Method for high-throughput AFLP-based polymorphism detection

• US 8,815,512 B2
• Filed: 11/01/2012
• Issued: 08/26/2014
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia, Srensen, Anker Preben, Van Schriek, Marco Geradus Maria
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US13/666,385
• Publication Number:
  US 20130059739A1
• Time in Patent Office: 663 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 506/2, 536/23.1, 536/24.2, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2535/138 Amplified fragment length polymorphism [AFLP]
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The invention relates to a method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of restriction endonuclease digest of DNA, adaptor-ligation, optional pre-amplification, selective amplification, pooling of the amplified products, sequencing the libraries with sufficient redundancy, clustering followed by identification of the genetic markers within the library and/or between libraries and determination of (co-)dominant genotypes of the genetic markers.

Claims

1. A method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of:

(a) providing DNA from one or more samples;

(b) restricting the DNA with at least one restriction endonuclease to produce at least one restriction fragment comprising sticky remains of a restriction site;

(c) ligating at least one adaptor to the at least one restriction fragment to produce at least one adaptor-ligated restriction fragment, wherein one end of the adaptor comprises an overhang that is compatible with the sticky remains of the restriction site of the restriction fragment;

(d) amplifying by solution PCR the at least one adaptor-ligated restriction fragment with at least one primer pair to produce a library of at least one amplified adaptor-ligated double stranded restriction fragment, wherein the amplified adaptor-ligated restriction fragment comprises an identifier tag;

(e) sequencing a pool of two or more libraries of tagged amplified adaptor-ligated restriction fragments using high throughput sequencing technology, wherein each library comprises a unique identifier tag, wherein the sequencing comprises amplifying by PCR on a solid support;

(f) clustering the sequences per library, using the identifier tag;

(g) identifying genetic markers within the library and/or between libraries;

(h) determining dominant or co-dominant genotypes of the genetic markers in the two or more libraries.

2. The method according to claim 1, wherein the genetic marker is an AFLP marker or an SNP marker.

3. The method according to claim 1, wherein sequencing is based on sequencing by synthesis.

4. The method according to claim 1, wherein sequencing is performed on a solid support.

5. The method according to claim 1, wherein the sequencing comprises the steps of:

annealing amplified adaptor-ligated restriction fragments to beads, each bead annealing with a single adaptor-ligated fragment;

emulsifying the beads in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead;

performing emulsion PCR to amplify the adaptor-ligated restriction fragments on the surface of the beads;

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

generating a pyrophosphate signal.

6. The method according to claim 1, wherein the average sequencing redundancy of the tagged amplified adaptor-ligated restriction fragments is at least 6.

7. The method according to claim 1, wherein the sequence of each adaptor-ligated restriction fragment is determined at least 6 fold.

8. The method according to claim 1, wherein between endonuclease restriction and adaptor ligation a size selection is performed by a denaturation step.

9. The method according to claim 1, wherein the DNA is selected from the group consisting of genomic DNA, cDNA, BACs, YACs, whole-genome amplified DNA, PCR product.

10. The method according to claim 1, wherein the adaptor is a double stranded synthetic oligonucleotide adaptor having one end that is compatible with one or both ends of the restriction fragments.

11. The method according to claim 1, wherein the DNA is restricted with two or more restriction endonucleases.

12. The method according to claim 1, wherein the DNA is restricted with two restriction endonucleases.

13. The method according to claim 1, wherein at least one of the restriction endonucleases is a rare cutter.

14. The method according to claim 1, wherein at least one of the restriction endonucleases is a frequent cutter.

15. The method according to claim 1, wherein the primer contains from one to 10 (preferably randomly selected from amongst A, C, T or G) selective nucleotides, more preferably from one to 5 nucleotides.

16. The method according to claim 1 wherein the DNA is restricted using a combination of three or more restriction endonucleases.

17. The method accordingly to claim 1 wherein determining a co-dominant genotype comprises co-dominant scoring of AFLP and/or SNP marker sequences.

18. The method accordingly to claim 1, wherein the genotyping is used for at least one application selected from the group consisting of genetic mapping, quantitative trait loci mapping, fine mapping genes/traits, linkage disequilibrium (LD) mapping, marker-assisted back-crossing, genetic distance analysis, discovery of markers linked to traits or phenotypes, and diagnostic genotyping of patient samples.