4.5.1 Check

Purpose

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

This is usually the first quality-control step after alignment. The quality-control workflow is divided into three steps:

Step 1: Run rpf_Check for each sample
Step 2: Merge read-length distributions
Step 3: Merge gene/read saturation

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.

Step 1: Run rpf_Check

1.1 Parameters

Parameter	Required	Description
-t	Yes	Input gene/transcript annotation file generated by rpf_Reference, usually gene.norm.txt.
-b	Yes	Input BAM/SAM alignment file.
-o	Yes	Output prefix.
--thread	No	Number of threads. Default is 4. When greater than 1, an indexed BAM is prepared and the transcript reference is fetched by multiple processes.
-g	No	Filter reads by mapping uniqueness. Use 0 to keep all mapped reads or 1 to keep uniquely mapped reads only. Default is 0.
-a	No	Aligner used to generate the BAM/SAM file, controlling unique-read tag interpretation. Choices are star, hisat2, and bowtie2. Default is star.
-r	No	Backward-compatible option retained for old commands. The current rpf_Check always retains and reports plus/minus strands separately.
--secondary	No	Keep secondary alignments. Disabled by default.
--supplementary	No	Keep supplementary alignments. Disabled by default.
--duplicate	No	Keep duplicate alignments. Disabled by default.
-l	No	Keep only the representative/longest transcript for each gene. Disabled by default.
-s	No	Calculate RPF saturation (gene-level and read-level curves). Takes extra memory, especially when multi-mapping reads are retained. Disabled by default.

1.2 Example

cd ./sce/4.ribo-seq/01.qc/

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

1.3 Output

Output	Description
<prefix>.bam	Filtered and sorted BAM file.
<prefix>.bam.bai	BAM index file.
<prefix>_rpf_check.summary.json	Machine-readable run summary (JSON format).
<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 output figures

The read-length distribution plot shows the footprint-length profile and the dominant RPF length of the sample:

The gene and read saturation curves evaluate whether sequencing depth is sufficient, generated when -s is used:

Step 2: Merge read-length distributions

merge_length merges the read-length distribution tables from multiple samples into a single table.

2.1 Parameters

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.

2.2 Example

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

2.3 Output

File	Description
RIBO_length_distribution.txt	Merged read-length distribution table.
RIBO_length_distribution_dotline.png / RIBO_length_distribution_dotline.pdf	Dot-line plot of read-length distribution across samples.
RIBO_length_distribution_heatmap.png / RIBO_length_distribution_heatmap.pdf	Heatmap of read-length distribution across samples.

The dot-line plot and heatmap visualize the read-length profiles of all samples in a single figure:

Step 3: Merge gene/read saturation

merge_saturation merges the gene saturation files from multiple samples into a single table.

3.1 Parameters

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.

3.2 Example

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

3.3 Output

File	Description
RIBO_gene_saturation.txt	Merged gene saturation table.
RIBO_gene_saturation_dotline.png / RIBO_gene_saturation_dotline.pdf	Dot-line plot of gene saturation across samples.

The dot-line plot shows the gene saturation trend of all samples:

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.
• Secondary, supplementary, and duplicate alignments are removed by default; use --secondary, --supplementary, or --duplicate to keep them.