Improved methods for isothermal complementary DNA and library preparation.

Abstract

Described herein are compositions and methods for preparing double-stranded complementary DNA (cDNA) from RNA. In some embodiments, these methods allow for isothermal preparation of cDNA. In some embodiments, these methods allow for mesophilic or thermostable preparation of cDNA. Also described herein are compositions and methods for preparing libraries of cDNA and double-stranded cDNA fragments in a single reaction vessel.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 167,909, filed March 30, 2021, and Application No. 63 / 234,114, filed August 17, 2021, each of which is incorporated by reference in its entirety for any purpose.

[0002] Sequence Listing This application has been filed with a sequence listing in electronic format. The sequence listing is provided as a file entitled "2022-03-25_01243-0026-00PCT_Seq_List_ST25.txt" (created on March 25, 2022, size 6,160 bytes). The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to improved compositions and methods for double-stranded complementary DNA (ds-cDNA) preparation. These compositions can enable an isothermal method for preparing cDNA from RNA. The present disclosure also relates to compositions and methods for preparing libraries of ds-cDNA and ds-cDNA fragments. [Background technology]

[0004] RNA is an important biological molecule because its study facilitates the understanding of functional biological processes in cells (i.e., transcriptome or transcriptomics studies) and regulatory elements (such as long non-coding RNA (lncRNA) or microRNA (miRNA)). Analysis of RNA can also be useful for the detection of infectious agents (e.g., RNA viruses). A prerequisite for the study of RNA is often the conversion of the RNA to a DNA copy, since DNA has properties that enhance its chemical stability and make it amenable to manipulation using common molecular biology tools and reagents. Furthermore, for many types of sequencing library preparation, double-stranded DNA is required for the ligases used for adapter ligation or transposomes used for adapter addition by tagmentation. Thus, single-stranded RNA often must undergo conversion to double-stranded complementary DNA (ds-cDNA) prior to library preparation, which significantly increases the turnaround time and hands-on time in the RNA workflow.

[0005] Furthermore, the conversion of RNA to ds-cDNA requires coordinated temperature regulation in a programmable thermal cycler, as shown in FIG. 1. In conventional methods, the conversion of RNA to DNA is performed through a process of first-strand cDNA synthesis by reverse transcription, where an RNA molecule is directly copied by reverse transcriptase. The second-strand cDNA is formed by direct displacement of the original RNA molecule. In most embodiments, this is accomplished through a procedure similar to that developed by Gubler and Hoffman Gene 25:263-269 (1983). Many library preparation protocols (e.g., Illumina RNA-Seq library preparation) use methods similar to Gubler and Hoffman to produce blunt-ended double-stranded cDNA suitable for adapter addition.

[0006] The Gubler and Hoffman procedure was designed for efficient full-transcript-length double-stranded cDNA with blunt ends for easy ligation into cloning vectors to enable further studies using methods available in the 1980s. However, the goal of many next generation sequencing (NGS) library preparations (such as Illumina RNA-Seq library preparation) is different, and fragmented representations of RNA molecules (not single long cDNAs) are required to enable efficient sequencing. Furthermore, newer library construction methods such as tagmentation (such as Illumina DNA Flex PCR-Free (for research use only, RUO) (Illumina) technology, formerly known as Illumina's Nextera technology, and related products that use Tn5 transposomes) do not require end-repair molecules for adapter addition. Therefore, new ds-cDNA synthesis procedures that save time and simplify workflows would be beneficial for RNA library preparation protocols.

[0007] Therefore, more rapid conversion of RNA to a form compatible with library preparation is of great interest. A recent publication by Di et al., Proc. Natl. Acad. Sci. USA 117:2886-2893 (2020) suggests that RNA:DNA hybrids (products of first strand cDNA synthesis in about 40 min) can be tagmented by Tn5 transposomes and rapidly converted into RNA sequencing libraries, but that the use of RNA:DNA hybrids may result in lower library yields.

[0008] The methods described herein facilitate the conversion of a single-stranded RNA sample into double-stranded cDNA. The methods may be performed using a composition that includes a mixture of enzymes, including (1) a reverse transcriptase to prepare the first strand of cDNA and generate a DNA:RNA duplex, and (2) an RNA nickase (such as RNAse H) that can "nick" the RNA strand of this DNA:RNA duplex, allowing the RNA fragment to be used as a primer to initiate synthesis of the second strand of cDNA.

[0009] These methods of the present invention may eliminate the need for a computer-controlled programmable thermal cycler, reducing hands-on steps, improving total turnaround time, and simplifying the automation of library preparation from RNA samples. Furthermore, these simplified methods may enable isothermal cDNA preparation, and mesophilic and thermostable compositions including enzymes are described. Some methods described herein enable cDNA preparation in a single 10-minute reaction performed at a single temperature (i.e., by an isothermal reaction). In addition, the methods described herein enable library preparation from RNA samples with shorter incubation times and simpler workflows. In some cases, the inventive methods of cDNA preparation use random primers (i.e., randomers) and omit a step of PCR-like amplification to avoid the introduction of sequence-specific bias that can be found in other methods, such as EP 1929045. Furthermore, certain RNA nickases, such as RNAse H, are known to randomly nick RNA strands in a DNA:RNA duplex, and therefore the step of nicking the RNA also does not introduce sequence-specific bias. Applications of the ds-cDNA and library preparation methods of the present invention include disease surveillance and other assays for rapid quantitative identification of RNA molecules, where a simplified workflow and easily automated procedure is highly desirable. For example, workflows for applications such as enriching metagenomic RNA for viruses or pathogens of interest can be simplified by this procedure, making it more suitable for pathogen surveillance. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] European Patent No. 1929045 [Non-patent literature]

[0011] [Non-Patent Document 1] Gubler and Hoffman Gene25:263-269(1983) [Non-Patent Document 2] Di et al.,Proc.Natl.Acad.Sci.USA117:2886-2893(2020) Summary of the Invention [Means for solving the problem]

[0012] According to this description, compositions and methods for preparing double-stranded complementary DNA (cDNA) are described herein. In some embodiments, these compositions and methods can allow for the isothermal preparation of cDNA from RNA contained in a sample. In some embodiments, the compositions and methods allow for the preparation of a library of double-stranded DNA fragments from RNA contained in a sample.

[0013] Embodiment 1. A composition for preparing double-stranded cDNA from RNA by isothermal reaction, A reverse transcriptase; B RNA nickase; c. a DNA polymerase having strand displacement activity or 5'-3' exonuclease activity; d. A composition comprising dNTPs.

[0014] Embodiment 2. The composition of embodiment 1, wherein the activity of the reverse transcriptase is greater than the activity of the RNA nickase.

[0015] Embodiment 3. The composition of embodiment 1 or embodiment 2, wherein the reverse transcriptase and the RNA nickase are comprised in a single enzyme.

[0016] Embodiment 4. The composition of any one of embodiments 1 to 3, wherein the reverse transcriptase and the DNA polymerase are comprised in a single enzyme having both RNA-dependent and DNA-dependent polymerase activity.

[0017] Embodiment 5. The composition of embodiment 4, wherein the single enzyme reduces competition between reverse transcriptase and DNA polymerase.

[0018] Embodiment 6. The composition of any one of embodiments 1 to 5, wherein the DNA polymerase has strand displacement activity.

[0019] Embodiment 7. The composition of any one of embodiments 1 to 6, wherein the DNA polymerase has 5'-3' exonuclease activity.

[0020] Embodiment 8. The composition of any one of embodiments 1 to 7, wherein the reverse transcriptase is a polymerase having RNA-dependent DNA polymerase activity, and optionally, the reverse transcriptase is Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, a retrotransposon-derived reverse transcriptase, or a group II intron reverse transcriptase.

[0021] Embodiment 9. A composition according to any one of embodiments 1 to 8, wherein the RNA nickase is RNAse H.

[0022] Embodiment 10. A composition according to any one of embodiments 1 to 9, wherein the RNAse H is from Thermus thermophilus.

[0023] Embodiment 11. The composition according to any one of embodiments 1 to 10, wherein the DNA polymerase is E. coli DNA polymerase I or Bst DNA polymerase.

[0024] Embodiment 12. The composition of any one of embodiments 1 to 11, wherein the reverse transcriptase, RNA nickase, and / or DNA polymerase are mesophilic enzymes.

[0025] Embodiment 13. The composition of embodiment 12, wherein the mesophilic enzyme is active at 37°C to 49°C.

[0026] Embodiment 14. The composition of embodiment 13, wherein the mesophilic enzyme is active at 37° C.

[0027] Embodiment 15. The composition of any one of embodiments 12 to 14, wherein the mesophilic reverse transcriptase is MMLV reverse transcriptase.

[0028] Embodiment 16. A composition according to any one of embodiments 12 to 15, wherein the mesophilic RNA nickase is E. coli RNAse H.

[0029] Embodiment 17. A composition according to any one of embodiments 12 to 16, wherein the mesophilic polymerase is E. coli DNA polymerase I.

[0030] Embodiment 18. A composition according to any one of embodiments 1 to 11, wherein the reverse transcriptase, RNA nickase, and / or DNA polymerase are thermostable enzymes.

[0031] Embodiment 19. The composition of embodiment 18, wherein the thermostable enzyme has activity at 50°C to 72°C.

[0032] Embodiment 20. The composition of embodiment 19, wherein the thermostable enzyme has activity at 50° C.

[0033] Embodiment 21. The composition of any one of embodiments 18 to 20, wherein the thermostable reverse transcriptase is a thermostable variant of MMLV reverse transcriptase, or a thermostable reverse transcriptase derived from a retrotransposon, or a group II intron reverse transcriptase.

[0034] Embodiment 22. A composition according to any one of embodiments 18 to 21, wherein the thermostable RNA nickase is RNAse H from Thermus thermophilus.

[0035] Embodiment 23. The composition of any one of embodiments 18 to 22, wherein the thermostable DNA polymerase is Bst DNA polymerase.

[0036] Embodiment 24. A composition according to any one of embodiments 1 to 23, wherein the RNA is bound to a primer prior to preparing the double-stranded cDNA.

[0037] Embodiment 25. The composition of any one of embodiments 1 to 24, wherein the composition further comprises one or more additives selected from DTT, BSA, Tris pH 7.5, KCl, and / or MgCl2.

[0038] Embodiment 26. A composition according to any one of embodiments 1 to 25, wherein the composition has lower units / μl of RNA nickase compared to units / μl of reverse transcriptase and / or DNA polymerase.

[0039] Embodiment 27. A composition according to any one of embodiments 1 to 26, wherein the composition further comprises an RNA nickase inhibitor.

[0040] Embodiment 28 The composition of embodiment 27, wherein the RNA nickase inhibitor reduces the activity of RNA nickase.

[0041] Embodiment 29. A composition according to any one of embodiments 1 to 28, wherein the units / μl of RNA nickase and DNA polymerase in the composition overlap.

[0042] Embodiment 30. A composition according to any one of embodiments 1 to 29, wherein the activity of the DNA polymerase in the composition is 2-fold to 100-fold higher than the activity of the RNA nickase in the composition.

[0043] Embodiment 31. A composition according to any one of embodiments 1 to 30, wherein the activity of the reverse transcriptase in the composition is 10 to 1,000 times higher than the activity of the RNA nickase in the composition.

[0044] Embodiment 32. A composition according to any one of embodiments 1 to 31, wherein the reverse transcriptase activity in the composition is between 0.32 U / μl and 4.8 U / μl.

[0045] Embodiment 33. A composition according to any one of embodiments 1 to 32, wherein the DNA polymerase activity in the composition is between 0.04 U / μl and 0.37 U / μl.

[0046] Embodiment 34. A composition according to any one of embodiments 1 to 33, wherein the RNA nickase activity in the composition is between 0.004 U / μl and 0.04 U / μl.

[0047] Embodiment 35. A composition according to any one of embodiments 1 to 33, wherein the RNA nickase activity in the composition is greater than 0.04 U / μl.

[0048] Embodiment 36. The composition of embodiment 35, wherein the RNA nickase activity in the composition is between 0.05 U / μl and 0.3 U / μl.

[0049] Embodiment 37. A method for preparing double-stranded cDNA, comprising: a. combining a primer with a sample containing RNA, allowing the primer to bind to the RNA; b. Combining the sample with the composition of any one of embodiments 1 to 36, and preparing double-stranded cDNA by an isothermal reaction.

[0050] Embodiment 38. The method of embodiment 37, wherein the primer comprises a randomer primer.

[0051] Embodiment 39. The method of embodiment 37 or 38, wherein the primer comprises a primer that specifically binds to a sequence contained in the RNA.

[0052] Embodiment 40. The method of any one of embodiments 37 to 39, wherein the primer comprises a hexamer primer.

[0053] Embodiment 41. The method of any one of embodiments 37 to 40, wherein the primer comprises a primer containing a chemically modified nucleotide.

[0054] Embodiment 42 The method of embodiment 41, wherein the primer comprises a chemically modified nucleotide that renders the RNA bound by the primer resistant to cleavage by RNA nickase.

[0055] Embodiment 43 The method of embodiment 42, wherein the RNA nickase is RNAse H and the RNA bound by the primer is resistant to cleavage by RNAse H.

[0056] Embodiment 44 The method of any one of embodiments 41 to 43, wherein the chemically modified nucleotide comprises a methylphosphonate residue.

[0057] Embodiment 45. The method of any one of embodiments 37 to 44, wherein the reverse transcriptase produces a first strand of cDNA.

[0058] Embodiment 46 The method of embodiment 45, wherein the reverse transcriptase produces a DNA:RNA duplex comprising a first strand of cDNA and a strand of RNA.

[0059] Embodiment 47 The method of embodiment 46, wherein RNAse H nicks the RNA strand in a DNA:RNA duplex to produce RNA fragments.

[0060] Embodiment 48 The method of embodiment 47, wherein the DNA polymerase extends the second strand of DNA by priming from the RNA fragment.

[0061] Embodiment 49. The method of embodiment 47 or embodiment 48, wherein the 5'-3' activity of an RNA nickase and / or a DNA polymerase removes RNA fragments and 3' RNA overhangs.

[0062] Embodiment 50. The method according to any one of embodiments 37 to 49, wherein the DNA polymerase has 5'-3' exonuclease activity and / or 3'-5' exonuclease activity, which produces blunt-ended double-stranded cDNA.

[0063] Embodiment 51. The method of any one of embodiments 37 to 50, wherein dNTPs are used by both the reverse transcriptase and the DNA polymerase.

[0064] Embodiment 52. The method of any one of embodiments 37 to 51, wherein the isothermal reaction is at a temperature of 30°C to 49°C.

[0065] Embodiment 53. The method of embodiment 52, wherein the isothermal reaction is at a temperature of 37° C.

[0066] Embodiment 54. The method of any one of embodiments 37 to 51, wherein the isothermal reaction is at a temperature of 50°C to 72°C.

[0067] Embodiment 55. The method of embodiment 54, wherein the isothermal reaction is at a temperature of 50° C.

[0068] Embodiment 56 The method of embodiment 54 or embodiment 55, wherein the RNA exhibits a secondary structure that normally inhibits first strand synthesis at temperatures below 50° C.

[0069] Embodiment 57. The method of any one of embodiments 37 to 56, wherein the rate of production of the first strand of cDNA by reverse transcriptase is greater than the rate of nicking of RNA by RNA nickase.

[0070] Embodiment 58 The method of embodiment 57, wherein the activity of the reverse transcriptase exceeds the activity of the RNA nickase.

[0071] Embodiment 59. The method of any one of embodiments 37 to 58, wherein the isothermal reaction is incubated for 60 minutes or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less.

[0072] Embodiment 60 The method of embodiment 59, wherein the isothermal reaction is incubated for 15 minutes or less.

[0073] Embodiment 61. The method of any one of embodiments 37 to 60, wherein incubation for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes provides double-stranded cDNA for library preparation.

[0074] Embodiment 62. The method of any one of embodiments 37 to 61, further comprising depleting off-target RNA or enriching mRNA using a sample containing RNA prior to combining the primer with the sample containing RNA.

[0075] Embodiment 63 The method of embodiment 62, wherein the off-target RNA is a ribosomal RNA.

[0076] Embodiment 64. The method of embodiment 62 or embodiment 63, wherein enriching for mRNA comprises binding of mRNA to amplification or capture beads using poly-T primers.

[0077] Embodiment 65. The method of embodiment 64, wherein the capture beads comprise a surface having capture oligonucleotides comprising a poly-T sequence.

[0078] Embodiment 66. A composition for preparing a library of double-stranded cDNA fragments from RNA, comprising: A reverse transcriptase; B RNA nickase; c. a DNA polymerase having strand displacement activity or 5'-3' exonuclease activity; dNTPs, e. A transposome complex, the transposome complex being i. transposase, ii. a first transposon comprising a transposon end sequence; iii. a transposome complex comprising a second transposon comprising a sequence that is fully or partially complementary to the transposon end sequence.

[0079] Embodiment 67. The composition comprises Mg 2+ 67. The composition of embodiment 66, further comprising:

[0080] Embodiment 68. Mg 2+ Concentration of 1 mM to 50 mM, optionally Mg 2+ The composition of embodiment 67, wherein the concentration is 5 mM to 20 mM, and further optionally, the Mg2+ concentration is 8 mM.

[0081] Embodiment 69. The composition of any one of embodiments 66 to 68, wherein the library is prepared by an isothermal reaction.

[0082] Embodiment 70. The composition of any one of embodiments 66 to 69, wherein the RNA is bound to the primer before preparing the library.

[0083] Embodiment 71. A composition according to any one of embodiments 66 to 70, wherein the transposome complex is immobilized on a solid support.

[0084] Embodiment 72. The composition of embodiment 71, wherein the solid support is a bead.

[0085] Embodiment 73 The composition of embodiment 71 or embodiment 72, wherein the first transposon comprises an affinity element.

[0086] Embodiment 74 The composition of embodiment 73, wherein the affinity element is attached to the 5' end of the first transposon.

[0087] Embodiment 75. The composition of embodiment 71 or 72, wherein the first transposon comprises a linker.

[0088] Embodiment 76. The composition of embodiment 75, wherein the linker has a first end attached to the 5' end of the first transposon and a second end attached to the affinity element.

[0089] Embodiment 77 The composition of embodiment 71 or 72, wherein the second transposon comprises an affinity element.

[0090] Embodiment 78 The composition of embodiment 77, wherein the affinity element is attached to the 3' end of the second transposon.

[0091] Embodiment 79. The composition of embodiment 71 or 72, wherein the second transposon comprises a linker.

[0092] Embodiment 80. The composition of embodiment 79, wherein the linker has a first end attached to the 3' end of the second transposon and a second end attached to the affinity element.

[0093] Embodiment 81. The composition of any one of embodiments 73-74, 76-78, or 80, wherein the affinity element is biotin or double biotin.

[0094] Embodiment 82. The transposome complex is 1 mm 2 At least 10 per 3 , 10 4 , 10 5 , or 10 6 The composition according to any one of embodiments 66 to 81, wherein the complex is present on the solid support at a density of about 100 μg / ml.

[0095] Embodiment 83. The composition of embodiments 66 to 82, wherein the first transposon further comprises one or more adapter sequences.

[0096] Embodiment 84. The composition of embodiment 83, wherein the first transposon comprises a 3' transposon end sequence and a 5' adapter sequence.

[0097] Embodiment 85. The composition of any one of embodiments 66 to 84, wherein the transposase is a Tn5 transposase.

[0098] Embodiment 86 The composition of embodiment 85, wherein the Tn5 transposase is a hyperactive Tn5 transposase.

[0099] Embodiment 87. A composition according to any one of embodiments 66 to 86, wherein the activity of reverse transcriptase is greater than the activity of RNA nickase.

[0100] Embodiment 88. A composition according to any one of embodiments 66 to 87, wherein the reverse transcriptase and the RNA nickase are contained in a single enzyme.

[0101] Embodiment 89. The composition according to any one of embodiments 66 to 88, wherein the reverse transcriptase and the DNA polymerase are comprised in a single enzyme having both RNA-dependent polymerase activity and DNA-dependent polymerase activity.

[0102] Embodiment 90. The composition of embodiment 89, wherein the single enzyme reduces the competition between reverse transcriptase and DNA polymerase.

[0103] Embodiment 91. The composition of any one of embodiments 66 to 90, wherein the DNA polymerase has strand displacement activity.

[0104] Embodiment 92. The composition of any one of embodiments 66 to 91, wherein the DNA polymerase has 5'-3' exonuclease activity.

[0105] Embodiment 93. The composition of any one of embodiments 66 to 92, wherein the reverse transcriptase is a polymerase having RNA-dependent DNA polymerase activity, and optionally, the reverse transcriptase is an MMLV reverse transcriptase, a retrotransposon-derived reverse transcriptase, or a group II intron reverse transcriptase.

[0106] Embodiment 94. A composition described in any one of embodiments 66 to 93, wherein the RNA nickase is RNAse H.

[0107] Embodiment 95. The composition of any one of embodiments 66 to 94, wherein the DNA polymerase is E. coli DNA polymerase I.

[0108] Embodiment 96. The composition of any one of embodiments 66 to 95, wherein the reverse transcriptase, RNA nickase, and / or DNA polymerase are mesophilic enzymes.

[0109] Embodiment 97. The composition of embodiment 96, wherein the mesophilic enzyme is active at 37°C to 49°C.

[0110] Embodiment 98. The composition of embodiment 97, wherein the mesophilic enzyme is active at 37° C.

[0111] Embodiment 99. The composition of any one of embodiments 96 to 98, wherein the mesophilic reverse transcriptase is MMLV reverse transcriptase.

[0112] Embodiment 100. The composition of any one of embodiments 96 to 99, wherein the mesophilic RNA nickase is E. coli RNase H.

[0113] Embodiment 101. The composition of any one of embodiments 96 to 100, wherein the mesophilic polymerase is E. coli DNA polymerase I.

[0114] Embodiment 102. The composition of any one of embodiments 66 to 95, wherein the reverse transcriptase, RNA nickase, and / or DNA polymerase are thermostable enzymes.

[0115] Embodiment 103. The composition of embodiment 102, wherein the thermostable enzyme has activity at 50°C to 72°C.

[0116] Embodiment 104. The composition of embodiment 103, wherein the thermostable enzyme has activity at 50°C.

[0117] Embodiment 105. The composition of any one of embodiments 102 to 104, wherein the thermostable reverse transcriptase is a thermostable variant of MMLV reverse transcriptase, or a thermostable reverse transcriptase derived from a retrotransposon, or a group II intron reverse transcriptase.

[0118] Embodiment 106. The composition of any one of embodiments 102 to 105, wherein the thermostable RNA nickase is RNAse H from Thermus thermophilus.

[0119] Embodiment 107. The composition of any one of embodiments 102 to 106, wherein the thermostable DNA polymerase is Bst DNA polymerase.

[0120] Embodiment 108. (1) the reverse transcriptase, RNA nickase, and / or DNA polymerase are thermostable enzymes, and (2) the Mg 2+ Concentration is 1 mM to 50 mM, optionally containing Mg 2+ The concentration is 5 mM to 20 mM, and optionally Mg 2+ The composition of any one of embodiments 102 to 107, wherein the concentration is 8 mM.

[0121] Embodiment 109. A method for preparing a library of double-stranded cDNA fragments, comprising: a. combining a primer with a sample containing RNA, allowing the primer to bind to the RNA; b. A method comprising combining a sample with any one of the compositions of embodiments 66 to 108, (i) preparing double-stranded cDNA by an isothermal reaction, and (ii) preparing double-stranded cDNA fragments.

[0122] Embodiment 110. The method according to embodiment 109, wherein no solid-phase reversible immobilization purification is performed between the preparation of double-stranded cDNA by isothermal reaction and the preparation of double-stranded cDNA fragments.

[0123] Embodiment 111. The method of claim 109 or 110, wherein combining the primer with the sample and combining the sample with the composition of any one of embodiments 66 to 108 are performed in the same step.

[0124] Embodiment 112. The method according to any one of embodiments 109 to 111, wherein both (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are carried out in a single isothermal reaction.

[0125] Embodiment 113. The method according to any one of embodiments 109 to 111, wherein (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are performed at different temperatures.

[0126] Embodiment 114. The method according to any one of embodiments 109 to 113, wherein (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are carried out in a single reaction vessel.

[0127] Embodiment 115. The method of any one of embodiments 109 to 114, wherein combining the primer with the sample comprising RNA comprises mixing the sample comprising RNA with an elution solution, a primer, and a fragmentation mix.

[0128] Embodiment 116. The method of any one of embodiments 109 to 115, wherein combining the primer with the sample containing RNA is performed at 55° C. or higher.

[0129] Embodiment 117. The method of embodiments 109 to 116, wherein combining the primer with the sample containing RNA is performed at 65°C.

[0130] Embodiment 118. The method of any one of embodiments 109 to 117, wherein the primer comprises a randomer primer.

[0131] Embodiment 119. The method of any one of embodiments 109 to 118, wherein the primer comprises a primer that specifically binds to a sequence contained in the RNA.

[0132] Embodiment 120. The method of any one of embodiments 109 to 119, wherein the primer comprises a hexamer primer.

[0133] Embodiment 121. The method of any one of embodiments 109 to 120, wherein the primer comprises a primer comprising a chemically modified nucleotide.

[0134] Embodiment 122. The method of embodiment 121, wherein the primer comprises a chemically modified nucleotide that renders the RNA bound by the primer resistant to cleavage by RNA nickase.

[0135] Embodiment 123. The method of embodiment 122, wherein the RNA nickase is RNAse H and the RNA bound by the primer is resistant to cleavage by RNAse H.

[0136] Embodiment 124. The method of any one of embodiments 121 to 123, wherein the chemically modified nucleotide comprises a methylphosphonate residue.

[0137] Embodiment 125. The method of embodiments 109 to 124, wherein the reverse transcriptase produces a first strand of cDNA.

[0138] Embodiment 126 The method of embodiment 45, wherein the reverse transcriptase produces a DNA:RNA duplex comprising a first strand of cDNA and a strand of RNA.

[0139] Embodiment 127. The method of embodiment 46, wherein RNAse H nicks the RNA strand in a DNA:RNA duplex to produce RNA fragments.

[0140] Embodiment 128. The method of embodiment 127, wherein the DNA polymerase extends the second strand of DNA by priming from the RNA fragment.

[0141] Embodiment 129. The method of embodiment 127 or embodiment 128, wherein the 5'-3' activity of an RNA nickase and / or a DNA polymerase removes RNA fragments and 3' RNA overhangs.

[0142] Embodiment 130. The method of any one of embodiments 109 to 129, wherein the DNA polymerase has 5'-3' and / or 3'-5' exonuclease activity, which produces blunt-ended double-stranded cDNA.

[0143] Embodiment 131. The method of any one of embodiments 109 to 130, wherein dNTPs are used by both the reverse transcriptase and the DNA polymerase.

[0144] Embodiment 132. The method according to any one of embodiments 109 to 131, wherein the isothermal reaction for preparing double-stranded cDNA is at a temperature of 30 ° C to 49 ° C.

[0145] Embodiment 133. The method according to embodiment 132, wherein the isothermal reaction for preparing double-stranded cDNA is at a temperature of 37° C. or higher.

[0146] Embodiment 134. The method according to embodiment 133, wherein the isothermal reaction for preparing double-stranded cDNA is at a temperature of 37° C.

[0147] Embodiment 135. The method according to embodiment 133, wherein the isothermal reaction for preparing double-stranded cDNA is at a temperature of 55° C.

[0148] Embodiment 136. The method according to embodiment 134, wherein both (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are carried out in a single isothermal reaction at 37°C.

[0149] Embodiment 137. The method of embodiment 133, wherein preparing double-stranded cDNA and / or preparing double-stranded cDNA fragments is performed at above 37°C.

[0150] Embodiment 138. The method of embodiment 137, wherein preparing the double-stranded cDNA fragments is carried out at 55°C.

[0151] Embodiment 139. The method of embodiment 138, wherein preparing double-stranded cDNA fragments is carried out in 30 minutes or less, or 15 minutes or less.

[0152] Embodiment 140. The method according to embodiment 138 or 139, wherein preparing double-stranded cDNA is carried out at 37° C. and preparing double-stranded cDNA fragments is carried out at 55° C.

[0153] Embodiment 141. The composition used in the method comprises Mg 2+ Concentration of 1 mM to 50 mM, optionally Mg 2+ Concentration is 5 mM to 20 mM, and optionally, Mg 2+ The method of any one of embodiments 109 to 140, wherein the concentration is 8 mM.

[0154] Embodiment 142. The method of any one of embodiments 109 to 141, wherein the rate of production of the first strand of cDNA by reverse transcriptase is greater than the rate of nicking of RNA by RNA nickase.

[0155] Embodiment 143. The method of any one of embodiments 109 to 142, wherein the activity of the reverse transcriptase exceeds the activity of the RNA nickase.

[0156] Embodiment 144. The method according to any one of embodiments 109 to 143, wherein (i) preparing double-stranded cDNA by isothermal reaction and (ii) preparing double-stranded cDNA fragments are carried out with a total incubation time of 60 minutes or less or 30 minutes or less.

[0157] Embodiment 145. The method of any one of embodiments 109 to 144, further comprising depleting off-target RNA or enriching mRNA using a sample containing RNA prior to combining the primer with the sample containing RNA.

[0158] Embodiment 146. The method of embodiment 145, wherein the off-target RNA is ribosomal RNA.

[0159] Embodiment 147. The method of embodiment 145 or 146, wherein enriching for mRNA comprises binding of mRNA to amplification or capture beads using poly-T primers.

[0160] Embodiment 148. The method of embodiment 147, wherein the capture beads comprise a surface having capture oligonucleotides comprising a poly-T sequence.

[0161] Embodiment 149. The method according to any one of embodiments 109 to 148, wherein preparing double-stranded cDNA fragments is carried out by enrichment.

[0162] Embodiment 150. The method of embodiment 149, wherein enrichment is performed using hybrid capture.

[0163] Embodiment 151. The method of embodiment 150, wherein hybrid capture is performed using a target-specific biotinylated probe.

[0164] Embodiment 152. The method of embodiment 151, wherein the target-specific biotinylated probe binds to sequences from one or more infectious diseases.

[0165] Embodiment 153. The method of embodiment 152, wherein the one or more infectious diseases include one or more respiratory viruses.

[0166] Embodiment 154. The method according to any one of embodiments 109 to 153, wherein the method further comprises amplifying the double-stranded cDNA fragments to prepare amplicons.

[0167] Embodiment 155. The method of embodiment 154, wherein the amplification is carried out using target-specific primers.

[0168] Embodiment 156. The method of embodiment 155, wherein the target-specific primers bind to sequences from one or more infectious diseases.

[0169] Embodiment 157. The method of embodiment 156, wherein the one or more infectious diseases include one or more respiratory viruses.

[0170] Embodiment 158. The method of any one of embodiments 154 to 157, wherein the amplicons are subjected to solid-phase reversible immobilization purification.

[0171] Embodiment 159. The method of embodiment 158, wherein the total reaction time from combining the primer with the sample containing RNA to amplicon purification is 2 hours or less, 2.5 hours or less, or 3 hours or less.

[0172] Embodiment 160. A first transposon comprises a modified transposon end sequence comprising a mosaic end sequence, the mosaic end sequence comprising one or more mutations compared to the wild-type mosaic end sequence, the mutations being Uracil, B. Inosine, C. ribose, d. 8-oxoguanine, e. Thymine glycol, f. a modified purine, or g. The method of any one of embodiments 109-159, comprising substitution with a modified pyrimidine.

[0173] Embodiment 161. The method of embodiment 160, wherein the wild-type mosaic end sequence comprises SEQ ID NO:1, and further wherein the one or more mutations comprise substitutions at A16, C17, A18, and / or G19.

[0174] 162. the substitution at A16 is A16T, A16C, A16G, A16U, A16 inosine, A16 ribose, A16-8-oxoguanine, A16 thymine glycol, A16 modified purine, or A16 modified pyrimidine; b. the substitution at C17 is C17T, C17A, C17G, C17U, C17 inosine, C17 ribose, C17-8-oxoguanine, C17 thymine glycol, C17 modified purine, or C17 modified pyrimidine; c. the substitution at A18 is A18G, A18T, A18C, A18U, A18 inosine, A18 ribose, A18-8-oxoguanine, A18 thymine glycol, A18 modified purine, or A18 modified pyrimidine; and / or d. The method of embodiment 161, wherein the substitution at G19 is G19T, G19C, G19A, G19U, G19 inosine, G19 ribose, G19-8-oxoguanine, G19 thymine glycol, G19 modified purine, or G19 modified pyrimidine.

[0175] 163. a. combining the double stranded cDNA fragment with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites to cleave the first transposon end at uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within the mosaic end sequence to remove all or a portion of the first transposon end from the fragment; The method of any one of embodiments 160 to 162, further comprising: b. ligating an adaptor to the 5' and / or 3' ends of the fragments.

[0176] Embodiment 164. The method of embodiment 163, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0177] Embodiment 165. The method of embodiment 163, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0178] Embodiment 166. The method according to any one of embodiments 163 to 165, wherein all or part of the first transposon end to be cleaved is partitioned from the remainder of the sample.

[0179] Embodiment 167. The method according to any one of embodiments 163 to 166, further comprising filling in the 3' ends of the fragments and phosphorylating the 3' ends of the fragments with a kinase prior to ligation.

[0180] Embodiment 168. The method of embodiment 167, wherein filling is carried out using T4 DNA polymerase.

[0181] Embodiment 169. The method of embodiment 168, further comprising adding a single A overhang to the 3' end of the fragment.

[0182] Embodiment 170. The method of embodiment 169, wherein the polymerase adds a single A overhang.

[0183] Embodiment 171. The method of embodiment 170, wherein the polymerase is (i) Taq or (ii) Klenow fragment, exo-.

[0184] Embodiment 172. The method of any one of embodiments 163 to 171, wherein the fragment comprises 0 to 3 bases of a mosaic end sequence.

[0185] Embodiment 173. The method of any one of embodiments 163 to 172, further comprising sequencing the fragments after ligating the adaptors.

[0186] Embodiment 174 The method according to embodiment 173, wherein the method does not require amplification of the fragment prior to sequencing.

[0187] Embodiment 175. The method of embodiment 174, wherein the fragment is amplified prior to sequencing.

[0188] Embodiment 176. The method according to any one of embodiments 163 to 175, wherein the modified transposon end sequence comprises uracil, and the combination of DNA glycosylase and endonuclease / lyase that recognizes abasic sites is a uracil-specific excision reagent (USER).

[0189] Embodiment 177. The method of embodiment 176, wherein the USER is a mixture of uracil DNA glycosylase and endonuclease VIII or endonuclease III.

[0190] Embodiment 178. The method of any one of embodiments 163 to 175, wherein the modified transposon end sequences comprise inosine and the endonuclease is endonuclease V.

[0191] Embodiment 179. The method of any one of embodiments 163 to 175, wherein the modified transposon end sequences comprise ribose and the endonuclease is RNAse HII.

[0192] Embodiment 180. The method of any one of embodiments 163 to 175, wherein the modified transposon end sequence comprises 8-oxoguanine and the endonuclease is formamidopyrimidine-DNA glycosylase (FPG) or oxoguanine glycosylase (OGG).

[0193] Embodiment 181. The method of any one of embodiments 163 to 175, wherein the modified transposon end sequence comprises thymine glycol and the DNA glycosylase is endonuclease EndoIII (Nth) or Endo VIII.

[0194] Embodiment 182. The method of any one of embodiments 163 to 175, wherein the modified transposon end sequence comprises a modified purine, the DNA glycosylase is human 3-alkyladenine DNA glycosylase, and the endonuclease is endonuclease III or VIII.

[0195] Embodiment 183. The method of embodiment 182, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0196] Embodiment 184. The modified transposon end sequence comprises a modified pyrimidine; a. the DNA glycosylase is thymine-DNA glycosylase (TDG) or mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4), and the abasic site-recognizing endonuclease / lyase is an endonuclease, endonuclease III or VIII; or b. The method of any one of embodiments 163 to 175, wherein the endonuclease is DNA glycosylase / lyase ROS1 (ROS1).

[0197] Embodiment 185. The method of embodiment 184, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0198] Embodiment 186. The method of any one of embodiments 163 to 175, wherein the first transposon comprises a modified transposon end sequence comprising more than one mutation selected from uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, or modified pyrimidine, and wherein (1) an endonuclease, or (2) a combination of a DNA glycosylase and an endonuclease / lyase that recognizes heat, basic conditions, or abasic sites, is an enzyme mixture.

[0199] Embodiment 187. The method of embodiment 186, wherein the modified purine is 3-methyladenine or 7-methylguanine.

[0200] Embodiment 188. The method of embodiment 186, wherein the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0201] Embodiment 189. The method of any one of embodiments 172 to 188, wherein cleaving the first transposon end generates a sticky end for ligating an adapter.

[0202] Embodiment 190. The method of embodiment 189, wherein the sticky end is longer than one base.

[0203] Embodiment 191. The method of any one of embodiments 163 to 190, wherein the adaptor comprises a double-stranded adaptor.

[0204] Embodiment 192. The method of any one of embodiments 163 to 191, wherein adaptors are added to the 5' and 3' ends of the fragments.

[0205] Embodiment 193. The method of embodiment 192, wherein the adaptors added to the 5' and 3' ends of the fragments are different.

[0206] Embodiment 194. The method of any one of embodiments 163 to 193, wherein the adapter comprises a unique molecular identifier (UMI), a primer sequence, an anchor sequence, a universal sequence, a spacer region, an index sequence, a capture sequence, a barcode sequence, a cleavage sequence, a sequencing-related sequence, and combinations thereof.

[0207] Embodiment 195. The method of any one of embodiments 163 to 194, wherein the adapter comprises a UMI.

[0208] Embodiment 196 The method of embodiment 195, wherein an adaptor containing a UMI is ligated to both the 3' and 5' ends of the fragment.

[0209] Embodiment 197. The method of any one of embodiments 163 to 196, wherein the adapter is a forked adapter.

[0210] Embodiment 198. The method according to any one of embodiments 163 to 197, wherein ligation is carried out using a DNA ligase.

[0211] Embodiment 199. The method of any one of embodiments 109 to 198, wherein a tagmentation stop buffer is added after preparing the double-stranded cDNA fragments.

[0212] Embodiment 200. The method according to any one of embodiments 109 to 199, wherein the prepared double-stranded cDNA fragments are purified.

[0213] Embodiment 201. The method of any one of embodiments 109 to 200, wherein the double-stranded cDNA fragments are sequenced.

[0214] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0215] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0216] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0217] [Figure 1] Provides the times and steps of cDNA synthesis in conventional protocols (e.g., Illumina RNA Preparation with Enrichment, as described in RNA Prep with Enrichment(L) Tagmentation Reference Guide, Document # 1000000124435v02, Illumina, 2020 ("Document 1000000124435")) compared to the methods of the present invention. The X-axis reflects the time scale of manual user intervention for the addition of reagents required at the start of each step, indicated by horizontal bars. Temperature profiles are shown as small horizontal lines below each method. [Diagram 2] A comparative cDNA synthesis protocol similar to the method of Gubler and Huffman, 1983, is presented in which first-strand cDNA synthesis is separated in time from second-strand cDNA synthesis to generate a ds-cDNA copy of the original RNA. [Diagram 3] 1 provides an overview of the present method of near-simultaneous isothermal generation of double-stranded cDNA from RNA and the proposed mechanism of action. [Figure 4]Shown are the percentage of replicate read performance for the single-step protocol (1-step, denoted as "method of the invention") and the standard procedure (in this example, Illumina RNA Prep with Enrichment as described in Illumina document 470-2020-001-A (2020)). Black dots represent replicates of 10, 20, 30, 45, and 60 minutes of incubation for the single-step method. [Diagram 5] Figure 1 shows the insert size performance of the single-step isothermal protocol (1 step, denoted as "method of the invention") and the standard Illumina RNA Prep with Enrichment procedure. Black dots represent replicates of 10, 20, 30, 45, or 60 minutes of incubation using the single-step method. [Figure 6] The median coefficient of variance (CV) of coverage performance of the single-step isothermal protocol (1 step, denoted as "method of the invention") and the standard Illumina RNA Prep with Enrichment procedure is shown. Black dots represent replicates of 10, 20, 30, 45, and 60 minutes of incubation of the single-step method. [Figure 7A]Fragments per kilobase per million mapped reads (FPKM) scatter plots are shown. (A) Correlation of technical replicates for the "single-step" method of the present invention with 20 min incubation. Both libraries were generated with 12 ng of a mixture of Universal Human Reference RNA (UHR, Agilent PN 740000) and genomic RNA from bacteriophage MS2 (MS2, Roche PN 10165948001, GenBank accession NC_001417.2) at 80% UHR / 20% MS2. (B) Comparison of a library prepared from 10 ng UHR by the Illumina RNA Prep with Enrichment method with a library prepared from 12 ng of a mixture of 80% UHR / 20% MS2 by the single-step method. [Figure 7B] Fragments per kilobase per million mapped reads (FPKM) scatter plots are shown. (A) Correlation of technical replicates for the "single-step" method of the present invention with 20 min incubation. Both libraries were generated with 12 ng of a mixture of Universal Human Reference RNA (UHR, Agilent PN 740000) and genomic RNA from bacteriophage MS2 (MS2, Roche PN 10165948001, GenBank accession NC_001417.2) at 80% UHR / 20% MS2. (B) Comparison of a library prepared from 10 ng UHR by the Illumina RNA Prep with Enrichment method with a library prepared from 12 ng of a mixture of 80% UHR / 20% MS2 by the single-step method. [Figure 8]Box plots of FPKM R2 values ​​are shown. FPKM comparison includes only genes targeted by the TruSight® RNA Pan-Cancer Panel kit panel. Left panel: Single-step method comparison between different incubation times. Center panel: Comparison between Illumina RNA Prep with Enrichment libraries and Single-step method libraries. Right panel: Comparison between replicates of Illumina RNA Prep with Enrichment libraries performed during DVT. Key indicates RNA input, x-axis indicates RNA method incubation time (in minutes) where applicable. NA=Standard cDNA procedure and incubation time used in Project Illumina RNA Prep with Enrichment. [Figure 9] Shown is an Integrated Genomics Viewer (IGV) visualization of normalized read coverage across MS2 for Project Illumina RNA Prep with Enrichment (top track), the 20 min single-step procedure (middle track), and the standard method released with the 10 min single-step procedure (bottom track). Additionally, the bottom boxed tracks represent the percentage of G and C (%GC) in a 24 bp window (black = high %GC, white = low %GC), well-characterized short hairpin sequences in MS2, and protein-coding regions of MS2 (MS2 genome). [Figure 10] Density plots of MS2 read-depth normalized per-base coverage for the single-step (top) and Illumina RNA Prep with Enrichment (bottom) cDNA preparation protocols. Note that the single-step protocol has a broader distribution and more extreme values, which is consistent with the larger CV of coverage presented in Table 2. [Figure 11] 1 shows the primary alignment and performance measures for six replicates of a thermostable formulation of the method of the present invention. [Figure 12A]Performance of thermostable formulations is shown. cDNA was prepared using thermostable formulations, tagmented with enrichment bead-linked transposon or BLT (enrichment bead-linked transposon, eBLT), enriched with TruSight® RNA Fusion Panel (as described in TruSight RNA® Fusion Panel Protocol Guide, Illumina Document # 1000000009155 v00 (2016)), and assessed for reproducibility by FPKM analysis. (A) Correlation of replicates. (B) Comparison with the released standard protocol using Illumina RNA Prep with Enrichment. R2 values ​​are shown. [Figure 12B] Performance of thermostable formulations is shown. cDNA was prepared using thermostable formulations, tagmented with enrichment bead-linked transposon or BLT (enrichment bead-linked transposon, eBLT), enriched with TruSight® RNA Fusion Panel (as described in TruSight RNA® Fusion Panel Protocol Guide, Illumina Document # 1000000009155 v00 (2016)), and assessed for reproducibility by FPKM analysis. (A) Correlation of replicates. (B) Comparison with the released standard protocol using Illumina RNA Prep with Enrichment. R2 values ​​are shown. [Figure 13] A summary of a one-pot method combining cDNA synthesis and tagmentation in which a library of double-stranded DNA fragments is prepared in a single reaction vessel from a sample containing RNA is shown. The summary shows that the reaction temperature can be optionally increased to 55° C. at the end of the incubation to improve tagmentation efficiency. [Figure 14A]Figure 1 shows an outline of an exemplary double stranded cDNA preparation (using the method of the invention) and one-pot tagmentation preparation (combined cDNA and library fragment preparation). (A) Representative mesophilic and thermostable preparations. (B) Overview of the method of double stranded cDNA synthesis by either two steps or the method of the invention. (C) Comparison of different methods involving tagmentation to prepare library fragments. EPH3=Eluate, Primer, Fragmentation Mix, FSA=First Strand Synthesis Actinomycin D Mix, FSM=First Strand Synthesis Mix, SMM=Second Strand Mix, ST2=Tagmentation Stop Buffer 2, SPRI=Solid Phase Reversible Immobilization Purification. [Figure 14B] Figure 1 shows an outline of an exemplary double stranded cDNA preparation (using the method of the invention) and one-pot tagmentation preparation (combined cDNA and library fragment preparation). (A) Representative mesophilic and thermostable preparations. (B) Overview of the method of double stranded cDNA synthesis by either two steps or the method of the invention. (C) Comparison of different methods involving tagmentation to prepare library fragments. EPH3=Eluate, Primer, Fragmentation Mix, FSA=First Strand Synthesis Actinomycin D Mix, FSM=First Strand Synthesis Mix, SMM=Second Strand Mix, ST2=Tagmentation Stop Buffer 2, SPRI=Solid Phase Reversible Immobilization Purification. [Figure 14C] Figure 1 shows an outline of an exemplary double stranded cDNA preparation (using the method of the invention) and one-pot tagmentation preparation (combined cDNA and library fragment preparation). (A) Representative mesophilic and thermostable preparations. (B) Overview of the method of double stranded cDNA synthesis by either two steps or the method of the invention. (C) Comparison of different methods involving tagmentation to prepare library fragments. EPH3=Eluate, Primer, Fragmentation Mix, FSA=First Strand Synthesis Actinomycin D Mix, FSM=First Strand Synthesis Mix, SMM=Second Strand Mix, ST2=Tagmentation Stop Buffer 2, SPRI=Solid Phase Reversible Immobilization Purification. [Figure 15]Library results using two-step cDNA preparation followed by tagmentation with either BLT (e.g., Illumina® DNA Prep, as included in the (S) Tagmentation Kit, or Illumina® RNA Prep, as included in the (L) Tagmentation Kit) are shown. [Figure 16A] Library results using different types of cDNA preparations and tagmentation reactions using samples with 100 ng of RNA. (A) Results with a two-step cDNA preparation (standard method) followed by a separate tagmentation reaction. (B) Results with a one-step cDNA preparation (method of the present invention) followed by a separate tagmentation reaction. (C) Results with a combined one-pot cDNA and tagmentation preparation. (D) Overlay of results under different conditions. All conditions resulted in the preparation of a library of fragments. [Figure 16B] Library results using different types of cDNA preparations and tagmentation reactions using samples with 100 ng of RNA. (A) Results with a two-step cDNA preparation (standard method) followed by a separate tagmentation reaction. (B) Results with a one-step cDNA preparation (method of the present invention) followed by a separate tagmentation reaction. (C) Results with a combined one-pot cDNA and tagmentation preparation. (D) Overlay of results under different conditions. All conditions resulted in the preparation of a library of fragments. [Figure 16C] Library results using different types of cDNA preparations and tagmentation reactions using samples with 100 ng of RNA. (A) Results with a two-step cDNA preparation (standard method) followed by a separate tagmentation reaction. (B) Results with a one-step cDNA preparation (method of the present invention) followed by a separate tagmentation reaction. (C) Results with a combined one-pot cDNA and tagmentation preparation. (D) Overlay of results under different conditions. All conditions resulted in the preparation of a library of fragments. [Figure 16D]Library results using different types of cDNA preparations and tagmentation reactions using samples with 100 ng of RNA. (A) Results with a two-step cDNA preparation (standard method) followed by a separate tagmentation reaction. (B) Results with a one-step cDNA preparation (method of the present invention) followed by a separate tagmentation reaction. (C) Results with a combined one-pot cDNA and tagmentation preparation. (D) Overlay of results under different conditions. All conditions resulted in the preparation of a library of fragments. [Figure 17A] Results are shown using a combination one-pot cDNA preparation and tagmentation reaction. (A) Results from the reaction in the presence of reverse transcriptase (RT). (B) Results from a no template control reaction (NTC, no RNA in the starting sample). (C) Results from the reaction in the absence of RT. As expected, fragments were only produced under condition (A), where the reaction included an RNA template and RT. [Figure 17B] Results are shown using a combination one-pot cDNA preparation and tagmentation reaction. (A) Results from the reaction in the presence of reverse transcriptase (RT). (B) Results from a no template control reaction (NTC, no RNA in the starting sample). (C) Results from the reaction in the absence of RT. As expected, fragments were only produced under condition (A), where the reaction included an RNA template and RT. [Figure 17C] Results are shown using a combination one-pot cDNA preparation and tagmentation reaction. (A) Results from the reaction in the presence of reverse transcriptase (RT). (B) Results from a no template control reaction (NTC, no RNA in the starting sample). (C) Results from the reaction in the absence of RT. As expected, fragments were only produced under condition (A), where the reaction included an RNA template and RT. [Figure 18A] A comparison of BLT results using a combined one-pot cDNA preparation and tagmentation reaction is shown for either 100 ng (A) or 10 ng (B) of starting RNA. [Figure 18B]A comparison of BLT results using a combined one-pot cDNA preparation and tagmentation reaction is shown for either 100 ng (A) or 10 ng (B) of starting RNA. [Figure 19A] Results are shown using ribosomal RNA (rRNA)-depleted UHR libraries with different cDNA preparation and BLT tagmentation protocols: (A) Results with 1:10 diluted samples prepared with a two-step cDNA preparation followed by a separate tagmentation reaction, or with a one-step cDNA preparation (method of the invention) followed by a separate tagmentation reaction, (B) Results with undiluted samples using a combination of one-pot cDNA and tagmentation preparation, as well as a no reverse transcriptase control (No RVT). [Figure 19B] Results are shown using ribosomal RNA (rRNA)-depleted UHR libraries with different cDNA preparation and BLT tagmentation protocols: (A) Results with 1:10 diluted samples prepared with a two-step cDNA preparation followed by a separate tagmentation reaction, or with a one-step cDNA preparation (method of the invention) followed by a separate tagmentation reaction, (B) Results with undiluted samples using a combination of one-pot cDNA and tagmentation preparation, as well as a no reverse transcriptase control (No RVT). [Figure 20A] Alignment of the different preparations and general performance measures are shown. (A) Aligned percentages are all around 94-95%. (B) Median CV is increased for the combined reactions. (C) The duplicate percentage is higher for the combined reactions. 2st = 2-step cDNA preparation followed by a separate tagmentation reaction. 1stC = 1-step cDNA preparation (method of the invention) followed by a separate tagmentation reaction. 1potLib = 1-pot combined cDNA and tagmentation preparation. [Figure 20B]Alignment of the different preparations and general performance measures are shown. (A) Aligned percentages are all around 94-95%. (B) Median CV is increased for the combined reactions. (C) The duplicate percentage is higher for the combined reactions. 2st = 2-step cDNA preparation followed by a separate tagmentation reaction. 1stC = 1-step cDNA preparation (method of the invention) followed by a separate tagmentation reaction. 1potLib = 1-pot combined cDNA and tagmentation preparation. [Figure 20C] Alignment of the different preparations and general performance measures are shown. (A) Aligned percentages are all around 94-95%. (B) Median CV is increased for the combined reactions. (C) The duplicate percentage is higher for the combined reactions. 2st = 2-step cDNA preparation followed by a separate tagmentation reaction. 1stC = 1-step cDNA preparation (method of the invention) followed by a separate tagmentation reaction. 1potLib = 1-pot combined cDNA and tagmentation preparation. [Figure 21A] Using the same sample nomenclature as in Figures 20A-C, insert length (A) and alignment distribution (B) after library preparation under different conditions are shown. [Figure 21B] Using the same sample nomenclature as in Figures 20A-C, insert length (A) and alignment distribution (B) after library preparation under different conditions are shown. [Figure 22A] Gene expression correlation between different reaction conditions: (A) Correlation between two-step cDNA preparation (2-step) and one-pot cDNA preparation (1-pot) on 100ng starting RNA. (B) Correlation between two-step and one-pot on 10ng starting RNA. (C) Correlation between one-step cDNA preparation followed by a separate tagmentation reaction (1-step cDNA) and combined one-pot cDNA and tagmentation library preparation on 100ng starting RNA. [Figure 22B]Gene expression correlation between different reaction conditions: (A) Correlation between two-step cDNA preparation (2-step) and one-pot cDNA preparation (1-pot) on 100ng starting RNA. (B) Correlation between two-step and one-pot on 10ng starting RNA. (C) Correlation between one-step cDNA preparation followed by a separate tagmentation reaction (1-step cDNA) and combined one-pot cDNA and tagmentation library preparation on 100ng starting RNA. [Figure 22C] Gene expression correlation between different reaction conditions: (A) Correlation between two-step cDNA preparation (2-step) and one-pot cDNA preparation (1-pot) on 100ng starting RNA. (B) Correlation between two-step and one-pot on 10ng starting RNA. (C) Correlation between one-step cDNA preparation followed by a separate tagmentation reaction (1-step cDNA) and combined one-pot cDNA and tagmentation library preparation on 100ng starting RNA. [Figure 23] Shown are 10X gene coverage for 2st, 1stC, and 1potLib preps using either 100ng or 10ng of starting RNA. Sample nomenclature is the same as in Figures 20A-C. [Figure 24] 5' to 3' read distribution for 2st, 1stC, and 1potLib preparations using 10 ng of starting RNA. Sample nomenclature is the same as in Figures 20A-C. [Diagram 25] A comparison of times and steps is shown for a two-step cDNA followed by tagmentation (standard library prep (stdLP)), a one-step cDNA preparation followed by a separate tagmentation reaction (one-step cDNA+tagmentation), and a combined one-pot cDNA and tagmentation library preparation. The one-pot combined protocol can reduce library preparation (library prep, LP) time by about 50%, to roughly 2 hours. The one-pot combined protocol can also have only a single "clean-up" step (e.g., using SPRI beads). [Figure 26]Results from various different library preparation (LP) protocols using none (NoTC), 1k (1kTC), 10k (10kTC), or 100k (100kTC) Twist controls (TC, Twist Control Synthetic SARS-CoV-2 RNA Control 2 from Twist Bioscience (#102024 Control 2* MN908947.3 Wuhan-Hu-1)) are shown. The "A" and "B" samples for each group refer to two separate samples. Enrichment was performed using Respiratory Virus Oligos Panel Version 1 (Illumina) under the conditions summarized below: Standard LP with enrichment (i.e., two-step cDNA preparation followed by tagmentation as control, StdLP group). · 1-pot LP: 1 hour at 37°C (4mM Mg2+) - conditions outlined in Example 5 (1-pot group) · 1-pot LP: 45 minutes at 37°C followed by 15 minutes at 55°C (55C group) · 1-pot LP: 1 hour at 37°C, increasing Mg2+ to a final of 8mM (Mg group) · 1-pot LP: 1 hour at 37°C, omitting washes and addition of ST2 tagmentation stop solution with SDS (NoST2 group). [Figure 27] Median coverage of Twist control at 1 million (1M) reads for the different protocols outlined in Figure 26. Results were performed using enrichment with Respiratory Virus Oligos Panel Version 1 (Illumina). Arrows indicate that best performance was seen for the control (standard library prep) and the 1-pot LP with increased Mg2+ to a final of 8 mM (Mg group). [Figure 28] Results from various conditions of library preparation (LP) are shown. Test groups included incubation at 55°C and 8 mM Mg2+ concentration, as well as several combination protocols with one-pot LP at 37°C with 8 mM Mg2+. These one-pot protocols were compared to standard library preparation (StdLP) with two-step cDNA preparation followed by tagmentation. [Figure 29]Coverage results for Twist control at different incubation times using enrichment with Respiratory Virus Oligos Panel Version 2 (Illumina) are shown: 37-1h = 1h at 37°C, 45min + 55C = 45min at 37°C and 15min at 55°C, 30min + 55C = 30min at 37°C and 15min at 55°C, 14min + 55C = 15min at 37°C and 15min at 55°C. [Figure 30A] Results of experiments with rRNA-depleted samples are shown. (A) Percentage of duplicates for 2st, CTL, 1stC, and 1potLib samples with either 4 mM Mg2+ at 37°C for 1 hour (left group) or 8 mM Mg2+ at 37°C for 1 hour and 55°C for 15 minutes (3715) or 30 minutes (3730). The conditions for the 10ngCTL and 2st groups were the same, with separate first and second strand reactions to prepare cDNA, cleanup, and then tagmentation by BLT. (B) Number of genes detected with the different protocols. [Figure 30B] Results of experiments with rRNA-depleted samples are shown. (A) Percentage of duplicates for 2st, CTL, 1stC, and 1potLib samples with either 4 mM Mg2+ at 37°C for 1 hour (left group) or 8 mM Mg2+ at 37°C for 1 hour and 55°C for 15 minutes (3715) or 30 minutes (3730). The conditions for the 10ngCTL and 2st groups were the same, with separate first and second strand reactions to prepare cDNA, cleanup, and then tagmentation by BLT. (B) Number of genes detected with the different protocols. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0218] Array Description The following table provides a list of the specific sequences referenced herein.

[0219] [Table 1] [Mode for carrying out the invention]

[0220] I. Composition for preparing double-stranded cDNA from RNA by isothermal reaction As described herein, a mixture of specialized enzymes can achieve efficient ds-cDNA conversion suitable for Illumina library preparation techniques such as tagmentation with Tn5 (e.g., Illumina DNA Flex PCR-Free technology and bead-linked transposomes). The method can be performed in a single step as short as 10 minutes at a single temperature. Such single-step preparation of cDNA can be referred to as a one-step cDNA method (also described herein as the "inventive method" of cDNA preparation). Formulations with mesophilic (about 37°C) and thermostable (about 50°C) enzymes are possible. The advantage of the shorter time over conventional methods is shown in Figure 1 (where the process of converting RNA to ds-cDNA is shortened from 110 minutes in conventional models to 15 minutes in the inventive method). Potential applications of the inventive method are rapid infectious disease surveillance, as well as rapid simultaneous RNA and DNA assays for genotyping.

[0221] In some embodiments, the method of cDNA preparation does not amplify the nucleic acid content, but instead converts RNA into double-stranded cDNA, hi some embodiments, the method does not include a step of PCR amplification or a PCR-like process to prepare multiple amplicons from a given sequence contained in the RNA.

[0222] In some embodiments, the RNA is attached to a primer prior to preparing double-stranded cDNA.

[0223] In some embodiments, the composition is for the preparation of ds-cDNA by isothermal reaction. As used herein, "isothermal reaction" refers to a reaction that is carried out at a substantially constant temperature, i.e., without changing the reaction temperature at which the enzymatic reaction occurs by more than 15% from the baseline temperature. Thus, an isothermal reaction is carried out at a constant temperature or with a change from baseline of 15% or less. In some further embodiments, the reaction may be carried out at a temperature with even less temperature fluctuation, with a change from baseline of 10% or less, or 5% or less. In some embodiments, the reaction is carried out without a change in temperature. In some embodiments, the reaction can be carried out isothermally without the need for computer-controlled temperature regulation in a thermal cycler. In some cases of reactions that can be carried out isothermally without the need for computer-controlled regulation in a thermal cycler, the temperature may be 37°C or 50°C. In some embodiments, the primer is attached to the RNA before the composition is added to the sample containing the RNA. In some embodiments, the reaction is a mesophilic or thermostable reaction.

[0224] In some embodiments, a composition for preparing double-stranded cDNA from RNA by an isothermal reaction includes (i) a reverse transcriptase, (ii) an RNA nickase, (iii) a DNA polymerase with strand displacement or 5'-3' exonuclease activity, and (iv) dNTPs. Such a composition may be used in methods of ds-cDNA as described below and in FIG. 3. In some embodiments, the blend of enzymes in the composition act in concert to generate ds-cDNA suitable for tagmentation (e.g., bead-linked transposome (BLT) and Illumina DNA Flex PCR-Free (Research Use Only, RUO) technology products.

[0225] In some embodiments, the composition further comprises an RNA nickase inhibitor. In some embodiments, the RNA nickase inhibitor is an RNAse inhibitor.

[0226] In some embodiments, the reverse transcriptase can bind to a primer that is bound to the RNA prior to adding the composition to a sample containing the RNA. In some embodiments, the reverse transcriptase can generate a first strand of cDNA from the RNA.

[0227] In some embodiments, the RNA nickase can nick RNA, which may be referred to as an "RNA nickase." In some embodiments, the RNA nickase is a ribonuclease. In some embodiments, the ribonuclease is RNAse H. Thus, because RNAse is a representative RNA nickase, the RNA nickase may be referred to as an "enzyme having RNAse-H-like activity."

[0228] In some embodiments, an RNA nickase is included among reverse transcriptases that have RNAse H activity. Many commercially available reverse transcriptases have been engineered to lack RNAse H activity (which may improve cDNA synthesis yields), but many unengineered reverse transcriptases have RNAse H activity.

[0229] In some embodiments, the RNA nickase can nick the RNA contained in the RNA:DNA hybrid. In some embodiments, the RNA nickase can nick the strand of RNA hybridized to the first strand of the cDNA. For example, RNase H has been reported to digest RNA from DNA:RNA hybrids approximately every 7-21 bases (Schultz et al., J. Biol. Chem. 2006, 281:1943-1955; Champoux and Schultz, FEBS J. 2009, 276:1506-1516). In some embodiments, the RNA fragments generated by the RNA nickase can be used to prime the second strand of the cDNA from the first strand of the cDNA.

[0230] In some embodiments, RNAse H can nick the RNA strand in an RNA:DNA hybrid, and then a DNA polymerase can use the 3' OH end of the nicked RNA fragment as a primer to initiate synthesis of a second cDNA strand. Such a process cannot be performed with degraded or fragmented RNA prepared by other methods, because the RNA fragments have a 3' phosphate and the polymerase cannot use such an end to prime synthesis of a second cDNA strand.

[0231] In some embodiments, RNA nickase can also remove the RNA primer and the 3' overhangs after generation of the second strand of cDNA.

[0232] In some embodiments, the activity of the reverse transcriptase is greater than the activity of the RNA nickase. In some embodiments, the generation of the first strand of cDNA by the reverse transcriptase is faster than the rate of nicking of RNA by the RNA nickase. In this way, the first strand of cDNA can be generated before nicking the RNA template. In some embodiments, the reverse transcriptase and the RNA nickase are included in a single enzyme. In some embodiments, the single enzyme that includes the reverse transcriptase and the RNA nickase is Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT), Moloney Murine Leukemia Virus (MMLV reverse transcriptase), or Group II intron reverse transcriptase. Alternatively, in some embodiments, MMLV that does not have RNA nickase activity or has significantly reduced RNA nickase activity may be used.

[0233] In some embodiments, the reverse transcriptase and DNA polymerase are comprised in a single enzyme that has both RNA-dependent polymerase activity and DNA-dependent polymerase activity. In some embodiments, the single enzyme reduces competition between the reverse transcriptase and the DNA polymerase. In some embodiments, the single enzyme that contains both RNA-dependent polymerase activity and DNA-dependent polymerase activity eliminates steric inhibition of DNA polymerase binding by the reverse transcriptase.

[0234] In some embodiments, the DNA polymerase mediates second strand cDNA synthesis, hi some embodiments, the DNA polymerase destroys the RNA strand.

[0235] In some embodiments, the DNA polymerase has strand displacement activity, hi some embodiments, the DNA polymerase is capable of displacing the first strand of the generated cDNA.

[0236] In some embodiments, the DNA polymerase has a 5' to 3' exonuclease activity. In some embodiments, the DNA polymerase can remove RNA by its 5' to 3' exonuclease activity. In some embodiments, the DNA polymerase can produce blunt-ended ds-cDNA by its 5' to 3' exonuclease activity and / or 3' to 5' exonuclease activity.

[0237] In some embodiments, the reverse transcriptase is a polymerase with RNA-dependent DNA polymerase activity. Any such reverse transcriptase that is a polymerase with RNA-dependent DNA polymerase activity may be used, and those skilled in the art are aware of a wide variety of such enzymes. In some embodiments, the reverse transcriptase is MMLV reverse transcriptase, or a retrotransposon-derived reverse transcriptase, or a group II intron reverse transcriptase. In some embodiments, the reverse transcriptase is Protoscript II (New England Biolabs (NEB)), a recombinant MMLV reverse transcriptase with limited RNAse H activity and increased thermostability.

[0238] In some embodiments, the RNA nickase is RNAse H. In some embodiments, the RNAse H is from Thermus thermophilus. In some embodiments, the DNA polymerase is E. coli DNA polymerase I or Bst DNA polymerase.

[0239] In some embodiments, the composition comprises one or more additives in addition to the enzyme and dNTP. In some embodiments, the composition comprises one or more additives selected from dithiothreitol (DTT), bovine serum albumin (BSA), Tris pH 7.5, KCl, and / or MgCl2. In some embodiments, the composition comprises DTT and BSA. Those skilled in the art are well aware that such additives may improve the function of various enzyme compositions, and analysis of different additives is included in routine assay development. Therefore, this list of representative additives serves merely as an example, and those skilled in the art may exclude or substitute such additives.

[0240] In some embodiments, the compositions are for isothermal mesophilic or isothermal thermostable reactions.

[0241] A. Compositions for preparing mesophilic ds-cDNA In some embodiments, the composition may be for preparing ds-cDNA by an isothermal mesophilic reaction. As used herein, mesophilic reaction refers to a reaction carried out at 37°C to 49°C. In some embodiments, the mesophilic reaction can be carried out isothermally at 37°C without the need for computer-controlled temperature regulation in a thermal cycler. The data in Figures 4-10 show the results of using the composition for mesophilic ds-DNA preparation in a one-step process (i.e., the method of the present invention).

[0242] In some embodiments, the reverse transcriptase, RNA nickase, and / or DNA polymerase are mesophilic enzymes. In some embodiments, the mesophilic enzymes are active between 37° C. and 49° C. In some embodiments, the mesophilic enzymes are active at 37° C.

[0243] In some embodiments, the mesophilic reverse transcriptase is MMLV reverse transcriptase. In some embodiments, the mesophilic RNA nickase is E. coli RNAse H. In some embodiments, the mesophilic polymerase is E. coli DNA polymerase I.

[0244] Table 1 provides representative enzymes included in compositions for mesophilic ds-cDNA preparation.

[0245] [Table 2]

[0246] B. Compositions for preparing thermostable ds-cDNA In some embodiments, the composition may be for preparing ds-cDNA by an isothermal thermostable reaction. As used herein, a thermostable reaction refers to a reaction carried out at 50° C. or higher. In some embodiments, the thermostable reaction can be carried out isothermally at 50° C. or higher without the need for computer-controlled temperature regulation in a thermal cycler. The data in FIG. 11-FIG. 12B show the results of using the composition for thermostable ds-DNA preparation in a one-step process (i.e., the method of the present invention).

[0247] In some embodiments, the reverse transcriptase, RNA nickase, and / or DNA polymerase are thermostable enzymes. In some embodiments, the thermostable enzyme is active between 50° C. and 72° C. In some embodiments, the thermostable enzyme is active at 50° C. In some embodiments, the thermostable reverse transcriptase is a thermostable variant of MMLV reverse transcriptase, or a thermostable reverse transcriptase derived from a retrotransposon, or a group II intron reverse transcriptase. In some embodiments, the thermostable variant of MMLV reverse transcriptase is ProtoScript II (New England Biolabs). In some embodiments, the thermostable RNA nickase is RNAse H from Thermus thermophilus.

[0248] In some embodiments, the thermostable DNA polymerase is Bst DNA polymerase. In some embodiments, the DNA polymerase from the mesophilic composition is replaced with Bst DNA polymerase. In some embodiments, the Bst DNA polymerase is Bst 3.0 DNA polymerase (New England Biolabs).

[0249] Table 2 provides representative enzymes that are included in compositions for preparing thermostable ds-cDNA.

[0250] [Table 3]

[0251] C. Balance of enzyme units in the composition The composition of the present invention improves double-stranded cDNA preparation in a single isothermal reaction using a balance of enzyme activities.For example, if the activity of RNase is relatively high compared to other enzymes in the composition, the RNA template (from the original sample) will be degraded before a sufficient amount of the first strand of cDNA is prepared.In such a scenario, little cDNA is produced.

[0252] A composition having the appropriate balance of enzymatic activities for a one-step cDNA preparation (ie, the method of the present invention) can be referred to as a "master mix."

[0253] Thus, a composition with the proper balance of enzymatic activities improves the yield of double-stranded cDNA preparations.

[0254] The unit ranges of commercially available enzymes are well known to those of skill in the art and can be easily reviewed from the supplier's website or technical documentation provided with the commercially available enzymes. Table 3 provides information on specific enzymes and their characteristics from New England Biolabs (NEB) along with unit definitions. Although the method of the present invention is not limited to these specific enzymes, this table is useful for providing information on the well-known characteristics of representative commercially available enzymes (such as those described for the enzymes commercially available from NEB). Those of skill in the art will recognize a wide range of different enzymes that can be used in the present compositions and can select an enzyme based on the desired conditions for carrying out the methods described herein. In Table 3, RNAse H represents RNA nickase.

[0255] [Table 4]

[0256] In some embodiments, the correct balance of reverse transcriptase, RNA nickase, and DNA polymerase allows for the preparation of double-stranded cDNA from RNA using the compositions described herein in a single isothermal reaction.

[0257] In some embodiments, the composition has lower units / μl of RNA nickase (such as RNAse H) compared to units / μl of reverse transcriptase and / or DNA polymerase. In some embodiments, the composition includes an RNA nickase inhibitor that functions to reduce the activity of the RNA nickase. In some embodiments, the RNA nickase inhibitor is an RNAse inhibitor.

[0258] In some embodiments, the composition may include an RNAse inhibitor that functions to limit contamination with exogenous RNAses. In some embodiments, the RNAse inhibitor functions to inhibit RNAse A, a common laboratory contaminant that does not function as an RNA nickase.

[0259] In some embodiments, the units / μl of RNA nickase and DNA polymerase in the composition overlap with the DNA polymerase, hi some embodiments, the DNA polymerase has an activity that is 2-100 times higher than the activity of the RNA nickase.

[0260] In some embodiments, the units / μl of reverse transcriptase is 10-1,000 times higher than the units / μl of RNA nickase.

[0261] In some embodiments, the units / μl of reverse transcriptase and DNA polymerase in the composition overlap, hi some embodiments, the units / μl of reverse transcriptase in the composition is higher than the units / μl of DNA polymerase.

[0262] In some embodiments, the reverse transcriptase activity in the composition is between 0.32 U / μl and 4.8 U / μl. In some embodiments, the DNA polymerase activity in the composition is between 0.04 U / μl and 0.37 U / μl. In some embodiments, the RNA nickase activity in the composition is between 0.004 U / μl and 0.04 U / μl. In some embodiments, the RNA nickase is RNAse H.

[0263] In some embodiments, the RNA nickase activity in the thermostable composition is relatively higher than the RNA nickase activity in the mesophilic composition. In some embodiments, the RNA nickase activity in the thermostable composition is greater than 0.04 U / μl. In some embodiments, the RNA nickase activity in the thermostable composition is between 0.05 U / μl and 0.3 U / μl.

[0264] II. Compositions for Preparing Libraries from RNA As described herein, a special mixture of enzymes can be used to prepare a library of double-stranded cDNA fragments from the RNA in a sample. In some embodiments, the composition allows for the preparation of a library of cDNA fragments in a single reaction vessel. In some embodiments, these fragments can be used for sequencing.

[0265] In some embodiments, the composition for one-pot library preparation (for the combination of cDNA and library preparation) can be a cDNA preparation "master mix" together with BLT.

[0266] In some embodiments, the library is prepared by isothermal reaction. In some embodiments, a single temperature is used for the reaction to prepare double-stranded cDNA and the reaction to prepare the library of cDNA fragments. Thus, any composition used to prepare cDNA described herein can also be used to prepare a library of cDNA fragments, as long as the transposome complex is included in the composition.

[0267] In some embodiments, the RNA is conjugated to a primer prior to preparing the library.

[0268] In some embodiments, a composition for preparing a library of double-stranded cDNA fragments from RNA comprises a reverse transcriptase, an RNA nickase, a DNA polymerase having strand displacement or 5'-3' exonuclease activity, dNTPs, and a transposome complex. In some embodiments, the transposome complex comprises a transposase, a first transposon comprising a transposon end sequence, and a second transposon comprising a sequence that is fully or partially complementary to the transposon end sequence.

[0269] In some embodiments, the composition allows for first and second strand cDNA preparation followed by fragmentation of this double stranded DNA. In some embodiments, the composition comprises an enzyme for mesophilic cDNA synthesis. In some embodiments, the composition comprises an enzyme for thermostable cDNA synthesis.

[0270] In some embodiments, the composition for preparing a library comprises the components described for the composition for preparing double-stranded cDNA and further comprises a transposome complex. In some embodiments, the transposome complex comprises a transposase, a first transposon comprising a transposon end sequence, and a second transposon comprising a sequence that is fully or partially complementary to the transposon end sequence. In some embodiments, the transposome complex allows the generation of fragments of double-stranded cDNA generated by preparing double-stranded cDNA from RNA.

[0271] In some embodiments, the cDNA can be fragmented by the transposome complex as it is prepared from the RNA without the need to modify the components of the composition or the reaction vessel, hi some embodiments, the cDNA does not need to be purified before fragments of the cDNA are prepared using the composition.

[0272] In some embodiments, the composition for preparing a library of double-stranded cDNA from RNA contains magnesium. Magnesium is known to promote transposase activity (see Picelli et al., Genome Research 24:2033-2040 (2014)). In some embodiments, the composition contains Mg. 2+ In some embodiments, the Mg concentration of the composition is 1 mM to 50 mM. 2+ In some embodiments, the Mg concentration of the composition is 5 mM to 20 mM. 2+ The concentration is 8 mM.

[0273] A. Transposome Complex Transposon-based techniques can be utilized to fragment DNA, where a target nucleic acid, such as genomic DNA, is treated with a transposome complex that simultaneously fragments and tags ("tagmentation") the target, thereby generating a population of fragmented nucleic acid molecules tagged with unique adapter sequences at the ends of the fragments. Tagmentation involves modification of DNA with a transposome complex that includes a transposase enzyme complexed with one or more tags (such as adapter sequences) that include transposon end sequences (referred to herein as transposons). Thus, tagmentation can result simultaneously in fragmentation of DNA and ligation of adapters to the 5' ends of both strands of a double-stranded fragment.

[0274] A transposition reaction is a reaction in which one or more transposons are inserted into a target nucleic acid at random or near-random sites. 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 end sequence that binds to the enzyme, and an adapter sequence bound to one of the two transposon end sequences. One strand of the double-stranded transposon end sequence is transferred to a single strand of the target nucleic acid, and the complementary transposon end sequence strand is not transferred (i.e., non-transferred transposon sequence). The adapter sequence may include one or more functional sequences (e.g., primer sequences) as necessary or desired.

[0275] A "transposome complex" is composed 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 a transposition reaction. In some aspects, the transposon recognition sequence is a double-stranded transposon end sequence. The transposase binds to a transposase recognition site in a target nucleic acid and inserts the transposon recognition sequence into the target nucleic acid. In some such insertion events, one strand of the transposon recognition sequence (or end sequence) is transcribed into the target nucleic acid and a cleavage event occurs. Exemplary transposition procedures and systems can be readily adapted for use with transposases.

[0276] "Transposase" refers to an enzyme that can form a functional complex with a transposon end-containing composition (e.g., a transposon, a transposon end, a transposon end composition) and catalyze the insertion or transposition of the transposon end-containing composition into a double-stranded target nucleic acid. Transposases as provided herein can also include integrases from retrotransposons and retroviruses.

[0277] Exemplary transposases that may be used in certain embodiments provided herein include (or are encoded by) Tn5 transposase, Sleeping Beauty (SB) transposase, Vibrio harveyi, MuA transposase and Mu transposase recognition sites including R1 and R2 end sequences, Staphylococcus aureus Tn552, Ty1, Tn7 transposase, Tn / O and IS10, Mariner transposase, Tc1, P Element, Tn3, bacterial insertion sequences, retroviruses, and yeast retrotransposons. More examples include IS5, Tn10, Tn903, IS911, and engineered versions of transposase family enzymes. The methods described herein also include combinations of transposases, not just a single transposase.

[0278] In some embodiments, the transposase is a Tn5, Tn7, MuA, or Vibrio harveyi transposase, or an active variant thereof. In other embodiments, the transposase is a Tn5 transposase or a variant thereof. In other embodiments, the transposase is a Tn5 transposase or a variant thereof. In other embodiments, the transposase is a Tn5 transposase or an active variant thereof. In some embodiments, the Tn5 transposase is a hyperactive Tn5 transposase or an active variant thereof. In some aspects, the Tn5 transposase is a Tn5 transposase described in PCT Publication WO 2015 / 160895, which is incorporated herein by reference. In some aspects, the Tn5 transposase is a hyperactive Tn5 having mutations at positions 54, 56, 372, 212, 214, 251, and 338 relative to wild-type Tn5 transposase. In some aspects, the Tn5 transposase is a hyperactive Tn5 with the following mutations relative to wild-type Tn5 transposase: E54K, M56A, L372P, K212R, P214R, G251R, and A338V. In some embodiments, the Tn5 transposase is a fusion protein. In some embodiments, the Tn5 transposase fusion protein comprises a fusion elongation factor Ts (Tsf) tag. In some embodiments, the Tn5 transposase is a hyperactive Tn5 transposase that comprises mutations at amino acids 54, 56, and 372 relative to the wild-type sequence. In some embodiments, the hyperactive Tn5 transposase is a fusion protein, and optionally, the fusion protein is an elongation factor Ts (Tsf). In some embodiments, the recognition site is a Tn5-type transposase recognition site (Goryshin and Reznikoff, J. Biol. Chem., 273:7367, 1998). In one embodiment, a transposase recognition site that forms a complex with a hyperactive Tn5 transposase is used (e.g., EZ-Tn5TM transposase, Epicentre Biotechnologies, Madison, Wis.). In some embodiments, the Tn5 transposase is a wild-type Tn5 transposase.

[0279] In some embodiments, the transposome complex comprises a dimer of two molecules of transposase. In some embodiments, the transposome complex is a homodimer, where the two molecules of transposase are each bound to a first and a second transposon of the same type (e.g., the sequences of the two transposons bound to each monomer are the same, forming a "homodimer"). In some embodiments, the compositions and methods described herein employ two populations of transposome complexes. In some embodiments, the transposons in each population are the same. In some embodiments, the transposome complexes in each population are homodimers, where the first population has a first adaptor sequence in each monomer and the second population has a different adaptor sequence in each monomer.

[0280] The term "transposon end" refers to a double-stranded nucleic acid DNA that exhibits only the necessary nucleotide sequences ("transposon end sequences") to form a complex with a transposase or integrase enzyme that functions in an in vitro transposition reaction. In some embodiments, the transposon end can form a functional complex with a transposase in a transposition reaction. As a non-limiting example, the transposon end can include a 19-bp outer end ("outer end, OE") transposon end, an inner end ("inner end, IE") transposon end, or a "mosaic end ("mosaic end, ME") transposon end recognized by a wild-type or mutant Tn5 transposase, or an R1 and R2 transposon end, as described in the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, which is incorporated by reference in its entirety. The transposon end can include any nucleic acid or nucleic acid analogue that is suitable for forming a functional complex with a transposase or integrase enzyme in an in vitro transposition reaction. For example, transposon ends can comprise DNA, RNA, modified bases, non-natural bases, modified backbones, and can contain nicks in one or both strands. Although the term "DNA" is used throughout this disclosure in reference to transposon end compositions, it should be understood that any suitable nucleic acid or nucleic acid analogue can be utilized in the transposon ends.

[0281] Similarly, the term "transferred strand" refers to the transferred portions of both transposon ends. Similarly, the term "non-transferred strand" refers to the non-transferred portions of both "transposon ends." The 3' end of the transferred strand is bound or transferred to target DNA in an in vitro transposition reaction. The non-transferred strand, which exhibits a transposon end sequence complementary to the transferred transposon end sequence, is not bound or transferred to target DNA in an in vitro transposition reaction.

[0282] In some embodiments, the transferred strand and the non-transferred strand are covalently linked. For example, in some embodiments, the transferred and non-transferred strand sequences are provided on a single oligonucleotide, for example in a hairpin configuration. Thus, the free end of the non-transferred strand is not directly attached to the target DNA by the translocation reaction, but is indirectly attached to the DNA fragment because the non-transferred strand is linked to the transferred strand by the loop of the hairpin structure. Further examples of transposome structures and methods of preparing and using transposomes can be found in the disclosure of U.S. Patent Application Publication No. 2010 / 0120098, the contents of which are incorporated herein by reference in their entirety.

[0283] In some embodiments, the transposome complexes are engineered to incorporate a unique molecular identifier (UMI) or index sequence, hi some embodiments, the transposome complexes comprise modified mosaic end sequences.

[0284] In some embodiments, compositions for library preparation are optimized to increase library yield. In some embodiments, the compositions increase yield by including a thermostable enzyme, such that incubation above 37° C., e.g., incubation at 55° C., can be performed to increase tagmentation yield. In some embodiments, the compositions include 8 mM or more Mg to increase tagmentation yield. 2+ In some embodiments, (1) the reverse transcriptase, RNA nickase, and / or DNA polymerase are thermostable enzymes, and (2) the Mg 2+ Concentration is 1 mM to 50 mM, optionally containing Mg 2+ The concentration is 5 mM to 20 mM, and optionally Mg 2+ In some embodiments, the composition comprises a thermostable enzyme and 8 mM or more of Mg. 2+ Including,

[0285] B. Transposomes for integrating UMI or index sequences In some embodiments, the first transposon further comprises a UMI or index sequence that is incorporated into the fragment when the double-stranded cDNA fragment is prepared.

[0286] Unique molecular identifiers (UMIs) are sequences of nucleotides applied to or identified in nucleic acid molecules that can be used to distinguish individual nucleic acid molecules from one another. UMIs can be sequenced along with the nucleic acid molecules to which they are associated to determine whether the read sequences are one source nucleic acid molecule or another. The term "UMI" can be used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide itself. UMIs are similar to barcodes that are commonly used to distinguish reads of one sample from reads of other samples, but UMIs are instead used to distinguish a nucleic acid template fragment from another fragment when many fragments from individual samples are sequenced together. UMIs can be defined in many ways, as described in WO2019 / 108972 and WO2018 / 136248, which are incorporated herein by reference.

[0287] Unique molecular identifiers (UMIs) are sequences of nucleotides applied to or identified in nucleic acid molecules that can be used to distinguish individual nucleic acid molecules from one another. UMIs can be sequenced along with the nucleic acid molecules to which they are associated to determine whether the read sequences are one source nucleic acid molecule or another. The term "UMI" can be used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide itself. UMIs are similar to barcodes that are commonly used to distinguish reads of one sample from reads of other samples, but UMIs are instead used to distinguish a nucleic acid template fragment from another fragment when many fragments from individual samples are sequenced together. UMIs can be defined in many ways, as described in WO2019 / 108972 and WO2018 / 136248, which are incorporated herein by reference.

[0288] Unique molecular identifiers (UMIs) are sequences of nucleotides applied to or identified in nucleic acid molecules that can be used to distinguish individual nucleic acid molecules from one another. UMIs can be sequenced along with the nucleic acid molecules to which they are associated to determine whether the read sequences are one source nucleic acid molecule or another. The term "UMI" can be used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide itself. UMIs are similar to barcodes that are commonly used to distinguish reads of one sample from reads of other samples, but UMIs are instead used to distinguish a nucleic acid template fragment from another fragment when many fragments from individual samples are sequenced together. UMIs can be defined in many ways, as described in WO2019 / 108972 and WO2018 / 136248, which are incorporated herein by reference.

[0289] In some embodiments, the library of UMIs includes nonrandom sequences. In some embodiments, nonrandom UMIs (nrUMIs) are predefined for a particular experiment or application. In certain embodiments, rules are used to generate a set of sequences or to select samples from a set to obtain nrUMIs. For example, the set of sequences 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 particular number of nucleotides (e.g., 2, 3, or 4). That is, an nrUMI sequence cannot be converted to any other available nrUMI sequence by replacing fewer than a particular number of nucleotides. In some implementations, the set of UMIs used in the sequencing process includes fewer UMIs than all possible UMIs given a particular sequence length. For example, a set of nrUMIs with 6 nucleotides will have a total of 4 UMIs. A Instead of 6=4096 possible different sequences, a total of 96 different sequences may be included, in some embodiments, the library of UMIs includes 120 non-random sequences.

[0290] In some implementations where the nrUMIs are selected from a set having fewer than all possible distinct sequences, the number of nrUMIs is less than the number of source DNA molecules, sometimes significantly less. In such implementations, the nrUMI information can be combined with other information, such as virtual UMIs, read positions on a reference sequence, and / or sequence information of the reads, to identify sequence reads that originate from the same source DNA molecule.

[0291] In some embodiments, a library of rUMIs may include random UMIs (rUMIs) selected as random samples, with or without replacement, from a set of UMIs consisting of all possible different oligonucleotide sequences given one or more sequence lengths. For example, if each UMI in a set of UMIs has n nucleotides, the set may include 4 UMIs with sequences that differ from each other. n Contains 4 UMIs. A A random sample selected from the n UMIs constitutes the rUMI.

[0292] In some embodiments, the library of UMIs is pseudo-random or partially random and may include a mixture of nrUMIs and rUMIs.

[0293] In some embodiments, an adapter sequence or other nucleotide sequence may be present between the UMI and the insert DNA.

[0294] In some embodiments, an adapter sequence or other nucleotide sequence may be present between each UMI and the insert DNA.

[0295] In some embodiments, the UMI is located 3' to the insert DNA. In some embodiments, a sequence of nucleic acid representing one or more adapter sequences may be located between the UMI and the insert DNA.

[0296] In some embodiments, the UMI is added to the target double-stranded nucleic acid using an oligonucleotide or polynucleotide during or after tagmentation of the nucleic acid. In many embodiments, the UMI is added to the target double-stranded nucleic acid prior to the library amplification step.

[0297] In some embodiments, UMI reagents from the TruSight® Oncology workflow (Illumina catalog number 20024586) may be utilized in accordance with the present disclosure.

[0298] In some embodiments, each double-stranded nucleic acid molecule in a UMI library contains one unique UMI sequence, or a single UMI. In some embodiments, the UMI may be located on either side of the insert DNA. In some embodiments, an adapter sequence or other nucleotide sequence may be present between the UMI and the insert DNA.

[0299] In some embodiments, the UMI library includes a double-stranded UMI, which may reduce the detection error range compared to using a single UMI. The double-stranded UMI allows the skilled artisan to pair the plus strand with its minus strand despite errors that may occur in the sequencing reaction. Such sequencing mismatches are identified during sequencing, and the sequence of the nucleic acid fragment can still be accurately reconstructed despite having a mismatch. In some embodiments, the method for making a UMI library that includes a double-stranded UMI includes a forked adaptor. In some embodiments, the forked adaptor is a BLT fork adaptor.

[0300] In some embodiments, each double-stranded nucleic acid fragment in a UMI library contains two, three, or four UMI sequences, which may have complementary sequences to one another or may each have a different sequence.

[0301] In some embodiments, an adapter sequence or other nucleotide sequence may be present between each UMI and the insert DNA.

[0302] In some embodiments, the UMI is located 5' of the insert DNA. In some embodiments, the UMI is located 3' of the insert DNA. In some embodiments, a sequence of nucleic acid representing one or more adapter sequences may be located between the UMI and the insert DNA. In some embodiments, the UMI is located between the adapter sequence and the transposon end sequence.

[0303] In some embodiments, the UMI may be on the first strand, the second strand, or both strands of the double-stranded target nucleic acid fragment. In some embodiments, the UMI is on the first strand. In some embodiments, the first copy of the UMI is on the first strand of the double-stranded target nucleic acid fragment, and the second copy of the UMI is on the second strand of the double-stranded target nucleic acid fragment. In some embodiments, the first UMI is on the first strand, and the second UMI is on the second strand.

[0304] 1. Inline UMI and index array The UMI may be located anywhere on the double-stranded nucleic acid molecule. In many embodiments, the location of the UMI on the double-stranded nucleic acid molecule varies. In some embodiments, the UMI is located directly adjacent to the insert DNA, i.e., the UMI is an "in-line UMI." In some embodiments, the in-line UMI is adjacent to the 3' end of the insert DNA. In some embodiments, the in-line UMI is adjacent to the 5' end of the insert DNA.

[0305] While UMIs are useful for removing PCR duplicates and detecting low frequency variants in double stranded nucleic acids, UDIs are useful for mitigating sample inaccuracy assignment due to index hopping in library sequencing and demultiplexing. UDIs are unique i5 and i7 index sequences that are added to the ends of target nucleic acids such that both ends contain UDIs. UDIs are used with patterned flow cells such as Illumina's NovaSeq 6000 system (see, e.g., WO 2018 / 204423, WO 2018 / 208699, WO 201 / 9055715, and WO 2016 / 176091, which are incorporated herein by reference in their entirety). Those skilled in the art will understand that in-line UMIs allow compatibility of UMI libraries with standard downstream library preparations that utilize UDIs, such as sample multiplexed PCR and sequencing chemistry recipes in Illumina's TruSeq™ and AmpliSeq™ workflows. In some embodiments, the sequencing method used with in-line UMIs does not require custom primers or custom reads.

[0306] In some embodiments, the UMI library is sequenced using in-line UMIs using standard sequencing methods. In these embodiments, the UMIs are directly adjacent to the 3' end of the insert nucleic acid. Thus, each UMI and insert nucleic acid sequence is captured using standard sequencing primers without the need to sequence additional sequences between them.

[0307] In some embodiments, the "in-line UMI" is located between the insert DNA and the adapter sequence. In some embodiments, the adapter sequence is a second adapter sequence.

[0308] C. Transposomes for integrating modified transposon ends with mutations in the mosaic end sequences Described herein are modified transposon end sequences that contain mosaic end sequences, including those disclosed in U.S. Patent Application Nos. 63 / 224,201, 63 / 167,150, and International Application PCT / US22 / 22167, each of which is incorporated herein in its entirety. In some embodiments, these modified transposon end sequences contain mosaic end sequences that allow for the cleavage and removal of the mosaic end sequence after transposition. A key requirement for transposition is the "mosaic end" (ME), which is specifically recognized by Tn5 and is required for its transposition activity. Tn5 naturally recognizes "outer end" (OE) and "inner end" (IE) sequences that have been shown to be highly intolerant of mutations (as shown in Table 4), with most mutations causing reduced activity (see JC Makris et al. PNAS 85(7):2224-28 (1988)). Subsequent studies showed that chimeric sequences derived from IE and OE, called "mosaic ends" (Table 4), along with mutant Tn5 enzymes, increased transposition activity by approximately 100-fold compared to the native system (see Maggie Zhou et al., Journal of Molecular Biology 276(5):913-25 (1998)). This highly active system is used by Illumina in their Illumina DNA Flex PCR-Free (RUO) product. The crystal structure of Tn5 in complex with a DNA substrate shows that 13 of the 19 base pairs have nucleobase-specific crystal contacts (see Douglas R. Davies et al., Science 289 5476:77-85 (2000)), although other bases have been shown to play a role in catalysis (see Mindy Steiniger-White et al., Journal of Molecular Biology 322(5):971-82 (2002)). Typically, the activity of Tn5 is assessed by an in vivo reporter system (papillation assay such as that described in Zhou et al. J. Mol. Biol. 276:913-925 (1998)).

[0309] [Table 5]

[0310] In Table 4, sequences in normal font indicate the shared sequences, double underlined italicized sequences are derived from natural OE substrates, and bold italicized sequences are derived from natural IE substrates.

[0311] A representative wild-type mosaic end sequence (transferred strand) is SEQ ID NO: 1. Various mutant Tn5 and transposon ends are described in WO 2015 / 160895 and U.S. Pat. No. 9,080,211, each of which is incorporated by reference in its entirety, and may be suitable for use in the methods described herein.

[0312] Several DNA enzymes or combinations of enzymes can mediate the selective removal of modified bases, such as uracil, inosine, ribose bases, 8-oxoG, thymine glycol, modified purines, and modified pyrimidines, among others (see Table 5, and Properties of DNA Repair Enzymes and Structure-specific Endonucleases, New England Biolabs, downloaded on January 20, 2022 from www.international.neb.com / tools-and-resources / selection-charts, and Jacobs and Schar Chromosoma 121:1-20 (2012)). Such enzymes include modification-specific endonucleases or modification-specific glycosylases. Modified purines for use with modification-specific glycosylases include 3-methyladenine (3mA) and 7-methylguanine (7mG). Modified pyrimidines for use with modification-specific glycosylases may include 5-methylcytosine (5mC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). Selective removal of uracil and 8-oxoG using DNA repair enzymes is already used in certain sequencing platforms.

[0313] Because only one strand of the mosaic end, called the "transferred strand," is covalently added to the library insert during transposition, incorporation of such modified bases, specifically into the mosaic end transferred strand, may allow for selective cleavage and removal of the mosaic end transferred strand. However, this type of mosaic end cleavage and removal requires mutation of the mosaic end sequence from its canonical sequence (SEQ ID NO:1).

[0314] [Table 6] * N-glycosylases can be paired with AP lyases / endonucleases (e.g., EndoIII or EndoVIII). Alternatively, abasic sites are chemically labile and can be cleaved under heat and / or basic conditions.

[0315] In Table 5, Endo = endonuclease, FPG = formamidopyrimidine-DNA glycosylase, OGG = oxoguanine glycosylase (OGG), hAAG = human 3-alkyladenine DNA glycosylase, UNG = uracil-N-glycosylase, Nth = cloned nth gene, TDG = thymine-DNA glycosylase, MBD4 = mammalian DNA glycosylase-methyl-CpG binding domain protein 4, and ROS1 = endonuclease ROS1 (with bifunctional DNA glycosylase / lyase activity).

[0316] Disclosed herein are modified transposon end sequences that include a mosaic end sequence, the mosaic end sequence comprising one or more mutations compared to a wild-type mosaic end sequence, the mutations comprising substitutions with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines (e.g., 3mA or 7mG), or modified pyrimidines. In some embodiments, these substitutions are used in methods for truncating transposon ends after transposition, as described below.

[0317] In some embodiments, the mosaic end sequence can be a mosaic end sequence for use with a Tn5 transposase. In some embodiments, the modified transposon end sequence has a mutation in the mosaic end sequence compared to SEQ ID NO:1.

[0318] In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising one or more mutations compared to SEQ ID NO:1, the one or more mutations comprising a substitution at A16, C17, A18, and / or G19. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at A16. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at C17. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at A18. In some embodiments, the modified transposon end sequence comprises a mosaic end sequence comprising a substitution at G19. In some embodiments, the modified transposon end sequence comprises SEQ ID NO:5, 7, 9, or 16-24.

[0319] In some embodiments, the mosaic end sequence comprises more than one mutation, hi some embodiments, the mosaic end sequence comprises eight or fewer mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO: 1).

[0320] In addition to one or more mutations at A16, C17, A18, and / or G19, further mutations may also be present in the mosaic end sequence. In some embodiments, the mosaic end sequence comprises one or more mutations compared to SEQ ID NO:1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence comprises one to four substitution mutations compared to SEQ ID NO:1 in addition to one or more mutations at A16, C17, A18, and / or G19.

[0321] In some embodiments, the mosaic end sequence has one substitution mutation compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence has two substitution mutations compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence has three substitution mutations compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19. In some embodiments, the mosaic end sequence has four substitution mutations compared to SEQ ID NO: 1 in addition to one or more mutations at A16, C17, A18, and / or G19.

[0322] In some embodiments, the substitution at A16 is A16T, A16C, A16G, A16U, A16 inosine, A16 ribose, A16-8-oxoguanine, A16 thymine glycol, A16 modified purine, or A16 modified pyrimidine, and the substitution at C17 is C17T, C17A, C17G, C17U, C17 inosine, C17 ribose, C17-8-oxoguanine, C17 thymine glycol, C17 modified purine, or C17 modified pyrimidine. and the substitution at A18 is A18G, A18T, A18C, A18U, A18 inosine, A18 ribose, A18-8-oxoguanine, A18 thymine glycol, A18 modified purine, or A18 modified pyrimidine, and / or the substitution at G19 is G19T, G19C, G19A, G19U, G19 inosine, G19 ribose, G19-8-oxoguanine, G19 thymine glycol, G19 modified purine, or G19 modified pyrimidine. In some embodiments, the modified purine is 3mA or 7mG. In some embodiments, the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine.

[0323] In some embodiments, the mutations include substitutions with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines. In some embodiments, these mutations allow for a process to cleave the mosaic terminal sequence after transposition.

[0324] In some embodiments, the modified transposon end sequence comprises a mutation at A16, C17, A18, or G19.

[0325] In some embodiments, the modified transposon end sequence comprises two mutations selected from mutations at A16, C17, A18, or G19. In some embodiments, the modified transposon end sequence comprises three mutations selected from mutations at A16, C17, A18, or G19. In some embodiments, the modified transposon end sequence comprises four mutations at A16, C17, A18, and G19.

[0326] In some embodiments, the modified transposon end sequence has one to four substitution mutations at A16, C17, A18, and / or G19 compared to SEQ ID NO:1. In some embodiments, the modified transposon end sequence has one substitution mutation compared to the wild-type sequence (in some embodiments, SEQ ID NO:1). In some embodiments, the modified transposon end sequence has two substitution mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO:1). In some embodiments, the modified transposon end sequence has three substitution mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO:1). In some embodiments, the modified transposon end sequence has four substitution mutations compared to the wild-type sequence (in some embodiments, SEQ ID NO:1).

[0327] D. Immobilized Transposomes In the methods and compositions presented herein, the transposome complex is immobilized on a solid support. In some embodiments, the transposome complex is immobilized on a support via one or more polynucleotides, such as a polynucleotide that includes a transposon end sequence. In some embodiments, the transposome complex may be immobilized via a linker that connects the transposase enzyme to the solid support. In some embodiments, both the transposase enzyme and the polynucleotide are immobilized on the solid support. When referring to the immobilization of a molecule (e.g., a nucleic acid) to a solid support, the terms "immobilized" and "attached" are used interchangeably herein, and both terms are intended to encompass direct or indirect, covalent or non-covalent attachment, unless otherwise indicated, either explicitly or by context. In some embodiments, covalent attachment may be used, but generally, what is required is that the molecule (e.g., a nucleic acid) remains immobilized or attached to the support under conditions under which the support is intended to be used, for example, in applications requiring nucleic acid amplification and / or nucleic acid sequencing.

[0328] In some embodiments, a 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.

[0329] In some embodiments, the transposome complex comprises a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion comprising a transposon end sequence and a first tag.

[0330] In some embodiments, the transposome complex comprises a transposase bound to a first polynucleotide, the first polynucleotide comprising a 3' portion comprising a transposon end sequence and a second tag.

[0331] Thus, in some embodiments, the transposon composition comprises a transferred strand having other nucleotide sequences, such as tag or adapter sequences, at one or more 5' of the transferred transposon sequence. In some embodiments, in addition to the transferred transposon sequence, the transposon may have one or more other tag moieties or tag domains. In some embodiments, in addition to the transferred transposon sequence, the transposon comprises one or more adapters.

[0332] In some embodiments, the transposome complex is immobilized to a solid support via the first polynucleotide.

[0333] In some embodiments, the transposome complex comprises a second polynucleotide that comprises a region that is complementary to a transposon end sequence. In some embodiments, the transposome complex is immobilized to a solid support via the second polynucleotide.

[0334] In some embodiments, the length of the double-stranded fragments in the immobilized library is adjusted by increasing or decreasing the density of transposome complexes on the solid support.

[0335] Means for immobilizing transposome complexes are described in US Patent No. 10,920,219, which is incorporated herein in its entirety. In some embodiments, the first transposon comprises an affinity element. In some embodiments, the affinity element is attached to the 5' end of the first transposon. In some embodiments, the first transposon comprises a linker. In some embodiments, the linker has a first end attached to the 5' end of the first transposon and a second end attached to the affinity element.

[0336] In some embodiments, the transposome complex comprises a second transposon that is complementary to at least a portion of the first transposon end sequence. In some embodiments, the second transposon comprises an affinity element. In some embodiments, the affinity element is attached to the 3' end of the second transposon. In some embodiments, the second transposon comprises a linker. In some embodiments, the linker has a first end attached to the 3' end of the second transposon and a second end attached to the affinity element.

[0337] In some embodiments, the affinity element is biotin or dual biotin, hi some embodiments, the solid support is coated with streptavidin.

[0338] In some embodiments, the solid support is a bead and the method uses DNA BLT (bead-linked transposome). Surface-bound transposomes (e.g., BLT) can tagment long molecules of double-stranded DNA and generate template libraries on beads or other surfaces (US Pat. No. 9,683,230). Immobilizing transposomes on beads provides novel properties such as controllable insert size and yield. This is the basis of Illumina DNA Flex PCR-Free technology, formerly known as Illumina's Nextera technology.

[0339] Because transposon ends have an affinity for DNA, DNA BLT does not require capture oligonucleotides for immobilization on beads; instead, DNA can be immobilized using a polynucleotide containing the transposon end sequence. Alternatively, capture oligonucleotides can be used to capture DNA molecules.

[0340] Representative products that use immobilized transposomes (i.e., bead-linked transposomes) include Illumina® DNA Prep, (S) Tagmentation and Illumina® RNA Prep, (L) Tagmentation or Illumina® RNA Prep, (L) Tagmentation.

[0341] 1. Solid Support for Immobilizing Transposomes Certain embodiments may utilize solid supports comprised of an inert substrate or matrix (e.g., glass slides, polymeric beads, etc.) that have been functionalized by application of a layer or coating of an intermediate material that contains reactive groups that allow for covalent attachment to, for example, biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly those described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein by reference in their entireties. In such embodiments, the biomolecule (e.g., polynucleotide) may be covalently attached directly to the intermediate material (e.g., hydrogel), although the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g., glass substrate). The term "covalent attachment to a solid support" should be interpreted accordingly to encompass this type of sequence.

[0342] The terms "solid surface", "solid support" and other grammatical equivalents herein refer to any material that is suitable or can be modified to be suitable for binding transposome complexes. As will be appreciated by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, plastics, fiber optic bundles, and various other polymers. In some embodiments, particularly useful solid supports and solid surfaces are placed in a flow cell device. Exemplary flow cells are shown in more detail below.

[0343] In some embodiments, the solid support comprises a patterned surface suitable for immobilizing transposome complexes in a regular pattern. A "patterned surface" refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more of the regions can be features in which one or more transposome complexes are present. The features can be separated by interstitial regions in which no transposome complexes are present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some implementations, the pattern can be a random arrangement of features and / or interstitial regions. In some embodiments, the transposome complexes are randomly distributed on the solid support. In some embodiments, the transposome complexes are distributed on the patterned surface. Exemplary patterned surfaces that can be used in the methods and compositions provided herein are described in U.S. Application Serial No. 13 / 661524 or U.S. Patent Application Publication No. 2012 / 0316086, each of which is incorporated herein by reference.

[0344] In some embodiments, the solid support comprises an array of wells or depressions on its surface, which can be fabricated as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As will be understood in the art, the technique used will depend on the composition and shape of the array substrate.

[0345] The composition and geometry of the solid support may vary depending on its use. In some embodiments, the solid support is a planar structure, such as a slide, a chip, a microchip, and / or an array. Thus, the surface of the substrate may be in the form of a planar layer. In some embodiments, the solid support comprises one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber that comprises a solid surface through which one or more fluidic reagents can flow. Examples of flow cells and associated fluidic systems and detection platforms that can readily be used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. No. 7,329,492, U.S. Pat. No. 7,211,414, U.S. Pat. No. 7,315,019, U.S. Pat. No. 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0346] In some embodiments, the solid support or its surface is non-planar, such as the interior or exterior surface of a tube or container. In some embodiments, the solid support comprises a microsphere or bead. By "microsphere" or "bead" or "particle" or grammatical equivalents herein is meant a small discrete particle. Suitable bead compositions include, but are not limited to, plastic, ceramic, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex, or cross-linked dextran such as Sepharose, cellulose, nylon, cross-linked micelles, and Teflon, as well as any other material outlined herein for solid supports can all be used. The "Microsphere Selection Guide" from Bangs Laboratories, Fishers, Ind. is a useful guide. In certain embodiments, the microsphere is a magnetic microsphere or bead.

[0347] The beads need not be spherical. Irregular particles may be used. Alternatively or additionally, the beads may be porous. Bead sizes range from nanometers, i.e., 100 nm, to millimeters, i.e., 1 mm, with beads being about 0.2 microns to about 200 microns, or about 0.5 to about 5 microns, although in some embodiments smaller or larger beads may be used.

[0348] The density of these surface-bound transposomes can be adjusted by varying the density of the first polynucleotide or by the amount of transposase added to the solid support. For example, in some embodiments, transposome complexes can be assembled at a density of 1 mm 2 At least 10 per 3 , 10 4 , 10 5 , or 10 6 The complexes are present on the solid support at a density of 100 .mu.m.

[0349] When double-stranded DNA is synthesized for tagmentation on transposomes on a solid support (e.g., BLT), the transposome complex generates a ds fragment bound to the surface at both ends by tagmentation. In some embodiments, the length of the crosslinked fragment can be varied by changing the density of the transposome complex on the surface. In certain embodiments, the length of the resulting crosslinked fragment can be 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp, 1900bp, 2000bp, 2100bp, 2200bp, 2300bp, 2400bp, 2500bp, 2600bp, 2700bp, 2800bp, 2900bp, 3000bp, 3100bp, 3200bp, 3300bp, 3400bp, 3500bp, 3600bp, 3700bp, 3800bp, 3900bp, 4000bp, 4100bp, 4200bp, 4300bp, 4400bp, 4500bp, 4600bp, 4700bp, 4800bp, 4900bp, 5000bp, 5100bp, 5200bp, 5300bp, 5400bp, 5500bp, 5600bp, 5700bp, 5800bp, 5900bp, 6000bp, 6 In some embodiments, the length of the cross-linked fragments may be less than 300 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 less than 100000 bp. In such embodiments, the cross-linked fragments may then be amplified into clusters using standard cluster chemistry, as exemplified by the disclosures in U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are incorporated herein by reference in their entirety.

[0350] The binding of nucleic acid to the support, whether rigid or semi-rigid, can occur via covalent or non-covalent bonds. Exemplary linkages are described in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, and 7,427,678, and U.S. Patent Publication No. 2011 / 0059865(A1), each of which is incorporated herein by reference. In some embodiments, nucleic acid or other reaction components can be bound to a gel or other semi-solid support, which is then bound or attached to a solid phase support. In such embodiments, nucleic acid or other reaction components are considered to be solid phase.

[0351] In some embodiments, the solid support comprises a microparticle, a bead, a planar support, a patterned surface, or a well. In some embodiments, the planar support is the interior or exterior surface of a tube.

[0352] In some embodiments, the solid support is for tagmentation of DNA and is referred to as a DNA bead-linked transposome (DNA BLT).

[0353] In some embodiments, the solid support further comprises a transposase bound to the first polynucleotide to form a transposome complex.

[0354] In some embodiments, the solid support comprises a library of tagged fragments immobilized thereon, prepared according to any of the methods described herein.

[0355] In some embodiments, the kit comprises a solid support as described herein. In some embodiments, the kit further comprises a transposase. In some embodiments, the kit further comprises a composition as described herein.

[0356] In some embodiments, the transposome complex may be a solution-phase transposome complex, such as that described in U.S. Patent Application No. 9,683,230, the entirety of which is incorporated herein by reference. In some embodiments, the liquid-phase transposome complex is used to generate tagged fragments in solution. In some embodiments, the method may further include contacting the liquid-phase transposome complex with the immobilized DNA fragment under conditions in which the DNA fragment is further fragmented by the liquid-phase transposome complex, thereby obtaining an immobilized nucleic acid fragment with one end in solution.

[0357] 2. Exemplary BLT As used herein, BLT can refer to any type of bead on whose surface transposome complexes are immobilized. A variety of BLTs are known in the art.

[0358] An exemplary BLT is the Illumina RNA Prep with Enrichment(L) product (see RNA Prep with Enrichment(L) Tagmentation Reference Guide, Document # 1000000124435 v02, Illumina, 2020 ("Document 1000000124435"). Such BLTs are often currently the preferred method for preparation of RNA libraries (i.e., libraries of cDNA fragments generated from an RNA sample) due to their ability to produce fragments with larger inserts.

[0359] Another exemplary BLT is Illumina® DNA Prep, (S)Tagmentation. In some embodiments, such a BLT produces fragments with smaller inserts based on a higher density of transposomes on the beads compared to other BLTs.

[0360] In some embodiments, BLT is used in one-pot library preparation (combining cDNA and library preparation). In some embodiments, BLT with higher transposomal activity improves library yield in one-pot library preparation (e.g., higher density of transposomes on beads). Those skilled in the art can use standard experiments to determine the best conditions for using a given BLT in the methods of the present invention.

[0361] E. Adapters and Tags In some embodiments, the first transposon comprises one or more adaptor sequences. In some embodiments, the first transposon comprises a 3' transposon end sequence and a 5' adaptor sequence. In some embodiments, the 5' adaptor sequence is a tag sequence. The fragmentation mediated by the transposome complex comprising the first transposon comprising a 3' transposon end sequence and a 5' tag can be used in a method for generating a library of tagged fragments.

[0362] In some embodiments, the tag is an adapter sequence. 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, a sequencing-related sequence can be any sequence that is related to a subsequent sequencing step. A sequencing-related sequence can act to simplify downstream sequencing steps. For example, a sequencing-related sequence can be a sequence that is otherwise incorporated via a step of ligating an adapter to a nucleic acid fragment. In some embodiments, the adapter sequence includes a P5 or P7 sequence (or their complements) to facilitate binding to a flow cell in a particular sequencing method. The present disclosure is not limited to the types of adapter sequences that can be used, and one of skill in the art will recognize additional sequences that can be used for library preparation and next-generation sequencing.

[0363] As used herein, the term "tag" refers to a portion or domain of a polynucleotide that exhibits a sequence for a desired intended purpose or use. A tag domain may include any sequence provided for any desired purpose. For example, in some embodiments, a tag domain includes one or more restriction endonuclease recognition sites. In some embodiments, a tag domain includes one or more regions suitable for hybridization with primers for a cluster amplification reaction. In some embodiments, a tag domain includes one or more regions suitable for hybridization with primers for a sequencing reaction. It will be understood that any other suitable features may be incorporated into the tag domain. In some embodiments, a tag domain includes a sequence having a length of 5 bp to 200 bp. In some embodiments, a tag domain includes a sequence having a length of 10 bp to 100 bp. In some embodiments, a tag domain includes a sequence having a length of 20 bp to 50 bp. In some embodiments, a tag domain includes a sequence having a length of 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 bp.

[0364] A tag can include one or more functional sequences or components (eg, a primer sequence, an anchor sequence, a universal sequence, a spacer region, or an index tag sequence) as necessary or desired.

[0365] In some embodiments, the tag comprises a region for cluster amplification, hi some embodiments, the tag comprises a region for priming a sequencing reaction.

[0366] In some embodiments, the method further comprises amplifying the fragment on the solid support by reacting a polymerase with an amplification primer corresponding to a portion of the first transposon. In some embodiments, the portion of the first transposon comprises an amplification primer. In some embodiments, the tag of the first transposon comprises an amplification primer.

[0367] In some embodiments, the tag comprises the A14 primer sequence (SEQ ID NO: 11). In some embodiments, the tag comprises the B15 primer sequence (SEQ ID NO: 12).

[0368] In some embodiments, transposomes on individual beads carry a unique index, and when large numbers of such indexed beads are used, phased transcripts result.

[0369] III. cDNA Preparation Method A difficulty in RNA sequencing (e.g., RNA-Seq from Illumina) analysis is the need to convert RNA to DNA prior to library preparation, adding significant time and complexity to the procedure. However, RNA is an important molecule, providing quantitative functional information about the transcriptome and metatranscriptome. Importantly, in many cases for disease surveillance, RNA is of primary concern, since the most pathogenic families of viruses have RNA genomes (see "Select Agents and Toxins List," CDC / USDA (2020) or www.selectagents.gov). The easier method for RNA library preparation presented herein may be preferred by users, especially those who may be less familiar with NGS protocols. Furthermore, the method of the present invention may allow for simple and cost-effective automation to process multiple samples. Figures 4-10 show data from the inventive mesophilic method of cDNA preparation in a one-step process (i.e., the method of the present invention). Figures 11-12B show data from the inventive thermostable method of cDNA preparation in a one-step process.

[0370] The method described herein can convert RNA from a sample into double-stranded cDNA in as little as 15 minutes. This is a significant reduction compared to 110 minutes for conventional protocols (such as Illumina Stranded Total RNA Prep or Illumina Stranded mRNA Prep, as shown in FIG. 1). Additionally, the number of touch points (i.e., user actions) is reduced, making the protocol easier for end users. A more detailed overview of the timeline differences for the standard two-step method of cDNA preparation versus the one-step method of the present invention is shown in FIG. 14B.

[0371] Described herein is a method for preparing double-stranded cDNA, the method comprising: (i) combining a primer with a sample containing RNA and allowing binding of the primer to the RNA; and (ii) combining the sample with a composition for preparing ds-cDNA described herein and preparing double-stranded cDNA by an isothermal reaction.

[0372] Figure 3 shows a model of the method. In some embodiments, a composition with a balance of individual enzyme components aids in the preparation of ds-cDNA. In some embodiments, the production of first strand cDNA exceeds the rate at which RNA is nicked by RNAse H. In some embodiments, the activity of reverse transcriptase exceeds the activity of RNAse H.

[0373] In some embodiments, the sample comprises 10 ng or more of RNA. In some embodiments, the sample comprises less than 10 ng of RNA.

[0374] In some embodiments, the reverse transcriptase produces a first strand of cDNA. In some embodiments, the reverse transcriptase produces a DNA:RNA duplex comprising the first strand of cDNA and a strand of RNA. In some embodiments, RNAse H nicks the RNA strand in the DNA:RNA duplex, producing an RNA fragment. In some embodiments, a DNA polymerase extends the second strand of DNA by priming from the RNA fragment. In some embodiments, the 5'-3' and 3' to 5' activity of the RNA nickase and / or DNA polymerase removes the RNA fragment and the RNA overhang.

[0375] In some embodiments, the rate of production of the first strand of cDNA by reverse transcriptase is greater than the rate of nicking of RNA by RNA nickase. In some embodiments, the activity of reverse transcriptase exceeds the activity of RNA nickase. In this way, the first strand of cDNA is produced before RNA is degraded by RNA nickase.

[0376] In some embodiments, the DNA polymerase has 5'-3' exonuclease activity and / or 3'-5' exonuclease activity, which produces blunt-ended double-stranded cDNA. In some embodiments, the DNA polymerase has strand displacement activity.

[0377] In some embodiments, dNTPs are used by both the reverse transcriptase and the DNA polymerase.

[0378] In some embodiments, depletion of undesired RNA is performed before preparing cDNA. In some embodiments, the undesired RNA is ribosomal RNA (rRNA). In some embodiments, the method includes depleting off-target RNA with a sample containing RNA before combining the primer with the sample containing RNA.

[0379] In some embodiments, enrichment of the desired RNA is performed prior to preparing cDNA. In some embodiments, the desired RNA is mRNA.

[0380] A. Primer A variety of different primers can be used in this method.

[0381] In some embodiments, the primer comprises a random primer (also known as a randomer primer). In some embodiments, the randomer primer does not target a specific sequence (i.e., the primer is not targeted). In some embodiments, the randomer primer allows for unbiased production of ds-cDNA from RNA because the user has not selected a specific sequence for the randomer primer to bind. In some embodiments, the method using the randomer primer avoids biased or uneven preparation of cDNA because the method does not include a primer designed to bind to a specific sequence within the RNA. In some embodiments, gene-specific or transcript-specific primers are not required for the method. Instead, random primers can be used to prime the synthesis of the first strand of cDNA to initiate the coordinated process of transforming RNA into double-stranded cDNA as described herein. In some embodiments, the random primer avoids the need for specialized primers, such as AT-rich primers, that may be required to facilitate binding of the targeted primer.

[0382] In some embodiments, the primer specifically binds to one or more sequences contained in the RNA. Such primers that specifically bind to one or more sequences contained in the RNA can be called "targeting primers". In some embodiments, the targeting primer allows the production of ds-cDNA from a specific region of the RNA. This targeted production of ds-cDNA is based on the fact that the first strand of cDNA is only generated based on the region that the targeting primer binds to.

[0383] In some embodiments, the primer comprises a hexamer primer, hi some embodiments, the primer comprises a random hexamer primer.

[0384] In some embodiments, the primers comprise a mixture that includes randomer primers and targeted primers.

[0385] In some embodiments, the primer comprises a primer comprising a chemically modified nucleotide. In some embodiments, the primer comprising a chemically modified nucleotide renders the RNA bound by the primer resistant to cleavage by an RNA nickase. In some embodiments, the RNA nickase is RNAse H, and the RNA bound by the primer comprising a chemically modified nucleotide is resistant to cleavage by RNAse H.

[0386] In some embodiments, the chemically modified nucleotide comprises a methylphosphonate residue.

[0387] B. Isothermal reaction Compared to methods known in the art, the advantage of the method of the present invention is the ability to produce ds-cDNA from RNA without the need for temperature changes. As shown in Figure 1, prior art methods require up to five temperature changes. These temperature changes require more sophisticated equipment (such as programmable thermocyclers) and user input compared to current isothermal methods.

[0388] In some embodiments, the isothermal reaction for preparing ds-cDNA may be carried out using a mesophilic or thermostable composition, as described above.

[0389] In some embodiments, the method does not require barcode. In some embodiments, the method does not require computer-controlled temperature regulation in a thermal cycler. In some embodiments, the reaction temperature does not need to be changed after the composition described herein is added. In some embodiments, primer binding may be performed at different temperatures, and the reaction temperature changes when the composition is added, but the reaction does not require temperature change when preparing ds-cDNA from the RNA bound by primer.

[0390] In some embodiments, the isothermal reaction is at a temperature between 30° C. and 49° C. In some embodiments, the isothermal reaction is at a temperature of 37° C. In some embodiments, the isothermal reaction at a temperature between 30° C. and 49° C. is performed using a composition for mesophilic ds-cDNA preparation, as described herein.

[0391] In some embodiments, the isothermal reaction is at a temperature between 50° C. and 72° C. In some embodiments, the isothermal reaction is at a temperature of 50° C. In some embodiments, the isothermal reaction at a temperature between 50° C. and 72° C. is performed using a composition for preparing thermostable ds-cDNA as described herein.

[0392] In some embodiments, the RNA exhibits secondary structures that normally inhibit first strand synthesis at temperatures below 50° C. In some embodiments, isothermal reactions performed at 50° C.-72° C. exhibit improved ds-cDNA yields or coverage compared to isothermal reactions performed at temperatures below 50° C. RNA secondary structures, such as hairpins, will be well known to those of skill in the art.

[0393] C. Incubation time An advantage of the method of the present invention may be the reduced reaction time compared to methods known in the art: as shown in Figure 1, the prior art method requires up to 110 minutes, whereas the method of the present invention can be carried out in 15 minutes or less.

[0394] In some embodiments, the isothermal reaction is incubated for 60 minutes or less, 45 minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes or less, or 10 minutes or less. In some embodiments, the isothermal reaction is incubated for 15 minutes or less. In some embodiments, the isothermal reaction is incubated for 10 minutes or less.

[0395] In some embodiments, incubation for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes provides ds-cDNA for library preparation.

[0396] D. rRNA depletion or mRNA enrichment In some embodiments, before starting cDNA preparation (e.g., before primers are bound to RNA), desired RNA is enriched or undesired RNA is depleted. In this way, cDNA is produced only from desired RNA. Such a method can avoid waste of reagents and unnecessary data analysis.

[0397] In some embodiments, the method comprises removing undesired RNA before preparing cDNA.In this way, cDNA is not produced from undesired RNA.In some embodiments, undesired RNA is an abundant RNA that would otherwise result in the production of a significant amount of cDNA associated with undesired RNA.

[0398] In some embodiments, removal of undesired RNA is by enzymatic depletion. In some embodiments, the remaining RNA after depletion of undesired RNA is then converted to cDNA by the methods described herein.

[0399] In some embodiments, the undesired RNA comprises a ribosomal RNA or a beta globin transcript. Several different types of rRNA depletion methods are known, including those disclosed in U.S. Patent No. 9,745,570 and WO 2020 / 132304, each of which is incorporated herein by reference in its entirety.

[0400] In some embodiments, the rRNA is cytoplasmic or mitochondrial. In some embodiments, the rRNA is human, mouse, rat, gram (-) bacterial, or gram (+) bacterial rRNA. In some embodiments, the undesired RNA comprises a beta globin transcript. In some embodiments, the undesired RNA is a human beta globin transcript.

[0401] In some embodiments, rRNA depletion can be performed using the Illumina® Ribo-Zero Plus rRNA Depletion kit or other similar kits or methods.

[0402] In some embodiments, the method includes enriching the desired RNA prior to preparing the cDNA, hi some embodiments, the desired RNA is mRNA.

[0403] In some embodiments, enriching for mRNA comprises amplifying with poly-T primers or binding of the mRNA to capture beads, hi some embodiments, the capture beads comprise a surface having capture oligonucleotides that include a poly-T sequence.

[0404] IV. Library Preparation Methods In some embodiments, the method allows for preparing a library of double-stranded DNA fragments in a single reaction vessel from a sample containing RNA. In some embodiments, the double-stranded DNA fragments comprise double-stranded cDNA prepared as described above. In some embodiments, the method of library preparation comprises a method of preparing double-stranded cDNA from RNA as described herein. In some embodiments, the combination of cDNA and library preparation may be performed in a single reaction vessel, which may be referred to herein as a "one-pot" method. In some embodiments, the one-pot method of cDNA and library preparation comprises a one-step method of cDNA preparation combined with the preparation of library fragments in the same reaction vessel. In some embodiments, the preparation of library fragments in the one-pot method is by tagmentation using BLT.

[0405] A representative overview of a one-pot method of library preparation, combining cDNA and library preparation, is shown in Figure 13. As shown in Figures 14A and 14B, library preparation by BLT can be included in a method that also includes mesophilic or thermostable double-stranded cDNA synthesis. In this way, users can generate libraries for sequencing from RNA in a single reaction vessel. The time advantage of one-pot library preparation is shown in Figure 14C, where one-pot library preparation can save 1-1.5 hours of time over other preparation methods and avoid multiple hands-on steps for the user. Figure 16C shows that comparable fragments are generated in one pot (combined cDNA preparation and tagmentation) compared to two-step cDNA preparation (Figure 16A) or one-step cDNA preparation (Figure 16B) followed by separate tagmentation, as summarized in Figure 16D. Figures 17A-18B and Figures 20A-24 provide further data on one-pot tagmentation libraries and comparative data with other library preparations.

[0406] Figure 25 outlines the advantages of one-pot library preparation, which reduces the time from 4 hours for two-step cDNA preparation followed by separate tagmentation, or 3 hours for one-step cDNA preparation followed by separate tagmentation, to approximately 2 hours. In some embodiments, one-pot library preparation methods require only a single cleanup step (such as with SPRI beads). In some embodiments, no SPRI or other cleanup is performed between the preparation of double-stranded cDNA and the preparation of double-stranded cDNA fragments by isothermal reaction. Thus, one-pot library preparation can save overall time and hands-on time for the user.

[0407] The number of steps required to convert a target nucleic acid, such as DNA, into an adaptor-modified template ready for next-generation sequencing can be minimized by tagmentation. Tagmentation results in simultaneous fragmentation of the target nucleic acid and ligation of adaptors to the 5'-ends of both strands of the double-stranded nucleic acid fragment. If the transposome complex is support-bound (i.e., immobilized on a solid support), the resulting fragments are bound to the solid support after the tagmentation reaction (either directly in the case of 5'-linked transposome complexes or via hybridization in the case of 3'-linked transposome complexes).

[0408] In some embodiments, tagmentation is performed after preparing double-stranded cDNA. In some embodiments, tagmentation is performed on bead-linked transposome (BLT). BLT does not bind to RNA itself, therefore, BLT in the reaction fragments the double-stranded cDNA prepared from RNA. In some embodiments, the method using BLT results in a library of cDNA fragments immobilized on beads.

[0409] In some embodiments, the reactions for preparing cDNA from RNA and the reactions for preparing fragments of the cDNA are performed simultaneously. In some embodiments, the preparation of the library fragments can be performed as the cDNA is being prepared without the need to change reaction vessels. In some embodiments, the cDNA can be fragmented as it is being prepared without purifying the cDNA prior to fragmentation. In some embodiments, all steps of the library preparation are performed in a single reaction vessel.

[0410] In some embodiments, a method for preparing a library of double-stranded cDNA fragments includes combining a primer with a sample containing RNA and allowing binding of the primer to the RNA, combining the sample with a composition described herein, (i) preparing double-stranded cDNA by an isothermal reaction, and (ii) preparing the double-stranded cDNA fragments.

[0411] In some embodiments, undesired RNAs are depleted or desired RNAs are enriched prior to library preparation. In some embodiments, the undesired RNAs are rRNAs and the desired RNAs are mRNAs. Representative data from rRNA depletion using one-pot library preparation is shown in Figures 19A-B.

[0412] In some embodiments, combining the primer with the sample and combining the sample with the composition are performed in the same step.

[0413] In some embodiments, both (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are performed in a single isothermal reaction.

[0414] In some embodiments, (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are performed at different temperatures.

[0415] In some embodiments, (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are performed in a single reaction vessel.

[0416] In some embodiments, combining the primer with the sample comprising RNA comprises mixing the sample comprising RNA with an eluate, primers, and fragmentation mix. An exemplary eluate, primer, and fragmentation mix would be EPH3 (Illumina®).

[0417] In some embodiments, combining the primers with the sample comprising RNA is performed at or above 55° C. In some embodiments, combining the primers with the sample comprising RNA is performed at 65° C.

[0418] In some embodiments, the fragments of the cDNA library are prepared by tagmentation. In some embodiments, the reaction temperature is increased after double-stranded cDNA preparation to increase the efficiency of tagmentation. In some embodiments, RNA is converted into double-stranded cDNA in a single reaction vessel, and then converted into a library of double-stranded DNA fragments, where double-stranded cDNA is prepared through an isothermal reaction, and the temperature of the reaction vessel is increased to improve the efficiency of tagmentation to prepare fragments. In some embodiments, RNA is converted into double-stranded cDNA in a single reaction vessel through a single isothermal reaction, and then converted into a library of double-stranded DNA fragments.

[0419] In some embodiments, the isothermal reaction for preparing double-stranded cDNA is at a temperature between 30° C. and 49° C. In some embodiments, the isothermal reaction for preparing double-stranded cDNA is at a temperature of 37° C. or higher. In some embodiments, the isothermal reaction for preparing double-stranded cDNA is at a temperature of 37° C. In some embodiments, the isothermal reaction for preparing double-stranded cDNA is at a temperature of 55° C.

[0420] In some embodiments, both (i) preparing double-stranded cDNA and (ii) preparing double-stranded cDNA fragments are performed in a single isothermal reaction at 37° C. In some embodiments, preparing double-stranded cDNA fragments and / or preparing double-stranded cDNA fragments is performed above 37° C. In some embodiments, preparing double-stranded cDNA fragments is performed at 55° C. In some embodiments, preparing double-stranded cDNA is performed at 37° C. and preparing double-stranded cDNA fragments is performed at 55° C.

[0421] In some embodiments, the Mg content of the composition used in the method is 2+ The concentration is 1 mM to 50 mM, and optionally, Mg 2+ The concentration is 5 mM to 20 mM, and optionally, Mg 2+ In some embodiments, (1) the reverse transcriptase, RNA nickase, and / or DNA polymerase are thermostable enzymes, and (2) the Mg content of the composition used in the method is 8 mM. 2+ The concentration is 1 mM to 50 mM, and optionally Mg 2+ The concentration is 5 mM to 20 mM, and optionally Mg 2+ The concentration is 8 mM.

[0422] In some embodiments, the rate of production of the first strand of cDNA by the reverse transcriptase is greater than the rate of nicking of RNA by the RNA nickase. In some embodiments, the activity of the reverse transcriptase exceeds the activity of the RNA nickase. In some embodiments, (i) preparing double-stranded cDNA by an isothermal reaction and (ii) preparing double-stranded cDNA fragments are performed in a total incubation of 60 minutes or less, or 30 minutes or less.

[0423] In some embodiments, the gap-filling ligation is performed after preparation of the double-stranded DNA fragments. In some embodiments, the double-stranded DNA fragments are amplified.

[0424] In some embodiments, double-stranded DNA fragments are sequenced.In some embodiments, tagmentation incorporates adaptors for sequencing double-stranded DNA fragments.In some embodiments, double-stranded DNA fragments are amplified before sequencing.In some embodiments, double-stranded DNA fragments are not amplified before sequencing.

[0425] In some embodiments, the methods of the present invention allow for faster and simpler preparation of libraries for sequencing from starting samples containing RNA.For example, the methods of the present invention can allow for enhanced pathogen surveillance of RNA viruses, although sequencing from any type of RNA sample can be enhanced using the methods of the present invention.

[0426] In some embodiments, depletion of undesired RNAs or enrichment of desired RNAs can be performed prior to library preparation, as described above.

[0427] In some embodiments, the tagmentation stop buffer is added after preparing double-stranded cDNA fragments. In some embodiments, the prepared double-stranded cDNA fragments are purified. In some embodiments, the prepared double-stranded cDNA fragments are sequenced. In some embodiments, the prepared double-stranded cDNA fragments are purified and then sequenced.

[0428] In some embodiments, the method of library preparation includes fragmenting the modified transposon ends. In some embodiments, the method of library preparation includes incorporating UMIs. In some embodiments, the method of library preparation further includes steps such as gap-filling ligation, amplification, and / or sequencing of the fragments.

[0429] In some embodiments, the method of library preparation comprises targeted enrichment. In some embodiments, the step of preparing double-stranded cDNA fragments comprises enrichment of target fragments. In some embodiments, the amplification of double-stranded cDNA fragments is carried out using target-specific primers that bind to and allow amplification of target fragments.

[0430] A. Optimization of the Tagmentation Reaction In some embodiments, the method for one-pot library preparation includes optimization to increase the yield of the tagmentation reaction. The results of such optimization are shown in Figures 26-30B.

[0431] In some embodiments, a step of incubation above 37° C. is included in the one-pot library preparation. It is known in the art that tagmentation preferably occurs above 37° C., for example at 55° C. In some embodiments, the one-pot library method includes incubation of the reaction at 55° C. In some embodiments, the incubation at 55° C. is for 15 minutes or 30 minutes. In some embodiments, the incubation at 55° C. is performed after incubation at 37° C. (i.e., the reaction temperature is increased after allowing for double-stranded cDNA preparation at 37° C.).

[0432] In some embodiments, the entire one-pot library preparation is performed by isothermal reactions above 37° C. In some embodiments, the entire one-pot library preparation is performed by isothermal reactions at 40° C. or higher, 45° C. or higher, 50° C. or higher, or 55° C. or higher. In some embodiments, the entire one-pot library preparation is performed at 55° C. Such library preparations at 55° C. can be performed using thermostable enzymes described herein.

[0433] In some embodiments, the one-pot library preparation involves the use of relatively high Mg 2+The experiments in Figures 26 to 30B were performed at 1000 ng / mL concentration. Transposases such as Tn5 are known to use magnesium ions as a cofactor in the tagmentation reaction. 2+ It has been shown that increasing Mg helps improve library yields in one-pot library preparations. 2+ The concentration is 1 mM to 50 mM. 2+ The concentration is 5 mM to 20 mM. 2+ The concentration is 8 mM. In some embodiments, users may choose the Mg concentration that provides optimal yields. 2+ The concentration is determined empirically. In some embodiments, Mg 2+ The concentration is optimized to increase the tagmentation reaction while minimizing the potential for RNA degradation.

[0434] B. Rearrangement reaction for fragmentation Transposition is an enzyme-mediated process by which DNA sequences are inserted, deleted, and duplicated within a genome. This process has been adapted for widespread use in fragmenting double-stranded nucleic acids (such as double-stranded DNA and DNA:RNA duplexes). Transposition can generate DNA fragments without the use of standard fragmentase protocols.

[0435] The well-studied E. coli Tn5 transposon mobilizes by a "cut-and-paste" transposition mechanism. First, the Tn5 transposase Tnp (hereafter referred to as Tn5) recognizes conserved substrate sequences on either side of the transposon DNA, which is then excised or "cut" from the genome. Tn5 then inserts or "pastes" this transposon DNA into the target DNA.

[0436] Tn5 is exploited in many library preparation reagents (such as those from Illumina) for its ability to "tagment" genomic DNA, i.e., simultaneously "tag" and "fragment," thus greatly reducing the time and complexity involved in traditional sonication / ligation-based library preparation protocols. To aid its use in library preparation, Tn5 is preloaded with a transposon consisting of conserved substrate sequences, called "mosaic ends" or "end sequences," appended to an adapter sequence (e.g., Illumina's A14 and B15 adapter sequences). This transposome complex, containing Tn5 transposase and the transposon sequence with the adapter, is then mixed with the genomic DNA sample. The resulting library preparation transposon has only the short adapter sequence, thus simultaneously resulting in fragmentation of genomic DNA and tagging with the short adapter sequence.

[0437] In some embodiments, transposition with the modified transposon ends described herein provides results equivalent to transposition with wild-type (i.e., transposon ends that do not contain a mutation described herein). In some embodiments, preparing fragments with a transposome complex described herein results in preparation of at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the number of fragments compared to preparing fragments with a transposome complex comprising a first transposon that comprises a transposon end sequence that comprises a wild-type mosaic end sequence comprising SEQ ID NO:1.

[0438] 1. Removal of mosaic ends In some embodiments, selective cleavage of mosaic ends using an enzyme is a very attractive mechanism for transforming Tn5 into a fragmentase system (i.e., generating fragments lacking mosaic ends). As used herein, "base modification" or "DNA base modification" refers to the position of a modified base (e.g., those listed in Table 3) in a double-stranded nucleic acid that is recognized by an enzyme (e.g., (a) an endonuclease, or (b) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites) and triggers cleavage at this modified base. In some embodiments, the endonuclease or DNA glycosylase is modification-specific.

[0439] In some embodiments, the base modification is cleaved using (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an abasic site-recognizing endonuclease / lyase. For example, a DNA glycosylase can generate an abasic site that is then acted upon by heat, basic conditions, or an abasic site-recognizing endonuclease / lyase. The USER Reagent is an exemplary enzyme mixture that includes a DNA glycosylase and an abasic site-recognizing endonuclease / lyase. Users can choose the method of cleaving at the abasic site depending on their preferred workflow. A modification-specific endonuclease can cleave the modified base in a one-step reaction, or a modification-specific glycosylase followed by an AP lyase / endonuclease or heat can cleave the modified base in a two-step reaction.

[0440] Fragments prepared from such a rearrangement reaction followed by cleavage at the modified bases contain a 5' overhang having a 5' phosphate and a 3'-OH, and an insert having 0 to 3 bases of the ME sequence, depending on one or more of the modification sites at positions 16 to 19 of SEQ ID NO:1.

[0441] In some embodiments, cleavage of the modified mosaic end sequence is mediated by (a) an endonuclease, or (b) a combination of a DNA glycosylase and heat, basic conditions, or an abasic site-recognizing endonuclease / lyase. In some embodiments, (a) an endonuclease, or (b) a combination of a DNA glycosylase and heat, basic conditions, or an abasic site-recognizing endonuclease / lyase may mediate cleavage at uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines.

[0442] In some embodiments, the (a) endonuclease or (b) combination of DNA glycosylase and heat, basic conditions, or abasic site recognizing endonuclease / lyase is USER, endonuclease V, RNAse HII, formamidopyrimidine-DNA glycosylase (FPG), oxoguanine glycosylase (OGG), endonuclease III (Nth), endonuclease VIII, a mixture of human alkyladenine DNA glycosylase and endonuclease VIII or endonuclease III, a mixture of thymine-DNA glycosylase (TDG) or mammalian DNA glycosylase-methyl-CpG binding domain protein 4 (MBD4) and endonuclease VIII or endonuclease III, or DNA glycosylase / lyase ROS1 (ROS1). In some embodiments, ROS1 can function as a modified endonuclease based on its bifunctional glycosylase / lyase activity.

[0443] In some embodiments, the modified transposon end sequence comprises uracil, the mixture is an N-glycosylase, and the apurinic or apyrimidinic site (AP) lyase / endonuclease is a Uracil-Specific Excision Reagent (USER). In some embodiments, the USER is a mixture of uracil DNA glycosylase and endonuclease VIII or endonuclease III.

[0444] In some embodiments, the modified transposon end sequence comprises inosine and the endonuclease is endonuclease V. In some embodiments, the modified transposon end sequence comprises ribose and the endonuclease is RNAse HII.

[0445] In some embodiments, the modified transposon end sequence comprises 8-oxoguanine and the endonuclease is formamidopyrimidine-DNA glycosylase (FPG) or oxoguanine glycosylase (OGG).

[0446] In some embodiments, the modified transposon end sequence comprises thymine glycol and the DNA endonuclease is endonuclease III (Nth) or endonuclease VIII.

[0447] In some embodiments, the modified transposon end sequence comprises a modified purine and the DNA glycosylase and endonuclease / lyase that recognizes the abasic site is a mixture of human alkyl adenine DNA glycosylase (hAAG) and endonuclease VIII or endonuclease III.

[0448] In some embodiments, the modified transposon end sequence comprises a modified pyrimidine, the DNA glycosylase is TDG or MBD4, and the abasic site-recognizing endonuclease / lyase is endonuclease VIII or endonuclease III. An alternative modification-specific endonuclease for use with modified transposon ends that contain modified pyrimidines is ROS1.

[0449] In some embodiments, the first transposon comprises a modified transposon end sequence comprising more than one mutation selected from uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine, and an endonuclease, or DNA glycosylase, and an endonuclease / lyase that recognizes an abasic site are included in the mixture. In some embodiments, the endonuclease, or DNA glycosylase, and the endonuclease / lyase that recognizes abasic sites comprises more than one enzyme selected from USER, endonuclease V, RNAse HII, formamidopyrimidine-DNA glycosylase (FPG), oxoguanine glycosylase (OGG), endonuclease III (Nth), endonuclease VIII, a mixture of hAAG and endonuclease VIII / endonuclease III, or a mixture of TDG or MBD4 combined with endonuclease VIII / endonuclease III, or ROS1. In some embodiments, methods using modified transposon end sequences containing more than one mutation and a combination of an endonuclease, and / or a DNA glycosylase, and an abasic site-recognizing endonuclease / lyase improve the cleavage efficiency of mosaic end sequences compared to methods using modified transposon end sequences containing a single mutation and a single endonuclease, or a combination of a DNA glycosylase, and an abasic site-recognizing endonuclease / lyase. For ROS1, a single endonuclease has both glycosylase and lyase functions.

[0450] In some embodiments, a method of fragmenting double-stranded nucleic acid includes combining a sample containing double-stranded nucleic acid with a transposome complex and preparing fragments.

[0451] In some embodiments, a method for preparing double-stranded nucleic acid fragments lacking all or a portion of a first transposon end includes combining a sample containing nucleic acid with a transposome complex, preparing a fragment, combining the sample with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes an abasic site, and cleaving the first transposon end with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purines, and / or modified pyrimidines in a mosaic sequence to remove all or a portion of the first transposon end from the fragment. In some embodiments, the modified purine is 3-methyladenine or 7-methylguanine. In some embodiments, the modified pyrimidine is 5-methylcytosine, 5-formylcytosine, or 5-carboxycytosine. In some embodiments, the method cleaves all or a portion of the first transposon end (transferred strand) from the fragment.

[0452] In some embodiments, cleaving the first transposon end generates a sticky end for adapter ligation. As used herein, a "sticky end" is an end of a double-stranded fragment where one strand is longer than the other (i.e., there is an overhang) and the overhang allows for ligation of an adapter that contains a complementary overhang.

[0453] In some embodiments, the adaptors are added after removing all or part of the first transposon end from the fragment. In some embodiments, the adaptors are added by ligation. In some embodiments, an end repair and A-tailing mix allows for ligation of the adaptors. Those skilled in the art will be aware of other means for adding adaptors (e.g., PCR amplification or click chemistry).

[0454] 2. Adapter Ligation In some embodiments, a method of preparing double-stranded nucleic acid fragments containing adaptors includes combining a sample containing nucleic acid with a transposome complex described herein, preparing fragments, combining the sample with (1) an endonuclease, or (2) a combination of a DNA glycosylase and heat, basic conditions, or an endonuclease / lyase that recognizes abasic sites, cleaving the first transposon end with uracil, inosine, ribose, 8-oxoguanine, thymine glycol, modified purine, and / or modified pyrimidine within the mosaic end sequence to remove all or a portion of the first transposon end from the fragment, and ligating an adaptor to the 5' and / or 3' end of the fragment.

[0455] In some embodiments, adaptors containing sequences are ligated onto the library fragments after removal of all or part of the mosaic end sequences. Fragments that have been subjected to adaptor ligation to the 5' and / or 3' ends of the fragments may be referred to as "tagged fragments."

[0456] In some embodiments, ligation is performed with a DNA ligase.

[0457] In some embodiments, the adaptor comprises a double-stranded adaptor.

[0458] In some embodiments, adaptors are added to the 5' and 3' ends of the fragments. In some embodiments, the adaptors added to the 5' and 3' ends of the fragments are different.

[0459] A wide variety of library preparation methods that include a step of adapter ligation are known in the art, such as TruSeq and TruSight Oncology 500 (see, e.g., TruSeq® RNA Sample Preparation v2 Guide, 15026495 Rev.F, Illumina, 2014). Adapters used with other ligation methods may also be used in the methods of the invention (see, e.g., Illumina Adapter Sequences, Illumina, 2021). Adapters for use in the present invention also include those described in WO 2008 / 093098, WO 2008 / 096146, WO 2018 / 208699, and WO 2019 / 055715, each of which is incorporated herein by reference in its entirety.

[0460] In some embodiments, adapter ligation may allow for more flexible incorporation of adapters (such as adapters with longer lengths) compared to methods that tag fragments via tagmentation (wherein adapter sequences are incorporated into the fragments during the transposition reaction). In some methods involving tagmentation, additional adapter sequences can be incorporated by PCR reactions (such as those described in U.S. Patent Application Publication No. 2018 / 0201992(A1)), and the methods of the present invention may eliminate the need for an additional PCR step to incorporate additional adapter sequences.

[0461] Ligation techniques are commonly used to prepare NGS libraries for sequencing. In some embodiments, the ligation step uses enzymes to attach specialized adapters to both ends of the DNA fragments. In some embodiments, A bases are added to the blunt ends of each strand to prepare them for ligation to the sequencing adapters. In some embodiments, each adapter contains a T base overhang, providing a complementary overhang for ligating the adapter to the A-tailed fragmented DNA.

[0462] Adapter ligation protocols are known to have advantages over other methods. For example, adapter ligation can be used to generate a perfect complement of sequencing primer hybridization sites for single reads, paired-end reads, and index reads. In some embodiments, adapter ligation eliminates the need for an additional PCR step to add index tags and index primer sites.

[0463] In some embodiments, the adapter comprises a unique molecular identifier (UMI), a primer sequence, an anchor sequence, a universal sequence, a spacer region, an index sequence, a capture sequence, a barcode sequence, a cleavage sequence, a sequencing-related sequence, and combinations thereof. As used herein, a "barcode sequence" refers to a sequence that can be used to distinguish samples. As used herein, a sequencing-related sequence can be any sequence that is associated with a subsequent sequencing step. A sequencing-related sequence can act to simplify downstream sequencing steps. For example, a sequencing-related sequence can be a sequence that would otherwise be incorporated via a step of ligating an adapter to a nucleic acid fragment. In some embodiments, the adapter sequence comprises a P5 or P7 sequence (or their complements) to facilitate binding to a flow cell in a particular sequencing method.

[0464] In some embodiments, the adaptor comprises a UMI. In some embodiments, an adaptor comprising a UMI is ligated to both the 3' and 5' ends of the fragment.

[0465] In some embodiments, the adaptor may be a forked adaptor. As used herein, a "forked adaptor" refers to an adaptor that includes two strands of nucleic acid, each of which includes a region that is complementary to the other strand and a region that is not complementary to the other strand. In some embodiments, the two strands of nucleic acid in the forked adaptor are annealed together prior to ligation, and the annealing is based on the complementary regions. In some embodiments, the complementary regions each include 12 nucleotides. In some embodiments, the forked adaptor is ligated to both strands at the ends of the double-stranded DNA fragment. In some embodiments, the forked adaptor is ligated to one end of the double-stranded DNA fragment. In some embodiments, the forked adaptor is ligated to both ends of the double-stranded DNA fragment. In some embodiments, the forked adaptors on opposite ends of the fragment are different. In some embodiments, one strand of the forked adaptor is phosphorylated at its 5' to facilitate ligation to the fragment. In some embodiments, one strand of the forked adaptor has a phosphorothioate bond immediately preceding the 3'T. In some embodiments, the 3'T is an overhang (i.e., not paired with a nucleotide in the other strand of the forked adaptor). In some embodiments, the 3'T overhang can base pair with an A-tail present on the library fragment. In some embodiments, the phosphorothioate bond blocks exonuclease digestion of the 3'T overhang. In some embodiments, PCR with partially complementary primers is used after adaptor ligation to extend the ends and resolve the forks.

[0466] In some embodiments, the adaptor may include a tag. As used herein, the term "tag" refers to a portion or domain of a polynucleotide that exhibits a sequence for a desired intended purpose or use. A tag domain may include any sequence provided for any desired purpose. For example, in some embodiments, a tag domain includes one or more restriction endonuclease recognition sites. In some embodiments, a tag domain includes one or more regions suitable for hybridization with primers for a cluster amplification reaction. In some embodiments, a tag domain includes one or more regions suitable for hybridization with primers for a sequencing reaction. It will be understood that any other suitable features may be incorporated into the tag domain. In some embodiments, a tag domain includes a sequence having a length of 5 bp to 200 bp. In some embodiments, a tag domain includes a sequence having a length of 10 bp to 100 bp. In some embodiments, a tag domain includes a sequence having a length of 20 bp to 50 bp. In some embodiments, the tag domain comprises a sequence having a length of 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 bp.

[0467] A tag can include one or more functional sequences or components (eg, a primer sequence, an anchor sequence, a universal sequence, a spacer region, or an index tag sequence) as necessary or desired.

[0468] In some embodiments, the tag comprises a region for cluster amplification, hi some embodiments, the tag comprises a region for priming a sequencing reaction.

[0469] In some embodiments, the method further comprises amplifying the fragment on the solid support by reacting a polymerase with an amplification primer corresponding to a portion of the tag. In some embodiments, the portion of the adaptor ligated onto the fragment after removal of all or a portion of the mosaic end sequence comprises an amplification primer. In some embodiments, the tag of the first transposon comprises an amplification primer.

[0470] In some embodiments, the tag comprises an A14 primer sequence. In some embodiments, the tag comprises a B15 primer sequence.

[0471] In some embodiments, transposomes on individual beads carry a unique index, and when large numbers of such indexed beads are used, phased transcripts result.

[0472] Adapters ligated onto library fragments may have advantages over adapters incorporated during tagmentation. For example, unique molecular identifiers (UMIs) can be used to enable highly sensitive variant detection by labeling single fragments with unique sequence tags prior to PCR (see Jesse J. Salk, et al., Nature Reviews Genetics 19(5):269-85(2018)). Some library preparation products, such as TSO 500 (Illumina), include ligation-based UMI offerings in which UMI sequences are incorporated adjacent to the library insert, allowing for simultaneous sequencing as part of the insert read. Thus, the development of fBLT allows existing ligation-based products to be leveraged (such as the use of existing adapters and protocols), while at the same time allowing compatibility with existing enrichment workflows and on-board sequencing primers.

[0473] C. Gap-filling ligation In some embodiments, gaps in the DNA sequence remaining after the translocation event can also be filled using a strand displacement extension reaction, such as one that includes Bst DNA polymerase and a dNTP mix. In some embodiments, the gap filling ligation is performed using an extension-ligation mix buffer.

[0474] The library of double-stranded DNA fragments can then optionally be amplified (such as by cluster amplification) and sequenced using a sequencing primer.

[0475] D. Amplification The present disclosure further relates to the amplification of DNA fragments (i.e., cDNA fragments) produced according to the methods provided herein. In some embodiments, the immobilized DNA fragments produced by surface-bound transposomes can be amplified according to any suitable 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 sample preparation to proceed on the same solid support from the initial sample introduction step, through amplification, and optionally through the sequencing step.

[0476] For example, in some embodiments, the immobilized DNA fragments are amplified using cluster amplification methodology, as exemplified by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are incorporated herein by reference in their entirety. The incorporated materials of U.S. Patent Nos. 7,985,565 and 7,115,400 describe a method of solid-phase nucleic acid amplification that allows the amplification products to be immobilized on a solid support to form an array consisting of clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. Arrays so formed are generally referred to herein as "clustered arrays." The product of a solid-phase amplification reaction such as described in U.S. Patent Nos. 7,985,565 and 7,115,400 is a so-called "bridged" structure formed by annealing a pair of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on a solid support at their 5' ends, in some embodiments, via covalent bonds. The cluster amplification methodology is an example of a method for producing immobilized amplicons using an immobilized nucleic acid template. Other suitable methodologies can also be used to produce immobilized amplicons from the immobilized DNA fragments produced according to the methods provided herein. For example, one or more clusters or colonies can be formed by solid-phase PCR, regardless of whether one or both primers of each pair of amplification primers are immobilized.

[0477] In other embodiments, DNA fragments are amplified in solution. For example, in some embodiments, DNA fragments are cleaved or otherwise released from solid support, and then amplification primers are hybridized to the released molecules in solution. In other embodiments, amplification primers are hybridized to immobilized DNA fragments for one or more initial amplification steps, followed by subsequent amplification steps in solution. Thus, in some embodiments, immobilized nucleic acid templates can be used to produce liquid phase amplicons.

[0478] It should be understood that any of the amplification methodologies described herein or generally known in the art can be utilized with universal or target-specific primers to amplify the immobilized DNA fragments. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence based amplification (NASBA), as described in U.S. Pat. No. 8,003,354, which is incorporated herein by reference in its entirety. The above amplification methods can be used to amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR, SDA, TMA, NASBA, etc., can be utilized to amplify the immobilized DNA fragments. In some embodiments, primers specifically directed to the nucleic acid of interest are included in the amplification reaction.

[0479] 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 described in U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907, EP 0320308(B1), EP 0336731(B1), EP 0439182(B1), WO 90 / 01069, WO 89 / 12696, and WO 89 / 09835, all of which are incorporated by reference) techniques. It should be understood that these amplification methodologies can be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method can include a ligation probe amplification or oligonucleotide ligation assay (OLA) reaction that includes a primer specifically directed to the nucleic acid of interest. In some embodiments, the amplification method can include a primer extension ligation reaction that includes a primer specifically directed to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify the nucleic acid of interest, the amplification can include primers used in the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified in U.S. Patent Nos. 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.

[0480] Exemplary isothermal amplification methods that can be used in the methods of the present disclosure include, but are not limited to, Multiple Displacement Amplification (MDA), as exemplified, for example, by Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification, as exemplified, for example, by U.S. Pat. No. 6,214,587, each of which is incorporated herein by reference in its entirety. Other non-PCR-based methods that can be used in the present disclosure include strand displacement amplification (SDA), as described, for example, in Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, U.S. Patent Nos. 5,455,166 and 5,130,238, and Walker et al., Nucl. Acids Res. 20:1691-96 (1992), or hyperbranched strand displacement amplification, as described, for example, in Lage et al., Genome Research 13:294-307 (2003), each of which is incorporated herein by reference in its entirety. Isothermal amplification methods can be used with strand-displacing Phi 29 polymerase or large fragment 5'→3' exo- of Bst DNA polymerase for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacement activity. High processivity allows the polymerase to produce fragments of 10-20 kb in length. As noted above, polymerases with low processivity and strand displacement activity, such as Klenow polymerase, can be used to produce smaller fragments under isothermal conditions. Further description of the amplification reaction, conditions, and components are described in detail in the disclosure of U.S. Patent No. 7,670,810, which is incorporated herein by reference in its entirety.

[0481] Another nucleic acid amplification method useful in the present disclosure is tagged PCR, which uses a population of two-domain primers with a constant 5' region followed by a random 3' region, as described, for example, in Grothues, et al. Nucleic Acids Res. 21(5):1321-2 (1993), which is incorporated herein by reference in its entirety. The first round of amplification is carried out to allow multiple initiations on heat-denatured DNA based on individual hybridization from the randomly synthesized 3' region. Due to the nature of the 3' region, the initiation sites are considered to be random throughout the genome. Unbound primers can then be removed, and further replication can be carried out using primers that are complementary to the constant 5' region.

[0482] In some embodiments, amplification functions by adding one or more secondary adapter sequences to fully duplexed 5'-tagged target fragments to form sequencing fragments. Amplification is accomplished by incubating fully duplexed 5'-tagged target fragments, which include primer sequences at each end, with a secondary adapter carrier, a single nucleotide, and a polymerase under conditions sufficient to amplify the target fragments and incorporate a secondary adapter carrier (or its complement), which includes a primer sequence and a complement to the secondary adapter sequence.

[0483] In some embodiments, the secondary adapter carrier comprises a primer sequence, an index sequence, a barcode sequence, a purification tag, or a combination thereof. In some embodiments, the secondary adapter carrier comprises a primer sequence. In some embodiments, the secondary adapter carrier comprises an index sequence. In some embodiments, the secondary adapter carrier comprises an index sequence and a primer sequence.

[0484] In some embodiments, the fully duplexed 5' tagged target fragment comprises a different primer sequence at each end. In such embodiments, each secondary adapter carrier comprises a complement to one of the two primer sequences. In some embodiments, the two primer sequences are the A14 primer sequence and the B15 primer sequence.

[0485] In some embodiments, multiple secondary adapters are added by amplification. In some embodiments, the secondary adapter carriers each comprise one of two primer sequences. In some embodiments, the secondary adapter carriers each comprise one of multiple index sequences. In some embodiments, the secondary adapter carriers comprise a secondary adapter having a P5 primer sequence (SEQ ID NO: 13) and a secondary adapter having a P7 primer sequence (SEQ ID NO: 14), or a complement thereof.

[0486] In some embodiments, the sequencing fragment is attached to a flow cell. In some embodiments, the sequencing fragment is hybridized to a complementary primer grafted to a flow cell or a surface. In some embodiments, the sequence of the sequencing fragment is detected by a next generation sequencing method such as array sequencing or sequencing while synthesis.

[0487] P5 and P7 primers are used on the surface of commercially available flow cells sold by Illumina, Inc. for sequencing on various Illumina platforms. Such primer sequences are described in U.S. Patent Application Publication No. 2011 / 0059865(A1), which is incorporated by reference in its entirety. Although P5 and P7 primers are illustrated, it should be understood that any suitable amplification primers can be used in the examples presented herein.

[0488] In some embodiments, the amplifying step of the method comprises PCR or isothermal amplification. In some embodiments, the amplifying step of the method comprises PCR.

[0489] In some embodiments, sequencing is performed after amplification. In some embodiments, amplification is not performed before sequencing. Many different sequencing methods are known to those of skill in the art and are described, for example, in U.S. Patent Nos. 9,683,230 and 10,920,219, each of which is incorporated herein by reference in its entirety.

[0490] In some embodiments, the method includes amplifying the double-stranded cDNA fragment to prepare an amplicon thereof. In some embodiments, the amplicon is subjected to solid-phase reversible immobilization purification.

[0491] In some embodiments, the total reaction time from combining the primers with the sample containing RNA to purification of the amplicon is 2 hours or less, 2.5 hours or less, or 3 hours or less.

[0492] As described below, amplification can also be performed to enrich for library fragments of interest as a type of target enrichment.

[0493] E. Target enrichment In some embodiments, library preparation is performed in one or more steps to enrich for fragments containing the target of interest (i.e., target library fragments). Such methods can reduce the number of library fragments of low interest in the library. In this way, users can reduce the time and cost associated with sequencing library fragments that are not of interest.

[0494] For example, a user may wish to prepare and sequence library fragments from a patient sample, where the library fragments of interest are generated from nucleic acids of an agent causing one or more infectious diseases. In such an embodiment, the patient sample generates many library fragments (i.e., library fragments from the host) from the patient's own nucleic acids, which are not of interest to the user. By enriching for sequences from one or more infectious diseases, the user can reduce sequencing of library fragments (or their amplicons) that contain patient-specific sequences, allowing deeper sequencing of library fragments (or their amplicons) that contain sequences from one or more infectious diseases. For example, a user may wish to determine COVID-19-related sequences from a patient (to determine the presence or absence of COVID-19 and / or to evaluate a particular COVID-19 variant) and may have little interest in sequences from the patient (i.e., the host).

[0495] In some embodiments, the infectious disease is caused by a virus, bacteria, parasite, or fungus.

[0496] Target enrichment may occur at a variety of different steps within library preparation and downstream processing. In some embodiments, target enrichment occurs simultaneously with or immediately after the preparation of double-stranded cDNA fragments in the library preparation method. In some embodiments, target enrichment occurs during amplification after preparation of double-stranded cDNA fragments.

[0497] In some embodiments, double-stranded cDNA fragments are prepared using enrichment. In some embodiments, enrichment is performed during or immediately after tagmentation in library preparation. In some embodiments, enrichment is performed using hybrid capture. The benefits and applications of hybrid capture to human infectious diseases are well known (see, e.g., Gaudin and Desnues, Frontiers in Microbiology, 9:Article 2924 (2018)).

[0498] In some embodiments, hybrid capture is performed using a target-specific biotinylated probe. In some embodiments, the target-specific biotinylated probe binds sequences from one or more infectious diseases. In some embodiments, the one or more infectious diseases include one or more respiratory viruses. Such enrichment workflows have been described for respiratory viruses, such as COVID-19. In some embodiments, the method incorporates a virus-targeting panel, such as described in Enrichment workflow for detecting coronavirus using Illumina NGS systems, Illumina Document 1270-2020-002-A (2020). In some embodiments, the virus-targeting panel is the Respiratory Virus Oligo Panel (RVOP, Illumina). Multiple versions of RVOP are known in the literature. Figures 26-30B show data for experiments performed with RVOP enrichment (either version 1 or version 2).

[0499] Target enrichment may also be performed during amplification of double-stranded cDNA fragments. In some embodiments, the method of library preparation further comprises amplifying the double-stranded cDNA fragments to prepare amplicons. In some embodiments, the amplification is performed using target-specific primers. In some embodiments, the target-specific primers bind to sequences from one or more infectious diseases. In some embodiments, the one or more infectious diseases include one or more respiratory viruses. EXAMPLES

[0500] Example 1. Overview of the isothermal method for ds-cDNA preparation Isothermal method can be used to prepare ds-cDNA from RNA in a single isothermal reaction.Many different methods of carrying out this reaction are presented, where similar methods are carried out after primer binding to RNA (e.g., random hexamer).After primer binding, reaction mix (which can be called "master mix") containing the following components can be added. 1. Reverse transcriptase to form the first strand of cDNA; 2. RNAse H to nick the RNA and generate priming sites for second strand cDNA synthesis; 3. A DNA polymerase with either strand displacement or 5'-3' exonuclease activity to generate second strand cDNA, and 4. dNTPs that are shared between reverse transcriptase and DNA polymerase to generate the first and second strands.

[0501] Such a method may be referred to as a "single-step" or "one-step" method (i.e., the method of the present invention) since first-strand and second-strand cDNA are generated simultaneously in a single reaction vessel. An appropriate formulation that allows single-step double-stranded cDNA synthesis may be required. A hypothetical mechanism for this reaction is shown in FIG. 3.

[0502] Example 2. Preparation of mesophilic ds-cDNA The preparation method of mesophilic ds-cDNA was evaluated in the context of Illumina RNA Prep with Enrichment(L) product (see RNA Prep with Enrichment(L) Tagmentation Reference Guide, Document # 1000000124435 v02, Illumina, 2020 (Document 1000000124435)). In the baseline experiment, 10-12 ng of Universal Human Reference RNA (UHR, Agilent PN 740000) enriched using Illumina TruSight® RNA Pan-Cancer Panel kit was used. In some cases, approximately 3.5 kb genomic RNA from bacteriophage MS2 (MS2, Roche PN 10165948001, GenBank accession NC_001417.2) was used as a control material to evaluate library preparation performance. Libraries were prepared using either pure UHR, MS2, or a mixture of UHR and MS2 RNA (80% UHR / 20% MS2 by mass) as described below, with approximately 10-12 ng of total RNA input.

[0503] Equal volumes of sample RNA and EPH3 (Illumina) buffer (8.5 μl each) were mixed and heated to 65° C. for 5 min, then cooled to 4° C. in a thermal cycler. This step allowed hybridization of the primer contained in the EPH3 buffer to the RNA.

[0504] After the primer binding incubation, 33 μl of approximately 1.5× “single-step” cDNA synthesis buffer (i.e., cDNA synthesis master mix) was added directly to the 17 μl RNA+EPH3 mix. To determine how incubation time affects performance, the single-step formulations were incubated for either 10 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes. The single-step cDNA synthesis reaction may be referred to as “Method of the Invention” in the figures.

[0505] cDNA was generated from 12 ng of 80% UHR / 20% MS2 RNA using a single-step procedure, followed by tagmentation with eBLT and enrichment with the TruSight® RNA Pan-Cancer Panel kit panel as described in Illumina RNA Prep with Enrichment (Document 1000000124435 (2020)). Sequenced libraries were compared to similar libraries generated from 10 ng or 100 ng of UHR using Illumina RNA Prep with Enrichment with enrichment with RNA Pan-Cancer Oligos, included as part of the TruSight® RNA Pan-Cancer Panel kit (see TruSight® RNA Pan-Cancer Panel Reference Guide, Illumina Document # 1000000001632-v01 (2016)). Data were analyzed using the Illumina BaseSpace Sequence Hub (BSSH) RNA-Seq Alignment App v.1.1.1.

[0506] A. Analysis of Percentage of Duplicate Reads The percentage of replicate reads is a measure of the conversion of samples to libraries. A lower percentage of replicate reads is preferable. With respect to replicates, the results suggest that the single-step method has comparable or better performance than the standard procedure (Figure 4). In the single-step workflow, incubation times between 10 and 60 min had little effect on performance.

[0507] B. Insert size analysis. As expected according to the model (Figure 3), our single-step method produces shorter library fragments than the standard procedure using Illumina RNA Prep with Enrichment (Figure 5). The insert size average of approximately 150-170 bp generated by our single-step method is acceptable for most RNA-Seq applications. Incubation times (up to 10 min) had little impact on performance in the single-step workflow.

[0508] C. Median coverage CV The median coefficient of variation (CV) of coverage reflects library quality, with more uniform conversion of samples to libraries resulting in lower CV. Results are shown in Table 6. Incubation time had little effect on the single-step workflow, as measured by incubations of 10, 20, 30, 45, and 60 minutes.

[0509] D. Gene Expression Correlation An important feature of RNA-Seq products is the high reproducibility of gene expression estimates between technical replicates. Gene expression reproducibility is high for technical replicates of the single-step method using only a 20 min enzyme incubation (Figure 7A, exemplary data shown, R of 0.998). 2 Similar results were obtained with a 10 min incubation (not shown).

[0510] As a quantitative assay, RNA-Seq is highly sensitive to changes in protocols and reagents. Not surprisingly, the single-step method shows a lower correlation to the standard Illumina RNA Prep with Enrichment protocol, as shown in a comparative example (Figure 7B). This cross-procedure comparison shows a lower R of fragments per kilobase per million mapped reads (FPKM, a measure to normalize for sequencing depth and gene length). 2value (0.846), but the Spearman ρ (rank) was high (ρ = 0.934), indicating that the single-step method retains quantitative information.

[0511] R of FPKM comparison across multiple library preparations using both the standard Illumina RNA Prep with Enrichment cDNA procedure and a single-step cDNA method with different incubation times. 2 Box plots comparing values ​​show high reproducibility within each method and, as expected, lower agreement between the two methods (Figure 8).

[0512] E. Coverage characteristics of MS2 control RNA MS2 genomic RNA is a useful template in exploring alternative library preparations because assay performance is easy to visualize and analyze without the complexities of human transcriptomics and enrichment analysis. Here, bases within the MS2 genome were assessed for their raw read coverage, allowing calculation of the CV of coverage across the MS2 genome and a normalized value of read depth of coverage for comparison across experiments. Visually, MS2 read coverage is similar between the standard and single-step protocols (Figure 9), but the data suggests that the single-step protocol has a slightly higher CV of coverage (Figure 10 and Table 6).

[0513] [Table 7] * All replicates were of a mixture of 80% UHR, 20% MS2. 12ng input. ** Two libraries were 100% MS2 and two libraries were an 80% UHR / 20% MS2 mixture. Inputs varied from 10 ng to 100 ng.

[0514] Thus, the single-step protocol of the present invention provided sufficient cDNA quality with a faster preparation time.

[0515] Example 3. Method for preparing thermostable ds-cDNA Coordinate synthesis of double-stranded cDNA can also occur using alternative formulations composed of thermostable enzymes rather than their mesophilic counterparts (Table 2). The thermostable enzymes in such formulations can be referred to as a thermostable master mix.

[0516] Equal volumes of sample RNA and EPH3 (Illumina) buffer (8.5 μl each) were mixed and heated to 65° C. for 5 minutes, then cooled to 4° C. in a thermal cycler. This step allowed hybridization of the primers contained in the EPH3 buffer to the RNA. The 15 μl reaction with the hybridized primers was then added to the thermostable enzyme and buffer components for single-step cDNA preparation.

[0517] The thermostable formulation was performed isothermally at approximately 50° C. Tagmentation of the resulting ds-cDNA results and enrichment with TruSight® RNA Fusion Panel (as described in TruSight RNA® Fusion Panel Protocol Guide, Illumina Document # 1000000009155 v00 (2016)) results in quality libraries (FIG. 11). As shown in FIG. 11, the thermostable formulation produced an insert size of approximately 250 bp, which is similar to the insert size produced using standard Illumina RNA Prep with Enrichment methodology (e.g., approximately 210 bp, as shown in FIG. 5). The libraries were quantitative, both with reasonable inter-replicate technical reproducibility and with reasonable agreement with the method using Illumina RNA Prep with Enrichment (FIGS. 12A and 12B). Thermostable formulations may be preferred when there is concern about RNA secondary structure, which may otherwise inhibit first strand cDNA synthesis, which occurs at 37°C.

[0518] In this experiment, incubation was for 60 min, although much shorter incubation times are possible. Furthermore, replacement of the MMLV reverse transcriptase with alternative thermostable reverse transcriptases derived from retrotransposons may improve performance and assay speed.

[0519] Example 4. Summary of two-step versus one-pot cDNA preparation. Figure 13 provides an overview of a representative one-pot tagmentation protocol. Representative two-step and one-step cDNA preparation protocols are shown in Figure 14A, with the option of adding BLT to either protocol to prepare a library of cDNA fragments. Figure 14B highlights an overview of how the one-step cDNA preparation protocol (also referred to herein as the method of the present invention) reduces reaction time by approximately one hour and also avoids multiple temperature changes. ST2 (tagmentation stop buffer) is not required if the user only wishes to prepare cDNA, but is included as it can be used to stop tagmentation if BLT is included in the protocol outlined in Figure 14B for library preparation.

[0520] Figure 14C shows the protocol for the current method of two-step cDNA preparation followed by separate tagmentation, along with a shorter protocol for one-step cDNA (i.e., the method of the present invention) followed by separate tagmentation, or for one-pot library preparation, which combines cDNA and library preparation. The one-pot library can save one hour compared to the current method.

[0521] To compare cDNA yields, cDNA prepared by the one-pot or two-step cDNA protocol was purified with solid-phase reversible immobilization (SPRI) beads and eluted. As shown in Figure 4, cDNA was obtained from both the inventive (one-step) and two-step (standard) protocols.

[0522] Example 5. Comparison of one-pot library preparation to methods using separate tagmentation A variety of different library preparation protocols, summarized in Figure 14C, were evaluated.

[0523] The experiment evaluated fragments generated using BLT after a two-step cDNA preparation with 10 ng of input RNA (no rRNA depletion). These results showed that BLT successfully generated fragments with a size of approximately 325 base pairs after a two-step cDNA preparation (Figure 15).

[0524] The one-pot library preparation allowed the preparation of a library of double-stranded DNA fragments from a starting sample containing RNA. In this protocol, the RNA-containing sample was mixed with a mix containing a primer (EPH3). The reaction mix (total volume 17 μl) was as follows: 7.5 μl nuclease-free water 1 μl of 10 ng / μl UHR total RNA 8.5 μl EPH3.

[0525] The reaction mix was placed in a thermocycler with a heated lid and the reactions were heated to 65°C for 5 minutes and then held at 4°C.

[0526] The plate was removed from the thermocycler and 33 μl of 1-pot master mix was added to produce a total volume of 50 μl. The 1-pot master mix contained the balance of enzyme units in the ranges discussed in section IC along with BLT. The enzymes in the 1-pot master mix used were E coli DNA polymerase I (in the range of 0.04 U / μl to 0.37 U / μl), RNAse H (in the range of 0.004 U / μl to 0.04 U / μl), and Protoscript II reverse transcriptase (in the range of 0.32 U / μl to 4.8 U / μl). The reactions were mixed well.

[0527] A total of 10 μl of BLT (Illumina Catalog No. 20024594) was aliquoted into the wells of the plate. The plate was spun down to collect the BLT at the bottom of the wells. The plate was then placed on a magnet for approximately 2 minutes. Approximately 10 μl of the supernatant was then removed. This step removed the buffer that was supplied with the BLT.

[0528] The BLT was then resuspended in 50 μl of sample with 1-pot master mix. The plate was incubated at 37° C. for 1 hour in a thermocycler with the heated lid set at 45° C. Then, 10 μl of ST2 buffer was added to stop the tagmentation reaction. The plate was left at room temperature for approximately 5 minutes. The plate was then placed on a magnet for 2 minutes, after which the supernatant was removed and discarded. The beads were resuspended in 100 μl of TWB wash buffer. The wash with TWB was repeated a total of three times, and then the excess TWB was removed.

[0529] PCR was then performed. To each sample, 20 μl of Enhanced PCR Mix (EPM, Illumina) buffer and 20 μl of nuclease-free water were added. Then, 10 μl of unique dual index PCR primers were added (total volume 50 μl). The plate was placed in a thermocycler and amplified for 15 cycles at 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds.

[0530] Samples were purified with 81 μl of Illumina Purification Beads (IPB). DNA was eluted from the beads with 30 μl of resuspension buffer (RSB, Illumina). Library yields were quantified using Qubit (Thermo Fisher Scientific).

[0531] Results from different protocol conditions are shown in Figure 16A-D. One-pot library preparation (combined cDNA and tagmentation reactions in one pot, Figure 16C) yielded similar results as two-step cDNA preparation with separate tagmentation (Figure 16A) and one-pot cDNA preparation with separate tagmentation (Figure 16B). Comparison of results from different protocols is summarized in Figure 16D. In summary, one-pot library preparation yielded similar library yields in a significantly faster and simpler protocol compared to other tested protocols.

[0532] Figure 17A shows the results using 100 ng of UHR and one-pot library preparation (including cDNA preparation and tagmentation in a single reaction). In comparison, Figure 17B shows the results of a no template control (NTC, no starting RNA) and Figure 17C shows the results of a control lacking reverse transcriptase (RT). These results show that the one-pot library results are based on the successful preparation of double-stranded cDNA and tagmentation of this cDNA.

[0533] BLT successfully prepared library fragments for both starting samples containing 100 ng of RNA (FIG. 18A) and 10 ng of RNA (FIG. 18B).

[0534] Example 6. Evaluation of library preparation with rRNA depletion The experiment evaluated different library preparations using BLT with rRNA depletion using standard conditions of the Illumina® Ribo-Zero Plus rRNA Depletion Kit prior to primer binding. For 100 ng samples, 13 cycles of PCR were used. For 10 ng samples, 15 cycles of PCR were used.

[0535] The results of the experiment with rRNA depletion are shown in Figures 19A-B. The yields in the one-pot library preparation were lower than those in the other preparations, which may be due to the relatively small amount of starting RNA after rRNA depletion. The control experiment without reverse transcriptase (No RVT) showed no library yield.

[0536] Alignments and general performance measures are shown in Figures 20A-C. Alignment percentages were all around 94-95% (Figure 20A). Median coefficient of variation (CV) of coverage and overlap percentages were higher for the one-pot library preparations compared to protocols using separate tagmentation reactions (Figures 20B and 20C). These results may indicate a relatively low efficiency of tagmentation using one-pot library preparations compared to other conditions. Presence percentages were around 1-2% for all preparations (data not shown).

[0537] Insert length and alignment distribution were similar for all library preparations (Figures 21A-B). Furthermore, gene expression correlation was good between the control library preparation (i.e., two-step cDNA preparation with separate tagmentation) and the one-pot library preparation using both 100 ng (Figure 22A) and 10 ng (Figure 22B) of starting material. Gene expression correlation was also good when comparing the one-pot library preparation with the one-pot cDNA protocol with separate tagmentation (Figure 22C).

[0538] Several outlier genes with relatively large differences in expression using one-pot library prep versus two-step cDNA prep with separate tagmentation are listed in Table 7. However, these genes represent only a small fraction of the total library.

[0539] [Table 8]

[0540] The number of genes detected at 10x coverage (18 million reads) was lower in the one-pot library preparation protocol with 10 ng starting RNA, but was not different when 100 ng starting RNA was used (Figure 23). These results indicate that one-pot library preparation can result in relatively inefficient tagmentation at lower input ranges. Read distribution was normal for all conditions, suggesting the absence of 5' or 3' bias in the library preparation (Figure 24).

[0541] These results indicate that tagmentation efficiency may be a limiting factor in determining the yield of one-pot library preparation.

[0542] To test the effect of improving tagmentation efficiency, reactions were performed using RNA Prep with Enrichment and Ribo-Zero Plus rRNA Depletion Kits (Illumina) using conditions with almost twice the amount of MgCl2 (8.25 mM MgCl2 versus the standard 4.3 mM MgCl2), and these conditions improved yields (data not shown). These results indicate that a one-pot library preparation composition with a higher magnesium concentration can increase the yield of library fragments, likely by increasing transposase efficiency.

[0543] Similarly, increasing the temperature at the end of the reaction incubation for one-pot library preparation may result in higher library yields. Since 55°C is known to be the optimum temperature for Tn5, a higher reaction temperature may increase the transposase activity.

[0544] Example 7.1 Optimization of pot library preparation conditions The results (as described in Example 5) showed that one-pot library preparation using simultaneous cDNA synthesis and tagmentation can robustly generate robust libraries. Next, considering its ability to reduce library preparation time and hands-on steps (as shown in Figure 25), various different conditions were evaluated to potentially improve library yield, focusing on protocol modifications to try to increase tagmentation efficiency by one-pot library preparation. However, it should be noted that in many applications (such as library preparation with enrichment), the potential reduction in library preparation efficiency by one-pot protocol is unlikely to affect downstream processes such as sequencing.

[0545] Enrichment experiments were performed with a variety of different protocols using 10 ng of UHR as "host RNA" along with synthetic controls. Synthetic controls ("Twist") were 0, 1k, 10, or 100k Twist Control Respiratory Virus Controls (Twist Bioscience) spiked into the UHR samples. Enrichment was performed using Respiratory Virus Oligos Panel V1 (RVOP, Illumina). Protocols for the data shown in Figure 26 included the following: Standard library preparation (LP) with enrichment (i.e., two-step cDNA preparation followed by tagmentation as a control, StdLP). 1 pot LP: 1 hour at 37°C (4 mM Mg 2+ ) - Conditions outlined in Example 5 (1 pot) 1 pot LP: 37°C for 45 minutes followed by 55°C for 15 minutes (55C) 1 pot LP: 1 hour at 37℃, Mg 2+ to a final concentration of 8 mM (Mg) 1-pot LP: 1 hour at 37°C, omit adding ST2 (tagmentation stop solution containing SDS) and omit washing (NoST2).

[0546] The results in Figure 26 show that standard LP (StdLP group) had the best yield. Adding incubation at 55°C (55C group) or additional Mg2+ (Mg) improved yield over the original one-pot LP protocol (1-pot). Omitting SDS and washing (NoST2) was detrimental to yield.

[0547] Coverage was also assessed by determining the median coverage of the Twist control at 1 million reads using the Respiratory Virus Oligos Panel Version 1 (Illumina) (Figure 27). The one-pot library prep protocol generally did not perform as well as the standard library prep protocol, although the addition of additional Mg 2+ Addition of Mg (to a final concentration of 8 mM) provided a significant enhancement to the performance of the one-pot library preparation. The greatest increase in activity was observed with 2+ This is likely due to the increased concentration, with little coverage seen after omitting SDS and washing (NoST2 sample in FIG. 27). In general, if target enrichment is performed during library preparation, the user may not need high coverage of the desired target sequence for the unenriched library. Thus, the coverage shown in FIG. 27 may be sufficient for many types of sequencing analysis.

[0548] Next, experiments were conducted to combine conditions that could enhance the performance of one-pot library preparation (Figure 28). 2+ The combination protocols included several with incubation at concentrations of 4 mM Mg 2+ The library yield was higher than that of one-pot LP at 37°C using

[0549] Better coverage was seen with enrichment in Respiratory Virus Oligos Panel V2 (Illumina) (Figure 29) compared to Respiratory Virus Oligos Panel Version 1 (Illumina) (Figure 27). Methods involving incubation at 55°C for 15 minutes generally had better results than incubation at 37°C, although all reactions were performed with 8 mM Mg 2+ When the concentrations were the same, there was little difference between the results for different times of incubation (Figure 29). These data indicate that library preparation can be performed in as little as 30 minutes (e.g., 15 minutes at 37°C and 15 minutes at 55°C).

[0550] The library preparation conditions were also investigated for ribosomal RNA (rRNA) depletion samples with 10 ng UHR. The different library preparation conditions were as follows: 1 hour at 37°C Incubation at 37°C followed by 15 or 30 minutes at 55°C Additional Mg 2+ Conditions (8 mM)

[0551] FIG. 30A shows a 8 mM Mg 2+ Incubation at 55°C for 15 minutes (3715) or 30 minutes (3730) can be added with 4 mM Mg 2+ The results show that the percentage of duplicates was dramatically reduced compared to a standard one-pot library preparation for 1 h at 37 °C using 4 mM Mg. 2+ Approximately 50% of the one-pot library preparation was performed at 37 °C for 1 h in 8 mM Mg 2+ After a 1 hour total reaction run at 37°C, the reaction yield dropped to approximately 6% for the one-pot library prep with incubation at 55°C for 15 minutes (3715) or 30 minutes (3730).

[0552] The number of genes detected after different library preparations was also evaluated (Figure 30B).2+ The 3715 and 3730 protocols (1 h at 37°C) detected approximately 2200 fewer genes compared to standard 2-step cDNA preparation followed by tagmentation (2st). However, the 3715 and 3730 protocols detected approximately 750 fewer genes compared to the 2st protocol. Thus, the 55°C incubation and the higher Mg2 + The protocol using the concentration showed the highest number of genes detected for the one-pot protocol compared to the standard two-step cDNA preparation followed by tagmentation protocol.

[0553] Therefore, for the determination of the number of duplicates and detected genes, 55°C incubation and higher Mg 2+ The one-pot protocol was improved by incorporating concentrations (such as 8 mM). These improvements were seen in these conditions for both rRNA-depleted and enriched samples. These results may indicate that tagmentation efficiency is the limiting step for library yield for the one-pot protocol.

[0554] If the user does not require maximum yield for library preparation of a given sample, one-pot protocols may be preferred due to their shorter time for library preparation, up to 30 minutes total. Furthermore, users may also prefer one-pot protocols to reduce the number of hands-on steps. For example, one-pot protocols can omit the cleanup step with SPRI beads between cDNA synthesis and tagmentation (as shown in Figure 25). One-pot protocols can also eliminate sample loss inherent in steps that require sample pipetting.

[0555] Equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples set forth certain embodiments in detail and explain the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in the text, it will be understood that the embodiments can be embodied in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0556] As used herein, the term about refers to numerical values, including, for example, integers, ratios, and percentages, whether or not explicitly stated. The term generally refers to a range of numerical values ​​(e.g., ±5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When a term such as at least and about precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges provided in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. A composition for preparing double-stranded cDNA from RNA by isothermal reaction, a. a reverse transcriptase; b. RNA nickase; c. a DNA polymerase having strand displacement activity or 5'-3' exonuclease activity; d. dNTPs.

2. The composition of claim 1 , wherein the reverse transcriptase activity is greater than the RNA nickase activity.

3. The composition of claim 1 or 2, wherein the reverse transcriptase and the RNA nickase are comprised in a single enzyme.

4. The composition of any one of claims 1 to 3, wherein the reverse transcriptase and the DNA polymerase are comprised in a single enzyme having both RNA-dependent and DNA-dependent polymerase activity.

5. a. The reverse transcriptase is a polymerase having RNA-dependent DNA polymerase activity, and optionally, the reverse transcriptase is Moloney Murine Leukemia Virus (MMLV) reverse transcriptase, a retrotransposon-derived reverse transcriptase, or a group II intron reverse transcriptase; b. the RNA nickase is RNAse H; c. the RNAse H is from Thermus thermophilus, and / or d. The DNA polymerase is E. coli DNA polymerase I or Bst DNA polymerase; The composition according to any one of claims 1 to 4.

6. The composition of any one of claims 1 to 5, wherein the reverse transcriptase, the RNA nickase, and / or the DNA polymerase are mesophilic enzymes.

7. a. The mesophilic enzyme has activity at 37°C to 49°C. b. The mesophilic reverse transcriptase is MMLV reverse transcriptase; c. the mesophilic RNA nickase is E. coli RNAse H, and / or d. The mesophilic polymerase is E. coli DNA polymerase I; The composition of claim 6.

8. The composition of any one of claims 1 to 5, wherein the reverse transcriptase, the RNA nickase, and / or the DNA polymerase are thermostable enzymes.

9. a. The thermostable enzyme has activity at 50°C to 72°C. b. The thermostable reverse transcriptase is a thermostable variant of MMLV reverse transcriptase, or a thermostable reverse transcriptase derived from a retrotransposon, or a group II intron reverse transcriptase; c. the thermostable RNA nickase is RNAse H from Thermus thermophilus, and / or d. The thermostable DNA polymerase is Bst DNA polymerase; The composition of claim 8.

10. a. The RNA is bound to a primer prior to preparing the double-stranded cDNA; b. the composition further comprises one or more additives selected from DTT, BSA, Tris pH 7.5, KCl, and / or MgCl2; c. the composition has lower units / μl of the RNA nickase compared to units / μl of the reverse transcriptase and / or DNA polymerase; and / or d. the composition further comprises an RNA nickase inhibitor; The composition according to any one of claims 1 to 9.

11. a. The reverse transcriptase activity in the composition is 0.32 U / μl to 4.8 U / μl. b. the DNA polymerase activity in the composition is between 0.04 U / μl and 0.37 U / μl; and / or c. the RNA nickase activity in the composition is between 0.004 U / μl and 0.04 U / μl, greater than 0.04 U / μl, or between 0.05 U / μl and 0.3 U / μl; The composition according to any one of claims 1 to 10.

12. 1. A method for preparing double-stranded cDNA, comprising: a. combining a primer with a sample containing RNA and allowing the primer to bind to the RNA; b) combining said sample with the composition of any one of claims 1 to 11 and preparing double-stranded cDNA by an isothermal reaction.

13. The primer is a. Contains random primers; b. A primer that specifically binds to a sequence contained in the RNA; c. Contains a hexamer primer; d. comprising a primer comprising a chemically modified nucleotide, optionally rendering the RNA bound by the primer resistant to cleavage by RNA nickase; The method of claim 12.

14. The method of claim 12 or 13, wherein a reverse transcriptase produces a DNA:RNA duplex comprising a first strand of cDNA and a strand of RNA, optionally the RNAse H nicks the RNA strand in the DNA:RNA duplex to produce an RNA fragment, and optionally the DNA polymerase extends a second strand of DNA by priming from the RNA fragment.

15. 15. The method of any one of claims 12 to 14, further comprising depleting off-target RNA or enriching mRNA with the sample comprising RNA prior to combining primers with the sample comprising RNA, optionally enriching mRNA comprises binding mRNA to amplification or capture beads using poly-T primers.

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