Comparing the translational potential of open reading frames within individual transcripts

Ribosome profiling is routinely used for discovering actively translated ORFs. Standard ribosome profiling involves RNase digestion of ribosome-protected mRNA fragments, followed by sucrose gradient fractionation, and RNA sequencing (RNA-seq).

As ribosomes progress along the mRNA codon-by-codon, they generate a characteristic triplet periodicity profile in the footprint data. Triplet periodicity can be used to determine the correct reading frame for the translated ORFs and distinguish true translation events from background noise.

RIBOSS is a Python package that integrates long- and short-read RNA sequencing data for reference-guided transcriptome assembly with ribosome profiling data to identify and characterise novel translational events beyond annotated regions. See the use cases and benchmarking results here.

Key Features:

• Long-Read Integration: RIBOSS integrates long-read RNA sequencing data to improve transcriptome assembly and enhance the identification of novel translational events.
• Assessment of Translational Potential: RIBOSS quantitatively assesses the relative translational potential of non-canonical ORFs compared to annotated ORFs, enabling users to infer their regulatory roles.
• Peptide Identification: Users can optionally enable BLASTP searches and efetch to identify peptides encoded by non-canonical ORFs.
• Prokaryotic and Eukaryotic Support: RIBOSS is designed to handle ribosome profiling data from both prokaryotic and eukaryotic species.

User guide

Install Miniforge3 and create a conda environment

wget https://github.com/conda-forge/miniforge/releases/download/24.7.1-2/Miniforge3-24.7.1-2-Linux-x86_64.sh
bash Miniforge3-24.7.1-2-Linux-x86_64.sh -b -p $HOME/miniforge3
eval "$(/$HOME/miniforge3/bin/conda shell.bash hook)" # your terminal prompt will show (base) bash-5.1$

Install RIBOSS

git clone https://github.com/lcscs12345/riboss.git
cd riboss
conda env create -f environment.yml
conda activate riboss # your terminal prompt will show (riboss) bash-5.1$
DIRNAME=`which python | xargs dirname`
cp bin/riboprof $DIRNAME
chmod +x $DIRNAME/riboprof
which python | awk 'sub(/python/,"pip3") {print $1, "install -e ."}' | sh # editable mode

Activate the conda environment for next time

eval "$(/$HOME/miniforge3/bin/conda shell.bash hook)"
conda activate riboss

Basic usage

from riboss.orfs import translate
na='ATGGTCTGA'
translate(na)

You should see 'MV'.

References:

• Ingolia, N. T., Hussmann, J. A., & Weissman, J. S. (2019) Ribosome Profiling: Global Views of Translation. Cold Spring Harb. Perspect. Biol., 11. DOI: 10.1101/cshperspect.a032698
• Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. S., & Weissman, J. S. (2009) Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science, 324: 218–223. DOI: 10.1126/science.1168978
• Lim, C. S., & Brown, C. M. (2024). RIBOSS detects novel translational events by combining long- and short-read transcriptome and translatome profiling. BioRxiv. DOI: 10.1101/2024.11.07.622529
• Lim, C.S., Wardell, S.J.T., Kleffmann, T. & Brown, C.M. (2018) The exon-intron gene structure upstream of the initiation codon predicts translation efficiency. Nucleic Acids Res, 46:4575-4591. DOI: 10.1093/nar/gky282