title: Differential RNA-seq (dRNA-seq) for annotation of transcriptional start sites and small RNAs in Helicobacter pylori.
abstract: 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.
authors.affiliation: Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Bischler
authors.affiliation: Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg, Germany.
authors.forename: Hock Siew
authors.initials: HS
authors.lastname: Tan
authors.affiliation: Integrative Transcriptomics, ZBIT (Center for Bioinformatics Tübingen), University of Tübingen, Sand 14, D-72076 Tübingen, Germany.
authors.forename: Kay
authors.initials: K
authors.lastname: Nieselt
authors.affiliation: Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg, Germany. Electronic address: cynthia.sharma@uni-wuerzburg.de.
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Sharma
mesh_terms: D016680:Genome, Bacterial; D016480:Helicobacter pylori / Q000235:genetics* / Q000472:pathogenicity; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D058977:Molecular Sequence Annotation; D024363:Transcription Initiation Site; D059467:Transcriptome / Q000235:genetics
keywords: 5′UTR; Comparative transcriptomics; Differential RNA-seq; Gene regulation; Promoter motifs; Small RNAs; Transcriptional start sites