Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution

Abstract

Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and that many functional properties of transcripts are based not 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 few experimental conditions. Here we applied direct high-throughput sequencing of complementary DNAs (RNA-Seq), supplemented with 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 rapid proliferation, meiotic differentiation and environmental stress, as well as 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 sample transcriptomes deeply at maximal resolution. In contrast to 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 robustly transcribed or rapidly degraded. The combined sequencing and strand-specific array data provide rich condition-specific information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus improving the existing genome annotation. Sequence reads spanning exon-exon or exon-intron junctions give unique insight into a surprising variability in splicing efficiency across introns, genes and conditions. Splicing efficiency was largely coordinated with transcript levels, and increased transcription led to increased splicing in test genes. Hundreds of introns showed such regulated splicing during cellular proliferation or differentiation.

Power sequencing

“…1) to sequence at varying levels of detail the transcriptomes of four organisms -the fission yeast Saccharomyces pombe 1 , the budding yeast Saccharomyces cerevisiae 2 , the plant Arabidopsis thaliana 3 and the mouse 4,5 ; for the sequencing step, four of the groups used the Illumina Genome Analyzer system [1][2][3][4] and one used the ABI SOLiD system 5 . Between 30 and 125 million sequences 25 to 39-base-pairs in length were obtained in each study.…”

“…Technological improvements such as longer reads, paired-end reads (the ability to obtain sequence from both ends each molecule and the distance between those sequences) 8 , enrichment for sequences of interest 9 , DNA-strand-specific sequencing of the mRNA transcripts 1 , and methods to sequence all RNAs 1 and not just mRNA, will all make mRNASeq even more powerful. Algorithms that can accurately assemble short-sequence reads into longer stretches 10 will further allow sequencing of the transcriptome of organisms for which a reference genome is not available.…”

“…Together, these advances will provide even greater insight into the transcriptional landscapes, regulation of gene expression and alternative splicing. Most importantly, next-generation sequencing has the potential to turn individual laboratories into small genome centres and to allow an individual scientist to determine the entire transcriptome of any source (any organism, tumour samples, tissues from patients with neurodegenerative disorders and so on) in a matter of days, and for only a few thousand In this technique, which was used for analysis of transcriptomes of five organisms [1][2][3][4][5] , the isolated mRNA is analysed by one of three procedures. a, In the first procedure, mRNAs are randomly sheared, linker molecules are attached to their ends, and they are then converted to DNA.…”

“…So studying the transcribed portion of the genome -the transcriptome -significantly aids gene identification, as well as providing insight into the inner workings of the genome and the biology of an organism. Five papers 1-5 , including one on page XXX of this issue by Wilhelm et al 1 , describe how advances in DNA-sequencing technology can be harnessed to explore transcriptomes in remarkable detail.…”

“…So studying the transcribed portion of the genome -the transcriptome -significantly aids gene identification, as well as providing insight into the inner workings of the genome and the biology of an organism. Five papers 1-5 , including one on page XXX of this issue by Wilhelm et al 1 , describe how advances in DNA-sequencing technology can be harnessed to explore transcriptomes in remarkable detail.The concept of sequencing large numbers of randomly selected mRNAs is not new. It forms the basis of the controversial, yet revolutionary EST method 6 , which was originally used to identify genes in the reference copy of the human genome.…”