Methods for adding adapters to nucleic acids and compositions for practicing the same
View Patent ↗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 method comprising:
(a) combining:
a template nucleic acid;
a template switch oligonucleotide comprising a 3′ hybridization domain and one or more ribonucleotides 5′ of the 3′ hybridization domain;
a polymerase; and
dNTPs;
in a reaction mixture under conditions sufficient to produce a first strand cDNA complementary to the template nucleic acid and the template switch oligonucleotide;
b) degrading the template switch oligonucleotide; and
c) amplifying the first strand cDNA.
2. The method according to claim 1 , wherein the template switch oligonucleotide comprises two or more ribonucleotides 5′ of the 3′ hybridization domain.
3. The method according to claim 1 , wherein the template switch oligonucleotide is degraded using a ribonuclease.
4. The method according to claim 1 , wherein the amplifying is performed with a primer comprising a barcode.
5. The method according to claim 1 , wherein said template nucleic acid is from a single cell.
6. The method according to claim 1 , wherein the 3′ hybridization domain comprises a homo-trinucleotide.
7. The method according to claim 1 , wherein the 3′ hybridization domain comprises a hetero-trinucleotide.
8. The method according to claim 1 , wherein the template switch oligonucleotide comprises a sequencing platform 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.
9. The method according to claim 1 , wherein the template nucleic acid is sheared or fragmented template nucleic acid.
10. The method according to claim 9 , wherein the method further comprises shearing or fragmenting the template nucleic acid prior to the combining.
11. The method according to claim 1 , wherein the method comprises heating the reaction mixture.
12. The method according to claim 1 , wherein the reaction mixture includes a first strand synthesis primer comprising at least one exo-sample nucleotide.
13. The method according to claim 1 , wherein the degrading comprises degrading dissociated template switch oligonucleotide.
14. A method comprising:
(a) combining:
a template nucleic acid;
a template switch oligonucleotide comprising one or more exo-sample nucleotides selected from the group consisting of: bromodeoxyuridine, 7-methylguanine, 5,6-dihydroxydeoxythymidine, and 3-methyldeoxadenosine, or any combination thereof and a 3′ hybridization domain;
a polymerase; and
dNTPs;
in a reaction mixture under conditions sufficient to produce a first strand cDNA complementary to the template nucleic acid and the template switch oligonucleotide;
b) degrading a portion of the template switch oligonucleotide that is 5′ of the 3′ hybridization domain; and
c) amplifying the first strand cDNA.
15. The method according to claim 14 , wherein the template switch oligonucleotide is degraded using one or more enzymes.
16. The method according to claim 15 , wherein the one or more enzymes are selected from the group consisting of: a uracil-N-glycosylase, a lyase, a DNA glycosylase-lyase and combinations thereof.
17. The method according to claim 16 , wherein the DNA glycosylase-lyase is Endonuclease VIII.
18. The method according to claim 14 , wherein the template switch oligonucleotide further comprises a ribonucleotide 5′ of the 3′ hybridization domain.
19. The method according to claim 14 , wherein the template switch oligonucleotide comprises a sequencing platform 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.