4.5.2 Enzymatic bias

Function

rpf_Digest detects 5' and 3' end sequence preferences around read ends. These patterns can reflect nuclease digestion, ligation, or library-construction bias.

This step is useful because strong end-sequence bias can affect local RPF density and may mimic codon-level pausing signals.

Input files

Input	Description
BAM file	Filtered BAM file generated by rpf_Check.
Transcript annotation	Normalized transcript annotation file, usually gene.norm.txt.
Transcript FASTA	Normalized transcript sequence file, usually gene.norm.rna.fa.
Read length range	RPF read length range to retain. For Ribo-seq, this is often around 27--33 nt.

Parameters

Parameter	Required	Description
-t	Yes	Input transcript annotation file in TXT format.
-s	Yes	Input transcript sequence file in FASTA format.
-b	Yes	Input BAM file.
-o	Yes	Output prefix. Main digestion table is <prefix>_digestion_sites.txt.
-l	No	Retain only the transcript with the longest CDS for each gene. Disabled by default.
--scale	No	Scale the motif matrix and generate scaled digestion profiles. Disabled by default.
-m	No	Minimum read length to keep. Default is 20.
-M	No	Maximum read length to keep. Default is 100.

Output files

Output	Description
<prefix>_digestion_sites.txt	Digestion-site distribution table.
<prefix>_5end_counts.txt	5' end nucleotide count table.
<prefix>_5end_pwm.txt	5' end nucleotide PWM table.
<prefix>_5end_seqlogo2.pdf	5' end sequence logo.
<prefix>_3end_counts.txt	3' end nucleotide count table.
<prefix>_3end_pwm.txt	3' end nucleotide PWM table.
<prefix>_3end_seqlogo2.pdf	3' end sequence logo.
<prefix>_scaled_digestion_sites_plot.pdf	Scaled digestion plot when --scale 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_Digest \
        -b ${bam} \
        -t ../../../1.reference/norm/gene.norm.txt \
        -s ../../../1.reference/norm/gene.norm.rna.fa \
        -o ${prefix_name} \
        -m 27 \
        -M 33 \
        --scale \
        &> ${prefix_name}.log
done

Merge related results

merge_digestion

Merge 5' or 3' digestion PWM files from multiple samples.

Parameter	Required	Description
-l / --list	Yes	Input digestion PWM files, such as *_5end_pwm.txt or *_3end_pwm.txt. Multiple files can be provided.
-o	Yes	Output prefix. The output table is <prefix>_reads_digestion.txt.

merge_digestion \
    -l *_5end_pwm.txt *_3end_pwm.txt \
    -o RIBO

Notes

• Strong nucleotide preference around read ends suggests digestion or ligation bias.
• Check both 5' and 3' end patterns before interpreting codon-level pausing signals.
• Use the same read-length range as downstream Ribo-seq density generation whenever possible.