Circular RNAs (circRNAs), covalently closed continuous RNA loops, are generated from cognate linear RNAs through back splicing events, and alternative splicing events may generate different circRNA isoforms at the same locus. However, the challenges of reconstruction and quantification of alternatively spliced full-length circRNAs remain unresolved. On the basis of the internal structural characteristics of circRNAs, we developed CircAST, a tool to assemble alternatively spliced circRNA transcripts and estimate their expression by using multiple splice graphs. Simulation studies showed that CircAST correctly assembled the full sequences of circRNAs with a sensitivity of 85.63%–94.32% and a precision of 81.96%–87.55%. By assigning reads to specific isoforms, CircAST quantified the expression of circRNA isoforms with correlation coefficients of 0.85–0.99 between theoretical and estimated values. We evaluated CircAST on an in-house mouse testis RNA-seq dataset with RNase R treatment for enriching circRNAs and identified 380 circRNAs with full-length sequences different from those of their corresponding cognate linear RNAs. RT-PCR and Sanger sequencing analyses validated 32 out of 37 randomly selected isoforms, thus further indicating the good performance of CircAST, especially for isoforms with low abundance. We also applied CircAST to published experimental data and observed substantial diversity in circular transcripts across samples, thus suggesting that circRNA expression is highly regulated. CircAST can be accessed freely at https://github.com/xiaofengsong/CircAST.
环形RNA(circRNA)具有共价闭合环状结构，由同源线性RNA通过反向剪接事件产生，且可由其内部可变剪接事件形成不同的环形转录本。然而环形RNA全长转录本的组装和定量问题仍未得到解决。根据环形RNA内部结构的特点，我们开发了一种工具CircAST，通过构建多剪接图模型来重建环形RNA转录本并估计其表达量。模拟结果表明CircAST在重建环形RNA转录本全长序列时敏感度为85.63%–94.32%，准确度为81.96%–87.55%。在转录本定量方面，CircAST通过算法把reads分配到具体的转录本来计算该转录本的表达丰度，可使估算值和真实值的相关系数达到0.85–0.99。我们还将CircAST应用于小鼠睾丸RNase R处理后的RNA-seq测序数据，结果发现380个环形RNA全长转录本与其同源的线性RNA转录本的全长序列不同。最后我们采用RT-PCR和Sanger测序对随机选择的37个环形转录本进行了验证，其中32个环形转录本成功验证，这说明CircAST具有较好的性能，特别是其针对低丰度的环形RNA转录本。我们还将CircAST应用于已公开发表的实验数据中，结果表明不同样本表达的环形RNA转录本具有显著多样性，提示其表达是受到高度调控的。CircAST可在以下网址免费下载使用：https://github.com/xiaofengsong/CircAST。





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