Fig. 3

Novel quantitative assay for circRNA conservation analysis. a Workflow of the conserved circRNA identification. First, orthologous gene pairs were downloaded from the OMA database. Only orthologous gene pairs expressing circRNAs were retained. Next, the boundary of the cirexon pairs, defined as the exons involved in circRNA formation, in the orthologous gene pairs was determined based on whole genome alignments. To detect conserved circRNAs derived from these cirexons, 50-bp fragments on both sides of the circRNA BSJ were extracted and used to represent the BSJ sequence. All circRNA BSJ sequences from a single species were aligned to those from the other species using BLAT, followed by the reciprocal best hits strategy, to identify the orthologous circRNAs. Then, the MultiMSOAR tool was employed to integrate the reciprocal best pairwise orthologous circRNAs and obtain the best-matching orthologous circRNAs. b Scheme for the MCS calculation, integrating detailed conservation profiles in species, tissue, and individual cellular levels. The solid circles in each layer denote the tissues or individuals expressing the circRNA. c The distribution of MCS on highly conserved circRNAs with MCS scores ranging from 5 to 6. The scheme was able to successfully distinguish between the conservation of circRNAs either in species, tissues, or individual cells. Each circle denotes a circRNA and the circle size represents the number of individuals expressing this circular transcript. d Distribution of MCS for the conserved circRNAs identified in the six species. e Illustration of the improved candidate circRNA prioritization based on MCS using the YAP1 gene. Top: illustration of the genomic region of the circRNAs derived from the human YAP1 gene. Note that a total of 65 circRNAs were expressed from this gene but only the top 3 circRNAs (based on their MCS) were plotted. Curved gray lines indicate the BSJs of the three circRNAs. Bottom: conservation profiles of the 65 circRNAs derived from the YAP1 gene. Each dot represents a circRNA. The gray, yellow, orange, and red colors represent increased MCS