Sequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Therefore, examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies. Thus, direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. Here, we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CIRCexplorer3-CLEAR). A new quantitation parameter, fragments per billion mapped bases (FPB), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression level using linear RNA expression level as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation. CIRCexplorer3-CLEAR is publically available at https://github.com/YangLab/CLEAR.
除了反向剪接位点，环形RNA与其对应的线形RNA在一级序列上完全重复，因此从转录组测序数据中系统发现环形RNA和线形RNA的策略不同。现有对环形RNA的全转录组检测和定量主要依赖对跨反向剪接位点读序的分析，这与线形RNA定量依赖对覆盖基因外显子和跨外显子读序的分析不一致，导致现有计算方法大多无法直接用来比较环形RNA与其对应线形RNA的表达，这是环形RNA全转录组分析的难点之一。在这项最新的研究中，我们升级开发了环形RNA与其对应线形RNA直接定量比较的分析系统CIRCexplorer3-CLEAR（circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq, https://github.com/YangLab/CLEAR），用于开展环形RNA的精准定量研究。CIRCexplorer3-CLEAR使用统一的定量参数FPB（fragments per billion mapped bases），利用跨反向剪接位点和跨正常剪接位点的读序数据分别计算环形RNA （FPBcirc）和线形RNA（FPBlinear）的表达；并进一步通过直接比较环形RNA及其对应线形RNA的FPB值获得CIRCscore（FPBcirc vs FPBlinear），用来表征环形RNA相对于线形RNA的表达水平。相对于原有的FPM，新建立的FPB不受读序长度与测序策略的影响，而CIRCscore值则能进一步去除线形RNA的表达背景，因此基于FPB和CIRCscore的定量分析将有更广的适应性和更高的鲁棒性。利用CIRCexplorer3-CLEAR，研究人员可筛选获得相对于线形RNA高表达的环形RNA，并开展后续环形RNA功能及作用机制等研究。





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