Methods and compositions for preparing sequencing libraries
Embodiments provided herein relate to methods and compositions for next generation sequencing. Some embodiments include the preparation of a template library from a target nucleic acid in contact with a surface, and sequencing the library on the surface.
1. A method of preparing a population of target nucleic acids for sequencing comprising:
(a) providing a substrate having a surface comprising capture probes;
(b) contacting the surface with a reaction volume comprising a plurality of template nucleic acids and transposomes, wherein each transposome comprises a transposon sequence and a transposase, wherein the template nucleic acids are prepared by contacting a target nucleic acid with a plurality of transposomes; and
(c) associating the template nucleic acids with the capture probes in the presence of a recombinase.
2. The method of claim 1 , comprising (d) sequencing the associated template nucleic acids.
3. The method of claim 2 , comprising amplifying the associated template nucleic acids after (c) and before (d).
4. The method of claim 3 , wherein the amplifying comprises bridge amplification.
5. The method of claim 1 , wherein step (a) comprises providing a sample comprising the target nucleic acid and the transposomes.
6. The method of claim 5 , wherein the sample comprises a polymerase and/or a ligase.
7. The method of claim 1 , wherein the template nucleic acids are prepared by contacting the target nucleic acid with the plurality of transposomes in the presence of the surface.
8. The method of claim 1 , wherein the probes comprise nucleic acids.
9. The method of claim 8 , wherein the capture probes comprise the recombinase.
10. The method of claim 1 , wherein the template nucleic acids comprise an affinity moiety selected from the group consisting of biotin, avidin, and streptavidin.
11. The method of claim 1 , wherein the transposon sequence comprises a fragmentation site and wherein the method comprises the step of cleaving the fragmentation site.
12. The method of claim 11 , wherein the fragmentation site is cleaved after step (c).
13. The method of claim 1 , wherein the transposase is removed by contacting the transposase with a protease or SDS.
14. The method of claim 1 , wherein at least one transposome is different from at least one other transposome.
15. The method of claim 1 , comprising amplifying the associated template nucleic acids after (c) using an amplification technique facilitated by the recombinase.
16. The method of claim 15 , wherein the recombinase binds regions of double-stranded nucleic acids comprising non-complementary strands during the amplifying.
17. The method of claim 1 , wherein the capture probes are immobilized on the substrate.