Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5’ and 3’ untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.

SUMMARY Clostridium difficile infection (CDI) is the leading cause of antimicrobial and health care-associated diarrhea in humans, presenting a significant burden to global health care systems. In the last 2 decades, PCR- and sequence-based techniques, particularly whole-genome sequencing (WGS), have significantly furthered our knowledge of the genetic diversity, evolution, epidemiology, and pathogenicity of this once enigmatic pathogen. C. difficile is taxonomically distinct from many other well-known clostridia, with a diverse population structure comprising hundreds of strain types spread across at least 6 phylogenetic clades. The C. difficile species is defined by a large diverse pangenome with extreme levels of evolutionary plasticity that has been shaped over long time periods by gene flux and recombination, often between divergent lineages. These evolutionary events are in response to environmental and anthropogenic activities and have led to the rapid emergence and worldwide dissemination of virulent clonal lineages. Moreover, genome analysis of large clinically relevant data sets has improved our understanding of CDI outbreaks, transmission, and recurrence. The epidemiology of CDI has changed dramatically over the last 15 years, and CDI may have a foodborne or zoonotic etiology. The WGS era promises to continue to redefine our view of this significant pathogen.

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.

High-throughput sequencing of cDNA has been used to study eukaryotic transcription on a genome-wide scale to single base pair resolution. In order to compensate for the high ribonuclease activity in bacterial cells, we have devised an equivalent technique optimized for studying complete prokaryotic transcriptomes that minimizes the manipulation of the RNA sample. This new approach uses Illumina technology to sequence single-stranded (ss) cDNA, generating information on both the direction and level of transcription throughout the genome. The protocol, and associated data analysis programs, are freely available from http://www.sanger.ac.uk/Projects/Pathogens/Transcriptome/. We have successfully applied this method to the bacterial pathogens Salmonella bongori and Streptococcus pneumoniae and the yeast Schizosaccharomyces pombe. This method enables experimental validation of genetic features predicted in silico and allows the easy identification of novel transcripts throughout the genome. We also show that there is a high correlation between the level of gene expression calculated from ss-cDNA and double-stranded-cDNA sequencing, indicting that ss-cDNA sequencing is both robust and appropriate for use in quantitative studies of transcription. Hence, this simple method should prove a useful tool in aiding genome annotation and gene expression studies in both prokaryotes and eukaryotes.