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 nucleic acid, a template switch oligonucleotide, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template nucleic acid and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid including a region polymerized from the dNTPs by the polymerase. The methods further include attaching sequencing platform adapter constructs to ends of the product nucleic acid or a derivative thereof. Aspects of the invention further include compositions and kits.
1. A kit comprising:
a template switch oligonucleotide comprising a 3′ hybridization domain and one or more exo-sample nucleotides 5′ of the 3′ hybridization domain;
a sequencing platform adapter construct; and
a template switching polymerase.
2. The kit of claim 1 , wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.
3. The kit of claim 1 , comprising a first-strand synthesis primer comprising a first domain that hybridizes to a template nucleic acid and a second domain that does not hybridize to the template RNA.
4. The kit of claim 3 , wherein the second domain comprises a sequencing platform adapter construct comprising a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.
5. The kit of claim 1 , wherein the template switching polymerase is a reverse transcriptase.
6. The kit of claim 1 , wherein the exo-sample nucleotides comprise a ribonucleotide.
7. The kit of claim 6 , wherein the ribonucleotide is cleavable by a ribonuclease.
8. The kit of claim 1 , wherein the exo-sample nucleotides comprise a deoxyuridine.
9. The kit of claim 8 , wherein the deoxyuridine is cleavable by a uracil-N-glycosylase.
10. The kit of claim 1 , wherein the exo-sample nucleotides comprise a nucleotide selected from the group consisting of: bromodeoxyuridine, 7-methylguanine, 5,6-dihydroxydeoxythymidine, 3-methyldeoxyadenosine, and any combination thereof.
11. A kit comprising:
a template switch oligonucleotide comprising a 3′ hybridization domain and one or more ribonucleotides 5′ of the 3′ hybridization domain;
a sequencing platform adapter construct;
a template switching polymerase; and
a ribonuclease.
12. The kit of claim 11 , wherein the template switch oligonucleotide comprises two or more ribonucleotides 5′ of the 3′ hybridization domain.
13. The kit of claim 11 , wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.
14. The kit of claim 11 , comprising a first-strand synthesis primer comprising a first domain that hybridizes to a template nucleic acid and a second domain that does not hybridize to the template nucleic acid.
15. The kit of claim 14 , wherein the second domain comprises a sequencing platform adapter construct comprising a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.
16. A kit comprising:
a template switch oligonucleotide comprising a 3′ hybridization domain and one or more exo-sample nucleotides selected from the group consisting of: bromodeoxyuridine, 7-methylguanine, 5,6-dihydroxydeoxythymidine, and 3-methyldeoxyadenosine, or any combination thereof;
a sequencing platform adapter construct;
a template switching polymerase; and
an enzyme for degrading a portion of the template switch oligonucleotide that is 5′ of the 3′ hybridization domain.
17. The kit of claim 16 , wherein the enzyme is selected from the group consisting of: a uracil-N-glycosylase, a lyase, a DNA glycosylase-lyase and combinations thereof.
18. The kit of claim 16 , wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.
19. The kit of claim 16 , comprising a first-strand synthesis primer comprising a first domain that hybridizes to a template nucleic acid and a second domain that does not hybridize to the template nucleic acid.
20. The kit of claim 19 , wherein the second domain comprises a sequencing platform adapter construct comprising a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.