Strategies for high throughput identification and detection of polymorphisms

Abstract

The invention relates to a method for identifying one or more polymorphisms in nucleic acid samples, comprising: (a) performing a reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of the nucleic acid samples comprising amplified fragments, wherein the reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using one or more primers to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence to indicate origin of each library obtained by the reproducible complexity reduction; (b) combining the plurality of libraries to obtain a combined library and sequencing at least a portion of the combined library to obtain sequences; (c) aligning the sequences to obtain an alignment; and (d) identifying one or more polymorphisms in the plurality of nucleic acid samples.

1 . A kit for use in a method for detecting one or more polymorphisms in a plurality of nucleic acid samples, comprising:
 (a) an adaptor comprising an overhang that can be ligated to a protruding end of a restriction fragment produced by digestion with one or more endonucleases; and   (b) a set of primers for PCR amplification,   wherein at least one of the adaptor and the primers comprises an identifier sequence.   
     
     
         2 . The kit of  claim 1 , wherein the identifier sequence identifies the origin of a sample. 
     
     
         3 . The kit of  claim 1 , wherein at least one primer of the set of primers comprises at least one selective oligonucleotide at its 3′-end for hybridizing to a subset of restriction fragments. 
     
     
         4 . The kit according of  claim 1 , wherein the adaptor comprises the identifier sequence. 
     
     
         5 . The kit according of  claim 1 , wherein at least one of the primers comprises the identifier sequence. 
     
     
         6 . The kit according of  claim 1 , wherein the adaptor comprises a PCR primer-binding sequence for hybridizing to a PCR primer. 
     
     
         7 . The kit of  claim 1 , wherein the adaptor comprises a sequencing primer-binding sequence for hybridizing to a sequencing primer. 
     
     
         8 . The kit of  claim 1 , wherein the adaptor comprises sequences complementary to sequences attached to a solid support for annealing the adaptor to the solid support. 
     
     
         9 . The kit of  claim 1 , wherein the adaptor comprises sequences complementary to sequences attached to a bead for annealing the adaptor to the bead. 
     
     
         10 . The kit of  claim 1 , wherein at least one of the primers is phosphorylated. 
     
     
         11 . The kit of  claim 1 , wherein the kit further comprises a biotinylated capture oligonucleotide hybridization probe for capturing a subset of nucleic acid restriction fragments. 
     
     
         12 . The kit of  claim 1 , wherein the kit further comprises a polymerase for a polymerase chain reaction (PCR). 
     
     
         13 . The kit of  claim 12 , wherein the polymerase substantially lacks 3′-5′ exonuclease activity. 
     
     
         14 . The kit of  claim 1 , wherein the adaptor comprises a non-ligatable 5′-end. 
     
     
         15 . The kit of  claim 1 , wherein the adaptor comprises two at least partly complementary synthetic oligonucleotides. 
     
     
         16 . The kit of  claim 1 , wherein the length of the adapter is between 10 and 30 base pairs. 
     
     
         17 . The kit of  claim 1 , wherein the adaptor comprises a PCR primer-binding sequence for hybridizing to a primer of the set of primers for PCR amplification. 
     
     
         18 . The kit of  claim 1 , further comprising one or more endonucleases for producing a plurality of nucleic acid fragments having protruding ends. 
     
     
         19 . The kit of  claim 18 , comprising the endonuclease EcoRI and/or MseI.