Bio Protein Clustering Pangenome

Cluster proteins into orthogroups and derive pangenome matrices.

Instructions

Tool guides and versions: docs/README.md.

1. After clustering, build and validate the complete small-to-large comparison bundle with:

   uv run --script skills/bio-protein-clustering-pangenome/scripts/build_pangenome_artifacts.py \
     orthogroups.tsv --genomes genomes.tsv --marker-catalog marker_catalog.tsv \
     --marker-hits marker_hits.tsv --ncrna ncRNA_census.tsv \
     --out results/bio-protein-clustering-pangenome
   

   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.

2. 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.

3. Build presence/absence matrix AND an integer copy-number matrix (orthogroup × genome) covering the query AND the close relatives produced by /bio-phylogenomics.

4. Compute core/accessory/cloud/singleton partitions.

5. Identify single-copy orthologs for phylogenetic analysis.

6. Discriminate paralogs from orthologs in multi-copy gene families.

7. Calculate pangenome statistics (completeness, orthogroup occupancy).

8. 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.

9. 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.

10. Synteny / conserved neighborhoods — for each pair (query, relative) compute conserved gene neighborhoods (e.g., ≥2 collinear orthologs). Tool selection:

• Pairwise / classical: MCScanX (Nature Protocols 2024 updated protocol).
• 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.

11. 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.
12. Annotate candidate orthogroups with /bio-annotation; for high-value unknowns, route representatives to /bio-structure-annotation when structure-based inference is appropriate.
13. 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 | |------|--------| | 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

Output

• results/bio-protein-clustering-pangenome/orthogroups.tsv
• results/bio-protein-clustering-pangenome/presence_absence.parquet
• results/bio-protein-clustering-pangenome/copy_number_matrix.parquet
• results/bio-protein-clustering-pangenome/relative_genome_metrics.tsv
• results/bio-protein-clustering-pangenome/family_copy_number_comparison.tsv (status is one of query_specific, missing_expected, expanded, contracted, conserved, or present_in_reference_minority; the last means the reference median is 0 while at least one reference carries the family, so no fold change is reported and it is not a query-specific discovery)
• results/bio-protein-clustering-pangenome/conserved_neighborhoods.tsv
• results/bio-protein-clustering-pangenome/closest_relative_comparison.tsv
• results/bio-protein-clustering-pangenome/query_specific_candidates.tsv
• results/bio-protein-clustering-pangenome/pangenome_report.md
• results/bio-protein-clustering-pangenome/logs/
• stdout: the last line is one JSON envelope {ok, skill, out, warnings} (driver stdout contract in AGENTS.md)

Quality Gates

• [ ] Cluster size distributions meet project thresholds.
• [ ] Matrix completeness meets project thresholds.
• [ ] The tested artifact bundle contains marker-gene and ncRNA censuses alongside copy-number and synteny matrices for the same genome set.
• [ ] On execution failure, preserve logs and report the failed command; retry only after diagnosing the cause and recording the changed parameters. Report unmet biological thresholds as results; never tune parameters solely to pass a gate.
• [ ] 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.