Systems and methods for total nucleic acid library preparation by template switching

Abstract

The present disclosure provides a method for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA. The method includes performing a first template switching reaction on the nucleic acid sample in the absence of at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, thereby forming a first nucleic acid product comprising double-stranded DNA having at least one extended 3' end complementary to a first template switch oligonucleotide. The method further includes performing a second template switching reaction on the nucleic acid sample, thereby forming a second nucleic acid product comprising a first primer extension product complementary to at least a portion of the RNA, the first primer extension product having an extended 3' end complementary to a second template switch oligonucleotide.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 63 / 386,725, filed December 9, 2022, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH Not applicable.

[0003] background The present disclosure relates generally to library preparation for next-generation sequencing of nucleic acids, and more particularly to systems and methods for whole nucleic acid library preparation and targeted sequencing by template switching. [Background technology]

[0004] To analyze a nucleic acid sample using existing sequencing technology, it is generally necessary to first prepare and optionally enrich the nucleic acids in the sample using one or more library preparation schemes, target enrichment schemes, or a combination thereof. Library preparation schemes are often used to make a nucleic acid sample compatible with a given sequencing technology, for example, by adding a common nucleic acid adapter sequence to the terminal nucleic acid fragments derived from the sample. In comparison, target enrichment schemes are often used to selectively isolate specific genomic regions of interest before sequencing. Such enrichment methods are suitable for experiments where it may be desirable to study fewer than the entire nucleic acid sequence from a biological source, but more than just a small number of nucleic acid sequences (e.g., more than 1,000).

[0005] In one aspect, it may be advantageous to generate both RNA and DNA sequencing libraries from a sample, but existing library preparation and target enrichment schemes are generally not widely applicable to different types of nucleic acids.For example, certain schemes may be applicable to the preparation of libraries starting from either DNA or RNA (but not both).Furthermore, if it is desired to prepare nucleic acid libraries from both DNA and RNA from the same sample, additional steps may be required to first separate DNA from RNA for separate processing.

[0006] Previous approaches have provided limited solutions for integrating DNA and RNA library generation. For example, U.S. Patent Application No. 2018 / 0080021 to Reuter et al. describes a method for simultaneous sequencing of RNA and DNA from the same sample. The approach taught by Reuter et al. is based on i) adding adapters to whole genomic DNA using Tn5 transposase and ii) producing a transcriptome library in the same reaction using RNA ligase. While this protocol is effective for preparing whole genome and transcriptome libraries in a single tube, the protocol is characterized by a high degree of complexity.

[0007] Therefore, new schemes for the integration of DNA and RNA library preparation and target enrichment are needed. Summary of the Invention

[0008] overview The present invention overcomes the aforementioned drawbacks by providing systems and methods for total nucleic acid library preparation via template switching, as described in the enumerated list below. 1. In a first reaction mixture: i) a nucleic acid sample comprising at least one double-stranded DNA, the double-stranded DNA having a first strand and a second strand, the second strand being at least partially complementary to the first strand, and each of the first strand and the second strand having a 5' end and a 3' end; ii) a first reverse transcriptase; and iii) a first template switch oligonucleotide that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; and iv) combining with a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to at least one nucleotide excluded from the first template switch oligonucleotide; conducting a first template switching reaction in a first reaction mixture; adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA with a reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing a first template switch oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; extending the non-templated 3' overhang of the double-stranded DNA with a reverse transcriptase, thereby forming a first nucleic acid product comprising double-stranded DNA having at least one extended 3' end that is complementary to the first template switch oligonucleotide; A method comprising: 2. The method according to item 1, wherein the nucleic acid sample further comprises at least one RNA, the RNA having a 5' end and a 3' end. 3. To a second reaction mixture: i) a first nucleic acid product; ii) RNA; iii) a second reverse transcriptase; and iv) a second template switch oligonucleotide; and v) a second mixture of dNTPs; conducting a second template switching reaction in a second reaction mixture; synthesizing a polynucleotide complementary to the RNA with a second reverse transcriptase, thereby forming a first primer extension product complementary to at least a portion of the RNA; adding at least one non-templated nucleotide to the 3' end of the first primer extension product with a second reverse transcriptase, thereby forming a non-templated 3' overhang on the first primer extension product; annealing a second template switch oligonucleotide to the non-templated 3' overhang of the first primer extension product; extending the non-templated 3' overhang of the first primer extension product with a second reverse transcriptase, thereby forming a second nucleic acid product comprising the first primer extension product having an extended 3' end that is complementary to a second template switch oligonucleotide; The method according to item 2, further comprising: 4. further comprising combining in the second reaction mixture a first oligonucleotide primer having a 3' end complementary to the RNA; conducting a second template switching reaction in the second reaction mixture; annealing the 3' end of a first oligonucleotide primer to the RNA; 4. The method of claim 3, further comprising extending the first oligonucleotide primer with a second reverse transcriptase, thereby forming a first primer extension product. 5. The method of item 3, wherein the nucleotide sequence of the first template switch oligonucleotide differs from the nucleotide sequence of the second template switch oligonucleotide by at least one nucleotide. 6. The method of item 2, wherein the first template switching reaction is unable to form a by-product comprising at least a partial complement of an RNA having a 3' end complementary to the first template switch oligonucleotide. 7. The method of item 1, further comprising terminating at least one of the 3' ends of the first nucleic acid product. 8. The method according to item 7, wherein the termination step comprises incorporating a dideoxynucleotide at at least one 3' end of the first nucleic acid product. 9. The method of item 1, wherein the first template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence. 10. The method of item 3, wherein the second template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence. 11. The method of item 3, further comprising purifying the first nucleic acid product and the nucleic acid sample containing RNA prior to combining into the second reaction mixture. 12. To the first reaction mixture: i) a nucleic acid sample comprising at least one double-stranded DNA, the double-stranded DNA having a first strand and a second strand, the second strand being at least partially complementary to the first strand, and each of the first strand and the second strand having a 5' end and a 3' end; ii) a reverse transcriptase; and iii) a first template switch oligonucleotide having a 5' domain and a 3' domain, wherein the 3' domain of the first template switch oligonucleotide excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; and iv) a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide; and v) combining a ddNTP complementary to at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide; conducting a first template switching reaction in a first reaction mixture; adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA with a reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing a first template switch oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; extending the non-templated 3' overhang of the double-stranded DNA with a reverse transcriptase, thereby forming a first nucleic acid product comprising double-stranded DNA having at least one extended 3' end that is complementary to the 3' domain of the first template switch oligonucleotide; A method comprising: 13. The method according to item 12, wherein the nucleic acid sample further comprises at least one RNA, the RNA having a 5' end and a 3' end. 14. To the second reaction mixture: i) a first nucleic acid product; ii) RNA; iii) a second reverse transcriptase; and iv) a second template switch oligonucleotide; and v) a second mixture of dNTPs; conducting a second template switching reaction in a second reaction mixture; synthesizing a polynucleotide complementary to the RNA with a second reverse transcriptase, thereby forming a first primer extension product complementary to at least a portion of the RNA; adding at least one non-templated nucleotide to the 3' end of the first primer extension product with a second reverse transcriptase, thereby forming a non-templated 3' overhang on the first primer extension product; annealing a second template switch oligonucleotide to the non-templated 3' overhang of the first primer extension product; extending the non-templated 3' overhang with a second reverse transcriptase, thereby forming a second nucleic acid product comprising a first primer extension product having an extended 3' end complementary to a second template switch oligonucleotide; Item 14. The method of item 13, further comprising: 15. Further comprising combining in the second reaction mixture a first oligonucleotide primer having a 3' end complementary to the RNA; conducting a second template switching reaction in the second reaction mixture; annealing the 3' end of a first oligonucleotide primer to the RNA; 15. The method of claim 14, further comprising extending the first oligonucleotide primer with a second reverse transcriptase, thereby forming a first primer extension product. 16. The method of item 14, wherein the nucleotide sequence of the first template switch oligonucleotide differs from the nucleotide sequence of the second template switch oligonucleotide by at least one nucleotide. 17. The method of item 13, wherein the first template switching reaction is unable to form a by-product comprising at least a partial complement of an RNA having a 3' end complementary to the first template switch oligonucleotide. 18. The method of item 12, wherein the 5' domain of the first template switch oligonucleotide comprises at least one nucleotide that is excluded from the 3' domain of the first template switch oligonucleotide, and the first nucleic acid product comprises at least one 3' end that terminates with a ddNTP. 19. The method of item 12, wherein the 3' domain of the first template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence. 20. The method of item 14, wherein the second template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence. 21. The method of item 14, further comprising purifying the first nucleic acid product and the nucleic acid sample containing RNA prior to the step of combining into the second reaction mixture. 22. The second nucleic acid product comprises a second target sequence, and the method comprises: a second amplification reaction mixture comprising: i) a second nucleic acid product; and ii) a first primer having a 3' end corresponding to at least the 5' end of the second template switch oligonucleotide; and iii) a second primer having a 3' end corresponding to a second target sequence, wherein the second primer is a primer having a 3' end corresponding to a second target sequence. 23. The first nucleic acid product comprises a first target sequence, and the method comprises: A first amplification reaction mixture comprising: i) a first nucleic acid product; ii) a first primer having a 3' end corresponding to at least the 5' end of the first template switch oligonucleotide; and 23. The method of any one of items 1 to 22, further comprising amplifying at least a portion of the first nucleic acid product in a first amplification reaction mixture comprising: 24. The method of any one of items 1 to 23, wherein each of the 3' ends of the first nucleic acid products comprises an extended 3' end that is complementary to the first template switch oligonucleotide. 25. The method of any one of items 1 to 24, wherein the second template switching reaction is unable to form a by-product comprising a first nucleic acid product having a 3' end complementary to the second template switch oligonucleotide. 26. The method of any one of items 1 to 25, wherein the nucleic acid sample comprises a plurality of DNAs. 27. The method of any one of items 1 to 26, wherein the nucleic acid sample comprises a plurality of RNAs. 28. The method of any one of items 1 to 27, wherein the reverse transcriptase is selected from Moloney murine leukemia virus (MMLV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, and mutants thereof. 29. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is a polydT sequence. 30. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is a target-specific sequence. 31. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is a random sequence. 32. The method of any one of items 1 to 31, wherein the first template switch oligonucleotide comprises a stuffer region. 33. The method of item 4 or 15, wherein the second template switch oligonucleotide comprises a stuffer region. 34. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is complementary to an exon region of the RNA. 35. The method according to item 7, wherein the termination step comprises incorporating a dideoxynucleotide into at least one 3' end of the first nucleic acid product using a terminal transferase. 36. The method according to item 35, wherein the terminal transferase is Taq DNA polymerase. 37. The method of any one of items 1 to 36, wherein performing the first template switching reaction in the first reaction mixture further comprises adding at least three non-templated nucleotides to at least one of the 3' ends of the double-stranded DNA with a reverse transcriptase. 38. The method of item 3 or 14, wherein performing a second template switching reaction in the second reaction mixture comprises adding at least three non-templated nucleotides to the 3' end of the first primer extension product with a reverse transcriptase. 39. The method of item 2 or 13, further comprising recovering a nucleic acid sample comprising the first nucleic acid product and at least one RNA as a second nucleic acid sample. 40. To the first reaction mixture: i) a nucleic acid sample comprising at least one double-stranded DNA, the double-stranded DNA having a first strand and a second strand, the second strand being at least partially complementary to the first strand, each of the first strand and the second strand having a 5' end and a 3' end; and at least one RNA, the RNA having a first strand having a 5' end and a 3' end; ii) a reverse transcriptase; and iii) a first template switch oligonucleotide that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; and iv) combining with a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to at least one nucleotide excluded from the first template switch oligonucleotide; conducting a first template switching reaction in a first reaction mixture; adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA with a reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing a first template switch oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; extending the non-templated 3' overhang of the double-stranded DNA with a reverse transcriptase, thereby forming a first nucleic acid product comprising double-stranded DNA having at least one extended 3' end that is complementary to the first template switch oligonucleotide; A method comprising: 41. A method for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA, comprising: performing a first template switching reaction on the nucleic acid sample in the absence of at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, thereby forming a first nucleic acid product comprising double-stranded DNA having at least one extended 3' end complementary to the first template switch oligonucleotide; performing a second template switching reaction on the nucleic acid sample, thereby forming a second nucleic acid product comprising a first primer extension product complementary to at least a portion of the RNA, wherein the first primer extension product has an extended 3' end complementary to a second template switch oligonucleotide. 42. A kit for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA, comprising: a first template switch oligonucleotide that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to at least one nucleotide excluded from the first template switch oligonucleotide; and Includes a kit. 43. A second template switch oligonucleotide; and a second mixture of dNTPs; and Item 43. The kit of item 42, further comprising: 44. A kit for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA, comprising: a first template switch oligonucleotide having a 5' domain and a 3' domain, wherein the 3' domain of the first template switch oligonucleotide excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide; and a ddNTP complementary to at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide; Includes a kit. 45. A second template switch oligonucleotide; and a second mixture of dNTPs; and Item 45. The kit of item 44, further comprising: 46. ​​The method of item 3 or 14, wherein at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises a ribonucleotide. 47. The method of item 46, further comprising contacting at least one of the first template switch oligonucleotide and the second template switch oligonucleotide with a ribonuclease. 48. The method of item 3 or 14, wherein at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises a 5' modification selected from a nucleotide analog, a linkage modification, a terminal modification, and a fluorescent label. 49. The method of item 1 or 3, wherein the 3' end of at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises a homopolymer sequence of at least three nucleotides. 50. The method of item 49, wherein the homopolymer sequence is selected from polyriboguanosine, polyguanosine, polyribocytidine, and polycytidine. 51. The method of item 12, wherein at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises at least one 2'-O-methyl nucleoside modification. 52. The method of item 1 or 12, wherein the first template switch oligonucleotide further excludes uracil. 53. The method according to item 1 or 12, wherein the first mixture of dNTPs further excludes dUTP. 54. The method of item 12, wherein the 5' domain of the first template switch oligonucleotide comprises at least one nucleotide that is excluded from the 3' domain of the first template switch oligonucleotide. 55. The method of item 12 or 54, wherein the ddNTP further comprises a capture moiety. 56. The method of claim 55, wherein the capture moiety is selected from biotin and desthiobiotin. 57. The method of item 1 or 12, wherein the first mixture of dNTPs comprises at least one dNTP having a capture moiety. 58. The method of claim 57, wherein the capture moiety is selected from biotin and desthiobiotin. 59. The method of item 3 or 14, wherein the second mixture of dNTPs comprises at least one capture moiety. 60. The method of claim 59, wherein the capture moiety is selected from biotin and desthiobiotin. 61. The method of item 4 or 15, wherein the first oligonucleotide primer comprises at least one capture moiety. 62. The method of claim 61, wherein the capture moiety is selected from biotin and desthiobiotin.

[0009] The above and other aspects and advantages of the present invention will emerge from the following description. In the description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown, by way of example, preferred embodiments of the invention. Such embodiments do not necessarily represent the full scope of the invention, however, and reference is therefore made to the claims and this specification for interpreting the scope of the invention. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of a method for preparing a total nucleic acid sample according to the present disclosure.

[0011] [Figure 2A] FIG. 1 is a schematic diagram showing the components of a total nucleic acid sample according to the present disclosure.

[0012] [Figure 2B] FIG. 2B is a schematic diagram showing the components of the total nucleic acid sample of FIG. 2A after the addition of non-templated 3′ overhangs to the 3′ ends of the double-stranded DNA.

[0013] [Figure 2C] FIG. 2C is a schematic diagram showing the components of the total nucleic acid sample of FIG. 2B after annealing of the first template switch oligonucleotide to the non-templated 3′ overhang.

[0014] [Figure 2D] FIG. 2D is a schematic diagram showing the components of the total nucleic acid sample of FIG. 2C after extending the non-templated 3' overhang of the double-stranded DNA, thereby forming a first nucleic acid product comprising double-stranded DNA having at least one extended 3' end that is complementary to a first template switch oligonucleotide.

[0015] [Figure 3A] FIG. 2E is a schematic diagram showing the first nucleic acid product and RNA of FIG. 2D in combination with a second template switch oligonucleotide and primer.

[0016] [Figure 3B] FIG. 3B is a schematic diagram showing the components of FIG. 3A after synthesizing a polynucleotide complementary to the RNA, thereby forming a first primer extension product, and adding a non-templated 3′ overhang to the 3′ end of the first primer extension product.

[0017] [Figure 3C] FIG. 3C is a schematic diagram showing the components of FIG. 3B after annealing of a second template switch oligonucleotide to the non-templated 3′ overhang.

[0018] [Figure 3D] FIG. 2D is a schematic diagram showing the components of FIG. 2C after extending the non-templated 3′ overhang of the first primer extension product, thereby forming a second nucleic acid product comprising the first primer extension product having an extended 3′ end that is complementary to a second template switch oligonucleotide.

[0019] [Figure 4A] FIG. 3D is a schematic diagram showing the components of FIG. 3D combined with primer pairs for amplification.

[0020] [Figure 4B] FIG. 4B is a schematic diagram showing the products of the amplification reaction shown in FIG. 4A.

[0021] Like numbers are used to describe like parts from figure to figure throughout the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description I. Definition In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to encompass the itemized element or step, whether by itself or together with one or more additional elements or steps, (iv) the terms "about" and "approximately" may be understood to allow for standard variations that will be appreciated by one of ordinary skill in the art, and (v) when ranges are provided, the endpoints are included.

[0023] Approximately: As used herein, the term "approximately" or "about" refers to a value similar to the stated reference value when applied to one or more values ​​of interest. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise specified or otherwise clear from the context (unless such number would exceed 100% of possible values).

[0024] Associated: As used herein, two events or entities are "associated" with one another if the presence, level, and / or form of one event or entity correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly, thereby bringing them into and / or remaining in close physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently bound by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0025] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained from or derived from a biological source of interest (e.g., a tissue or organism or cell culture), as described herein. In some embodiments, the source of interest comprises or consists of an organism, such as an animal or a human. In some embodiments, the biological sample comprises or consists of a biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; free-floating nucleic acid; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymphatic fluid; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washings or lavage fluids, such as ductal lavage or bronchoalveolar lavage; aspirate; scraping; bone marrow specimen; tissue biopsy specimen; surgical specimen; other body fluids, secretions and / or excretions; and / or cells derived therefrom. In some embodiments, a biological sample comprises or consists of cells obtained from an individual. In some embodiments, the obtained cells are or comprise cells derived from the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.), etc. In some embodiments, as the context will dictate, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., removing one or more components of the primary sample and / or adding one or more agents to the primary sample), e.g., filtering using a semipermeable membrane. Such a "processed sample" may comprise, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0026] Comprising: Compositions or methods described herein as "comprising" one or more named elements or steps are open-ended, meaning that the named elements or steps are essential, but that other elements or steps may be added within the composition or method. A composition or method described as "comprising" (or "comprises") one or more named elements or steps should also be understood to describe a corresponding more limited composition or method that "consistes essentially of" (or "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. Any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps is also understood to describe a corresponding, more limited, and close-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of other, unnamed elements or steps. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0027] Designed: As used herein, the term "designed" refers to (i) an agent whose structure has been or is selected by the hand of man, (ii) an agent that is produced by a process requiring the hand of man, and / or (iii) an agent that differs from natural substances and other known agents.

[0028] Determining: Those of skill in the art reading this specification will understand that "determining" can utilize or can be accomplished through the use of any of a variety of techniques available to those of skill in the art, including, for example, the specific techniques expressly mentioned herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves reviewing and / or manipulating data or information, e.g., utilizing a computer or other processing device adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information and / or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0029] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, calculating the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment; non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.The nucleotides at corresponding positions are then compared.When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identity between two sequences can be achieved using a mathematical algorithm.For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons conducted with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0030] Sample: As used herein, the term "sample" refers to a substance that is or contains a composition of interest for qualitative and / or quantitative evaluation. In some embodiments, the sample is a biological sample (i.e., from a living thing (e.g., a cell or organism)). In some embodiments, the sample is derived from a geological, aquatic, astronomical, or agricultural source. In some embodiments, the source of interest comprises or consists of a living organism, such as an animal or a human. In some embodiments, a sample for forensic analysis is or comprises biological tissue, biological fluid, organic or non-organic matter, such as clothing, dirt, plastic, water. In some embodiments, an agricultural sample comprises or consists of organic matter, such as leaves, petals, bark, wood, seeds, plants, fruit, etc.

[0031] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a total or near-total extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or progress to completion, or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the possibility of lack of completion that is inherent in many biological and chemical phenomena.

[0032] Synthetic: As used herein, the word "synthetic" means produced by the hand of man and therefore in a form that does not occur in nature, either by having a structure that is not found in nature, or by being associated with one or more other components that are not associated with them in nature, or by being unassociated with one or more other components that are associated with them in nature.

[0033] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity, but that structurally differs from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a variant also differs functionally from the reference entity. Generally, whether a particular entity is properly considered a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To name just a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties, such that variants of the small molecule share the core structural element and the characteristic pendant moieties but differ in other pendant moieties and / or in the type of linkages (single vs. double, E vs. Z, etc.) present within the core; a polypeptide may have a characteristic sequence element comprised of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or that contribute to a particular biological function; and a nucleic acid may have a characteristic sequence element comprised of multiple nucleotide residues that have designated positions relative to each other in linear or three-dimensional space. For example, a variant polypeptide can differ from a reference polypeptide as a result of one or more differences in amino acid sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide exhibits an overall sequence identity with the reference polypeptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively, or in addition, in some embodiments, the variant polypeptide does not share at least one characteristic sequence element with the reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, the variant polypeptide shares one or more of the biological activities of the reference polypeptide.In some embodiments, a variant polypeptide lacks one or more of the biological activities of a reference polypeptide. In some embodiments, a variant polypeptide exhibits a reduced level of one or more biological activities compared to a reference polypeptide. In many embodiments, a polypeptide of interest is considered a "variant" of a parent or reference polypeptide when it has an amino acid sequence identical to that of the parent except for a small number of sequence changes at specific positions. Typically, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted compared to the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the parent. In many cases, a variant has a very small number (e.g., less than 5, 4, 3, 2, or 1) of functional residues (i.e., residues involved in a specific biological activity) substituted. Furthermore, a variant typically has no more than 5, 4, 3, 2, or 1 additions or deletions compared to the parent, and often no additions or deletions. Furthermore, any additions or deletions are typically less than about 25, 20, 19, 18, 17, 16, 15, 14, 13, 10, 9, 8, 7, or 6, and generally less than about 5, 4, 3, or 2 residues. In some embodiments, a variant may also have one or more functional defects and / or otherwise be considered a "mutant." In some embodiments, the parent or reference polypeptide is one found in nature. As will be understood by those skilled in the art, multiple variants of a particular polypeptide of interest may commonly be found in nature, particularly when the polypeptide of interest is an infectious agent polypeptide.

[0034] II. Detailed Description of Certain Embodiments As mentioned above, it can be useful in various situations to provide an integrated DNA and RNA library preparation and target enrichment scheme. In one aspect, simultaneous generation of RNA and DNA sequencing libraries allows for the collection of both DNA and RNA sequencing data from the same sample using next-generation sequencing (NGS). RNA sequencing data can provide validation of DNA variant calls and aid in the identification of driver mutations by quantifying expressed transcripts, allele-specific expression, and RNA editing. However, generating paired DNA and RNA sequencing libraries for NGS from the same biological specimen is not without challenges. Library construction from DNA and RNA from a single sample is typically achieved by purifying total nucleic acids (TNA), which are then split into two separate samples and treated with either DNase I to recover RNA or RNase A to recover DNA. This approach results in the loss of half the RNA and half the DNA. Furthermore, the addition of DNase I or RNase A can result in degradation of the desired TNA fraction, which is problematic when only small amounts of sample are available. Other existing commercial products allow for the sequential isolation of RNA and DNA fractions in a single protocol, but once DNA and RNA are isolated, the samples are processed separately, requiring additional time and effort.

[0035] Therefore, there is a need for a system and method for generating RNA and DNA sequencing libraries from a single sample originating from TNA.In this approach, DNA and RNA parts are never physically separated, but instead prepared for sequencing in a single tube.In another aspect, there is a need for a scheme that i) is compatible with automation platforms (for example, liquid handling robots), ii) can accommodate high-quality or low-quality TNA samples, and iii) can distinguish the reads that originate from either DNA or RNA after sequencing.

[0036] The present disclosure provides methods and kits for efficiently adding unique adapters to RNA, DNA, or both, where DNA and RNA are present in a single sample and are never separated. The disclosed approach further enables the selection and enrichment of DNA, RNA, or both, for example, through the use of amplification and sequencing. Furthermore, sequencing reads derived from either DNA or RNA strands can be easily distinguished with high reliability using the disclosed systems and methods. The methods disclosed herein are expected to provide a simpler workflow with fewer steps than existing workflows. Finally, the disclosed methods are expected to be compatible with a variety of nucleic acid sample types, including both fragmented and high-quality TNAs.

[0037] In one aspect, the present disclosure provides a method for integrated TNA library preparation based on the terminal transferase activity and template switching ability of reverse transcriptase (RT) enzymes, such as MMLV RT. The use of RT enzymes is an effective method for adding known sequences or adapters to the ends of complete cDNA sequences. This mechanism involves the ability of RT to add non-templated nucleotides to the 3' end of a complementary DNA (cDNA) strand. Upon reaching the end of the template (usually the 5' end of an RNA molecule), the terminal transferase activity of the RT enzyme catalyzes the addition of non-templated nucleotides to the 3' end of the growing cDNA strand. The resulting 3' overhang facilitates the annealing of a complementary 3' oligo, referred to herein as a template-switching oligo (TSO). The 3' non-template overhang is typically a polycytosine (e.g., CCC) with a complementary TSO containing a 3' polyriboguanosine (e.g., rGrGrG, where "r" represents a ribonucleotide base); however, it will be understood that terminal transferase can generate alternative 3' overhangs depending on the composition of the dNTP pool and the specificity of the enzyme. Once the TSO anneals to the non-template overhang, the RT enzyme then switches templates, moving from the initially reverse-transcribed RNA template to the new TSO template. The end result is the attachment of a 3' new sequence to the 3' cDNA, which is the reverse complement of the TSO. Exemplary template switching applications are described, for example, in U.S. Patent No. 5,962,271 to Chenchik et al., incorporated herein by reference in its entirety.

[0038] Although the template switching mechanism of RT is known to be compatible with both DNA and RNA templates, existing integrated library preparation schemes do not utilize the template switching mechanism of RT for the preparation of both DNA and RNA present in the same sample. One challenge in achieving the use of the template switching mechanism of RT for integrated library preparation relates to controlling the selective addition of different TSO-derived sequences to RNA and DNA when both nucleic acids are present in the same sample. Nevertheless, the present disclosure provides systems and methods for TNA library preparation via template switching.

[0039] In general, the present disclosure is based on the surprising discovery that a library preparation scheme involving a reverse transcriptase with template switching activity can be applied to the preparation of TNA samples (i.e., samples containing both DNA and RNA). Advantageously, the library preparation scheme is a one-pot approach that allows for selective and unambiguous tagging of both DNA and RNA present in the same sample. Selective and distinguishable tagging is achieved by two separate template switching reactions performed sequentially on the TNA sample. Despite both dsDNA and RNA being present in the same reaction, each template switching reaction is selective for the preparation of either dsDNA or RNA. The resulting products of the scheme include DNA-derived nucleic acid products bearing a first adapter sequence and RNA-derived nucleic acid products bearing a second adapter sequence that is different from the first adapter sequence. Thus, after sequencing of the nucleic acid products, the resulting reads readily associate with either the original RNA template(s) or the original DNA template(s) present in the TNA sample.

[0040] Referring now to FIG. 1 , an embodiment of method 10 according to the present disclosure includes step 12 of preparing a TNA sample. In one aspect, the TNA sample includes all nucleic acids extracted and isolated from a biological sample. The TNA sample can include genomic DNA, messenger RNA (mRNA), ribosomal RNA (rRNA), etc. In one aspect, step 12 can include preparing fragmented, blunt-ended DNA. As will be appreciated by those skilled in the art, blunt-ended dsDNA can be prepared in a variety of ways. DNA, including genomic DNA, can be fragmented using any suitable approach, including enzymatic fragmentation, mechanical shearing, sonication, etc. DNA fragments can be made blunt-ended using any suitable approach, including both fill-in reactions (e.g., via polymerases) and Chewbach reactions (e.g., via exonucleases). In another aspect, it may be desirable to enrich for a portion of the total RNA present in the TNA. One enrichment method includes, for example, rRNA reduction using RNase H. As will be appreciated by those skilled in the art, further preparation steps can be applied to the TNA sample in step 12.

[0041] After preparation in step 12, the TNA sample contains both blunt dsDNA and RNA. The TNA sample prepared in step 12 is then subjected to two separate template switching reactions performed sequentially. Step 14 of method 10 involves performing a first template switching reaction. In the first template switching reaction, reaction conditions are provided such that the template switching reaction occurs only on the blunt dsDNA portion of the TNA. In one embodiment, the first template switching reaction mixture includes a nucleic acid sample, a first reverse transcriptase, a first TSO (TSO-A) that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine, and a first mixture of dNTPs that excludes at least one dNTP selected from dATP, dCTP, dGTP, and dTTP. The at least one dNTP excluded from the mixture of dNTPs is complementary to at least one nucleotide excluded from the first template switch oligonucleotide. For example, if TSO-A excludes only nucleotides containing the nucleobase thymine, the first mixture of dNTPs will exclude the complementary dATP. By excluding at least one type of nucleobase from the first TSO (i.e., TSO-A) and the complementary dNTP from the first mixture of dNTPs in the reaction, we ensure that only dsDNA, not RNA, undergoes template switching. Notably, this design eliminates nonspecific priming and extension associated with the RNA template present in the reaction because replication of the RNA template halts when the RT encounters a base complementary to the missing dNTP. Thus, the absence of all four canonical dNTPs prevents the RT from fully extending the 5' end of the RNA and catalyzing the template switching reaction associated with the RNA present in the TSO sample. Notably, we observed that the template switching reaction appears to be highly efficient for dsDNA present in the sample, with the majority (i.e., greater than 50%) of blunt dsDNA undergoing template switching.More particularly, other schemes can be implemented to achieve template switching with only dsDNA templates. For example, dideoxynucleotide triphosphates (ddNTPs) can be used as described below.

[0042] It will be understood that, in general, the nucleobases uracil and thymine can be used interchangeably. For example, if the first TSO excludes the nucleobase adenine, the first mixture of dNTPs excludes dTTP, which is complementary to adenine. In this case, it may be useful to further exclude dUTP from the first mixture of dNTPs. Similarly, if the first TSO excludes thymine, it may be useful to further exclude the nucleobase uracil from the first TSO. Thus, in one embodiment of the present disclosure, if the first template switch oligonucleotide excludes thymine, the first template switch oligonucleotide further excludes uracil. In another embodiment of the present disclosure, if the first mixture of dNTPs excludes dTTP, the first mixture of dNTPs further excludes dUTP.

[0043] Continuing with reference to step 14 of method 10 in FIG. 1 , the composition obtained from the first reaction mixture includes a first nucleic acid product comprising dsDNA having at least one extended 3' end complementary to the first TSO. The composition also includes any remaining reagents, such as untreated RNA, TSO-A, RT enzyme, and dNTPs. In preparation for the second template switching reaction of the disclosed method, method 10 can include step 16, in which the reaction mixture is subjected to a cleanup step to recover the first nucleic acid product and RNA from other components in the composition obtained from the first reaction mixture. Step 16 can include any suitable cleanup scheme for recovering nucleic acids from other components of the reaction. Exemplary cleanup schemes include column- and bead-based nucleic acid recovery methods, such as solid-phase reversible immobilization (SPRI) on carboxylated paramagnetic beads, solvent (e.g., ethanol)-based extraction protocols, and combinations thereof.

[0044] The next step 18 of method 10 involves performing a second template switching reaction. The second template switching reaction is configured to achieve template switching of the remaining TNA (i.e., RNA in this example) that did not undergo template switching in the first template switching reaction step. The second template switching reaction includes a first nucleic acid product, RNA, a reverse transcriptase having template switching activity, a second template switch oligonucleotide (TSO-B), and a second mixture of dNTPs. TSO-B has a sequence distinguishable from that of TSO-A to distinguish between nucleic acid products derived from dsDNA template(s) and RNA template(s). In one embodiment, the nucleotide sequence of the first template switch oligonucleotide differs from the nucleotide sequence of the second template switch oligonucleotide by at least one nucleotide. In another embodiment, the nucleotide sequence of the first template switch oligonucleotide differs from the nucleotide sequence of the second template switch oligonucleotide by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides.

[0045] In another embodiment, the second mixture of DNA contains all four canonical dNTPs to allow the reverse transcriptase to completely replicate the RNA template, allowing the template switching reaction to occur at the 5' end of the RNA template. In particular, the second template switching reaction can optionally include at least one primer designed to allow the reverse transcriptase to prime the replication of the RNA template. The primer can be an oligo-dT primer, a target-specific primer, a randomizer, or a combination thereof.

[0046] With continued reference to step 18 of method 10 in Figure 1, the second template switching reaction facilitates priming off of the RNA template(s), reverse transcription for cDNA synthesis, and template switching using TSO-B. It should be understood that any unreacted blunt dsDNA that did not undergo template switching in the first reaction can undergo template switching in the second reaction. Therefore, if it is desirable to prevent the addition of sequences complementary to the second TSO (i.e., TSO-B) to the dsDNA, steps should be taken to ensure that complete conversion of the dsDNA to the first nucleic acid product occurs in the first template switching reaction. Alternatively, or in addition, steps can be taken to remove unreacted dsDNA from the second reaction. This and other approaches are described in further detail herein.

[0047] Method 10 further includes a step 20 of cleaning up the second template switching reaction. The composition obtained from the second reaction mixture includes the first nucleic acid product and a second nucleic acid product comprising cDNA derived from the RNA template(s). The cDNA has an extended 3' end complementary to TSO-B. The composition also includes unprocessed RNA, TSO-B, RT enzyme, and any remaining reagents, such as dNTPs. In preparation for downstream processing (if any) prior to sequencing, the composition obtained from the second reaction mixture can be subjected to a cleanup step to recover the first and second nucleic acid products from other components in the composition obtained from the second reaction mixture.

[0048] Method 10 further includes step 22 of amplifying the TSO sequence-tagged nucleic acid. In one embodiment, the first and second nucleic acid products can be amplified by PCR using a variety of methods. Amplification step 22 can further include ligation of adapters compatible with the selected sequencing platform. The amplification reaction can be designed to amplify DNA-derived products, RNA-derived products, or both by including primers specific to one or both of the TSO-A and TSO-B sequences. The TSO-specific primers can further be paired with target-specific primers to enable enrichment of specific nucleic acid sequences. It will be understood that the two-step template switching approach and subsequent amplification method disclosed herein are susceptible to various modifications to accommodate different desired results, as will become apparent from this disclosure.

[0049] After the first and second template switching reactions in steps 14 and 18, step 24 of method 10 involves performing a sequencing reaction on the product nucleic acid. In one embodiment, the product of the template switching reaction is sequenced directly without amplification. In another embodiment, the product resulting from the amplification in step 22 is sequenced. Any suitable sequencing platform can be used, including short-read and long-read platforms, sequencing by synthesis platforms, and nanopore-based sequencing platforms. Method 10 further includes step 26 of assigning the sequencing read. Based on the detected TSO sequence, a given sequencing read can be precisely and accurately assigned as originating from either DNA or RNA molecules originally present in the TNA sample.

[0050] Referring now to Figure 2A, a TNA sample can include at least one double-stranded DNA 100 and at least one RNA 200. The double-stranded DNA 100 has a first strand 102 and a second strand 104. The second strand 104 is at least partially complementary to the first strand 102. Furthermore, the first strand 102 has a 5' end 106 and a 3' end 108, and the second strand 104 has a 5' end 110 and a 3' end 112. Notably, the orientation of the first strand 102 and the second strand 104, along with all other illustrated nucleic acids, is indicated by the use of arrows throughout the figure. The double-stranded DNA 100 further defines a first target sequence 114.

[0051] Referring to Figure 2B, double-stranded DNA 100 is combined in a first reaction mixture with a first reverse transcriptase (not shown), a first template switch oligonucleotide or TSO 116, and a first mixture of dNTPs (not shown). The first TSO 116 excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine. The first mixture of dNTPs excludes at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, where the at least one dNTP excluded from the mixture of dNTPs is complementary to the at least one nucleotide excluded from the first TSO 116.

[0052] The first reaction mixture contains components necessary to perform a first template switching reaction in the first reaction mixture. In one embodiment, the first template switching reaction uses a reverse transcriptase to add at least one non-templated nucleotide to at least one of the 3' ends of double-stranded DNA 100, thereby forming a non-templated 3' overhang 118 on double-stranded DNA 100. Non-templated 3' overhang 118 is complementary to the 5' end of TSO 116, allowing annealing of first TSO 116 to non-templated 3' overhang 118 of double-stranded DNA 100 (FIG. 2C).

[0053] After annealing of the TSO 116, the non-templated 3' overhang 118 of the double-stranded DNA 100 is extended with a reverse transcriptase, thereby forming a first nucleic acid product 120 comprising the double-stranded DNA 100 having at least one extended 3' end 122 complementary to the first template switch oligonucleotide 116.

[0054] 3A , a second template switching reaction can be performed on at least one RNA 200. RNA 200 has a 5′ end 202 and a 3′ end 204 and further defines a second target sequence 206. RNA 200 is combined with a second reaction mixture including first nucleic acid product 120, a second reverse transcriptase (not shown), a second template switch oligonucleotide 208, and a second mixture of dNTPs (not shown). In one embodiment, the nucleotide sequence of first template switch oligonucleotide 116 differs from the nucleotide sequence of second template switch oligonucleotide 208 by at least one nucleotide. The second reaction mixture can optionally include a first oligonucleotide primer 210 that is at least partially complementary to RNA 200.

[0055] 3B and 3C, performing a second template switching reaction in a second reaction mixture includes synthesizing a polynucleotide complementary to RNA 200 with a second reverse transcriptase, thereby forming a first primer extension product 212 complementary to at least a portion of RNA 200. In one embodiment, performing a second template switching reaction in the second reaction mixture further includes annealing a 3′ end of a first oligonucleotide primer 210 to RNA 200 and extending the first oligonucleotide primer with a second reverse transcriptase, thereby forming first primer extension product 212. Furthermore, at least one non-templated nucleotide can be added to the 3′ end of first primer extension product 212 with the second reverse transcriptase, thereby forming a non-templated 3′ overhang 214 on first primer extension product 212. A second template switch oligonucleotide 208 can be annealed to the non-templated 3' overhang 214 of the first primer extension product 212, and the non-templated 3' overhang 214 of the first primer extension product 212 can be extended with a second reverse transcriptase, thereby forming a second nucleic acid product 216 comprising the first primer extension product 212 having an extended 3' end 218 that is complementary to the second template switch oligonucleotide (Figure 3D).

[0056] Referring to Figure 4, first nucleic acid product 120 and second nucleic acid product 216 can be amplified using various methods for attaching sequencing adaptors to each end. In one embodiment, first nucleic acid product 120 includes top strand 124 and bottom strand 126. Each of top strand 124 and bottom strand 126 can be selectively amplified using a separate primer pair. For example, a first primer pair for amplifying top strand 124 can include target-specific primer 128 and primer 130 specific to extended 3' end 122, while a second primer pair for amplifying bottom strand 126 can include target-specific primer 132 and primer 134 specific to extended 3' end 122. In another embodiment, second nucleic acid product 216 can be selectively amplified using a separate primer pair that is different from that used to amplify first nucleic acid product 120. For example, a first primer pair for amplifying second nucleic acid product 216 can include a target-specific primer 220 and a primer 222 specific to extended 3′ end 218 .

[0057] In particular, each of primers 128, 130, 132, and 134 can include a 5' tail 136 that defines an adapter sequence. The 5' tail 136 can define the same or a different sequence and can include a sequencing platform-specific sequence, a sample identifier sequence, a molecular identifier sequence, etc., and combinations thereof. Similarly, each of primers 220 and 222 can include a 5' tail 224 that defines an adapter sequence. The 5' tail 224 can define the same or a different sequence and can include a sequencing platform-specific sequence, a sample identifier sequence, a molecular identifier sequence, etc., and combinations thereof.

[0058] Referring to Figure 4B, the products of the amplification step shown in Figure 4A are uniquely identified by the corresponding TSO sequence and, due to the addition of an adapter sequence, are further suitable for sequencing on a selected platform. In a first example, a first product 138 is derived from the top strand 124 after amplification with primers 128 and 130. The first product 138 includes the first target sequence 114, which includes the TSO-derived sequence and a consensus sequence corresponding to the 5' tail 136, and an extended 3' end 122. In a second example, a second product 140 is derived from the bottom strand 126 after amplification with primers 132 and 134. The second product 140 includes the first target sequence 114, which includes the TSO-derived sequence and a consensus sequence corresponding to the 5' tail 136, and an extended 3' end 122. In a third example, a third product 226 is derived from the second nucleic acid product 216 after amplification with primers 220 and 222. The third product 226 comprises the second target sequence 206 , which includes the TSO-derived sequence and the consensus sequence corresponding to the 5′ tail 224 , and an extended 3′ end 218 .

[0059] It will be understood that various modifications can be made to the disclosed method to improve the template switching reaction on DNA or to ensure that only DNA undergoes template switching in the first template switching reaction. In one embodiment, the first TSO or the second TSO-B can include one or more of: i) nucleotide analogs such as locked nucleic acid (LNA), fluorobeta-D-arabinonucleic acid (FANA), 2'-O-methyl RNA, 2'-fluoro RNA, etc.; ii) linkage modifications such as phosphorothioates, 3'-3', and 5'-5' reverse linkages; iii) 5'-end modifications, 3'-end modifications, or combinations thereof, such as amino, biotin, digoxigenin 11dUTP, phosphate, thiol, dye, and quencher modifications; iv) one or more fluorescently labeled nucleotides; or v) any other features that provide a desired functionality to the template switch oligonucleotide.

[0060] In another embodiment, the first TSO can contain a unique sequence that unambiguously identifies the reads derived from the first template switching reaction and thus indicates which products or sequencing reads were derived from the dsDNA portion of the TNA.

[0061] In another embodiment, buffer conditions can be optimized to favor template switching onto DNA rather than onto RNA.

[0062] In another embodiment, template switching reactions can be improved by using TSOs with 3'-terminal sequences selected from NNN and rNrNrN (where r represents an RNA base and N represents a nucleic acid base). In one example, the 3'-terminal sequence of the TSO is a homopolymer (e.g., AAA, rArArA, CCC, rCrCrC, TTT, rTrTrT, GGG, or rGrGrG). In another example, the 3'-terminal sequence of the TSO is a heteropolymer (e.g., CGC, rCrGrC, etc.). In yet another example, a composition is prepared having multiple TSOs with different 3'-terminal sequences. For example, a TSO composition can contain equal portions of TSOs with two different 3'-terminal sequences.

[0063] In another embodiment, dNTPs that are excluded from the mixture of dNTPs are selected to enhance template switching.

[0064] In another embodiment, the TSO can include a unique molecular identifier (UMI), also known as a unique molecular identifier (UID) or barcode sequence. The UMI can have variable length and sequence. The TSO can include a UMI at the 5' end, 3' end, or in an intermediate position. In one embodiment, the UMI is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length.

[0065] In another embodiment, a template switching enhancer region can be located adjacent to the 5' region of the 3' end of the TSO. The enhancer region includes the first base incorporated after successful template switching. The sequence of the enhancer region can be selected to enhance the template switching reaction.

[0066] In another embodiment, the disclosed method can include the use of a mixture of at least two different first or second TSOs, each containing a variable stuffer region located 5' of the 3'-end sequence. The stuffer region contains a nucleotide sequence selected to reduce the loss of complexity that can occur when reading sequences from the 3'-end of the TSO (because this sequence can be identical for all TSOs used in the template switching reaction). As will be appreciated, certain sequencing instruments benefit from greater sequence complexity in the template during initial sequencing cycles. Therefore, a stuffer region can be used to improve sequence diversity and, therefore, overall results after sequencing.

[0067] In another embodiment, enhancer regions, stuffer regions, or a combination thereof can serve as a key to aid in the identification of TSO elements during data analysis, for example, identification of sequences that align with stuffer regions can be used to identify the location of other sequences such as UIDs / UMIs.

[0068] In another embodiment, the disclosed method can include a heat denaturation step. For example, heat denaturation can be performed after the template switching reaction to denature some or all of the dsDNA in the TNA sample.

[0069] In another embodiment, the oligonucleotide primers used in the PCR amplification step can be designed to be complementary to sequences located in intron regions to ensure that only DNA is amplified.

[0070] In another embodiment, the method according to the present disclosure can include a terminal transferase step using ddNTPs after the DNA template switching step. For example, ddATP can be added in excess to the product of the template switching reaction along with Taq DNA polymerase. This adds terminators to all blunt dsDNA molecules, excluding them from subsequent reactions targeting RNA. The use of ddNTPs prevents the products of the first template switching reaction from being unavailable as templates in the second template switching reaction targeting RNA in the sample. This prevents the formation of DNA-derived products with multiple adapters or concatemers at the 3' end. Furthermore, the original DNA template present in the sample that did not undergo template switching in the first template switching reaction is tailed with a terminator and cannot be further extended in the second template switching reaction.

[0071] In another aspect, the disclosed method can include a nuclease treatment step. For example, a nuclease enzyme can be added to the first template switching reaction to degrade both blunt dsDNA and ssDNA that have not undergone the template switching reaction, effectively eliminating these molecules from the second template switching reaction. In some embodiments, a nuclease treatment step can be used in place of an alternative cleanup step between the first and second template switching reactions. One example nuclease is E. coli Exonuclease I, which is a 3'-5' exonuclease that degrades all ssDNA in the reaction when added before the first template switching reaction cleanup. Another example nuclease is E. coli ExoIII, which is a 3'-5' exonuclease that degrades blunt double-stranded dsDNA. Notably, E. coli ExoIII does not digest protruding 3' overhangs on dsDNA that can be generated by melting or removing TSO-A.

[0072] In yet another embodiment, the TSO can be designed to include a sequencing platform-specific adapter sequence.

[0073] In one embodiment, the method of the present disclosure can further include the use of a capture moiety. Examples of capture moieties include biotin and desthiobiotin. In one approach, dNTPs are labeled with a capture moiety, so that when template switching reaction occurs, the capture moiety is incorporated into the first nucleic acid product derived from a dsDNA template or the second nucleic acid product derived from an RNA template. The resulting labeled product can be captured using streptavidin beads. The use of a capture moiety further enables the recovery of oligonucleotide primers (e.g., RNA-specific, randomer, or oligo-dT primers) for generating first primer extension products from RNA by incorporating the capture moiety into the oligonucleotide primer.

[0074] The use of capture moieties also facilitates subsequent cleanup or purification steps. In one example, blunt dsDNA that has not undergone template switching is removed from the reaction during a streptavidin cleanup step prior to the second template switching reaction, thereby reducing carryover of blunt dsDNA into the second template switching reaction. In another embodiment, when desthiobiotin-labeled dNTPs are used, biotin can be added to the subsequent PCR reaction to facilitate the release of any template molecules bound to a capture surface, such as streptavidin-coated beads.

[0075] In one embodiment of the present disclosure,

[0076] The nucleic acid sample comprises at least one double-stranded DNA combined with a reverse transcriptase, a first template switch oligo, a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, and 2',3' dideoxynucleotides (ddNTPs). In one embodiment, the ddNTPs include the nucleic acid bases excluded from the first mixture of dNTPs. When performing a first template switching reaction, the DNA template is replicated by the reverse transcriptase until the ddNTP is reached. The termination point can be controlled by selecting where the complementary base of the ddNTP is located within the first template switch oligo. Notably, the incorporation of the ddNTP prevents any priming-off of RNA templates that may be pre-established in the nucleic acid sample, thereby limiting template switching to the DNA portion of the nucleic acid sample.

[0077] In some embodiments, the ddNTP is labeled with a capture moiety. For example, the ddNTP can be labeled with biotin or desthiobiotin. During the first template switching reaction, the reaction terminates when the ddNTP is incorporated into the complementary position within the first template switch oligo template. In particular, the complementary base can be positioned within the first template switch oligo at a defined position. The resulting product incorporates a capture moiety (in this case, a single biotin or desthiobiotin) at the 3' end of the first nucleic acid product resulting from the template switching reaction. The first nucleic acid product can then be recovered, for example, using streptavidin beads. To achieve recovery of RNA-derived products resulting from the template switching reaction according to the present disclosure, an oligonucleotide primer (e.g., a sequence-specific primer, randomer, or oligo-dT primer) can be labeled with or otherwise include at least one capture moiety. In one aspect, the use of a capture moiety can enable both purification between the template switching reaction and other downstream processing steps.

[0078] The schematic flowcharts depicted in the figures are generally presented as logical flowchart diagrams. Thus, the depicted order and labeled steps represent one embodiment of the presented method. Other steps and methods may be envisioned that are equivalent in function, logic, or effect to one or more steps, or portions thereof, of the depicted method. Furthermore, the format and symbols used in the figures are provided to illustrate the logical steps of the method and are understood not to limit the scope of the method. While various arrow and line types may be used, they are understood not to limit the scope of the corresponding method. In fact, some arrows or other connectors may be used to indicate only the logical flow of the method. For example, arrows may indicate wait or monitoring periods of unspecified duration between listed steps of the depicted method. Furthermore, the order in which a particular method occurs may or may not strictly adhere to the order of the corresponding steps shown.

[0079] The present invention is presented in several various embodiments in the following description with reference to the figures, where like numbers represent the same or similar elements. Throughout this specification, reference to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, throughout this specification, appearances of the phrases "in one embodiment," "in an embodiment," and similar language may, but do not necessarily, all refer to the same embodiment.

[0080] The described features, structures, or characteristics of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are set forth to provide a thorough understanding of system embodiments. However, those skilled in the art will recognize that the system and method may both be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of the invention. Therefore, the foregoing description is intended to be illustrative, and not to limit the scope of the inventive concepts.

[0081] Each reference identified in this application is incorporated herein by reference in its entirety.

Claims

1. The first reaction mixture comprises: i) a nucleic acid sample comprising at least one double-stranded DNA, the double-stranded DNA having a first strand and a second strand, the second strand being at least partially complementary to the first strand, and each of the first strand and the second strand having a 5' end and a 3' end; ii) a first reverse transcriptase; iii) a first template switch oligonucleotide that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; and iv) a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to the at least one nucleotide excluded from the first template switch oligonucleotide; Combining conducting a first template switching reaction in the first reaction mixture; adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA with the reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing the first template switch oligonucleotide to the non-templated 3′ overhang of the double-stranded DNA; and extending the non-templated 3' overhang of the double-stranded DNA with the reverse transcriptase, thereby forming a first product nucleic acid comprising the double-stranded DNA having at least one extended 3' end complementary to the first template switch oligonucleotide; A method comprising:

2. 2. The method of claim 1, wherein the nucleic acid sample further comprises at least one RNA, the RNA having a 5' end and a 3' end.

3. Into a second reaction mixture: i) the first nucleic acid product; ii) the RNA; iii) a second reverse transcriptase; iv) a second template switch oligonucleotide, and v) a second mixture of dNTPs; Combining conducting a second template switching reaction in the second reaction mixture; synthesizing a polynucleotide complementary to said RNA with said second reverse transcriptase, thereby forming a first primer extension product complementary to at least a portion of said RNA; adding at least one non-templated nucleotide to the 3' end of the first primer extension product with the second reverse transcriptase, thereby forming a non-templated 3' overhang on the first primer extension product; annealing the second template switch oligonucleotide to the non-templated 3' overhang of the first primer extension product; and extending the non-templated 3' overhang of the first primer extension product with the second reverse transcriptase, thereby forming a second nucleic acid product comprising the first primer extension product having an extended 3' end complementary to the second template switch oligonucleotide; The method of claim 2 further comprising:

4. further comprising combining in said second reaction mixture a first oligonucleotide primer having a 3' end complementary to said RNA; and performing the second template switching reaction in the second reaction mixture; annealing the 3' end of the first oligonucleotide primer to the RNA; and 4. The method of claim 3, further comprising extending the first oligonucleotide primer with the second reverse transcriptase, thereby forming the first primer extension product.

5. 4. The method of Claim 3, wherein the nucleotide sequence of the first template switch oligonucleotide differs from the nucleotide sequence of the second template switch oligonucleotide by at least one nucleotide.

6. 3. The method of Claim 2, wherein the first template switching reaction is unable to form a by-product comprising a complement of at least a portion of the RNA having a 3' end complementary to the first template switch oligonucleotide.

7. 10. The method of claim 1, further comprising terminating at least one of the 3' ends of the first nucleic acid product.

8. 8. The method of claim 7, wherein the terminating step comprises incorporating a dideoxynucleotide at the at least one 3' end of the first nucleic acid product.

9. 2. The method of claim 1, wherein the first template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence.

10. 4. The method of claim 3, wherein the second template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence.

11. 4. The method of claim 3, further comprising purifying the nucleic acid sample containing the first nucleic acid product and the RNA prior to combining into the second reaction mixture.

12. The first reaction mixture comprises: i) a nucleic acid sample comprising at least one double-stranded DNA, the double-stranded DNA having a first strand and a second strand, the second strand being at least partially complementary to the first strand, and each of the first strand and the second strand having a 5' end and a 3' end; ii) reverse transcriptase; iii) a first template switch oligonucleotide having a 5' domain and a 3' domain, wherein the 3' domain of the first template switch oligonucleotide excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; iv) a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to the at least one nucleotide excluded from the 3′ domain of the first template switch oligonucleotide; and v) combining a ddNTP complementary to the at least one nucleotide excluded from the 3′ domain of the first template switch oligonucleotide; and conducting a first template switching reaction in the first reaction mixture; adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA with the reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing the first template switch oligonucleotide to the non-templated 3′ overhang of the double-stranded DNA; and extending the non-templated 3' overhang of the double-stranded DNA with the reverse transcriptase, thereby forming a first product nucleic acid comprising the double-stranded DNA having at least one extended 3' end that is complementary to the 3' domain of the first template switch oligonucleotide; A method comprising:

13. 13. The method of claim 12, wherein the nucleic acid sample further comprises at least one RNA, the RNA having a 5' end and a 3' end.

14. Into a second reaction mixture: i) the first nucleic acid product; ii) the RNA; iii) a second reverse transcriptase; iv) a second template switch oligonucleotide, and v) a second mixture of dNTPs; and conducting a second template switching reaction in the second reaction mixture; synthesizing a polynucleotide complementary to said RNA with said second reverse transcriptase, thereby forming a first primer extension product complementary to at least a portion of said RNA; adding at least one non-templated nucleotide to the 3' end of the first primer extension product with the second reverse transcriptase, thereby forming a non-templated 3' overhang on the first primer extension product; annealing the second template switch oligonucleotide to the non-templated 3' overhang of the first primer extension product; and extending the non-templated 3' overhang with the second reverse transcriptase, thereby forming a second product nucleic acid comprising the first primer extension product having an extended 3' end complementary to the second template switch oligonucleotide; 14. The method of claim 13, further comprising:

15. further comprising combining in said second reaction mixture a first oligonucleotide primer having a 3' end complementary to said RNA; and performing the second template switching reaction in the second reaction mixture; annealing the 3' end of the first oligonucleotide primer to the RNA; and 15. The method of claim 14, further comprising extending the first oligonucleotide primer with the second reverse transcriptase, thereby forming the first primer extension product.

16. 15. The method of Claim 14, wherein the nucleotide sequence of the first template switch oligonucleotide differs from the nucleotide sequence of the second template switch oligonucleotide by at least one nucleotide.

17. 14. The method of Claim 13, wherein the first template switching reaction is unable to form a by-product comprising a complement of at least a portion of the RNA having a 3' end complementary to the first template switch oligonucleotide.

18. 13. The method of Claim 12, wherein the 5' domain of the first template switch oligonucleotide comprises the at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide, and the first nucleic acid product comprises at least one 3' end that terminates with the ddNTP.

19. 13. The method of Claim 12, wherein the 3' domain of the first template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence.

20. 15. The method of Claim 14, wherein the second template switch oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence.

21. 15. The method of claim 14, further comprising purifying the nucleic acid sample comprising the first nucleic acid product and the RNA prior to combining into the second reaction mixture.

22. the second nucleic acid product comprises a second target sequence; and The method comprises: i) the second nucleic acid product; and ii) a first primer having a 3′ end corresponding to at least the 5′ end of the second template switch oligonucleotide; and iii) a second primer having a 3' end corresponding to the second target sequence; 15. The method of claim 3 or 14, further comprising amplifying at least a portion of the second nucleic acid product in a second amplification reaction mixture comprising:

23. the first nucleic acid product comprises a first target sequence; and The method comprises: i) the first nucleic acid product; ii) a first primer having a 3′ end corresponding to at least the 5′ end of the first template switch oligonucleotide; and iii) a second primer having a 3' end corresponding to the first target sequence; 23. The method of any one of claims 1 to 22, further comprising amplifying at least a portion of the first nucleic acid product in a first amplification reaction mixture comprising:

24. 24. The method of any one of claims 1 to 23, wherein each of the 3' ends of the first nucleic acid products comprises an extended 3' end that is complementary to the first template switch oligonucleotide.

25. 25. The method of any one of claims 1 to 24, wherein the second template switching reaction is unable to form a by-product comprising the first nucleic acid product having a 3' end complementary to a second template switch oligonucleotide.

26. The method of any one of claims 1 to 25, wherein the nucleic acid sample comprises a plurality of DNAs.

27. The method of any one of claims 1 to 26, wherein the nucleic acid sample comprises a plurality of RNAs.

28. 28. The method of any one of claims 1 to 27, wherein the reverse transcriptase is selected from Moloney murine leukemia virus (MMLV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase, and mutants thereof.

29. The method of claim 4 or 15, wherein the 3' end of the first oligonucleotide primer is a polydT sequence.

30. 16. The method of claim 4 or 15, wherein the 3' end of the first oligonucleotide primer is a target-specific sequence.

31. The method of claim 4 or 15, wherein the 3' end of the first oligonucleotide primer is a random sequence.

32. 32. The method of any one of claims 1 to 31, wherein the first template switch oligonucleotide comprises a stuffer region.

33. 16. The method of Claim 4 or 15, wherein the second template switch oligonucleotide comprises a stuffer region.

34. 16. The method of claim 4 or 15, wherein the 3' end of the first oligonucleotide primer is complementary to an exon region of the RNA.

35. 8. The method of claim 7, wherein the terminating step comprises incorporating a dideoxynucleotide onto the at least one 3' end of the first nucleic acid product with a terminal transferase.

36. 36. The method of claim 35, wherein the terminal transferase is Taq DNA polymerase.

37. 37. The method of any one of claims 1 to 36, wherein performing the first template switching reaction in the first reaction mixture further comprises adding at least three non-templated nucleotides to at least one of the 3' ends of the double-stranded DNA with the reverse transcriptase.

38. 15. The method of claim 3 or 14, wherein performing the second template switching reaction in the second reaction mixture comprises adding at least three non-templated nucleotides to the 3′ end of the first primer extension product with the reverse transcriptase.

39. 14. The method of claim 2 or 13, further comprising recovering the nucleic acid sample comprising the first nucleic acid product and the at least one RNA as a second nucleic acid sample.

40. The first reaction mixture comprises: i) a nucleic acid sample, at least one double-stranded DNA, the double-stranded DNA having a first strand and a second strand, the second strand being at least partially complementary to the first strand, and the first strand and the second strand each having a 5' end and a 3' end; and the nucleic acid sample comprising at least one RNA, the RNA having a first strand with a 5' end and a 3' end; ii) reverse transcriptase; iii) a first template switch oligonucleotide that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; and iv) combining a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to the at least one nucleotide excluded from the first template switch oligonucleotide; and conducting a first template switching reaction in the first reaction mixture; adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA with the reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing the first template switch oligonucleotide to the non-templated 3′ overhang of the double-stranded DNA; and extending the non-templated 3' overhang of the double-stranded DNA with the reverse transcriptase, thereby forming a first product nucleic acid comprising the double-stranded DNA having at least one extended 3' end complementary to the first template switch oligonucleotide; A method comprising:

41. 1. A method for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA, comprising: performing a first template switching reaction on the nucleic acid sample in the absence of at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to a first template switch oligonucleotide; and performing a second template switching reaction on the nucleic acid sample, thereby forming a second nucleic acid product comprising a first primer extension product complementary to at least a portion of the RNA, wherein the first primer extension product has an extended 3' end complementary to a second template switch oligonucleotide; The method comprising:

42. 1. A kit for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA, comprising: a first template switch oligonucleotide that excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; and a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to the at least one nucleotide excluded from the first template switch oligonucleotide.

43. a second template switch oligonucleotide, and a second mixture of dNTPs, 43. The kit of claim 42, further comprising:

44. 1. A kit for performing a template switching reaction on a nucleic acid sample comprising at least one double-stranded DNA and at least one RNA, comprising: a first template switch oligonucleotide having a 5' domain and a 3' domain, wherein the 3' domain of the first template switch oligonucleotide excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine; a first mixture of dNTPs excluding at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the mixture of dNTPs is complementary to the at least one nucleotide excluded from the 3′ domain of the first template switch oligonucleotide; and a ddNTP complementary to the at least one nucleotide excluded from the 3′ domain of the first template switch oligonucleotide; The kit comprises:

45. a second template switch oligonucleotide; and a second mixture of dNTPs; 45. The kit of claim 44, further comprising:

46. 15. The method of Claim 3 or 14, wherein at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises a ribonucleotide.

47. 47. The method of Claim 46, further comprising contacting at least one of the first template switch oligonucleotide and the second template switch oligonucleotide with a ribonuclease.

48. 15. The method of Claim 3 or 14, wherein at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises a 5' modification selected from a nucleotide analog, a linkage modification, a terminal modification, and a fluorescent label.

49. 4. The method of Claim 1 or 3, wherein the 3′ end of at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises a homopolymer sequence of at least three nucleotides.

50. 50. The method of claim 49, wherein the homopolymer sequence is selected from polyriboguanosine, polyguanosine, polyribocytidine, and polycytidine.

51. 13. The method of claim 12, wherein at least one of the first template switch oligonucleotide and the second template switch oligonucleotide comprises at least one 2'-O-methyl nucleoside modification.

52. 13. The method of Claim 1 or 12, wherein the first template switch oligonucleotide further excludes uracil.

53. 13. The method of claim 1 or 12, wherein the first mixture of dNTPs further excludes dUTP.

54. 13. The method of Claim 12, wherein the 5' domain of the first template switch oligonucleotide comprises the at least one nucleotide that is excluded from the 3' domain of the first template switch oligonucleotide.

55. 55. The method of claim 12 or 54, wherein the ddNTP further comprises a capture moiety.

56. 56. The method of claim 55, wherein the capture moiety is selected from biotin and desthiobiotin.

57. 13. The method of claim 1 or 12, wherein the first mixture of dNTPs comprises at least one dNTP having a capture moiety.

58. 58. The method of claim 57, wherein the capture moiety is selected from biotin and desthiobiotin.

59. 15. The method of claim 3 or 14, wherein the second mixture of dNTPs comprises at least one capture moiety.

60. 60. The method of claim 59, wherein the capture moiety is selected from biotin and desthiobiotin.

61. 16. The method of claim 4 or 15, wherein the first oligonucleotide primer comprises at least one capture moiety.

62. 62. The method of claim 61 , wherein the capture moiety is selected from biotin and desthiobiotin.

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