CIRCexplorer3:A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression

Sequences of circular RNAs(circ RNAs)produced from back-splicing of exon(s)completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction(BSJ)sites.Therefore,examination of global circ RNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites,which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies.Thus,direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging.Here,we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq(CIRCexplorer3-CLEAR).A new quantitation parameter,fragments per billion mapped bases(FPB),is applied to evaluate circular and linear RNA expression individually by fragments mapped to circ RNA-specific BSJ sites or to linear RNA-specific splicing junction(SJ)sites.Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcircby FPBlinearto generate a CIRCscore,which indicates the relative circ RNA expression level using linear RNA expression level as the background.Highlyexpressed circ RNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation.CIRCexplorer3-CLEAR is publically available at https://github.com/Yang Lab/CLEAR.