FL-circAS: an integrative resource and analysis for full-length sequences and alternative splicing of circular RNAs with nanopore sequencing

Abstract Circular RNAs (circRNAs) are RNA molecules with a continuous loop structure characterized by back-splice junctions (BSJs). While analyses of short-read RNA sequencing have identified millions of BSJ events, it is inherently challenging to determine exact full-length sequences and alternatively spliced (AS) isoforms of circRNAs. Recent advances in nanopore long-read sequencing with circRNA enrichment bring an unprecedented opportunity for investigating the issues. Here, we developed FL-circAS (https://cosbi.ee.ncku.edu.tw/FL-circAS/), which collected such long-read sequencing data of 20 cell lines/tissues and thereby identified 884 636 BSJs with 1 853 692 full-length circRNA isoforms in human and 115 173 BSJs with 135 617 full-length circRNA isoforms in mouse. FL-circAS also provides multiple circRNA features. For circRNA expression, FL-circAS calculates expression levels for each circRNA isoform, cell line/tissue specificity at both the BSJ and isoform levels, and AS entropy for each BSJ across samples. For circRNA biogenesis, FL-circAS identifies reverse complementary sequences and RNA binding protein (RBP) binding sites residing in flanking sequences of BSJs. For functional patterns, FL-circAS identifies potential microRNA/RBP binding sites and several types of evidence for circRNA translation on each full-length circRNA isoform. FL-circAS provides user-friendly interfaces for browsing, searching, analyzing, and downloading data, serving as the first resource for discovering full-length circRNAs at the isoform level.