Trans-splicing rarely contributed to back-spliced junctions. (A) Schematic illustration of the analysis of paired-end reads used to distinguish trans-spliced products from circRNAs. Depending on the insert size, mate reads of trans-spliced but not back-spliced junction-spanning reads could potentially map to adjacent linear exons. Based on the insert sizes of the ENCODE paired-end RNA-seq libraries, we only considered circRNAs that were <400 nucleotides. (B) Distances of all mapped mate reads from the acceptors (left) and donors (right). Two possible trans-spliced events are indicated. (C) The identified trans-spliced event from the ANKRD28 locus. (D) Circular fractions of 598 circRNAs detected in non-poly(A)-selected RNA-seq data from U2OS cells, analyzed using non-poly(A)-selected RNA-seq data (Ribo-Zero) and poly(A)-selected RNA-seq data (poly(A)+).