METHOD FOR HIGH-THROUGHPUT AFLP-BASED POLYMORPHISM DETECTION

• US 20090269749A1
• Filed: 12/20/2006
• Published: 10/29/2009
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Schriek, Marco Gerardus Maria, Van Eijk, Michael Josephus Theresia, Sorensen, Anker Preben
• Granted Patent: US 8,481,257 B2
• 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 1/6855 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 restriction fragments;

(c) ligating adaptors to the restriction fragments to produce adaptor-ligated restriction fragments;

(d) optionally, amplifying the adaptor-ligated restriction fragments with a primer pair that is complementary to the adaptors to produce pre-amplified adaptor-ligated restriction fragments;

(e) amplifying the (optionally pre-amplified) adaptor-ligated restriction fragments with a primer pair, wherein at least one of the primers contains an identifier tag at the 5′

end of the primer to produce a library of tagged amplified subsets of adaptor-ligated restriction fragments for each sample;

(f) optionally, pooling the libraries;

(g) sequencing the libraries using high throughput sequencing technology;

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

(i) identify genetic markers within the library and/or between libraries

(j) determine (co-)dominant genotypes of the genetic markers in one 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, preferably pyrosequencing.

4. The method according to claims 1, wherein sequencing is performed on a solid support such as a bead.

5. The method according to claims 1-4, 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 claims 1-4, wherein the average redundancy of the tagged amplified adaptor-ligated restriction fragments is at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9.

7. The method according to claims 1, wherein the sequence of each adaptor-ligated restriction fragment is determined at least 6, preferably at least 7, more preferably at least 8 and most preferably at least 9 fold.

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

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

10. The method according to claims 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 claims 1, wherein the DNA is restricted with two, preferably three, or more restriction endonucleases.

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

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

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

15. The method according to claims 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 according to claim 1 wherein genotype is co-dominant scoring of AFLP and/or SNP marker sequences.

18. The method according to claim 1, wherein the genotyping is selected from the group consisting of genetic mapping, QTL 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 etc.