Method for the identification of the clonal source of a restriction fragment

1. 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.

2. The method according to claim 1 , wherein the restriction fragments are assigned to the corresponding clone by clustering identical sequences that are in part of the restriction fragments, which 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: (a) annealing adapter-ligated restriction fragments to beads, each bead annealing with a single adapter-ligated restriction fragments; (b) emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead; (c) performing emulsion PCR to amplify adapter-ligated restriction fragments on the surface of beads; (d) optionally, selecting or enriching the beads containing adaptor-ligated restriction fragments; (e) loading the beads in wells, each well comprising a single bead; and (f) generating a pyrophosphate signal.

7. The method according to claim 3 , wherein the high-throughput sequencing comprises the steps of: (a) annealing the adapter-ligated restriction fragments to a surface containing first and second primers or first and second primer binding sequences respectively, (b) performing bridge amplification to provide clusters of amplified adapter-ligated restriction fragments, and (c) determining the nucleotide sequence of the amplified adapter-ligated restriction fragments using labeled 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 8 , wherein the identifier is from 4-10 base pairs.

12. The method according to claim 8 , wherein the identifier is from 4-8 base pairs.

13. The method according to claim 8 , wherein the identifier is from 4-6 base pairs.

14. The method according to claim 1 , further comprising combining the adaptor-ligated restriction fragments of step (b).

15. The method of claim 1 , further comprising amplifying the adaptor-ligated restriction fragments of step (b) with at least one primer that comprises (i) a pool-specific section corresponding to the pool-specific identifier section in the adaptor or (ii) a pool-specific identifier at the position of the degenerate identifier section, respectively, to provide amplified adaptor-ligated restriction fragments.

16. The method of claim 15 , further comprising combining the amplified adaptor-ligated restriction fragments.

17. The method of claim 16 , further comprising sequencing at least the pool-specific identifier and part of the restriction fragment of the amplified adaptor-ligated restriction fragments.

18. The method of claim 1 , further comprising ordering the restriction fragments derived from the same clone to build a contig.

19. The method of claim 18 , further comprising ordering the contigs of the clones to thereby build a clone-contig and generate a physical map.

20. The method of claim 15 , wherein the restriction fragments are assigned to the corresponding clone by clustering amplified adaptor-ligated restriction fragments that contain identical sequences in part of the restriction fragments but carry different pool-specific identifiers.

21. The method of claim 15 , wherein the at least one primer carries 1-10 selective nucleotides at it 3′ end to provide for a random subset of amplified adaptor-ligated restriction fragments.

22. The method of claim 21 , wherein the at least one primer carries 1-4 selective nucleotides at it 3′ end to provide for a random subset of amplified adaptor-ligated restriction fragments.

23. A method for identifying the clonal source of a restriction fragment, comprising:

(a) ligating adaptors to one or both sides of restriction fragments obtained from digesting artificial chromosome clones in a plurality of pools, wherein each clone is present in more than one pool, with one or more restriction endonucleases, wherein at least one adaptor contains a pool-specific identifier or a degenerate identifier section, respectively, to provide adaptor-ligated restriction fragments;

(b) sequencing at least the pool-specific identifier and part of the restriction fragment; and

(c) assigning the restriction fragment sequences of step (b) to the corresponding clonal sources using the pool-specific identifiers.