Methods for determining a nucleotide sequence contiguous to a known target nucleotide sequence
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 preparing a nucleic acid for sequencing, the method comprising:
(i) ligating a universal oligonucleotide tail adaptor that comprises a first ligatable duplex end and a second unpaired end to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, the universal oligonucleotide tail adaptor comprising an amplification strand and a blocking strand, wherein a 3′ duplex portion of the amplification strand and a 5′ duplex portion of the blocking strand are substantially complementary and form the first ligatable duplex end;
(ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a first adaptor primer having a nucleotide sequence identical to a first portion of the amplification strand; and
(iii) amplifying an amplification product of (ii) using a second target-specific primer that specifically anneals to the amplification product of (ii) and a second adaptor primer having a nucleotide sequence identical to a second portion of the amplification strand, wherein the second target-specific primer is nested relative to the first target-specific primer.
2. The method of claim 1 , wherein the entirety of the blocking strand is a duplex portion that is substantially complementary to the 3′ duplex portion of the amplification strand.
3. The method of claim 1 , wherein the blocking strand further comprises a 3′ unpaired portion which is not substantially complementary to the 5′ unpaired portion of the amplification strand.
4. The method of claim 1 , wherein the duplex portions of the amplification and blocking strands are at least 95% complementary, at least 98% complementary, at least 99% complementary, or 100% complementary.
5. The method of claim 1 , wherein the nucleic acid is a deoxyribonucleic acid.
6. The method of claim 5 , wherein the method further comprises subjecting a ribonucleic acid to a reverse transcriptase regimen to generate the deoxyribonucleic acid.
7. 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.
8. The method of claim 7 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.
9. The method of claim 7 further comprising, prior to step (i), phosphorylating the nucleic acid.
10. The method of claim 1 further comprising, prior to step (i), adenylating the nucleic acid to produce a 3′-adenosine overhang on the nucleic acid.
11. The method of claim 1 , wherein ligating in step (i) comprises performing an overhang ligation reaction.
12. The method of claim 1 , wherein ligating in step (i) comprises performing a TA ligation reaction.
13. The method of claim 1 , wherein the first target-specific primer can specifically anneal to the known target nucleotide sequence at an annealing temperature in a range of 61° C. to 72° C.
14. The method of claim 1 , wherein the known target nucleotide sequence comprises a sequence associated with a gene rearrangement.
15. The method of claim 1 , wherein the universal oligonucleotide tail adaptor further comprises a barcode portion.
16. A method for preparing a nucleic acid for sequencing, the method comprising:
(i) ligating a universal oligonucleotide tail adaptor that comprises a first ligatable duplex end and a second unpaired end to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, the universal oligonucleotide tail adaptor comprising an amplification strand and a blocking strand, wherein a 3′ duplex portion of the amplification strand and a 5′ duplex portion of the blocking strand are substantially complementary and form the first ligatable duplex end;
(ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a first adaptor primer having a nucleotide sequence identical to a first portion of the amplification strand; and
(iii) amplifying an amplification product of (ii) using a second target-specific primer that specifically anneals to the amplification product of (ii) and a second adaptor primer having a nucleotide sequence identical to a second portion of the amplification strand, wherein ligating in step (i) comprises performing an overhang ligation reaction, and wherein the universal oligonucleotide tail adaptor further comprises a barcode portion.
17. The method of claim 16 further comprising, prior to step (i), phosphorylating the nucleic acid.
18. The method of claim 16 further comprising, prior to step (i), adenylating the nucleic acid to produce a 3′-adenosine overhang on the nucleic acid.
19. The method of claim 16 , wherein ligating in step (i) comprises performing a TA ligation reaction.
20. The method of claim 16 further comprising sequencing an amplification product of (iii) using a first sequencing primer and a second sequencing primer.