified fragment identifies the beads from which the fragment was generated.

[0239] Embodiment 227. The method according to any one of Embodiments 210 to 226, wherein the sample comprises a single cell.

[0240] Embodiment 228. Any one of Embodiments 210 to 227, wherein preparing double-stranded cDNA from RNA and combining the sample with a second solid support to immobilize the cDNA includes any one of Embodiments 145.

[0241] Additional objectives and benefits are partially described below, some of which are evident from the description or can be learned through practice. These objectives and benefits will be realized and achieved by the elements and combinations specifically indicated in the attached claims.

[0242] Please understand that the general explanation above and the detailed explanation below are illustrative and explanatory only, and do not limit the scope of the claims.

[0243] The accompanying drawings incorporated herein and constituting part of this specification illustrate one (or several) embodiments and serve to illustrate the principles described herein together with the specification. [Brief explanation of the drawing]

[0244] [Figure 1A]This paper compares current methods of RNA:RNA double helix fragmentation using immobilized transpososomes (A) versus DNA:RNA double helix fragmentation using transpososomes in solution via capture and tagging (Cap-Tag, B, and C). In both Figure 1A and Figure 1B, the target RNA is immobilized by a poly-T capture oligonucleotide that binds to the poly-A tail of the mRNA. In Figures 1B and 1C, it is proposed that reverse transcriptase initiates second strand synthesis via nicks created in the RNA strand, and that these dsDNA double helixes are substrates for transpososomes. However, as a result of using transpososomes from solution, reads are generated only from the 3' end of the transcript. In contrast, this method using immobilized transpososomes (Figure 1A) allows for library generation corresponding to the full length of the target RNA. [Figure 1B] This paper compares current methods of RNA:RNA double helix fragmentation using immobilized transpososomes (A) versus DNA:RNA double helix fragmentation using transpososomes in solution via capture and tagging (Cap-Tag, B, and C). In both Figure 1A and Figure 1B, the target RNA is immobilized by a poly-T capture oligonucleotide that binds to the poly-A tail of the mRNA. In Figures 1B and 1C, it is proposed that reverse transcriptase initiates second strand synthesis via nicks created in the RNA strand, and that these dsDNA double helixes are substrates for transpososomes. However, as a result of using transpososomes from solution, reads are generated only from the 3' end of the transcript. In contrast, this method using immobilized transpososomes (Figure 1A) allows for library generation corresponding to the full length of the target RNA. [Figure 1C]This paper compares current methods of RNA:RNA double helix fragmentation using immobilized transpososomes (A) versus DNA:RNA double helix fragmentation using transpososomes in solution via capture and tagging (Cap-Tag, B, and C). In both Figure 1A and Figure 1B, the target RNA is immobilized by a poly-T capture oligonucleotide that binds to the poly-A tail of the mRNA. In Figures 1B and 1C, it is proposed that reverse transcriptase initiates second strand synthesis via nicks created in the RNA strand, and that these dsDNA double helixes are substrates for transpososomes. However, as a result of using transpososomes from solution, reads are generated only from the 3' end of the transcript. In contrast, this method using immobilized transpososomes (Figure 1A) allows for library generation corresponding to the full length of the target RNA. [Figure 2] This describes how DNA:RNA double helixes in solution can be tagged by transpososomes immobilized on a surface. Target RNA is used for cDNA synthesis to generate DNA:RNA double helixes, which are then tagged via an immobilized transpososome complex. Tagmentation produces double-stranded DNA:RNA double helixes crosslinked to two immobilized transpososome complexes on a solid support. The transposase activity of the transpososome complex (e.g., Tn5 as shown herein) is then terminated, or the transposase is removed. Strand exchange occurs, followed by gap-filling ligation. A library of fragments can be released in either a tube or a flow cell. [Figure 3A]Representative sequencing results from RNA sequencing libraries generated using cDNA synthesis from universal human reference RNA to produce DNA:RNA double helixes are shown. These DNA:RNA double helixes were tagmented by immobilized transposome complexes such as BLTs (shown in Figure 2). Following tagmentation with immobilized transposome complexes on BLTs, gap-filling ligation was performed. The libraries were released into tubes and seeded directly into flow cells. The libraries were sequenceable (A). The distribution of results shows sequencing of coding, untranslated region (UTR), introns, and intergenetic sequences (B), thus indicating that different regions of RNA could be sequenced using this method. Normalized coverage along transcript length is also shown (C), which supports the relative absence of 3' bias in the sequencing results. [Figure 3B] Representative sequencing results from RNA sequencing libraries generated using cDNA synthesis from universal human reference RNA to produce DNA:RNA double helixes are shown. These DNA:RNA double helixes were tagmented by immobilized transposome complexes such as BLTs (shown in Figure 2). Following tagmentation with immobilized transposome complexes on BLTs, gap-filling ligation was performed. The libraries were released into tubes and seeded directly into flow cells. The libraries were sequenceable (A). The distribution of results shows sequencing of coding, untranslated region (UTR), introns, and intergenetic sequences (B), thus indicating that different regions of RNA could be sequenced using this method. Normalized coverage along transcript length is also shown (C), which supports the relative absence of 3' bias in the sequencing results. [Figure 3C]Representative sequencing results from RNA sequencing libraries generated using cDNA synthesis from universal human reference RNA to produce DNA:RNA double helixes are shown. These DNA:RNA double helixes were tagmented by immobilized transposome complexes such as BLTs (shown in Figure 2). Following tagmentation with immobilized transposome complexes on BLTs, gap-filling ligation was performed. The libraries were released into tubes and seeded directly into flow cells. The libraries were sequenceable (A). The distribution of results shows sequencing of coding, untranslated region (UTR), introns, and intergenetic sequences (B), thus indicating that different regions of RNA could be sequenced using this method. Normalized coverage along transcript length is also shown (C), which supports the relative absence of 3' bias in the sequencing results. [Figure 4] This describes the simultaneous encapsulation of cells and capture beads in a droplet, followed by cell lysis, cDNA synthesis, and library preparation. Beads with immobilized fragment libraries can be delivered to a solid surface (e.g., a flow cell) for sequencing before the library fragments are released. Alternatively, beads with immobilized target nucleic acids can be delivered to a solid surface for sequencing, followed by fragment preparation on the beads and release of the fragments onto the solid surface for sequencing. Release of fragments onto a flow cell can enable on-flow cell spatial reads to identify fragments likely to originate from the same cell. [Figure 5]The image shows beads containing capture oligonucleotides. In this representative example, the capture oligonucleotides include a polyT sequence, a barcode (which can be used as a bead code), and a P5 adapter sequence. The polyT sequence can be used to capture mRNA, and the P5 and barcode can be incorporated during the preparation of DNA:RNA double helix. Other adapters (shown here as B'-P7') can be incorporated during or after tagmentation. After tagmentation, strand transposition and ligation can be performed to prepare library fragments for sequencing. [Figure 6] The diagram shows beads having a capture oligonucleotide (here, a poly-T sequence for capturing mRNA) and an immobilized oligonucleotide containing a sequence ("short A") for hybridizing to a hybridization sequence contained in a second transposon in the transposome complex. In this way, the immobilized oligonucleotide can be used to assemble the transposome complex. The immobilized oligonucleotide may further contain a P5 adapter sequence and a bead code. [Figure 7] This describes a workflow for producing a library from full-length RNA using activatable BLTs with immobilized oligonucleotides for assembling transpososomes, including steps such as mRNA capture in a droplet, cDNA synthesis in bulk, hybridization (Hyb) of transpososomes to immobilized oligonucleotides, and tagmentation. The BLTs can be captured on a flow cell before or after tagmentation. [Figure 8] This paper describes a method for single-cell resolution of a library, either for use in droplets or for capture in microwells of a flow cell. [Figure 9] This describes a method for cDNA synthesis, followed by tagmentation and library preparation on a BLT, and then release of the fragments in a flow cell or tube. This method prepares a full-length mRNA library using a poly-T primer that binds to the poly-A tail of mRNA. [Figure 10] This describes a method for cDNA synthesis, followed by tagmentation and library preparation on a BLT, and then release of the fragments in a flow cell or tube. This method prepares a total RNA library using random primers that bind to all RNAs. [Figure 11A] The following are different beads that may be used in this method for preparing a library from RNA: (A) Beads for preparing a full-length mRNA library, having a capture oligonucleotide (here, a poly-T sequence for capturing mRNA) and an immobilized oligonucleotide containing a sequence (e.g., "short A") for hybridizing to a hybridization sequence contained in a second transposon included in the transposomal complex, which may be used to assemble the transposomal complex. (B) Hybridization of the transposomal to the short A sequence. (C) Preparation of multiple fragments of full-length mRNA on the beads. [Figure 11B] The following are different beads that may be used in this method for preparing a library from RNA: (A) Beads for preparing a full-length mRNA library, having a capture oligonucleotide (here, a poly-T sequence for capturing mRNA) and an immobilized oligonucleotide containing a sequence (e.g., "short A") for hybridizing to a hybridization sequence contained in a second transposon included in the transposomal complex, which may be used to assemble the transposomal complex. (B) Hybridization of the transposomal to the short A sequence. (C) Preparation of multiple fragments of full-length mRNA on the beads. [Figure 11C]The following are different beads that may be used in this method for preparing a library from RNA: (A) Beads for preparing a full-length mRNA library, having a capture oligonucleotide (here, a poly-T sequence for capturing mRNA) and an immobilized oligonucleotide containing a sequence (e.g., "short A") for hybridizing to a hybridization sequence contained in a second transposon included in the transposomal complex, which may be used to assemble the transposomal complex. (B) Hybridization of the transposomal to the short A sequence. (C) Preparation of multiple fragments of full-length mRNA on the beads. [Figure 12] A typical bead for preparing a full-length mRNA library is shown. The bead contains multiple capture oligonucleotides (here, poly-T sequences for capturing mRNA) and multiple immobilized oligonucleotides containing sequences for hybridizing to a hybridization sequence contained in a second transposon. Before and after the preparation of DNA:RNA double helixic structures, the immobilized oligonucleotides could be used to hybridize and immobilize the transposome complex onto the bead. [Figure 13] This shows a typical example of how multiple transposomes on a BLT prepare fragments from the full length of a DNA:RNA double helix generated from mRNA. [Figure 14] This shows a symmetric tagging event for BLT after 3'UMI was incorporated during cDNA synthesis. [Figure 15] This shows tagmentation of a double-stranded cDNA containing 3'UMI after preparation. In this example, each bead contains an immobilized first transposon with a bead code, enabling a UMI counting assay across the entire mRNA transcript. [Figure 16] This describes tagmentation using beads after preparation of double-stranded cDNA having a 3'UMI. The method includes first-strand cDNA synthesis, template switching, and PCR amplification before tagmentation. [Figure 17]This document describes a primer extension strand-specific BLT (PRESS-BLT) workflow involving strand-specific cDNA synthesis. [Figure 18] The PRESS-BLT workflow is outlined, where the first transposon is immobilized on a bead using dual biotin. [Figure 19] This section summarizes how dual biotin and shortened ME' sequences can improve workflow yields. For example, shortened ME' sequences may be useful in the manner shown in Figure 14, or in other methods in which the non-transferred ME' chain is replaced with a different oligonucleotide. [Figure 20] A diagram illustrates an exemplary workflow for preparing an RNA sequencing library. mRNA is captured from a total RNA sample using an RNA BLT containing polyT-capture oligonucleotides. The sample is then washed, and cDNA is generated using reverse transcriptase to form a DNA:RNA double helix. The DNA:RNA double helix is ​​then fragmented via a transposomal complex immobilized on the RNA BLT. In embodiments shown in Figures 20–28, double-stranded cDNA can also be prepared (instead of a DNA:RNA double helix) and fragmented via a transposomal complex immobilized on the RNA BLT. [Figure 21]This section outlines how DNA and RNA in a mixed sample (i.e., containing both DNA and RNA) can bind to RNA BLTs and DNA BLTs. Since RNA does not have affinity for the transposomal complex, it binds only to RNA BLTs containing poly-T captured oligonucleotides. However, DNA could bind to either DNA BLTs or RNA BLTs based on its affinity for the transposon terminal sequences contained in the immobilized transposomal complex in both DNA and RNA BLTs. Therefore, methods for use with samples containing both RNA and DNA must selectively separate DNA into DNA BLTs. Methods for use with mixed samples containing both DNA and RNA can use different tags during the tagmentation reaction, such that iDNA is the index for identifying DNA BLTs and iRNA is the index for identifying RNA BLTs. In this way, fragments derived from DNA can be distinguished from fragments derived from RNA. [Figure 22] This paper outlines a method for preparing RNA and DNA sequencing samples from RNA and DNA-containing samples using RNA BLTs that are reversibly inactivated before application. In this way, DNA in the sample binds to the DNA BLT, while only RNA binds to the RNA BLT (via poly-T capture oligonucleotides). The DNA bound to the DNA BLT is tagged, and the beads are washed. The RNA BLT transposomal complex is then activated (i.e., inactivation is reversed). Reverse transcription is performed to generate DNA:RNA double helix immobilized on the RNA BLT, followed by fragmentation of the DNA:RNA double helix. [Figure 23]This paper outlines a method for preparing RNA and DNA sequencing samples from RNA and DNA-containing samples using "naked" RNA BLTs. These "naked" RNA-BLTs do not contain transposase. First, DNA is tagged using DNA-BLTs, and RNA is captured on poly-T capture oligonucleotides so that cDNA synthesis can be performed. The sample is then washed, and transposases are added to assemble functional transpososomes on the previously "naked" RNA-BLTs. Reverse transcriptase polymerase is added, and DNA:RNA double-strand tagging is performed using RNA BLTs. Transpososomes can be assembled on beads after cDNA synthesis by hybridization of the transposomal complex to the oligonucleotides immobilized on the RNA-BLTs. [Figure 24] This paper outlines a method for preparing RNA and DNA sequencing samples from RNA and DNA-containing samples using a three-bead approach. In the three-bead approach, the method begins with DNA-BLTs and poly-T beads (lacking transpososomes). DNA is tagged by DNA BLTs, and RNA is captured by poly-T beads. The beads are washed. RNA-BLTs are then added along with a reagent to replace the captured RNA from the poly-T beads onto the RNA-BLTs. Reverse transcriptase polymerase is added, and DNA:RNA double-strand tagging is performed using RNA BLTs. [Figure 25] This paper outlines a method for preparing RNA and DNA sequencing samples from RNA and DNA-containing samples using DNA capture, followed by the physical separation of DNA-binding DNA BLTs from RNA BLTs. Excess DNA BLTs can be used to bind all double-stranded (ds) DNA in the sample, allowing for DNA tagging. RNA can then be separated from the tagged DNA BLTs and bound to RNA BLTs. RNA tagging is performed after generating DNA:RNA double helixes via reverse transcriptase polymerase. The RNA BLTs and DNA BLTs can then be combined. [Figure 26] This paper describes a method for preparing a library of tagged fragments prepared from DNA or RNA (converted to cDNA) using DNA-specific tagging and cDNA-specific tagging, as well as splitting. A sample containing all nucleic acids (including DNA and RNA, TNA) is combined with a DNA BLT, and DNA-specific tagging is performed to incorporate DNA-specific barcodes into the fragments. The DNA BLT can then be split, while the DNA fragments remain associated with the BLT. Double-stranded cDNA (ds-cDNA) can then be generated from RNA, tagged via RNA(cDNA)-specific tagging, and RNA-specific barcodes can be incorporated into the fragments. In this way, the fragments are tagged so that downstream sequencing and bioinformatics can use the barcodes to distinguish RNA-derived samples from DNA-derived samples. [Figure 27]A method for preparing a library of tagged fragments prepared from DNA or RNA (converted to cDNA) is described using DNA-specific tagmentation and RNA (cDNA)-specific tagmentation. This method can be carried out in a single reaction vessel (i.e., a one-pot reaction). After DNA-specific tagmentation is performed and DNA-specific barcodes are incorporated using DNA BLTs, “suicide” synthetic DNA can be added to these DNA BLTs. Such synthetic DNA may be uracil-containing double-stranded DNA (dsDNA), which cannot be amplified if uracil-intolerant DNA polymerase is used for amplification. In this way, all DNA BLTs are saturated with either sample-derived dsDNA or synthetic dsDNA, and the DNA BLTs (containing transposome complexes with DNA-specific barcodes) are not available for further tagmentation. ds-cDNA can then be prepared from RNA in the sample, and cDNA-specific tagmentation can be used to incorporate RNA barcodes using RNA BLTs containing transposome complexes with RNA-specific barcodes. After cleanup and amplification, the tagged fragments can be sequenced, and the barcodes are used to determine whether the sample originated from DNA or RNA. [Figure 28]This paper describes a method for preparing libraries of tagged fragments prepared from DNA or RNA (converted to DNA:RNA double helix) using DNA-specific tagmentation and DNA:RNA double-strand specific tagmentation. After DNA-specific tagmentation is performed and the DNA barcode is incorporated using DNA BLTs containing transposomal complexes with DNA-specific barcodes, "suicide" synthetic DNA can be added to these DNA BLTs. Then, single strands of cDNA can be prepared from RNA in the sample to generate DNA:RNA double helix, and the RNA barcode can be incorporated using RNA BLTs containing transposomal complexes with RNA-specific barcodes, using DNA:RNA double-strand tagmentation. An example of RNA BLT is tedRNA beads from xGEN USA, which have improved efficacy in tagging DNA:RNA double helix. After cleanup and amplification, the tagged fragments can be sequenced, and the barcodes are used to determine DNA-derived samples versus RNA-derived samples. [Modes for carrying out the invention]

[0245] I. Method for preparing an RNA sequencing library using bead-linked transposomes containing captured oligonucleotides. Many protocols for sequencing RNA samples employ sample preparation that converts the RNA in the sample into a double-stranded cDNA format before sequencing. A method is provided herein for sequencing RNA samples using DNA:RNA double helix to avoid 3' bias when tagging mRNA. In some embodiments, this method allows for uniform 5' to 3' coverage of the target RNA in the resulting library product (shown in Figure 1A).

[0246] As used herein, “DNA:RNA” double helix refers to a double helix of RNA and DNA. A DNA:RNA double helix contains RNA with some amount of DNA associated with it. The DNA in a DNA:RNA double helix allows for fragmentation, and the amount of DNA in a DNA:RNA double helix is ​​sufficient for a transposase to fragment the DNA:RNA double helix.

[0247] In all embodiments, it is understood that the DNA:RNA double helix may also be converted to double-stranded DNA before fragmentation (i.e., tagmentation). In some embodiments, all or part of the second strand of the cDNA is generated before fragmentation. In some embodiments, the DNA:RNA double helix may be converted to double-stranded DNA in solution or after the DNA:RNA double helix has been bound to the BLT. Converting to double-stranded DNA means that some or all of the RNA in the DNA:RNA double helix is ​​converted to DNA. In such embodiments, fragmentation may occur in the region of the double-stranded DNA.

[0248] In some embodiments, a sample containing RNA from one or more species is attached to a surface, where the mRNA transcript is captured via a capture oligonucleotide, such as by its 3' poly-A tail on the surface of the beads. Reverse transcriptase (RT) polymerase is then attached to produce a first strand of cDNA. Thus, in this method, cDNA synthesis can occur after the RNA has been bound to the capture oligonucleotide. The DNA:RNA double helix is ​​tagged by a surface-bound transposome, thus generating a library template from the full length of the mRNA transcript.

[0249] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from target RNA is described as follows: The method involves applying a sample containing target RNA to a solid support on which a transposomal complex and a capture oligonucleotide are immobilized, The transpososome complex contains a transposase bound to a first polynucleotide, and the first polynucleotide has a 3' portion containing the transposon terminal sequence, The first tag, and, The sample is applied to a solid support under conditions where the 3' end of the target RNA is bound to the capture oligonucleotide. Under conditions that synthesize cDNA and generate a DNA:RNA double helix immobilized on a captured oligonucleotide, reverse transcriptase polymerase is added. The method comprises fragmenting a DNA:RNA duplex using a transposome complex under conditions in which the DNA:RNA duplex is tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a 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 the DNA strand.

[0250] In some embodiments, an activatable BLT may be used, and the transposase is bound to the first polynucleotide during the process. In some embodiments, the transposase is bound to the first polynucleotide before or after the reverse transcriptase is added. In other words, the transposase may be added after the DNA:RNA double helix is ​​formed, or before the DNA:RNA double helix is ​​formed.

[0251] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from target RNA is described as follows: The method involves applying a sample containing target RNA to a solid support on which a capture oligonucleotide and a first polynucleotide are immobilized, wherein the first polynucleotide comprises a 3' portion containing a transposon terminal sequence and a first tag, and the sample is applied to the solid support under conditions that the 3' end of the target RNA is bound to the capture oligonucleotide. Add a transposase under conditions where the transposase binds to a first polynucleotide to form a transposome complex, and add reverse transcriptase polymerase under conditions for synthesizing cDNA and generating a DNA:RNA duplex immobilized on a capture oligonucleotide, and fragment the DNA:RNA duplex using the transposome complex under conditions where the DNA:RNA duplex is tagged at the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5'-tagged with a first tag, and

[0252] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA is applying a sample containing the target RNA to a solid support to which a capture oligonucleotide and a first polynucleotide are immobilized, wherein the first polynucleotide comprises a 3' portion comprising a transposon end sequence and a first tag, and the sample is applied to the solid support under conditions where the 3' end of the target RNA binds to the capture oligonucleotide, and add reverse transcriptase polymerase under conditions for synthesizing cDNA and generating a DNA:RNA duplex immobilized on a capture oligonucleotide, and add a transposase under conditions where the transposase binds to the first polynucleotide to form a transposome complex, and fragment the DNA:RNA duplex using the transposome complex under conditions where the DNA:RNA duplex is tagged at the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5'-tagged with a first tag, and

[0253] In some embodiments, the method further comprises washing the solid support to remove any unbound target RNA after applying the sample to the solid support.

[0254] When a DNA:RNA double helix is ​​attached to a solid support, the transposome complex tags the double helix, thus generating fragments bound to the surface at both ends. In some embodiments, the length of the crosslinked fragments can be varied by changing the density of the transposome complex on the surface. In certain embodiments, the lengths of the resulting crosslinked fragments are 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, 2 The bp values ​​are 700 bp, 2800 bp, 2900 bp, 3000 bp, 3100 bp, 3200 bp, 3300 bp, 3400 bp, 3500 bp, 3600 bp, 3700 bp, 3800 bp, 3900 bp, 4000 bp, 4100 bp, 4200 bp, 4300 bp, 4400 bp, 4500 bp, 4600 bp, 4700 bp, 4800 bp, 4900 bp, 5000 bp, 10000 bp, 30000 bp, or 100,000 bp or less. In such embodiments, the crosslinked fragments may then be amplified within clusters using standard cluster chemistry, as illustrated by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of these are incorporated herein by reference in their entirety.

[0255] In some embodiments, fragmentation produces double-stranded DNA:RNA double helix crosslinked to two immobilized transposome complexes on a solid support. In some embodiments, the length of the crosslinked double helix is ​​between 100 and 1500 base pairs.

[0256] In some embodiments, a DNA:RNA double helix generated from the 3' end of mRNA is bound to a capture oligonucleotide at one end and to an immobilized transposome complex at the other end. In some embodiments, the capture oligonucleotide contains a sequence similar to or identical to a sequence contained in one or more transposon ends. In some embodiments, the capture oligonucleotide may contain a first tag.

[0257] In some embodiments, DNA:RNA double-stranded fragments are It can be used to generate the coding, untranslated regions (UTRs), introns, and / or intergenetic sequences of target RNA.

[0258] In some embodiments, an in vitro transposition reaction for tagging a target DNA:RNA duplex and generating an immobilized tagged DNA:RNA duplex comprises a transposase, a transposon sequence composition, and suitable reaction conditions.

[0259] As described herein, certain modifications to the protocol may improve yield. However, these specific methods are not intended to limit the scope of the claims, but rather to provide guidance to those skilled in the art who wish to optimize the method for their particular samples. For example, a user working with a sample containing single cells may wish to use the methods described herein to improve library yield, since single cells have limited amounts of RNA and DNA.

[0260] A. Sample and target mRNA In some embodiments, the sample includes target RNA. In some embodiments, the sample includes RNA and DNA. In some embodiments, the target RNA is mRNA. In some embodiments, the target RNA includes coding, untranslated region (UTR) sequences, intron sequences, and / or intergene sequences.

[0261] In some embodiments, the step of applying a sample containing target RNA or a sample containing RNA and DNA includes attaching the biological sample to a solid support. The biological sample can be any type that contains RNA or RNA and DNA and can be deposited on a solid surface for tagmentation. For example, the sample may contain RNA or RNA and DNA in various purified states, including purified RNA or RNA and DNA. However, the sample does not need to be completely purified and may contain RNA or RNA and DNA contaminated with, for example, proteins, other nucleic acid species, other cellular components, and / or any other contaminants. In some embodiments, the biological sample contains a mixture of RNA or RNA and DNA, proteins, other nucleic acid species, other cellular components, and / or any other contaminants present in proportions similar to those found in vivo. For example, in some embodiments, the components are found in proportions similar to those 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. RNA or RNA and DNA by the method provided herein. Because it becomes possible to bind to a solid support, other contaminants can be removed simply by washing the solid support after surface-bound tagmentation has occurred. Biological samples may include, for example, crude cell lysates or whole cells. For example, crude cell lysates applied to a solid support in the method described herein do not require one or more of the conventional separation steps used to isolate nucleic acids from other cellular components. Exemplary separation steps are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., which are incorporated herein by reference.

[0262] In some embodiments, the sample applied to the solid support has an absorbance ratio of 260 / 280 which is 1.7 or less.

[0263] Therefore, in some embodiments, the 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 RNA or RNA and DNA.

[0264] In some embodiments, the sample applied to the solid support is blood. In some embodiments, the sample applied to the solid support is cell lysate.

[0265] In some embodiments, the cell lysate is a crude cell lysate. In some embodiments, the method further includes applying the sample to a solid support and then lysing the cells in the sample to produce a cell lysate.

[0266] In some embodiments, DNA:RNA double helixes are prepared in solution and then immobilized on BLT (see Figure 2).

[0267] One advantage of the methods and compositions presented herein is that a biological sample can be added to the flow cell, and subsequent lysis and purification steps can all occur within the flow cell by simply flowing the necessary reagents through the flow cell, without further transfer or handling steps.

[0268] In some embodiments, the target RNA comprises a sequence that is complementary to at least a portion of one or more capture oligonucleotides.

[0269] 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.

[0270] In some embodiments, the 3' end of the target RNA binds to the capture oligonucleotide.

[0271] In some embodiments, the target RNA is mRNA. In some embodiments, the target RNA is polyadenylated (i.e., contains a stretch of RNA containing only adenine bases). In some embodiments, the mRNA contains a poly-A tail. In some embodiments, the 3' end of the mRNA contains a poly-A tail.

[0272] In some embodiments, the target mRNA contains a poly-A sequence and binds to a capture oligonucleotide containing a poly-T sequence.

[0273] The DNA and RNA contained in a sample may be referred to as "total nucleic acids." The method described herein may be useful in preparing DNA and RNA libraries from a sample containing total nucleic acids, wherein each fragment in the DNA library contains a DNA-specific barcode, and each fragment in the RNA library contains an RNA-specific barcode. Such a method may avoid the need to separate DNA and RNA before preparing DNA and RNA libraries from a single sample.

[0274] B. Transposomal complex In some embodiments, the transposomal complex comprises a transposase bound to one or more polynucleotides.

[0275] A “transposomal complex” consists of at least one transposase (or other enzyme 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 the transposition reaction. In some aspects, the transposon recognition sequence is a double-stranded transposon terminal sequence. The transposase binds to a transposase recognition site in the target nucleic acid and inserts the transposon recognition sequence into the target nucleic acid. In some such insertion events, one strand of the transposon recognition sequence (or terminal sequence) is transcribed into the target nucleic acid, resulting in a cleavage event. Exemplary transposition procedures and systems can be readily adapted for use with transposases.

[0276] "Transposase" means an enzyme that forms a functional complex having a transposon end-containing composition (e.g., a transposon, a transposon end, or a transposon end composition) and can catalyze the insertion or rearrangement of the transposon end-containing composition into a double-stranded target nucleic acid. The transposases presented herein may also include integrases from retrotransposons and retroviruses.

[0277] Transposon-based technologies can be used to fragment DNA, where target nucleic acids, such as genomic DNA, are treated with a transposomal complex that simultaneously fragments and tags the target ("tagmentation"), thereby generating a population of fragmented nucleic acid molecules tagged with adapter sequences specific to the ends of the fragments. Tagmentation involves the modification of DNA by a transposomal complex containing a transposase enzyme complexed with one or more tags (such as adapter sequences) containing transposon end sequences (hereinafter referred to as transposons). Tagmentation can result in simultaneous fragmentation of DNA and ligation of adapters to the 5' ends of both strands of the double-stranded fragments.

[0278] A transposition reaction is a reaction in which one or more transposons are inserted into a target nucleic acid at a random or nearly random site. Components of a transposition reaction may include a transposase (or other enzymes capable of fragmenting and tagging nucleic acids as described herein, e.g., integrase), a transposon element comprising a double-stranded transposon terminal sequence bound to the enzyme, and an adapter sequence bound to one of the two transposon terminal sequences. One strand of the double-stranded transposon terminal sequence is transposed to one strand of the target nucleic acid, while the complementary transposon terminal sequence strand is not transposed (i.e., a non-transposition transposon sequence). The adapter sequence may optionally or desiredly include one or more functional sequences (e.g., primer sequences).

[0279] 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 terminal 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 designed versions of transposase family enzymes. The methods described herein also include combinations of transposases, not just single transposases.

[0280] In some embodiments, the transposase is Tn5, Tn7, MuA, or Vibrioharvey transposase, or active variants thereof. In other embodiments, the transposase is Tn5 transposase or a variant thereof. In other embodiments, the transposase is Tn5 transposase or a variant thereof. In other embodiments, the transposase is Tn5 transposase or an active variant thereof. In some embodiments, the Tn5 transposase is a highly active Tn5 transposase, or an active variant thereof. In some embodiments, the Tn5 transposase is a Tn5 transposase described in PCT International Publication 2015 / 160895, which is incorporated herein by reference. In some embodiments, the Tn5 transposase is a highly active Tn5 having mutations at positions 54, 56, 372, 212, 214, 251, and 338 relative to wild-type Tn5 transposase. In some embodiments, the Tn5 transposase is a highly active Tn5 having the following mutations from the wild-type Tn5 transposases: 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 contains a fusion elongation factor Ts(Tsf) tag. In some embodiments, the Tn5 transposase is a highly active Tn5 transposase containing mutations at amino acids 54, 56, and 372 from the wild-type sequence. In some embodiments, the highly active 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 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 highly active Tn5 transposase is used (e.g., EZ-Tn5™ transposase, Epicentre Biotechnologies, Madison, Wis.). In some embodiments, the Tn5 transposase is a wild-type Tn5 transposase.

[0281] In some embodiments, the transposome complex comprises a dimer of two molecules of transposase. In some embodiments, the transposome complex is homodimer, and the two molecules of transposase are each bound to a first and second transposon of the same type (for example, the sequences of the two transposons bound to each monomer are the same and form 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 homodimer, and the first population has a first adapter sequence in each monomer, while the second population has different adapter sequences in each monomer.

[0282] The term “transposon terminus” refers to a double-stranded nucleic acid DNA that exhibits only the nucleotide sequence (“transposon terminus sequence”) necessary to form a complex with a transposase or integrase enzyme that functions in an in vitro rearrangement reaction. In some embodiments, a transposon terminus can form a functional complex with a transposase in a rearrangement reaction. As a non-limiting example, as described in the disclosure of U.S. Patent Application Publication 2010 / 0120098, which is incorporated entirely herein by reference, a transposon terminus may include a 19-bp outer end (“outer end, OE”) transposon terminus, an inner end (“inner end, IE”) transposon terminus, or a “mosaic end, ME”) transposon terminus recognized by wild-type or mutant Tn5 transposase, or R1 and R2 transposon terminus. A transposon terminus may include any nucleic acid or nucleic acid analog suitable for forming a functional complex with a transposase or integrase enzyme in an in vitro rearrangement reaction. For example, transposon ends may contain DNA, RNA, modified bases, non-native bases, or modified backbones, and may contain nicks in one or both strands. The term "DNA" is used throughout this disclosure in reference to transposon end compositions, but it should be understood that any suitable nucleic acid or nucleic acid analog may be used at the transposon ends.

[0283] Similarly, the term “transfer strand” refers to the transposed portion of both transposon ends. Similarly, the term “non-transfer strand” refers to the non-transfer portion of both “transposon ends.” The 3' end of the transfer strand is bound to or transferred to the target DNA in the in vitro transposition reaction. The non-transfer strand, which exhibits a transposon end sequence complementary to the transposed transposon end sequence, is not bound to or transferred to the target DNA in the in vitro transposition reaction.

[0284] In some embodiments, the transfer and non-transfer strands are covalently bonded. For example, in some embodiments, the transfer and non-transfer strand sequences are provided on a single oligonucleotide, for example, in a hairpin configuration. Thus, although the free end of the non-transfer strand is not directly bound to the target DNA by the transposition reaction, the non-transfer strand is indirectly attached to the DNA fragment because it is linked to the transfer strand by the loop of the hairpin structure. Further examples of transposome structures and methods for preparing and using transpososomes 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.

[0285] In some embodiments, the transpososome complex includes a transposase bound to a first polynucleotide. In some embodiments, the first polynucleotide includes a 3' portion containing the transposon terminal sequence and a first tag.

[0286] In some embodiments, the transposomal complex includes a second polynucleotide containing a region complementary to the transposon terminal sequence.

[0287] 1. Transposase As used throughout, the term transposase refers to an enzyme that forms a functional complex with a transposon-containing composition (e.g., a transposon, a transposon composition) and can catalyze the insertion or rearrangement of the transposon-containing composition into a double-stranded target nucleic acid to which it is incubated in an in vitro rearrangement reaction. The transposases of the methods provided also include integrases from retrotransposons and retroviruses. Exemplary transposases that may be used in the methods provided include wild-type or mutant forms of Tn5 transposase and MuA transposase.

[0288] A transposition reaction is a reaction in which one or more transposons are inserted into a target nucleic acid at a random or nearly random site. Essential components of a transposition reaction are transposases and DNA oligonucleotides that represent the transposon's nucleotide sequence, including the transposition transposon sequence and its complement (i.e., the non-transposition transposon terminal sequence), as well as other components necessary for functional transposition or the formation of a transposomal complex. The methods of this disclosure are exemplified by using a transposition complex formed by a highly active Tn5 transposase and a Tn5-type transposon terminus, or by a MuA or HYPERMu transposase and a Mu transposon terminus containing R1 and R2 terminal sequences (see, for example, 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 that can insert transposon terminus in a random or near-random manner with sufficient efficiency to tag a target nucleic acid for the intended purpose may be used in a manner that provides this.Other examples of known transposition systems that may be used in the provided method include Staphylococcus aureus Tn552, Tyl, transposons Tn7, Tn / O and IS10, Mariner transposase, Tel, P element, Tn3, bacterial insertion sequences, retroviruses, and yeast retrotransposons (e.g., Colegio OR et al, J. Bacteriol., 183:2384-8, 2001; Kirby C et al, Mol. Microbiol., 43:173-86, 2002; Devine SE, and Boeke J D., Nucleic Acids Res., 22:3765-72, 1994; 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. See, but are not limited to, Sci USA, 86:2525-9, 1989 and Boeke JD and Corces VG, Annu Rev Microbiol. 43:403-34, 1989 (these are incorporated herein by reference in their entirety).

[0289] Methods for inserting a transposon into a target sequence can be carried out in vitro using any suitable transposon system, either by utilizing a suitable in vitro transposition system or by developing a suitable transposon system based on the knowledge of the art. Generally, a suitable in vitro transposition system for use in the methods of the present disclosure 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 capable of catalyzing the transposition reaction. Suitable transposase transposon terminal sequences that can be used include, but are not limited to, wild-type, derivative, or mutant transposon terminal sequences that form a complex with a transposase selected from the wild-type, derivative, or mutant of the transposase.

[0290] In some embodiments, the transposase comprises a Tn5 transposase. In some embodiments, the Tn5 transposase is a highly active Tn5 transposase. In some embodiments, the transposase has increased activity toward DNA:RNA double helix compared to Tn5.

[0291] 2. Inactivated transpososomes In some embodiments, the transposome complex is reversibly inactivated during the process. In some embodiments, the transposome complex is reversibly inactivated when a sample containing target RNA is applied to a solid support having the transposome complex and the captured oligonucleotide, and is activated before or at the time of fragmentation of the DNA:RNA complex. In some embodiments, the transposome complex is activated before or at the time of fragmentation by removing a transposome inactivator. Any of the methods of reversible inactivation may be used in conjunction with the methods described herein, which involve fragmentation of either DNA:RNA double helix or double-stranded DNA. In other words, any transposome complex described herein can be reversibly inactivated. In some embodiments, tagmentation involves activating a transposome complex that was previously in a reversibly inactivated state.

[0292] In some embodiments, the transposome complex is reversibly inactivated by a transposome inactivator bound to the transposome complex. In some embodiments, the transposome inactivator is bound to the Tn5 binding site of the transposome complex.

[0293] A wide range of methods have been able to inactivate transpososomes. In some embodiments, the transpososome inactivator includes dephosphorylated ME', extra bases, inhibitory double helixes, and / or thermolabile antibodies.

[0294] In some embodiments, the dephosphorylated ME' sequence can be phosphorylated to generate a phosphorylated ME' sequence, thereby activating the transposome.

[0295] In some embodiments, extra bases (i.e., nucleotides) are located adjacent to the transposon terminal sequence, blocking association with the transposase. In some embodiments, the extra bases are separated from the transposon terminal sequence by a cleavable linker. In some embodiments, treatment with a drug to cleave the cleavable linker generates an active transposome complex.

[0296] In some embodiments, the inhibitor duplex is bound to the transposase of the transposomal complex.

[0297] In some embodiments, the thermolabile antibody is a complex of the transposase on the DNA binding site of the transposome complex. In some embodiments, a reaction solution containing inactivated transpososomes with the thermolabile antibody is heated so that the thermolabile antibody is inactivated. Once the thermolabile antibody is inactivated, the transpososome can be activated.

[0298] 3. Liquid-phase transposome complex The methods presented herein may further include the steps of providing a transposome complex in solution and contacting the solution-phase transposome complex with an immobilized fragment under conditions in which DNA:RNA is fragmented by the transposome complex solution, thereby obtaining an immobilized nucleic acid fragment having one end in solution. In some embodiments, the transposome complex in solution may include a second tag, and as a result, the method produces an immobilized nucleic acid fragment having the second tag, the second tag being in solution. The first and second tags may be different or the same.

[0299] In some embodiments, the method may further include contacting a liquid-phase transposome complex with an immobilized DNA:RNA fragment under conditions in which the DNA:RNA fragment is further fragmented by the liquid-phase transposome complex, thereby obtaining an immobilized nucleic acid fragment having one end in solution.

[0300] In some embodiments, the liquid-phase transposome complex includes a second tag, thereby generating an immobilized nucleic acid fragment having the second tag in solution. 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 complex includes the second tag.

[0301] In some embodiments, one form of surface-bound transposomes is primarily present 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 the initial 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 structure contain the same tag domain at each end of the bridge. A second tagmentation reaction may be performed by adding transposomes from a solution that further fragment the bridge. In some embodiments, most or all of the liquid-phase transposomes contain tag domains different from those 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 transposome include tag domains different from the tag domains present on the bridge structure generated in the first tagmentation reaction.

[0302] In some embodiments, the template length is longer than that which can be suitably amplified using standard cluster chemistry. For example, in some embodiments, the template length 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 These are 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 may then be carried out by adding transposomes from a solution that further fragments the bridge, as described in U.S. Patent No. 9683230, which is incorporated entirely herein. Therefore, the second tagmentation reaction leaves short, surface-fixed cuts that can be converted into clusters ready for further sequencing steps, by removing the internal spans of the bridges. In certain embodiments, the template length may be within a range defined by upper and lower limits selected from those illustrated above.

[0303] In some embodiments, sequences that can hybridize to a capture probe can be incorporated using transposition in solution (i.e., using a liquid-phase transposome complex). Thus, tagmentation can be used to generate fragments that can specifically bind to a solid support containing the capture probe on its surface. In some embodiments, a DNA:RNA double helix generated from cDNA or RNA is tagged in solution to incorporate a tag containing a sequence that can hybridize to a capture probe. After this tagmentation, the fragment generated from cDNA or DNA:RNA double helix can bind to a solid support containing the capture probe. In some embodiments, the capture probe contains a P5 or P7 sequence (or their complements).

[0304] C. Beads containing captured oligonucleotides In some embodiments, the capture oligonucleotide is immobilized on a solid support. In some embodiments, the 3' end of the target RNA is bound to the capture oligonucleotide. In some embodiments, the capture oligonucleotide may function to immobilize the target RNA on a solid support. An exemplary workflow using beads containing a capture oligonucleotide for capturing mRNA is shown in Figure 20.

[0305] In some embodiments, the capture probe includes a poly-T array.

[0306] In some embodiments, the target RNA is mRNA, which binds to a capture oligonucleotide containing a poly-T sequence.

[0307] In some embodiments, the captured oligonucleotide does not contain a poly-T sequence.

[0308] In some embodiments, the captured oligonucleotide is immobilized on the beads via a P5 or P7 sequence.

[0309] In some embodiments, the captured oligonucleotide further includes a bead code or other types of barcode. As used herein, “bead code” is a nucleic acid sequence present on a bead that is distinct from all or most other beads in the pool of beads. In some embodiments, the bead code can be used to identify fragments generated on the same bead.

[0310] In some embodiments, the sequence contained in the captured oligonucleotide is incorporated into the cDNA during synthesis.

[0311] In some embodiments, the captured oligonucleotide includes a tag that is also present in the first tag contained in the first polynucleotide of the immobilized transposome.

[0312] In some embodiments, the solid support does not include capture oligonucleotides for immobilizing target nucleic acids. For example, double-stranded DNA and DNA:RNA double helix can be immobilized on the solid support via binding to an immobilized transposome complex.

[0313] D. Polynucleotide and immobilized transposome complex In some embodiments, the transposome complex composition comprises or consists of at least one transposon having one or more other nucleotide sequences in addition to the transposon sequence. Such nucleotide sequences may be referred to as polynucleotides.

[0314] In the methods and compositions presented herein, transposomal complexes are immobilized on a solid support. In some embodiments, the transposomal complex is immobilized on the support via one or more polynucleotides, such as polynucleotides containing transposon terminal sequences. In some embodiments, the transposomal complex may be immobilized via a linker that binds a transposase enzyme to the solid support. In some embodiments, both the transposase enzyme and the polynucleotide are immobilized on the solid support. When referring to the immobilization of molecules (e.g., nucleic acids) on a solid support, the terms “immobilized” and “bound” are used interchangeably herein, and both terms are intended to encompass direct or indirect, covalent or non-covalent bonding unless otherwise indicated either explicitly or by context. In some embodiments, covalent bonding may be used, but generally, what is required is that the molecule (e.g., nucleic acid) remains immobilized or bound to the support under conditions in which the support is intended to be used, for example in applications requiring nucleic acid amplification and / or nucleic acid sequencing.

[0315] In some embodiments, the transpososome complex comprises a transposase conjugated to a first polynucleotide, the first polynucleotide comprising a 3' portion containing the transposon terminal sequence and a first tag. In some embodiments, the first tag is a DNA-specific barcode.

[0316] In some embodiments, the transpososome complex comprises a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion containing the transposon terminal sequence and a second tag. In some embodiments, the second tag comprises an RNA-specific barcode.

[0317] Therefore, in some embodiments, the transposon composition includes a transposition chain having one or more other nucleotide sequences 5' of the transposition transposon sequence, for example, a tag sequence. In addition to the transposition transposon sequence, the tag may have one or more other tag portions or tag domains.

[0318] "Tagmentation" refers to the use of transposases on fragments and tagged nucleic acids. Tagmentation involves the modification of DNA by a transposomal complex containing a transposase enzyme complexed with one or more tags (such as adapter sequences) containing transposon terminal sequences (hereinafter referred to as transposons). Tagmentation can result in both DNA fragmentation and ligation of adapters to the 5' ends of both strands of a double-stranded fragment.

[0319] In some embodiments, the transposome complex is immobilized on a solid support via a first polynucleotide.

[0320] In some embodiments, the transposome complex includes a second polynucleotide containing a region complementary to the transposon terminal sequence. In some embodiments, the transposome complex is immobilized on a solid support via the second polynucleotide.

[0321] In some embodiments, the transposome complex is 1 mm 2 At least 10 3 , 10 4 , 10 5 , or 10 6 They exist on a solid support at a density of individual composites.

[0322] 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 a solid support.

[0323] Several different types of immobilized transposomes can be used in these ways, as described in U.S. Patent No. 9,683,230, which is incorporated in its entirety herein.

[0324] E. Spatially separated capture of nucleic acids on beads In some embodiments, a solid support containing a capture oligonucleotide is used to capture nucleic acids. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, a capture oligonucleotide containing a polyT sequence is used to capture mRNA.

[0325] In some embodiments, RNA is captured on capture beads. In some embodiments, RNA is captured on RNA-BLT.

[0326] In some embodiments, the RNA is captured on a solid support within a droplet. In some embodiments, the application of the sample containing the target RNA to the solid support is performed within the droplet.

[0327] In some embodiments, the DNA is captured on a solid support within a droplet. In some embodiments, the application of the sample containing the target DNA to the solid support is performed within the droplet.

[0328] 1.Droplet In some embodiments, applying a sample containing target RNA to a solid support includes providing a single cell in a droplet along with beads, lysing the cell in the droplet, releasing the target RNA from the single cell, and capturing the target RNA on the beads. In some embodiments, the droplet is removed before synthesizing cDNA. Various methods using droplets are known in the art, such as in International Publication Nos. 2015 / 168161 and 2017 / 040306, each of which is incorporated herein in whole. Target DNA from a sample may similarly be captured on beads (such as BLTs), and then the droplet is removed.

[0329] In some embodiments, after the capture of RNA in a droplet, cDNA synthesis is performed on beads, in bulk, to produce the first strand of cDNA (i.e., to produce a DNA:RNA double helix). As used herein, “in bulk” is used to indicate that the step is performed on beads, but these beads are in solution and not separated by a droplet. Thus, if the step of the method is performed in bulk after the capture of nucleic acids onto beads, these immobilized nucleic acids are not in solution but remain immobilized on the beads. In general, all methods described herein allow for the capture of RNA or DNA in a droplet, but subsequent steps may be performed either in the droplet or in bulk. In some embodiments, tagmentation is performed in bulk.

[0330] For example, Figure 5 shows a typical example in which a bead code (i.e., a barcode) is incorporated during the synthesis of the first strand of cDNA for preparing a DNA:RNA double helix in a droplet. The bead code is contained in a capture oligonucleotide that also includes a poly-T sequence (for binding to the poly-A tail of mRNA) and a P5 adapter sequence that can be used to immobilize the fragment on a solid surface. The immobilized DNA:RNA double helix can be tagged in bulk, followed by strand exchange and ligation. Adapters (such as B' and P7') can be added to the free ends (not bound to beads) of the fragment using tagging in solution or PCR.

[0331] 2. Barcoding within liquid droplets In some embodiments, barcoding may be performed by separating individual cells into droplets and incorporating bead codes. In some embodiments, bead codes can be incorporated into library fragments based on the bead codes contained in the beads within the droplets. In some embodiments, droplets are used to spatially separate the sample.

[0332] In some embodiments, the BLT includes an immobilized oligonucleotide containing a bead cord. In some embodiments, the immobilized oligonucleotide includes a first transposon containing a bead cord. In some embodiments, the immobilized oligonucleotide includes a hybridization sequence for binding to the bead cord and a second transposon.

[0333] In some embodiments, the bead code can be incorporated into cDNA generated from RNA derived from a sample. In some embodiments, the bead code can be incorporated into a library fragment during tagmentation.

[0334] In some embodiments, droplets are separated from each other in an emulsion. In some embodiments, droplets are formed and / or manipulated using a droplet actuator. In some embodiments, one or more droplets contain different sets of first chain synthesis primers containing barcodes. In some embodiments, each droplet contains a number of first chain synthesis primers, each of which has the same sequence containing the same barcode, and the barcode from one droplet is different from that of another, but the rest of the first chain synthesis primers remain the same across droplets. Thus, in these embodiments, the barcodes serve as identifiers for the droplets and the single cells contained within them.

[0335] In some embodiments, one or more droplets contain different sets of UMI-containing first chain synthesis primers. Thus, each individual cell lysed in each droplet is identifiable by the barcode in each droplet. In some embodiments, droplet-based barcoding can be performed by merging a droplet containing a single cell with other droplets containing a unique set of barcodes. This format allows for additional multiplexing beyond what is available in the multi-wall format. The first chain synthesis and template switching take place within each individual droplet. Additionally or alternatively, in some embodiments, droplets may be merged before tagmentation. Additionally or alternatively, in some embodiments, droplets may be merged after PCR and before tagmentation. For example, in some embodiments, tagged cDNA can be merged after the first chain synthesis has taken place in individual droplets, and thus the cDNA can be pooled.

[0336] In some embodiments, sample and UMI barcoding can be performed by separating individual cells using beads having UMI and / or barcode-tagged primers for first chain synthesis. In some embodiments, the beads are separated into droplets in an emulsion. In some embodiments, the beads are separated and manipulated using a droplet actuator. In some embodiments, bead-based barcoding can be performed by preparing a set of beads, each having its own set of barcodes.

[0337] 3. Spatial separation using nucleic acid capture on BLT in droplets In some embodiments, RNA is captured on a BLT in a droplet, followed by cDNA preparation and fragmentation, after which the fragments are released from the BLT. Cells and capture beads can be co-encapsulated in a droplet, followed by lysis and mRNA capture in the droplet. Then, cDNA synthesis, tagmentation, and ligation can also be performed in bulk, and the resulting library fragments are retained on the beads. A typical method is shown in Figure 7, where the BLT is an activatable BLT, and the transposome is assembled after bulk cDNA synthesis.

[0338] In some embodiments, DNA is captured on a BLT in a droplet, and fragmentation occurs before the fragments are released from the BLT.

[0339] In some embodiments, library fragments are retained on beads due to the association of the fragment with a transposase (contained in an immobilized transpososome complex on the BLT). In some embodiments, the fragments remain immobilized on the beads until a protease or SDS is added to release the fragment from the transposase. In some embodiments, the beads with the immobilized library fragments are delivered to a solid support (such as a flow cell) for sequencing, and the library is released from the beads. Such release from beads captured on a flow cell allows for "on-flow cell spatial reads," as shown in Figure 4, where fragments from a single bead are released in close proximity to each other. In this way, fragments that are spatially close on the flow cell can be determined to be likely to originate from nucleic acids from the same cell.

[0340] In some embodiments, the capture of nucleic acids in droplets and subsequent library preparation on BLTs allows for the separation of fragments from different cells, even in the absence of barcoding.

[0341] 4. Spatial isolation using partitioned solid supports In some embodiments, compartmentalization allows for the spatial separation of library fragments for sequencing. In some embodiments, compartmentalization allows fragments from the original DNA or RNA in the sample to be in close proximity after library preparation. In some embodiments, sequencing data and proximity data can be used together to determine which fragments were present in the starting DNA or RNA molecule in the sample.

[0342] In some embodiments, compartmentalization is performed using a solid support containing microwells. In some embodiments, single cells are compartmentalized within the microwells. In some embodiments, the sample can be diluted using cell density and volume calculations so that most cells are compartmentalized within microwells that do not contain other cells.

[0343] Figure 8 shows a typical example where poly-T RNA capture can be performed on a flow cell containing microwells that allow for the capture of single cells per microwell.

[0344] In some embodiments, the application of a sample containing target RNA to a solid support is performed in microwells on the solid support. In some embodiments, the application of a sample containing target RNA to a solid support includes lysing cells and releasing target RNA from single cells in microwells. In some embodiments, the method further includes releasing an immobilized library of DNA:RNA fragments and sequencing the fragments in the same microwells. Thus, fragments localized in a given microwell can be characterized as originating from a single cell. In some embodiments, sequencing data enables resolution of fragments immobilized on the same solid support based on the spatial proximity of the fragments on the surface for sequencing.

[0345] In some embodiments, the solid support is a flow cell containing microwells.

[0346] In some embodiments, the flow cell containing microwells includes a polymer coating. In some embodiments, the flow cell is coated with a covalently bonded polymer. In some embodiments, the covalently bonded polymer is PAZAM. In some embodiments, the polymer coating includes reaction sites for reacting with oligonucleotides. Such covalently bonded polymers are described in International Publication No. 2013 / 184796, which is incorporated herein by reference in whole. In some embodiments, polymers such as PAZAM are crosslinked using ultraviolet light.

[0347] The method using a flow cell containing microwells may also use special hybridization buffers and adapter blockers, as described in International Publication No. 2020 / 036991, which is incorporated herein by reference in its entirety.

[0348] F. Solid support In the methods and compositions presented herein, the transposome complex is immobilized on a solid support. In some embodiments, the transposome complex and / or the capture oligonucleotide is immobilized on the support via one or more polynucleotides, such as a polynucleotide containing the transposon terminal sequence. In some embodiments, the transposome complex may be immobilized via a linker that binds a transposase enzyme to the solid support. In some embodiments, both the transposase enzyme and the polynucleotide are immobilized on the solid support. When referring to the immobilization of molecules (e.g., nucleic acids) on a solid support, the terms “immobilized” and “bound” are used interchangeably herein, and both terms are intended to encompass direct or indirect, covalent or non-covalent bonding unless otherwise indicated either explicitly or by context. In some embodiments, covalent bonding may be used, but generally, what is required is that the molecule (e.g., nucleic acid) remains immobilized or bound to the support under conditions in which the support is intended to be used, for example in applications requiring nucleic acid amplification and / or nucleic acid sequencing.

[0349] Certain embodiments may utilize a solid support comprising an inert substrate or matrix (e.g., a glass slide, polymer beads, etc.) functionalized by the application of a layer or coating of an intermediate material containing reactive groups that enable covalent bonding 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 the polyacrylamide hydrogels described in International Publication No. 2005 / 065814 and U.S. Patent Application Publication No. 2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecules (e.g., polynucleotides) may be directly covalently attached to the intermediate material (e.g., hydrogel), or the intermediate material may be noncovalently bonded to the substrate or matrix (e.g., a glass substrate) itself. The term “covalent bonding to a solid support” should be interpreted as appropriate to encompass this type of arrangement.

[0350] The terms “solid surface,” “solid support,” and other grammatical equivalents herein refer to any material suitable for, or that can be modified to be suitable for, the binding of transposome complexes. As will be understood 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., acrylic, 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, optical fiber bundles, and various other polymers. In some embodiments, particularly useful solid supports and solid surfaces are placed within a flow cell apparatus. Exemplary flow cells are described in further detail below.

[0351] In some embodiments, the solid support includes a patterned surface suitable for immobilizing transposome complexes in a regular pattern. “Patterned surface” refers to an arrangement of different regions within or on the exposed layer of the solid support. For example, one or more of the regions may be features containing one or more transposome complexes. This feature may be separated by interstitial regions where transposome complexes are not present. In some embodiments, the pattern may be an xy format of features in rows and columns. In some embodiments, the pattern may be a repeating arrangement of features and / or interstitial regions. In some embodiments, the pattern may 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 may be used in the methods and compositions described herein are described in U.S. Patent Application No. 13 / 661524 or U.S. Patent Publication No. 2012 / 0316086(A1), each of which is incorporated herein by reference.

[0352] In some embodiments, the solid support comprises an array of wells or depressions on its surface. This can be processed using a variety of techniques, including but not limited to photolithography, stamping, molding, and microetching, as is generally known in the art. As is understood in the art, the technique used depends on the composition and shape of the array substrate.

[0353] The composition and geometric shape of the solid support may vary depending on its use. In some embodiments, the solid support is a planar structure such as a slide, chip, microchip, and / or array. Thus, the surface of the substrate may be in the form of a planar layer. In some embodiments, the solid support includes one or more surfaces of a flow cell. As used herein, the term “flow cell” refers to a chamber containing a solid surface through which one or more fluid reagents can flow. Examples of flow cells and associated fluid systems and detection platforms readily usable in the methods of this disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), International Publication No. 04 / 018497, U.S. Patent No. 7,057,026, International Publication No. 91 / 06678, International Publication No. 07 / 123744, U.S. Patent No. 7,329,492, International Publication No. 7,211,414, International Publication No. 7,315,019, International Publication No. 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0354] In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the solid support comprises microspheres or beads. "Microsphere," "beads," "particles," or grammatical equivalents as used herein mean small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, triasol (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 materials outlined herein for the solid support. Bangs Laboratories, Fishers, Ind.'s "Microsphere Selection Guide" is a useful guide. In certain embodiments, the microspheres are magnetic microspheres or beads.

[0355] The beads do not need to be spherical. Irregular particles may be used. Alternatively or additionally, the beads may be porous. The bead size ranges from nanometers, i.e., 100 nm, to millimeters, i.e., 1 mm, and the beads are approximately 0.2 microns to approximately 200 microns, or approximately 0.5 to approximately 5 micrometers, although in some embodiments smaller or larger beads may be used.

[0356] The density of these surface-bound transpososomes can be adjusted by changing the density of the first polynucleotide or by the amount of transposase added to the solid support. For example, in some embodiments, the transpososome complex is 1 mm 2 At least 10 3 , 10 4 , 10 5 , or 10 6 They exist on a solid support at a density of individual composites.

[0357] The binding of nucleic acids to a support, whether rigid or semi-rigid, can occur via covalent or non-covalent bonds. Exemplary bindings are described in U.S. Patents 6,737,236, 7,259,258, 7,375,234, and 7,427,678, and U.S. Patent Publication 2011 / 0059865, each incorporated herein by reference. In some embodiments, nucleic acids or other reactive components may be bound to a gel or other semi-solid support, which is then bound or adhered to a solid-phase support. In such embodiments, the nucleic acids or other reactive components are understood to be the solid phase.

[0358] In some embodiments, the solid support includes microparticles, beads, a planar support, a patterned surface, or a well. In some embodiments, the planar support is the inner or outer surface of a tube.

[0359] In some embodiments, a solid support has a library of tagged RNA fragments immobilized and prepared thereon. In some embodiments, a solid support has a library of tagged DNA fragments immobilized and prepared thereon. In some embodiments, two or more solid supports are used to generate a library of tagged RNA fragments on one solid support and a library of tagged DNA fragments on another solid support.

[0360] In some embodiments, the solid support comprises an immobilized capture oligonucleotide and a first polynucleotide, the first polynucleotide comprising a 3' portion containing the transposon terminal sequence and a first tag. In some embodiments, the first tag is a DNA-specific barcode. In some embodiments, this solid support is for tagging DNA and is referred to as a DNA bead-linked transposome (DNA BLT).

[0361] In some embodiments, the solid support further comprises a transposase conjugated to a first polynucleotide to form a transposomal complex.

[0362] In some embodiments, the solid support comprises an immobilized capture oligonucleotide and a second polynucleotide, the second polynucleotide comprising a 3' portion containing the transposon terminal sequence and a second tag. In some embodiments, the second tag comprises an RNA-specific barcode. In some embodiments, this solid support is for tagging nucleic acids generated from RNA (either ds-cDNA or DNA:RNA double helix) and is referred to as an RNA bead-linked transposome (RNA BLT).

[0363] In some embodiments, the solid support further comprises a transposase conjugated to a second polynucleotide to form a transposomal complex.

[0364] In some embodiments, the solid support comprises a library of tagged fragments immobilized thereon, prepared according to one of the methods described herein.

[0365] In some embodiments, the kit includes a solid support as described herein. In some embodiments, the kit further includes a transposase. In some embodiments, the kit further includes a reverse transcriptase polymerase. In some embodiments, the kit further includes a second solid support for immobilizing DNA, comprising a second transpososome complex comprising a transposase, and a third polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a second tag.

[0366] G.DNA BLT and RNA BLT In some embodiments, these methods utilize BLTs (bead-linked transposomes). Transpososomes bound to a surface (e.g., BLTs) can tagment long molecules of double-stranded DNA, allowing for the creation of template libraries on beads or other surfaces (U.S. Patent No. 9683230). Immobilizing transpososomes on beads yields novel properties such as controllable insertion size and yield. This forms the basis of the Illumina DNA Flex PCR-Free technology, formerly known as Illumina's Nextera technology. BLTs capable of tagging double-stranded DNA may be referred to as DNA-BLTs.

[0367] Because transposon ends have an affinity for DNA, DNA BLTs do not require capture oligonucleotides for immobilization onto beads; instead, DNA can be immobilized using polynucleotides containing the transposon end sequence. Alternatively, capture oligonucleotides can be used to capture DNA molecules.

[0368] In some embodiments, a solid support for immobilizing DNA includes a first transposome complex immobilized thereon. In some embodiments, the first transposome complex includes a transposase and a first polynucleotide, the first polynucleotide including a 3' portion containing the transposon terminal sequence. In some embodiments, the first polynucleotide further includes a first tag. In some embodiments, the first tag is a DNA-specific barcode.

[0369] In some embodiments, these methods utilize RNA BLTs. As used herein, “RNA BLT” refers to a BLT used to prepare tagged fragments derived from RNA in a sample. Thus, RNA BLTs can produce fragments from nucleic acids produced from RNA. In some embodiments, RNA BLTs produce fragments from ds-cDNA produced from RNA (after the synthesis of two strands of cDNA). In some embodiments, RNA BLTs produce fragments from DNA:RNA double helix (only a single strand of cDNA is produced). In some embodiments, as shown in Figures 26–28, RNA BLTs function to incorporate RNA-specific barcodes, while DNA BLTs function to incorporate DNA-specific barcodes.

[0370] In some embodiments, a solid support for immobilizing nucleic acids generated from RNA (e.g., ds-cDNA or DNA:RNA double helix) includes a second transpososome complex immobilized thereon. In some embodiments, the second transpososome complex includes a transposase and a first polynucleotide, the first polynucleotide including a 3' portion containing the transposon terminal sequence. In some embodiments, this transposase has increased activity for generating fragments from DNA:RNA double helix. In some embodiments, the first polynucleotide further includes a second tag. In some embodiments, the second tag includes an RNA-specific barcode.

[0371] 1. Activatable BLT In some embodiments, beads can be used to allow a user to generate activated transpososomes at select time. For example, beads that allow a user to capture transpososomes from a solution at select time may be referred to as “activatable BLTs.” A common embodiment of activatable BLTs is that they can be applied to a sample in a state in which they cannot fragment nucleic acids (e.g., DNA:RNA double helix or double-stranded cDNA), but the user can activate the BLTs at select time. In this way, the user controls the timing of tagmentation in a multi-step manner. A range of different types of activatable transpososomes are described herein and can be used in different ways. Figures 11A–11C show beads containing a capture oligonucleotide and an oligonucleotide that can bind to transpososomes from solution via a short A sequence. Figure 12 shows an embodiment in which first and second transposons are bound to the beads via the short A sequence.

[0372] In some embodiments, activatable BLTs can avoid undesirable tagmentation. For example, the use of activatable BLTs can allow the user to ensure that cDNA synthesis is complete (e.g., the generation of a DNA:RNA double helix) before tagmentation by transpososomes. In this way, the user can avoid fragmentation of "partial" DNA:RNA double helixes (i.e., tagmentation of an incomplete DNA:RNA double helix where the cDNA is generated from only a portion of the RNA).

[0373] Activatable BLTs offer several advantages, including allowing users to control the timing of tagging in multi-step methods. In some embodiments, activatable BLTs can sequence the tagging process of cDNA or DNA:RNA double helix generated from sample-derived DNA versus sample-derived RNA. Such sequencing can enable tagging of DNA-derived fragments versus RNA-derived fragments.

[0374] Activatable BLTs can be used in any of the methods described herein, together with transposomes immobilized on a solid support (e.g., beads). In some embodiments, the activatable BLT is DNA-BLT or RNA-BLT.

[0375] In some embodiments, the activatable RNA BLT is for capturing RNA, preparing DNA:RNA double helixes, and then tagging the DNA:RNA double helixes. In some embodiments, the activatable BLT includes an immobilized polyT sequence for capturing mRNA.

[0376] In some embodiments, the solid support comprises a capture oligonucleotide and an immobilized oligonucleotide, the immobilized oligonucleotide comprising a sequence for hybridizing to a hybridization sequence contained in a second transposon contained in a transposome complex. In some embodiments, the solid support is a bead. In some embodiments, the immobilized oligonucleotide further comprises a bead code and / or one or more adapter sequences. In some embodiments, the beads are contained in a pool of beads, and each bead comprises an immobilized oligonucleotide comprising a different bead code compared to the bead code contained in the immobilized oligonucleotide contained in the other beads in the pool.

[0377] In some embodiments, the activatable BLT comprises an immobilized oligonucleotide capable of capturing transpososomes from solution. In some embodiments, the activatable BLT comprises an immobilized oligonucleotide comprising a hybridization sequence capable of binding to transposons contained in the transpososome complex.

[0378] In some embodiments, an immobilized oligonucleotide capable of capturing transpososomes from solution includes an adapter sequence (such as P5 or P7, or their complements), a bead barcode, and a sequence for hybridizing to a sequence contained in a second transposon of the transpososome complex. As shown in representative examples in Figures 6-7, the immobilized oligonucleotide may include P5, a bead barcode (BC), and a sequence (short A) for hybridizing to a sequence (A') contained in a second transposon (the second transposon contains A'-ME'). The immobilized oligonucleotide may also include a sequencing adapter sequence (e.g., A14 or B15, or their complements). In this way, the user can capture mRNA, prepare a DNA:RNA double helix on a bead, and then hybridize the transpososome to the bead to prepare a cDNA fragment on the same bead. The use of an activatable BLT means that the user can prepare a full-length DNA:DNA double helix before preparing a DNA fragment. In this way, partial DNA:RNA double helix fragmentation can be avoided because the user can optimize the DNA:RNA double helix conditions and has no concerns about fragmentation starting before the synthesis of the first strand of cDNA is complete.

[0379] In some embodiments, the activatable RNA-BLT comprises an immobilized polyT sequence for capturing mRNA and an immobilized oligonucleotide for capturing transposomes from solution. In some embodiments, such an activatable BLT can capture mRNA and enable the preparation of DNA:RNA double helix on beads, after which the transposomes can be hybridized to the immobilized oligonucleotide, enabling tagmentation. In some embodiments, only one type of transposome is hybridized to the immobilized oligonucleotide, thus enabling symmetric tagmentation on the BLT.

[0380] 2. BLT for symmetric tagging In some embodiments, all transposome complexes contain the same sequencing adapter sequence. In some embodiments, the first transposon contained in all transposome complexes is identical. In some embodiments, all transposome complexes immobilized on beads (such as on DNA-BLT or RNA-BLT) contain the same first and second transposons. The use of such transposome complexes, in which the same adapter sequence can be attached to both ends of a fragment, can be referred to as "symmetric tagging" because these transposome complexes result in the same adapter being attached to the 5' ends of both strands of the double-stranded fragment produced by tagging.

[0381] In some embodiments, fragmentation of DNA:RNA double helix using a transposome complex is performed using a transposome complex containing a first transposon with the same adapter sequence. In some embodiments, all transposome complexes are identical.

[0382] In some embodiments, a BLT for preparing an RNA sequencing library includes a transposome complex containing the same one or more adapter sequences. In some embodiments, all transposons in the transposome complexes included in the pool of BLTs are identical. In some embodiments, a BLT containing the same one or more adapter sequences means that both ends of a double-stranded cDNA fragment are tagged with the same adapter sequence. In some embodiments, both 5' ends of a double-stranded cDNA fragment of DNA:RNA double-stranded DNA incorporate the same one or more adapters. In some embodiments, both ends of the double-stranded cDNA contain the same 5' tag.

[0383] In some embodiments, methods using a symmetric tagmentation step increase the yield of sequenceable fragments (i.e., each fragment having a different sequencing adapter sequence at each end) compared to standard asymmetric tagmentation steps where two or more types of transposome complexes are used for tagmentation. Uncontrolled tagmentation using two types of transposomes with different tags (e.g., A and B, with a first read sequencing adapter and a second read sequencing adapter) results in the loss of nearly half of the reads from the amplified tagmentation product, as control and uncontrolled tagged products (AA, BB, AB, BA) are produced, but only AB and BA are suitable for subsequent amplification and sequencing. In contrast, current methods using BLT for symmetric tagmentation can increase the probability that the resulting fragments contain both the first read and the second read sequencing adapter.

[0384] In some embodiments, methods using symmetric tagging can increase library yield compared to other library preparation methods.

[0385] Several different methods for adding a second adapter after tagmentation are described herein. For example, the first sequencing adapter may be incorporated into double-stranded DNA or DNA:RNA double helix during tagmentation, and the second read sequencing adapter may be incorporated in a later step (e.g., by ligation). Exemplary methods are described herein. In some embodiments, the method can improve library yield (compared to methods using asymmetric tagmentation) by incorporating one sequencing adapter sequence by symmetric tagmentation and another sequencing adapter sequence via the use of a primer or oligonucleotide containing a second sequencing adapter sequence.

[0386] 3. BLT for Asymmetric Tag Mention In some embodiments, the fragmentation of DNA:RNA double helix using a transposome complex is performed using two different transposome complexes, each containing a first transposon with a different adapter sequence.

[0387] The use of two different transposomal complexes, each containing a first transposon with a different adapter sequence, can be referred to as "asymmetric tagging" because these transposomal complexes may result in different adapters being attached to the 5' ends of the two strands of the double-stranded fragment produced by tagging. In some embodiments, asymmetric tagging can enable a faster or easier workflow by incorporating two different adapters during the tagging process.

[0388] In some embodiments, at least some fragments are tagged with a first read sequence adapter sequence at the 5' end of one strand and with a second read sequence adapter sequence at the 5' end of the other strand using asymmetric tagging.

[0389] 4. BLT containing captured oligonucleotides In some embodiments, the solid support comprises transposomes and captured oligonucleotides. In some embodiments, the solid support is a bead. In some embodiments, the bead comprises a captured oligonucleotide and a first polynucleotide that may be contained within the transposome.

[0390] In some embodiments, the first polynucleotide further comprises a bead cord. In some embodiments, the beads are contained in a pool of beads, and each bead contains an immobilized first polynucleotide which comprises a different bead cord compared to the bead cords contained in the other beads in the pool.

[0391] In some embodiments, the BLT further comprises a capture oligonucleotide, such beads may be used to capture RNA and subsequently prepare a first strand of cDNA to generate immobilized DNA:RNA double helix and beads, and finally to prepare immobilized fragments from the DNA:RNA double helix. In some embodiments, the capture oligonucleotide is a poly-T sequence for capturing mRNA, and the beads allow for the preparation of the library on the same beads used to capture the mRNA. In any of these embodiments, a second strand of cDNA may also be prepared after mRNA capture to prepare double-stranded cDNA.

[0392] In some embodiments, the BLT containing the captured oligonucleotide is an activatable BLT containing an immobilized oligonucleotide that includes a hybridization sequence which can be used to hybridize to a transposome complex.

[0393] Figures 11A–11C show the preparation of DNA:RNA double helix (Figure 11A), hybridization of transposons to immobilized oligonucleotides containing hybridization sequences for the formation of a transpososome complex (Figure 11B), and multiple tagmentation reactions mediated by the transpososome complex (Figure 11C). After the preparation of multiple fragments by tagmentation, additional adapters can be incorporated and sequenceable fragments prepared using the methods described herein (e.g., primer extension after symmetric tagmentation).

[0394] Figure 12 shows beads for a full-length mRNA preparation having multiple capture oligonucleotides and multiple immobilized oligonucleotides for immobilizing transposomes. Figure 13 shows how multiple transposomes can enable the preparation of fragments from full-length DNA of DNA:RNA double helix generated from mRNA.

[0395] Delivery of library fragments to the surface for sequencing by H.BLT In some embodiments, a solid support with immobilized library fragments is used to deliver the library fragments to a surface for sequencing. In some embodiments, the surface for sequencing is a flow cell. Figures 9 and 10 summarize how tagmentation and library preparation may be performed on a BLT and how the fragments may then be released in a flow cell or tube. Figure 9 uses poly-T primers to prepare a library from full-length mRNA, while Figure 10 uses random primers to prepare full-length total RNA for the library.

[0396] In some embodiments, a library of fragments on a solid support is delivered to a surface for sequencing, while the fragments are immobilized on the solid support and then released and captured on the surface for sequencing. In some embodiments, a solid support having immobilized nucleic acids is delivered to a surface for sequencing, and a library of immobilized fragments is generated on the solid support by tagmentation, and then the fragments are released and captured on the surface for sequencing.

[0397] In some embodiments, the method includes, after delivery, capturing a solid support with an immobilized library of DNA:RNA fragments on the surface for sequencing, releasing the immobilized fragments from the solid support, and capturing the fragments on the surface for sequencing. In some embodiments, the method further includes sequencing the fragments on the surface for sequencing.

[0398] In some embodiments, BLT can be used as a solid-phase support. An exemplary use of BLT as a solid-phase support is described in International Publication No. 2015 / 095226, which is incorporated herein by reference in its entirety. In some embodiments, fragments released from the BLT can be recaptured on a region of the surface adjacent to the area where the beads were captured on the surface for sequencing. In this way, all fragments from a given bead are captured in close proximity to the surface for sequencing.

[0399] In some embodiments, BLTs having immobilized library fragments can be delivered to a surface for sequencing. In some embodiments, the DNA or DNA:RNA double helix on the surface of the BLT may already be tagged before the BLT is delivered to the surface for sequencing. For example, tagging of the BLT may occur in a reaction vessel, the BLT is then delivered to a flow cell, and the fragments are then released and captured on the flow cell.

[0400] In some embodiments, BLTs having immobilized nucleic acids can be delivered to a surface for sequencing. Following this delivery, library fragments can be generated. For example, double-stranded DNA or DNA:RNA double helix can be immobilized on the surface of the BLT, and tagmentation occurs after the BLT has been delivered to the surface for sequencing. For example, tagmentation of the BLT can be performed using a flow cell, after which the fragments are released and captured on the flow cell.

[0401] The release of fragments from BLTs can occur by several means. For example, fragments may be released by protease or SDS treatment. Alternatively, fragments may be produced by disrupting the beads that release fragments from the BLTs. In any of these mechanisms for fragment release, fragments from a given bead may be released and captured onto a nearby sequencing surface (such as a flow cell) to help prepare a physical map of the sequence of the original DNA or RNA molecule.

[0402] In some embodiments, the BLT may include hydrogel beads. In some embodiments, the hydrogel beads can be melted or dissolved to release the fragments bound to the beads, such as in the methods disclosed in International Publication Nos. 2019 / 028047 and 2019 / 028166, which are incorporated entirely herein.

[0403] In some embodiments, the BLT may comprise biodegradable polyester beads. In some embodiments, the polyester beads can be degraded to release fragments bound to the beads, such as in the method disclosed in international application PCT / US2021 / 040612, which is incorporated herein in whole. For example, each transposome complex may comprise a polynucleotide binding moiety that enables the binding of a polynucleotide to another drug. In some embodiments, the polynucleotide binding moiety functions to bind a polynucleotide to a bead comprising a bead binding moiety. In some embodiments, each bead binding moiety is covalently bound to the polyester bead via a linker. In some embodiments, the linker comprises -N=CH-(CH2)3-CH=N-, -C(O)NH-(CH2)6-N=, or -C(O)NH-(CH2)6-N=CH-(CH2)3CH=N-.

[0404] In some embodiments, the biodegradable polyester beads decompose at temperatures above 50°C, above 60°C, above 70°C, or above 80°C. In some embodiments, the biodegradable polyester beads decompose at a temperature of 60°C. In some embodiments, the biodegradable polyester beads decompose with an aqueous base. In some embodiments, the aqueous base is NaOH. In some embodiments, the NaOH is 1M to 5M NaOH. In some embodiments, the NaOH is 3M NaOH (see Yeo et al., J Biomed Mater Res B Appl Biomater 87(2):562-9 (2008)). In some embodiments, the biodegradable polyester beads decompose with an aqueous NaOH solution at temperatures between 50°C and 90°C.

[0405] In some embodiments, BLTs having immobilized nucleic acids or fragments thereof can be brought into contact with a surface for sequencing by gravity sedimentation. In some embodiments, BLTs having immobilized nucleic acids or fragments thereof can be bound to a surface for sequencing using receptors and ligands.

[0406] In some embodiments, since all fragments from DNA or cDNA generated from RNA are produced on the same beads, proximity data can be used together with sequencing data on the fragments to generate information about a given DNA or RNA contained in the sample. The use of proximity data to prepare a physical map of immobilized polynucleotides can be carried out using the methods described herein.

[0407] I. Primer extension after symmetric tagging of BLT In some embodiments, symmetric tagging of BLT results in both ends of a double-stranded DNA fragment having the same one or more adapter sequences.

[0408] In some embodiments, the non-transferred ME' sequence (from the second transposon) is thawed and gap-filled by PCR extension after tagmentation. In some embodiments, the ME' sequence is removed by increasing the reaction temperature.

[0409] In some embodiments, gap filling is performed after the non-transitioned ME' sequence is removed. In some embodiments, a primer is annealed to the gap-filled ME' sequence. In some embodiments, this primer is used for extension and may be referred to as an extension primer. In some embodiments, the extension primer contains an ME sequence. In some embodiments, the ME sequence contained in the extension primer hybridizes to the gap-filled ME' sequence.

[0410] In some embodiments, the extension primer also includes a sequencing adapter sequence. In some embodiments, the sequencing adapter sequence included in the extension primer was not included in the transposome complex. In some embodiments, extension by the extension primer generates a fragment in which each end of the double-stranded fragment contains a different sequencing adapter sequence.

[0411] In some embodiments, uracil-intolerant DNA is used for primer extension. In some embodiments, the second strand of cDNA is not extended because it contains uracil, based on the strand-specific cDNA preparation described above.

[0412] In some embodiments, if the transposome complex contains the A14 sequence (or its complement), the extension primer contains the B15 sequence (or its complement). In some embodiments, if the transposome complex contains the B15 sequence (or its complement), the extension primer contains the A14 sequence (or its complement). In these representative examples, A14 and A15 represent exemplary sequencing adapter sequences, and the method is not limited to such adapter sequences. Any set of paired adapter sequences of interest may be used in the transposome complex and extension primer, and those skilled in the art are well aware of how sequencing is performed on different platforms and how such platforms may evolve over time.

[0413] 1. Saturation of DNA BLT by synthetic DNA In some embodiments, after a DNA BLT (which may be used as a first solid support in some methods) is used to perform tagmentation to generate a fragment containing a DNA-specific barcode, the DNA BLT is bound with synthetic double-stranded DNA. In some embodiments, the DNA BLT is saturated with synthetic double-stranded DNA. In some embodiments, the method includes adding synthetic double-stranded DNA to the first solid support after performing tagmentation on the first solid support.

[0414] In some embodiments, the DNA BLT cannot bind to (and tag) nucleic acids after this saturation. Such saturation of the DNA BLT may allow for a stepwise method of subsequently generating either cDNA or DNA:RNA double helix from RNA for tagging on a second solid support. In some embodiments, the cDNA or DNA:RNA double helix cannot be tagged by the saturated DNA BLT. Instead, the cDNA or DNA:RNA double helix can be tagged on a second solid support (i.e., the RNA BLT), allowing for the generation of fragments from the cDNA or DNA:RNA double helix tagged with RNA-specific barcodes. In some embodiments, the synthetic double-stranded DNA contains uracil, which blocks amplification of the tagged fragment by certain high-fidelity DNA polymerases.

[0415] In some embodiments, saturation of DNA BLT with synthetic double-stranded DNA may enable a method that can be completed in a single reaction vessel. In some embodiments, saturation of DNA BLT with synthetic double-stranded DNA eliminates the need to split the DNA BLT after tagging the DNA from the sample.

[0416] J. Tags and DNA-specific barcodes and RNA-specific barcodes As used herein, the terms “tag” and “tag domain” refer to a portion or domain of a polynucleotide that represents a sequence for a desired intended purpose or use. Several embodiments presented herein include a transposome complex comprising a polynucleotide having a 3' portion containing a transposon terminal sequence and a tag containing a tag domain. The tag domain may contain any sequence provided for any desired purpose. For example, in some embodiments, the tag domain contains one or more restriction endonuclease recognition sites. In some embodiments, the tag domain contains one or more regions suitable for hybridization with primers for cluster amplification reactions. In some embodiments, the tag domain contains one or more regions suitable for hybridization with primers for sequencing reactions. It will be understood that any other suitable features may be incorporated into the tag domain. In some embodiments, the tag domain contains a sequence having a length of 5 bp to 200 bp. In some embodiments, the tag domain contains a sequence having a length of 10 bp to 100 bp. In some embodiments, the tag domain contains a sequence having a length of 20 bp to 50 bp. In some embodiments, the tag domain includes an array having a length of 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 bp.

[0417] The tag may include one or more functional sequences or components (e.g., primer sequences, anchor sequences, universal sequences, spacer regions, or index tag sequences) as needed or desired.

[0418] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first tag includes the same tag domain. In some embodiments, the tag includes a region for cluster amplification. In some embodiments, the tag includes a region for priming a sequencing reaction.

[0419] In some embodiments, the method further comprises amplifying a fragment on a solid support by reacting a polymerase with an amplification primer corresponding to a portion of a first polynucleotide. In some embodiments, the portion of the first polynucleotide comprises the amplification primer. In some embodiments, the first tag of the first polynucleotide comprises the amplification primer.

[0420] In some embodiments, the transposomes on individual beads hold a unique index, and when a large number of such indexed beads are used, a phased transcript is produced. In some embodiments for use with samples containing RNA and DNA, RNA BLTs include a tag that is an index for identifying RNA BLTs ("iRNA"). In some embodiments for use with samples containing RNA and DNA, DNA BLTs include a tag that is an index for identifying DNA BLTs ("iDNA"). The index for identifying RNA BLTs may be referred to as an "RNA-specific barcode," and the index for identifying DNA BLTs may be referred to as a "DNA-specific barcode." Exemplary methods of using DNA BLTs and RNA BLTs are shown in Figures 21-25.

[0421] In some embodiments, the first polynucleotide includes a first tag. In some embodiments, the second polynucleotide includes a second tag.

[0422] In some embodiments, the first and second tags include an A14 primer sequence. In some embodiments, the first and second tags include a B15 primer sequence.

[0423] In some embodiments, the tag incorporated during tagmentation includes an adapter sequence. In some embodiments, the adapter sequence may be added via a primer during tagmentation, during first-strand cDNA synthesis, or after tagmentation.

[0424] In some embodiments, the adapter sequence includes a primer sequence, an index tag sequence, a capture sequence, a barcode sequence, a cleavage sequence, or a sequencing-related sequence, or a combination thereof. As used herein, the sequencing-related sequence may be any sequence relevant to a later sequencing step. The sequencing-related sequence may act to simplify downstream sequencing steps. For example, the sequencing-related sequence may be a sequence incorporated otherwise via a step of ligating the adapter to a nucleic acid fragment. In some embodiments, the adapter sequence includes a P5 or P7 sequence (or its complement) to facilitate binding to a flow cell in a particular sequencing method. This disclosure is not limited to the types of adapter sequences that may be used, and those skilled in the art will recognize additional sequences that may be used for library preparation and next-generation sequencing.

[0425] In some embodiments, the first read sequencing adapter sequence is incorporated during tagmentation, and the second read sequencing adapter sequence is incorporated via primers after tagmentation. Different sequencing protocols use different “first read sequencing adapters” and “second read sequencing adapters,” and these adapters vary by manufacturer and instrument. In other words, the order and identity of sequencing reads are arbitrary for a given sequencing method. Therefore, the “first read” and “second read” sequencing adapters used herein simply require the presence of two read sequencing adapters, and they do not require that a specific adapter be used for the first sequencing read versus the second sequencing read in any downstream sequencing method after the preparation of the sequencing library. Those skilled in the art may choose to perform the downstream sequencing reaction first using the “second read” sequencing adapter, and then, if so, may choose the “first read” sequencing adapter.

[0426] In some embodiments, the first read and / or second read sequencing adapter sequences include different primer binding sites.

[0427] K. Reverse transcriptase polymerase and cDNA synthesis reaction In some embodiments, reverse transcriptase polymerase is used for cDNA synthesis. As used herein, “reverse transcriptase polymerase” refers to any RNA-dependent DNA polymerase capable of catalyzing DNA synthesis using RNA as a template. Reverse transcriptase polymerase can be used to synthesize the first strand of complementary DNA (cDNA) from RNA. In some embodiments, an immobilized RNA molecule is converted to a DNA:RNA double helix via reverse transcriptase polymerase.

[0428] In some embodiments, the reverse transcriptase polymerase produces only a single strand of cDNA. In some embodiments, the single strand of cDNA is used to bind to a target RNA, i.e., to produce a DNA:RNA double helix from the target RNA.

[0429] In some embodiments, the reverse transcriptase polymerase produces two strands of cDNA, i.e., the reverse transcriptase produces double-stranded (ds) cDNA.

[0430] In some embodiments, the reverse transcriptase polymerase is M-MLV reverse transcriptase.

[0431] In some embodiments, the reagents for cDNA synthesis include reverse transcriptase, random primers, oligo-dT primers, dNTPs, and / or a RNase inhibitor. In some embodiments, both random primers and oligo-dT primers are used in the cDNA synthesis reaction.

[0432] In some embodiments, reverse transcription is performed after the RNA has been immobilized (i.e., captured) on the beads. In some embodiments, reverse transcription is performed in solution.

[0433] 1. Preparation of cDNA using template switching In some embodiments (e.g., Figure 16), the preparation of double-stranded cDNA from RNA is carried out by template switching, as described in International Publications 2017 / 040306, 2015 / 168161, and 2010 / 117620, which are incorporated herein in their entirety by reference. In short, oligo(dT) and / or randomer primers prime the first strand cDNA synthesis reaction. When reverse transcriptase (e.g., SMARTSCRIBE®) reaches the 5' end of the mRNA, the terminal transferase activity of this enzyme adds several additional non-template nucleotides to the 3' end of the cDNA. A template-switch oligonucleotide (TSO), designed to form base pairs with this non-template nucleotide stretch, anneals and creates an elongation template, allowing the reverse transcriptase to continue replicating to the end of the oligonucleotide.

[0434] In some embodiments, the second strand of cDNA is synthesized using a template-switching oligonucleotide primer (TSO primer). In some embodiments, the TSO primer further includes a second amplification primer binding site. In some embodiments, the first strand synthesis primer is extended beyond the mRNA template to further copy the TSO primer strand. In some embodiments, the second strand of cDNA is synthesized using a TSO primer. In some embodiments, the second strand of cDNA is synthesized using a second amplification primer that is complementary to the first strand of cDNA, which is extended beyond the mRNA template to encompass the complementary TSO strand.

[0435] An exemplary system using a template switch oligonucleotide and compatible reverse transcriptase is SMRT-seq (Takara Bio).

[0436] 2. Preparation of stratified cDNA Various methods are known in the art that enable sequencing data to identify the mRNA strand from which a library fragment originated. The use of such “strand” methods may allow the user to determine the sequence of the original mRNA strand using the sequence of the first strand of cDNA (without interfering with data from the second strand of cDNA).

[0437] Exemplary methods for preparing strand cDNA are outlined in the "TruSeq Stranded Total RNA Reference Guide," Illumina, 2017. mRNA is copied to the first strand of cDNA using reverse transcriptase in a first-strand synthesis actinomycin mix that enables RNA-dependent synthesis and prevents undesirable DNA-dependent synthesis. The first-strand synthesis actinomycin mix can improve strand specificity when generating the first strand of cDNA. Second-strand cDNA synthesis is performed using DNA polymerase I and RNase H in a second-strand marking mix, where dTTP is replaced by dUTP. Incorporation of dUTP into the second strand of cDNA can quench amplification of this strand if uracil-intolerant DNA polymerase is used (as described in the amplification section below).

[0438] In some embodiments, the nucleoside triphosphates included in the composition for first-strand cDNA synthesis include dCTP, dATP, dGTP, and dTTP.

[0439] In some embodiments, dTTP is replaced with dUTP in the second strand cDNA synthesis reaction due to strand specificity. In some embodiments, the composition for second strand cDNA synthesis includes dCTP, dATP, dGTP, and dUTP. In some embodiments, the incorporation of dUTP in the second strand of cDNA suppresses the amplification of the second strand of cDNA in the index PCR reaction during library preparation. In some embodiments, the suppression of the amplification of the second strand of cDNA enables a strand-specific method.

[0440] In some embodiments, cDNA preparation is performed using a non-strand method that preserves strand information from mRNA.

[0441] L. Chain replacement and gap filling ligation After fragmentation of DNA:RNA double helix using immobilized transposomes, a strand-substitution polymerase (e.g., Bst polymerase) can be used to substitute the RNA strand of the DNA:RNA fragment to generate a second cDNA strand. In some embodiments, strand exchange is used to generate a double-stranded DNA fragment. In some embodiments, the strand exchange step removes the RNA strand of a fragment from the DNA:RNA double helix and replaces the RNA with the second strand of DNA. In some embodiments, strand exchange via strand-substitution polymerase transforms the DNA:RNA-containing fragment within the double-stranded DNA fragment.

[0442] In some embodiments, gaps in the DNA sequence remaining after a transposition event may also be filled using a strand displacement extension reaction, such as one comprising Bst DNA polymerase and a dNTP mixture. In some embodiments, gap-filling ligation is performed using an extension-ligation mixed buffer.

[0443] Subsequently, the library of double-stranded DNA fragments can be amplified of any choice (e.g., using cluster amplification) and sequenced using sequencing primers.

[0444] M. Amplification This disclosure further relates to the amplification of immobilized DNA fragments produced according to the methods provided herein. Immobilized DNA fragments produced by surface-bound transposomes can be amplified according to any preferred amplification methodology known in the art. In some embodiments, the immobilized DNA fragments are amplified on a solid support. In some embodiments, the solid support is the same solid support on which surface-bound tagmentation occurs. In such embodiments, the methods and compositions provided herein allow the sample preparation to proceed on the same solid support from the initial sample introduction step to amplification and optionally to the sequencing step.

[0445] For example, in some embodiments, immobilized DNA fragments are amplified using cluster amplification methodologies, as illustrated by the disclosures of U.S. Patents 7,985,565 and 7,115,400, the contents of each of these are incorporated herein by reference in their entirety. The incorporated material of U.S. Patents 7,985,565 and 7,115,400 describes a solid-phase nucleic acid amplification method that enables the immobilization of amplification products onto 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 multiple identical immobilized polynucleotide chains and multiple identical immobilized complementary polynucleotide chains. The array thus formed is generally referred herein to as a “clustered array.” Products of solid-phase amplification reactions, such as those described in U.S. Patents 7,985,565 and 7,115,400, are so-called "bridged" structures formed by annealing a pair of immobilized polynucleotide chains and an immobilized complementary chain, both chains being immobilized on a solid support at their 5' ends via covalent bonds in some embodiments. Cluster amplification methodologies are an example of methods for producing immobilized amplicons using immobilized nucleic acid templates. Immobilized amplicons can also be produced from immobilized DNA fragments produced according to the methods provided herein using other suitable methodologies. 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.

[0446] In other embodiments, the immobilized DNA fragment is amplified in solution. For example, in some embodiments, the immobilized DNA fragment is cleaved or otherwise released from the solid support, and then the amplification primer is hybridized to the free molecule in solution. In other embodiments, the amplification primer is hybridized to the immobilized DNA fragment for one or more initial amplification steps, and then the subsequent amplification steps are performed in solution. Thus, in some embodiments, an immobilized nucleic acid template can be used to produce a liquid-phase amplicon.

[0447] It should be understood that any amplification methodology described herein or commonly known in the art may be used with universal or target-specific primers to amplify immobilized DNA fragments. Preferred amplification methods 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. Patent No. 8,003,354, which is incorporated herein by reference in its entirety. One or more nucleic acids of interest can be amplified using these amplification methods. For example, immobilized DNA fragments can be amplified using PCR, including multiplex PCR, SDA, TMA, and NASBA. In some embodiments, primers specifically directed to the nucleic acid of interest are included in the amplification reaction.

[0448] Other suitable methods for amplifying polynucleotides 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) (generally U.S. Patents Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907; European Patents Nos. 0320308(B1), 0336731(B1), and 0439182(B1); International Publications Nos. 90 / 01069, 89 / 12696, and 89 / 09835, all incorporated herein by reference) techniques. It should be understood that these amplification methodologies can be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method may include a ligation probe amplification or oligonucleotide ligation assay (OLA) reaction containing a primer specifically directed to the nucleic acid of interest. In some embodiments, the amplification method may include a primer extension ligation reaction containing a primer specifically directed to the nucleic acid of interest. As non-limiting examples of primer extension and ligation primers that can be specifically designed to amplify the nucleic acid of interest, the amplification may include primers used in GoldenGate assays (Illumina, Inc., San Diego, CA), as exemplified in U.S. Patents 7,582,420 and 7,611,869, each of which is incorporated herein by reference in whole.

[0449] Examples of isothermal amplification methods that may be used in the methods of this disclosure include, but are not limited to, multiple displacement amplification (MDA) as exemplified by Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal chain displacement nucleic acid amplification as exemplified by, for example, U.S. Patent No. 6,214,587 (each of which is incorporated herein by reference in whole). Other non-PCR methods that may be used in this disclosure include, for example, strand displacement amplification (SDA) as described in Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, U.S. Patents 5,455,166 and 5,130,238, and Walker et al., Nucl. Acids Res. 20:1691-96 (1992), or superbranched strand displacement amplification as described in, for example, Lage et al., Genome Research 13:294-307 (2003), each of which is incorporated herein by reference in its entirety. Isothermal amplification can be used with large fragments of strand displacement Phi 29 polymerase or Bst DNA polymerase, 5'→3' exo-, for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processability and strand displacement activity. Due to its high processability, the polymerase is capable of producing fragments 10–20 kb in length. As described above, smaller fragments can be produced under isothermal conditions using polymerases with low processability and chain displacement activity, such as Klenow polymerase. Further descriptions of the amplification reaction, conditions, and components are detailed in the disclosure of U.S. Patent No. 7,670,810, which is incorporated herein by reference in its entirety.

[0450] Another nucleic acid amplification method useful in this disclosure is tagged PCR using a population of two-domain primers, each consisting of 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 performed to enable numerous initiations on thermally denatured DNA based on individual hybridization from randomly synthesized 3' regions. Due to the nature of the 3' region, the start sites are considered to be random throughout the genome. Subsequently, unbound primers can be removed, and further replication can be performed using primers complementary to the constant 5' region.

[0451] 1. Amplification by uracil-intolerant DNA polymerase In some embodiments, the synthetic double-stranded DNA is attached to the first solid support (such as a DNA bead-linked transposome (BLT)) after tagging the DNA on the DNA fragment to incorporate a DNA-specific barcode. In some embodiments, the synthetic double-stranded DNA binds to any transposomal complex on the first solid support that is not yet bound by a DNA fragment. In some embodiments, the synthetic double-stranded DNA is "suicide DNA" that cannot be amplified later. In some embodiments, the synthetic double-stranded DNA contains uracil, and uracil-intolerant DNA polymerase is used for amplification in a later step (see, for example, the methods outlined in Figures 27 and 28).

[0452] In some embodiments, the uracil-intolerant DNA polymerase is a high-fidelity or proofreading DNA polymerase. Those skilled in the art know that many proofreading DNA polymerases are unable to amplify uracil-containing templates due to a "uracil-binding pocket" that detects uracil residues in the template strand and stalls further DNA synthesis (see "Thermo Scientific Phusion DNA Polymerases," Thermo Fisher 2015). In some embodiments, the high-fidelity DNA polymerase is KAPA HiFi HotStart (Roche) or Phusion (Thermo Fisher). The use of uracil-intolerant polymerase is also described in international application PCT / US2021 / 036599 and Mulqueen et al., High-content single-cell combinatorial indexing, bioRxiv preprint (available at doi.org / 10.1101 / 2021.01.11.425995, published on January 12, 2021), each of which is incorporated herein by reference in whole. In some embodiments, the transposomal complex contains a uracil base immediately following the mosaic terminal sequence, and the use of uracil-intolerant polymerase prevents extension beyond the mosaic terminal, as described by Mulqueen et al.

[0453] Similarly, uracil-intolerant DNA polymerases can be used in the strand method of cDNA preparation. In some embodiments, the presence of uracil in the second strand of cDNA prepared from RNA in a sample can quench the amplification of this second strand when uracil-intolerant DNA polymerase is used. In this way, the amplified cDNA is limited to that generated from the first strand of cDNA, allowing for the identification of the mRNA strand that was present in the sample.

[0454] 2. Selective amplification In some embodiments, DNA-specific and RNA-specific barcodes enable selective amplification. “Selective amplification” refers to the amplification of fragments derived from DNA in a sample (i.e., fragments tagged with DNA-specific barcodes) or fragments derived from RNA in a sample (i.e., fragments tagged with RNA-specific barcodes). Selective amplification allows the user to amplify (and potentially sequence) only the DNA-derived or RNA-derived fragments. Alternatively, the user may choose to amplify both DNA-derived and RNA-derived fragments.

[0455] Depending on the user's objectives for the experiment, only DNA or RNA derived from a given sample may be the target. Alternatively, the user may desire multi-omics analysis of the sample to evaluate both DNA and RNA.

[0456] In some embodiments, the DNA-specific barcode and the RNA-specific barcode include different primer-binding sequences. In some embodiments, the method further includes amplifying a tagged fragment containing a DNA-specific barcode using a primer that binds to a primer-binding sequence contained in the DNA-specific barcode. In some embodiments, the method further includes amplifying a tagged fragment containing an RNA-specific barcode using a primer that binds to a primer-binding sequence contained in the RNA-specific barcode. In some embodiments, the method further includes amplifying a tagged fragment containing a DNA-specific barcode and a tagged fragment containing an RNA-specific barcode using a primer mix that includes a primer that binds to a primer-binding sequence contained in the DNA-specific barcode and a primer that binds to a primer-binding sequence contained in the RNA-specific barcode.

[0457] N. Sequence determination This disclosure further relates to sequencing of immobilized DNA fragments produced according to methods provided herein. In some embodiments, the methods include sequencing tagged fragments or amplified tagged fragments. The immobilized DNA fragments may be generated from dsDNA contained in a sample, ds-cDNA generated from RNA contained in a sample, or ds-DNA generated from DNA:RNA double-stranded strand exchange after tagging of RNA contained in a sample. In some embodiments, the immobilized DNA fragments may include DNA-specific barcodes or RNA-specific barcodes such that the bioinformatics resolution after sequencing can distinguish between fragments derived from DNA in the sample and fragments derived from RNA in the sample.

[0458] In some embodiments, DNA:RNA double-stranded fragments are directly sequenced without strand exchange.

[0459] The immobilized DNA fragments produced by surface-bound transposome-mediated tagmentation can be sequenced according to any preferred sequencing methodology, including direct sequencing, such as synthetic sequencing, ligation sequencing, hybridization sequencing, and nanopore sequencing. In some embodiments, the immobilized DNA fragments are 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.

[0460] One exemplary sequencing methodology is sequencing-by-synthesis (SBS). In SBS, the sequence of nucleotides in a nucleic acid template is determined by monitoring the extension of nucleic acid primers along a nucleic acid template (e.g., a target nucleic acid or its amplicon). The underlying chemical process may be polymerization (e.g., catalyzed by a polymerase enzyme). In certain polymer-based embodiments of SBS, fluorescently labeled nucleotides are attached to the primers in a template-dependent manner (thus extending the primers) so that the sequence of the template can be determined by detecting the order and type of nucleotides attached to the primers.

[0461] The flow cell provides a convenient solid support for containing amplified DNA fragments produced by the methods of the present disclosure. One or more amplified DNA fragments in such format can be subjected to SBS or other detection techniques, which involve repeated delivery of reagents during a cycle. For example, to initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc., can be introduced into / passed through a flow cell containing one or more amplified nucleic acid molecules. The site where the labeled nucleotide is incorporated by primer extension can be detected. Optionally, the nucleotide may further include reversible termination properties, which terminate further primer extension once the nucleotide is attached to the primer. For example, a nucleotide analog with a reversible terminator moiety can be attached to the primer so that further extension does not occur until a deblocking agent is delivered and that portion is removed. Thus, in embodiments using reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection is performed). Washing can be performed between various delivery steps. Next, the cycle is repeated n times to extend the primer with n nucleotides, thereby enabling the detection of a sequence of length n. Exemplary SBS procedures, fluid systems, and detection platforms that can be readily adapted for use with the amplicons produced by the method of this disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), International Publication No. 04 / 018497, U.S. Patent No. 7,057,026, International Publication No. 91 / 06678, International Publication No. 07 / 123744, U.S. Patent No. 7,329,492, International Publication No. 7,211,414, International Publication No. 7,315,019, International Publication No. 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0462] Other sequencing procedures that use cyclic reactions, such as pyrosequencing, can be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when specific nucleotides are incorporated into the 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. Patents 6,210,891, 6,258,568, and 6,274,320, each incorporated herein by reference). In pyrosequencing, emitted PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of the generated ATP can be detected via luciferase-produced photons. Thus, the sequencing reaction can be monitored via a luminescence detection system. Excitation radiation sources used in fluorescence-based detection systems are not required for the pyrosequencing procedure. Useful fluid systems, detectors, and procedures that can be adapted for the application of pyrosequencing to amplicons produced by this disclosure are described, for example, in International Patent Application No. PCT / US11 / 57111, U.S. Patent Application Publication No. 2005 / 0191698(A1), U.S. Patent No. 7,595,883, and U.S. Patent No. 7,244,559, each of which is incorporated herein by reference.

[0463] Some embodiments can utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between fluorophore-supported polymerase and γ-phosphate-labeled nucleotides, or using a zero-mode waveguide (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), and Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference.

[0464] Some SBS embodiments include the detection of protons released during the incorporation of nucleotides into the extension product. For example, sequencing based on the detection of released protons can use electrodetectors and related technologies commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or sequencing methods and systems described in U.S. Patent Publications 2009 / 0026082(A1), 2009 / 0127589(A1), 2010 / 0137143(A1), or 2010 / 0282617(A1), each of which is incorporated herein by reference. The methods herein for amplifying target nucleic acids using dynamic exclusion can be readily applied to substrates used for proton detection. More specifically, the methods herein can be used to produce a clonal population of amplicons used for proton detection.

[0465] Another useful sequencing technique is nanopore sequencing (see, for example, Deamer et al. Trends Biotechnol. 18, 147-151 (2000), Deamer et al. Acc. Chem. Res. 35: 817-825 (2002), and Li et al. Nat. Mater. 2: 611-615 (2003), the disclosures of which are incorporated herein by reference). In some embodiments of nanopores, the target nucleic acid or individual nucleotides removed from the target nucleic acid passes through the nanopore. As the nucleic acid or nucleotides pass through the nanopore, the type of each nucleotide can be identified by measuring the variation in the electrical conductance of the pore. (U.S. Patent 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), these disclosures are incorporated herein by reference).

[0466] Exemplary methods for array-based expression and genotyping analysis applicable to the detections described herein are described in U.S. Patent Nos. 7,582,420, 6,890,741, 6,913,884, or 6,355,431, or U.S. Patent Publication Nos. 2005 / 0053980(A1), 2009 / 0186349(A1), or 2005 / 0181440(A1), each of which is incorporated herein by reference.

[0467] An advantage of the methods described herein is that they provide rapid and efficient parallel detection of multiple target nucleic acids. Therefore, this disclosure provides an integrated system that allows for the preparation and detection of nucleic acids using techniques known in the art, such as those exemplified above. Accordingly, the integrated system of this disclosure may include a fluid component capable of delivering amplification reagents and / or sequencing reagents to one or more immobilized DNA fragments, and the system may include components such as pumps, valves, reservoirs, and fluid lines. A flow cell may constitute and / or be used in the integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Application Publication 2010 / 0111768(A1) and U.S. Patent Application 13 / 273,666, each of which is incorporated herein by reference. As exemplified with respect to flow cells, one or more fluid components of the integrated system may be used in amplification and detection methods. Taking an embodiment of nucleic acid sequencing as an example, one or more fluid components of the integrated system may be used for the delivery of sequencing reagents in the amplification method described herein and in sequencing methods such as those exemplified above. Alternatively, the integrated system may include separate fluid systems for carrying out amplification and detection methods. Examples of integrated sequencing systems capable of producing amplified nucleic acids and determining nucleic acid sequences include, but are not limited to, the MiSeq® platform (Illumina Inc., San Diego, CA) and the device described in U.S. Patent Application No. 13 / 273,666, incorporated herein by reference.

[0468] O. Physical map of immobilized polynucleotide molecules Furthermore, this specification also presents a method for generating a physical map of immobilized polynucleotides. The method can advantageously be used to identify clusters that are likely to contain linked sequences (i.e., first and second portions from the same target polynucleotide molecule). Thus, the relative proximity of any two clusters obtained from the immobilized polynucleotides provides useful information for aligning the sequence information obtained from the two clusters. Specifically, the distance between any two given clusters on a solid surface is positively correlated with the probability that the two clusters originate from the same target polynucleotide molecule, as described in detail in International Publication No. 2012 / 025250, which is incorporated herein by reference in its entirety.

[0469] For example, in some embodiments, long DNA:RNA double-strand molecules spread across the surface of a flow cell are tagged in situ, resulting in a line of connected DNA:RNA bridges traversing the surface of the flow cell. Furthermore, a physical map of the immobilized DNA:RNA can then be generated before or after strand exchange generates the immobilized DNA. Thus, the physical map correlates the physical relationships of clusters after the immobilized DNA has been amplified. Specifically, the physical map calculates the probability that sequence data obtained from any two clusters are concatenated, as described in the incorporated material of International Publication No. 2012 / 025250.

[0470] In some embodiments, a physical map is generated by imaging the DNA to establish the position of immobilized DNA molecules across a solid surface. In some embodiments, the immobilized DNA is imaged by adding an imaging agent to a solid support and detecting a signal from the imaging agent. In some embodiments, the imaging agent may be a detectable label. Suitable detectable labels include, but are not limited to, protons, haptens, radionuclides, enzymes, fluorescent labels, chemiluminescent labels, and / or chromogens. For example, in some embodiments, the imaging agent is an insertion dye or a non-insertion DNA binder. Any suitable insertion dye or non-insertion DNA binder known in the art may be used, including, but are not limited to, those described in U.S. Patent Application Publication 2012 / 0282617, which is incorporated herein by reference in its entirety.

[0471] In some embodiments, the immobilized DNA:RNA double helix is ​​further fragmented to release free ends before strand exchange and cluster formation. Breaking of the bridge structure can be performed using any suitable methodology known in the art, as exemplified by the incorporated material in International Publication No. 2012 / 025250. For example, breakage may occur by incorporating a modified nucleotide such as uracil, as described in International Publication No. 2012 / 025250, by incorporating a restriction endonuclease site, or by applying a liquid-phase transposome complex to the bridge DNA structure, as described elsewhere herein.

[0472] In certain embodiments, multiple RNAs are flowed onto a flow cell comprising multiple nanochannels, each nanochannel having multiple transposome complexes immobilized thereon. As used herein, the term nanochannel refers to a narrow channel through which long linear nucleic acid molecules flow. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 900 or fewer, or 1000 or fewer individual long-chain target RNAs flow into each nanochannel. In some embodiments, individual nanochannels are separated by a physical barrier that prevents individual long chains of target RNA from interacting with multiple nanochannels. In some embodiments, the solid support comprises at least 10, 50, 100, 200, 500, 1000, 3000, 5000, 10000, 30000, 50000, 80000, or 100000 nanochannels. In some embodiments, transposomes bound to the surface of the nanochannels tag the RNA. Sequential mapping can then be performed, for example, by tracking clusters along the length of one of these channels. In some embodiments, the long chain of the target RNA is at least 0.1kb, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, 80kb, 85kb, 90kb, 95kb, 100k b, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb, 1000kb, 5000kb, 10000kb, 20000kb, 30000kb length, or 50000kb length.In some embodiments, the long chain of the target RNA is 0.1kb, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 15kb, 20kb, 25kb, 30kb, 35kb, 40kb, 45kb, 50kb, 55kb, 60kb, 65kb, 70kb, 75kb, 80kb, 8 The lengths are 5kb, 90kb, 95kb, 100kb, 150kb, 200kb, 250kb, 300kb, 350kb, 400kb, 450kb, 500kb, 550kb, 600kb, 650kb, 700kb, 750kb, 800kb, 850kb, 900kb, 950kb or less, or 1000kb or less. As an example, a flow cell having 1000 or more nanochannels containing immobilized tagmentation products mapped within nanochannels can be used to sequence the genome of an organism with short, "positioned" reads. In some embodiments, the immobilized tagmentation products mapped within nanochannels can be used to analyze haplotypes. In some embodiments, the immobilized tagmentation products mapped within nanochannels can be used to analyze phasing challenges.

[0473] II. Method for preparing an RNA sequencing library using bead-linked transpososomes and cDNA synthesis in solution In some embodiments, cDNA is synthesized in solution from an RNA-containing sample as the first step in library preparation. In other words, the DNA:RNA double helix can be generated in solution before tagmentation by BLT (as shown in Figure 2). In some embodiments, the DNA:RNA double helix is ​​then captured on the BLT by a capture oligonucleotide. In some embodiments, the DNA:RNA double helix binds directly to the BLT based on its affinity for the transposase contained in the transposomal complex.

[0474] In some embodiments, cDNA synthesis is carried out by reverse transcriptase. In some embodiments, this cDNA synthesis yields a DNA:RNA double helix, producing a DNA strand that can hybridize to an RNA strand. In some embodiments, reverse transcriptase polymerase is added to an RNA-containing sample under conditions for cDNA synthesis. In some embodiments, the conditions for cDNA synthesis include the presence of nucleotides and / or primers (such as poly-T primers and / or randomer primers) that can bind to RNA. In some embodiments, the reaction mixture for preparing the DNA:RNA double helix comprises oligo-dT primers, reverse transcriptase, and nucleotides. In some embodiments, the DNA:RNA double helix synthesizes the first strand of cDNA at a reaction temperature of 42°C.

[0475] In some embodiments, the reverse transcriptase simply prepares DNA from RNA (without generating further copies of the DNA to obtain double-stranded DNA).

[0476] In some embodiments, cDNA preparation is carried out in solution to generate DNA:RNA double helix or double-stranded cDNA.

[0477] In some embodiments, DNA:RNA double helixes prepared in solution can then be bound to BLTs and tagged. After stopping or removing the transposase, strand exchange can be performed, followed by gap filling and ligation, after which the library can be released. In some embodiments, if double-stranded cDNA is prepared in solution and then tagged, strand exchange is not required. In some embodiments, these methods can be carried out in a tube or a flow cell.

[0478] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA includes: adding reverse transcriptase polymerase to a sample containing the target RNA under conditions that synthesize cDNA and produce a DNA:RNA double helix; immobilizing the DNA:RNA double helix onto a solid support on which a transposomal complex is immobilized, wherein the transposomal complex comprises a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and a first tag, and the sample is applied to the solid support under conditions that the DNA:RNA double helix directly binds to the capture oligonucleotide or transposase; and fragmenting the DNA:RNA double helix using the transposomal complex under conditions that the DNA:RNA double helix is ​​tagged at the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 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 the DNA strand.

[0479] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a target RNA comprises: applying a sample containing the target RNA to a solid support on which a capture oligonucleotide is immobilized; adding reverse transcriptase polymerase under conditions that synthesize cDNA and produce DNA:RNA double helix immobilized on the capture oligonucleotide; and fragmenting the DNA:RNA double helix in solution using a transposome complex under conditions that the DNA:RNA double helix is ​​tagged at the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a first tag. In some embodiments, the RNA is mRNA, and the capture oligonucleotide contains a poly-T sequence. In some embodiments, the library contains DNA:RNA fragments generated from the 3' ends of one or more RNAs. In some embodiments, the capture oligonucleotide further comprises a first read sequencing adapter sequence, a bead code, and / or one or more additional adapter sequences. In some embodiments, the transposome complex in solution comprises a first transposome containing a second read sequence adapter sequence and / or one or more additional adapter sequences. In some embodiments, the DNA:RNA double-strand library is sequenced without amplifying the fragments before sequencing.

[0480] III.3' Method for preparing RNA sequencing libraries using UMI Current UMI sequencing methods alone allow for quantitative sequencing of transcript fragments only, which hinders high-resolution isoform identification. In some embodiments, combining ligated-length read techniques with the 3'UMI binding described herein enables quantitative sequencing of RNA libraries, as well as isoform identification by ligating all fragments of the original transcript together with the 3'UMI. In some embodiments, bead-ligated transposomes (BLTs) with a common bead code identifier can generate ligated-length reads for full-length RNA. While UMI-based methods may have many applications, they may be particularly useful in alternative splicing studies where identifying sequences derived from different RNA molecules is crucial.

[0481] In some embodiments, the method combines 3'UMI tagging for accurate quantification with a linked-length lead bead code, as illustrated by Figures 15 and 16. In some embodiments, 3'UMI tagging is performed before amplification. In some embodiments, the incorporation of 3'UMI pre-amplification of cDNA and pre-tagmentation adapts the method to single-cell and ultra-low RNA inputs. In some embodiments, the method enables a full-length RNA counting assay for single cells. In some embodiments, the method includes template switching.

[0482] In some embodiments, a method for preparing a library of double-stranded DNA fragments from RNA comprises: preparing a first strand of cDNA from full-length RNA in a sample using a poly-T primer containing UMI and a first read sequencing adapter sequence; preparing a second strand of cDNA to generate double-stranded cDNA; and applying the double-stranded cDNA to beads on which transposome complexes are immobilized, wherein each transposome complex comprises a transposase, a first transposon containing a 3' transposon terminal sequence, and a second transposon containing a sequence and a hybridization sequence that are completely or partially complementary to the transposon terminal sequence, and the transposome complex hybridizes oligonucleotides immobilized on the beads. The method involves immobilizing a transposition sequence by binding, wherein the oligonucleotide includes a sequence that is fully or partially complementary to the 5' affinity element, the first read sequencing adapter sequence, the bead code, and the hybridization sequence; immobilizing the double-stranded cDNA and performing tagmentation on the beads to prepare a double-stranded DNA fragment; removing the second transposon; hybridizing a primer including the second read sequencing adapter sequence and a sequence that is fully or partially complementary to the transposon terminal sequence to the transposon terminal sequence; and performing gap filling and extension to prepare a double-stranded DNA fragment including the first read sequencing adapter and the second read sequencing adapter.

[0483] In some embodiments, a method for preparing a library of double-stranded DNA fragments from RNA involves preparing a first strand of cDNA from full-length RNA in a sample using a poly-T primer containing a UMI and a first read sequencing adapter sequence, preparing a second strand of cDNA to generate double-stranded cDNA, and applying the double-stranded cDNA to beads on which transposome complexes are immobilized, wherein each transposome complex is a first transposon containing a transposase, a 3' transposon end sequence, a bead code, and a second read sequencing adapter sequence, and further comprising a 5' affinity element for immobilizing the transposome complexes on a solid support. The method includes applying a first transposon and a second transposon containing a sequence that is completely or partially complementary to the transposon terminal sequence; immobilizing double-stranded cDNA and performing tagmentation on beads to prepare a double-stranded DNA fragment; removing the second transposon; hybridizing a primer containing a second read sequencing adapter sequence and a sequence that is completely or partially complementary to the transposon terminal sequence to the transposon terminal sequence; and performing gap filling and extension to prepare a double-stranded DNA fragment containing the first read sequencing adapter and the second read sequencing adapter.

[0484] In some embodiments, the primer includes a 5' portion containing a second read sequence adapter and a 3' portion containing a sequence that is fully or partially complementary to the transposon terminal sequence.

[0485] In some embodiments, when a sequence that is completely or partially complementary to the transposon terminal sequence is removed, the fragment remains bound to the transposome at one or both ends.

[0486] In some embodiments, each primer contains a different UMI. In some embodiments, the full-length RNAs include a pool of different full-length RNAs, and the poly-T primers include a pool of different poly-T primers containing different UMIs. In some embodiments, each poly-T primer in the pool of different poly-T primers contains a different UMI.

[0487] In some embodiments, each fragment contains a unique UMI. In some embodiments, the full-length RNA comprises a pool of different full-length RNAs, and a 3' double-stranded DNA fragment prepared from a single full-length RNA contains a different UMI than a 3' double-stranded DNA fragment prepared from other full-length RNAs in the pool.

[0488] In some embodiments, each bead contains a unique bead code. In some embodiments, the full-length RNAs comprise a pool of different full-length RNAs, and the beads comprise a pool of beads. In some embodiments, each bead immobilizes a transposome complex containing a different bead code compared to the bead code contained in the transposome complex immobilized on other beads in the pool.

[0489] In some embodiments, all fragments prepared from double-stranded cDNA prepared from a single full-length RNA are tagged on the same beads.

[0490] In some embodiments, all double-stranded fragments, including a first read sequencing adapter and a second read sequencing adapter prepared from double-stranded cDNA, are placed on the same solid support after gap filling and extension.

[0491] In some embodiments, the full-length RNA comprises a pool of different full-length RNAs, and all double-stranded fragments, including a first read sequencing adapter and a second read sequencing adapter prepared from a single full-length RNA in the pool, are placed on the same solid support after gap filling and extension.

[0492] In some embodiments, the method further includes amplifying a double-stranded fragment containing a first read sequencing adapter and a second read sequencing adapter to prepare an amplified fragment.

[0493] In some embodiments, the double-stranded cDNA preparation is performed by the strand method. In some embodiments, the presence of a bead code in the sequence obtained from a double-stranded fragment containing a first read sequencing adapter and a second read sequencing adapter or an amplified fragment identifies the bead from which the fragment was generated.

[0494] In some embodiments, the sample is a single cell.

[0495] In some embodiments, the method includes the preparation of a strand RNA library.

[0496] In some embodiments, one sequencing adapter sequence is incorporated into the fragment during the tagmentation process, and different sequencing adapter sequences are incorporated into the fragment via primers used for extension after tagmentation. In some embodiments, the primers for incorporating the sequencing adapter sequences are tagged primers containing the sequencing adapter sequences.

[0497] In some embodiments, the method includes a symmetric tagging step, in which all transposome complexes contain the same adapter sequence.

[0498] In some embodiments, the method is compatible with 3'UMI tagging, pre-amplification, and / or full-length RNA isoform detection.

[0499] In some embodiments, the method further includes sequencing an amplified fragment or double-stranded fragment comprising a first read sequencing adapter and a second read sequencing adapter.

[0500] In some embodiments, sequencing enables the detection of full-length RNA isoforms.

[0501] In some embodiments, the 3' UMI (contained in the 3' fragment generated during tagmentation) can be used during the analysis of sequencing results to identify cDNAs that are different from other cDNAs (based on other cDNAs having other UMIs). Figure 14 outlines how the 3' fragment incorporates the UMI during first strand synthesis (based on the UMI contained in the first strand synthesis primer), followed by a single tagmentation event after the capture of the 3' end of the cDNA. Since all fragments from a given cDNA are fragmented on the same DNA BLT, all fragments incorporate the same bead code. Figure 15 summarizes this aspect of the embodiment, where all fragments are generated by tagmentation on the bead containing this code, so "UMI1" is incorporated into the cDNA from isoform #1 during cDNA synthesis, and all fragments of this isoform incorporate "bead code A". In contrast, since all fragments were generated by tagmentation on the beads containing this code, "UMI2" is incorporated into the cDNA from isoform #2 during cDNA synthesis, and all fragments of this isoform incorporate "bead code B". A method using template switching and PCR amplification is shown in Figure 16, and an adapter (such as P5) can be incorporated using template switching primers.

[0502] Using these methods, all fragments generated from cDNA from a given mRNA isoform can be classified during the analysis of the sequencing results. This analysis allows for the distinction between different mRNA isoforms within the sequencing results.

[0503] A. Determination of linked lead length arrangement Standard short-read sequencing provides accurate base-level sequences to offer short-range information, but it may not provide long-range genomic information. Furthermore, haplotype information is not preserved for the sequenced genome or references with short-read data, making long-range haplotype reconstruction difficult with standard methods. Therefore, while standard sequencing and analytical approaches can generally be called single nucleotide variants (SNVs), these methods may not identify the full spectrum of structural variations found in individual genomes. As used herein, "structural variation" of a genome refers to events larger than SNVs, including events of 50 base pairs or more. Representative structural variants include copy number variations, inversions, deletions, and duplications.

[0504] "Linked-read sequencing" or "linked-read sequencing" refers to a sequencing method that provides long-range information about the genome sequence.

[0505] In some embodiments, ligated read sequencing can be used for haplotype reconstruction. In some embodiments, ligated read sequencing improves the calling of structural variants. In some embodiments, ligated read sequencing improves access to regions of the genome with limited accessibility. In some embodiments, ligated read sequencing is used for de novo diploid assembly. In some embodiments, ligated read sequencing improves the sequencing of highly polymorphic sequences (such as human leukocyte antigen genes) that require de novo assembly.

[0506] In some embodiments, linked-length read sequence determination may be based on spatial separation of fragments before emission from the BLT, or on bead barcoding.

[0507] 1. Determination of linked-length read sequences based on spatial separation In some embodiments, full-length nucleic acids are "wrapped" onto a single bead, such as a BLT, which means that the full-length nucleic acid can associate with multiple transposomal complexes on the single bead. As used herein, nucleic acids may be DNA, cDNA, or DNA:RNA double helix.

[0508] In some embodiments, beads are delivered to a surface for sequencing using full-length nucleic acids bound to the beads. For example, nucleic acids can be bound to an activatable BLT and delivered to a flow cell. The BLT can be activated after being bound to the flow cell to enable the preparation of fragments. Fragments can then be released such that fragments produced from a given full-length nucleic acid (prepared on the same bead) are released in much closer proximity to fragments prepared on other beads.

[0509] In some embodiments, the BLT is delivered to the surface for sequencing along with the fragments bound to the BLT.

[0510] In some embodiments, ligated read sequencing uses molecular barcodes to tag reads derived from the same long DNA fragment. If a unique barcode is attached to every short read generated from an individual DNA molecule, the short reads can ligate that DNA molecule together. In other words, reads sharing a barcode can be classified as originating from a single long input molecule, enabling the assembly of long-range information from the short reads.

[0511] B. First chain synthesis primer In some embodiments, a first strand synthesis primer can incorporate one or more tags into the first strand of cDNA generated from RNA contained in the sample. In some embodiments, the first strand synthesis primer includes a poly-T sequence. In some embodiments, this poly-T sequence can hybridize to a poly-A tail on the 3' end of the RNA. In some embodiments, the RNA is mRNA. In some embodiments, the use of a primer containing a poly-T sequence enables tagging of the first strand of cDNA by 3'UMI.

[0512] In some embodiments, the first chain synthesis primer further comprises a UMI, an index sequence (or its complement), a first read sequencing adapter sequence (or its complement), and / or one or more additional adapter sequences.

[0513] In some embodiments, the first chain synthesis primer is included in a pool of first chain synthesis primers. In some embodiments, the first chain synthesis primer in the pool of first chain synthesis primers contains a unique UMI that is different from all or most of the other primers in the mix.

[0514] In some embodiments, the first strand synthesis primer includes an oligo-dT sequence, a UMI, an index sequence, and an adapter sequence. A typical first strand of cDNA produced using such a primer is shown in Figure 14, where the first strand of cDNA includes an oligo-dT sequence, a UMI, an i7 sequence (index sequence), and a P7 sequence (i.e., an adapter sequence used as a primer landing site). In some embodiments, a sequencing surface, such as a flow cell, is coated with a lawn of the primer landing site. In some embodiments, the primer landing site is P5 or P7. In some embodiments, the primer landing site (such as P5 or P7) facilitates the binding of the fragment to the flow cell.

[0515] In some embodiments, the oligo dT sequence and the first read sequencing adapter sequence are identical for each first strand synthesis primer in the primer mixture. In some embodiments, the UMI is unique for each first strand synthesis primer. In this way, downstream sequencing events can distinguish fragments generated from different RNA molecules contained in samples containing different RNAs.

[0516] In some embodiments, the index sequence included in the first chain synthesis primer is one of a known pool of index sequences, such as the i7 or i5 sequence (see, for example, Illumina Document #1000000002694 v10, Illumina, Inc. 2019).

[0517] In some embodiments, the first chain synthesis primer includes one or more adapter sequences. In some embodiments, the adapter sequences include a primer sequence, an index tag sequence, a capture sequence, a barcode sequence, a cleavage sequence, or a sequencing-related sequence, or a combination thereof.

[0518] C. Unique Molecular Identifier (UMI) A unique molecular identifier (UMI) is a nucleotide sequence applied to or used to identify a nucleic acid molecule, which can be used to distinguish individual nucleic acid molecules from one another. UMIs may be sequenced together with the relevant nucleic acid molecules to determine whether a read sequence belongs to one source nucleic acid molecule or another. The term "UMI" may be used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide itself. While a UMI is similar to a barcode commonly used to distinguish a read from one sample from a read from another, a UMI is instead used to distinguish a nucleic acid template fragment from another fragment when many fragments from an individual sample are sequenced together. UMIs can be defined in many ways, as described in International Publications 2019 / 108972 and 2018 / 136248, which are incorporated herein by reference.

[0519] A unique molecular identifier (UMI) is a nucleotide sequence applied to or used to identify a nucleic acid molecule, which can be used to distinguish individual nucleic acid molecules from one another. UMIs may be sequenced together with the relevant nucleic acid molecules to determine whether a read sequence belongs to one source nucleic acid molecule or another. The term "UMI" may be used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide itself. While a UMI is similar to a barcode commonly used to distinguish a read from one sample from a read from another, a UMI is instead used to distinguish a nucleic acid template fragment from another fragment when many fragments from an individual sample are sequenced together. UMIs can be defined in many ways, as described in International Publications 2019 / 108972 and 2018 / 136248, which are incorporated herein by reference.

[0520] In some embodiments, the UMI library includes non-random sequences. In some embodiments, non-random 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, a set of sequences may 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 specific number (e.g., 2, 3, or 4) nucleotides. That is, an nrUMI sequence cannot be converted to any other available nrUMI sequence by replacing fewer than a specific 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 each would have a total of 4 A Instead of 6 = 4096 possible different sequences, it may contain a total of 96 different sequences. In some embodiments, the UMI library contains 120 non-random sequences.

[0521] In some implementations, where nrUMIs are selected from a set of sequences fewer than all possible different sequences, the number of nrUMIs is less than, and sometimes significantly less than, the number of source DNA molecules. In such implementations, nrUMI information can be combined with other information, such as virtual UMIs, read positions on reference sequences, and / or read sequence information, to identify sequence reads originating from the same source DNA molecule.

[0522] In some embodiments, a library of rUMIs may include random UMIs (rUMIs) selected as random samples, with or without substitution, from a set of UMIs consisting of all possible different oligonucleotide sequences given one or more sequence lengths. For example, if each UMI in the set of UMIs has n nucleotides, the set may have 4 sequences that are different from each other. A Contains n UMIs. 4 A A random sample selected from n UMIs constitutes an rUMI.

[0523] In some embodiments, the UMI library may be pseudo-random or partially random and may include a mixture of nrUMI and rUMI.

[0524] In some embodiments, an adapter sequence or other nucleotide sequence may be present between the UMI and the inserted DNA.

[0525] In some embodiments, an adapter sequence or other nucleotide sequence may be present between each UMI and the inserted DNA.

[0526] In some embodiments, the UMI is located at 3' of the inserted DNA. In some embodiments, sequences of nucleic acids representing one or more adapter sequences may be located between the UMI and the inserted DNA.

[0527] In some embodiments, UMI is located on the first strand of the synthesized cDNA. In some embodiments, the first copy of UMI is located on the first strand of the synthesized cDNA, and the second copy of UMI (i.e., its complement) is located on the second strand of the synthesized cDNA.

[0528] D. Primers for incorporating one or more adapters after tagging. In some embodiments, the primer is hybridized after tagmentation to the BLT to incorporate one or more adapter sequences. In some embodiments, the primer includes a sequencing adapter sequence and a sequence that is fully or partially complementary to the transposon terminal sequence. In some embodiments, the primer includes a sequencing adapter sequence different from the sequencing adapter included in the first chain synthesis primer. In some embodiments, the sequence of the first chain synthesis primer is different from the sequence of the primer used after tagmentation.

[0529] In some embodiments, the primer hybridized after tagmentation is not entirely complementary to the sequence in the first transposon. In other words, the hybridization of the primer to the fragment immobilized on the BLT generates a "Y-shaped adapter" or "fork-shaped adapter"-like structure.

[0530] In some embodiments, the primer hybridized after tagging includes a sequence that is fully or partially complementary to the sequence contained in the first transposon. In some embodiments, the primer hybridized after tagging includes a sequence that is fully or partially complementary to the mosaic end (ME) sequence (or its complement) contained in the first transposon. In some embodiments, the primer hybridized after tagging includes a sequence that is not contained in the first transposon.

[0531] In some embodiments, the ME' sequence in the non-transposition chain is dissociated from the fragment before hybridizing a primer containing a sequence that is fully or partially complementary to the sequence contained in the first transposon. In some embodiments, the sequence that is fully or partially complementary to the transposon terminal sequence is shorter than the transposon terminal sequence. Such embodiments are shown as a shortened ME' in Figure 19. In some embodiments, when the sequence that is fully or partially complementary to the transposon terminal sequence is shorter than the transposon terminal sequence (i.e., a shortened ME' sequence is used), fewer adapter dimers are produced.

[0532] In some embodiments, the second transposon contains a shorter ME' sequence to facilitate the dissociation of the ME' sequence from the ME sequence after tagmentation. In some embodiments, the shortened ME' sequence is useful for exchanging the non-transferred ME' sequence with a different oligonucleotide (e.g., a primer). In some embodiments, the shortened ME' sequence may also reduce the occurrence of blunt-end ligation.

[0533] E. Hybridization sequence In some embodiments, the beads include oligonucleotides that can be used to bind to transposomes in solution. In this way, the beads can be "activatable BLTs," and the user can control the timing of BLT generation.

[0534] In some embodiments, the beads contain oligonucleotides comprising a hybridization sequence. In some embodiments, the hybridization sequence binds to a sequence that is fully or partially complementary to the sequence contained in the transposome complex. In some embodiments, the hybridization sequence binds to a second transposon contained in the transposome, and the transposome from solution may be bound via a hybridization sequence that is fully or partially complementary to the sequence contained in the second transposon (to form a BLT).

[0535] F. Bead Cord A given 3' fragment of cDNA can incorporate a UMI sequence (as described above). In this way, the UMI sequence in the 3' fragment generated from a given cDNA can be used to distinguish this cDNA from others generated. This 3' fragment (shown in Figure 15) or DNA:RNA double helix generated from a given cDNA also contains a bead code incorporated during tagmentation. In this way, sequencing data from the 3' fragment (including the UMI) can be sorted together with sequencing data from other fragments generated on the same bead to generate a complete sequence of the starting RNA from the sample. In other words, cDNA generated from a full-length RNA transcript is tagged by a single bead, which tags all segments of the cDNA derived from the original full-length RNA that have the same bead code sequence. In some embodiments, by fragmenting a single cDNA on a given bead, all fragments can be linked back to the original transcript and the UMI introduced during reverse transcription. Thus, this method can provide data on a unique RNA molecule.

[0536] While all fragments of a given cDNA are generally generated on the same BLT, this BLT can also generate fragments from other cDNAs bound to it. In other words, fragments derived from different RNAs in the original sample may share the same bead code. However, sequence alignment can be used to identify which fragments on a given bead originate from a given sequence.

[0537] IV. Additional methods for preparing RNA or DNA libraries after symmetric tagging. Several different methods for generating sequenceable fragments may be used after symmetric tagmentation of BLTs. These methods may be combined with any of the protocols described herein. Several exemplary methods are disclosed in U.S. Provisional Application No. 63 / 168,802, which is incorporated herein in whole.

[0538] In some embodiments, the method includes releasing a double-stranded target nucleic acid fragment from a transposome complex after tagmentation of DNA or DNA:RNA double helix, hybridizing a polynucleotide comprising an adapter sequence and a sequence that is fully or partially complementary to the first 3' terminal transposon sequence such that the adapter sequence in the polynucleotide is different from the adapter sequence in the transposome complex, optionally extending a second strand of the double-stranded target nucleic acid fragment, optionally ligating the polynucleotide or the extended polynucleotide with the double-stranded target nucleic acid fragment, and producing the double-stranded target nucleic acid fragment. In some embodiments, the polynucleotide further comprises a UMI. In some embodiments, the fragment comprises a UMI located directly adjacent to the 3' end of the inserted DNA.

[0539] In some embodiments, the method includes releasing a double-stranded target nucleic acid fragment from a transposomal complex after tagmentation of DNA or DNA:RNA double helix, hybridizing a first polynucleotide adapter sequence such that the adapter in the first transposon is different from the adapter in the first polynucleotide, optionally adding a second polynucleotide containing a region complementary to the first polynucleotide to produce a double-stranded adapter, optionally extending the second strand of the double-stranded target nucleic acid fragment, optionally ligating the double-stranded adapter with the double-stranded target nucleic acid fragment to produce the double-stranded target nucleic acid fragment. In some embodiments, the first polynucleotide further comprises a UMI. In some embodiments, the fragment comprises a UMI located between the double-stranded target nucleic acid fragment and the adapter sequence derived from the first polynucleotide.

[0540] In some embodiments, the fragment is tagged at the 5' end of one strand with a first read sequence adapter sequence from a first transposon and at the 5' end of the other strand with a second read sequence adapter sequence from a first polynucleotide.

[0541] V. Preparation method for strand-specific cDNA preparation by tagmentation for library preparation (PRESS-BLT) In some embodiments, the strand-specific cDNA preparation method is combined with tagmentation to prepare the library. A primer-extended strand-specific BLT approach (PRESS-BLT) may be used to describe a strand-specific method comprising cDNA synthesis, BLT formation, and library preparation, as shown in Figures 17 and 18.

[0542] In some embodiments, a method for preparing a strand-specific library of single-stranded DNA from RNA via PRESS-BLT involves: preparing a first strand of cDNA from RNA contained in a sample using a nucleotide comprising reverse transcriptase, primers, and dTTP under conditions that inhibit DNA-dependent DNA synthesis; preparing a second strand of cDNA from the first strand of cDNA using a nucleotide comprising DNA polymerase, primers, and dUTP to prepare double-stranded cDNA; and applying the double-stranded cDNA to a solid support on which a transposome complex is immobilized, wherein each transposome complex comprises a transposase and a first transposon comprising a 3' portion containing a transposon terminal sequence and a first read sequencing adapter sequence, and a first transposon comprising a 5' affinity element for immobilizing the transposome complex to a solid support, and a complete transposon with respect to the transposon terminal sequence. A method comprising: applying a second transposon sequence containing a sequence that is or partially complementary to the first transposon sequence; fragmenting double-stranded DNA using a transposome complex to prepare tagged double-stranded DNA fragments containing a first read sequencing adapter sequence; removing the second transposon, filling the gap and extending the fragment; separating the strands of the double-stranded DNA fragment; hybridizing a primer containing the second read sequencing adapter sequence to a transposon terminal sequence or a sequence that is fully or partially complementary to the transposon terminal sequence, amplifying the resulting DNA strands that are not bound to a solid support and contain the first and second read sequencing adapters; and releasing the resulting strands from the solid support, wherein the release is the release of single-stranded DNA fragments containing the first and second read sequencing adapters.

[0543] In some embodiments, the condition that inhibits DNA-dependent DNA synthesis is the presence of a buffer containing actinomycin D. In some embodiments, the primers are one or more randomizer primers. In some embodiments, the primers are a mixture of randomizer primers and poly-T primers. In some embodiments, the primers for preparing the second strand of cDNA are the same as the primers for preparing the first strand of cDNA. In some embodiments, the RNA is a long non-coding RNA or antisense transcript.

[0544] In some embodiments, amplification is performed using uracil-intolerant polymerase. In some embodiments, amplification is not performed from a DNA strand containing uracil.

[0545] In some embodiments, the unique molecular identifier (UMI) is contained in a primer comprising a second read sequencing adapter sequence. In some embodiments, the UMI is located between the second read sequencing adapter sequence and a sequence that can bind to the transposon terminal sequence or a sequence that is completely or partially complementary to the transposon terminal sequence. In some embodiments, the UMI is contained within a first transposon. In some embodiments, the UMI is located between the transposon terminal sequence and the first read sequencing adapter sequence.

[0546] In some embodiments, different fragments in the resulting library contain different UMIs. In some embodiments, the RNA contains a pool of different RNAs, and the single-stranded fragments containing a first read sequencing adapter and a second read sequencing adapter contain a pool of different fragments, and each fragment contains a different UMI from other fragments in the pool of different fragments.

[0547] The PRESS-BLT method allows the use of a range of different affinity elements. In some embodiments, the affinity element is biotin or desthiobiotin, and the solid support contains streptavidin or avidin on its surface. In some embodiments, the affinity element is bibiotin, as shown in Figure 19.

[0548] The chains generated by amplification can be released from the solid support in several ways. In some embodiments, the release is carried out using heat or sodium hydroxide treatment. In some embodiments, the single-stranded fragments containing the first and second read sequencing adapters are separated from the solid support after release.

[0549] In some embodiments, the method further includes preparing an indexed fragment by performing index-primer amplification using a single-stranded DNA fragment containing a first read sequencing adapter and a second read sequencing adapter after release. Such index-primer amplification is well known in the art for indexing sequencing data. In some embodiments, the index-primer amplification is performed in a reaction vessel separate from the solid support.

[0550] In some embodiments, index primer amplification is performed using uracil-intolerant polymerase. In this way, the second strand of cDNA is not amplified if it contains uracil.

[0551] In some embodiments, the method further includes sequencing a single-stranded DNA fragment or indexed fragment comprising a first read sequencing adapter and a second read sequencing adapter. In some embodiments, the sequencing data is generated from a first strand of cDNA derived from RNA. In some embodiments, the sequencing data is not generated from a second strand of cDNA derived from RNA. In some embodiments, the method does not require ligation.

[0552] PRESS-BLT offers several advantages for mRNA analysis. In some embodiments, the method demarcates duplicate sequences in RNA. In some embodiments, the method enables transcription expression estimation. In some embodiments, transcription expression estimation is based on UMI analysis. A general overview of PRESS-BLT is that it includes the steps of strand-specific cDNA synthesis, symmetric tagging by BLT, and primer extension for preparing library fragments.

[0553] A. Strand-specific cDNA synthesis in PRESS-BLT In some embodiments, total RNA is copied to a first-strand cDNA using an FSA buffer containing reverse transcriptase, random primers, nucleoside triphosphate, and actinomycin D. In some embodiments, actinomycin D specifically inhibits DNA-dependent DNA synthesis and improves strand specificity. A typical method of strand-specific cDNA synthesis is shown in Figure 17.

[0554] Such strand-specific cDNA synthesis can be used within the PRESS-BLT method, but can also be used in conjunction with other library preparation methods.

[0555] BLT for use in B.PRESS-BLT In some embodiments, double-stranded cDNA prepared using a strand-specific protocol is then tagged to generate tagged double-stranded DNA fragments.

[0556] In some embodiments, the tagmentation in the PRESS-BLT method is symmetric tagmentation, where all transposomal complexes contain a first transposon with the same adapter sequence. In some embodiments, the method using symmetric tagmentation increases the yield of sequenceable fragments (i.e., each fragment having a different sequencing adapter sequence at each end) compared to an asymmetric tagmentation step in which two or more types of transposomal complexes are used for tagmentation.

[0557] In some embodiments, cDNA is tagged such that the fragment incorporates the same tag at the 5' ends of both strands. In some embodiments, the tagged double-stranded cDNA fragment produced by tagging has the same tag at both ends of the fragment. In some embodiments, cDNA is tagged with a tag that includes a single sequencing adapter sequence. In some embodiments, the sequencing adapter sequence is A14 or B15.

[0558] Primer extension in C.PRESS-BLT In some embodiments, the non-transferred ME' sequence (from the second transposon) is thawed and gap-filled by PCR extension after tagmentation. In some embodiments, the ME' sequence is removed by increasing the reaction temperature. These steps of the method are outlined in Figure 18.

[0559] In some embodiments, gap filling is performed after the non-transitioned ME' sequence is removed. In some embodiments, a primer is annealed to the gap-filled ME' sequence. In some embodiments, this primer is used for extension and may be referred to as an extension primer. In some embodiments, the extension primer contains an ME sequence. In some embodiments, the ME sequence contained in the extension primer hybridizes to the gap-filled ME' sequence.

[0560] In some embodiments, the extension primer also includes a sequencing adapter sequence. In some embodiments, the sequencing adapter sequence included in the extension primer was not included in the transposome complex. In some embodiments, extension by the extension primer generates a fragment in which each end of the double-stranded fragment contains a different sequencing adapter sequence.

[0561] In some embodiments, uracil-intolerant DNA is used for primer extension. In some embodiments, primer extension occurs only from the first strand of the cDNA. In some embodiments, the second strand of the cDNA is not extended because it contains uracil, based on the strand-specific cDNA preparations described above.

[0562] In some embodiments, if the transposome complex contains the A14 sequence (or its complement), the extension primer contains the B15 sequence (or its complement). In some embodiments, if the transposome complex contains the B15 sequence (or its complement), the extension contains the A14 sequence (or its complement). In these representative examples, A14 and A15 represent exemplary sequencing adapter sequences, and the method is not limited to such adapter sequences. Any set of paired adapter sequences of interest may be used in the transposome complex and extension primer, and those skilled in the art are well aware of how sequencing is performed on different platforms and how such platforms may evolve over time.

[0563] In some embodiments, the extension primer includes a UMI. In some embodiments, the UMI marks an mRNA transcript unique to the copy produced by PCR amplification. In other words, amplicon copies from the same cDNA (e.g., derived from a single mRNA transcript) from any downstream amplification step will contain the same UMI. Thus, analysis of sequencing results can identify multiple copies of the fragment produced from the same mRNA.

[0564] As shown in Figure 19, certain modified transposons can improve the results of press-BLT. These modifications may also be useful in other methods described herein. In some embodiments, double biotin is used to immobilize transposon sequences onto beads to generate BLT. In some embodiments, double biotin has a stronger affinity for streptavidin compared to biotin, which improves the binding and release of transpososomes from BLT. Such modifications may be used in press-BLT methods in conjunction with any other methods described herein.

[0565] VI. Method for preparing RNA and DNA sequencing libraries using bead-linked transposomes In some embodiments, the methods involve applying a sample containing RNA and DNA. These methods may be carried out using components similar to those enumerated for using RNA sequencing libraries. Any of the methods for RNA sequencing shown in Figures 2 to 18 and described herein may be used when preparing RNA and DNA sequencing libraries from the same sample. Figures 22 to 28 also show exemplary methods in which RNA may be converted to DNA:RNA double-stranded or double-stranded DNA during the method.

[0566] In some embodiments, the method can resolve sequencing of RNA-derived and DNA-derived samples of total nucleic acid (TNA) samples by library preparation based on tagmentation. In some embodiments, the method allows a single reaction vessel to generate RNA and DNA libraries.

[0567] In some embodiments, the method enables “directional” library preparation that can distinguish the nucleic acid strands from which the sequenced fragments originated.

[0568] In some embodiments, different methods can be combined to generate “strand” RNA and DNA tagging-based sequencing libraries.

[0569] In some embodiments, DNA and RNA sequencing libraries can be generated from a single sample reaction. In some embodiments, DNA and RNA sequencing libraries can be generated in a single reaction vessel. In some embodiments, the method can capture genomic and transcriptome or other information in a single reaction, which may be referred to as a multi-omics assay.

[0570] Various methods can be used with samples containing RNA and DNA, enabling the preparation of RNA and DNA sequencing libraries from the same sample. In some embodiments, these methods avoid DNA tagging by RNA-BLTs. Instead, RNA in a sample containing RNA and DNA is tagged by a BLT designed for DNA:RNA double-strand fragmentation (RNA BLT), and DNA in the sample is tagged by a BLT designed for DNA fragmentation (DNA BLT).

[0571] Figure 21 illustrates a challenge in these methods, namely, that DNA itself may bind to transpososomes that may be present on the RNA BLT. This application describes several methods for identifying DNA:RNA double-stranded tagging by RNA BLT. These methods avoid DNA tagging by RNA BLT. Figures 22–28 provide exemplary workflows that enable DNA tagging by DNA BLT (e.g., for embedding DNA-specific barcodes) and DNA:RNA double-stranded or double-stranded DNA prepared from RNA by RNA BLT (e.g., for embedding RNA-specific barcodes).

[0572] In some embodiments, the first and second tags are different. In some embodiments, the first and second tags allow for the distinction between fragments of an RNA sequencing library and fragments of a DNA sequencing library. In some embodiments, the index for identifying fragmentation by DNA BLT is referred to as "iDNA" or the index for identifying DNA BLT (see Figure 21). In some embodiments, the index for identifying fragmentation by RNA BLT is referred to as "iRNA" or the index for identifying RNA BLT (see Figure 21).

[0573] The challenge of such workflows is to guide double-stranded DNA substrates to DNA BLTs only, and not to RNA BLTs. Figure 21 summarizes that DNA molecules have affinity for RNA BLTs due to their ability to directly bind to the transposomal complex. This method describes various ways to efficiently guide RNA fragmentation to RNA BLTs and DNA fragmentation to DNA BLTs when the sample contains both RNA and DNA.

[0574] A. How to use 3 beads In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA uses three beads. An exemplary method using three beads is shown in Figure 24. In some embodiments, these three beads may be an RNA BLT, a DNA BLT, and an "RNA capture bead." The RNA capture bead may contain a poly-T capture oligonucleotide or another agent for capturing RNA without resulting in RNA tagging (i.e., the RNA capture bead lacks an active transpososome complex). Following DNA tagging and RNA capture by the RNA capture bead, the RNA is transferred from the RNA capture bead to the RNA BLT.

[0575] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA is described as follows: A sample containing RNA and DNA is immobilized on a first solid support for DNA, comprising a first transposome complex immobilized thereon, wherein the first transposome complex comprises a transposase and a first polynucleotide, and the first polynucleotide comprises a 3' portion containing the transposon terminal sequence and optionally a first tag, the first solid support. The method is applied to a second solid support having a first captured oligonucleotide immobilized thereon, The sample is applied to a mixture of first and second solid supports under the conditions that DNA is bound to a first transposome complex on a first solid support, fragmented, optionally tagged, and RNA is bound to a first capture oligonucleotide on a second solid support. The method involves transferring RNA bound to a second solid support to a third solid support, wherein the third solid support has a second capture oligonucleotide immobilized thereon that binds to the transferred RNA, and a second transpososome complex, the second transpososome complex comprising a transposase and a second polynucleotide, the second polynucleotide comprising a 3' portion containing the transposon terminal sequence and a second tag, and the transfer is performed accordingly. Under conditions for synthesizing cDNA and generating a DNA:RNA double helix immobilized on a second capture oligonucleotide, reverse transcriptase polymerase is added under conditions for generating an RNA double helix. The method comprises fragmenting a DNA:RNA double helix using a second transposome complex under conditions in which the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 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 the DNA strand.

[0576] In some embodiments, the first and / or second capture oligonucleotides include a poly-T sequence. In some embodiments, the RNA includes a sequence complementary to at least one portion of the first and / or second capture oligonucleotides. In some embodiments, the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotides. In some embodiments, the method further includes washing the solid support after applying the sample to the solid support to remove any unbound DNA or RNA.

[0577] B. Two Beads: A Method Using DNA BLT and RNA Beads Lacking Functional Transpososomes In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA uses two solid supports. In some embodiments, the solid supports are beads. In some embodiments, the two beads are DNA BLTs and "naked" RNA beads. As used herein, "naked" BLTs refer to beads that can ligate transposome complexes but do not have ligated active transposome complexes. For example, the transposome complex may lack the transposase or other essential component required for the activity of the transposome complex. Thus, "naked" BLTs allow for the addition of further components during a later step to enable fragmentation. The use of "naked" RNA BLTs allows for control over the timing of RNA fragmentation.

[0578] In some embodiments, a first solid support for immobilizing DNA comprises a first transposomal complex immobilized thereon, the first transposomal complex comprising a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing the transposon terminal sequence. In some embodiments, the first solid support further comprises a first tag.

[0579] In some embodiments, the second solid support has a capture oligonucleotide and a second polynucleotide immobilized thereon, the second polynucleotide comprising a 3' portion containing a transposon terminal sequence and a second tag. In some embodiments, the second tag comprises an RNA-specific barcode.

[0580] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA is described as follows: A sample containing RNA and DNA is immobilized on a first solid support for DNA, comprising a first transposome complex immobilized thereon, wherein the first transposome complex comprises a transposase and a first polynucleotide, and the first polynucleotide comprises a 3' portion containing the transposon terminal sequence and optionally a first tag, the first solid support. The present invention relates to applying a sample to a second solid support on which a capture oligonucleotide and a second polynucleotide are immobilized, wherein the second polynucleotide comprises a 3' portion containing a transposon terminal sequence and a second tag, and the sample is applied to a mixture of the first and second solid supports under conditions where DNA is bound to a first transposome complex on the first solid support, fragmented, optionally tagged, and RNA is bound to the capture oligonucleotide on the second solid support. The transposase is added under conditions in which the transposase binds to a second polynucleotide and forms a transposome complex on a second solid support. Under conditions for synthesizing cDNA and generating a DNA:RNA double helix immobilized on a second capture oligonucleotide, reverse transcriptase polymerase is added under conditions for generating an RNA double helix. The method comprises fragmenting a DNA:RNA double helix using a second transposome complex under conditions in which the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 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 the DNA strand.

[0581] In some embodiments, the first and / or second capture oligonucleotides include a poly-T sequence. In some embodiments, the RNA includes a sequence complementary to at least one portion of the first and / or second capture oligonucleotides. In some embodiments, the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotides. In some embodiments, the method further includes washing the solid support after applying the sample to the solid support to remove any unbound DNA or RNA.

[0582] C. Two Beads: Method Using DNA BLT and Inactivated RNA BLT In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA uses two solid supports. In some embodiments, the solid supports are beads. In some embodiments, the two beads are DNA BLTs and "inactivated" RNA BLTs. As used herein, "inactivated" RNA BLTs refer to RNA BLTs that have been reversibly inactivated. Thus, "inactivated" RNA BLTs allow for activation in a later step to enable fragmentation. Therefore, the use of "inactivated" RNA beads allows for control over the timing of RNA fragmentation.

[0583] In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA is described as follows: The present invention relates to applying a sample containing RNA and DNA to a mixture of the first and second solid supports, on which a first solid support for immobilizing DNA comprises a first transposome complex immobilized thereon, wherein the first transposome complex comprises a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a first tag; and a second solid support for immobilizing RNA, on which a second transposome complex is immobilized thereon, wherein the transposome complex comprises a transposase bound to a second polynucleotide, the second polynucleotide comprising a 3' portion containing a transposon terminal sequence and a second tag, wherein the sample is applied to a mixture of the first and second solid supports under conditions that the DNA binds to the first transposome complex on the first solid support, is fragmented, optionally tagged, and the RNA binds to the capture oligonucleotide on the second solid support. Under conditions that synthesize cDNA and generate a DNA:RNA double helix immobilized on a second capture oligonucleotide, reverse transcriptase polymerase is added. Activating the second transposomal complex, The method comprises fragmenting a DNA:RNA double helix using an activated second transposome complex under conditions in which the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a 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 the DNA strand.

[0584] In some embodiments, the transposome complex is reversibly inactivated by a transposome inactivator bound to the transposome complex. In some embodiments, the transposome inactivator is bound to the Tn5 binding site of the transposome complex. In some embodiments, the transposome inactivator comprises dephosphorylated ME', an extra base, an inhibitory double helix, and / or a thermolabile antibody. In some embodiments, the transposome complex is activated by the removal of the transposome inactivator.

[0585] In some embodiments, the capture probe comprises a poly-T sequence. In some embodiments, the RNA comprises a sequence complementary to at least one portion of the capture oligonucleotide. In some embodiments, the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotide. In some embodiments, the method further comprises washing the solid support after the application of the sample to remove any unbound DNA or RNA.

[0586] D. A method using two beads with sequential immobilization of DNA and RNA. In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA uses two solid supports. In some embodiments, the solid supports are beads. In some embodiments, the DNA and RNA in the sample are immobilized sequentially on separate solid supports.

[0587] In some embodiments, a method for preparing a tagged DNA:RNA fragment immobilized library from a sample containing RNA and DNA comprises applying the sample containing RNA and DNA to a first solid support for immobilizing DNA, wherein the first solid support comprises a first transposomal complex immobilized thereon, the first transposomal complex comprises a transposase and a first polynucleotide, the first polynucleotide comprising a 3' portion containing a transposon terminal sequence and optionally a first tag, and the sample is applied under conditions that the DNA binds to the first transposomal complex on the first solid support, is fragmented, and optionally tagged. The first solid support containing bound DNA is separated from the RNA, The method of applying RNA to a second solid support for immobilizing RNA immobilized on a capture oligonucleotide and a second transposomal complex, wherein the second transposomal complex comprises a transposase bound to a second polynucleotide, the second polynucleotide comprising a 3' portion containing the transposon terminal sequence and a second tag, and the RNA is bound to the capture oligonucleotide on the second solid support. Under conditions that synthesize cDNA and generate a DNA:RNA double helix immobilized on a second capture oligonucleotide, reverse transcriptase polymerase is added. The method comprises fragmenting a DNA:RNA double helix using an activated second transposome complex under conditions in which the DNA:RNA double helix is ​​tagged on the 5' end of one strand, thereby producing an immobilized library of DNA:RNA fragments, wherein at least one strand is 5' tagged with a 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 the DNA strand.

[0588] In some embodiments, the capture probe comprises a poly-T sequence. In some embodiments, the RNA comprises a sequence complementary to at least one portion of the capture oligonucleotide. In some embodiments, the first and / or second transposome complex is immobilized on a solid support via the first and / or second polynucleotide. In some embodiments, the method further comprises washing the solid support after the RNA application step to remove any unbound RNA. In some embodiments, the method further comprises recombining the first solid support having bound DNA with the immobilized library of tagged DNA:RNA fragments.

[0589] Method using two beads for serial immobilization of E.DNA and RNA and preparation of double-stranded cDNA In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA uses two solid supports. In some embodiments, the solid supports are beads. In some embodiments, DNA and double-stranded cDNA (ds-cDNA) generated from RNA are immobilized sequentially on separate solid supports.

[0590] In some embodiments, a method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, involves combining the sample containing RNA and DNA with a first solid support to immobilize the DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode, and further involves immobilizing the DNA and tagging on the first solid support. The method comprises performing tagging to prepare a tagged fragment containing a DNA-specific barcode, preparing double-stranded cDNA from RNA, and combining the sample with a second solid support to immobilize the cDNA, wherein the second solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and an RNA-specific barcode; and immobilizing the cDNA and performing tagging on the second solid support to prepare a tagged fragment containing an RNA-specific barcode.

[0591] In some embodiments, the ds-cDNA is generated in solution. In some embodiments, the first and second solid supports are combined after tagging on the second solid support, and each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0592] In some embodiments, the method involves splitting the first solid support having immobilized tagged fragments containing DNA-specific barcodes from the remainder of the sample, after tagmentation has been performed on the first solid support and before preparing double-stranded cDNA from RNA.

[0593] In some embodiments, the method includes separating the first solid support having the immobilized DNA from the rest of the sample after the DNA has been immobilized and before performing tagmentation on the first solid support to prepare tagged fragments containing DNA-specific barcodes.

[0594] In some embodiments, the preparation of double-stranded cDNA from RNA is performed by template switching.

[0595] F. A method using two beads for serial immobilization of DNA and RNA and preparation of DNA:RNA double helix. In some embodiments, a method for preparing an immobilized library of tagged DNA:RNA fragments from a sample containing RNA and DNA uses two solid supports. In some embodiments, the solid supports are beads. In some embodiments, the DNA and DNA:RNA double helix generated from the RNA are immobilized sequentially on separate solid supports.

[0596] In some embodiments, a method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, involves combining the sample containing RNA and DNA with a first solid support to immobilize the DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; immobilizing the DNA; and performing tagmentation on the first solid support to obtain the DNA-specific barcode. The method comprises preparing a tagged fragment containing a transposase, preparing a single strand of cDNA from RNA to produce a DNA:RNA double helix, and combining the sample with a second solid support to immobilize the DNA:RNA double helix, wherein the second solid support contains a transposomal complex immobilized thereon, and the transposomal complex contains a transposase having activity against the DNA:RNA double helix, and a transposon containing a transposon terminal sequence and an RNA-specific barcode; and immobilizing the DNA:RNA double helix and performing tagmentation on the second solid support to prepare a tagged fragment containing an RNA-specific barcode.

[0597] In some embodiments, the method further comprises combining the first and second solid supports after performing tagmentation on a second solid support, wherein each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0598] In some embodiments, the method further comprises splitting the first solid support having immobilized tagged fragments containing DNA-specific barcodes from the remainder of the sample, after tagmentation has been performed on the first solid support and before preparing a single strand of cDNA from RNA to produce a DNA:RNA double helix.

[0599] In some embodiments, the method further includes separating the first solid support having the immobilized DNA from the rest of the sample after immobilizing the DNA and before performing tagmentation on the first solid support to prepare tagged fragments containing DNA-specific barcodes.

[0600] In some embodiments, the DNA:RNA double helix is ​​generated in solution. In some embodiments, the first and second solid supports are combined after tagging on the second solid support, and each solid support has an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0601] G. Method using two beads: DNA tagging or DNA:RNA double-strand tagging using DNA BLT and liquid-phase cDNA In some embodiments, DNA in a sample may be tagged using DNA BLT, and then double-stranded cDNA or DNA:RNA double helix prepared from RNA may be tagged in solution. In other words, the cDNA or DNA:RNA double helix may react with a liquid-phase transposome complex after preparing the tagged DNA fragment. In some embodiments, the tagging of the double-stranded cDNA or DNA:RNA double helix incorporates a sequence that can hybridize to a capture probe. In some embodiments, the tagged fragment generated from the cDNA or DNA:RNA double helix may be bound to its surface by a solid support containing a capture probe.

[0602] In some embodiments, a method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, involves combining the sample containing RNA and DNA with a first solid support to immobilize the DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; immobilizing the DNA; and performing tagmentation on the first solid support to tag the DNA-specific barcode. The method comprises preparing a tagged fragment, preparing double-stranded cDNA from RNA, and preparing a tagged fragment of double-stranded cDNA by performing tagmentation on the double-stranded DNA in solution, wherein the transposome complex in solution comprises a transposase and a transposon containing a transposon terminal sequence, an RNA-specific barcode, and a sequence that hybridizes to a capture probe, and the tagged fragment contains an RNA-specific barcode and a sequence that hybridizes to a capture probe, and further comprising combining the sample with a second solid support having a surface containing a capture probe, and immobilizing the tagged fragment of double-stranded cDNA on the second solid support.

[0603] In some embodiments, the method further comprises immobilizing a tagged fragment of double-stranded cDNA on a second solid support, and then combining the first and second solid supports, each solid support having an immobilized tagged fragment containing either a DNA-specific barcode or an RNA-specific barcode.

[0604] In some embodiments, the method further comprises splitting the first solid support having immobilized tagged fragments containing DNA-specific barcodes from the remainder of the sample, after tagmentation on the first solid support and before double-stranded cDNA from RNA.

[0605] In some embodiments, the method further includes separating the first solid support having the immobilized DNA from the rest of the sample after immobilizing the DNA and before performing tagmentation on the first solid support to prepare tagged fragments containing DNA-specific barcodes.

[0606] In some embodiments, a method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragments contain either a DNA-specific barcode or an RNA-specific barcode, comprises: combining the sample containing RNA and DNA with a first solid support for DNA immobilization, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode; immobilizing the DNA; and performing tagmentation on the first solid support to prepare tagged fragments containing a DNA-specific barcode. The method involves preparing a single strand of cDNA from RNA to produce a DNA:RNA double helix, and preparing a tagged fragment of the DNA:RNA double helix by performing tagmentation on the DNA:RNA double helix in solution, wherein the transposome complex in solution comprises a transposase and a transposon containing a transposon terminal sequence, an RNA-specific barcode, and a sequence that hybridizes to a capture probe, and the tagged fragment contains an RNA-specific barcode and a sequence that hybridizes to a capture probe, and the method involves combining the sample with a second solid support having a surface containing a capture probe, and immobilizing the tagged fragment of the DNA:RNA double helix on the second solid support.

[0607] In some embodiments, the capture probe includes nucleic acid. [Examples]

[0608] Examples Example 1. Preparation of an RNA sequencing library using BLT containing captured oligonucleotides. RNA sequencing libraries can be prepared from full-length total RNA derived from RNA-containing samples using the methods described herein.

[0609] mRNA derived from a sample can be immobilized on RNA bead-linked transposomes (BLTs) by binding the mRNA's poly(A) tail to a poly(T) capture oligonucleotide on a bead.

[0610] Next, reverse transcriptase is used for cDNA synthesis. Reverse transcriptase polymerase is used to generate DNA:RNA double helix from the target RNA bound to the beads. Exemplary reagents for cDNA synthesis include reverse transcriptase, random primers, oligo-dT primers, dNTPs, and / or RNase inhibitors. Both random primers and oligo-dT primers may be used in the cDNA synthesis reaction.

[0611] The cDNA synthesis reaction can be carried out at 42°C for 90 minutes, followed by 85°C for 5 minutes. Washing of the sample after cDNA synthesis is not necessary.

[0612] DNA:RNA double-strand tagging can then be performed using RNA BLTs. Various BLTs that can be used to generate RNA BLTs are described. DNA:RNA double-strand tagging functions to generate DNA:RNA fragments that are immobilized onto beads by transpososomes.

[0613] The BLT transposomal complex may contain a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion containing the transposon terminal sequence and a first tag. In this way, the first tag is incorporated during DNA:RNA fragment generation.

[0614] After fragmentation, strand exchange and gap-filling ligation are performed. In some embodiments, a second tagmentation reaction is performed to generate a double-stranded DNA fragment with one end submerged in solution. The second tagmentation reaction may incorporate a second tag.

[0615] The library can then be released for further procedures, which may be carried out in a tube or flow cell. This method enables a method for providing the full-length sequence of mRNA.

[0616] Such a method can also be used with an activatable BLT containing an immobilized oligonucleotide that can bind to a transposome in solution. Such beads are shown in Figure 13, where the A' sequence in the second transposon can bind to a short A sequence in the immobilized polynucleotide used to bind to the transposome. The immobilized oligonucleotide may also include an adapter (such as a P5 sequence) and a bead code (BC), as shown in Figure 13.

[0617] Example 2. Preparation of a multi-omics sequencing library using three beads As shown in Figure 24, an approach using three beads can be used to generate RNA and DNA sequencing libraries from a sample containing RNA and DNA. This method utilizes RNA capture beads that can bind to RNA but do not have transpososomes.

[0618] In this method, DNA BLTs can be used for DNA tagging while RNA is captured by RNA capture beads. After washing, the RNA is transferred from the RNA capture beads to the RNA BLTs. After reverse transcription, the DNA:RNA double helix can be fragmented and tagged by the active RNA BLTs. After strand exchange and gap-filling ligation, the RNA and DNA sequencing libraries can then be released from their respective BLTs.

[0619] Example 3. Preparation of a multi-omics sequencing library using DNA BLT and RNA beads lacking the active transpososome complex. As shown in Figure 23, RNA and DNA sequencing libraries can be generated from RNA and DNA-containing samples using DNA BLTs and RNA BLTs lacking transposase, followed by the addition of transposase to produce RNA BLTs. BLTs that bind to RNA but lack transposase may be referred to as "naked" RNA BLTs.

[0620] In this method, DNA can be tagged using a DNA BLT while the RNA is captured by a naked RNA BLT. After washing, a transposase is added to activate the RNA BLT. After reverse transcription, the DNA:RNA double helix can be tagged by the activated RNA BLT. After strand exchange and gap-filling ligation, the RNA and DNA sequencing libraries can then be released from their respective BLTs.

[0621] Example 4. Preparation of a multi-omics sequencing library using DNA BLT and inactivated RNA BLT. Using DNA BLTs and reversibly "inactivated" RNA BLTs, RNA and DNA sequencing libraries can be generated from RNA and DNA-containing samples, as shown in Figure 22.

[0622] In this method, DNA BLTs can be used to tag DNA, while RNA is captured by inactivated RNA BLTs. After washing, the inactivation of RNA BLTs is reversed (i.e., the RNA BLT transposomal complex is activated). After activation, reverse transcription can be performed to fragment the DNA:RNA double helix and tag it with the activated RNA BLTs. After strand exchange and gap-filling ligation, RNA and ...

Claims

1. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragment includes either a DNA-specific barcode or an RNA-specific barcode, and the method is a. Combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode. b. Immobilizing the DNA, c. Performing tagging on the first solid support to prepare a tagged fragment containing a DNA-specific barcode, d. Preparing double-stranded cDNA from the RNA, e. Combining the sample with a second solid support in order to immobilize cDNA, wherein the second solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and an RNA-specific barcode. f. A method comprising immobilizing the cDNA and performing tagmentation on the second solid support to prepare a tagged fragment containing an RNA-specific barcode.

2. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragment includes either a DNA-specific barcode or an RNA-specific barcode, and the method is a. Combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode. b. Immobilizing the DNA, c. Performing tagging on the first solid support to prepare a tagged fragment containing a DNA-specific barcode, d. Preparing a single strand of cDNA from the RNA to produce a DNA:RNA double helix, e. Combining the sample with a second solid support in order to immobilize a DNA:RNA double helix, wherein the second solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase having activity toward DNA:RNA double helix, and a transposon containing a transposon terminal sequence and an RNA-specific barcode. f. A method comprising immobilizing the DNA:RNA double helix and performing tagmentation on the second solid support to prepare a tagged fragment containing an RNA-specific barcode.

3. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragment includes either a DNA-specific barcode or an RNA-specific barcode, and the method is a. Combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode. b. Immobilizing the DNA, c. Performing tagging on the first solid support to prepare a tagged fragment containing a DNA-specific barcode, d. Preparing double-stranded cDNA from the RNA, e. Preparing a tagged fragment of the double-stranded cDNA by performing tagmentation on the double-stranded cDNA in solution, wherein the transposome complex in solution comprises a transposase and a transposon containing a transposon terminal sequence, an RNA-specific barcode, and a sequence that hybridizes to a capture probe, and the tagged fragment comprises the RNA-specific barcode and the sequence that hybridizes to the capture probe. f. The sample is combined with a second solid support having a surface containing a capture probe, g. A method comprising immobilizing the tagged fragment of the double-stranded cDNA onto the second solid support.

4. A method for preparing an immobilized library of tagged fragments from a sample containing RNA and DNA, wherein the tagged fragment includes either a DNA-specific barcode or an RNA-specific barcode, and the method is a. Combining a sample containing RNA and DNA with a first solid support in order to immobilize DNA, wherein the first solid support includes a transposomal complex immobilized thereon, and the transposomal complex includes a transposase and a transposon containing a transposon terminal sequence and a DNA-specific barcode. b. Immobilizing the DNA, c. Performing tagging on the first solid support to prepare a tagged fragment containing a DNA-specific barcode, d. Preparing a single strand of cDNA from the RNA to produce a DNA:RNA double helix, e. Preparing a tagged fragment of the DNA:RNA double helix by performing tagmentation on the DNA:RNA double helix in solution, wherein the transposome complex in solution comprises a transposase and a transposon containing a transposon terminal sequence, an RNA-specific barcode, and a sequence that hybridizes to a capture probe, and the tagged fragment contains the RNA-specific barcode and the sequence that hybridizes to the capture probe. f. The sample is combined with a second solid support having a surface containing a capture probe, g. A method comprising immobilizing the tagged DNA:RNA double helix fragment onto the second solid support.

5. The method according to any one of claims 1 to 4, further comprising adding synthetic double-stranded DNA to the first solid support after performing tagmentation on the first solid support, wherein optionally the synthetic double-stranded DNA contains uracil and the amplification is performed using uracil-intolerant DNA polymerase.

6. The DNA-specific barcode and the RNA-specific barcode include different primer-binding sequences, and optionally the method may be: a. Amplifying a tagged fragment containing the DNA-specific barcode using a primer that binds to the primer-binding sequence contained in the DNA-specific barcode. b. Amplifying a tagged fragment containing the RNA-specific barcode using a primer that binds to the primer-binding sequence contained in the RNA-specific barcode, or c. The method according to any one of claims 1 to 5, further comprising amplifying a tagged fragment containing the DNA-specific barcode and a tagged fragment containing the RNA-specific barcode using a primer mix comprising a primer that binds to the primer-binding sequence contained in the DNA-specific barcode and a primer that binds to the primer-binding sequence contained in the RNA-specific barcode.

7. The transpososome complex is reversibly inactivated before tagmentation, and tagmentation includes activating the transpososome complex, optionally, a. The transposome complex is reversibly inactivated by a transposome inactivator bound to the transposome complex, and optionally, i. The transposome inactivator is bound to the Tn5 binding site of the transposome complex, or ii. The transposome inactivator comprises a dephosphorylated ME', an extra base, an inhibitory double helix, and / or a thermolabile antibody, and / or b. The method according to any one of claims 1 to 6, wherein the transposome complex is activated by removing the transposome inactivator.

8. The method according to any one of claims 1 to 7, wherein the solid support is a bead.

9. The method according to claim 2 or 4, wherein tagmentation produces double-stranded DNA:RNA double helix crosslinked to two immobilized transposome complexes on the solid support, and optionally, the second strand of DNA is synthesized to prepare the double-stranded DNA before tagmentation.

10. The method according to claim 9, wherein the length of the crosslinked double helix is ​​100 base pairs to 1500 base pairs.

11. The method according to any one of claims 1 to 10, wherein the sample is blood.

12. The method according to any one of claims 1 to 11, wherein the sample is a cell lysate.

13. The method according to claim 12, wherein the cell lysate is a crude cell lysate.

14. The method according to claim 3 or 4, wherein the capture probe includes a polyT array.

15. The method according to claim 3 or 4, wherein the capture probe includes a P5 or P7 sequence.

16. The method according to claim 1 or 2, further comprising combining the first and second solid supports after performing tagmentation on the second solid support, wherein each solid support has an immobilized tagged fragment containing either the DNA-specific barcode or the RNA-specific barcode.

17. The method according to claim 1 or 2, further comprising splitting the first solid support having the immobilized tagged fragment containing a DNA-specific barcode from the remainder of the sample, after performing tagmentation on the first solid support and before preparing double-stranded cDNA from the RNA.

18. The method according to claim 1 or 2, further comprising, after immobilizing the DNA and before performing tagmentation on the first solid support, separating the first solid support having the immobilized DNA from the rest of the sample to prepare a tagged fragment containing a DNA-specific barcode.

19. The method according to claim 1 or 3, wherein the preparation of double-stranded cDNA from the RNA is performed by template switching.

20. The method according to claim 2 or 4, wherein the tagged fragment containing the RNA-specific barcode includes at least one strand that is 5' tagged with the RNA-specific barcode.

21. The method according to claim 2, further comprising providing a third solid support, wherein the third solid support comprises a capture oligonucleotide configured to bind to the RNA.

22. The method according to claim 21, further comprising transferring the RNA bound to the third solid support to the second solid support.

23. The method according to claim 21 or 22, wherein the transposome complex on the second solid support includes a 3' portion having the transposon terminal sequence.

24. The method according to claim 1 or 2, wherein the transposome complex of the first solid support, the transposome complex of the second solid support, or both, include a unique molecular identifier (UMI).

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