High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations

Abstract

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline—MoNaS (Mosquito Na⁺ channel mutation Search)—which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests. The Voltage Gated Sodium Channel (VGSC) in insect is targeted by pyrethroid insecticides and genetic variations in the protein are known to confer pyrethroid resistance. Since the VGSC gene in genome consists of many exons and long introns, there is no simple method to genotype whole of coding regions from the genomic DNA of insect. Here, we designed hybridization capture probe set to concentrate VGSC coding exons in NGS library from individual genomic DNA of the arbovirus vector mosquito Aedes albopictus. The probe set we designed was able to capture VGSC exons not only from A. albopictus genomic DNA but also from genomic DNA of two other mosquito species belonging to the same subfamily only with slight decrease of efficiency. The technology will allow unbiased analysis of the VGSC gene in multiple mosquito species with relatively low sequencing cost and enhance discovery of new resistance mutations.