Schematic description of RAP workflow. Quality check and filtering step for quality assessment (Steps 1 and 2). High quality reads are aligned to the reference genome using TopHat (Step 3). Alignments are assembled into full-length transcripts and their relative abundances are estimated by Cufflinks to (Step 4) and raw-counted by HTSeq (Step 5). Unspliced reads are filtered out after an ungapped alignment to the genome (Step 6). Remaining reads (potentially spliced) are mapped to a custom built junction library (Step 7). Reads still unmapped are scanned to identify poly(A) tags (Step 8). Cassette exons are identified and quantified by adopting a statistical tool, SpliceTrap (Step 9), and chimeric transcripts are detected by means of ChimeraScan (Step 10). After the completion of the main analysis, several differential analyses can be executed. Cuffdiff at transcript level (Step A), based on expression levels calculated by Cufflinks. DESeq at gene level (Step B), based on gene raw counts calculated by HTSeq. Differential exons (Step C), differential junctions usage (Step D) and differential polyadenylation sites (Step E) can also be calculated.