High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols
<p>Abstract</p> <p>Background</p> <p>Association mapping approaches are dependent upon discovery and validation of single nucleotide polymorphisms (SNPs). To further association studies in <it>Anopheles gambiae </it>we conducted a major resequencing programm...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2009-07-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/10/320 |
| _version_ | 1818757138664652800 |
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| author | Steen Keith Weetman David Wilding Craig S Donnelly Martin J |
| author_facet | Steen Keith Weetman David Wilding Craig S Donnelly Martin J |
| author_sort | Steen Keith |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Association mapping approaches are dependent upon discovery and validation of single nucleotide polymorphisms (SNPs). To further association studies in <it>Anopheles gambiae </it>we conducted a major resequencing programme, primarily targeting regions within or close to candidate genes for insecticide resistance.</p> <p>Results</p> <p>Using two pools of mosquito template DNA we sequenced over 300 kbp across 660 distinct amplicons of the <it>An. gambiae </it>genome. Comparison of SNPs identified from pooled templates with those from individual sequences revealed a very low false positive rate. False negative rates were much higher and mostly resulted from SNPs with a low minor allele frequency. Pooled-template sequencing also provided good estimates of SNP allele frequencies. Allele frequency estimation success, along with false positive and negative call rates, improved significantly when using a qualitative measure of SNP call quality. We identified a total of 7062 polymorphic features comprising 6995 SNPs and 67 indels, with, on average, a SNP every 34 bp; a high rate of polymorphism that is comparable to other studies of mosquitoes. SNPs were significantly more frequent in members of the cytochrome p450 mono-oxygenases and carboxy/cholinesterase gene-families than in glutathione-S-transferases, other detoxification genes, and control genomic regions. Polymorphic sites showed a significantly clustered distribution, but the degree of SNP clustering (independent of SNP frequency) did not vary among gene families, suggesting that clustering of polymorphisms is a general property of the <it>An. gambiae </it>genome.</p> <p>Conclusion</p> <p>The high frequency and clustering of SNPs has important ramifications for the design of high-throughput genotyping assays based on allele specific primer extension or probe hybridisation. We illustrate these issues in the context of the design of Illumina GoldenGate assays.</p> |
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| format | Article |
| id | doaj.art-a83480e215564e0f8c113ace304b7f11 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-18T06:06:10Z |
| publishDate | 2009-07-01 |
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| series | BMC Genomics |
| spelling | doaj.art-a83480e215564e0f8c113ace304b7f112022-12-21T21:18:32ZengBMCBMC Genomics1471-21642009-07-0110132010.1186/1471-2164-10-320High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocolsSteen KeithWeetman DavidWilding Craig SDonnelly Martin J<p>Abstract</p> <p>Background</p> <p>Association mapping approaches are dependent upon discovery and validation of single nucleotide polymorphisms (SNPs). To further association studies in <it>Anopheles gambiae </it>we conducted a major resequencing programme, primarily targeting regions within or close to candidate genes for insecticide resistance.</p> <p>Results</p> <p>Using two pools of mosquito template DNA we sequenced over 300 kbp across 660 distinct amplicons of the <it>An. gambiae </it>genome. Comparison of SNPs identified from pooled templates with those from individual sequences revealed a very low false positive rate. False negative rates were much higher and mostly resulted from SNPs with a low minor allele frequency. Pooled-template sequencing also provided good estimates of SNP allele frequencies. Allele frequency estimation success, along with false positive and negative call rates, improved significantly when using a qualitative measure of SNP call quality. We identified a total of 7062 polymorphic features comprising 6995 SNPs and 67 indels, with, on average, a SNP every 34 bp; a high rate of polymorphism that is comparable to other studies of mosquitoes. SNPs were significantly more frequent in members of the cytochrome p450 mono-oxygenases and carboxy/cholinesterase gene-families than in glutathione-S-transferases, other detoxification genes, and control genomic regions. Polymorphic sites showed a significantly clustered distribution, but the degree of SNP clustering (independent of SNP frequency) did not vary among gene families, suggesting that clustering of polymorphisms is a general property of the <it>An. gambiae </it>genome.</p> <p>Conclusion</p> <p>The high frequency and clustering of SNPs has important ramifications for the design of high-throughput genotyping assays based on allele specific primer extension or probe hybridisation. We illustrate these issues in the context of the design of Illumina GoldenGate assays.</p>http://www.biomedcentral.com/1471-2164/10/320 |
| spellingShingle | Steen Keith Weetman David Wilding Craig S Donnelly Martin J High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols BMC Genomics |
| title | High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols |
| title_full | High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols |
| title_fullStr | High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols |
| title_full_unstemmed | High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols |
| title_short | High, clustered, nucleotide diversity in the genome of <it>Anopheles gambiae </it>revealed through pooled-template sequencing: implications for high-throughput genotyping protocols |
| title_sort | high clustered nucleotide diversity in the genome of it anopheles gambiae it revealed through pooled template sequencing implications for high throughput genotyping protocols |
| url | http://www.biomedcentral.com/1471-2164/10/320 |
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