Computational protocol: RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages

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Protocol publication

[…] Total RNA was isolated as described previously [] and subjected to DNase treatment to remove residual genomic DNA contamination. Illumina cDNA libraries were prepared using TruSeq RNA sample preparation from a poly(A)-selected RNA. Sequencing of cDNA libraries was performed at Fasteris, Geneva, using Illumina Hiseq sequencing systems with 100 or 125 bp read lengths and sequence depths of > 40 million reads per sample. Reads were mapped to the T. b. brucei 927 reference genome version 5 (either coding sequences or putative 3’UTRs), using the bowtie tool available in Galaxy Interface (usegalaxy.org) with default parameters that allow a maximum of 2 mismatches per 28 bp seed (Galaxy version 1.1.2). Sequencing depth and mapping coverage are provided in Additional file . Mapping to the genome was used to visualise the data on Gbrowse; to estimate transcript abundance, reads were first mapped to coding sequences and unmapped reads were re-mapped to 3’ UTRs. Read counts for the annotated genes or 3’UTR were extracted using SAMTools pileup and RPM values were calculated. Bioconductor package DESeq [] was used to identify the differentially expressed genes from biological replicates. […]

Pipeline specifications

Software tools Bowtie, Galaxy, GBrowse, SAMtools, DESeq
Application RNA-seq analysis
Organisms Trypanosoma brucei, Homo sapiens, Toxoplasma gondii, Drosophila melanogaster
Diseases Parasitic Diseases
Chemicals Inositol