Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
View Patent ↗The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.
1. A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising:
(i) ligating a universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence to produce a ligation product;
(ii) amplifying the ligation product by polymerase chain reaction using a first adaptor primer that specifically anneals to the universal oligonucleotide tail-adaptor, and a first target-specific primer that specifically anneals to the known target nucleotide sequence;
(iii) amplifying an amplification product of (ii) by polymerase chain reaction using a second adaptor primer and a second target-specific primer, wherein the second adaptor primer and the second target-specific primer are nested relative to the first adaptor primer and the first target-specific primer, respectively; and
(iv) sequencing an amplification product of (iii) using a first sequencing primer and a second sequencing primer, wherein the first sequencing primer and the second sequencing primer are complementary to opposite strands of the amplification product of (iii).
2. The method of claim 1 further comprising, prior to step (i), mechanically shearing a nucleic acid preparation to produce the nucleic acid comprising the known target nucleotide sequence.
3. The method of claim 2 further comprising, prior to step (i), end-repairing the nucleic acid to blunt the ends of the nucleic acid produced by mechanically shearing the nucleic acid preparation.
4. The method of claim 3 further comprising, prior to step (i), phosphorylating the nucleic acid.
5. The method of claim 4 further comprising, prior to step (i), adenylating the nucleic acid to produce a 3′-adenosine overhang on the nucleic acid.
6. The method of claim 1 , wherein the nucleic acid comprises cDNA.
7. The method of claim 1 , wherein the nucleic acid comprises genomic DNA.
8. The method of claim 1 , wherein the nucleic acid comprises RNA.
9. The method of claim 8 , wherein the RNA is mRNA.
10. The method of claim 1 , wherein the nucleic acid is prepared from a formalin-fixed and paraffin-embedded (FFPE) biological specimen.
11. The method of claim 1 , wherein the nucleic acid is prepared from a fresh biological specimen.
12. The method of claim 2 , wherein, after mechanically shearing the nucleic acid preparation, the resulting nucleic acid is subjected to a cleanup procedure.
13. The method of claim 12 , wherein the cleanup procedure is a Solid Phase Reversible Immobilization (SPRI) cleanup procedure.
14. The method of claim 2 , wherein the nucleic acid is subjected to end-repair followed by ligation of the universal oligonucleotide tail-adaptor to the nucleic acid in step (i) without an intervening cleanup procedure.
15. The method of claim 2 , wherein the nucleic acid is further subjected to end-repair and phosphorylation followed by ligation of the universal oligonucleotide tail-adaptor to the nucleic acid in step (i) without an intervening cleanup procedure.
16. The method of claim 2 , wherein the nucleic acid is further subjected to end-repair, phosphorylation and dA-tailing followed by ligation of the universal oligonucleotide tail-adaptor to the nucleic acid in step (i) without an intervening cleanup procedure.
17. The method of claim 1 , wherein the universal oligonucleotide tail-adaptor is Y-shaped and has a truncated 3′ arm.
18. The method of claim 1 , wherein the universal oligonucleotide tail-adaptor is a half-truncated Y-shaped adaptor.
19. The method of claim 1 , wherein the universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein the duplex portion comprises up to 17 base pairs (bp).
20. The method of claim 1 , wherein the universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein the duplex portion comprises at least 7 bp.
21. The method of claim 19 , wherein the universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein the duplex portion comprises at least 7 and up to 17 bp.
22. The method of claim 20 , wherein the universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein duplex portion is not so long as to substantially suppress amplification in step (ii).
23. The method of claim 20 , wherein the universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein the duplex portion is of limited length to avoid the formation of intramolecular hairpins during amplification.
24. The method of claim 1 , wherein the duplex portion of the universal oligonucleotide tail-adaptor is of sufficient length to remain in duplex form during ligation in step (i).
25. The method of claim 1 , wherein ligating in step (i) comprises performing a blunt-end ligation reaction.
26. The method of claim 1 , wherein ligating in step (i) comprises performing an overhang ligation reaction.
27. The method of claim 1 , wherein ligating in step (i) comprises performing a TA ligation reaction.
28. The method of claim 1 , wherein the first target-specific primer comprises a nucleic acid that can specifically anneal to the known target nucleotide sequence at an annealing temperature in a range of 61° C. to 72° C.
29. The method of claim 28 , wherein the annealing temperature is about 65° C.
30. The method of claim 1 , wherein the second adaptor primer is nested with respect to the first adaptor primer by at least 3 nucleotides.
31. The method of claim 30 , wherein the second adaptor primer is nested with respect to the first adaptor primer by at least 3 and up to 10 nucleotides.
32. The method of claim 1 , wherein the PCR of step (ii) or step (iii) comprises 5 cycles to 20 cycles.
33. The method of claim 1 , wherein the amplification product of step (ii) is subjected to a cleanup procedure prior to being amplified in step (iii).
34. The method of claim 1 , wherein sequencing in step (iv) comprises performing a next-generation sequencing assay.
35. The method of claim 1 , wherein the known target nucleotide sequence comprises a sequence associated with a gene rearrangement.
36. The method of claim 35 , wherein the gene rearrangement results in a fusion oncogene.
37. The method of claim 1 , wherein the nucleic acid is prepared from a sample obtained from a subject suspected of having a gene rearrangement.
38. The method of claim 1 , wherein the universal oligonucleotide tail-adaptor further comprises a barcode portion.
39. The method of claim 1 further comprising pooling multiple samples of ligation products prior to step (ii), and amplifying each of the multiple samples in step (ii) to produce the amplification product of step (ii), each sample having a ligated universal oligonucleotide tail-adaptor containing a unique barcode portion.
40. A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising:
(i) ligating a truncated Y-shaped universal oligonucleotide tail-adaptor to a nucleic acid comprising the known target nucleotide sequence to produce a ligation product;
(ii) amplifying the ligation product by polymerase chain reaction using a first adaptor primer that specifically anneals to the truncated Y-shaped universal oligonucleotide tail-adaptor, and a first target-specific primer that specifically anneals to the known target nucleotide sequence;
(iii) amplifying an amplification product of (ii) by polymerase chain reaction using a second adaptor primer and a second target-specific primer, wherein the second adaptor primer and the second target-specific primer are nested relative to the first adaptor primer and the first target-specific primer, respectively; and
(iv) sequencing an amplification product of (iii) using a first sequencing primer and a second sequencing primer, wherein the first sequencing primer and the second sequencing primer are complementary to opposite strands of the amplification product of (iii).
41. The method of claim 40 , wherein the truncated Y-shaped universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein the duplex portion comprises up to 17 base pairs (bp).
42. The method of claim 41 , wherein the truncated Y-shaped universal oligonucleotide tail-adaptor comprises a ligatable duplex portion and an unpaired end, wherein the duplex portion comprises at least 7 and up to 17 bp.
43. The method of claim 40 , wherein the second adaptor primer is nested with respect to the first adaptor primer by at least 3 and up to 10 nucleotides.
44. The method of claim 41 , wherein the known target nucleotide sequence comprises a sequence associated with a gene rearrangement.
45. The method of claim 44 , wherein the gene rearrangement results in a fusion protein.
46. The method of claim 41 , wherein the nucleic acid is prepared from a sample obtained from a subject suspected of having a gene rearrangement.
47. The method of claim 40 , wherein the second adaptor primer is nested with respect to the first adaptor primer by at least 3 nucleotides.