High throughput detection of molecular markers based on aflp and high through-put sequencing

Abstract

The present invention relates to a high throughput method for the identification and detection of molecular markers wherein restriction fragments are generated and suitable adaptors comprising (sample-specific) identifiers are ligated. The adapter-ligated restriction fragments may be selectively amplified with adaptor compatible primers carrying selective nucleotides at their 3′ end. The amplified adapter-ligated restriction fragments are, at least partly, sequenced using high throughput sequencing methods and the sequence parts of the restriction fragments together with the sample-specific identifiers serve as molecular marker.

1 .- 22 . (canceled) 
     
     
         23 . A kit for use in a method for detecting one or more polymorphisms in a plurality of nucleic acid samples, comprising:
 one or more adaptors comprising a primer-compatible sequence and an sample-specific identifier sequence, and   one or more primers comprising sequences at its 3′-end a sequence for hybridizing to one or more sample nucleic acid sequences of the plurality of nucleic acid samples.   
     
     
         24 . The kit according to  claim 23 , wherein the kit further comprises one or more primers comprising sequences for hybridizing to the primer-compatible sequence of the one or more adaptors. 
     
     
         25 . The kit according to  claim 23 , wherein the one or more primers comprising sequences at its 3′-end a sequence for hybridizing to one or more sample nucleic acids the plurality of nucleic samples sequence further comprises sequences for hybridizing to the primer-compatible sequence of the one or more adaptors. 
     
     
         26 . The kit according to  claim 23 , wherein the one or more adapters comprise from 5′-end to 3′-end the primer-compatible sequence, the sample-specific identifier sequence, and an end that can be ligated to the blunt or protruding end of a restriction fragment. 
     
     
         27 . The kit according to  claim 23 , wherein the sample-specific identifier sequence comprises from 4-16 nucleotides. 
     
     
         28 . The kit according to  claim 23 , wherein the sample-specific identifier sequence does not contain 2 or more consecutive identical bases. 
     
     
         29 . The kit according to  claim 23 , wherein the adaptor comprises sequences for annealing to a solid support. 
     
     
         30 . The kit according to  claim 23 , wherein the adaptor comprises sequences for annealing to a bead. 
     
     
         31 . The kit according to  claim 23 , wherein at least one of the primers comprises a sequence complementary to the sample specific-identifier sequence of the one or more adaptors. 
     
     
         32 . The kit according to  claim 23 , wherein the one or more primers comprise at its 3′-end sequences for hybridizing to a subset of nucleic acids of the plurality of nucleic acid samples. 
     
     
         33 . The kit according to  claim 23 , wherein the one or more primers further comprise nucleotides selected from locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). 
     
     
         34 . The kit according to  claim 23 , wherein at least one of the primers is phosphorylated. 
     
     
         35 . The kit according to  claim 23 , wherein the kit further comprises a polymerase for a polymerase chain reaction (PCR). 
     
     
         36 . The kit according to  claim 23 , wherein the kit further comprises a ligase for ligating the one or more adaptors to the nucleic acid samples. 
     
     
         37 . The kit according to  claim 23 , wherein the kit further comprises a restriction endonuclease.