4.5.7 Metaplot

Function

rpf_Metaplot generates metagene profiles around translation initiation sites (TIS) and translation termination sites (TTS).

Metaplot analysis helps evaluate positional density patterns, offset quality, initiation/termination enrichment, and possible boundary artifacts.

Input files

Input	Description
Merged density file	Usually RIBO_merged.txt or RNA_merged.txt generated by rpf_Merge.
Transcript annotation	Normalized transcript annotation file, usually gene.norm.txt.
Read-count threshold	Minimum RPF/read count used to retain transcripts.

Parameters

Parameter	Required	Description
-t	Yes	Input transcript annotation file in TXT format.
-r	Yes	Input merged density file in TXT format.
-o	Yes	Output prefix.
-m	No	Remove transcripts with fewer than this number of RPFs/reads. Default is 50.
--utr5	No	Number of codons/bins used in the 5' UTR window. Default is 20.
--cds	No	Number of codons/bins used in the CDS window. Default is 50.
--utr3	No	Number of codons/bins used in the 3' UTR window. Default is 20.
-n	No	Normalize read counts to RPM. Disabled by default.
--mode	No	Metaplot mode. Choices are line and bar. Default is bar.

Output files

Output	Description
<prefix>_tis_tts_metaplot.txt	Merged TIS/TTS metaplot table.
<prefix>_*_tis_tts_metaplot.txt	Sample-level metaplot table.
<prefix>_*_meta_bar_plot.pdf / <prefix>_*_meta_bar_plot.png	Bar-mode metaplot figures when --mode bar is used.
<prefix>_*_meta_line_plot.pdf / <prefix>_*_meta_line_plot.png	Line-mode metaplot figures when --mode line is used.
<prefix>.log	Running log if redirected by the user.

Example

rpf_Metaplot \
    -t ../../../1.reference/norm/gene.norm.txt \
    -r ../05.merge/RIBO_merged.txt \
    -o RIBO \
    -m 50 \
    --mode bar \
    &> RIBO.log

Merge related results

merge_metagene

Merge metagene tables from multiple samples or analyses.

Parameter	Required	Description
-l / --list	Yes	Input metagene files, usually *_tis_tts_metaplot.txt or *_metaplot.txt. Multiple files can be provided.
-o	Yes	Output prefix. The output table is <prefix>_metagene.txt.

merge_metagene \
    -l *_tis_tts_metaplot.txt \
    -o RIBO

Notes

• Ribo-seq metaplots should show interpretable signal around TIS/TTS when offset correction is reliable.
• Strong boundary artifacts may indicate nuclease bias, offset problems, or transcript-end mapping artifacts.
• Use the same -m, --utr5, --cds, and --utr3 settings across samples when comparing metaplots.