4.5.1 Check

Function

rpf_Check inspects transcriptome-aligned BAM files, extracts reads mapped to transcript regions, generates filtered and indexed BAM files, summarizes read-length distribution, and optionally calculates read/gene saturation curves.

This is usually the first quality-control step after alignment.

Input files

Input	Description
BAM file	Transcriptome-aligned BAM file, such as Aligned.toTranscriptome.out.bam.
Transcript annotation	Normalized transcript annotation file, usually gene.norm.txt generated by rpf_Reference.
Output prefix	Sample-specific prefix used for filtered BAM, tables, figures, and logs.

Parameters

Parameter	Required	Description
-t	Yes	Input transcript annotation file in TXT format, usually gene.norm.txt.
-b	Yes	Input BAM file.
-o	Yes	Output prefix.
--thread	No	Number of threads. Default is 1. Large BAM files may require more memory.
-g	No	Mapping-locus filter. Use 0 to keep all reads or 1 to keep uniquely mapped reads only. Default is 0.
-a	No	Aligner source used to interpret BAM tags. Choices are star, hisat2, and bowtie2. Default is star.
-r	No	Also count reads aligned to the negative strand. Disabled by default.
-l	No	Keep only the longest transcript for each gene. Disabled by default.
-s	No	Calculate read and gene saturation. This step can be time- and memory-consuming. Disabled by default.

Output files

Output	Description
<prefix>.bam	Filtered and sorted BAM file.
<prefix>.bam.bai	BAM index file.
<prefix>_length_distribution.txt	Read-length distribution table.
<prefix>_length_distribution.pdf / <prefix>_length_distribution.png	Read-length distribution plots.
<prefix>_gene_saturation.txt	Gene saturation table, generated when -s is used.
<prefix>_gene_saturation.pdf / <prefix>_gene_saturation.png	Gene saturation plots, generated when -s is used.
<prefix>_reads_saturation.txt	Read saturation table, generated when -s is used.
<prefix>_reads_saturation.pdf / <prefix>_reads_saturation.png	Read saturation plots, generated when -s is used.
<prefix>.log	Running log if redirected by the user.

Example

for bam in ../../3.star/*Aligned.toTranscriptome.out.bam
do
    prefix_name=$(basename ${bam} Aligned.toTranscriptome.out.bam)

    rpf_Check \
        -b ${bam} \
        -t ../../../1.reference/norm/gene.norm.txt \
        -o ${prefix_name} \
        -g 1 \
        -a star \
        --thread 10 \
        -s \
        &> ${prefix_name}.log
done

Merge related results

merge_length

Merge read-length distribution tables from multiple samples.

Parameter	Required	Description
-l / --list	Yes	Input *_length_distribution.txt files. Multiple files can be provided.
-o	Yes	Output prefix. The output table is <prefix>_length_distribution.txt.

merge_length \
    -l *_length_distribution.txt \
    -o RIBO

merge_saturation

Merge gene saturation files from multiple samples.

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

merge_saturation \
    -l *_gene_saturation.txt \
    -o RIBO

Notes

• Ribo-seq libraries should show a dominant footprint length, commonly within a narrow RPF length range.
• Use -s when you need to evaluate whether sequencing depth is sufficient; skip it for a fast initial check.
• Use -g 1 when downstream analysis should be based on uniquely mapped reads only.