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 transposase and a transposon sequence comprising a sequence that hybridizes to the capture probes, wherein the template nucleic acids are prepared by contacting a target nucleic acid with the plurality of transposomes in the presence of the surface;
(c) associating the template nucleic acids with the capture probes; and
(d) sequencing the template nucleic acids on the surface.
2. The method of claim 1 , comprising amplifying the associated template nucleic acids after (c) and before (d).
3. The method of claim 1 , wherein the amplifying comprises bridge amplification on the surface.
4. The method of claim 1 , wherein (a) comprises providing a sample comprising the target nucleic acid and the transposomes.
5. The method of claim 4 , wherein the sample comprises a polymerase and/or a ligase.
6. The method of claim 1 , wherein the capture probes comprise nucleic acids.
7. The method of claim 6 , wherein (c) comprises hybridizing the template nucleic acids with the capture probes.
8. The method of claim 7 , wherein (c) comprises preparing single-stranded template nucleic acids prior to hybridizing.
9. The method of claim 1 , wherein either the plurality of template nucleic acids or the capture probes comprises an affinity moiety that is biotin and the other of the plurality of template nucleic acids and the capture probes comprises an affinity moiety that is avidin or streptavidin.
10. The method of claim 1 , wherein the transposon sequence comprises a fragmentation site and the method comprises cleaving the fragmentation site.
11. The method of claim 10 , wherein the fragmentation site is cleaved after (c).
12. The method of claim 1 , wherein the transposase is removed from the template nucleic acids after (b) or after (c) by contacting the transposase with a protease or SDS.
13. The method of claim 1 , wherein at least one transposome is different from at least one other transposome.
14. The method of claim 1 , wherein sequencing on the surface comprises determining the proximity of sequences obtained from the template nucleic acids in a linear representation of the target nucleic acid sequence by determining the physical proximity of the captured template nucleic acids on the surface.
15. The method of claim 1 , wherein the associated template nucleic acids are not amplified before the sequencing.