Method for high-throughput AFLP-based polymorphism detection

• US 9,702,004 B2
• Filed: 12/01/2016
• Issued: 07/11/2017
• 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:
  US15/366,417
• Publication Number:
  US 20170081718A1
• Time in Patent Office: 222 Days
• Field of Search: 435 61, 435 612, 435 911, 435 912, 435 9152, 536 231, 536 242, 536 2433, 506 16, 506 31, 506 32
• 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 determining dominant or co-dominant genotypes of one or more polymorphisms, comprising:

(a) ligating an adaptor to a nucleic acid fragment to produce an adaptor-ligated nucleic acid fragment;

(b) amplifying the adaptor-ligated nucleic acid fragment to produce a library of amplified adaptor-ligated nucleic acid fragments, wherein the amplified adaptor-ligated nucleic acid fragments comprise an identifier tag;

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

(d) clustering the sequences per library and determining dominant or co-dominant genotypes of one or more polymorphisms in the two or more libraries using the identifier tag.

2. The method according to claim 1, wherein step (d) comprises determining genotypes of one or more SNPs.

3. The method according to claim 1, wherein step (d) comprises co-dominant scoring of SNP sequences to determine SNP genotypes.

4. The method according to claim 1, wherein the one or more polymorphisms are genotyped from the amplified adaptor-ligated nucleic acid fragments that are sequenced with an average sequencing redundancy of at least 6.

5. The method according to claim 1, wherein the one or more polymorphisms are genotyped from the amplified adaptor-ligated nucleic acid fragments that are sequenced with an average sequencing redundancy of at least 9.

6. The method according to claim 1, wherein step (c) comprises sequencing by synthesis.

7. The method according to claim 1, wherein step (c) comprises:

(1) annealing amplified adaptor-ligated nucleic acid fragments to beads, each bead annealing with a single fragment;

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

(3) performing emulsion PCR to amplify the fragments on the surface of the beads;

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

(5) generating a pyrophosphate signal.

8. The method according to claim 1, wherein the nucleic acid fragment is genomic DNA or cDNA.

9. The method according to claim 1, wherein the nucleic acid fragment is of mammalian origin.

10. The method according to claim 1, wherein the nucleic acid fragment is of human origin.

11. A method for determining dominant or co-dominant genotypes of one or more polymorphisms, comprising:

(a) ligating an adaptor to a nucleic acid fragment to produce a library of adaptor-ligated nucleic acid fragment;

(b) sequencing a pool of two or more libraries of adaptor-ligated nucleic acid fragments using high throughput sequencing, wherein each library comprises a unique identifier tag, and wherein the sequencing comprises amplifying by PCR on a solid support;

(c) clustering the sequences per library and determining dominant or co-dominant genotypes of one or more polymorphisms in the two or more libraries using the identifier tag.