E-MTAB-5 - High throughput sequenceing of fission yeast to survey the dynamic repertoire of a eukaryotic transcriptome at the single nucleotide resolution
Released on 10 December 2008, last updated on 3 May 2014
Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and many functional properties of transcripts are not based on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or a few conditions. We applied direct high-throughput sequencing of cDNAs, complemented with different expression data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including exponential proliferation, meiotic differentiation and environmental stress, and in RNA processing mutants, to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental, and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to deeply sample transcriptomes at unprecedented resolution. Unlike hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not actively transcribed or rapidly degraded. The combined sequencing and strand-specific array data provided rich information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus refining the existing genome annotation. Sequence trans-reads spanning exon-exon or exon-intron junctions gave unique insight into a surprising variability in splicing efficiency across introns and genes. Splicing efficiency was largely coordinated with transcriptional efficiency, and hundreds of introns showed regulated splicing as a function of cellular proliferation or differentiation.
co-expression, growth condition, replicate
Dynamic repertoire of a eukaryotic transcriptome surveyed at single nucleotide resolution. Brian Wilhelm, Samuel Marguerat, Stephen Watt, Falk Schubert, Valerie Wood, Ian Goodhead, Christopher J. Penkett, Jane Rogers and Jürg Bähler.