Comparing the translatability of open reading frames within individual transcripts

RIBOSS consists of Python modules for analysis of ribosome profiling data for prokaryotes and eukaryotes. See styphimurium.ipynb where RIBOSS detects new ORFs in S. Typhimurium operons. This example starts from transcriptome assembly using long- and short-read RNA-seq data. It leverages the newly assembled transcriptome and highly phased ribosome profiling data to discover novel translation events.

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)"
conda env create -f environment.yml

Install RIBOSS

conda activate riboss
git clone https://github.com/lcscs12345/riboss.git
DIRNAME=`which python | xargs dirname`
cp riboss/bin/riboprof $DIRNAME
chmod +x $DIRNAME/riboprof

Activate the conda environment for next time

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

Test instructions

The alignment files for transcriptome assembly and ribosome profiling are available at Zenodo. Download the RNA-seq alignment files and mv D23005*.bam doc/styphimurium/rnaseq.

• D23005.sorted.bam: Illumina short-read alignment file.
• D23005-sc-1962750.sorted.bam: PacBio long-read alignment file.

Download the Ribosome profiling alignment files and mkdir doc/styphimurium/riboseq; mv ERR913094*.out.bam doc/styphimurium/riboseq.

• ERR9130942Aligned.out.bam: RNase I, 1000 U.
• ERR9130943Aligned.out.bam: RNase I, 500 U.
• ERR9130946Aligned.out.bam: matched RNA-seq.

Follow the steps in test.ipynb.

References:

• 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
• Bryant, O.J., Lastovka, F., Powell, J. et al. (2023) The distinct translational landscapes of gram-negative Salmonella and gram-positive Listeria. Nat Commun, 14:8167. DOI: 10.1038/s41467-023-43759-1
• Ondari, E.M., Klemm, E.J., Msefula, C.L. et al. (2019) Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Salmonella Typhimurium ST313 [version 1; peer review: 2 approved]. Wellcome Open Res, 4:74. DOI: 10.12688/wellcomeopenres.15059.1