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4.8.3 smORF evidence

Function

smorf_evidence evaluates translation evidence for filtered smORFs using Ribo-seq P-site density tracks.

For each ORF and sample, it quantifies RPF signal, coverage, frame-specific density, frame periodicity, start/pre-stop pausing, post-stop release signal, and coverage shape. The output is a long-format ORF-by-sample evidence table.

Input files

Input Required Description
Filtered ORF table Yes *.passed.message.txt generated by smorf_filter.
P-site density input Yes Provide --density-list, --density, or both --density-plus and --density-minus.
genePred file No ORF exon-block annotation, usually *.genePred from smorf_scanner.
Chromosome sizes No Two-column chromosome size file. If omitted, sizes are inferred from density files when possible.

Density input modes

Density list mode

Use --density-list for multiple samples or strand-specific files. The file must be tab-delimited and must contain at least sample and path columns. Optional columns are strand and format.

sample  strand  path    format
sample1 +   sample1_plus.bedgraph   bedgraph
sample1 -   sample1_minus.bedgraph  bedgraph
sample2 +   sample2_plus.bedgraph   bedgraph
sample2 -   sample2_minus.bedgraph  bedgraph

Allowed strand values include +, -, ., plus, minus, forward, reverse, and unstranded.

Parameters

Parameter Required Description
-i, --orf-table Yes Filtered smORF table generated by smorf_filter.
-o, --output Yes Output evidence table in TSV format. Gzip output is supported when the file name ends with .gz.
-a, --genepred No Optional genePred file used for ORF exon blocks.
-z, --chrom-sizes No Optional two-column chromosome size file.
-l, --density-list Conditional Density file list with columns sample, path, and optional strand, format.
-p, --density-plus Conditional Plus-strand P-site density file.
-n, --density-minus Conditional Minus-strand P-site density file.
-d, --density Conditional Unstranded P-site density file.
-s, --sample No Sample name used for direct density input. Default: sample1.
-f, --density-format No Density file format: auto, wig, or bedgraph. Default: auto.
-c, --coord-mode No Coordinate mode for ORF table and genePred-like blocks: 0based-half-open or 1based-closed. Default: 0based-half-open.
-r, --post-stop-codons No Number of codons after the stop codon used for release-signal calculation. Default: 10.
-P, --pseudocount No Pseudocount used for ratio calculations. Default: 0.1.
-R, --min-rpf-sum No Minimum ORF-level RPF sum required for evidence scoring. Default: 3.0.
-C, --min-covered-codon No Minimum covered codon count required for evidence scoring. Default: 2.
-v, --min-coverage-ratio No Minimum nucleotide-level coverage ratio required for evidence scoring. Default: 0.10.
--strong-periodicity No Frame-0 ratio threshold for strong periodicity. Default: 0.70.
--moderate-periodicity No Frame-0 ratio threshold for moderate periodicity. Default: 0.55.
--strong-start-pause No Start-pausing ratio threshold for strong signal. Default: 1.50.
--moderate-start-pause No Start-pausing ratio threshold for moderate signal. Default: 1.20.
--strong-stop-pause No Pre-stop pausing ratio threshold for strong signal. Default: 1.50.
--moderate-stop-pause No Pre-stop pausing ratio threshold for moderate signal. Default: 1.20.
--strong-release No Release ratio threshold for strong signal. Default: 3.0.
--moderate-release No Release ratio threshold for moderate signal. Default: 1.5.
--uniform-coverage-ratio No Coverage-ratio threshold for the Uniform coverage-shape label. Default: 0.40.
--uniform-gini No Gini threshold for the Uniform coverage-shape label. Default: 0.50.
--uniform-max-to-mean No Max-to-mean threshold for the Uniform coverage-shape label. Default: 5.0.
--skewed-max-to-mean No Max-to-mean threshold for the Skewed coverage-shape label. Default: 10.0.
--skewed-top-fraction No Top-position density fraction threshold for the Skewed coverage-shape label. Default: 0.70.
--disperse-coverage-ratio No Coverage-ratio threshold below which coverage is labeled Disperse. Default: 0.20.
-E, --progress-every No Print progress every N ORFs per chromosome. Default: 10000.
-N, --keep-no-evidence No Keep ORFs labeled NoEvidence in the output. Default: False.

Output files

Output Description
User-defined --output file Long-format ORF-by-sample evidence table.

The output table contains annotation columns, quantitative RPF metrics, frame-specific density metrics, pausing/release metrics, coverage-shape metrics, and the final translation_evidence label.

Example

smorf_evidence \
  -i smorf.filtered.passed.message.txt \
  -a smorf.raw.genePred \
  -l ribo_density.list \
  -f bedgraph \
  -o smorf.riboseq_evidence.txt

Notes

  • The command requires one of these density input styles: --density-list, --density, or both --density-plus and --density-minus.
  • If --density-format auto is used, file suffixes such as .bedgraph, .bdg, .wig, or .wiggle are used to infer the format.
  • By default, ORFs labeled NoEvidence are removed from the output. Use --keep-no-evidence to retain them.
  • Strand-specific bedGraph files can be generated by rpf_Bam2bw and used directly in the density list.