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

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
pip install -e . # 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'. For detail usage, see styphimurium.ipynb

To reproduce plots in the manuscript

Create new directories mkdir -p doc/ doc/metatranscriptome doc/styphimurium/ doc/styphimurium/rnaseq doc/styphimurium/riboseq.

The alignment files for transcriptome assembly and ribosome profiling are available at Zenodo.

• SRR11215003.bam and SRR11215004.bam: Nanopore long-read direct RNA-seq. Download and mv SRR24781620.bam doc/metatranscriptome.
• SRR11215663.bam and SRR11215664.bam: Illumina short-read RNA-seq. Download and mv SRR24781620.bam doc/metatranscriptome.
• SRR24781620.bam: Nanopore long-read cDNA sequencing. Download and mv SRR24781620.bam doc/styphimurium/rnaseq.

Download the Ribosome profiling alignment files and 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.

References:

• 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
• 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
• Yang, M., Cousineau, A., Liu, X., Luo, Y., Sun, D., Li, S., Gu, T., Sun, L., Dillow, H., Lepine, J., Xu, M., Zhang, B. (2020) Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION - Promising Strategies for Multiplex Identification of Viable Pathogens in Food. Front Microbiol, 11:514. DOI: 10.3389/fmicb.2020.00514