Showing papers by "Craig S. Wilding published in 2009"
TL;DR: It is asserted that the link between kdr genotype and DDT- and pyrethroid-susceptibility phenotype is clear and that diagnostic assays to test the importance of other resistance mechanisms in field populations are required.
153 citations
TL;DR: 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 as illustrated in the context of thedesign of Illumina GoldenGate assays.
Abstract: 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. 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. 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.
38 citations
TL;DR: It is shown that genomic DNA extractions from single Anopheles gambiae Giles using a standard commercial kit‐based methodology yield extracts with concentrations below the linear range of spectrophotometric absorbance at 260 nm, which is currently insufficient for many high‐throughput genome screening applications.
Abstract: Accurate estimates of DNA quantity are likely to become increasingly important for successful genomic screening of insect populations via recently developed, highly multiplexed genotyping assays and high-throughput sequencing methods Here we show that genomic DNA extractions from single Anopheles gambiae Giles using a standard commercial kit-based methodology yield extracts with concentrations below the linear range of spectrophotometric absorbance at 260 nm Concentrations determined by spectrophotometry were not reproducible, and are therefore neither accurate nor reliable However, DNA quantification using a fluorescent nucleic acid stain (PicoGreen((R))) gave highly reproducible concentration estimates, and indicated that, on average, single mosquitoes yielded approximately 300 ng of DNA Such a total yield is currently insufficient for many high-throughput genome screening applications, necessitating whole genome amplification of all or most individuals in a population prior to genotyping
12 citations