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:
(a) amplifying a single strand of a nucleic acid template comprising a known target nucleotide sequence, a first nucleotide sequence contiguous to the known target nucleotide sequence, and a 5′ portion comprising an amplification strand of a ligated universal oligonucleotide tail-adaptor, using (i) a first primer that anneals to a 3′ portion of the nucleic acid template comprising the known target nucleotide sequence and (ii) a second primer that comprises a nucleotide sequence identical to a portion of the amplification strand of the ligated universal oligonucleotide tail-adaptor, 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 primer and a nucleotide sequence that is identical to a portion of a first sequencing primer, and
(ii) a fourth primer that comprises a 3′ nucleotide sequence identical to a portion of the second primer and a 5′ portion comprising a nucleic acid sequence that is identical to a portion of a second sequencing primer,
thereby generating a second amplification product.
2 . The method of claim 1 , further comprising
(c) determining the sequence of the first nucleotide sequence in the second amplification product.
3 . The method of claim 2 , wherein step (c) comprises sequencing the second amplification product using at least the first sequencing primer.
4 . The method of claim 1 , wherein the second primer comprises a nucleotide sequence identical to a portion of the second sequencing primer.
5 . The method of claim 1 , wherein the amplification strand comprises a barcode portion and the second amplification product comprises the barcode portion.
6 . The method of claim 1 , wherein the first nucleotide sequence comprises a genetic alteration.
7 . The method of claim 1 , wherein the nucleic acid template comprises DNA.
8 . The method of claim 1 , wherein the first nucleotide sequence comprises an insertion or deletion.
9 . The method of claim 1 , wherein the nucleic acid template comprises cDNA.
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.
12 . The method of claim 10 , wherein the gene rearrangement comprises a fusion oncogene.
13 . 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.
14 . The method of claim 1 , wherein the method further comprises, prior to (a), ligating the universal oligonucleotide tail adaptor to the nucleic acid template comprising the known target nucleotide sequence to produce a ligation product.
15 . The method of claim 14 , wherein the universal oligonucleotide tail adaptor comprises a first ligatable duplex end and a second unpaired end.
16 . The method of claim 14 , wherein the universal oligonucleotide tail adaptor comprises a blocking strand.
17 . A method for preparing a nucleic acid for sequencing, the method comprising:
(a) amplifying a single strand of a nucleic acid template comprising a known target nucleotide sequence, a first nucleotide sequence contiguous to the known target nucleotide sequence, and a 5′ portion comprising an amplification strand of a ligated universal oligonucleotide tail-adaptor, using (i) a target-specific primer that anneals to the 3′ portion of the nucleic acid template comprising the known target nucleotide sequence and (ii) a first adaptor primer that comprises a nucleotide sequence identical to a portion of the amplification strand of the ligated universal oligonucleotide tail-adaptor, 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 second target-specific primer that comprises a nucleotide sequence identical to a portion of the first primer and anneals to the first amplification product of (a), and
(ii) a second adaptor primer that comprises a 3′ nucleotide sequence identical to a portion of the first adaptor primer,
thereby generating a second amplification product.
18 . The method of claim 17 , further comprising:
(d) determining the sequence of the first nucleotide sequence in the second amplification product by a process comprising next-generation sequencing.