Computational protocol: Genetic variation associated with increased insecticide resistance in the malaria mosquito, Anopheles coluzzii

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Protocol publication

[…] We generated strand-specific mRNA-seq libraries (KAPA Biosystems, Wilmington, USA) for 6 biological replicates of control and treatment conditions for both the 1995 An. coluzzii (cyp-2) and 2014 An. coluzzii (cyp-1) genotypes (Additional file: Table S2). These 24 libraries were sequenced on a single lane of HiSeq3000 SR50 at the UC Davis DNA Technologies Core. Poor quality bases and Illumina adapter sequences were trimmed from the raw reads using Trimmomatic version 0.30 [], with the following parameters: LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLEN:36. The trimmed reads were then assessed for general quality using FastQC version 0.10.1 (Babraham Bioinformatics, Cambridge, UK). [...] Single-end reads were mapped with STAR version 2.5 (--quantMode GeneCounts; []) to the An. coluzzii reference genome (scaffolds AcolM1) with gene annotation information (AcolM1.3.gtf; Additional file : Table S2). A detailed workflow of our differential expression analysis is available as a Jupyter notebook (Additional file : Table S3). In brief, gene level counts were converted to counts per million (cpm) using edgeR [] and we removed genes with very low expression levels by requiring at least 5 libraries with a cpm > 2 for each gene. After this filtering step, we were left with 8485 genes that we tested for differential expression. We further normalized individual samples using the trimmed mean of M-values (TMM) method. To estimate differential expression between genotypes and between treatments, we used voom [] in the limma package [] to perform a variance-stabilizing transformation on the raw counts and then fit a linear model using lmFit []. We applied a cell means model including genotype and treatment after accounting for potential batch effects at the dissection stage. To identify differentially expressed (DE) genes we used the eBayes function [] and Benjamini-Hochberg (FDR)-corrected P-values (< 0.05) from the decideTests function.We classified a gene as a putative detoxification gene if it was included in the detoxification chip [], which includes over 200 genes belonging to at least three major enzyme families: glutathione S-transferases, cytochrome P450 monooxygenases and esterases. [...] Structural variants were detected in whole genome sequencing data (Additional file : Table S4) using LUMPY version 0.2.13 []. Discordant paired-end and split-end reads were extracted using samtools. Library size, mean, and standard deviation were estimated using paired_distro.py script from LUMPY. The inferred breakpoints produced by LUMPY were genotyped using SVtyper version 0.1.2. Calls identified as break ends were removed and analyzed separately. Remaining inferred breakpoints were used if they were larger than 100 bp in size, were not homozygous reference in all samples, and had more than ten supporting reads in the two highest coverage samples. For remaining calls, the ratio of cyp-1 samples having either a heterozygous or homozygous alternate to the total number of cyp-1 samples possible was calculated. This was repeated for cyp-2 samples, and Fisher’s exact test was performed to determine which SVs differed in frequency between the two populations.Inferred breakpoints which corresponded to potential repeat elements and genic regions were determined using bedtools intersect version 2.17.0. For repeat elements, a 50% reciprocal overlap between structural variant call and repeat elements was required. For genic regions, one breakpoint must have occurred within 500 bp of gene. […]

Pipeline specifications

Software tools STAR, Jupyter Notebook, edgeR, voom, limma, LUMPY, SAMtools, SVTyper, BEDTools
Applications Miscellaneous, WGS analysis
Organisms Anopheles coluzzii
Diseases Malaria
Chemicals Organophosphates, Piperonyl Butoxide, Sodium, Permethrin