An upgraded computational pipeline (CIRCexplorer2) to systematically identify alternative (back-)splicing in back-spliced circular RNAs (circRNAs). (A) Schematic diagrams of two types of alternative back-splicing. Colored bars, exons. Black lines, introns. Red polylines, (canonical) collinear splicing. Red arc lines, back-splicing (circularization). (B) Schematic diagrams of four basic types of alternative splicing. Colored bars, exons. Black lines, introns. Red lines, splicing. Red arc lines, back-splicing (circularization). (C) The schematic diagram of CIRCexplorer2. The analysis was performed as described (Zhang et al. 2014) with modifications (Supplemental Methods). Alternative back-splicing and alternative splicing in circRNAs were determined with stringent criteria (Supplemental Methods). (D) Ten thousand circRNAs (gray bars) were detected by CIRCexplorer2. Thousands of novel exons (blue points) were identified in circRNAs in different human cell lines with de novo assembly (Supplemental Methods). (E) The identification and visualization of circRNAs in the CAMSAP1 locus from H9 (left panel) or PA1 (right panel) cell lines. Different types of RNA-seq data sets from ribo−, p(A)+, p(A)− or p(A)−/RNase R RNA populations were used for comparison. CAMSAP1 circRNAs could be determined from ribo−, p(A)−, and p(A)−/RNase R RNA-seq data sets by identifying back-splice junctions. Notably, ribo− RNA-seq is not suitable to study canonical splicing events (intron retention, in this case) that occur specifically within circRNAs, as ribo− RNAs contain both polyadenylated and nonpolyadenylated transcripts. Blues bars, exons. Black lines, introns. Black thick line, the retained intron. Red arc lines, back-splicing (circularization).
