PT  - JOURNAL ARTICLE
AU  - Kentaro Itokawa
AU  - Koji Yatsu
AU  - Tsuyoshi Sekizuka
AU  - Yoshihide Maekawa
AU  - Osamu Komagata
AU  - Masaaki Sugiura
AU  - Tomonori Sasaki
AU  - Takashi Tomita
AU  - Makoto Kuroda
AU  - Kyoko Sawabe
AU  - Shinji Kasai
TI  - 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
AID  - 10.1101/564609
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 564609
4099  - http://biorxiv.org/content/early/2019/06/10/564609.short
4100  - http://biorxiv.org/content/early/2019/06/10/564609.full
AB  - In insects, 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 of VGSC gene, 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 annotated 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 homologous 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 enhance efficiency of the downstream bioinformatic process, we developed an automated pipeline to genotype VGSC after capture sequencing—MoNaS (Mosquito Na+ channel mutation Search)—which calls amino acid substitutions 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 genetics studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.