Methods of producing nucleic acid libraries and compositions and kits for practicing same
View Patent ↗Provided are methods of producing nucleic acid libraries. The methods include combining single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA), an adapter oligonucleotide, and a splint oligonucleotide, to form complexes including the splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA and to the adapter oligonucleotide. An end of the first adapter oligonucleotide is adjacent to an end of the first terminal region of the SSB-bound ssNA, and the methods may further include covalently linking the adjacent ends. Also provided are compositions and kits that find use, e.g., in practicing the methods of the present disclosure.
1. A method of producing a nucleic acid library, comprising:
contacting single-stranded nucleic acid (ssNA) with single-stranded nucleic acid binding protein (SSB) to produce SSB-bound ssNA; and
combining:
the SSB-bound ssNA,
a first adapter oligonucleotide, and
a first splint oligonucleotide comprising an SSB-bound ssNA hybridization region and a first adapter oligonucleotide hybridization region;
to form complexes comprising the first splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA via the SSB-bound ssNA hybridization region, and the first splint oligonucleotide hybridized to the first adapter oligonucleotide via the first adapter oligonucleotide hybridization region, such that an end of the first adapter oligonucleotide is adjacent to an end of the terminal region of the SSB-bound ssNA, wherein the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a ligation-blocking modification.
2. The method according to claim 1 , further comprising covalently linking the adjacent ends of the first adapter oligonucleotide and SSB-bound ssNA.
3. The method according to claim 2 , wherein the covalently linking comprises ligating the adjacent ends by enzymatic ligation.
4. The method according to claim 1 , wherein the ssNA is single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA).
5. The method according to claim 1 , wherein the combining comprises:
(a) combining:
a complex comprising the first splint oligonucleotide hybridized to the first adapter oligonucleotide via the first adapter oligonucleotide hybridization region, and
the SSB-bound ssNA; or
(b) combining:
a complex comprising the first splint oligonucleotide hybridized to the SSB-bound ssNA via the SSB-bound ssNA hybridization region, and
the first adapter oligonucleotide.
6. The method according to claim 1 , wherein the combining further comprises:
combining:
the SSB-bound ssNA,
a second adapter oligonucleotide, and
a second splint oligonucleotide comprising an SSB-bound ssNA hybridization region and a second adapter oligonucleotide hybridization region,
wherein the formed complexes further comprise the second splint oligonucleotide hybridized via the SSB-bound ssNA hybridization region to the terminal region of the SSB-bound ssNA opposite the terminal region hybridized to the first splint oligonucleotide, and the second splint oligonucleotide hybridized to the second adapter oligonucleotide via the second adapter oligonucleotide hybridization region, such that an end of the second adapter oligonucleotide is adjacent to the end of the SSB-bound ssNA opposite the end adjacent to the first adapter oligonucleotide.
7. The method according to claim 6 , further comprising covalently linking the adjacent ends of the second adapter oligonucleotide and SSB-bound ssNA.
8. The method according to claim 7 , wherein the covalently linking comprises ligating the adjacent ends by enzymatic ligation.
9. The method according to claim 1 , wherein the SSB-bound ssNA hybridization region comprises a random sequence.
10. The method according to claim 1 , wherein the first adapter oligonucleotide comprises: (i) an adapter for PCR amplification or a complement thereof, (ii) a partial or complete sequencing adapter or a complement thereof, or (iii) a combination of (i) and (ii).
11. The method according to claim 1 , further comprising sequencing at least a portion of the ssNA or a derivative thereof.
12. The method of claim 1 , wherein the ligation-blocking modification comprises absence of a 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA.
13. The method of claim 1 , wherein the ligation-blocking modification comprises an inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA.
14. The method of claim 13 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises an amino modifier.
15. The method of claim 13 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a spacer.
16. The method of claim 13 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a dideoxy base.
17. The method of claim 13 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises an inverted dideoxy base.
18. The method of claim 13 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a 3′ phosphate.