High throughput detection of molecular markers based on AFLP and high through-put sequencing
First Claim
1. A method for detecting one or more polymorphisms in a plurality of nucleic acid samples, comprising:
- obtaining a pool of amplicons from the plurality of nucleic acid samples, which comprises amplifying the nucleic acid samples or fragments thereof using one or more primers, wherein the amplicons derived from each of the nucleic acid samples are tagged with a unique sample-specific identifier sequence;
sequencing the pool of amplicons to obtain sequence reads, wherein the sample-specific identifier sequence and at least part of the sequence of the nucleic acid samples are sequenced by high-throughput sequencing; and
detecting one or more polymorphisms in the plurality of nucleic acid samples by comparing the sequence reads and using the identifier sequence to assign the polymorphisms to the nucleic acid samples.
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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.
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Citations
12 Claims
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1. A method for detecting one or more polymorphisms in a plurality of nucleic acid samples, comprising:
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obtaining a pool of amplicons from the plurality of nucleic acid samples, which comprises amplifying the nucleic acid samples or fragments thereof using one or more primers, wherein the amplicons derived from each of the nucleic acid samples are tagged with a unique sample-specific identifier sequence; sequencing the pool of amplicons to obtain sequence reads, wherein the sample-specific identifier sequence and at least part of the sequence of the nucleic acid samples are sequenced by high-throughput sequencing; and detecting one or more polymorphisms in the plurality of nucleic acid samples by comparing the sequence reads and using the identifier sequence to assign the polymorphisms to the nucleic acid samples. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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Specification