Bio Gene Calling

Call genes and annotate basic features for prokaryotes, viruses, and eukaryotes.

Instructions

1. Select gene caller by organism class:

• Prokaryotes and viruses (including giant viruses): pyrodigal-gv v0.3+ (SIMD-accelerated Cython bindings around prodigal-gv; same model set, much faster, actively maintained).
• Eukaryotes: BRAKER3 (Genome Research 2024, DOI: 10.1101/gr.278090.123) as the fully-automated pipeline. BRAKER3 invokes AUGUSTUS internally; do not run AUGUSTUS as a standalone caller.

1. Run gene calling and produce GFF/FAA/FNA.
2. Always run tRNA detection and rRNA detection on every assembly, and report counts per class. Negative findings (zero hits at default and relaxed thresholds) are required results — never leave ncRNA presence/absence unstated.

• tRNA: tRNAscan-SE v2.0.12+ (preferred; isotype-specific covariance models) or ARAGORN v1.2.41+ for tmRNA where appropriate.
• rRNA: Infernal v1.1.5+ cmsearch against the relevant Rfam covariance models. Pick the model set by domain of life:
• Bacteria: RF00177 (SSU 16S), RF02541 (LSU 23S), RF00001 (5S).
• Archaea: RF01959 (SSU 16S), RF02540 (LSU 23S), RF00001 (5S).
• Eukaryotes: RF01960 (SSU 18S), RF02543 (LSU 28S), RF00002 (5.8S), RF00001 (5S).
• Metazoan mitochondria, when applicable: RF02555 (12S), RF02546 (16S).

cmsearch --rfam --cut_ga --nohmmonly is a sensible default; if no hits, rerun without --cut_ga and record both results.

1. For viral or otherwise specialized genomes, choose the gene caller and mode from tool documentation and the literature-derived analysis playbook for the inferred group; record the rationale.
2. Summarize gene count, gene density, coding fraction, ORF length distribution, unusually long ORFs, overlapping genes, tRNAs, rRNAs, and other features that may affect downstream discovery.
3. Flag gene-calling anomalies relative to the inferred group and data type, including patterns that could hide interesting biology or indicate artifacts.
4. Produce a ncRNA_census.tsv with columns: assembly, class (tRNA/rRNA/tmRNA/other), tool, model (Rfam accession when applicable), threshold (default/relaxed), count, notes. This file is required even when all counts are zero.

Quick Reference

Input Requirements

Prerequisites:

• Tools available in the active environment (Pixi/conda/system). See docs/README.md for expected tools.
• Input contigs or bins are available.

Inputs:

• contigs.fasta or bins/*.fasta

Output

• results/bio-gene-calling/genes.gff3
• results/bio-gene-calling/proteins.faa
• results/bio-gene-calling/cds.fna
• results/bio-gene-calling/gene_metrics.tsv
• results/bio-gene-calling/gene_calling_discovery_flags.tsv
• results/bio-gene-calling/ncRNA_census.tsv
• results/bio-gene-calling/logs/

Quality Gates

• [ ] Gene count sanity checks pass.
• [ ] Start/stop codon checks pass.
• [ ] On failure: retry with alternative parameters; if still failing, record in report and exit non-zero.
• [ ] Verify contigs are non-empty and DNA alphabet.
• [ ] Verify outputs contain expected feature types.
• [ ] Specialized inputs use a literature/tool-supported gene-calling mode or document why not.
• [ ] Gene metrics include discovery-relevant flags for unusual ORFs, gene density, coding fraction, and tRNA/RNA features.
• [ ] ncRNA_census.tsv exists and records both default-threshold and relaxed-threshold results for tRNA and rRNA, including explicit zero counts.

Examples

Example 1: Expected input layout

contigs.fasta or bins/*.fasta

Troubleshooting

Issue: Missing inputs or reference databases Solution: Verify paths and permissions before running the workflow.

Issue: Low-quality results or failed QC gates Solution: Review reports, adjust parameters, and re-run the affected step.