Gaffo Enrico, Bonizzato Annagiulia, Kronnie Geertruy Te, Bortoluzzi Stefania
Department of Molecular Medicine, University of Padova, Padova 35131, Italy.
Department of Women's and Children's Health, University of Padova, Padova 35128, Italy.
Noncoding RNA. 2017 Feb 10;3(1):8. doi: 10.3390/ncrna3010008.
Circular RNAs (circRNAs) are generated by backsplicing of immature RNA forming covalently closed loops of intron/exon RNA molecules. Pervasiveness, evolutionary conservation, massive and regulated expression, and posttranscriptional regulatory roles of circRNAs in eukaryotes have been appreciated and described only recently. Moreover, being easily detectable disease markers, circRNAs undoubtedly represent a molecular class with high bearing on molecular pathobiology. CircRNAs can be detected from RNAseq data using appropriate computational methods to identify the sequence reads spanning backsplice junctions that do not colinearly map to the reference genome. To this end, several programs were developed and critical assessment of various strategies and tools suggested the combination of at least two methods as good practice to guarantee robust circRNA detection. Here,we present CirComPara (http://github.com/egaffo/CirComPara), an automated bioinformatics pipeline, to detect, quantify and annotate circRNAs from RNAseq data using in parallel four different methods for backsplice identification. CirComPara also provides quantification of linear RNAs and gene expression, ultimately comparing and correlating circRNA and gene/transcript expression level. We applied our method to RNAseqdata of monocyte and macrophage samples in relation to haploinsufficiency of the RNAbinding splicing factor Quaking (QKI). The biological relevance of the results, in terms of number, types and variations of circRNAs expressed, illustrates CirComPara potential to enlarge the knowledge of the transcriptome, adding details on the circRNAome, and facilitating further computational and experimental studies.
环状RNA（circRNAs）由未成熟RNA的反向剪接产生，形成内含子/外显子RNA分子的共价闭合环。circRNAs在真核生物中的普遍性、进化保守性、大量且受调控的表达以及转录后调控作用，直到最近才得到认识和描述。此外，作为易于检测的疾病标志物，circRNAs无疑代表了一类对分子病理生物学具有重要意义的分子。可以使用适当的计算方法从RNA测序数据中检测circRNAs，以识别跨越反向剪接连接点的序列读数，这些读数不能线性映射到参考基因组。为此，人们开发了几个程序，对各种策略和工具的关键评估表明，至少结合两种方法是保证可靠检测circRNAs的良好做法。在这里，我们展示了CirComPara（http://github.com/egaffo/CirComPara），这是一个自动化的生物信息学管道，用于使用四种不同的反向剪接识别方法并行检测、定量和注释来自RNA测序数据的circRNAs。CirComPara还提供线性RNA和基因表达的定量，最终比较并关联circRNA与基因/转录本的表达水平。我们将我们的方法应用于与RNA结合剪接因子震颤（QKI）单倍体不足相关的单核细胞和巨噬细胞样本的RNA测序数据。结果在表达的circRNAs的数量、类型和变异方面的生物学相关性，说明了CirComPara在扩大转录组知识、增加circRNA组细节以及促进进一步的计算和实验研究方面的潜力。
