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 determining a nucleotide sequence, the method comprising:
(a) amplifying a nucleic acid template comprising a 3′-end comprising a known target nucleotide sequence, a first nucleotide sequence contiguous to the known target nucleotide sequence, and a 5-end comprising an amplification strand of a ligated universal oligonucleotide tail-adaptor, using (i) a first primer that comprises a nucleotide sequence identical to a 5′ portion of a first sequencing primer and (ii) a second primer that anneals to the 3-end of the nucleic acid template comprising the known target nucleotide sequence, thereby generating a first amplification product comprising the first nucleotide sequence and the known target nucleotide sequence;
(b) amplifying the first amplification product of (a) using (i) a third primer that comprises a nucleotide sequence identical to a portion of the first sequencing primer and is nested relative to the first primer and (ii) a fourth primer that comprises a 5′ portion comprising a nucleic acid sequence that is identical to a second sequencing primer, anneals to a 3′ portion of the first amplification product comprising the known target nucleotide sequence, and is nested relative to the second primer, thereby generating a second amplification product; and
(c) determining the sequence of the first nucleotide sequence in the second amplification product.
2. The method of claim 1 , wherein step (c) comprises sequencing the second amplification product using at least the first sequencing primer.
3. The method of claim 2 , wherein the sequencing of the second amplification product further comprises using a second sequencing primer substantially identical to a portion of the fourth primer.
4. The method of claim 1 , wherein the amplification strand comprises a barcode portion and the second amplification product comprises the barcode portion.
5. The method of claim 1 , wherein the first nucleotide sequence comprises a genetic alteration.
6. The method of claim 1 , wherein the nucleic acid template comprises DNA.
7. The method of claim 1 , wherein the first nucleotide sequence comprises an insertion or deletion.
8. The method of claim 1 , wherein the nucleic acid template comprises cDNA.
9. The method of claim 1 , wherein the second primer comprises a 3′-end substantially complementary to the known target nucleotide sequence and a 5′ tag sequence portion comprising a GC content of at least 65%.
10. The method of claim 1 , wherein the known target nucleotide sequence and/or the first nucleotide sequence comprises a sequence comprising a gene rearrangement.
11. The method of claim 10 , wherein the gene rearrangement comprises a fusion oncogene.
12. The method of claim 1 , wherein the nucleic acid template is prepared from a sample obtained from a subject having a condition associated with a genetic alteration.
13. The method of claim 1 , wherein the known target nucleotide sequence and/or the first nucleotide sequence comprises a sequence from a gene selected from the group of: anaplastic lymphoma kinase (ALK); c-ros oncogene 1 (ROS1); and rearranged during transfection (RET).
14. A method for determining a nucleotide sequence, the method comprising:
(a) ligating an amplification strand of a universal oligonucleotide tail-adaptor to a 5′-end of a nucleic acid template comprising (i) a 3-end comprising a known target nucleotide sequence and (ii) a first nucleotide sequence contiguous to the known target nucleotide sequence;
(b) amplifying the nucleic acid template using (i) a first primer that comprises a nucleotide sequence identical to a 5′ portion of a first sequencing primer and (ii) a second primer that anneals to the 3′-end of the nucleic acid template comprising the known target nucleotide sequence, thereby generating a first amplification product comprising the first nucleotide sequence and the known target nucleotide sequence;
(c) amplifying the first amplification product of (b) using (i) a third primer that comprises a nucleotide sequence identical to a portion of the first sequencing primer and that is nested relative to the first primer and (ii) a fourth primer that comprises a 5′ portion comprising a nucleic acid sequence that is identical to a second sequencing primer, anneals to a 3′ portion of the first amplification product comprising the known target nucleotide sequence and is nested relative to the second primer, thereby generating a second amplification product comprising the first nucleotide sequence; and
(d) determining the sequence of the first nucleotide sequence in the second amplification product by a process comprising next-generation sequencing.