Distinguishing rare variations in a nucleic acid sequence from a sample

Abstract

The invention generally relates to methods for distinguishing a rare genetic variation in a nucleic acid sequence.

What is claimed is: 
     
         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.