Method for genome sequencing using a sequence-based physical map

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 markers.

1 . A method for the generation of a physical map of at least part of a genome comprising the steps of:
 (a) generating an artificial chromosome clone bank wherein each artificial chromosome clone contains part of a sample DNA;   (b) combining the artificial chromosome clones in one or more pools, wherein each clone is present in more than one pool, to create a library;   (c) digesting the one or more pools with one or more restriction endonucleases to provide restriction fragments for each pool;   (d) ligating adaptors to one or both sides of the restriction fragments, wherein at least one adaptor contains a pool-specific identifier or a degenerate identifier section to provide adaptor-ligated restriction fragments;   (e) optionally, combining the adaptor-ligated restriction fragments;   (f) amplifying the adaptor-ligated restriction fragments of step (d) with at least one primer, which primer contains a pool-specific section corresponding to the pool-specific identifier section in the adaptor or contains a pool-specific identifier at the position of the degenerate identifier section, respectively, to provide amplified adaptor-ligated restriction fragments (amplicons);   (g) optionally, combining the amplicons in a set of combined amplicons;   (h) determining the sequence of at least the pool-specific identifier and part of the restriction fragment of the amplicons or set of combined amplicons;   (i) assigning the restriction fragment sequences determined in the amplicons of step (h) to the corresponding clones using the pool-specific identifiers;   (j) ordering the restriction fragments derived from the same clone to build a contig;   (k) ordering the contigs of the clones of step (j) to thereby build a clone-contig and generate a physical map.   
     
     
         2 . The method according to  claim 1 , wherein the restriction fragments are assigned to the corresponding clone by clustering amplicons that contain identical sequences in the restriction fragments but carry different pool-specific identifiers. 
     
     
         3 . The method according to  claim 1 , wherein the sequencing is carried out by means of high-throughput sequencing. 
     
     
         4 . The method according to  claim 3 , wherein the high-throughput sequencing is performed on a solid support. 
     
     
         5 . The method according to  claim 3 , wherein the high-throughput sequencing is based on Sequencing-by-Synthesis. 
     
     
         6 . The method according to  claim 3 , 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 and enriching beads containing amplified amplicons;   loading the beads in wells, each well comprising a single bead; and   generating a pyrophosphate signal.   
     
     
         7 . The method according to  claim 3 , 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.   
     
     
         8 . The method according to  claim 1 , wherein the identifier is from 4-16 bp. 
     
     
         9 . The method according to  claim 8 , wherein the identifier does not contain 2 or more identical consecutive bases. 
     
     
         10 . The method according to  claim 8 , wherein for two or more clones, the corresponding identifiers contain at least two different nucleotides. 
     
     
         11 . The method according to  claim 1 , wherein the at least one primer carries 1-10 selective nucleotides at it 3′ end to provide for a random subset of amplicons. 
     
     
         12 . A kit comprising one or more primers as defined in  claim 1 . 
     
     
         13 . A kit comprising one or more adaptors as defined in  claim 1 . 
     
     
         14 . A kit comprising primers and adaptors as defined in  claim 1 . 
     
     
         15 . A method for identifying the clonal source of a restriction fragment, comprising:
 (a) digesting artificial chromosome clones in a plurality of pools with one or more restriction endonucleases to provide for a set of restriction fragments for each pool, and wherein each clone is present in more than one pool;   (b) ligating adaptors to one or both sides of the restriction fragments, wherein at least one adaptor contains a pool-specific identifier or a degenerate identifier section, respectively, to provide adaptor-ligated restriction fragments;   (c) sequencing at least the pool-specific identifier and part of the restriction fragment; and   (d) assigning the restriction fragment sequences of step (c) to the corresponding clonal sources using the pool-specific identifiers.   
     
     
         16 . A kit comprising one or more adaptors as defined in  claim 15 .