STRATEGIES FOR SEQUENCING COMPLEX GENOMES USING HIGH THROUGHPUT SEQUENCING TECHNOLOGIES

• US 20090142758A1
• Filed: 06/23/2006
• Published: 06/04/2009
• Est. Priority Date: 06/23/2005
• Status: Abandoned Application

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Hogers, Rene Cornelis Josephus, Sorensen, Anker Preben, van Eijk, Michael Josephus Theresia
• Application Number:
  US11/993,961
• Publication Number:
  US 20090142758A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2539/107 Representational Difference Analysis [RDA]

Abstract

A method for determining a genome sequence comprising the steps of digesting the genome with at least one first restriction endonuclease, ligating at least one adaptor to the restriction fragments of the first subset, selectively amplifying the first set of adaptor-ligated restriction fragments using a first primer combination wherein at least a first primer contains a first selected sequence at the 3′ end of the primer sequence, comprising 1-10 selective nucleotides, repeating these steps with at least a second primer combinations wherein the primer contains a different second selected sequence, fragmenting each of the subsets of amplified adaptor-ligated restriction fragments to generate sequencing libraries, determine the nucleotide sequence of the fragments, aligning the sequence of the fragments in each of the libraries to generate contigs, repeating these steps for one second and/or further restriction endonucleases, aligning the contigs obtained for each of the second and/or further restriction endonucleases to provide for a sequence of the genome.

Claims

1. A method for determining a genome sequence comprising the steps of:

(a) providing a first subset of the genome by digesting the genome with at least one first restriction endonuclease to provide restriction fragments;

(b) ligating at least one adaptor to the restriction fragments of the first subset to provide a first set of adaptor-ligated restriction fragments;

(c) selectively amplifying the first set of adaptor-ligated restriction fragments using a first primer combination wherein at least a first primer contains a section that is complementary to the adaptor and to part of the recognition sequence of the restriction endonuclease and that further contains a first selected sequence at the 3′

end of the primer sequence, wherein the first selected sequence comprises 1-10 selective nucleotides, to provide a first subset of amplified adaptor-ligated restriction fragments;

(d) repeating step (c) with at least a second and/or further primer combinations wherein the primer contains a different second and/or further selected sequence at its 3′

end that contains the same number of selective nucleotides, to provide for second and/or further subsets of amplified adaptor-ligated restriction fragments;

(e) fragmenting each of the first, second and/or further subsets of amplified adaptor-ligated restriction fragments to generate first, second and/or further sequencing libraries, followed by optional pooling of the libraries;

(f) determine (at least part of) the nucleotide sequence of (at least part of) the fragments contained in each of the first, second and/or further libraries;

(g) aligning the sequence of the fragments in each of the first, second and/or further libraries to generate contigs of the amplified adaptor-ligated restriction fragments derived representing dispersed fractions of the genome;

(h) repeating steps (a)-(g) for at least one second and/or further restriction endonucleases;

(i) aligning the contigs obtained in step (g) and (h) for each of the second and/or further restriction endonucleases to provide for a sequence of the genome.

2. Method according to claim 1, wherein at least one of the first, second and/or further restriction endonucleases is a rare cutter.

3. Method according to claim 1, wherein at least one of the first, second and/or further restriction endonuclease is a frequent cutter.

4. Method according to claim 1, wherein at least two rare cutters are used.

5. Method according to, claim 1 wherein one rare cutter and one frequent cutter are used.

6. Method according to, claim 1, wherein the amplification method is PCR, optionally PCR.

7. Method according to claim 1 wherein the selected sequence at the 3′

end of the primer contains 1-8 selected nucleotides, optionally 1-5, more optionally 1-3.

8. Method according to claim 1, wherein the first, second and further selected sequence have the same number of nucleotides but differ in nucleotide sequence from each other in the selective sequence located at the 3′

-end of the primer.

9. Method according to claim 1, wherein sequencing is performed by Sanger dideoxy sequencing.

10. Method according to claim 1 wherein sequencing is performed on a solid support, optionally bead.

11. Method according to claim 1, wherein the sequencing is based on High Throughput Sequencing, optionally Sequencing-by-Synthesis.

12. Method according to claim 1, wherein the sequencing is based on Sequencing-by-Synthesis, optionally Pyrosequencing.

13. Method according to claim 1, wherein sequencing comprises the steps of:

(f1) ligating sequencing-adaptors to the fragments;

(f2) annealing sequencing-adaptor-ligated fragments to beads, each bead annealing with a single fragment;

(f3) emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead;

(f4) performing emulsion PCR to amplify adaptor-ligated fragments on the surface of beads

(f5) selecting/enriching beads containing amplified adaptor-ligated fragments

(f6) loading the beads in wells, each well comprising a single bead; and

(f7) generating a pyrophosphate signal.

14. Method according to claim 13, wherein contig building is further aided by the use of nucleotide sequences derived from other sources, including, but not limited to BAC-end sequences, BAC shotgun sequences, EST sequences or whole genome shotgun sequences.

15. Method according to claim 13, wherein genome subsets are prepared by digesting the genome with restriction endonucleases to provide restriction fragments, followed by non-selective amplification.

16. Method according to claim 13, wherein the method for reducing the complexity of the mixture is based on indexing linkers, CHIP or PCR primers directed against conserved motifs.