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The RAP pipeline workflow uses a cocktail of different tools/technologies in different steps.
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RAP performs a quality check and filtering step for quality assessment, producing a subset of high quality reads, used in the following steps.
These reads are aligned to the reference genome adopting a spliced aligner (TopHat) to identify exon-exon splice junctions.
Mapping results are then analyzed by Cufflinks to reconstruct putative full-length transcripts and estimate their relative abundance, measured in FPKM (Fragments Per Kilobase of exon per Million mapped fragments)
Unspliced reads are filtered out after an ungapped alignment to the genome
Remaining reads (potentially spliced) are mapped to a custom built junction library
Reads still unmapped are scanned to identify poly(A) tags. Following a trimming procedure, fragments are aligned again to the genome to identify polyadenylation sites
Cassette exons are identified by adopting a statistical tool, SpliceTrap, to detect exon skipping events and estimate their inclusion ratios.
Chimeric transcripts, derived from fusion genes or trans-splicing, are detected by means of ChimeraScan.
After the completion of the main analysis, several differential analyses can be executed. Differential expression can be calculated adopting two different procedures of analysis, to meet different demands.
At gene level, DESeq can be executed, based on gene raw counts calculated by HTSeq.
At transcript level, Cuffdiff is the other options, based on FPKM transcripts expression levels calculated by Cufflinks.
Other operations can be executed to calculate differential exon skipping events and differential polyadenylation sites.