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

High-throughput genotyping of a full voltage-gated sodium 1 channel gene via genomic DNA using target capture 2 sequencing and analytical pipeline MoNaS to discover 3 novel insecticide resistance mutations 4 Abstract 18Insects' voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid 19 insecticides. Various amino acid substitutions in the VGSC protein are known to confer 20 insecticide resistance and are selected under insecticide pressure. In the genome, the 21 VGSC gene consists of more than 30 exons sparsely distributed across a large genomic 22 region, which often exceeds 100 kbp. Due to this complex genomic structure of gene VGSC, 23 it is usually challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from 24 individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide 25 probes via annotated CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe 26 set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled 27 barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The 28 probe set also captured all homologous VGSC CDSs except tiny exons from the gDNA of 29 other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable 30 efficiency by virtue of the high conservation of VGSC at the nucleotide level. Furthermore, 31 we developed an automated bioinformatic analysis pipeline to genotype VGSC after capture 32 sequencing-MoNaS (Mosquito Na + channel mutation Search)-which conducts mapping 33 of reads, variant calling, and variant annotation for nonsynonymous mutations. The 34 proposed method and our bioinformatic tool should facilitate the discovery of novel amino 35 acid variants conferring insecticide resistance on VGSC and population genetics studies on 36 resistance alleles with respect to the origin, selection, and migration of both clinically and 37 agriculturally important insect pests. 38 39