RNA‐Seq‐Pop: Exploiting the sequence in RNA sequencing—A Snakemake workflow reveals patterns of insecticide resistance in the malaria vector Anopheles gambiae

Abstract

We provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides end-to-end analysis of RNA sequencing data sets. The workflow allows the user to perform quality control, perform 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 genome-wide selection scans, together with clear visualizations. RNA-Seq-Pop is applicable to any organism, and 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. The Busia laboratory 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 colonization 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 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%), supporting a previous link with pyrethroid resistance. RNA-Seq-Pop is hosted at: github.com/sanja ynagi/ rna-seq-pop. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An. gambiae and other taxa.

Genomic Profiling of Insecticide Resistance in Malaria Vectors: Insights into Molecular Mechanisms.

“…Integrating differential expression and single nucleotide polymorphism (SNP) data has the potential to enhance the discovery of novel resistance markers [ 46 , 47 ]. We integrated whole-genome sequence data from the Ag1000G with our transcriptomic meta-analysis, estimating signals of positive selection in wild mosquitoes, and locating the intersection of genes that both are highly overexpressed and are located at sites of selection signals.…”

Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii

“…To detect signatures of positive directional selection (selective sweeps) in the metabolic resistance genes of interest, a Snakemake RNA‐seq population genetics pipeline RNA‐seq‐Pop was utilized (Ibrahim et al., 2023 ; Nagi et al., 2022 ). The pipeline aligns RNA‐seq reads to the reference genome, then calls genomic variants with Freebayes, at a user‐provided level of ploidy (in this case, 16; from 8 diploid mosquitoes in each pool).…”

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector Anopheles coluzzii

“…To detect signature of selective sweeps in the major metabolic resistance genes of interest, a Snakemake RNAseq population genetics pipeline, the RNAseq-Pop was utilised [ 48 ]. The workflow aligns RNA-seq reads to the reference genome, and calls genomic variants with Freebayes , at a user-provided level of ploidy, in our case 16 (8 diploid pooled mosquitoes).…”