Method for the identification of the clonal source of a restriction fragment
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) providing a sample DNA;
(b) generating an artificial chromosome clone bank wherein each artificial chromosome clone contains part of the sample DNA;
(c) combining the artificial chromosome clones in a plurality of pools, wherein each clone is present in more than one pool of the plurality of pools, to create a library;
(d) digesting the DNA of the clones in the plurality of pools with one or more restriction endonucleases to provide for a set of restriction fragments for each pool;
(e) 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;
(f) determining the sequence of at least the pool-specific identifier and part of the restriction fragment of the adapter-ligated restriction fragments;
(g) assigning the restriction fragment sequences determined in the adapter-ligated restriction fragments to the corresponding clones using the pool-specific identifiers;
(h) building a contig based on sequence matching of the restriction fragment derived sections;
(i) ordering the restriction fragments of step (h) to thereby build a clone-contig and generating a physical map.
2. The method according to claim 1 , wherein the adaptor-ligated restriction fragments of step (e) are combined.
3. The method according to claim 1 , further comprising amplifying the adaptor-ligated restriction fragments of step (e) with at least one primer.
4. The method according to claim 3 , wherein the 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 amplicons.
5. The method according to claim 4 , wherein the amplicons are combined in a set of combined amplicons.
6. The method according to claim 1 , wherein the restriction fragments are assigned to the corresponding clone by clustering adapter-ligated restriction fragments that contain identical sequences in the restriction fragments but carry different pool-specific identifiers.
7. The method according to claim 1 , wherein the sequencing is carried out by means of high-throughput sequencing.
8. The method according to claim 7 , wherein the high-throughput sequencing is performed on a solid support.
9. The method according to claim 7 , wherein the high-throughput sequencing is based on Sequencing-by-Synthesis.
10. The method according to claim 7 , 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
generating a pyrophosphate signal.
11. The method according to claim 7 , 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.
12. The method according to claim 1 , wherein the identifier is from 4-16 bp.
13. The method according to claim 12 , wherein the identifier is from 4-10 bp.
14. The method according to claim 13 , wherein the identifier is from 4-8 bp.
15. The method according to claim 14 , wherein the identifier is from 4-6 bp.
16. The method according to claim 12 , wherein the identifier does not contain 2 or more identical consecutive bases.
17. The method according to claim 12 , wherein for two or more clones, the corresponding identifiers contain at least two different nucleotides.
18. The method according to claim 3 , wherein at least one primer carries 1-10 selective nucleotides at its 3′ end to provide for a random subset of amplicons.
19. The method according to claim 18 , wherein the at least one primer carries 1-4 selective nucleotides at it 3′ end.
20. The method according to claim 1 , wherein the artificial chromosome is a BAC or YAC.