Circular RNAs (circRNAs) produced from back-splicing of exon(s) are completely overlapped with their cognate linear RNAs in sequences, except back-splicing junction (BSJ) sites. Transcriptome-wide profiling and quantification of circRNAs from RNA-seq datasets is generally based on the detection of RNA-seq fragments spanning BSJ sites, while quantification of linear RNA expression relies on fragments mapped to whole gene bodies. In this scenario, direct comparison of circular and cognate linear RNA expression from the same gene loci has remained challenging.   

The research group led by Dr. YANG Li from CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health (SINH) of Chinese Academy of Sciences (CAS) recently developed an integrated toolkit for direct circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CIRCexplorer3-CLEAR, https://github.com/YangLab/CLEAR) updated from the previously-reported CIRCexplorer pipeline. 

To evaluate circular and linear RNA expression in a comparative way, a new quantitation parameter, fragments per billion mapped bases (FPB), is developed. Fragments mapped to circRNA-specific BSJ sites are used to calculate FPB_circ and those aligned to linear RNA-specific splicing junction (SJ) sites are applied to calculate FPB_linear. CIRCscore, which is dividing FPB_circ by FPB_linear, is defined to show relative circRNA expression normalized by cognate linear RNA expression. Importantly, FPB value is more resistant to different sequencing lengths and strategies than commonly-used fragments per million mapped fragments (FPM) for circular RNAs. Moreover, using CIRCscore to evaluate circRNA expression can further reduce differences that are caused by RNA-seq samples themselves. Taken together, the CIRCexplorer3-CLEAR pipeline is an accurate and robust method for circRNA quantification and can be applied for selection of highly expressed circRNAs relative to cognate linear RNAs for downstream circRNA analysis. 

The study entitled "CIRCexplorer3: A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression" was published online in Genomics, Proteomics & Bioinformatics on January 15, 2020. 

This work was mainly conducted at Dr. YANG Li’s lab at PICB, SINH with the help of Dr. CHEN Ling-Ling from Shanghai Institute of Biochemistry and Cell Biology (SIBCB) and Dr. CARMICHAEL Gordon G. from University of Connecticut. Computational analyses were performed by MA Xu-Kai, WANG Meng-Ran and DONG Rui from PICB, SINH. Experiments were performed by LIU Chu-Xiao from Dr. CHEN Ling-Ling’s lab at SIBCB. This work was financially supported by grants from the Strategic Priority Research Program of CAS, the National Natural Science Foundation of China, and the Howard Hughes Medical Institute International Program, the United States. 

New CIRCexplorer3-CLEAR pipeline for direct circular RNA and linear RNA expression comparison
(Image by Dr. YANG Li’s group) 

Media Contact:
WANG Jin (Ms.)
Shanghai Institute of Nutrition and Health,
Chinese Academy of Sciences
Email: sibssc@sibs.ac.cn