The ribosome profiling technique (Ribo-seq) allows the selective sequencing of translated RNA regions. Recently, the analysis of genomic sequences associated to Ribo-seq reads has been widely employed to assess their coding potential. These analyses led to the identification of differentially translated transcripts under different experimental conditions, and/or ribosome pausing on codon motifs. In the context of the ever-growing need for tools analyzing Ribo-seq reads, researchers from the University Nice Sophia Antipolis have developed ‘RiboProfiling’, a new Bioconductor open-source package. ‘RiboProfiling’ provides a full pipeline to cover all key steps for the analysis of ribosome footprints. This pipeline has been implemented in a single R workflow. The package takes an alignment (BAM) file as input and performs ribosome footprint quantification at a transcript level. It also identifies footprint accumulation on particular amino acids or multi amino-acids motifs. Report summary graphs and data quantification are generated automatically. The package facilitates quality assessment and quantification of Ribo-seq experiments. Its implementation in Bioconductor enables the modeling and statistical analysis of its output through the vast choice of packages available in R.
Workflow of ‘RiboProfiling’ Ribo-seq analysis from BAM to
quantification on genomic features and codon motifs
A second particularity in the handling of Ribo-seq data comes from the shift existing between the extremities of the read (i.e. 5’ or 3’) and the P-site position of the ribosome. Our package allows the identification of an offset from the 5’ end of the read, but also from the 3’ end. The function ‘readStartCov’ computes the read frequency distribution centered on the translation start site (TSS) of the most expressed protein coding transcripts (by default the 3% most expressed). Based on this frequency distribution, the ‘plotSummarizedCov’ function enables the visual quantification of the offset between the reads and the ribosome P-site. In this Ribo-seq example, the 5’ read end is shifted 13 bp from the TSS. The innovation of this feature consists in the visualization of read lengths independently and as a summary figure.
Availability – The package is built in R (>=3.3.0) and freely available from Bioconductor website: https://www.bioconductor.org/packages/release/bioc/html/RiboProfiling.html