Anopheles funestus pooled template whole genome sequencing for permethrin resistance GWAS

Pooled template whole genome sequencing (POOLseq) was carried out, using Anopheles funestus (sensu stricto) mosquitoes. Mosquitoes used in the study were insectary-reared F1 progeny of field-caught mosquitoes from two locations in Cameroon and Malawi. In all cases, 3-5 day old female F1 mosquitoes were subjected to WHO bioassays to expose them to permethrin for a given time. For the Cameroonian mosquitoes, a pool of mosquitoes (n=40) dead after 20 minutes' exposure formed the 'susceptible' group and a pool of mosquitoes (n=40) alive after 60 minutes' exposure formed the 'resistant' group. For the Malawian mosquitoes, two pools of mosquitoes (n=44 and n=43) dead after 60 minutes' exposure formed the 'susceptible' group (2 replicates) and three pools of mosquitoes (n=36, n=36, n=37) alive after 180 minutes' exposure formed the 'resistant' group (3 replicates). Total genomic DNA was extracted from individual mosquitoes using the QIAgen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Extracted genomic DNA was quantified per individual and combined in equal amounts to form pools. For each pool, whole genome DNAseq libraries were prepared using the TruSeq Nano (Low-Throughput) DNA Library Preparation Kit (Illumina). Libraries were pooled in equimolar amounts (with other libraries not included in this study) and each pool of libraries sequenced on one lane of the HiSeq 2500 (Illumina) at 2x150 bp paired-end sequencing with v4 chemistry.