Ontology highlight
ABSTRACT:
PROVIDER: PRJNA723358 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14294400_1.fastq.gz | Fastqsanger.gz | |||
| SRR14294400_2.fastq.gz | Fastqsanger.gz | |||
| SRR14294401_1.fastq.gz | Fastqsanger.gz | |||
| SRR14294401_2.fastq.gz | Fastqsanger.gz | |||
| SRR14294402_1.fastq.gz | Fastqsanger.gz |
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Project description:Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest. Targeted genomic enrichment followed by next-generation sequencing dramatically increased the efficiency of mutation discovery in human genomes. Here we demonstrate that these techniques also revolutionize traditional genetic approaches in model systems. We developed a two-step protocol utilizing a traditional bulk-segregant analysis (BSA) approach for positional cloning mutants in phenotype-driven forward genetic screens. First, BSA pools are 'light' sequenced for rough mapping, followed by targeted enrichment and deep-sequencing of the mutant BSA pool for the linked genomic region to fine-map and discover candidate mutations. We applied this method successfully to three Arabidopsis mutants and show that it can be scaled by multiplexing. Similarly, we applied these techniques to a gene-driven reverse genetics method (chemical driven target-selected mutagenesis or TILLING) that is used for generating gene knockouts in a wide range of organisms, including plants, invertebrates and vertebrates. We developed an efficient multiplexed genomic enrichment protocol for pre-barcoded samples. As a proof-of-principle, 770 genes were screened for induced mutations in 30 rats, which identified all but one known variants (30) as well as a large series of novel knockout and missense alleles. Mutations were retrieved at the expected frequency with a the false-positive rate of less than 1 in 6 million basepairs, which is much lower as compared to traditional mutation discovery approaches. Both methods are largely independent of the genome size due to the targeted enrichment and can thus be applied to any genetic model system of interest.
2010-09-12 | E-GEOD-22024 | biostudies-arrayexpress
Project description:Enrichment culture Targeted loci environmental
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Project description:Enrichment culture Targeted loci environmental
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Project description:Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we have explored the use of short fragment libraries (85-110bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. These short fragment libraries were enriched for 1.69 Mb of exonic sequences, using custom 244K microarrays, and sequenced using AB/SOLiD. High enrichment specificity (60 M-bM-^@M-^S 75%) was obtained at 67-213x average coverage, with 77-92% and 90-98% of targeted regions covered with more than 25% and 10% of the average coverage, respectively. As a more appropriate measure of the evenness of coverage, which is relatively independent of sequencing depth, we introduce the evenness of coverage parameter E. E values up to 75% were achieved. To verify the accuracy of SNP/mutation detection we evaluated 384 known non-reference SNPs in the targeted regions. At ~ 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls while 3.6% of targeted regions were marked as unsurveyed. A total of 1197 new variants were detected. Verification revealed only 8 false positive calls, resulting in an overall false positive rate of less than 1 per ~200,000 bp (0.0005%, equivalent to an overall phred score of 55). 4 samples + capture design file
2010-02-01 | E-GEOD-18542 | biostudies-arrayexpress