Fig. 1

Workflow of reverse overlap detection and full-length circular RNA reconstruction. RO, reverse overlap; BSJ, back-spliced junction; FSJ, forward-spliced junction. a RO is an overlapped region in amplified circular transcripts in which the 5′- or 3′- ends of paired reads are reversely overlapped with each other. The presence of a 5’ RO indicates that the paired reads are derived from a circular transcript. The presence of both 5′ and 3′ ROs indicates that a full-length circular transcript can be generated by merging the 5′ and 3′ overlapped sequences of the read. b Alignment of a read pair with 5′ RO and/or 3′ RO. c, d Candidate RO-merged reads are mapped to the reference genome to accurately determine the locations of the reads and to rule out contamination. The longest alignment is chosen as an anchor for determining the location of the reads (c). Unmapped and abnormally mapped fragments in the candidate RO-merged reads are realigned to the reference genome based on the location of the anchored alignment; the alignment boundaries are then adjusted based on the GT/AG splicing signal (d). e Workflow of full-length circRNA reconstruction. ROs, BSJs and cirexons are first detected from RNA-seq data. Full-length circRNAs can be reconstructed when both 5′ and 3′ RO are present or when the circRNAs are completely covered by BSJ reads. For circRNAs lacking 3′ RO or FSJs, a combined assembly is performed to integrate the 5′ RO reads and the BSJ reads. f Isoform-level quantification of circRNAs. The BSJ and RO-merged reads are aligned to the reference genome. A forward splice graph (FSG) that records the splicing and coverage information is built based on the alignments. Next, the resulting FSC is dissected into paths that represent putative circular isoforms of the circRNA (right panel). Paths that contain phasing FSJ, where the splicing event is exclusively occupied by only one circular isoform, or co-occurred FSJs, where the number of splicing events is supported by the same RO read, are classified as phased isoforms. The read coverage profile of each path is modeled by a Monte Carlo simulation (right middle panel). Expressed circular isoforms are dissected and quantified by employing an approximate exhaustive search algorithm (bottom)