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 a plurality of complementary deoxyribonucleic acid molecule comprising:
(a) depleting at least one ribosomal RNA from a sample comprising a template nucleic acid molecule;
(b) annealing a primer to said template nucleic acid molecule, thereby generating an annealed template nucleic acid molecule; and
(c) mixing, in presence of nucleotides, (i) said annealed template nucleic acid molecule, (ii) one or more acceptor nucleic acid molecules, (iii) a reverse transcriptase, under conditions sufficient to conduct one or more reactions to generate a plurality of complementary deoxyribonucleic acid molecules, wherein said one or more reactions comprise: 1) reverse transcribing a sequence of said annealed template nucleic acid molecule; 2) migrating to an acceptor nucleic acid molecule; and 3) reverse transcribing a sequence of said acceptor nucleic acid molecule, at a temperature of from about 12° C. to about 42° C. at an error rate of at most about 5% as measured by a number of nucleic acid bases that are misincorporated during said one or more reactions.
2. The method of claim 1 , wherein said migrating is independent of sequence identity between said template nucleic acid molecule and said acceptor nucleic acid molecule.
3. The method of claim 1 , wherein (a)-(c) are performed in a single vessel.
4. The method of claim 1 , wherein (a)-(c) are performed in absence of nucleic acid purification.
5. The method of claim 1 , wherein said plurality of complementary deoxyribonucleic acid molecules is prepared in a time period of at most about 3 hours.
6. The method of claim 1 , wherein said reverse transcriptase amplifies said annealed template nucleic acid molecule at a processivity of at least about 80% per nucleic acid base.
7. The method of claim 1 , wherein said reverse transcriptase is configured to prepare at least one complementary deoxyribonucleic acid molecule of said plurality of complementary deoxyribonucleic acid molecules with a performance index greater than 1.0 for at least one enzyme property selected from the group consisting of processivity and stability as compared to a purified enzyme of SEQ ID NO: 52.
8. The method of claim 7 , wherein said reverse transcriptase is an R2 reverse transcriptase.
9. The method of claim 8 , wherein said R2 reverse transcriptase is an R2 non-long terminal repeat retrotransposon enzyme.
10. The method of claim 1 , wherein said reverse transcriptase has at least 80% identity to 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, or SEQ ID NO: 67 and contains at least one substitution modification relative to SEQ ID NO: 52.
11. The method of claim 1 , wherein said plurality of complementary deoxyribonucleic acid molecules comprise detectable tags.
12. The method of claim 1 , further comprising, in (c), performing a polymerase chain reaction (PCR) amplification on a complementary deoxyribonucleic acid molecule generated by said reverse transcriptase, thereby forming one or more amplicons of said complementary deoxyribonucleic acid molecule.
13. The method of claim 12 , wherein (a)-(c) are performed in a same vessel.
14. The method of claim 1 , wherein said sample is a tissue sample.
15. The method of claim 1 , wherein said sample comprises one or more cell free nucleic acids.
16. A method for preparing a plurality of complementary deoxyribonucleic acid molecule comprising:
(a) annealing one or more primers to one or more template nucleic acid molecules derived from a single cell to generate one or more annealed template nucleic acid molecules; and
(b) mixing, in presence of nucleotides, (i) said one or more annealed template nucleic acid molecules, (ii) one or more acceptor nucleic acid molecules, (iii) a reverse transcriptase, under conditions sufficient to conduct one or more reactions to generate a plurality of complementary deoxyribonucleic acid molecules, wherein said one or more reactions comprise reverse transcribing a sequence of an annealed template nucleic acid molecule of said one or more annealed template nucleic acid molecules, migrating to an acceptor nucleic acid molecule, and reverse transcribing a sequence of said acceptor nucleic acid molecule, in a single reaction vessel at room temperature.
17. The method of claim 16 , wherein (a) and (b) are performed on up to about 100 nM of said one or more template nucleic acid molecules.
18. The method of claim 16 , wherein said migration is independent of sequence identity between the template and the acceptor nucleic acid molecule.
19. The method of claim 16 , wherein said plurality of complementary deoxyribonucleic acid molecules are prepared in a time period of at most about 3 hours in said single reaction vessel.
20. The method of claim 16 , wherein said one or more template nucleic acid molecules are selected from the group consisting of an artificially fragmented deoxyribonucleic acid template, a naturally fragmented deoxyribonucleic acid template, an artificially fragmented ribonucleic acid template, a naturally fragmented ribonucleic acid template, and a combination thereof.
21. The method of claim 16 , wherein said reverse transcriptase comprises at least one improved enzyme property relative to a wild type reverse transcriptase.
22. The method of claim 21 , wherein said at least one improved enzyme property is selected from the group consisting of: thermal stability, specific activity, processivity, strand displacement, end-to-end template jumping, affinity, and fidelity.
23. The method of claim 16 , wherein said reverse transcriptase is an R2 reverse transcriptase.
24. The method of claim 16 , wherein said reverse transcriptase has at least 80% identity to 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, or SEQ ID NO: 67 and contains at least one substitution modification relative to SEQ ID NO:52.
25. The method of claim 16 , wherein said plurality of complementary deoxyribonucleic acid molecules comprise detectable tags.
26. The method of claim 16 , further comprising sequencing said plurality of complementary deoxyribonucleic acid molecules.
27. The method of claim 16 , further comprising depleting at least one ribonucleic acid from said one or more template nucleic acid molecules prior to annealing one or more primers.
28. The method of claim 27 , wherein depleting said at least one ribonucleic acid comprises performing an oligonucleotide probe-guided endonucleolitic cleavage of said ribonucleic acid.
29. The method of claim 16 , wherein said one or more primers comprise one or more random primer(s).
30. The method of claim 16 , wherein said one or more primers hybridize to one or more adapter sequences of said one or more template nucleic acid molecules.