Methods of preparing nucleic acids for sequencing
Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
1. A method of preparing nucleic acids for analysis, the method comprising:
(a) contacting a first nucleic acid template comprising a sequence of a first strand of a double-stranded target nucleic acid with a complementary target-specific primer that comprises a target-specific hybridization sequence, under conditions to promote template-specific hybridization and extension of the target-specific primer;
(b) contacting a second nucleic acid template comprising a sequence of a second strand that is complementary to the sequence of the first strand of the double-stranded target nucleic acid with a plurality of different primers that share a common sequence that is 5′ to different hybridization sequences, under conditions to promote template-specific hybridization and extension of at least one of the plurality of different primers, wherein the different hybridization sequences have different 3′ ends, and wherein each primer of the plurality of different primers does not anneal to the same sequence of the double-stranded target nucleic acid as any other primer of the plurality of different primers,
wherein, following (a) and (b), an extension product is generated to contain both a sequence that is characteristic of the target-specific primer and a sequence that is characteristic of the at least one of the plurality of different primers; and
(c) subjecting the extension product to an amplification reaction comprising successive rounds of polymerase extension of i) a tail primer that comprises a 3′ sequence that specifically anneals to the complement of the common sequence and that comprises a 5′ tail sequence, and ii) a primer that specifically anneals to the complement of the target-specific hybridization sequence.
2. The method of claim 1 , wherein the first strand or the second strand of the double-stranded target nucleic acid is a ribonucleic acid.
3. The method of claim 1 , wherein the first strand or the second strand of the double-stranded target nucleic acid is a deoxyribonucleic acid.
4. The method of claim 1 , wherein steps (a) and (b) are performed sequentially.
5. The method of claim 1 , wherein the first nucleic acid template comprises an extension product resulting from the hybridization and extension of the at least one of the plurality of different primers in step (b).
6. The method of claim 1 , wherein the second nucleic acid template comprises an extension product resulting from the hybridization and extension of the target-specific primer in step (a).
7. The method of claim 2 , wherein the ribonucleic acid is a messenger RNA encoded from a chromosomal segment that comprises a genetic rearrangement.
8. The method of claim 3 , wherein the double-stranded target nucleic acid is a chromosomal segment that comprises a portion of a genetic rearrangement.
9. The method of claim 8 , wherein the genetic rearrangement is an inversion, deletion, or translocation.
10. The method of claim 1 , further comprising contacting the extension product or amplified extension product with an immobilized oligonucleotide under conditions in which hybridization occurs between the extension product and immobilized oligonucleotide.
11. The method of claim 1 , wherein the double-stranded target nucleic acid comprises a target portion having a known sequence and a flanking portion having an unknown sequence.
12. The method of claim 11 , wherein different hybridization sequences are complementary to the flanking portion.
13. The method of claim 11 , wherein the target-specific hybridization sequence is complementary to the target portion.
14. The method of claim 1 , wherein the target-specific primer further comprises, 5′ to the target-specific hybridization sequence, at least one of an index sequence, a barcode sequence and an adaptor sequence.
15. The method of claim 1 , wherein the common sequence comprises at least one of an index sequence, barcode sequence and an adaptor sequence.
16. The method of claim 14 , wherein the adaptor sequence is a cleavable adaptor sequence for immobilizing oligonucleotides in a flow cell.
17. The method of claim 1 , wherein the tail primer comprises a nucleic acid sequence identical to the common sequence.
18. The method of claim 1 , wherein the 5′ tail sequence comprises at least one of an index sequence, a barcode sequence, an adaptor sequence, and a sequencing primer hybridization sequence.
19. The method of claim 1 , wherein the primer that specifically anneals to the complement of the target-specific hybridization sequence comprises a sequence identical to the target-specific primer.
20. The method of claim 1 , wherein the primer that specifically anneals to the complement of the target-specific hybridization sequence comprises at least one modified internucleoside linkage.
21. The method of claim 1 , wherein the target-specific primer comprises at least one modified internucleoside linkage.
22. The method of claim 1 further comprising purifying the extension product prior to (c).
23. The method of claim 1 further comprising purifying the amplified product of (c).
24. The method of claim 1 , wherein the plurality of different primers can specifically anneal to sequences comprised by separate genes.
25. The method of claim 1 , wherein the plurality of different primers can specifically anneal to different portions of the first strand or the second strand of the double-stranded target nucleic acid comprised by a single gene.
26. The method of claim 1 , wherein the plurality of different primers can specifically anneal to different exons of a gene comprising the first strand or the second strand of the double-stranded target nucleic acid.
27. The method of claim 1 , wherein the first strand or the second strand of the double-stranded target nucleic acid is derived from a biological sample.
28. The method of claim 27 , wherein the first strand or the second strand of the double-stranded target nucleic acid is a cDNA produced from RNA in the biological sample.
29. The method of claim 1 , wherein the double-stranded target nucleic acid is derived from a sample comprising nucleic acids that have been ligated to an oligonucleotide comprising a barcode sequence.