High throughput screening of populations carrying naturally occurring mutations
First Claim
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1. A method for the detection of a mutation in a target sequence, comprising:
- (a) providing a plurality of pools of amplification products each comprising the target sequence amplified from a nucleic acid of a subset of the one or more members of the population, wherein each pool of the amplification products shares a unique pool identifier;
(b) performing high throughput sequencing to sequence the amplification products; and
(c) clustering/aligning the sequences of the amplified products without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis, and detecting the one or more members and/or pools carrying the mutation using the pool identifier.
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Abstract
Efficient methods are disclosed for the high throughput identification of mutations in genes in members of mutagenized populations. The methods comprise DNA isolation, pooling, amplification, creation of libraries, high throughput sequencing of libraries, preferably by sequencing-by-synthesis technologies, identification of mutations and identification of the member of the population carrying the mutation and identification of the mutation.
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Citations
20 Claims
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1. A method for the detection of a mutation in a target sequence, comprising:
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(a) providing a plurality of pools of amplification products each comprising the target sequence amplified from a nucleic acid of a subset of the one or more members of the population, wherein each pool of the amplification products shares a unique pool identifier; (b) performing high throughput sequencing to sequence the amplification products; and (c) clustering/aligning the sequences of the amplified products without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis, and detecting the one or more members and/or pools carrying the mutation using the pool identifier. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification