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.
• Standard Output Files: RIBOSS generates output files in standard formats (BED12, genePred, bigGenePred, and bedGraph) compatible with widely used tools like BEDTools, UCSC Genome Browser, IGV, and Artemis.

User guide

Install Miniforge3 on Linux

RIBOSS currently only supports Linux systems.

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 $HOME/miniforge3
eval "$(/$HOME/miniforge3/bin/conda shell.bash hook)" # your terminal prompt will show (base) bash-5.1$

Create a conda environment and install RIBOSS

git clone https://github.com/lcscs12345/riboss.git
cd riboss
conda config --set channel_priority flexible # required if channel_priority was set to strict. See https://docs.conda.io/projects/conda/en/latest/user-guide/tasks/manage-channels.html
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'.

Column description of the main RIBOSS output (riboss.boss.pkl)

column	name	description
1	tid	Transcript ID.
2	boss	ORF that have a greater translational potential compared to canonical ORFs.
3	start_codon_x	Start codon of x, where x is non-canonical ORF (see Figure below).
4	start_codon_y	Start codon of y, where x is canonical ORF (see Figure below).
5	ORF_range_x	Transcript level position of x [start, end].
6	ORF_type_x	ORF types of x.
7	start_rprofile_x	Ribosome profiles around the start codon
8	ORF_range_y	Transcript level position of x [start, end].
9	ORF_type_y	ORF types of y.
10	start_rprofile_y	Ribosome profiles around the start codon
11	tab	2 x 3 and 2 x 2 contingency table for footprint counts. See Notes below.
12	odds_ratio	The odds of x being translated.
13	statistical test	G-test with bootstrap and post hoc test for 2 x 3 contingency table or Boschloo exact test for 2 x 2 contingency table
14	result	G-tests' results (statistic, adjusted_pvalue, adjusted_bootstrap_pvalue, adjusted_residuals, adjusted_posthoc_pvalues) or Boschloo exact test result (statistic, pvalue).

Notes:

• The contingency tables in tab is always [Non-canonical ORFs, Canonical ORFs].
• The 2 x 3 contingency table summerises the footprint counts mapped to frames 0, 1, and 2 as [[F0, F1, F2], [F0, F1, F2]].
• The 2 x 2 contingency table summerises the footprint counts mapped to frames 0, 1 and 2 as [[F0, F1 + F2], [F0, F1 + F2]]. Pseudocount of 1 is added to zeroes to avoid odds ratio infinity when carrying out the Boschloo exact test.

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

Citing us:

• Lim, C. S., & Brown, C. M. (2024). RIBOSS detects novel translational events by combining long- and short-read transcriptome and translatome profiling. Brief Bioinform. DOI: 10.1093/bib/bbaf164
• 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