Fig. 2

Server-side RNA-seq analysis pipeline. a Installation and configuration. First the GUI transfers the pipeline scripts to the server or utilizes pre-installed scripts if Amazon Cloud instance is being used. Then the pipeline detects installed software and downloads and installs all the analysis tools required for the workflow using an update file on our website which is linked to the current version of CANEapp. After that the pipeline downloads required reference files from ENSEMBL. Reference indexes for STAR and TopHat, as well as gene classification files are prepared in the next step. b Parallel alignment and reconstruction module. Samples are analyzed in parallel; first the reads go through an optional trimming step and are aligned to the genome with either TopHat or STAR. Aligned reads are used to reconstruct transcripts with Cufflinks. This module includes a resource monitor that optimally distributes available resources between subprocesses. c Transcript filtering and classification module. ENSEMBL reference is used to classify genes generated from combining transcript files from all samples. Then the transcripts are filtered to remove potentially spurious single-exon transcripts, and unannotated transcripts and loci are analyzed to predict their ability to code for proteins. d Gene expression and results formatting module. Cuffdiff, edgeR and DESeq2 are used to quantify gene expression and identify differentially expression genes. The pipeline converts output files into fully annotated tab-delimited files, as well as GTF files containing differentially expressed genes. The module also contains primer design scripts that automate primer design for qRT-PCR validation of gene expression