Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori
Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori
复制标题
DOI：
10.1016/j.ymeth.2015.06.012
复制
发表时间：
2015-09-15
期刊：
影响因子：
4.8
通讯作者：
Sharma, Cynthia M.
中科院分区：
文献类型：
--
作者：
Bischler, Thorsten;Tan, Hock Siew;Sharma, Cynthia M.
转录边界的全局作图是阐明生物体转录特征的完整补充的关键步骤。它有助于操纵子和非翻译区以及新转录本的注释，包括顺式和反式编码的小RNA（sRNA）。基于cDNA的深度测序的所谓RNA测序（RNA-seq）极大地促进了具有单核苷酸分辨率的转录物作图。然而，传统的RNA-seq方法通常不能区分初级和加工的转录物。在这里，我们描述了最近开发的差异RNA-seq（dRNA-seq）的方法，这有利于注释的转录起始位点（TSS）的基础上深度测序的两个差异处理的cDNA文库对，一个库被富集的初级转录本。使用人类病原体幽门螺杆菌作为模式生物，我们描述了应用dRNA-seq与自动TSS注释方法一起生成细菌全基因组TSS图谱。除了可以通过这种方法确定的转录组和监管功能的描述，我们讨论了不同的库制备方案和测序平台，以及手动和自动TSS注释的影响。此外，我们已经建立了一个易于访问的在线浏览器的可视化的H。pylori转录组数据，以及我们之前的H. pylori dRNA-seq研究。(C)2015 Elsevier Inc. All rights reserved.
The global mapping of transcription boundaries is a key step in the elucidation of the full complement of transcriptional features of an organism. It facilitates the annotation of operons and untranslated regions as well as novel transcripts, including cis- and trans-encoded small RNAs (sRNAs). So called RNA sequencing (RNA-seq) based on deep sequencing of cDNAs has greatly facilitated transcript mapping with single nucleotide resolution. However, conventional RNA-seq approaches typically cannot distinguish between primary and processed transcripts. Here we describe the recently developed differential RNA-seq (dRNA-seq) approach, which facilitates the annotation of transcriptional start sites (TSS) based on deep sequencing of two differentially treated cDNA library pairs, with one library being enriched for primary transcripts. Using the human pathogen Helicobacter pylori as a model organism, we describe the application of dRNA-seq together with an automated TSS annotation approach for generation of a genome-wide TSS map in bacteria. Besides a description of transcriptome and regulatory features that can be identified by this approach, we discuss the impact of different library preparation protocols and sequencing platforms as well as manual and automated TSS annotation. Moreover, we have set up an easily accessible online browser for visualization of the H. pylori transcriptome data from this and our previous H. pylori dRNA-seq study. (C) 2015 Elsevier Inc. All rights reserved.