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 . Method for the identification of restriction fragments in a sample, comprising the steps of:
 (a) providing a sample nucleic acid;   (b) digesting the sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments;   (c) providing double stranded synthetic adaptors comprising
 a 5′ primer-compatible sequence, 
 a sample-specific identifier section, 
 a section that is complementary to the remains of the recognition sequence of the restriction endonuclease; 
   (d) ligating the double stranded synthetic adaptors to the restriction fragments in the set, to provide a set of adaptor-ligated restriction fragments;   (e) amplification of the set of adaptor-ligated restriction fragments, with one or more primers that are at least complementary to:
 the sample-specific identifier section, 
 the section that is complementary to the remains of the recognition sequence of the restriction endonuclease, 
   to provide for amplified adaptor-ligated restriction fragments (amplicons);   (f) determining the sequence of at least the sample-specific identifier section, the remains of the recognition sequence of the restriction endonuclease and of part of the sequence of the restriction fragment located adjacent thereto of (part of) the amplified adaptor-ligated restriction fragments,   (g) identifying the presence or absence of amplified adaptor-ligated restriction fragments in the sample.   
     
     
         2 . Method according to  claim 1 , wherein the restriction fragments are molecular markers. 
     
     
         3 . Method according to  claim 2 , wherein the molecular markers are AFLP markers. 
     
     
         4 . Method according to  claim 1 , wherein two or more samples are compared for the presence or absence of restriction fragments and/or molecular markers. 
     
     
         5 . Method according to  claim 1 , wherein two or more samples are combined in a pool after the step of ligating the adaptors. 
     
     
         6 . Method according to  claim 5 , wherein for each sample in the pool a sample-specific identifier is used that differs from the other sample-specific identifiers in the pool. 
     
     
         7 . Method according to  claim 1 , wherein the primers contain one or more selective nucleotides at the 3′ end. 
     
     
         8 . Method according to  claim 1 , wherein the restriction endonuclease is a type II restriction endonuclease. 
     
     
         9 . Method according to  claim 1 , wherein the restriction endonuclease is a type IIs restriction endonuclease. 
     
     
         10 . Method according to  claim 1 , wherein two or more restriction endonucleases are used. 
     
     
         11 . Method according to  claim 1 , wherein the sequencing is carried out by means of high-throughput sequencing. 
     
     
         12 . Method according to  claim 8 , wherein the high-throughput sequencing is performed on a solid support. 
     
     
         13 . Method according to  claim 8 , wherein the high-throughput sequencing is based on Sequencing-by-Synthesis. 
     
     
         14 . Method according to  claim 8 , wherein the high-throughput sequencing comprises the steps of:
 annealing the amplicons or adapter-ligated restriction fragments to beads, each bead annealing with a single adapter-ligated restriction fragments or amplicon;   emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead;   performing emulsion PCR to amplify adapter-ligated restriction fragments or amplicons on the surface of beads;   optionally, selecting/enriching beads containing amplified amplicons;   loading the beads in wells, each well comprising a single bead; and   determining the nucleotide sequence of the amplified adapter-ligated restriction fragments or amplified amplicons using generating a pyrophosphate signal.   
     
     
         15 . Method according to  claim 8 , wherein the high-throughput sequencing comprises the steps of:
 annealing the adapter-ligated restriction fragments or amplicons to a surface containing first and second primers or first and second primer binding sequences respectively,   performing bridge amplification to provide clusters of amplified adapter-ligated restriction fragments or amplified amplicons,   determining the nucleotide sequence of the amplified adapter-ligated restriction fragments or amplified amplicons using labelled reversible terminator nucleotides.   
     
     
         16 . Method according to  claim 1 , wherein the identifier is from 4-16 bp, preferably from 4-10, more preferably from 4-8, most preferably from 4-6 bp. 
     
     
         17 . Method according to  claim 13 , wherein the identifier does not contain 2 or more identical consecutive bases. 
     
     
         18 . Method according to  claim 13 , wherein for two or more samples, the corresponding identifiers contain at least two different nucleotides. 
     
     
         19 - 22 . (canceled) 
     
     
         23 . A composition comprising an EcoRI adaptor comprising a P5 sequence and an MseI adaptor comprising a P7 sequence. 
     
     
         24 . A kit comprising one or more PCR primers and one or more ligation adaptors;
 wherein the PCR primer comprises, from 5′ to 3′, a primer sequence, a sample identifier sequence, a degenerate type IIs cohesive end sequence, and selective nucleotides;   wherein the ligation adaptor comprises, from 5′ to 3′, a primer binding site, a sample identifier sequence, and a degenerate type IIs cohesive end sequence.