TableS4.xlsx from Exploring differential exon usage via short- and long-read RNA sequencing strategies

Alternative splicing produces various mRNAs, and thereby various protein products, from one gene, impacting a wide range of cellular activities. However, accurate reconstruction and quantification of full-length transcripts using short-reads is limited, due to their length. Long-reads sequencing technologies may provide a solution by sequencing full-length transcripts. We explored the use of both Illumina short-reads and two long Oxford Nanopore Technology (cDNA and Direct RNA) RNA-Seq reads for detecting global differential splicing during mouse embryonic stem cell differentiation, applying several bioinformatics strategies: gene-based, isoform-based and exon-based. We detected the strongest similarity among the sequencing platforms at the gene level compared to exon-based and isoform-based. Furthermore, the exon-based strategy discovered many differential exon usage (DEU) events, mostly in a platform-dependent manner and in non-differentially expressed genes. Thus, the platforms complemented each other in the ability to detect DEUs, i.e. long-reads exhibited an advantage in detecting DEUs at the 3׳ UTRs, and short-reads detected more DEUs. Exons within 20 genes, detected in one or more platforms, were here validated by PCR, including key differentiation genes, such as Mdb3 and Aplp1. We provide an important analysis resource for discovering transcriptome changes during stem cell differentiation and insights for analysing such data.可变剪接可从单个基因生成多种mRNA，进而产生多样的蛋白质产物，广泛影响细胞的各类生理活动。然而，受限于读长，基于短读长测序技术精准重建并定量全长转录本的能力存在显著局限。长读长测序技术可通过对全长转录本进行测序，为解决上述问题提供了可行方案。本研究针对小鼠胚胎干细胞分化过程中的全局差异剪接检测，同时采用Illumina短读长测序数据与牛津纳米孔科技（Oxford Nanopore Technology）的两类长读长RNA测序（RNA-Seq）数据（分别为互补DNA（cDNA）测序与直接RNA测序），并应用了基于基因、基于转录本亚型以及基于外显子的三种生物信息学分析策略。相较于基于外显子和基于转录本亚型的分析结果，各测序平台在基因层面的检测结果相似性最高。此外，基于外显子的分析策略鉴定出大量差异外显子使用（differential exon usage, DEU）事件，其中多数事件表现出平台依赖性，且多发生在无差异表达的基因中。由此可见，各测序平台在检测DEU事件的能力上具有互补性：长读长测序在检测3'非翻译区（3' UTR）内的DEU事件时更具优势，而短读长测序则可鉴定出更多的DEU事件。本研究通过聚合酶链式反应（polymerase chain reaction, PCR）验证了在一个或多个测序平台中鉴定得到的20个基因的外显子区域，其中涵盖Mdb3、Aplp1等核心干细胞分化相关基因。本研究为解析干细胞分化过程中的转录组变化提供了重要的分析资源，同时也为此类测序数据的分析提供了具有参考价值的研究思路。