High-throughput genotyping of Anopheles mosquitoes using intact legs by Agena Biosciences iPLEX

Recent developments in genotyping technologies coupled with the growing desire to characterise genome variation in Anopheles populations opens 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 Pre-amplification. 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% versus 89%, respectively), although this difference was not significant. We provide a proof of concept that WGA reactions can be performed directly on mosquito legs, the...