4.8.4 smORF quantification

Function

smorf_quant quantifies reliable smORFs from genomic P-site density tracks. It reads the reliable smORF genePred generated by smorf_evidence, intersects the spliced CDS of every ORF with the P-site density of each sample, and writes a wide ORF-by-sample raw P-site density count matrix.

The quantified region is the spliced CDS of each ORF. A complete terminal stop codon is excluded by default and can be included with --include-stop. The matrix is reported in smORF genePred order.

Input files

Input	Required	Description
Reliable smORF genePred (-g)	Yes	genePred file generated by smorf_evidence. Column 1 must contain unique ORF IDs.
Density list (-l)	Yes	Tab-delimited density design containing sample/name, strand, and path/file/density columns.
Output prefix (-o)	Yes	Output prefix. The count matrix is written as <prefix>.density_quant.txt.

Parameters

4.8.4.1 Required arguments

Parameter	Required	Default	Description
-g, --genepred	Yes		Reliable smORF genePred generated by smorf_evidence. Column 1 must contain unique ORF IDs.
-l, --density-list	Yes		Tab-delimited density design containing sample/name, strand, and path/file/density columns.
-o, --output	Yes		Output prefix. The count matrix is written as <prefix>.density_quant.txt.

4.8.4.2 Quantification arguments

Parameter	Required	Default	Description
-f, --frame	No	all	P-site frame retained relative to each spliced smORF. all matches the evidence rpf_sum definition. Choices: all, 0, 1, 2.
--include-stop	No	False	Include a complete terminal stop codon in quantification. By default the terminal stop is excluded.

4.8.4.3 Runtime arguments

Parameter	Required	Default	Description
-t, --thread	No	1	Number of samples quantified concurrently. Each density track is parsed only once.

Density list format

The density list is a tab-delimited design table with a header. The following columns are recognized (case-insensitive header names):

Column	Description
sample / name	Biological sample name. The matrix column header is taken from here.
strand	Track strand: + / plus / forward / fwd, - / minus / reverse / rev, or unstranded . / both / all / unstranded.
path / file / density	Density track path. Relative paths are resolved against the density list directory.
format	Optional density format: auto (default), wig, or bedgraph. Inferred from the file extension when auto.

Each sample must be described by either one unstranded track or a complete plus/minus pair:

sample  strand  path    format  group
ribo-1  +   ../ribo-1_plus.rpf.bedgraph bedgraph    wt  
ribo-1  -   ../ribo-1_minus.rpf.bedgraph    bedgraph    wt
ribo-2  +   ../ribo-2_plus.rpf.bedgraph bedgraph    treat
ribo-2  -   ../ribo-2_minus.rpf.bedgraph    bedgraph    treat

Output files

Output	Description
<prefix>.density_quant.txt	Wide ORF-by-sample raw P-site density count matrix.

The matrix contains 11 metadata columns followed by one column per sample (in density-list order):

Column	Description
orf_id	Unique smORF ID from the genePred column 1.
gene_id	Gene ID from the genePred column 12 (falls back to orf_id).
chrom	Chromosome.
strand	Strand of the smORF.
tx_start / tx_end	Transcript start / end.
cds_start / cds_end	CDS start / end.
exon_count	Number of exons.
coding_nt_length	Quantified coding nucleotide length.
coding_codon_count	Quantified coding length in codons.
<sample>	Raw P-site density count for the sample.

Examples

Quantify all-frame density across samples

cd ./sce/5.smorf/04.quant

smorf_quant \
  -g gmx4.smorf.reliable_smorf.genepred \
  -l ribo.bedgraph.list \
  -o gmx4.smorf \
  --frame all \
  --thread 16

Quantify frame-0 density and include the stop codon

smorf_quant \
  -g gmx4.smorf.reliable_smorf.genepred \
  -l ribo.bedgraph.list \
  -o gmx4.smorf \
  --frame 0 \
  --include-stop

Notes

• The quantified region is the spliced CDS. A complete terminal stop codon is removed unless --include-stop is enabled; the genePred status column (cmpl/complete/full) is used to decide whether the terminal stop is complete.
• --frame all counts P-sites in every frame and matches the rpf_sum definition used by smorf_evidence. --frame 0, 1, or 2 counts only P-sites in that frame relative to the spliced smORF.
• Density values are converted to absolute values internally, which supports negative minus-strand bedGraph conventions without changing the strand assigned in the density list.
• bedGraph files are checked for chromosome grouping and coordinate order. Files with separated chromosome blocks or decreasing coordinates are automatically sorted with GNU sort; WIG files retain their native step-block order requirement.
• Each density track is parsed only once. --thread controls how many samples are quantified concurrently (capped at the number of samples).
• The output matrix is written atomically (temporary file + rename), so an interrupted run does not leave a truncated matrix.