High throughput screening of populations carrying naturally occurring mutations

Abstract

Efficient methods are disclosed for the high throughput identification of mutations in genes in members of mutagenized populations. The methods comprise DNA isolation, pooling, amplification, creation of libraries, high throughput sequencing of libraries, preferably by sequencing-by-synthesis technologies, identification of mutations and identification of the member of the population carrying the mutation and identification of the mutation.

1 . A method of detecting a genetic variation, comprising:
 (a) providing a plurality of libraries of amplification products, wherein each library comprises one or more target sequences amplified from nucleic acid of a member of a population, and wherein the amplification products of each library are tagged with a tag sequence;   (b) generate sequence reads of the tagged amplification products using high throughput sequencing;   (c) detecting genetic variation by comparing the sequence reads; and   (d) identifying a member of the population carrying the genetic variation using the tag sequence.   
     
     
         2 . The method of  claim 1 , wherein the population is a mutagenized population. 
     
     
         3 . The method of  claim 1 , wherein the genetic variation is naturally occurring. 
     
     
         4 . The method of  claim 1 , wherein the genetic variation is a single nucleotide polymorphism (SNP), a small insertion or a deletion, or a variation in microsatellite repeat number. 
     
     
         5 . The method of  claim 1 , wherein the nucleic acid molecules are human derived. 
     
     
         6 . The method of  claim 1 , wherein the nucleic acid molecules are genomic DNA molecules. 
     
     
         7 . The method of  claim 1 , wherein both ends of each of the plurality of nucleic acid molecules are tagged. 
     
     
         8 . The method of  claim 1 , wherein the sequence reads are generated based on sequencing with a multi-fold redundancy. 
     
     
         9 . The method of  claim 8 , wherein the multi-fold redundancy comprises a redundancy of at least 2. 
     
     
         10 . The method of  claim 8 , wherein the multi-fold redundancy permits distinction between a sequencing error and a genetic variation. 
     
     
         11 . The method of  claim 1 , wherein the detection of the genetic variation comprises identifying a sequence change multiple times. 
     
     
         12 . The method of  claim 1 , wherein genetic variation is detected by comparing the sequence reads to a consensus sequence. 
     
     
         13 . The method of  claim 1 , wherein the identification is performed in silico. 
     
     
         14 . The method of  claim 1 , wherein the amplification products are produced by solution PCR. 
     
     
         15 . The method of  claim 1 , wherein the sequencing is bi-directional sequencing. 
     
     
         16 . The method of  claim 1 , wherein the sequencing is sequencing-by-synthesis. 
     
     
         17 . The method of  claim 1 , wherein the sequencing is performed on a solid support. 
     
     
         18 . The method of  claim 1 , wherein the genetic variation is identified without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis. 
     
     
         19 . The method of  claim 1 , wherein the nucleic acid molecules are tagged using amplification or ligation. 
     
     
         20 . A method of detecting a genetic variation as compared to a reference sequence, comprising:
 (a) providing a plurality of libraries of amplification products, wherein each library comprises one or more target sequences amplified from nucleic acid of a member of a population, and wherein the amplification products of each library are tagged with an tag sequence;   (b) generate sequence reads of the amplification products using high throughput sequencing;   (c) detecting genetic variation by comparing the sequence reads to a reference sequence; and,   (d) identifying a member of the population carrying the genetic variation using the tag sequence.