Computational protocol: Insecticide resistance mediated by an exon skipping event

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Protocol publication

[…] To generate a reference transcriptome for T. absoluta, RNA was extracted from eggs, larvae, pupae and adults of the TA1 strain using the Bioline Isolate II RNA mini kit (Bioline Reagents Ltd., UK). RNA was pooled and sent to Eurofins Genomics, Germany, for preparation of a random‐primed normalized cDNA library (with an insert size of 150–450 bp) to ensure the detection of transcripts expressed at even low levels. This was sequenced to high coverage on a single lane of an Illumina HiSeq 2000 using 100‐bp paired‐end sequencing. To identify changes in gene expression between the Spin and SpinSel strains, RNA was extracted from three replicates of 10 larvae from each as above, sent to The Genome Analysis CentreTM (TGAC) and multiplexed for sequencing on one lane of an Illumina HiSeq 2000 using 100‐bp paired‐end sequencing. FastQC was used to check the quality of the raw reads obtained (Andrews ) prior to de novo assembly using Trinity (release trinityrnaseq_r2013_08_14) (Grabherr et al. ) as no reference genome exists for T. absoluta. Two assemblies were performed: assembly 1 combined RNAseq data from Spin and SpinSel and assembly 2 combined data from TA1, Spin and SpinSel. Contigs obtained were annotated using Blast2Go (Conesa et al. ). Bowtie implemented in Trinity was used to map raw reads to assembled transcriptomes (Langmead et al. ), and the rsem software used to estimate gene expression for each biological replicate (Li & Dewey ). edger (version 3.14.0) and deseq2 (version 1.12.4) (Robinson et al. ; Love et al. ) were used to identify differentially expressed genes using default settings and a corrected P value cut‐off of 0.05. Both packages use the negative binomial model for analysing RNAseq count data but differ in their estimation of gene dispersal and genes were only considered differentially expressed if identified as so using both methods. […]

Pipeline specifications

Software tools FastQC, Trinity, Blast2GO, Bowtie, RSEM, edgeR, DESeq2
Application RNA-seq analysis
Chemicals Acetylcholine