title: High-throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX.
abstract: Recent developments in genotyping technologies coupled with the growing desire to characterize genome variation in Anopheles populations open the opportunity to develop more effective genotyping strategies for high-throughput screening. A major bottleneck of this goal is nucleic acid extraction. Here, we examined the feasibility of using intact portions of a mosquito's leg as sources of template DNA for whole-genome amplification (WGA) by primer-extension preamplification. We used the Agena Biosciences MassARRAY(®) platform (formerly Sequenom) to genotype 78 SNPs for 265 WGA leg samples. We performed nucleic acid extraction on 36 mosquito carcasses and compared the genotype call concordance with their corresponding legs and observed full concordance. Using three legs instead of one improved genotyping success rates (96% vs. 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, thereby eliminating the need to extract nucleic acid. This approach is straightforward and sensitive and allows both species determination and genotyping of Anopheles mosquitoes to be performed in a high-throughput manner. Our protocol also leaves the mosquito body intact facilitating other experimental analysis to be undertaken on the same sample. Based on our findings, this method would also be suitable for use with other insect species.
authors.affiliation: Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK.
authors.forename: Danica Joy
authors.initials: DJ
authors.lastname: Fabrigar
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK.
authors.forename: Christina
authors.initials: C
authors.lastname: Hubbart
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK.
authors.forename: Alistair
authors.initials: A
authors.lastname: Miles
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK.
authors.forename: Kirk
authors.initials: K
authors.lastname: Rockett
mesh_terms: D000818:Animals; D000852:Anopheles / Q000145:classification* / Q000235:genetics*; D005121:Extremities; D014644:Genetic Variation; D060005:Genotyping Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: Anopheles gambiae complex; DNA extraction; high-throughput genotyping; whole-genome amplification