Methods for processing paired end sequences
Recognized herein is the need for methods and processes for increasing the efficiency and accuracy of paired end sequencing.
1. A method for processing a template nucleic acid comprising:
a. providing a first set of beads and a second set of beads, wherein a first bead of the first set of beads comprises a first primer having sequence complementarity with a first adaptor coupled to a first strand of the template nucleic acid, wherein a second bead of the second set of beads comprises a second primer having sequence complementarity with a second adaptor coupled to a second strand of the template nucleic acid, wherein the first bead and the second bead are coupled;
b. coupling the template nucleic acid to the first bead and the second bead, thereby providing a bead-template complex; and
c. sequencing nucleic acid molecules attached to the first bead and the second bead to generate sequencing reads corresponding to the second stand and the first strand, respectively.
2. The method of claim 1 , wherein the nucleic acid molecules comprise one or more copies of the first strand and one or more copies of the second strand, and wherein a first sequence of the one or more copies of the first strand and a second sequence of the one or more copies of the second strand exhibit overlap with each other.
3. The method of claim 2 , wherein the overlap comprises at least 100 nucleotides.
4. The method of claim 1 , wherein the nucleic acid molecules comprise one or more copies of the first strand and one or more copies of the second strand, and wherein the one or more copies of the first strand have no overlap with the one or more copies of the second strand.
5. The method of claim 1 , wherein the first bead and the second bead are coupled via a covalent bond.
6. The method of claim 1 , wherein the first bead and the second bead are irreleasably coupled.
7. The method of claim 1 , wherein the first bead is releasably coupled to the second bead in a releasably coupled bead pair.
8. The method of claim 7 , wherein the first bead and the second bead are coupled through protein interactions or covalent bonds.
9. The method of claim 1 , wherein the sequencing reads for the first strand and the second strand are matched to generate paired end reads.
10. The method of claim 9 , wherein the nucleic acid molecules comprise one or more copies of the first strand and one or more copies of the second strand, and wherein the matching of the sequencing reads is based on overlap between a first sequence of the one or more copies of the first strand and a second sequence from the one or more copies of the second strand.
11. The method of claim 1 , further comprising, prior to (c) subjecting the bead-template complex to conditions sufficient to amplify the first strand on the second bead and the second strand on the first bead to generate one or more copies of the first strand coupled to the second bead and one or more copies of the second strand coupled to the first bead.
12. The method of claim 11 , wherein the amplification is performed in a partition.
13. The method of claim 11 , wherein the amplification comprises PCR.
14. The method of claim 13 , wherein the PCR comprises emulsion PCR (emPCR).
15. The method of claim 11 , wherein the amplification is linear.
16. The method of claim 11 , wherein the amplification is not performed in a partition.
17. The method of claim 1 , wherein the first bead comprises a plurality of first primers each having sequence complementarity to the first adapter, and wherein the second bead comprises a plurality of second primers each having sequence complementarity to the second adapter.
18. The method of claim 1 , wherein the sequencing comprises sequencing by synthesis.
19. The method of claim 1 , further comprising, prior to (a) coupling the first bead and the second bead.
20. The method of claim 1 , wherein the first primer and the second primer are different.