Prokaryotic whole‐transcriptome analysis: deep sequencing and tiling arrays

Hybridization to microarrays has been the standard for genome‐wide transcriptome analyses of prokaryotes in the past 10 years. Microarrays have several limitations, however, among which are a small dynamic range for detection of transcript levels due to problems with saturation, background noise, spot density and spot quality. Moreover, comparing different experiments requires complex normalization methods (Hinton et al., 2004) and comparing different strains requires designing pangenome arrays based on multiple sequenced genomes, leading to further problems in non‐specific or cross‐hybridization and complicated data analysis (Bayjanov et al., 2009). Most microarrays have a biased genome coverage, as they only contain a limited number of short probes for known or expected genes in sequenced genomes, and they rarely probe intergenic regions. Technological advances in array production and dropping costs have recently led to the design and use of high‐density tiling arrays based on overlapping short oligonucleotides covering both strands of entire genomes (Selinger et al., 2000; Mcgrath et al., 2007; Rasmussen et al., 2009; Toledo‐Arana et al., 2009). Tiling array and other studies have provided a first insight into far more complex transcriptomes than previously envisioned, including an ever‐expanding range of regulatory RNAs (Waters and Storz, 2009). To overcome the remaining limitations of microarrays, a totally new approach to whole‐transcriptome analysis was needed – and a much‐awaited breakthrough in DNA sequencing came to the rescue. Here, we describe the first whole‐transcriptome applications in prokaryotes and discover that a new treasure chest of regulation in prokaryotes is being opened.