Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori

• Published in: Methods (San Diego, Calif.) Vol. 86; pp. 89 - 101
• Main Authors: Bischler, Thorsten, Tan, Hock Siew, Nieselt, Kay, Sharma, Cynthia M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.2015
• Subjects: 5′UTR; Comparative transcriptomics; Differential RNA-seq; Gene regulation; Genome, Bacterial; Helicobacter pylori - genetics; Helicobacter pylori - pathogenicity; High-Throughput Nucleotide Sequencing - methods; Humans; Molecular Sequence Annotation; Promoter motifs; Small RNAs; Transcription Initiation Site; Transcriptional start sites; Transcriptome - genetics; Transcriptional start sites; Differential RNA-seq; Gene regulation; Comparative transcriptomics; 5′UTR; Promoter motifs; Small RNAs
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2015.06.012

•A differential RNA-seq (dRNA-seq) approach for primary transcriptome analysis.•dRNA-seq analysis of the gastric pathogen Helicobacter pylori as a model bacterium.•dRNA-seq for global annotation of transcriptional start sites (TSS) and small RNAs.•Comparison of automated and manual TSS annotation.•Variations due to sample and library preparations and sequencing protocols. 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.