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 FPB_circ by FPB_linear 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.

外显子反向剪接产生的环状 RNA (circRNA)序列与从相同基因位点转录的同源线性 RNA 的序列完全重叠，但反向剪接连接 (BSJ) 位点除外。因此，从RNA-seq数据集中检查全局circRNA表达通常依赖于跨BSJ位点的RNA-seq片段的检测，这不同于通过映射到整个基因体的归一化RNA-seq片段来量化线性RNA表达。因此，以全基因组方式直接比较相同基因位点的环状和线性 RNA 表达仍然具有挑战性。在这里，我们将之前报道的 CIRCexplorer 管道更新到版本 3，用于核糖体 RNA 耗尽的 RNA-seq (CIRCexplorer3-CLEAR) 的环状和线性 RNA 表达分析。新的定量参数，每十亿映射碱基的片段数 (FPB)，用于通过映射到 circRNA 特异性 BSJ 位点或线性 RNA 特异性剪接点 (SJ) 位点的片段单独评估环状和线性 RNA 表达。环状和线性RNA表达水平的比较直接通过将FPB _circ除以FPB_线性生成CIRCscore来实现，CIRCscore表示以线性RNA表达水平为背景的相对circRNA表达水平。CIRCexplorer3-CLEAR 可以轻松识别具有低同源线性 RNA 表达背景的高表达 circRNA，以进行进一步研究。CIRCexplorer3-CLEAR 可在 https://github.com/YangLab/CLEAR 上公开获取。