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4.7.2 Codon occupancy

Function

rpf_Occupancy calculates codon occupancy from a merged RPF density table. The command normalizes codon-level density by the average density of each gene, summarizes codon occupancy across genes, and generates correlation and heatmap plots.

Input files

Input Description
RPF density table Merged Ribo-seq density table, usually RIBO_merged.txt.
Gene list Optional transcript or gene list. If not provided, all genes in the density table are used.

Parameters

Parameter Required Description
-r Yes Input RPF density table in TXT format.
-l No Gene name or transcript ID list in TXT format. If omitted, all genes are used.
-o Yes Output prefix.
-s No Ribosomal site used for occupancy calculation. Choices: E, P, A. Default: P.
-f No Reading frame used for occupancy calculation. Choices: 0, 1, 2, all. Default: all.
-m No Retain transcripts with more than this minimum number of RPFs. Default: 30.
-n No Normalize RPF counts to RPM before calculating occupancy. Disabled by default.
--tis No Number of codons after the translation initiation site to discard. Default: 15.
--tts No Number of codons before the translation termination site to discard. Default: 5.
--scale No Scaling method for relative occupancy. Choices: zscore, minmax. Default: minmax.
--stop No Remove stop codons from codon-level summary tables and heatmaps. Disabled by default.
--all No Output all filtered RPF density and per-gene codon density tables. Disabled by default.

Output files

Output Description
<prefix>_codon_occupancy.txt Codon-level occupancy summary table containing total density, absolute occupancy, and relative occupancy.
<prefix>_occupancy_corr.txt Correlation matrix calculated from absolute codon occupancy.
<prefix>_occupancy_corrplot.pdf / .png Occupancy correlation heatmap.
<prefix>_occupancy_heatplot.pdf / .png Absolute codon occupancy heatmap.
<prefix>_occupancy_relative_heatplot.pdf / .png Relative codon occupancy heatmap.
<prefix>_occupancy_relative_lineplot.pdf / .png Relative codon occupancy line plot.
<prefix>_rpf_density.txt Filtered RPF density table. Generated only with --all.
<prefix>_codon_density.txt Per-gene and per-codon density table. Generated only with --all.

Example

for site in E P A
do
    rpf_Occupancy \
        -l ../../../1.reference/norm/gene.norm.txt \
        -r ../05.merge/RIBO_merged.txt \
        -m 30 \
        -s ${site} \
        -f 0 \
        --stop \
        --scale minmax \
        -o ${site}_site \
        &> ${site}_site.log
done

Notes

  • Occupancy values reflect codon-specific ribosome density after gene-level normalization.
  • Use --stop when stop codons should be excluded from summary plots.
  • Use --all when you need intermediate density tables for troubleshooting or downstream analysis.

Use merge_occupancy to merge multiple codon occupancy tables:

merge_occupancy -l *_codon_occupancy.txt -o RIBO