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.

中文翻译：

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CircAST：环状RNA中全长剪接异构体的全长组装和定量。

环状RNA（circRNA）是共价闭合的连续RNA环，是通过反向剪接事件从同源线性RNA产生的，并且可变剪接事件可能在同一位点产生不同的circRNA亚型。然而，重建和量化的选择性剪接的全长circRNA的挑战仍未解决。根据circRNA的内部结构特征，我们开发了CircAST，这是一种用于组装交替剪接的circRNA转录本并通过使用多个剪接图估计其表达的工具。仿真研究表明，CircAST正确组装了circRNA的完整序列，灵敏度为85.63％-94.32％，精度为81.96％-87.55％。通过将读段分配给特定的同工型，CircAST量化了相关系数为0的circRNA同工型的表达。理论值和估计值之间在85-0.99之间。我们在内部小鼠睾丸RNA-seq数据集上使用RNase R处理评估了CircAST，以丰富circRNA，并鉴定了380个circRNA，其全长序列与其相应同源线性RNA的序列不同。RT-PCR和Sanger测序分析验证了37种随机选择的同工型中的32种，从而进一步表明CircAST的良好性能，尤其是对于低丰度的同工型。我们还将CircAST应用于已发布的实验数据，并观察到整个样品的环形转录本具有相当大的多样性，从而表明circRNA的表达受到高度调节。可以从https://github.com/xiaofengsong/CircAST免费访问CircAST。我们在内部小鼠睾丸RNA-seq数据集上使用RNase R处理评估了CircAST，以丰富circRNA，并鉴定了380个circRNA，其全长序列与其相应同源线性RNA的序列不同。RT-PCR和Sanger测序分析验证了37种随机选择的同工型中的32种，从而进一步表明CircAST的良好性能，尤其是对于低丰度的同工型。我们还将CircAST应用于已发布的实验数据，并观察到整个样品的环形转录本具有相当大的多样性，从而表明circRNA的表达受到高度调节。可以从https://github.com/xiaofengsong/CircAST免费访问CircAST。我们在内部小鼠睾丸RNA-seq数据集上使用RNase R处理评估了CircAST，以丰富circRNA，并鉴定了380个circRNA，其全长序列与其相应同源线性RNA的序列不同。RT-PCR和Sanger测序分析验证了37种随机选择的同工型中的32种，从而进一步表明CircAST的良好性能，尤其是对于低丰度的同工型。我们还将CircAST应用于已发布的实验数据，并观察到整个样品的环形转录本具有相当大的多样性，从而表明circRNA的表达受到高度调节。可以从https://github.com/xiaofengsong/CircAST免费访问CircAST。RT-PCR和Sanger测序分析验证了37种随机选择的同工型中的32种，从而进一步表明CircAST的良好性能，尤其是对于低丰度的同工型。我们还将CircAST应用于已发布的实验数据，并观察到整个样品的环形转录本具有相当大的多样性，从而表明circRNA的表达受到高度调节。可以从https://github.com/xiaofengsong/CircAST免费访问CircAST。RT-PCR和Sanger测序分析验证了37种随机选择的同工型中的32种，从而进一步表明CircAST的良好性能，尤其是对于低丰度的同工型。我们还将CircAST应用于已发布的实验数据，并观察到整个样品的环形转录本具有相当大的多样性，从而表明circRNA的表达受到高度调节。可以从https://github.com/xiaofengsong/CircAST免费访问CircAST。