Computational protocol: Comparative Transcriptome Analyses of Deltamethrin-Resistant and -Susceptible Anopheles gambiae Mosquitoes from Kenya by RNA-Seq

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Protocol publication

[…] WHO guidelines were followed to analyze the results of the bioassays . Specifically, mortality was scored 24 hours after exposure and the Abbot’s formula was used to correct for control mortality when required. A population was classified as “resistant” if mortality was less than 80%, as “suspected resistant” if mortality ranged between 80–97% and as “susceptible” if mortality was higher than 98% . Fifty and 95% knock-down times (KT50 and KT95) were estimated by the logistic regression using the software STATISTICA (StatSoft, Tulsa, OK). The paired t-test was used to compare the mortality of female and male mosquitoes and to test for association between genotypes and phenotypes. [...] TopHat was used to map Illumina reads to the An. gambiae genome (version AgamP3). Cufflinks was used to quantify transcript abundance in terms of Fragment Per Kilobase of exon model per Million mapped fragment (FPKM). Differential accumulation of transcripts between deltemethrin-resistant (R) and –susceptible (S) mosquitoes was tested by the Cuffdiff program within Cufflinks. Cuffdiff was run requiring a minimum alignment count of 10 and the upper-quartile-norm option, which can improve robustness of differential accumulation calls for less abundant transcripts. The Cuflinks suite was run twice. The first run used the published annotation of the An. gambiae genome as a strict reference guide for transcript annotation. Results from this run were used to identify significant differentially accumulation of transcripts between R and S mosquitoes. In the second run, the An. gambiae genome annotation was used as a guide of transcript annotation. Under this option, Cuflinks also looks for read-coverage supporting novel transcriptional events (NTE). Enrichment of Gene Ontology (GO) terms among differentially accumulated transcripts was tested by g:Profiler . […]

Pipeline specifications

Software tools Statistica, TopHat, Cufflinks, g:Profiler
Applications Miscellaneous, RNA-seq analysis
Organisms Anopheles gambiae
Diseases Malaria
Chemicals Pyrethrins