Method for high-throughput AFLP-based polymorphism detection
First Claim
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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).
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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.
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Citations
31 Claims
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1. A high-throughput method for the discovery, detection and genotyping of a genetic marker in a plurality of samples, comprising the steps of:
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(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). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A high-throughput method for the discovery, detection and genotyping of a genetic marker in a plurality of samples, comprising the steps of:
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(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. - View Dependent Claims (31)
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Specification