Method for high-throughput AFLP-based polymorphism detection

• US 9,777,324 B2
• Filed: 04/22/2016
• Issued: 10/03/2017
• 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:
  US15/136,224
• Publication Number:
  US 20160258013A1
• Time in Patent Office: 529 Days
• 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 of one or more polymorphisms, comprising:

pooling a first library of amplified DNA fragments and a second library of amplified DNA fragments to obtain a pooled DNA library, the amplified DNA fragments of the first library comprising DNA sequences amplified from a first sample by solution PCR and a first unique identifier sequence, the amplified DNA fragments of the second library comprising DNA sequences amplified from a second sample by solution PCR and a second unique identifier sequence, wherein the first and second unique identifier sequences indicate different sample origins of the first and second libraries;

sequencing the pooled DNA library using high throughput sequencing technology, comprising amplifying on a solid support;

clustering sequences of the first and second libraries of amplified DNA fragments; and

determining dominant or co-dominant genotype(s) of one or more polymorphisms in the first and second libraries of amplified DNA fragments using the first and second unique identifier sequences.

2. The method according to claim 1, wherein the method comprises determining genotype(s) of one or more SNP polymorphisms.

3. The method according to claim 1, wherein the method comprises determining genotype(s) of one or more AFLP polymorphisms.

4. The method according to claim 1, wherein the method comprises co-dominant scoring of SNP polymorphism sequences to determine a co-dominant SNP genotype.

5. The method according to claim 1, wherein the method comprises co-dominant scoring of AFLP polymorphism sequences to determine a co-dominant AFLP genotype.

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

7. The method according to claim 1, wherein the one or more polymorphisms are genotyped from amplified DNA fragments that are sequenced with an average sequencing redundancy of at least 9.

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

9. The method according to claim 1, wherein the sequencing comprises:

(1) annealing amplified DNA fragments to beads, each bead annealing with a single DNA 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 DNA 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.

10. The method according to claim 1, wherein the first and second samples are genomic DNA or cDNA.

11. The method according to claim 1, wherein the first and second samples are mammalian DNA.

12. The method according to claim 1, wherein the first and second samples are human DNA.

13. A method for genotyping of one or more polymorphisms, comprising:

pooling a first library of amplified DNA fragments and a second library of amplified DNA fragments to obtain a pooled DNA library, the amplified DNA fragments of the first library comprising DNA sequences amplified from a first sample by solution PCR and a first unique identifier sequence, the amplified DNA fragments of the second library comprising DNA sequences amplified from a second sample by solution PCR and a second unique identifier sequence, wherein the first and second unique identifier sequences indicate different sample origins of the first and second libraries;

sequencing the pooled DNA library using high throughput sequencing technology, comprising amplifying on a solid support; and

determining dominant or co-dominant genotype(s) of one or more polymorphisms in the first and second libraries of amplified DNA fragments using the first and second unique identifier sequences.