Genotyping by next-generation sequencing

Abstract

Provided herein is technology relating to genotyping and particularly, but not exclusively, to methods for genotyping one or more organisms by genome sequencing.

We claim: 
     
         1 . A method for genotyping by sequencing, the method comprising:
 1) digesting a nucleic acid with a restriction enzyme to produce a fragment;   2) ligating a single-stranded barcode oligonucleotide to the fragment to produce a template;   3) amplifying the template to produce an amplicon; and   4) sequencing the amplicon to produce a sequence read.   
     
     
         2 . The method of  claim 1  wherein a template pool is produced by mixing a plurality of templates. 
     
     
         3 . The method of  claim 1  wherein a template pool is produced by mixing a plurality of templates from a plurality of individuals. 
     
     
         4 . The method of  claim 1  further comprising parsing the sequence read, mapping the sequence read, and assigning a genotype. 
     
     
         5 . The method of  claim 1  wherein the nucleic acid is digested with two different restriction enzymes. 
     
     
         6 . The method of  claim 1  wherein the nucleic acid is digested with NspI and BfuCI. 
     
     
         7 . The method of  claim 1  wherein the single-stranded barcode oligonucleotide identifies a subject that was the source of the nucleic acid. 
     
     
         8 . The method of  claim 1  wherein the amplifying comprises the use of a target specific primer. 
     
     
         9 . The method of  claim 1  wherein the amplifying selects an amplicon for sequencing. 
     
     
         10 . A method for genotyping by sequencing, the method comprising:
 1) providing a first plurality of nucleic acids from a first subject;   2) providing a second plurality of nucleic acids from a second subject;   3) digesting the first plurality of nucleic acids with a restriction enzyme to produce a first plurality of fragments;   4) digesting the second plurality of nucleic acids with the restriction enzyme to produce a second plurality of fragments;   5) ligating a first single-stranded barcode oligonucleotide to each fragment of the first plurality of fragments to produce a first plurality of templates;   6) ligating a second single-stranded barcode oligonucleotide to each fragment of the second plurality of fragments to produce a second plurality of templates;   7) mixing the first plurality of templates and the second plurality of templates to produce a template pool;   8) amplifying a subset of the template pool using a target specific primer to produce a plurality of amplicons; and   9) sequencing the plurality of amplicons to produce a plurality of sequence reads;   10) deconvoluting the sequence reads using a first sequence of the first barcode oligonucleotide and a second sequence of the second barcode oligonucleotide.   
     
     
         11 . A composition comprising a single-stranded barcode oligonucleotide. 
     
     
         12 . The composition of  claim 11  further comprising a second single-stranded oligonucleotide. 
     
     
         13 . A composition comprising a nucleic acid, wherein the nucleic acid sequence comprises a sequence of a single-stranded barcode oligonucleotide, a sequence of a target site, and a sequence of a second single-stranded oligonucleotide. 
     
     
         14 . The composition of  claim 13  further comprising a first amplification primer complementary to the single-stranded barcode oligonucleotide and a target specific amplification primer complementary to the second single-stranded oligonucleotide.