Methods for adding adapters to nucleic acids and compositions for practicing the same
Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template ribonucleic acid (RNA), a template switch oligonucleotide including a 3′ hybridization domain and a sequencing platform adapter construct, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template RNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.
1. A method comprising:
a) combining:
i) a template ribonucleic acid (RNA);
ii) a template switch oligonucleotide comprising a 3′ hybridization domain and a first primer binding domain;
iii) a first polymerase;
iv) a first strand primer comprising a first domain that hybridizes to the template RNA and a second domain comprising a second primer binding domain that is different from the first binding domain;
v) a second polymerase; and
vi) dNTPs;
in a single reaction mixture under conditions, thereby producing a product nucleic acid comprising the template RNA and the template switch oligonucleotide which are hybridized to adjacent regions of a single product nucleic acid comprising the first strand primer and a region polymerized from the dNTPs by the first polymerase; and
b) amplifying the product nucleic acid using the second polymerase with a forward primer and a reverse primer, wherein each of the forward primer and the reverse primer comprises a sequencing platform adapter construct comprising at least a portion of a capture sequence that is utilized by a sequencing platform, wherein the capture sequence specifically hybridizes to a surface-attached sequencing platform oligonucleotide on the sequencing platform, and wherein the forward primer and the reverse primer are different and hybridize to different sequences of the product nucleic acid; and
c) sequencing an amplified product nucleic acid produced in step (b) by the sequencing platform that comprises the surface-attached sequencing platform oligonucleotide that captures the at least portion of the capture sequence of the sequencing platform adapter construct; wherein the sequencing platform is a next generation sequencing platform and the sequencing platform adaptor construct comprises the sequence selected from the group consisting of SEQ ID NOs: 01 to 06, which are P5 domain, P7 domain, Read 1 primer domain, Read 2 primer domain, A adapter domain and P1 adapter domain.
2. The method according to claim 1 , wherein the method is performed in a single tube.
3. The method according to claim 1 , wherein the first polymerase comprises terminal transferase activity.
4. The method according to claim 1 , wherein the first polymerase comprises template switching activity.
5. The method according to claim 1 , wherein the first polymerase is a reverse transcriptase.
6. The method according to claim 5 , wherein the reverse transcriptase is a MMLV reverse transcriptase.
7. The method according to claim 1 , wherein the second polymerase is a thermostable polymerase.
8. The method according to claim 7 , wherein the thermostable polymerase is selected from the group consisting of: Taq polymerase, Pfu polymerase, Tfl polymerase, Tth polymerase, and Tli polymerase and combinations thereof.
9. The method according to claim 7 , wherein the amplifying step is performed under thermocycling conditions.
10. The method according to claim 1 , wherein the template RNA is a messenger RNA (mRNA).
11. The method according to claim 1 , wherein the template RNA is a non-polyadenylated RNA.
12. The method according to claim 11 , wherein the method, prior to the combining step (a), further comprises adding a nucleic acid sequence to the 3′ end of the non-polyadenylated RNA.
13. The method according to claim 12 , wherein the nucleic acid sequence added to the 3′ end of the non-polyadenylated RNA is a polyadenine sequence.
14. The method according to claim 11 , wherein the non-polyadenylated RNA is a microRNA (miRNA).
15. The method according to claim 1 , wherein the template RNA is a polyadenylated RNA having a polyadenylation sequence.
16. The method according to claim 1 , wherein the first strand primer comprises a modification that prevents a polymerase using the single product nucleic acid as a template from polymerizing a nascent strand beyond the modification in the first strand primer.
17. The method according to claim 1 , wherein the template switch oligonucleotide comprises a modification that prevents the first polymerase from switching from the template switch oligonucleotide to a different template nucleic acid after synthesizing the complement of the template switch oligonucleotide.
18. The method according to claim 17 , wherein the modification is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, and combinations thereof.
19. The method according to claim 1 , wherein the template switch oligonucleotide comprises one or more nucleotide analogs.
20. The method according to claim 19 , wherein the one or more nucleotide analogs comprise a locked nucleic acid (LNA).
21. The method according to claim 1 , wherein the template switch oligonucleotide comprises a linkage modification, an end modification, or both.
22. The method according to claim 1 , wherein at least one of the template switch oligonucleotide and the first strand primer comprises the sequencing platform adapter construct or portion thereof.
23. The method according to claim 22 , wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically binds to the surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof.
24. The method according to claim 22 , wherein the sequencing platform adaptor construct comprises a portion of a nucleic acid domain utilized by the sequencing platform.
25. The method according to claim 22 , wherein the sequencing platform adaptor construct comprises a complete nucleic acid domain utilized by the sequencing platform.
26. The method according to claim 1 , wherein each of the first strand primer and the template switch oligonucleotide comprises a 5′ domain that does not hybridize to the template RNA.
27. The method according to claim 1 , wherein the first primer binding domain and the second primer binding domain are 16 to 75 nucleotides in length.