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 . A method for the identification of restriction fragments in a plurality of samples, comprising the steps of:
 (a) providing a plurality of sample nucleic acids;   (b) digesting each sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments;   (c) providing at least two double stranded synthetic adaptors comprising a primer compatible sequence and a sample-specific identifier section;   (d) ligating the double stranded synthetic adaptors to the restriction fragments in the set, to provide a set of adaptor-ligated restriction fragments;   (e) amplifying the set of adaptor-ligated restriction fragments, with one or more primers that are complementary to at least a portion of the adapter to provide for amplified adaptor-ligated restriction fragments (amplicons),   (f) determining the sequence of at least the sample-specific identifier section, and part of the sequence of the restriction fragment of the amplified adaptor-ligated restriction fragments;   (g) identifying the presence or absence of amplified adaptor-ligated restriction fragments or fragment sequences in the samples; and   (h) comparing the identified fragments or fragment sequences between samples.   
     
     
         2 . The method according to  claim 1 , wherein the restriction fragments are molecular markers. 
     
     
         3 . The method according to  claim 2 , wherein the molecular markers are AFLP markers. 
     
     
         4 . The method according to  claim 1 , wherein two or more samples are compared for the presence or absence of restriction fragments or fragment sequences and/or molecular markers. 
     
     
         5 . The method according to  claim 1 , wherein two or more samples are combined in a pool after the step of ligating the adaptors to provide for pooled adaptor-ligated restriction fragments. 
     
     
         6 . The 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 . The method according to  claim 1 , wherein the primers contain one or more selective nucleotides at the 3′end. 
     
     
         8 . The method according to  claim 1 , wherein the restriction endonuclease is a type II restriction endonuclease. 
     
     
         9 . The method according to  claim 1 , wherein the restriction endonuclease is a type IIs restriction endonuclease. 
     
     
         10 . The method according to  claim 1 , wherein two or more restriction endonucleases are used. 
     
     
         11 . The method according to  claim 1 , wherein the sequencing is carried out by means of high-throughput sequencing. 
     
     
         12 . The method according to  claim 8 , wherein the high-throughput sequencing is performed on a solid support. 
     
     
         13 . The method according to  claim 8 , wherein the high-throughput sequencing is based on Sequencing-by-Synthesis. 
     
     
         14 . The 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 . The 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 labeled reversible terminator nucleotides. 
     
     
         16 . The method according to  claim 1 , wherein the identifier is from 4-16 bp. 
     
     
         17 . The method according to  claim 13 , wherein the identifier does not contain 2 or more identical consecutive bases. 
     
     
         18 . The method according to  claim 13 , wherein for two or more samples, the corresponding identifiers contain at least two different nucleotides. 
     
     
         19 . A method for the identification of molecular markers for genotyping, bulk segregant analysis, genetic mapping, marker-assisted back-crossing, mapping of quantitative trait loci, or linkage disequilibrium mapping, comprising the steps of:
 (a) providing a plurality of sample nucleic acids;   (b) digesting each sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments;   (c) providing double stranded synthetic adaptors comprising a primer-compatible sequence and a sample-specific identifier section;   (d) ligating the double stranded synthetic adaptors to the restriction fragments in the set, to provide a set of adaptor-ligated restriction fragments;   (e) amplifying the set of adaptor-ligated restriction fragments, with one or more primers that are complementary to at least a portion of the adaptor to provide for amplified adaptor-ligated restriction fragments (amplicons);   (f) determining the sequence of at least the sample-specific identifier section and part of the sequence of the restriction fragment of the amplified adaptor-ligated restriction fragments; and   (g) identifying the presence or absence of amplified adaptor-ligated restriction fragments or fragment sequences in the sample; and   (h) comparing the identified fragments or fragment sequences between samples.   
     
     
         20 . A kit comprising one or more primers as defined in  claim 1 . 
     
     
         21 . A kit comprising one or more adaptors as defined in  claim 1 . 
     
     
         22 . A kit comprising primers and adaptors as defined in  claim 1 . 
     
     
         23 . A method according to  claim 16 , wherein the identifier is from 4-10 bp. 
     
     
         24 . A method according to  claim 16 , wherein the identifier is from 4-8 bp. 
     
     
         25 . A method according to  claim 16 , wherein the identifier is from 4-6 bp.