Large-scale investigation of short read archives for disruption of transcription termination and circular splicing

我们和其他人最近报道了溶性单纯疱疹病毒1 （HSV-1）感染和盐胁迫和热胁迫都会导致大多数人类基因的转录终止中断（DoTT）。这导致大量下游基因转录（DoG转录）。考虑到应激诱导的转录变化已经被深入研究，令人惊讶的是，DoTT/DoG转录直到最近才被发现。最可能的原因是标准的RNA-seq分析只关注已知的转录本，从而忽略了注释基因之外的转录。然而，现在大多数期刊都要求在发表手稿之前，将原始测序数据提交给短读档案，如NCBI SRA。因此，即使原始研究没有调查DoTT/DoG转录，原始数据也可用于重新分析。虽然短读档案中RNA-seq数据的绝对规模以前使对所有可用的已发表数据进行大规模再分析变得令人望而却步，但最近的发展现在使这成为可能。另一方面，这些发展包括用于短读索引和搜索以及快速转录本定量的新颖的无比对方法。另一方面，在SRA的大多数人类RNA-seq实验中，全基因组读取覆盖率和剪接连接数据现在由recot2数据库提供。在这个项目中，我们建议利用这些新工具来解决我们之前对1型单纯疱疹病毒感染和热盐胁迫分析中产生的具体问题。首先，我们计划描述不同条件下DoTT/DoG转录的普遍性和相似性及其在物种间的保护性。其次，我们将研究环状rna的从头产生，并研究它们在不同条件下相对于线性rna的诱导。为此，我们将继续对recot2数据进行重新分析，以快速表征人类在广泛条件下的DoTT/DoG转录，并将其与无比对方法相结合，将该分析扩展到剩余的SRA和环状剪接。综上所述，本项目不仅旨在解决在sra范围内关于DoTT/DoG转录和环状剪接的重要生物学问题，而且还将作为一个概念证明，大规模研究特定转录事件的sra是可行和有用的。

We and others recently reported that both lytic herpes simplex virus 1 (HSV-1) infection and salt and heat stress lead to a disruption of transcription termination (DoTT) of the majority of human genes. This results in massive transcription downstream of genes (DoG transcription). Considering that stress-induced transcription changes have already been studied intensively, it is surprising that DoTT/DoG transcription has been discovered only so recently. The most likely reason for this is that standard RNA-seq analysis focuses only on known transcripts, thus ignoring transcription outside of annotated genes. However, most journals now require that before publication of manuscripts raw sequencing data is submitted to short read archives such as the NCBI SRA. Thus, even if the original studies did not investigate DoTT/DoG transcription, the raw data is available for reanalysis. While the sheer size of RNA-seq data in short read archives previously made it prohibitive to perform a large-scale reanalysis of all available published data, recent developments now make this feasible. On the on hand, these developments include novel alignment-free methods for both short read indexing and searching as well as fast transcript quantification. On the other hand, genome-wide read coverage and splice junction data is now provided by the recount2 database for the majority of human RNA-seq experiments in the SRA. In this project, we propose to leverage these new tools to address specific questions arising from our previous analysis of HSV-1 infection and heat and salt stress. First, we plan to characterize the prevalence and similarity of DoTT/DoG transcription in different conditions and its conservation between species. Second, we will study de novo production of circular RNAs as well as investigate their induction relative to linear RNAs in different conditions. For this purpose, we will pursue both a re-analysis of the recount2 data to quickly characterize DoTT/DoG transcription in a wide range of conditions in human as well as combine this with alignment-free approaches to extend this analysis to the remaining SRA and circular splicing. In summary, this project not only aims to address important biological questions on DoTT/DoG transcription and circular splicing in an SRA-wide manner, but it will also serve as a proof-of-concept that the large-scale investigation of SRAs for specific transcription events is both feasible and useful.