High, clustered, nucleotide diversity in the genome of Anopheles gambiae revealed through pooled-template sequencing: implications for high-throughput genotyping protocols

Wilding, Craig, Weetman, David ORCID: https://orcid.org/0000-0002-5820-1388, Steen, Keith ORCID: https://orcid.org/0000-0002-8933-8643 and Donnelly, Martin ORCID: https://orcid.org/0000-0001-5218-1497 (2009) 'High, clustered, nucleotide diversity in the genome of Anopheles gambiae revealed through pooled-template sequencing: implications for high-throughput genotyping protocols'. Bmc Genomics, Vol 10, p. 11.

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Abstract

Background: Association mapping approaches are dependent upon discovery and validation of single nucleotide polymorphisms (SNPs). To further association studies in Anopheles gambiae we conducted a major resequencing programme, primarily targeting regions within or close to candidate genes for insecticide resistance. Results: Using two pools of mosquito template DNA we sequenced over 300 kbp across 660 distinct amplicons of the An. gambiae 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 An. gambiae genome. Conclusion: 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.

Item Type:	Article
Additional Information:	ISI Document Delivery No.: 490NI Wilding, Craig S. Weetman, David Steen, Keith Donnelly, Martin J. BIOMED CENTRAL LTD
Uncontrolled Keywords:	QUANTITATIVE TRAIT LOCI MALARIA VECTOR RECOMBINATION RATE AEDES-AEGYPTI NULL ALLELES HONEY-BEE COMPLEX GENES POPULATION MOSQUITO
Subjects:	QX Parasitology > Insects. Other Parasites > QX 650 Insect vectors QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes QU Biochemistry > Genetics > QU 450 General Works QX Parasitology > Insects. Other Parasites > QX 515 Anopheles
Digital Object Identifer (DOI):	https://doi.org/10.1186/1471-2164-10-320
Depositing User:	Users 183 not found.
Date Deposited:	10 Mar 2010 14:17
Last Modified:	24 Jan 2022 15:11
URI:	https://archive.lstmed.ac.uk/id/eprint/412

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