HIGH THROUGHPUT DETECTION OF MOLECULAR MARKERS BASED ON AFLP AND HIGH THROUGH-PUT SEQUENCING

• US 20140303007A1
• Filed: 05/22/2014
• Published: 10/09/2014
• Est. Priority Date: 04/04/2006
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: VAN EIJK, Michael Josephus Theresia, Hogers, Rene Cornelis Josephus
• Granted Patent: US 10,023,907 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2535/138 Amplified fragment length polymorphism [AFLP]

Abstract

The present invention relates to a high throughput method for the identification and detection of molecular markers wherein restriction fragments are generated and suitable adaptors comprising (sample-specific) identifiers are ligated. The adapter-ligated restriction fragments may be selectively amplified with adaptor compatible primers carrying selective nucleotides at their 3′ end. The amplified adapter-ligated restriction fragments are, at least partly, sequenced using high throughput sequencing methods and the sequence parts of the restriction fragments together with the sample-specific identifiers serve as molecular marker.

Claims

1. A method for identifying one or more molecular markers in a plurality of samples, comprising:

(a) providing a plurality of sample nucleic acids;

(b) digesting each sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments;

(c) providing at least one double stranded synthetic adaptor comprising a primer compatible sequence and a double-stranded sample-specific identifier section, wherein said identifier does not comprise two or more consecutive identical nucleotides and each identifier differs by at least two nucleotides from any other identifier;

(d) ligating the double stranded synthetic adaptor to the restriction fragments in the set, to provide a set of adaptor-ligated restriction fragments;

(e) amplifying the set of adaptor-ligated restriction fragments, with one or more primers that are complementary to at least a portion of the adapter to provide for amplified adaptor-ligated restriction fragments;

(f) sequencing at least the sample-specific identifier section and part of the sequence of the restriction fragment of the amplified adaptor-ligated restriction fragments, wherein said sequencing of amplified adapter-ligated restriction fragments is redundant;

(g) identifying the presence or absence of amplified adaptor-ligated restriction fragments in the samples using the sample-specific identifier;

(h) comparing the identified fragments between samples; and

(i) identifying one or more molecular markers between samples.

2. The method according to claim 1, wherein the molecular markers are AFLP markers.

3. The method according to claim 1, wherein two or more samples are compared for the presence or absence of restriction fragments or fragment sequences and/or molecular markers.

4. The method according to claim 1, wherein two or more samples are combined in a pool after the step of ligating the adaptors to provide for pooled adaptor-ligated restriction fragments.

5. The method according to claim 4, wherein for each sample in the pool a sample-specific identifier is used that differs from the other sample-specific identifiers in the pool.

6. The method according to claim 1, wherein the primers contain one or more selective nucleotides at the 3′

end.

7. The method according to claim 1, wherein the restriction endonuclease is a type II restriction endonuclease.

8. The method according to claim 1, wherein the restriction endonuclease is a type IIs restriction endonuclease.

9. The method according to claim 1, wherein two or more restriction endonucleases are used.

10. The method according to claim 1, wherein the sequencing is carried out by means of high-throughput sequencing.

11. The method according to claim 10, wherein the high-throughput sequencing is performed on a solid support.

12. The method according to claim 10, wherein the high-throughput sequencing is based on Sequencing-by-Synthesis.

13. The method according to claim 10, wherein the high-throughput sequencing comprises the steps of:

annealing the amplicons or adapter-ligated restriction fragments to beads, each bead annealing with a single adapter-ligated restriction fragments or amplicon;

emulsifying the beads in water-in-oil micro reactors, each water-in-oil micro reactor comprising a single bead;

performing emulsion PCR to amplify adapter-ligated restriction fragments or amplicons on the surface of beads;

optionally, selecting/enriching beads containing amplified amplicons;

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

determining the nucleotide sequence of the amplified adapter-ligated restriction fragments or amplified amplicons using generating a pyrophosphate signal.

14. The method according to claim 10, wherein the high-throughput sequencing comprises the steps of:

annealing the adapter-ligated restriction fragments or amplicons to a surface containing first and second primers or first and second primer binding sequences respectively, performing bridge amplification to provide clusters of amplified adapter-ligated restriction fragments or amplified amplicons, determining the nucleotide sequence of the amplified adapter-ligated restriction fragments or amplified amplicons using labeled reversible terminator nucleotides.

15. The method according to claim 1, wherein the identifier is from 4-16 bp.

16. The method according to claim 1, wherein the identifier is from 4-10 bp.

17. The method according to claim 1, wherein the identifier is from 4-6 bp.

18. The method according to claim 1, wherein said sequencing is performed at least 6 times.

19. The method according to claim 1, wherein said sequencing is performed at least 9 times.

20. A method for the identification of molecular markers for genotyping, bulk segregant analysis, genetic mapping, marker-assisted back-crossing, mapping of quantitative trait loci, or linkage disequilibrium mapping, comprising the steps of:

(a) providing a plurality of sample nucleic acids;

(b) digesting each sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments;

(c) providing one or more double stranded synthetic adaptors comprising a primer-compatible sequence and a double-stranded sample-specific identifier section, wherein said identifier does not comprise two or more consecutive identical nucleotides and each identifier differs by at least two nucleotides from any other identifier;

(d) ligating the double stranded synthetic adaptors to the restriction fragments in the set, to provide a set of adaptor-ligated restriction fragments;

(e) amplifying the set of adaptor-ligated restriction fragments, with one or more primers that are complementary to at least a portion of the adaptor to provide for amplified adaptor-ligated restriction fragments;

(f) determining the sequence of at least the sample-specific identifier section and part of the sequence of the restriction fragment of the amplified adaptor-ligated restriction fragments;

(g) identifying the presence or absence of amplified adaptor-ligated restriction fragments or fragment sequences in the sample; and

(h) comparing the identified fragments or fragment sequences between samples.