4.5.3 Offset table

Offset tables define how read positions are shifted to assign RNA-seq or Ribo-seq signal to transcript coordinates.

For Ribo-seq, accurate P-site offsets are essential for 3-nt periodicity, codon-level analysis, pausing analysis, and smORF evidence evaluation. RNA-seq normally uses a constant offset table.

rna_Offset

Function

rna_Offset creates a simple RNA-seq offset table by assigning a constant offset to all read lengths in a specified range.

Input files

Input	Description
Read length range	Minimum and maximum read lengths used to construct the table.
Expected offset	Constant offset applied to each retained read length.
Output prefix	Prefix used to generate <prefix>_offset.txt.

Parameters

Parameter	Required	Description
-o	Yes	Output prefix. The output table is <prefix>_offset.txt.
-m	No	Minimum read length to keep. Default is 25.
-M	No	Maximum read length to keep. Default is 151.
-e	No	Expected constant offset. Default is 12.

Output files

Output	Description
<prefix>_offset.txt	RNA-seq offset table.
<prefix>.log	Running log if redirected by the user.

Example

rna_Offset \
    -m 25 \
    -M 150 \
    -e 12 \
    -o RNA \
    &> RNA.log

rpf_Offset

Function

rpf_Offset predicts Ribo-seq P-site offsets from transcriptome BAM files. The command outputs start/stop codon-based offsets and ribosome-structure-based offsets.

Input files

Input	Description
BAM file	Filtered transcriptome BAM file generated by rpf_Check.
Transcript annotation	Normalized transcript annotation file, usually gene.norm.txt.
Expected RPF length	Dominant footprint length used by the RSBM offset model.
Output prefix	Sample-specific prefix.

Parameters

Parameter	Required	Description
-t	Yes	Input transcript annotation file in TXT format.
-b	Yes	Input BAM file.
-o	Yes	Output prefix.
-a	No	Alignment region used for offset detection. Choices are both, tis, and tts. Default is both.
-l	No	Retain only the transcript with the longest CDS for each gene. Disabled by default.
-m	No	Minimum read length to keep. Default is 27.
-M	No	Maximum read length to keep. Default is 33.
-p	No	Expected RPF length fitted to ribosome structure. Default is 30.
-s	No	P-site shift for different RPF lengths. Default is 2.
--silence	No	Suppress warning information. Disabled by default.
-d	No	Output detailed offset end profiles. Disabled by default.

Output files

Output	Description
<prefix>_SSCBM_offset.txt	Start/stop codon-based offset table.
<prefix>_SSCBM_offset.pdf / <prefix>_SSCBM_offset.png	SSCBM offset plot.
<prefix>_SSCBM_offset_scale.pdf / <prefix>_SSCBM_offset_scale.png	Scaled SSCBM offset plot.
<prefix>_RSBM_offset.txt	Ribosome-structure-based offset table.
<prefix>_RSBM_offset.pdf / <prefix>_RSBM_offset.png	RSBM offset plot.
<prefix>_tis_5end.txt	TIS-aligned 5' end distribution when -d is used.
<prefix>_tis_3end.txt	TIS-aligned 3' end distribution when -d is used.
<prefix>_tts_5end.txt	TTS-aligned 5' end distribution when -d is used.
<prefix>_tts_3end.txt	TTS-aligned 3' end distribution when -d is used.
<prefix>.log	Running log if redirected by the user.

Example

for bam in ../01.check/*.bam
do
    prefix_name=$(basename ${bam} .bam)

    rpf_Offset \
        -b ${bam} \
        -t ../../../1.reference/norm/gene.norm.txt \
        -o ${prefix_name} \
        -m 27 \
        -M 33 \
        -p 30 \
        -d \
        &> ${prefix_name}.log
done

Merge related results

merge_offset

Merge SSCBM or RSBM offset tables from multiple samples.

Parameter	Required	Description
-l / --list	Yes	Input offset files, such as *_SSCBM_offset.txt or *_RSBM_offset.txt. Multiple files can be provided.
-o	Yes	Output prefix. The output table is <prefix>_offset.txt.

merge_offset \
    -l *_SSCBM_offset.txt \
    -o RIBO_SSCBM

merge_offset \
    -l *_RSBM_offset.txt \
    -o RIBO_RSBM

merge_offset_detail

Merge detailed TIS/TTS end-profile files generated with rpf_Offset -d.

Parameter	Required	Description
-l / --list	Yes	Input detailed offset end files, such as *_tis_5end.txt, *_tis_3end.txt, *_tts_5end.txt, and *_tts_3end.txt.
-o	Yes	Output prefix. The output table is <prefix>_offset_end.txt.

merge_offset_detail \
    -l *_tis_5end.txt *_tis_3end.txt *_tts_5end.txt *_tts_3end.txt \
    -o RIBO

Notes

• rpf_Offset currently calculates both SSCBM and RSBM outputs; the older --mode option is commented out in the script.
• A reliable offset table should show a stable dominant P-site offset for biologically meaningful read lengths.
• Weak or flat TIS/TTS profiles may indicate poor periodicity, low read depth, or poor library quality.