基于Nanopore测序的环形RNA数据挖掘方法

作为非编码RNA领域的研究热点，目前绝大多数的环形RNA功能仍不明确，需要更多新颖的理论假说和大量的功能验证。能否从海量转录组数据中精确识别和重建环形RNA转录本，则成为环形RNA功能探索的重要先决条件。现有的环形RNA识别方法都是基于二代测序数据上发展而来，对环形RNA全长重建有先天的局限性。三代测序虽然可以产生超长读段，但无法直接用于环形RNA的扩增和测序。本项目拟在前期自主开发的环形RNA识别和定量算法的基础上，建立基于Nanopore测序平台的环形RNA富集、扩增和测序技术；开发基于超长测序读段的环形RNA识别和全长重建算法；识别线粒体基因组来源和含有稀有可变剪接位点的环形RNA，以及统计环形RNA的真实长度分布；整合二代和三代的测序优势实现环形和线性转录本的无偏差估算；本研究将填补环形RNA计算方法学的空白，并为后续环形RNA的功能研究和进化探索提供便利的工具。

As essential regulator of various biological processes, circular RNAs (circRNAs) have been received extensive attention. However, our understanding of how circRNAs participate in biological processes remains preliminary, and novel mechanisms are required to elucidate their diversity. Thus, unbiased reconstruction approaches of circRNAs from high-throughput transcriptomic data become an essential prerequisite of circRNA functional study. Currently, most circRNA identification methods are development based on 2nd-generation sequencing technology and have inherent limitation on full-length sequence construction. 3rd-generation sequencing technology is an approach that has opened up a new era in transcriptomics studies by allowing ultra-long sequencing reads. However, the inclusion of a poly(A) selection step prevents the detection of circRNA. Therefore, we aim to develop an experimental and computational approach for effective reconstruction and quantification of full-length circRNAs using Nanopore sequencing technology. To achieve this, we incorporate several key innovations, notably (I) a modified version of circRNA enrichment approach to focus our sequencing on circRNAs; (II) new computational method to identify full-length circRNAs from ultra-long reads; (III) accurate identification of circRNAs from mitochondrial and circular transcripts containing rare splicing-signals, as well as their length distribution; (IV) unbiased expression quantification between circRNAs and linear transcripts by combing 2nd- and 3rd-generation sequencing technology. We believe this approach will be an important addition to the toolbox of circRNA identification and provide useful targets for functional screening, as well as expand our understanding of circRNAs.