Method for high-throughput AFLP-based polymorphism detection

• US 8,911,945 B2
• Filed: 06/27/2014
• Issued: 12/16/2014
• 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 Gerardus Maria
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/318,352
• Publication Number:
  US 20140315728A1
• Time in Patent Office: 172 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 genotyping genetic markers in one or more samples, comprising the steps of:

(a) reducing complexity of at least two nucleic acid samples using amplified fragment length polymorphism (AFLP) to produce at least two reproducible subsets of amplified adaptor-ligated restriction fragments from the at least two nucleic acid samples, wherein each reproducible subset comprises a unique identifier tag;

(b) sequencing a pool of the at least two reproducible subsets of amplified adaptor-ligated restriction fragments using sequencing by synthesis on a solid support;

(c) aligning the sequences of the reproducible subsets of amplified adaptor-ligated restriction fragments;

(d) identifying AFLP and/or single-nucleotide polymorphism (SNP) markers within a selection of amplified adaptor-ligated restriction fragments that are sequenced with a redundancy of at least 6; and

(e) determining dominant or co-dominant genotypes of the genetic markers in the selected amplified adaptor-ligated restriction fragments that are sequenced with the redundancy of at least 6.

2. The method according to claim 1, further comprising digesting the nucleic acid with two or more restriction enzymes.

3. The method according to claim 2, wherein two adaptors are ligated to the restriction fragments.

4. The method according to claim 3, wherein the redundancy is at least 7, 8 or 9.

5. The method according to claim 2, wherein the redundancy is at least 7, 8 or 9.

6. The method according to claim 1, wherein two adaptors are ligated to the restriction fragments.

7. The method according to claim 6, wherein the redundancy is at least 7, 8 or 9.

8. The method according to claim 1, wherein the redundancy is at least 7, 8 or 9.