Method for high-throughput AFLP-based polymorphism detection

• US 8,481,257 B2
• Filed: 12/20/2006
• Issued: 07/09/2013
• 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, Van Schriek, Marco Gerardus Maria, Srensen, Anker Preben
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US12/158,040
• Publication Number:
  US 20090269749A1
• Time in Patent Office: 2,393 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, 536/24.33
• US Class Current: 435/6.1
• 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 high-throughput method for the discovery, detection and genotyping of a genetic marker in a plurality of samples, comprising the steps of:

(a) providing DNA from a plurality of samples;

(b) restricting the DNA with at least one restriction endonuclease to produce restriction fragments;

(c) heating the restriction fragments to selectively dissociate the DNA fragments smaller than 120 bp;

(d) for each sample, ligating adaptors to the restriction fragments 120 bp or larger to produce adaptor-ligated restriction fragments, wherein for each sample at least one adaptor is tagged with a different identifier tag;

(e) amplifying the tagged adaptor-ligated restriction fragments with a primer pair, wherein at least one of the primers is complementary to at least part of the adaptor to produce a library of tagged amplified adaptor-ligated restriction fragments for each sample;

(f) subjecting at least one library to high-throughput sequencing;

(g) counting the number of sequences of each allele of a sequence polymorphism as a genetic marker in a library or between the libraries using the tag to distinguish between the libraries; and

(h) determining a dominant or a co-dominant genotype of the genetic marker in the libraries based on the presence or absence of sequences of each allele of the sequence polymorphism, based on the high-throughput sequencing data obtained from step (f).

2. The method according to claim 1, wherein the sequence polymorphism serving as the genetic marker is an amplified-fragment-length polymorphism marker or a single-nucleotide polymorphism marker.

3. The method according to claim 1, wherein the high-throughput sequencing is based on sequencing by synthesis.

4. The method according to claim 3, wherein the high-throughput sequencing is pyrosequencing.

5. The method according to claim 1, wherein the high-throughput sequencing is performed on a solid support.

6. The method according to claim 5, wherein the solid support is a bead.

7. The method according to claim 1, wherein the high-throughput sequencing comprises the steps of:

annealing amplified adaptor-ligated restriction fragments of step (e) to beads, each bead annealing with a single adaptor-ligated restriction fragment;

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

performing emulsion PCR in the microreactors 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.

8. The method according to claim 1, wherein the tagged amplified adaptor-ligated restriction fragments of step (e) have an average redundancy of at least 6.

9. The method according to claim 8, wherein the average redundancy of the tagged amplified adaptor-ligated restriction fragments of step (e) is at least 7.

10. The method according to claim 9, wherein the average redundancy of the tagged amplified adaptor-ligated restriction fragments of step (e) is at least 9.

11. The method according to claim 1, wherein the sequence of each adaptor-ligated restriction fragment is determined at least 6 fold.

12. The method according to claim 11, wherein the sequence of each adaptor-ligated restriction fragment is determined at least 7 fold.

13. The method according to claim 12, wherein the sequence of each adaptor-ligated restriction fragment is determined at least 9 fold.

14. The method according to claim 1, wherein the DNA is selected from the group consisting of genomic DNA, cDNA, bacterial artificial chromosome, yeast artificial chromosome, whole-genome amplified DNA, and PCR product.

15. The method according to claim 1, wherein the adaptor is a double stranded synthetic oligonucleotide adaptor comprising one end that is compatible with one or both ends of the restriction fragments.

16. The method according to claim 1, wherein the DNA is restricted with two or more restriction endonucleases.

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

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

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

20. The method according to claim 1, wherein at least one of the primers contains from one to ten selective nucleotides at the 3′

end of the primer.

21. The method according to claim 20, wherein the nucleotide are randomly selected from amongst A, C, T or G.

22. The method according to claim 20, wherein the primer contains from one to five selective nucleotides.

23. The method according to claim 20, wherein the one to five selective nucleotides are randomly selected from amongst A, C, T or G.

24. The method according to claim 1 wherein the DNA is restricted using a combination of three or more restriction endonucleases.

25. The method according to claim 1 wherein the genetic marker is an amplified-fragment-length polymorphism marker or a single-nucleotide polymorphism marker, and wherein the dominant or co-dominant genotype of the genetic marker is obtained by counting the number of sequences of each allele of the polymorphism.

26. The method according to claim 1, wherein the genotyping 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 markers linked to traits or phenotypes, and diagnostic genotyping of patient samples.

27. The method of claim 1, further comprising amplifying the adaptor-ligated restriction fragments before step (e) with a primer pair that is complementary to the adaptors to produce pre-amplified adaptor-ligated restriction fragments.

28. The method of claim 1, further comprising (a) producing multiple libraries from multiple samples;

and (b) then pooling the libraries prior to sequencing.

29. The method of claim 1, wherein both adaptors are tagged.

30. A high-throughput method for the discovery, detection and genotyping of a genetic marker in a plurality of samples, comprising the steps of:

(a) providing DNA from a plurality of samples;

(b) restricting the DNA with at least one restriction endonuclease to produce restriction fragments;

(c) heating the restriction fragments to selectively dissociate the DNA fragments smaller than 120 bp;

(d) for each sample, ligating adaptors to the restriction fragments 120 bp or larger to produce adaptor-ligated restriction fragments, wherein for each sample at least one adaptor is tagged with a different identifier tag;

(e) amplifying the tagged adaptor-ligated restriction fragments with a primer pair, wherein at least one of the primers is complementary to at least part of the adaptor to produce a library of tagged amplified adaptor-ligated restriction fragments for each sample;

(f) subjecting at least one library to high-throughput sequencing;

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

(h) assembling the clustered sequences into contigs;

(i) counting the number of sequences of each allele of a sequence polymorphism as a genetic marker in a library or between the libraries using the tag to distinguish between the libraries;

(j) determining a dominant or a co-dominant genotype of the genetic marker in the libraries based on the presence or absence of sequences of each allele of the sequence polymorphism, based on the high-throughput sequencing data obtained in step (f); and

(k) assigning a quality score to each polymorphism.

31. The method of claim 30, wherein the quality score is based on at least one of the following criteria:

number of reads per contig;

frequency of alleles per sample;

occurrence of homopolymer sequence; and

occurrence of neighbouring polymorphisms SNPs and indels with a quality score above the threshold.