Methods for processing nucleic acid samples
The present disclosure provides methods and systems for amplifying and analyzing nucleic acid samples. The present disclosure provides methods for preparing cDNA and/or DNA molecules and cDNA and/or DNA libraries using modified reverse transcriptases.
1. A method for preparing complementary deoxyribonucleic acid (cDNA), the method comprising:
(a) providing a single vessel comprising an R2 reverse transcription enzyme and one or more template ribonucleic acid molecules; and
(b) in said single vessel, using said R2 reverse transcription enzyme to perform an amplification reaction on said one or more template ribonucleic acid molecules to yield a plurality of complementary deoxyribonucleic acid molecules,
wherein said R2 reverse transcription enzyme comprises one of the following amino acid sequences: SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.
2. The method of claim 1 , wherein said one or more template ribonucleic acid molecules are from a single cell provided in said single vessel prior to (a).
3. The method of claim 1 , wherein said one or more template ribonucleic acid molecules are present in an amount up to about 100 nM.
4. The method of claim 1 , wherein said R2 reverse transcription enzyme is configured to perform said amplification reaction at a substantially constant temperature.
5. The method of claim 1 , wherein said R2 reverse transcription enzyme is configured to perform said amplification reaction at a temperature of from about 12° C. to about 42° C.
6. The method of claim 1 , wherein said R2 reverse transcription enzyme has a misincorporation error rate of at most about 1 base out of 100 bases.
7. The method of claim 6 , wherein said R2 reverse transcription enzyme has a misincorporation error rate of at most about 1 base out of 10,000 bases.
8. The method of claim 1 , wherein said R2 reverse transcription enzyme is configured to generate said plurality of complementary deoxyribonucleic acid molecules from said one or more template ribonucleic acid molecules with a specific activity of from about 20,000 units/mg to about 140,000 units/mg.
9. The method of claim 1 , wherein said one or more template ribonucleic acid (RNA) molecules comprise messenger RNA, ribosomal RNA, transfer RNA, micro RNA, long non-coding RNA, vault RNA, signal recognition particle RNA, small RNA, small nuclear, small nucleolar RNA, small conditional RNA, amall cajal body-specific RNA, macro long non-protein-coding RNA, long intergenic noncoding RNA, piwi-interacting RNA, small interfering RNA, and ribozymes.
10. The method of claim 1 , wherein said one or more template ribonucleic acid molecules comprise an artificially fragmented ribonucleic acid template, a naturally fragmented ribonucleic acid template, or a combination thereof.
11. The method of claim 1 , wherein using said R2 reverse transcription enzyme to perform the amplification reaction yields the plurality of complementary deoxyribonucleic acid molecules in a time period of less than three hours.
12. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises SEQ ID NO: 49.
13. The method of claim 11 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 50.
14. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 51.
15. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 53.
16. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 54.
17. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 55.
18. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 56.
19. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 57.
20. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 58.
21. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 59.
22. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 60.
23. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 61.
24. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 62.
25. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 63.
26. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 64.
27. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 65.
28. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 66.
29. The method of claim 1 , wherein said R2 reverse transcription enzyme comprises the amino acid sequence of SEQ ID NO: 67.