Cluster proteins into orthogroups and build pangenome matrices. Use when comparing gene-family presence, absence, expansion, contraction, or core and accessory content across genomes.
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Cluster proteins into orthogroups and build pangenome matrices. Use when comparing gene-family presence, absence, expansion, contraction, or core and accessory content across genomes.
Bio Protein Clustering Pangenome
Cluster proteins into orthogroups and derive pangenome matrices.
Instructions
After clustering, build and validate the complete small-to-large comparison bundle with:
The driver requires globally unique protein IDs, at least two reference genomes for a defensible median, and a fresh output directory. It persists marker and ncRNA censuses alongside copy-number, presence/absence, family-comparison, genome-frontier, and conserved-neighborhood artifacts. fixtures/ is a runnable three-genome contract test.
Cluster proteins. Choose the tool by dataset size and goal:
Default for orthology inference up to a few hundred genomes: OrthoFinder v3.1.5 (supports MSA-based gene trees; supersedes OrthoFinder v2 and OrthoMCL workflows).
Very large pangenomes where OrthoFinder is too RAM-heavy: ProteinOrtho v6.3.6.
Sequence clustering (not strict orthology) and similarity-search backbones: MMseqs2 v18-8cc5c. GPU search requires MMseqs2 v16 or newer plus a GPU-enabled build on CUDA Turing-or-newer hardware; full-speed kernels require Ampere or newer. Enable --gpu only for commands that expose it and record the CPU/GPU build used.
Build presence/absence matrix AND an integer copy-number matrix (orthogroup × genome) covering the query AND the close relatives produced by /bio-phylogenomics.
When a query genome or genome set is under study, use the literature-derived analysis playbook to choose an appropriate comparison baseline: closest relatives, a broader clade, environmental references, or a negative/control set.
Genome-property frontier table — produce relative_genome_metrics.tsv with one row per (query + relative) and columns for genome size, contig count, N50, gene count, coding density, GC, tRNA count, rRNA count, and any group-relevant property. Add a column that places the query in the relative distribution (percentile, min/median/max, "record-class" tag) and a column citing the literature reference defining the group's known range.
Synteny / conserved neighborhoods — for each pair (query, relative) compute conserved gene neighborhoods (e.g., ≥2 collinear orthologs). Tool selection:
Multi-genome at scale (>2 assemblies, up to >3 Gbp, >15% divergence): ntSynt (BMC Biology 2025, DOI: 10.1186/s12915-025-02455-w) — alignment-free minimizer-graph approach; does not detect duplications.
Strain-level work where duplication detection matters: SibeliaZ.
Save results as conserved_neighborhoods.tsv with columns: query_block_id, relative, relative_block_id, members (ortholog IDs), intergenic_spacing_query, intergenic_spacing_relative, spacing_ratio, notes. Flag conserved gene pairs and unusual spacing/expansions.
Identify discovery-relevant differences defined by the playbook, including query-specific families, missing expected families, expansions/contractions, unusual sharing patterns, and high-value unknowns. Persist as family_copy_number_comparison.tsv (query vs relative-median fold change per family) — coordinated with bio-annotation's family matrix.
Annotate candidate orthogroups with /bio-annotation; for high-value unknowns, route representatives to /bio-structure-annotation when structure-based inference is appropriate.
Produce a comparison summary that separates conserved lineage features from unusual or query-specific features and states the baseline used. The summary must report ALL of: genome-property frontier, marker-category presence/copy, family expansions/contractions, synteny conservation/breakage, and ncRNA counts side-by-side with relatives.
Quick Reference
Task
Action
Run workflow
Follow the steps in this skill and capture outputs.
Validate inputs
Confirm required inputs and reference data exist.
Review outputs
Inspect reports and QC gates before proceeding.
Tool docs
See docs/README.md.
References
See references.md.
Input Requirements
Prerequisites:
Tools are installed in the project's pinned Pixi environment. Commit pixi.toml and pixi.lock, and run tools through pixi run so the lockfile records exact builds. See docs/README.md for expected tools.
Protein FASTA inputs are available as one non-empty file per genome or species. OrthoFinder uses each filename as a taxon identifier, so filenames must be unique and stable.
Inputs:
protein_fastas/ with one amino-acid FASTA per genome, for example protein_fastas/genome_A.faa and protein_fastas/genome_B.faa
genomes.tsv mapping each stable genome identifier to its FASTA path and query/reference role
For MMseqs2 clustering of a concatenated FASTA, protein_to_genome.tsv mapping every unique protein ID back to exactly one genome; a merged proteins.faa without this mapping cannot produce a valid genome-by-family matrix
The tested artifact bundle contains marker-gene and ncRNA censuses alongside copy-number and synteny matrices for the same genome set.
On failure: retry with alternative parameters; if still failing, record in report and exit non-zero.
Verify every per-genome FASTA is non-empty, amino-acid encoded, and has protein IDs unique across the full dataset.
Verify the genome manifest covers every FASTA exactly once; if proteins were concatenated for MMseqs2, verify every clustered protein maps to exactly one genome.
Comparison baseline is justified from literature, phylogeny, taxonomy, or data availability.
Query-specific, missing, expanded, and conserved orthogroups are reported separately.
Candidate discovery orthogroups have annotation evidence or a recommended follow-up analysis.
relative_genome_metrics.tsv places each query in the distribution of relatives and notes the literature-defined extreme of the inferred group.
family_copy_number_comparison.tsv reports per-family fold change vs the relative median for the full annotated family set, not only top candidates.
conserved_neighborhoods.tsv is produced and includes intergenic spacing for both query and relative sides; broken synteny, unusual spacing, and expansions are flagged.
