Method for high-throughput AFLP-based polymorphism detection

• US 10,106,850 B2
• Filed: 08/22/2017
• Issued: 10/23/2018
• 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, Sorensen, Anker Preben, Van Schriek, Marco Gerardus Maria
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/683,252
• Publication Number:
  US 20170356042A1
• Time in Patent Office: 427 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 detection of genetic variation in two or more members of a population, comprising:

amplifying nucleic acid fragments of at least a first and second member of a population to obtain amplified nucleic acid fragments, wherein the amplified nucleic acid fragments of the first member of the population comprises sequences from a nucleic acid sample of the first member of the population and a first unique identifier sequence, wherein the amplified nucleic acid fragments of the second member of the population comprises sequences from a nucleic acid of the second member of the population and a second unique identifier sequence, and wherein the first and second unique identifier sequences indicate different sample origins of the nucleic acid fragments;

sequencing a pool of the amplified nucleic acid fragments using high throughput sequencing technology;

clustering the sequences of amplified nucleic acid fragments of the first and second members of the population and comparing the clustered sequences to a reference sequence to identify genetic variation; and

identifying the first and second member of the population as carrying the genetic variation using the first and second unique identifier sequences.

2. The method according to claim 1, wherein the amplifying step comprises amplifying by solution PCR.

3. The method according to claim 1, wherein the genetic variation comprises one or more SNP polymorphisms.

4. The method according to claim 1, wherein the genetic variation comprises one or more AFLP polymorphisms.

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

6. The method according to claim 5, wherein the sequencing is pyrosequencing.

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

8. The method according to claim 7, wherein the solid support is a bead.

9. The method according to claim 8, wherein the sequencing comprises:

(1) annealing amplified nucleic acid fragments to beads, each bead annealing with a single nucleic acid 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 nucleic acid 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 reference sequence is a consensus sequence.

11. The method according to claim 10, wherein the consensus sequence is established from the population.

12. The method according to claim 1, wherein the reference sequence is a genomic sequence of a species of interest.

13. The method according to claim 1, wherein the reference sequence is from a sequence database.

14. The method according to claim 1, wherein the genetic variation is determined by sequencing with an average sequencing redundancy of at least 6.

15. The method according to claim 1, wherein the genetic variation is determined by sequencing with an average sequencing redundancy of at least 9.

16. The method according to claim 1, wherein the first and second unique identifier sequences are used for identifying the member carrying the genetic variation.

17. The method according to claim 1, wherein the detection of genetic variation is used for applications selected from the group consisting of genetic mapping, quantitative trait loci mapping, fine mapping genes/traits, linkage disequilibrium mapping, marker-assisted back-crossing, genetic distance analysis, discovery of makers linked to trait or phenotypes, and diagnostic genotyping of patient samples.

18. The method according to claim 1, wherein the nucleic acid samples of the first and second members of the population are DNA.

19. The method according to claim 18, wherein the nucleic acid samples of the first and second members of the population are mammalian DNA.

20. The method according to claim 19, wherein the nucleic acid samples of the first and second members of the population are human DNA.

21. The method according to claim 1, wherein the method further comprises restricting the nucleic acid samples using at least one restriction endonuclease to produce nucleic acid fragments.

22. The method according to claim 1, wherein the unique identifier sequence is introduced into the nucleic acid fragments by primer extension.

23. The method according to claim 1, wherein the unique identifier sequence is introduced into the nucleic acid fragments by adapter ligation.