Mple shows a typical high-efficiency singleexon circularization event at scro. Most circles contain one or a few exons; OPC 8212 web however, a circular RNA from cyclic nucleotide-gated ion channel-like is supported by abundant back-spliced 
reads that specifically traverse 13 exons. A subset of genes generated multiple circular RNAs. For example, the guanylate kinase discs large 1 is not 
only alternatively spliced, it also yields two multi-exon circular RNAs. Finally, we highlight the Wnt pathway transcription factor pangolin for its complex circularization patterns. Of 18 circular products from this gene, the top 5 are depicted in NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Cell Rep. Author manuscript; available in PMC 2015 December 11. Westholm et al. Page 6 circle detection pipeline in D. melanogaster in the other species was complicated by shorter read length. To facilitate comparisons, we supplemented the output of our circular RNA annotation pipeline by directly mapping reads across potential back-splices using slightly relaxed parameters, requiring 15 instead of 20 nt mapping. We confirmed this procedure still specifically identified species of interest, since only 12% of matepairs to back-spliced reads mapped outside of inferred circles. Annotations of circular RNAs in D. yakuba and D. virilis are found in NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Westholm et al. Page 7 8. Amongst informative mate-pairs of back-spliced reads 64 were spliced while 127 contained intronic sequence. Thus, splicing is well-suppressed within this circle. We examined this issue comprehensively. The paired-end data contained 1590 circles for which mate pair reads were informative with respect to splicing status. We tabulated the number of spliced and intron-retained mate pairs for each of these circles, and observed that 90% of loci exhibited >90% of spliced mate pair reads. We summarized these data in Cell Rep. Author manuscript; available in PMC 2015 December 11. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Westholm et al. Page 8 small internal intron and 313348-27-5 web flanking >15kb introns. We therefore examined flanking intron lengths more systematically. D. melanogaster intron lengths are bimodal, with a predominant peak of 50150 bp followed by a broad distribution of longer introns. We observed that circularized exons were flanked by significantly longer introns than average, both upstream and downstream. Total Drosophila introns have a median length of 96 bp, and the median length of all >200 bp introns was 1099 bp. By contrast, the introns upstream and downstream of circular PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847196 RNAs had median lengths of 4662 and 2962 bp, respectively. Thus, introns flanking circularizing exons are much longer than expected, even when excluding the dominant class of short introns. The functional correlation of flanking intron length and circular RNA abundance was evident upon binning circular RNA levels. We observe independently for upstream and downstream introns that progressively higher-expressed RNA circles were associated with progressively longer average flanking intron lengths. Statistical analysis showed that not only were flanking intron lengths significantly different from background introns, for each of five progressively increasing bins of circular RNA expression, the average length distributions of both flanking upstream and downstream introns became significantly larger. Since first i.Mple shows a typical high-efficiency singleexon circularization event at scro. Most circles contain one or a few exons; however, a circular RNA from cyclic nucleotide-gated ion channel-like is supported by abundant back-spliced reads that specifically traverse 13 exons. A subset of genes generated multiple circular RNAs. For example, the guanylate kinase discs large 1 is not only alternatively spliced, it also yields two multi-exon circular RNAs. Finally, we highlight the Wnt pathway transcription factor pangolin for its complex circularization patterns. Of 18 circular products from this gene, the top 5 are depicted in NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Cell Rep. Author manuscript; available in PMC 2015 December 11. Westholm et al. Page 6 circle detection pipeline in D. melanogaster in the other species was complicated by shorter read length. To facilitate comparisons, we supplemented the output of our circular RNA annotation pipeline by directly mapping reads across potential back-splices using slightly relaxed parameters, requiring 15 instead of 20 nt mapping. We confirmed this procedure still specifically identified species of interest, since only 12% of matepairs to back-spliced reads mapped outside of inferred circles. Annotations of circular RNAs in D. yakuba and D. virilis are found in NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Westholm et al. Page 7 8. Amongst informative mate-pairs of back-spliced reads 64 were spliced while 127 contained intronic sequence. Thus, splicing is well-suppressed within this circle. We examined this issue comprehensively. The paired-end data contained 1590 circles for which mate pair reads were informative with respect to splicing status. We tabulated the number of spliced and intron-retained mate pairs for each of these circles, and observed that 90% of loci exhibited >90% of spliced mate pair reads. We summarized these data in Cell Rep. Author manuscript; available in PMC 2015 December 11. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Westholm et al. Page 8 small internal intron and flanking >15kb introns. We therefore examined flanking intron lengths more systematically. D. melanogaster intron lengths are bimodal, with a predominant peak of 50150 bp followed by a broad distribution of longer introns. We observed that circularized exons were flanked by significantly longer introns than average, both upstream and downstream. Total Drosophila introns have a median length of 96 bp, and the median length of all >200 bp introns was 1099 bp. By contrast, the introns upstream and downstream of circular PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847196 RNAs had median lengths of 4662 and 2962 bp, respectively. Thus, introns flanking circularizing exons are much longer than expected, even when excluding the dominant class of short introns. The functional correlation of flanking intron length and circular RNA abundance was evident upon binning circular RNA levels. We observe independently for upstream and downstream introns that progressively higher-expressed RNA circles were associated with progressively longer average flanking intron lengths. Statistical analysis showed that not only were flanking intron lengths significantly different from background introns, for each of five progressively increasing bins of circular RNA expression, the average length distributions of both flanking upstream and downstream introns became significantly larger. Since first i.