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

Abstract Background We provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides end-to- end analysis of RNA-Seq data sets. The workflow allows the user to perform quality control, differential expression analyses, call genomic variants and generate a range of summary statistics. Additional options include the calculation of allele frequencies of variants of interest, summaries of genetic variation and population structure (in measures such as nucleotide diversity, Watterson’s θ, and PCA), and genome wide selection scans ( F st , PBS), together with clear visualisations. We 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. Results The Busia lab-colony used for selections was collected in Busia, Uganda, in November 2018. We performed a comparative analysis of three groups: a parental G24 Busia strain; its deltamethrin-selected G28 offspring; and the susceptible reference strain Kisumu. Measures of genetic diversity reveal patterns consistent with that of laboratory colonisation and selection, with the parental Busia strain exhibiting the highest nucleotide diversity of 1.04·10 -3 , followed by the selected Busia offspring (7.1·10 -4 ), and finally, Kisumu (6.2·10 -4 ). Differential expression and variant analyses reveal that the selected Busia colony exhibits a number of distinct mechanisms of pyrethroid resistance, including the Vgsc-995S target-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%), suggesting a link with pyrethroid resistance, which was previously observed in field samples from the same region. RNA-Seq-Pop analysis also reveals that the most widely-used insecticide-susceptible An. gambiae strain, Kisumu, appears to be a hybrid strain of An. gambiae and its sibling species An. coluzzii , which should be taken into consideration in future research. RNA-Seq-Pop is designed for ease of use, does not require programming skills and integrates the package manager Conda to ensure that all dependencies are automatically installed for the user. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An. gambiae and other taxa.