Abstract
BackgroundRibosome profiling (or Ribo-seq) is currently the most popular methodology for studying translation; it has been employed in recent years to decipher various fundamental gene expression regulation aspects.The main promise of the approach is its ability to detect ribosome densities over an entire transcriptome in high resolution of single codons. Indeed, dozens of ribo-seq studies have included results related to local ribosome densities in different parts of the transcript; nevertheless, the performance of Ribo-seq has yet to be quantitatively evaluated and reported in a large-scale multi-organismal and multi-protocol study of currently available datasets.ResultsHere we provide the first objective evaluation of Ribo-seq at the resolution of a single nucleotide(s) using clear, interpretable measures, based on the analysis of 15 experiments, 6 organisms, and a total of 612, 961 transcripts. Our major conclusion is that the ability to infer signals of ribosomal densities at nucleotide scale is considerably lower than previously thought, as signals at this level are not reproduced well in experimental replicates. In addition, we provide various quantitative measures that connect the expected error rate with Ribo-seq analysis resolution.ConclusionsThe analysis of Ribo-seq data at the resolution of codons and nucleotides provides a challenging task, calls for task-specific statistical methods and further protocol improvements. We believe that our results are important for every researcher studying translation and specifically for researchers analyzing data generated by the Ribo-seq approach.ReviewersThis article was reviewed by Dmitrij Frishman, Eugene Koonin and Frank Eisenhaber.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0127-4) contains supplementary material, which is available to authorized users.
Highlights
Ribosome profiling is currently the most popular methodology for studying translation; it has been employed in recent years to decipher various fundamental gene expression regulation aspects
The experiment comprises of the following main steps: preparation of the biological samples; sample lysis; nuclease footprinting, in which mRNA that is not protected by ribosomes is digested; ribosome recovery; linker ligation; rRNA depletion; library sequencing, followed by bioinformatics analysis of the sequences [17]
We show that in most of the studied experiments to date, the level of reproducibility in measured ribosomal densities at nucleotide scale is considerably lower than previously thought, and argue that some of the aforementioned contradictions may be attributed to the resolution and relatively high ‘noise’ levels in ribosome profiling (RP) data when studying ribosome densities in short fragments of the coding regions
Summary
Ribosome profiling (or Ribo-seq) is currently the most popular methodology for studying translation; it has been employed in recent years to decipher various fundamental gene expression regulation aspects. Translation has a major role in the regulation of gene expression and significantly affects various fundamental intracellular processes and biomedical phenomena [1,2,3,4,5,6,7] It is an energetically most costly process, and each of its initiation, elongation and termination steps is tightly regulated [8, 9]. The most prominent experimental technique for studying translation in recent years has been ribosome profiling (RP; or Ribo-seq) [10] This approach enables high-throughput monitoring of ribosomal density along genes by utilizing deep sequencing methods and has been. Several methods have been applied for mapping the sequenced ribosome protected fragments, and the location of the A-site (or P-site) of the ribosome, to the genome [10, 17, 18, 21,22,23]
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