Circularization methods for single molecule sequencing sample preparation
The invention is a novel method of making and using a library such as a sequencing library of single stranded circular nucleic acid templates via splint ligation.
1. A method of forming a circular molecule from a target nucleic acid, the method comprising:
(a) attaching adaptor sequences to the ends of the target nucleic acid to form an adapted target nucleic acid, wherein the adaptor comprises one or more nuclease-resistant modifications, wherein the one or more nuclease-resistant modifications is located one or more bases from the 5′-end;
(b) contacting the adapted target nucleic acid with a double-stranded backbone nucleic acid comprising one or more nuclease-resistant modifications, wherein the one or more nuclease-resistant modifications is located one or more bases from each of the 5′-ends;
(c) generating single-stranded overhangs in both the backbone and the adapted target nucleic acid by exonuclease digestion of the 5′-end ending at the modifications;
(d) hybridizing the single-stranded overhangs of the backbone with the single-stranded overhangs of the adapted target nucleic acid; and
(e) ligating the ends of the backbone with the ends of the adapted target nucleic acid to form a circular molecule.
2. The method of claim 1 , wherein the adaptors are attached to the ends of the target nucleic acid by ligation.
3. The method of claim 1 , wherein the adaptors are attached to the ends of the target nucleic acid by extension of a target specific bipartite primer comprising adaptor sequences in the 5′-portion.
4. The method of claim 3 , wherein the adapted target nucleic acid is amplified prior to step (b).
5. The method of claim 1 , further comprising a polymerase fill in step between steps (d) and (e).
6. The method of claim 1 , wherein the adaptor or the backbone comprises at least one barcode.
7. The method of claim 1 , wherein the backbone comprises a ligand for a capture moiety.
8. A method of sequencing a target nucleic acid, the method comprising the steps of:
(a) forming circular nucleic acids according to claim 1 , wherein the adaptor comprises a sequencing primer-binding site;
(b) separating the strands of the circular molecule to generate single-stranded circular molecules;
(c) annealing a sequencing primer to the sequencing primer-binding site in each of the single-stranded circular molecule; and
(d) extending the annealed sequencing primer thereby sequencing the target nucleic acid.
9. The method of claim 8 , wherein the strands in step (b) are separated by heat or by chemical means.
10. The method of claim 8 , wherein the strands in step (b) are separated by nicking one strand and removing the nicked strand by exonuclease digestion.
11. The method of claim 8 , wherein sequencing utilizes a nanopore device.