A Strand-Specific RNA–Seq Analysis of the Transcriptome of the Typhoid Bacillus Salmonella Typhi

Abstract

High-density, strand-specific cDNA sequencing (ssRNA–seq) was used to analyze the transcriptome of Salmonella enterica serovar Typhi (S. Typhi). By mapping sequence data to the entire S. Typhi genome, we analyzed the transcriptome in a strand-specific manner and further defined transcribed regions encoded within prophages, pseudogenes, previously un-annotated, and 3′- or 5′-untranslated regions (UTR). An additional 40 novel candidate non-coding RNAs were identified beyond those previously annotated. Proteomic analysis was combined with transcriptome data to confirm and refine the annotation of a number of hpothetical genes. ssRNA–seq was also combined with microarray and proteome analysis to further define the S. Typhi OmpR regulon and identify novel OmpR regulated transcripts. Thus, ssRNA–seq provides a novel and powerful approach to the characterization of the bacterial transcriptome.

Next-generation transcriptome assembly

“…Reference-based transcriptome assembly is easier to perform for the simple transcriptomes of prokaryotic and lower eukaryotic organisms since these organisms have few introns and little alternative splicing. Transcription boundaries can be inferred from regions of contiguous read coverage 37,50,51 . Alternative transcription start and stop sites can also be inferred based upon the 5' cap or poly(A) signals within the mapped reads 50,52 .…”

“…Many genes often overlap, resulting in adjacent genes being assembled into one transcript, even though they are not from a polycistronic RNA. Strand-specific RNA-Seq has been used to successfully separate adjacent overlapping genes from opposite strands in the genome 50,51 . However, overlapping genes transcribed from the same strand with comparable expression levels cannot be easily separated without knowledge about their starts and ends.…”

Dual RNA-seq of pathogen and host

“…The first studies in prokaryotes reported the sequencing of rRNA-depleted samples to ~1-7 M reads/library (Oliver et al, 2009;Perkins et al, 2009;Yoder-Himes et al, 2009). This was sufficient to infer differential mRNA expression.…”

“…Many of the pioneering studies were performed using pathogenic bacteria, including S. Typhi (Perkins et al, 2009), Bacillus anthracis (Passalacqua et al, 2009), Burkholderia cenocepacia (Yoder-Himes et al, 2009), Helicobacter pylori or Chlamydia trachomatis (Albrecht et al, 2010), and identified a plethora of novel genes including many new sRNAs (for reviews see (Croucher and Thomson, 2010;Guell et al, 2011;Sorek and Cossart, 2010). The original RNA-seq protocol has since been further modified to increase the informational output: For instance, the usage of a specific exonuclease in a method referred to as differential RNA-seq (dRNA-seq) allowed the selective degradation of processed RNA molecules leaving only primary transcripts, thereby enabling the identification of TSSs on a genome-wide scale .…”

Transposase mediated construction of RNA-seq libraries

“…It enables the detection of noncanonical transcription start sites (Liu et al 2011) as well as termination sites , alternative splice isoforms Jiang and Wong 2009), transcript mutations/editing (Rosenberg et al 2011), and allelic biases in transcript abundance (Pickrell et al 2010). Methods that preserve the strand from which the transcript originated also allow for the identification of antisense transcription (He et al 2008;Perkins et al 2009), which can play a role in post-transcriptional regulation. Because of the power of RNA-seq and the prevalence of aberrant gene-expression patterns in many diseases, there is a growing need to construct libraries efficiently from low starting amounts of RNA in a high-throughput and reproducible fashion.…”