DISTINGUISHING RARE VARIATIONS IN A NUCLEIC ACID SEQUENCE FROM A SAMPLE
The invention generally relates to methods for distinguishing a rare genetic variation in a nucleic acid sequence.
1 . A method comprising the steps of:
producing a first strand product from a forward strand of a nucleic acid molecule and a first strand product from a reverse strand of the nucleic acid molecule, wherein the first strand products from the forward strand and the reverse strand each comprise a unique sequence tag that comprises a sequence composition different from other unique sequence tags;
compartmentalizing the first strand products from the forward strand and the reverse into compartmentalized portions, wherein a plurality of the compartmentalized portions comprise only a single first strand product; and
amplifying the forward and reverse strand amplification products in the compartmentalized portions.
2 . The method according to claim 1 , further comprising:
producing a plurality of first strand products from the forward strand and a plurality of first strand products from the reverse strand, wherein the first strand products from the forward strand and the reverse strand each comprise a unique sequence tag that comprises a sequence composition different from other unique sequence tags.
3 . The method according to claim 2 , wherein:
the plurality of first strand products from the forward strand and the plurality of first strand products from the reverse strand, comprise products from a plurality of different loci
4 . The method according to claim 1 , wherein:
the first strand products from the forward strand are produced in a first pool and the first strand products from the reverse strand are produced in a second pool.
5 . The method according to claim 1 , wherein:
the amplification is an exponential amplification.
6 . The method according to claim 5 , wherein:
the exponential amplification comprises PCR.
7 . The method according to claim 1 , wherein:
the first strand products are produced by a polymerase extension reaction.
8 . The method according to claim 7 , wherein:
the polymerase extension reaction employs primers comprising a nucleic acid molecule target specific region, a pool identification tag, a unique sequence tag, and a first universal portion.
9 . The method according to claim 1 , wherein:
the first strand products are produced by a ligation reaction
10 . The method according to claim 1 , further comprising:
sequencing products of the amplifying step to produce a plurality of sequence reads.
11 . The method according to claim 10 , further comprising:
analyzing the sequence reads to identify a variant from a consensus sequence in a plurality of the sequence reads that comprise a unique sequence tag sequence composition that is the same.
12 . The method according to claim 11 , further comprising:
correlating an identified variant from the forward strand with an identified variant from the reverse strand that is complementary to the forward strand.
13 . The method according to claim 11 , wherein the variant is associated with a disease.
14 . The method according to claim 13 , wherein the disease is cancer.
15 . The method according to claim 10 , wherein prior to the sequencing step, the method further comprises incorporating sequencing adaptors with the products of the amplifying step.