Systems and methods for total nucleic acid library preparation via template conversion

Abstract

The present disclosure provides a method for performing a template conversion reaction on a nucleic acid sample comprising at least one double stranded DNA and at least one RNA. The method comprises subjecting the nucleic acid sample to a first template conversion reaction in the absence of at least one dNTP selected from the group consisting of 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 conversion oligonucleotide. The method further includes performing a second template conversion reaction on the nucleic acid sample to form 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 the second template conversion oligonucleotide.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 386,725, filed on December 9, 2022, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Funded Research

[0004] not applicable. Background Art

[0005] The present disclosure relates generally to library preparation for next-generation sequencing of nucleic acids, and more particularly to systems and methods for total nucleic acid library preparation and targeted sequencing via template switching.

[0006] In order to analyze nucleic acid samples using existing sequencing technology, it is usually necessary to first prepare and optionally enrich the nucleic acid in the sample using one or more library preparation schemes, target enrichment schemes or its combination. The library preparation scheme is generally used to make nucleic acid samples compatible with given sequencing technology, for example, by adding a common nucleic acid adapter sequence to the terminal nucleic acid fragments derived from the sample. By contrast, the target enrichment scheme is generally used for selectively isolating specific genomic regions of interest before sequencing. This type of enrichment method is suitable for experiments in which it may be expected that research is less than all nucleic acid sequences derived from biological sources, but more than only a few (for example, more than 1000) nucleic acid sequences.

[0007] In one aspect, it may be advantageous to generate both RNA and DNA sequencing libraries from a sample; however, existing library preparation and target enrichment protocols are generally not widely applicable to different types of nucleic acids. For example, a particular protocol may be suitable for preparing a library starting with either DNA or RNA, but not both. Furthermore, in cases where it is desired to prepare a nucleic acid library from both DNA and RNA derived from the same sample, an additional step may be required to first separate the DNA from the RNA for separate processing.

[0008] Previous methods have provided limited solutions for the integration of DNA and RNA library generation. For example, U.S. Patent Application No. 2018 / 0080021 by Reuter et al. describes a method for simultaneous sequencing of RNA and DNA from the same sample. The method taught by Reuter et al. is based on the use of i) Tn5 transposase to add adapters to whole-genomic DNA and ii) RNA ligase to generate transcriptome libraries in the same reaction. Although this protocol is effective in preparing whole-genomic and transcriptome libraries in a single test tube, it is characterized by a high degree of complexity.

[0009] Therefore, new protocols that integrate DNA and RNA library preparation and target enrichment are needed. Summary of the Invention

[0010] The present invention overcomes the above-mentioned shortcomings by providing systems and methods for preparing total nucleic acid libraries via template switching, as described by the following enumerated list:

[0011] 1. A method comprising:

[0012] The following are combined into a first reaction mixture:

[0013] i) a nucleic acid sample comprising at least one 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,

[0014] ii) a first reverse transcriptase,

[0015] iii) a first template-switching oligonucleotide that excludes at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine, and

[0016] iv) a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the first template-switching oligonucleotide; and

[0017] Performing a first template switching reaction using the first reaction mixture comprises:

[0018] adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA using the reverse transcriptase, thereby forming a non-templated 3' overhang on the double-stranded DNA; annealing the first template-switching oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; and

[0019] The non-templated 3' overhang of the double-stranded DNA is extended with the reverse transcriptase, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to the first template-switching oligonucleotide.

[0020] 2. The method according to item 1, wherein the nucleic acid sample further comprises at least one RNA having a 5' end and a 3' end.

[0021] 3. The method according to item 2, further comprising:

[0022] The following were combined into a second reaction mixture:

[0023] i) the first nucleic acid product,

[0024] ii) said RNA,

[0025] iii) a second reverse transcriptase,

[0026] iv) a second template-switching oligonucleotide, and

[0027] v) a second dNTP mix; and

[0028] Performing a second template switching reaction using the second reaction mixture comprises:

[0029] synthesizing a polynucleotide complementary to the RNA using the second reverse transcriptase, thereby forming a first primer extension product complementary to at least a portion of the RNA;

[0030] adding at least one non-templated nucleotide to the 3' end of the first primer extension product using the second reverse transcriptase, thereby forming a non-templated 3' overhang on the first primer extension product;

[0031] annealing the second template switch oligonucleotide to the non-templated 3' overhang of the first primer extension product; and

[0032] The non-templated 3' overhang of the first primer extension product is extended 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-switching oligonucleotide.

[0033] 4. The method according to item 3, further comprising:

[0034] combining a first oligonucleotide primer having a 3' end complementary to the RNA into the second reaction mixture; and

[0035] Wherein performing the second template switching reaction with the second reaction mixture further comprises:

[0036] annealing the 3' end of the first oligonucleotide primer to the RNA; and

[0037] The first oligonucleotide primer is extended using the second reverse transcriptase to form the first primer extension product.

[0038] 5. The method according to item 3, wherein the nucleotide sequence of the first template-switching oligonucleotide differs from the nucleotide sequence of the second template-switching oligonucleotide by at least one nucleotide.

[0039] 6. The method according to item 2, wherein the first template switching reaction cannot form a by-product, wherein the by-product comprises a complement of at least a portion of the RNA having a 3' end complementary to the first template switching oligonucleotide.

[0040] 7. The method according to item 1, further comprising terminating at least one of the 3' ends of the first nucleic acid product.

[0041] 8. The method according to item 7, wherein the terminating step comprises incorporating a dideoxynucleotide at at least one 3' end of the first nucleic acid product.

[0042] 9. The method of item 1, wherein the first template-switching oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence.

[0043] 10. The method of item 3, wherein the second template switching oligonucleotide comprises at least one of a universal adapter sequence, a sample identification sequence, and a molecular identifier sequence.

[0044] 11. The method according to item 3, wherein the method further comprises purifying the nucleic acid sample comprising the first nucleic acid product and the RNA before the step of combining into the second reaction mixture.

[0045] 12. A method comprising:

[0046] The following are combined into a first reaction mixture:

[0047] i) a nucleic acid sample comprising at least one 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,

[0048] ii) reverse transcriptase,

[0049] iii) a first template-switching oligonucleotide having a 5' domain and a 3' domain, said 3' domain of said first template-switching oligonucleotide excluding at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine and thymine,

[0050] iv) a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, said at least one dNTP excluded from said dNTP mixture being complementary to said at least one nucleotide excluded from said 3' domain of said first template switch oligonucleotide; and

[0051] v) a ddNTP that is complementary to said at least one nucleotide excluded from said 3' domain of said first template switch oligonucleotide; and

[0052] Performing a first template switching reaction using the first reaction mixture comprises:

[0053] 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;

[0054] annealing the first template-switching oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; and

[0055] The non-templated 3' overhang of the double-stranded DNA is extended with the reverse transcriptase, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to the 3' domain of the first template-switching oligonucleotide.

[0056] 13. The method according to item 12, wherein the nucleic acid sample further comprises at least one RNA having a 5' end and a 3' end.

[0057] 14. The method according to item 13, further comprising:

[0058] The following were combined into a second reaction mixture:

[0059] i) the first nucleic acid product,

[0060] ii) said RNA,

[0061] iii) a second reverse transcriptase,

[0062] iv) a second template-switching oligonucleotide, and

[0063] v) a second dNTP mix; and

[0064] Performing a second template switching reaction using the second reaction mixture comprises:

[0065] synthesizing a polynucleotide complementary to the RNA using the second reverse transcriptase, thereby forming a first primer extension product complementary to at least a portion of the RNA;

[0066] adding at least one non-templated nucleotide to the 3' end of the first primer extension product using the second reverse transcriptase, thereby forming a non-templated 3' overhang on the first primer extension product;

[0067] annealing the second template switch oligonucleotide to the non-templated 3' overhang of the first primer extension product; and

[0068] The non-templated 3' overhang is extended with the 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 the second template-switching oligonucleotide.

[0069] 15. The method according to item 14, further comprising:

[0070] combining a first oligonucleotide primer having a 3' end complementary to the RNA into the second reaction mixture; and

[0071] Wherein performing the second template switching reaction with the second reaction mixture further comprises:

[0072] annealing the 3' end of the first oligonucleotide primer to the RNA; and

[0073] The first oligonucleotide primer is extended using the second reverse transcriptase to form the first primer extension product.

[0074] 16. The method according to item 14, wherein the nucleotide sequence of the first template-switching oligonucleotide differs from the nucleotide sequence of the second template-switching oligonucleotide by at least one nucleotide.

[0075] 17. The method according to item 13, wherein the first template switching reaction cannot form a by-product, the by-product comprising a complement of at least a portion of the RNA having a 3' end complementary to the first template switching oligonucleotide.

[0076] 18. The method of item 12, wherein the 5' domain of the first template-switching oligonucleotide comprises the at least one nucleotide that is excluded from the 3' domain of the first template-switching oligonucleotide, and wherein the first nucleic acid product comprises at least one 3' end that terminates in the ddNTP.

[0077] 19. The method of item 12, wherein the 3' domain of the first template switching oligonucleotide comprises at least one of a universal adaptor sequence, a sample identification sequence, and a molecule identifier sequence.

[0078] 20. The method of item 14, wherein the second template-switching oligonucleotide comprises at least one of a universal adaptor sequence, a sample identification sequence, and a molecular identifier sequence.

[0079] 21. The method according to item 14, wherein the method further comprises purifying the nucleic acid sample comprising the first nucleic acid product and the RNA before the step of combining into the second reaction mixture.

[0080] 22. The method according to item 3 or 14, wherein the second nucleic acid product comprises a second target sequence, and wherein the method further comprises:

[0081] amplifying at least a portion of the second nucleic acid product in a second amplification reaction mixture comprising:

[0082] i) the second nucleic acid product,

[0083] ii) a first primer having at least a 5' end corresponding to the second template switch oligonucleotide

[0084] 3' end, and

[0085] iii) a second primer having a 3' end corresponding to the second target sequence.

[0086] 23. The method according to any one of the preceding items, wherein the first nucleic acid product comprises a first target sequence, and wherein the method further comprises:

[0087] amplifying at least a portion of the first nucleic acid product in a first amplification reaction mixture comprising:

[0088] i) the first nucleic acid product,

[0089] ii) a first primer having at least a 5' end corresponding to the first template switch oligonucleotide

[0090] 3' end, and

[0091] iii) a second primer having a 3' end corresponding to the first target sequence.

[0092] 24. The method according to any one of the preceding items, wherein each of the 3' ends of the first nucleic acid product comprises an extended 3' end that is complementary to the first template switch oligonucleotide.

[0093] 25. The method according to any one of the preceding items, wherein the second template switching reaction fails to form byproducts comprising the first nucleic acid product having a 3' end complementary to the second template switching oligonucleotide.

[0094] 26. The method according to any one of the preceding items, wherein the nucleic acid sample comprises a plurality of DNAs.

[0095] 27. The method according to any of the preceding items, wherein the nucleic acid sample comprises a plurality of RNAs.

[0096] 28. The method according to any one of the preceding items, wherein the reverse transcriptase is selected from Moloney murine leukemia virus (MMLV) reverse transcriptase, avian myeloblastosis virus (AMV) reverse transcriptase and mutants thereof.

[0097] 29. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is a poly-dT sequence.

[0098] 30. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is a target-specific sequence.

[0099] 31. The method according to item 4 or 15, wherein the 3' end of the first oligonucleotide primer is a random sequence.

[0100] 32. The method according to any of the preceding items, wherein the first template-switching oligonucleotide comprises a stuffing region.

[0101] 33. The method of item 4 or 15, wherein the second template-switching oligonucleotide comprises a stuffing region.

[0102] 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.

[0103] 35. The method according to item 7, wherein the terminating step comprises incorporating a dideoxynucleotide at at least one 3' end of the first nucleic acid product using terminal transferase.

[0104] 36. The method according to item 35, wherein the terminal transferase is Taq DNA polymerase.

[0105] 37. The method according to any one of the preceding items, wherein performing the first template switching reaction with 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.

[0106] 38. The method according to item 3 or 14, wherein performing the second template switching reaction with 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.

[0107] 39. The method according to item 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.

[0108] 40. A method comprising:

[0109] The following are combined into a first reaction mixture:

[0110] i) a nucleic acid sample comprising at least one 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 having a first strand, the first strand having a 5' end and a 3' end,

[0111] ii) reverse transcriptase,

[0112] iii) a first template-switching oligonucleotide that excludes at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine, and

[0113] iv) a first dNTP mixture excluding dATP, dCTP, dGTP, and

[0114] at least one dNTP of a dTTP, said at least one dNTP excluded from said dNTP mixture being complementary to said at least one nucleotide excluded from said first template-switching oligonucleotide; and

[0115] Performing a first template switching reaction using the first reaction mixture comprises:

[0116] 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;

[0117] annealing the first template-switching oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; and

[0118] The non-templated 3' overhang of the double-stranded DNA is extended with the reverse transcriptase, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to the first template-switching oligonucleotide.

[0119] 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, the method comprising:

[0120] performing a first template switching reaction on the nucleic acid sample in the absence of at least one dNTP selected from the group consisting of 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 the first template switching oligonucleotide; and

[0121] The nucleic acid sample is subjected to a second template switching reaction, 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 the second template switching oligonucleotide.

[0122] 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, the kit comprising:

[0123] a first template-switching oligonucleotide that excludes at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine; and

[0124] A first dNTP mixture excludes at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the first template-switching oligonucleotide.

[0125] 43. The kit according to item 42, further comprising:

[0126] a second template-switching oligonucleotide; and

[0127] Second dNTP mix.

[0128] 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, the kit comprising:

[0129] a first template-switching oligonucleotide having a 5' domain and a 3' domain, the 3' domain of the first template-switching oligonucleotide excluding at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine;

[0130] a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide; and

[0131] a ddNTP that is complementary to the at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide.

[0132] 45. The kit according to item 44, further comprising:

[0133] a second template-switching oligonucleotide; and

[0134] Second dNTP mix.

[0135] 46. The method of item 3 or 14, wherein at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide comprises a ribonucleotide.

[0136] 47. The method of item 46, further comprising contacting at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide with a ribonuclease.

[0137] 48. The method according to item 3 or 14, wherein at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide comprises a 5' modification selected from a nucleotide analogue, a linkage modification, a terminal modification, and a fluorescent label.

[0138] 49. The method according to item 1 or 3, wherein the 3' end of at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide comprises a homopolymer sequence of at least three nucleotides.

[0139] 50. The method according to item 49, wherein the homopolymer sequence is selected from polynucleoguanosine, polyguanosine, polyribocytidine and polycytidine.

[0140] 51. The method of item 12, wherein at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide comprises at least one 2'-O-methyl nucleoside modification.

[0141] 52. The method according to item 1 or 12, wherein the first template-switching oligonucleotide further excludes uracil.

[0142] 53. The method of item 1 or 12, wherein the first dNTP mixture further excludes dUTP.

[0143] 54. The method according to item 12, wherein the 5' domain of the first template-switching oligonucleotide comprises the at least one nucleotide that is excluded from the 3' domain of the first template-switching oligonucleotide.

[0144] 55. A method according to item 12 or 54, wherein the ddNTP further comprises a capture moiety.

[0145] 56. A method according to item 55, wherein the capture moiety is selected from biotin and desthiobiotin.

[0146] 57. The method of item 1 or 12, wherein the first dNTP mixture comprises at least one dNTP having a capture moiety.

[0147] 58. A method according to item 57, wherein the capture moiety is selected from biotin and desthiobiotin.

[0148] 59. The method of item 3 or 14, wherein the second dNTP mixture comprises at least one capture moiety.

[0149] 60. The method of item 59, wherein the capture moiety is selected from biotin and desthiobiotin.

[0150] 61. The method according to item 4 or 15, wherein the first oligonucleotide primer comprises at least one capture portion.

[0151] 62. A method according to item 61, wherein the capture moiety is selected from biotin and desthiobiotin.

[0152] The foregoing and other aspects and advantages of the present invention will become apparent from the following description. In the specification, reference is made to the accompanying drawings which form a part hereof and in which there is shown by way of illustration a preferred embodiment of the invention. Such embodiments do not necessarily represent the full scope of the invention, however, and therefore, reference is made to the claims for interpreting the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 is a schematic diagram of a method for preparing a total nucleic acid sample according to the present disclosure.

[0154] Figure 2A is a schematic diagram depicting the components of a total nucleic acid sample according to the present disclosure.

[0155] Figure 2B is depicted after the addition of a non-templated 3' overhang to the 3' end of the double-stranded DNA Figure 2A Schematic diagram of the components of a total nucleic acid sample.

[0156] Figure 2C is depicted after annealing the first template-switching oligonucleotide to the non-templated 3' overhang Figure 2B Schematic diagram of the components of a total nucleic acid sample.

[0157] Figure 2Dis depicted after the non-templated 3' overhang of the double-stranded DNA is extended, thereby forming a first nucleic acid product comprising a double-stranded DNA having at least one extended 3' end complementary to the first template-switching oligonucleotide Figure 2C Schematic diagram of the components of a total nucleic acid sample.

[0158] Figure 3A is depicted with a second template switching oligonucleotide and primer combination Figure 2D Schematic diagram of the first nucleic acid product and RNA.

[0159] Figure 3B is depicted 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 Figure 3A Schematic diagram of the components.

[0160] Figure 3C is depicted after annealing the second template-switching oligonucleotide to the non-templated 3' overhang Figure 3B Schematic diagram of the components.

[0161] Figure 3D is depicted after the non-templated 3' overhang of the first primer extension product is extended, thereby forming a second nucleic acid product comprising the first primer extension product having an extended 3' end complementary to the second template-switching oligonucleotide. Figure 2C Schematic diagram of the components.

[0162] Figure 4A is depicted in combination with the primer pairs used for amplification Figure 3D Schematic diagram of the components.

[0163] Figure 4B It is a depiction Figure 4A Schematic representation of the products of the amplification reactions shown in .

[0164] In the detailed description that follows, like numbers will be used to describe like components from figure to figure. DETAILED DESCRIPTION

[0165] I. Definition

[0166] In this application, unless the context clearly indicates otherwise, (i) the term "a" or "an" will be understood to mean "at least one"; (ii) the term "or" will be understood to mean "and / or"; (iii) the terms "comprising" and "including" will be understood to cover the itemized components or steps, whether presented alone or with one or more other components or steps; (iv) the terms "about" and "approximately" will be understood to allow for standard variations, as will be understood by one of ordinary skill in the art; and (v) where ranges are provided, the endpoints are inclusive.

[0167] Approximately: As used herein, the term "approximately" or "about" as applied to one or more target values refers to a value that is similar to the reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that is 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 than or less than) of the reference value, unless otherwise specified or obvious from the context (unless the number exceeds 100% of the possible value).

[0168] Correlation: Two events or entities are "associated" with each other, as the term is used herein, if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, a particular entity (e.g., a polypeptide, genetic marker, metabolite, etc.) is considered to be associated with a particular disease, disorder, or condition (e.g., in a relevant population) if the presence, level, and / or form of the particular entity is associated with the incidence and / or susceptibility of the disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with each other if they interact, directly or indirectly, such that they are in physical proximity to each other and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically bound to each other are covalently attached to each other; in some embodiments, two or more entities that are physically bound to each other are not covalently attached to each other, but are non-covalently attached, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0169] Biological sample: As used herein, the term "biological sample" generally refers to a sample obtained or derived from a target biological source (e.g., a tissue or organism or cell culture), as described herein. In some embodiments, the target source includes an organism, such as an animal or a human, or consists thereof. In some embodiments, the biological sample includes or consists of a biological tissue or fluid. In some embodiments, the biological sample can be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; body fluids containing cells; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; stool; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; wash or lavage fluid, such as duct lavage fluid or bronchoalveolar lavage fluid; aspirate; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; other body fluids, secretions and / or excretions; and / or cells therefrom, etc. In some embodiments, the biological sample comprises or consists of cells obtained from an individual. In certain embodiments, the cell obtained is or includes cells from the individual from which the sample is obtained. In certain embodiments, sample is " the first sample " obtained directly from the target source by any appropriate means. For example, in some embodiments, the original biological sample is obtained by the method selected from the group consisting of the following items: biopsy (for example, fine needle aspiration or tissue biopsy), surgery, body fluid collection (for example, blood, lymph, feces, etc.). In certain embodiments, as can be clearly seen from the context, the term " sample " refers to the preparation obtained by processing the original sample (for example, by removing one or more components and / or by adding one or more medicaments thereto). For example, using a semipermeable membrane filter. Such " processed sample " can include, for example, nucleic acid or protein extracted from the sample, or nucleic acid or protein obtained by subjecting the first sample to amplification or reverse transcription, separation and / or purification of certain components such as mRNA.

[0170] 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 other elements or steps may be added within the scope of the composition or method. It should be understood that a composition or method described as “comprising” or “comprises” one or more named elements or steps also describes a corresponding, more limited composition or method “consisting essentially of” or “consists essentially of” the same named elements or steps, meaning that the composition or method includes the named basic elements or steps and may also include additional elements or steps that do not substantially affect the basic and novel characteristics of the composition or method. It should also be understood that any composition or method described herein as “comprising” or “consisting essentially of” one or more named elements or steps also describes a corresponding, more limited, closed composition or method “consisting of” or “consists of” the named elements or steps to exclude any other unnamed elements or steps. In any composition or method disclosed herein, a known or published equivalent of any named essential element or step may be substituted for that element or step.

[0171] Designed: As used herein, the term "designed" refers to an agent that: (i) has a structure that has been selected by the hand of man; (ii) has been produced by methods requiring human intervention; and / or (iii) is distinct from naturally occurring substances and other known agents.

[0172] Determining: As will be understood by those of ordinary skill in the art reading this specification, "determining" can be accomplished using or by using any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and / or manipulation of data or information, such as using a computer or other processing unit suitable for performing the relevant analysis. In some embodiments, determining involves receiving relevant information and / or material from a source. In some embodiments, determining involves comparing one or more characteristics of a sample or entity to a comparable reference.

[0173] Identity: As used herein, the term "identity" refers to the overall correlation between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, if the sequence of the polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, then these polymer molecules are considered to be "substantially identical" to each other. For example, for optimal comparison purposes, the percent identity calculation of two nucleic acid or polypeptide sequences can be performed by comparing the two sequences (for example, gaps can be introduced in one or both of the first and second sequences to achieve optimal comparison, and for comparison purposes, non-identical sequences can be ignored). In certain embodiments, the length of the sequence compared 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 of the corresponding positions are then compared. When the position in the first sequence is occupied by the residue (for example, nucleotide or amino acid) identical to the corresponding position in the second sequence, the molecule is identical at that position. Considering the number of each room and the length of the room, the identity percentage between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to realize the optimal comparison of the two sequences. The determination of the identity percentage between the comparison of the sequence and the two sequences can be completed using a mathematical algorithm. For example, the algorithm (CABIOS, 1989, 4:11-17) of Meyers and Miller can be used to determine the identity percentage between the two nucleotide sequences, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the NWSgapdna.CMP matrix can be used to determine the identity percentage between the two nucleotide sequences using the GAP program in the GCG software package.

[0174] Sample: As used herein, the term "sample" refers to a substance that is a target composition for qualitative and / or quantitative assessment or a substance containing the composition. 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 from a geological, aquatic, astronomical, or agricultural source. In some embodiments, the target source includes or consists of an organism, such as an animal or a human. In some embodiments, the sample used for forensic analysis is or includes biological tissue, biological fluid, organic or inorganic matter, such as, for example, clothing, dirt, plastic, water. In some embodiments, agricultural samples include or consist of organic matter, such as leaves, petals, bark, wood, seeds, plants, fruit, etc.

[0175] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. Those of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0176] Synthetic: As used herein, the term "synthetic" means produced by the hand of man, and therefore produced in a form not found in nature, either because it has a structure not found in nature, or because it is combined with one or more other components with which it is not combined in nature, or is not combined with one or more other components with which it is combined in nature.

[0177] Variant: As used herein, the term "variant" refers to an entity that displays significant structural identity to a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared to the reference entity. In many embodiments, a variant is also functionally different from its reference entity. Generally speaking, whether a particular entity is properly considered a "variant" of a reference entity is based on the degree of its structural identity to the reference entity. As understood by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a variant is a unique chemical entity that shares one or more such characteristic structural elements. To give just a few examples, a small molecule can have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic side group moieties such that variations in the small molecule are variations that share the core structural element and the characteristic side group moieties, but differ in other side group moieties and / or in the type of bond present in the core (single vs. double, E vs. Z, etc.), a polypeptide can have a characteristic sequence element consisting of a plurality of amino acids that have a specified position relative to each other in linear or three-dimensional space and / or contribute to a specific biological function, and a nucleic acid can have a characteristic sequence element consisting of a plurality of nucleotide residues that have a specified position relative to another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide due to one or more differences in the amino acid sequence and / or one or more differences in the chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, the variant polypeptide exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% overall sequence identity to the reference polypeptide. Alternatively or additionally, 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 has one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, the variant polypeptide shows one or more biological activity levels reduced compared to the reference polypeptide. In many embodiments, if the amino acid sequence of the target polypeptide is identical to the amino acid sequence of the parent except for a small amount of sequence changes at a specific position, the target polypeptide is considered to be a "variation" of the parent or reference polypeptide. Typically, compared to the parent, less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variation are substituted. In some embodiments, compared to the parent, the variation has 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substituted residues. Typically, the variation has a very small amount (e.g., less than 5, 4, 3, 2 or 1) of substituted functional residues (i.e., residues involved in a specific biological activity).In addition, a variant typically has no more than 5, 4, 3, 2, or 1 additions or deletions, and typically has no additions or deletions, compared to the parent. Furthermore, any additions or deletions are typically less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6 residues, and typically less than about 5, about 4, about 3, or about 2 residues. In some embodiments, a variant may also have one or more functional defects and / or may be otherwise considered a "mutant." In some embodiments, the parent or reference polypeptide is a polypeptide found in nature. As will be appreciated by one of ordinary skill in the art, multiple variations of a particular target polypeptide are commonly found in nature, particularly when the target polypeptide is an infectious agent polypeptide.

[0178] II. Detailed Description of Certain Embodiments

[0179] As also described above, in various situations, it may be useful to provide integrated DNA and RNA library preparation and targeted enrichment protocols. In one aspect, the simultaneous generation of RNA and DNA sequencing libraries enables the collection of both DNA and RNA sequencing data from the same sample using next-generation sequencing (NGS). RNA sequencing data provides validation of DNA variant calls and can help identify 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 of DNA and RNA from a single sample is typically achieved by purifying total nucleic acid (TNA), which is then divided into two different samples and treated with DNase I to recover RNA or with RNase A to recover DNA. This approach results in the loss of half of the RNA and half of the DNA. In addition, the addition of DNase I or RNase A can lead to degradation of the desired TNA fraction, which is problematic when only a small amount of sample is available for operation. Other existing commercial products allow the sequential separation of RNA and DNA fractions in a single protocol; however, once the DNA and RNA are separated, the samples are processed separately, requiring additional time and effort.

[0180] Therefore, there is a need for a system and method for generating RNA and DNA sequencing libraries from a single sample derived from TNA. In this approach, the DNA and RNA fractions are never physically separated, but rather prepared for sequencing in a single tube. Furthermore, there is a need for a solution that: i) is compatible with automated platforms (e.g., liquid handling robots), ii) accommodates high- or low-quality TNA samples, and iii) is capable of distinguishing reads derived from DNA and RNA after sequencing.

[0181] The present disclosure provides methods and kits for efficiently adding unique adapters to RNA, DNA, or both, wherein the DNA and RNA are present in a single sample and are never separated. The disclosed methods further allow for selection and enrichment of DNA, RNA, or both, for example, by using amplification and sequencing. In addition, sequencing reads derived from DNA or RNA chains are readily differentiated with high confidence using the disclosed systems and methods. It is expected that the methods disclosed herein provide a simpler workflow with fewer steps compared to existing workflows. Finally, it is expected that the disclosed methods are compatible with a variety of nucleic acid sample types, including both fragmented and high-quality TNAs.

[0182] In one aspect, the present disclosure provides a method for preparing an integrated TNA library based on the terminal transferase activity and template switching ability of a reverse transcriptase (RT) enzyme (such as MMLV RT). Using an RT enzyme is an effective way to add a known sequence or adapter to the end of a complete cDNA sequence. The mechanism involves the ability of RT to add non-templated nucleotides to the 3' end of a complementary DNA (cDNA) chain. Once the end of the template (usually the 5' end of an RNA molecule) is reached, the terminal transferase activity of the RT enzyme catalyzes the addition of non-templated nucleotides to the 3' end of the growing cDNA chain. The resulting 3' overhang facilitates annealing of a complementary 3' oligonucleotide (referred to herein as a template switching oligonucleotide (TSO)). The 3' non-templated overhang is typically a polycytosine (e.g., CCC), wherein the complementary TSO includes a 3' polynuclear guanosine (e.g., rGrGrG, where 'r' represents a ribonucleotide base); however, it should be understood that the terminal transferase can generate alternative 3' overhangs depending on the composition of the dNTP pool and the specificity of the enzyme. As TSO anneals to the non-template overhang, the RT enzyme then switches templates, transferring from the original reverse transcribed RNA template to the new TSO template. The end result is a 3' new sequence ligated to a 3' cDNA, which is the reverse complement of TSO. Exemplary template switching applications are described, for example, in U.S. Pat. No. 5,962,271 to Chenchik et al., the entire contents of which are incorporated herein by reference.

[0183] The template switching mechanism of RT is known to be compatible with both DNA and RNA templates; however, no existing integrated library preparation protocol utilizes the template switching mechanism of RT to prepare both DNA and RNA present in the same sample. One challenge in implementing the template switching mechanism of RT for integrated library preparation involves 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 a system and method for preparing TNA libraries via template switching.

[0184] In general, the present disclosure is based on the surprising discovery that a library preparation protocol involving a reverse transcriptase having template switching activity can be applied to prepare TNA samples (i.e., samples comprising both DNA and RNA). Advantageously, the library preparation protocol is a one-pot method capable of selectively and distinctly labeling both DNA and RNA present in the same sample. Selective and distinguishable labeling is achieved by sequentially performing two different template switching reactions on the TNA sample. Even if both dsDNA and RNA are present in the same reaction, each template switching reaction is selective for the preparation of dsDNA or RNA. The resulting products of the protocol include a DNA-derived nucleic acid product having a first adapter sequence and an RNA-derived nucleic acid product having a second adapter sequence different from the first adapter sequence. Therefore, after sequencing the nucleic acid products, the resulting reads are easily associated with the original RNA template or the original DNA template present in the TNA sample.

[0185] Now turn Figure 1 Embodiments of method 10 according to the present disclosure include step 12 of preparing a TNA sample. In one aspect, the TNA sample includes all nucleic acids extracted and isolated from the biological sample. The TNA sample may include genomic DNA, messenger RNA (mRNA), ribosomal RNA (rRNA), etc. In one aspect, step 12 may include preparing fragmented blunt-end DNA. As one of ordinary skill in the art will appreciate, blunt-end dsDNA can be prepared in a variety of ways. DNA, including genomic DNA, can be fragmented using any suitable method, including enzymatic fragmentation, mechanical shearing, sonication, etc. The DNA fragments can be blunt-ended using any suitable method, including both a fill-in reaction (e.g., via a polymerase) and a chewing reaction (e.g., via an exonuclease). In another aspect, it may be desirable to enrich for a portion of the total RNA present in the TNA. One enrichment method includes rRNA reduction, for example, using RNase H. As one of ordinary skill in the art will appreciate, other preparation steps may also be applied to the TNA sample in step 12.

[0186] After preparation in step 12, the TNA sample comprises both plain 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 comprises 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 plain dsDNA portion of the TNA. In one embodiment, the first template-switching reaction mixture comprises 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 dNTP mix that excludes at least one dNTP selected from dATP, dCTP, dGTP, and dTTP. The at least one dNTP excluded from the dNTP mix is complementary to the at least one nucleotide excluded from the first template-switching oligonucleotide. For example, if TSO-A excludes only nucleotides having the nucleobase thymine, the first dNTP mix will exclude the complementary dATP. Omitting at least one type of nucleobase from the first TSO (i.e., TSO-A) and excluding complementary dNTPs from the first dNTP mixture in the reaction ensures that only dsDNA, not RNA, undergoes template switching. In particular, this design eliminates nonspecific priming and extension associated with the RNA template present in the reaction because when RT encounters a base complementary to the missing dNTP, replication of the RNA template is blocked. Therefore, the lack of all four typical dNTPs prevents RT from fully extending to the 5' end of the RNA and prevents catalysis of the template switching reaction associated with the RNA present in the TNA sample. It is worth noting that the inventors have observed that the template switching reaction seems to be very effective for dsDNA present in the sample, with most (i.e., >50%) of the dsDNA undergoing template switching. More noteworthy is that other schemes can be implemented to achieve template switching only on dsDNA templates. For example, dideoxynucleotide triphosphates (ddNTPs) can be used as described below.

[0187] It will be understood that, in general, the nucleobases uracil and thymine are used interchangeably. For example, when the first TSO does not include the nucleobase adenine, the first dNTP mixture does not include dTTP, which is complementary to adenine. In this case, it may be useful to further exclude dUTP from the first dNTP mixture. Similarly, when the first TSO excludes thymine, it may be useful to further exclude the nucleobase uracil from the first TSO. Thus, in one aspect of the present disclosure, when the first template-switching oligonucleotide excludes thymine, the first template-switching oligonucleotide further excludes uracil. In another aspect of the present disclosure, when the first dNTP mixture excludes dTTP, the first mixture of dNTPs further excludes dUTP.

[0188] Continue to refer to Figure 1 In step 14 of method 10, the composition produced by the first reaction mixture includes a first nucleic acid product, the first nucleic acid product including dsDNA having at least one extended 3' end that is complementary to the first TSO. The composition also includes untreated RNA, TSO-A, RT enzyme, and any remaining reagents, such as dNTPs. In preparation for the second template switching reaction of the disclosed method, method 10 may include step 16, wherein the reaction mixture is subjected to a cleanup step to recover the first nucleic acid product and RNA from other components in the composition produced by the first reaction mixture. Step 16 may include any suitable cleanup protocol to recover nucleic acids from other components of the reaction. Exemplary cleanup protocols include column and bead-based nucleic acid recovery methods, such as solid phase reversible immobilization (SPRI) on carboxylated paramagnetic beads, solvent-based (e.g., ethanol) extraction protocols, and the like, and combinations thereof.

[0189] The next step 18 of method 10 includes performing a second template switching reaction. The second template switching reaction is configured to effect template switching on the remaining TNA (i.e., RNA in this example) that did not undergo a template switching reaction in the first template switching reaction step. The second template switching reaction comprises a first nucleic acid product, RNA, a reverse transcriptase having template switching activity, a second template switching oligonucleotide (TSO-B), and a second dNTP mixture. TSO-B has a sequence that is distinguishable from the sequence of TSO-A so as to distinguish nucleic acid products derived from dsDNA templates from nucleic acid products derived from RNA templates. In one aspect, the nucleotide sequence of the first template switching oligonucleotide differs from the nucleotide sequence of the second template switching oligonucleotide by at least one nucleotide. In another aspect, the nucleotide sequence of the first template switching oligonucleotide differs from the nucleotide sequence of the second template switching oligonucleotide by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides.

[0190] In another aspect, the second mixture of DNA includes all four typical dNTPs, enabling the reverse transcriptase to completely replicate the RNA template so that the template switching reaction occurs at the 5' end of the RNA template. Notably, the second template switching reaction may optionally include at least one primer designed to prime the RNA template with the reverse transcriptase. The primer may be an oligonucleotide dT primer, a target-specific primer, a random primer, or a combination thereof.

[0191] Continue to refer to Figure 1In step 18 of method 10, the second template switching reaction helps to initiate the reverse transcription of the RNA template for cDNA synthesis and template switching using TSO-B. It should be understood that any unreacted flat dsDNA that does not undergo template switching in the first reaction can undergo template switching in the second reaction. Therefore, if it is desired to prevent the addition of a sequence complementary to the second TSO (i.e., TSO-B) to the dsDNA, steps should be taken to ensure that the dsDNA is completely converted into the first nucleic acid product in the first template switching reaction. Alternatively, or in addition, steps can be taken to eliminate unreacted dsDNA from the second reaction. These and other methods will be further described herein.

[0192] Method 10 further includes a step 20 of clearing the second template switching reaction. The composition produced by the second reaction mixture includes a first nucleic acid product and a second nucleic acid product comprising a cDNA derived from the RNA template. The cDNA has an extended 3' end complementary to TSO-B. The composition also includes untreated RNA, TSO-B, RT enzyme, and any remaining reagents, such as dNTPs. In preparation for downstream processing (if any) prior to sequencing, the composition produced by 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 produced by the second reaction mixture.

[0193] Method 10 further includes a step 22 of amplifying the TSO sequence-tagged nucleic acid. In one aspect, the first and second nucleic acid products can be amplified by PCR using various methods. The amplification step 22 can further include ligating 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 for one or both of the TSO-A and TSO-B sequences. The TSO-specific primers can be further paired with target-specific primers to achieve enrichment of specific nucleic acid sequences. It will be understood that the two-step template switching method and subsequent amplification method disclosed herein can be modified in various ways to accommodate different desired results, as will be apparent from this disclosure.

[0194] Following the first and second template switching reactions in steps 14 and 18, respectively, step 24 of method 10 comprises performing a sequencing reaction on the product nucleic acid. In one aspect, the products of the template switching reactions are sequenced directly without amplification. In another aspect, the products amplified in step 22 are 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 comprises a step 26 of assigning sequenced reads. Based on the detected TSO sequence, a given sequenced read can be precisely and accurately assigned to be generated from a DNA or RNA molecule originally present in the TNA sample.

[0195] Now turn Figure 2A , a TNA sample may include at least one double-stranded DNA 100 and at least one RNA 200. Double-stranded DNA 100 has a first strand 102 and a second strand 104. Second strand 104 is at least partially complementary to first strand 102. Furthermore, first strand 102 has a 5' end 106 and a 3' end 108, and second strand 104 has a 5' end 110 and a 3' end 112. Notably, the directionality of first strand 102 and second strand 104, as well as all other depicted nucleic acids, is indicated by arrows throughout the figure. Double-stranded DNA 100 further defines a first target sequence 114.

[0196] refer to Figure 2B The double-stranded DNA 100 is combined with a first reverse transcriptase (not shown), a first template-switching oligonucleotide or TSO 116, and a first dNTP mixture (not shown) to form a first reaction mixture. The first TSO 116 excludes at least one nucleotide having a nucleobase selected from adenine, cytosine, guanine, and thymine. The first dNTP mixture excludes at least one dNTP selected from dATP, dCTP, dGTP, and dTTP, wherein the at least one dNTP excluded from the dNTP mixture is complementary to the at least one nucleotide excluded from the first TSO 116.

[0197] The first reaction mixture includes the necessary components for performing a first template switching reaction using the first reaction mixture. In one aspect, the first template switching reaction includes adding at least one non-templated nucleotide to at least one of the 3' ends of the double-stranded DNA 100 using a reverse transcriptase, thereby forming a non-templated 3' overhang 118 on the double-stranded DNA 100. The non-templated 3' overhang 118 is complementary to the 5' end of the TSO 116, allowing the first TSO 116 to anneal to the non-templated 3' overhang 118 of the double-stranded DNA 100 ( Figure 2C ).

[0198] After annealing of TSO 116 , the non-templated 3′ overhang 118 of double-stranded DNA 100 is extended with a reverse transcriptase, thereby forming a first nucleic acid product 120 comprising double-stranded DNA 100 having at least one extended 3′ end 122 complementary to first template-switching oligonucleotide 116 .

[0199] Now go to Figure 3AA 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 into a second reaction mixture that includes first nucleic acid product 120, a second reverse transcriptase (not shown), a second template-switching oligonucleotide 208, and a second dNTP mix (not shown). In one aspect, the nucleotide sequence of first template-switching oligonucleotide 116 differs from the nucleotide sequence of second template-switching 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.

[0200] refer to Figure 3B and Figure 3C Performing a second template switching reaction with the second reaction mixture includes synthesizing a polynucleotide complementary to RNA 200 with the second reverse transcriptase, thereby forming a first primer extension product 212 complementary to at least a portion of RNA 200. In one aspect, performing the second template switching reaction with the second reaction mixture further includes annealing the 3' end of the first oligonucleotide primer 210 to RNA 200 and extending the first oligonucleotide primer with the second reverse transcriptase, thereby forming the first primer extension product 212. In addition, the second reverse transcriptase can be used to add at least one non-templated nucleotide to the 3' end of the first primer extension product 212, thereby forming a non-templated 3' overhang 214 on the first primer extension product 212. The second template-switching oligonucleotide 208 can anneal 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 by 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 complementary to the second template-switching oligonucleotide. Figure 3D ).

[0201] 4 , various methods can be used to amplify the first nucleic acid product 120 and the second nucleic acid product 216 to attach sequencing adapters to each end. In one aspect, the first nucleic acid product 120 includes a top strand 124 and a bottom strand 126. Different primer pairs can be used to selectively amplify each of the top strand 124 and the bottom strand 126. For example, the first primer pair used to amplify the top strand 124 can include a target-specific primer 128 and a primer 130 specific for the extended 3' end 122, while the second primer pair used to amplify the bottom strand 126 can include a target-specific primer 132 and a primer 134 specific for the extended 3' end 122. In another aspect, the second nucleic acid product 216 can be selectively amplified using a different primer pair than the primer pair used to amplify the first nucleic acid product 120. For example, the first primer pair used to amplify the second nucleic acid product 216 can include a target-specific primer 220 and a primer 222 specific for the extended 3' end 218.

[0202] Notably, each of primers 128, 130, 132, and 134 may include a 5' tail 136 defining an adapter sequence. 5' tail 136 may have sequences that are identical or different, and may include sequencing platform-specific sequences, sample identifier sequences, molecule identifier sequences, and the like, and combinations thereof. Similarly, each of primers 220 and 222 may include a 5' tail 224 defining an adapter sequence. 5' tail 224 may have sequences that are identical or different, and may include sequencing platform-specific sequences, sample identifier sequences, molecule identifier sequences, and the like, and combinations thereof.

[0203] Steering Figure 4B , Figure 4A The products of the amplification steps shown are uniquely identified by their corresponding TSO sequences and, due to the addition of adapter sequences, are further compatible with sequencing on the selected platform. In a first example, after amplification with primers 128 and 130, a first product 138 is derived from top strand 124. First product 138 includes first target sequence 114, including an extended 3' end 122 of the TSO-derived sequence, and a common sequence corresponding to 5' tail 136. In a second example, after amplification with primers 132 and 134, a second product 140 is derived from bottom strand 126. Second product 140 includes first target sequence 114, including an extended 3' end 122 of the TSO-derived sequence, and a common sequence corresponding to 5' tail 136. In a third example, after amplification with primers 220 and 222, a third product 226 is derived from second nucleic acid product 216. The third product 226 includes the second target sequence 206 , including an extended 3′ end 218 of the TSO-derived sequence and a common sequence corresponding to the 5′ tail 224 .

[0204] It will be appreciated that various modifications can be made to the disclosed methods in order to improve the template switching reaction on the DNA or to ensure that only the DNA undergoes template switching in the first template switching reaction. In one aspect, the first TSO or the second TSO-B can include one or more i) nucleotide analogs, such as locked nucleic acid (LNA), fluoro-β-D-arabinoic acid (FANA), 2'-O-methyl RNA, 2'-fluoro RNA, ii) linkage modifications, such as phosphorothioate, 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 the desired functionality to the template switching oligonucleotide.

[0205] In another aspect, a unique sequence can be included in the first TSO that will unambiguously identify reads originating from the first template switching reaction and, therefore, indicate which products or sequencing reads are derived from the dsDNA portion of the TNA.

[0206] In another aspect, buffer conditions can be optimized such that template switching on DNA is preferred over template switching on RNA.

[0207] In another aspect, template switching reactions can be improved by using TSO having a 3' terminal sequence 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 embodiment, the 3' terminal sequence of the TSO is a heteropolymer (e.g., CGC, rCrGrC, etc.). In yet another embodiment, a composition is prepared having a plurality of TSOs with different 3' terminal sequences. For example, a TSO composition can include equal portions of TSOs with two different 3' terminal sequences.

[0208] In another aspect, dNTPs that are excluded from the dNTP mix are selected to enhance template switching.

[0209] In another aspect, a TSO can include a unique molecular identifier (UMI)—also referred to as a unique molecular identifier (UID) or a barcode sequence. A UMI can have variable length and order. A TSO can include a UMI at the 5' end, the 3' end, or an intermediate position. In one aspect, the UMI is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in length.

[0210] In another aspect, the template switching enhancer region can be positioned adjacent to the 5' region of the 3' end of the TSO. The enhancer region comprises the first base introduced after successful template switching. The enhancer region sequence can be selected to enhance the template switching reaction.

[0211] In another aspect, the disclosed method may include using a mixture of at least two different first or second TSOs, wherein the different TSOs include a variable stuffing region located 5' of the 3' terminal sequence. The stuffing region includes a nucleotide sequence selected to mitigate the complexity loss that may result when reading the sequence derived from the 3' end of the TSO (because the sequence may be the same for all TSOs used in the template switching reaction). As will be appreciated, some sequencing instruments benefit from greater sequence complexity of the template during the initial sequencing cycle. Therefore, the stuffing region can be used to improve sequence diversity and, therefore, improve the overall results after sequencing.

[0212] On the other hand, enhancer regions, filler regions, or a combination thereof can be used as keys to help identify TSO elements during data analysis. For example, identification of sequences aligned to the filler region can be used to identify the locations of other sequences (such as UIDs / UMIs).

[0213] In another aspect, the methods of the present disclosure 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.

[0214] In another aspect, the oligonucleotide primers used in the PCR amplification step can be designed to be complementary to sequences located in intronic regions to ensure that only DNA is amplified.

[0215] On the other hand, the method according to the present disclosure may include a terminal transferase step with ddNTPs after the DNA template switching step. For example, ddATP can be added to the product of the template switching reaction in excess together with Taq DNA polymerase. This will result in terminators being added to all flat ds DNA molecules and excluding them from subsequent reactions targeting RNA. The use of ddNTPs prevents the product of the first template switching reaction from being used as a template during 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. In addition, the original DNA template tail present in the sample that did not undergo template switching during the first template switching reaction carries a terminator and cannot be further extended during the second template switching reaction.

[0216] In another aspect, the methods of the present disclosure may include a nuclease treatment step. For example, a nuclease may be added to the first template-switching reaction such that both flat dsDNA and ssDNA that have not undergone the template-switching reaction will be degraded, thereby effectively eliminating these molecules from the second template-switching reaction. In some embodiments, a nuclease treatment step may be used in place of an alternative cleanup step between the first and second template-switching reactions. An example nuclease is E. coli exonuclease I, a 3' to 5' exonuclease that, when added prior to the first template-switching reaction cleanup, will degrade all ssDNA in the reaction. Another example nuclease is E. coli exonuclease III, a 3' to 5' exonuclease that degrades flat duplex dsDNA. Notably, E. coli exonuclease III does not digest protruding 3' overhangs on dsDNA, which can be generated by melting or removing TSO-A.

[0217] In yet another aspect, TSOs can be designed to include sequencing platform-specific adapter sequences.

[0218] On the one hand, the method of the present disclosure may further include the use of a capture moiety. Examples of capture moieties include biotin and desthiobiotin. In one method, dNTPs are labeled with a capture object so that when a template switching reaction occurs, the capture moiety is incorporated into the first nucleic acid product derived from the dsDNA template or the second nucleic acid product derived from the RNA template. The resulting labeled product can be captured using streptavidin beads. The use of the capture moiety further enables the oligonucleotide primer (e.g., RNA-specific primer, random primer, or oligonucleotide dT primer) for generating the first primer extension product from RNA to be recovered by incorporating the capture moiety into the oligonucleotide primer.

[0219] The use of the capture moiety additionally facilitates subsequent cleanup or purification steps. In one example, during the streptavidin cleanup step prior to the second template-switching reaction, flat dsDNA that has not undergone template switching will be removed from the reaction, thereby reducing flat dsDNA carryover into the second template-switching reaction. On the other hand, if desthiobiotin-labeled dNTPs are used, biotin can be added to subsequent PCR reactions to facilitate the release of any template molecules attached to the capture surface (such as streptavidin-coated beads).

[0220] In one embodiment of the present disclosure,

[0221] The nucleic acid sample includes at least one double-stranded DNA and a reverse transcriptase, a first template conversion oligonucleotide, a first dNTP mixture excluding at least one dNTP selected from dATP, dCTP, dGTP and dTTP, and a 2', 3' dideoxynucleotide (ddNTP) combination. On the one hand, ddNTP includes a core base excluded from the first dNTP mixture. When the first template conversion reaction is carried out, the DNA template is copied by the reverse transcriptase until the ddNTP is reached. The termination point can be controlled by selecting the position of the complementary base of the ddNTP in the first template conversion oligonucleotide. It is worth noting that when ddNTP is incorporated, any initiation of the RNA template that can be preset in the nucleic acid sample will stop, thereby limiting the template conversion of the DNA portion of the nucleic acid sample.

[0222] In some embodiments, ddNTP is labeled with a capture portion. For example, ddNTP can be labeled with biotin or desthiobiotin. During the first template switching reaction, when ddNTP is incorporated at the complementary position in the first template switching oligonucleotide template, the reaction terminates. It is worth noting that the complementary base can be positioned at a limited position in the first template switching oligonucleotide. The resulting product will incorporate a capture portion (in this case, a single biotin or desthiobiotin) at the 3' end of the first nucleic acid product produced by the template switching reaction. The first nucleic acid product can then be recovered using, for example, streptavidin beads. In order to achieve the recovery of RNA-derived products produced by the template switching reaction according to the present disclosure, oligonucleotide primers (e.g., sequence-specific primers, random primers, or oligonucleotide dT primers) can be labeled with at least one capture portion or otherwise include at least one capture portion. On the one hand, the use of capture portions can achieve purification between template switching reactions and other downstream processing steps.

[0223] The schematic flow charts shown in the accompanying drawings are generally listed in the form of logical flow charts. Therefore, the order depicted and the steps labeled indicate one embodiment of the method presented. Other steps and methods that are equivalent in function, logic or effect to one or more steps or portions thereof of the method shown can be conceived. In addition, the format and symbols used in the accompanying drawings are used to explain the logical steps of the method and should be understood not to limit the scope of the method. Although various arrow types and line types can be used, they should be understood not to limit the scope of the corresponding method. In fact, some arrows or other connectors can be used to indicate only the logical flow of the method. For example, an arrow can indicate a waiting or monitoring period of unspecified duration between the enumerated steps of the depicted method. In addition, the order in which a particular method occurs may or may not strictly adhere to the order of the corresponding steps shown.

[0224] In the following description with reference to the accompanying drawings, the present invention is presented in terms of several different embodiments, wherein like reference numerals represent like or similar elements. Reference throughout this specification to "one embodiment," "an embodiment," and similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0225] The features, structures or characteristics of the present invention can be combined in any suitable manner in one or more embodiments. In the following description, many specific details are listed to provide a thorough understanding of the embodiments of the system. However, those skilled in the relevant art will recognize that the system and method can be practiced without one or more of the specific details or using other methods, components, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid covering up various aspects of the present invention. Therefore, the foregoing description is intended to be exemplary and does not limit the scope of the inventive concept.

[0226] Each reference identified in this application is incorporated herein by reference in its entirety.

Claims

1. A method comprising: The following are combined into a first reaction mixture: i) a nucleic acid sample comprising at least one 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, ii) a first reverse transcriptase, iii) a first template-switching oligonucleotide that excludes at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine, and iv) a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the first template-switching oligonucleotide; as well as Performing a first template switching reaction using the first reaction mixture comprises: 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-switching oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; and The non-templated 3' overhang of the double-stranded DNA is extended with the reverse transcriptase, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to the first template-switching oligonucleotide. 2 . The method of claim 1 , wherein the nucleic acid sample further comprises at least one RNA having a 5′ end and a 3′ end.

3. The method according to claim 2, further comprising: The following were combined into a second reaction mixture: i) the first nucleic acid product, ii) said RNA, iii) a second reverse transcriptase, iv) a second template-switching oligonucleotide, and v) a second dNTP mix; as well as Performing a second template switching reaction using the second reaction mixture comprises: synthesizing a polynucleotide complementary to the RNA using the 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 using 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 The non-templated 3' overhang of the first primer extension product is extended 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-switching oligonucleotide.

4. The method according to claim 3, further comprising: combining a first oligonucleotide primer having a 3' end complementary to the RNA into the second reaction mixture; and Wherein performing the second template switching reaction with the second reaction mixture further comprises: annealing the 3' end of the first oligonucleotide primer to the RNA; as well as The first oligonucleotide primer is extended using the second reverse transcriptase to form the first primer extension product.

5. The method of claim 3, wherein the nucleotide sequence of the first template-switching oligonucleotide differs from the nucleotide sequence of the second template-switching oligonucleotide by at least one nucleotide.

6. The method of claim 2, wherein the first template switching reaction is unable to form a byproduct comprising a complement of at least a portion of the RNA having a 3' end that is complementary to the first template switching oligonucleotide.

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

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

9. The method of claim 1, wherein the first template-switching oligonucleotide comprises at least one of a universal adaptor sequence, a sample identification sequence, and a molecule identifier sequence.

10. The method of claim 3, wherein the second template-switching oligonucleotide comprises at least one of a universal adaptor sequence, a sample identification sequence, and a molecule identifier sequence.

11. The method of claim 3, wherein the method further comprises purifying the nucleic acid sample comprising the first nucleic acid product and the RNA before the step of combining into the second reaction mixture.

12. A method comprising: The following are combined into a first reaction mixture: i) a nucleic acid sample comprising at least one 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, ii) reverse transcriptase, iii) a first template-switching oligonucleotide having a 5' domain and a 3' domain, said 3' domain of said first template-switching oligonucleotide excluding at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine and thymine, iv) a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, said at least one dNTP excluded from said dNTP mixture being complementary to said at least one nucleotide excluded from said 3' domain of said first template switch oligonucleotide; and v) a ddNTP that is complementary to said at least one nucleotide excluded from said 3' domain of said first template switch oligonucleotide; as well as Performing a first template switching reaction using the first reaction mixture comprises: 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-switching oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; and The non-templated 3' overhang of the double-stranded DNA is extended with the reverse transcriptase, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to the 3' domain of the first template-switching oligonucleotide.

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

14. The method according to claim 13, further comprising: The following were combined into a second reaction mixture: i) the first nucleic acid product, ii) said RNA, iii) a second reverse transcriptase, iv) a second template-switching oligonucleotide, and v) a second dNTP mix; as well as Performing a second template switching reaction using the second reaction mixture comprises: synthesizing a polynucleotide complementary to the RNA using the 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 using 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 The non-templated 3' overhang is extended with the 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 the second template-switching oligonucleotide.

15. The method according to claim 14, further comprising: combining a first oligonucleotide primer having a 3' end complementary to the RNA into the second reaction mixture; and Wherein performing the second template switching reaction with the second reaction mixture further comprises: annealing the 3' end of the first oligonucleotide primer to the RNA; as well as The first oligonucleotide primer is extended using the second reverse transcriptase to form the first primer extension product.

16. The method of claim 14, wherein the nucleotide sequence of the first template-switching oligonucleotide differs from the nucleotide sequence of the second template-switching oligonucleotide by at least one nucleotide.

17. The method of claim 13, wherein the first template switching reaction is unable to form a byproduct comprising a complement of at least a portion of the RNA having a 3' end that is complementary to the first template switching oligonucleotide.

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

19. The method of claim 12, wherein the 3' domain of the first template switching oligonucleotide comprises at least one of a universal adaptor sequence, a sample identification sequence, and a molecule identifier sequence.

20. The method of claim 14, wherein the second template switching oligonucleotide comprises at least one of a universal adaptor sequence, a sample identification sequence, and a molecule identifier sequence.

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

22. The method of claim 3 or 14, wherein the second nucleic acid product comprises a second target sequence, and wherein the method further comprises: amplifying at least a portion of the second nucleic acid product in a second amplification reaction mixture comprising: i) the second nucleic acid product, 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.

23. The method of any one of the preceding claims, wherein the first nucleic acid product comprises a first target sequence, and wherein the method further comprises: amplifying at least a portion of the first nucleic acid product in a first amplification reaction mixture comprising: 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.

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

25. The method of any one of the preceding claims, wherein the second template switching reaction is unable to form a side product comprising the first nucleic acid product having a 3' end that is complementary to the second template switching oligonucleotide.

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

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

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

29. The method according to claim 4 or 15, wherein the 3' end of the first oligonucleotide primer is a poly-dT sequence.

30. 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. The method of any one of the preceding claims, wherein the first template-switching oligonucleotide comprises a stuffer region.

33. The method of claim 4 or 15, wherein the second template-switching oligonucleotide comprises a stuffer region.

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

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

37. The method of any one of the preceding claims, wherein performing the first template switching reaction with 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. The method of claim 3 or 14, wherein performing the second template switching reaction with 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. 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. A method comprising: The following are combined into a first reaction mixture: i) a nucleic acid sample comprising at least one 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 having a first strand, the first strand having a 5' end and a 3' end, ii) reverse transcriptase, iii) a first template-switching oligonucleotide that excludes at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine, and iv) a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the first template-switching oligonucleotide; as well as Performing a first template switching reaction using the first reaction mixture comprises: 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-switching oligonucleotide to the non-templated 3' overhang of the double-stranded DNA; and The non-templated 3' overhang of the double-stranded DNA is extended with the reverse transcriptase, thereby forming a first nucleic acid product comprising the double-stranded DNA having at least one extended 3' end complementary to the first template-switching oligonucleotide.

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, the method comprising: performing a first template switching reaction on the nucleic acid sample in the absence of at least one dNTP selected from the group consisting of 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 the first template switching oligonucleotide; as well as The nucleic acid sample is subjected to a second template switching reaction, 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 the second template switching 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, the kit comprising: a first template-switching oligonucleotide that excludes at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine; and A first dNTP mixture excludes at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the first template-switching oligonucleotide.

43. The kit of claim 42, further comprising: a second template-switching oligonucleotide; and Second dNTP mix.

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, the kit comprising: a first template-switching oligonucleotide having a 5' domain and a 3' domain, the 3' domain of the first template-switching oligonucleotide excluding at least one nucleotide having a nucleobase selected from the group consisting of adenine, cytosine, guanine, and thymine; a first dNTP mixture excluding at least one dNTP selected from the group consisting of dATP, dCTP, dGTP, and dTTP, the at least one dNTP excluded from the dNTP mixture being complementary to the at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide; and a ddNTP that is complementary to the at least one nucleotide excluded from the 3' domain of the first template switch oligonucleotide.

45. The kit of claim 44, further comprising: a second template-switching oligonucleotide; and Second dNTP mix.

46. The method of claim 3 or 14, wherein at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide comprises a ribonucleotide.

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

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

49. The method of claim 1 or 3, wherein the 3' end of at least one of the first template-switching oligonucleotide and the second template-switching oligonucleotide comprises a homopolymer sequence of at least three nucleotides.

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

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

52. The method of claim 1 or 12, wherein the first template-switching oligonucleotide further excludes uracil.

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

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

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

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

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

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

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

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

61. The method of claim 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 the group consisting of biotin and desthiobiotin.

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