RNA-Seq-Pop: Exploiting the sequence in RNA-Seq - a Snakemake workflow reveals patterns of insecticide resistance in the malaria vector Anopheles gambiae

We provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides e nd-to-end analysis of RNA-Seq data sets. The workflow allows the user to perform quality control, differential expression analyses, and call genomic variants. Additional options include the calculation of allele frequencies of variants of interest, summaries of genetic variation and population structure , and ge nome wide selection scans, together with clear visualisations. RNA-Seq-Po p is applicable to any organism, an d w e demonstrate the utility of the workflow by investigating pyrethroid resistance in selected strains of the major malaria mosquito, Anopheles gambiae. The workflow provides additional modules specifically for An. gambiae, including estimating recent ancestry and determining the karyotype of common chromosomal inversions.The Busia lab-colony used for selections was collected in Busia, Uganda, in November 2018. We perf orme d a comparative analysis of three groups: a parental G24 Busia strain; its deltamethrin-se lecte d G28 of f spri ng; and the susceptible reference strain Kisumu. Measures of genetic diversity reveal patterns cons is tent w i th that of laboratory colonisation and selection, with the parental Busia strain exhibiting the highest nucleotide diversity, followed by the selected Busia offspring, and finally, Kisumu. Differential expression and variant analyses reveal that the selected Busia colony exhibits a number of distinct mechanisms of pyrethroid resistance, including the Vgsc-995S targe t-site mutation, upregulation of SAP genes, P450s, and a cluster of carboxylesterases. During deltamethrin selections, the 2La chromosomal inversion rose in frequency (from 33% to 86%), supporting a previous link with pyrethroid resistance. RNA-Seq-Po p is hos ted here github.com/sanjaynagi/rna-seq-pop. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An. gambiae and other taxa.