4.7.1 Codon pausing score

Function

rpf_Pausing calculates relative codon pausing scores from a merged RPF density table. The command compares codon-level RPF density against either the gene-level average density or the local upstream/downstream background, then summarizes pausing scores by gene and codon.

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 pausing calculation. Choices: E, P, A. Default: P.
-f	No	Reading frame used for pausing calculation. Choices: 0, 1, 2, all. Default: all.
-b	No	Number of upstream and downstream codons used as local background. Default: 2. Set 0 to use the gene-level mean CDS density as background.
-m	No	Retain transcripts with more than this minimum number of RPFs. Default: 50.
--tis	No	Number of codons after the translation initiation site to discard. Default: 10.
--tts	No	Number of codons before the translation termination site to discard. Default: 5.
-n	No	Normalize RPF counts to RPM before calculating pausing scores. Disabled by default.
--scale	No	Scaling method for codon-level summary values. Choices: zscore, minmax. Default: minmax.
--stop	No	Remove stop codons from codon-level summary tables and heatmaps. Disabled by default.
--ind	No	Summarize valid pausing scores using codons with non-zero density in each individual sample. Disabled by default.
--fig	No	Draw per-gene pausing plots. Choices: none, png, pdf. Default: none. This can take a long time for large datasets.
--all	No	Output all codon-level pausing scores for each gene. Disabled by default.

Output files

Output	Description
<prefix>_cds_pausing_score.txt	Gene-level CDS pausing score table.
<prefix>_cds_codon_pausing_score.txt	Per-gene and per-codon pausing score summary.
<prefix>_sum_codon_pausing_score.txt	Codon-level summary table containing codon counts, RPF counts, absolute pausing scores, and relative pausing scores.
<prefix>_all_pausing_score.txt	Full codon-level pausing table. Generated only with --all.
<prefix>_total_pausing_heatplot.pdf / .png	Heatmap of total codon pausing scores.
<prefix>_valid_pausing_heatplot.pdf / .png	Heatmap of valid codon pausing scores.
<gene>_pausing_plot.pdf / .png	Per-gene pausing plot. Generated only when --fig pdf or --fig png is used.

Example

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

Notes

• High pausing scores indicate local RPF enrichment relative to the selected background.
• -b 0 uses the average CDS density of each gene as the background. Positive -b values use a local window around each codon.
• Interpret candidate pausing codons together with RPF coverage, digestion bias, periodicity, and replicate consistency.

Merge related results

Use merge_pausing to merge multiple pausing result tables:

merge_pausing -l *_sum_codon_pausing_score.txt -o RIBO