Bio Annotation

Functional annotation and taxonomy inference from sequence homology.

Instructions

1. Read docs/README.md and the relevant tool guides before running anything.
2. Normalize tool outputs and generate the complete comparison bundle with the schema-backed driver:

   uv run --script skills/bio-annotation/scripts/build_annotation_artifacts.py \
     raw_annotations.tsv --genomes genomes.tsv --markers marker_catalog.tsv \
     --out results/bio-annotation

raw_annotations.tsv is one row per protein and family hit with columns genome, protein_id, family_id, family_name, category, e_value, annotation, taxonomy, confidence, merged by you from the tool outputs below. genomes.tsv has genome, role (query|reference), domain, route; marker_catalog.tsv has category, family_id, family_name, expected. fixtures/ holds a runnable example. The driver refuses a non-empty destination, enforces globally unique protein identifiers, writes normalized Parquet tables, adds explicit absent-marker rows, and computes query-specific/missing/expanded/contracted families against the reference median. The artifact contract is in schemas/artifacts.schema.json.

1. When a nucleotide assembly, MAG, genome, or contig FASTA is available, run /tracking-taxonomy-updates first for the BBTools-container QuickClade percontig domain screen. Use that routing table to choose the right taxonomy/QC path before interpreting protein annotations.
2. For InterProScan, read docs/interproscan-usage.md and validate the exact CLI with --help or --version. InterProScan 5 (5.78-109.0) and the Nextflow-based InterProScan 6 (6.0.2.2) both have current releases (checked 2026-10-01). Pin one release and one InterPro data release per project.
3. Run InterProScan for domain/family annotation.
4. Run eggNOG-mapper v2.1.13+ for orthology-based annotation. v3.0.0 is in beta; use it only when the project pins it deliberately.
5. Run sequence-vs-database search and resolve taxonomy with TaxonKit v0.20.0+ (required for the March 2025 NCBI rank update that replaces "superkingdom" with "domain" and adds "realm" for viruses).

Backend choice (DIAMOND, clustered nr, MMseqs2-GPU): see docs/README.md.

1. For domain-specific taxonomy after QuickClade:

• Bacteria/Archaea -> run GTDB-Tk when genome/MAG-level sequence is available and cross-check NCBI/DIAMOND lineage assignments.
• Viral/phage -> route to /bio-viromics; use PHROG/NCVOG markers and vConTACT3 only for phage/prokaryotic-virus contexts.
• Giant-virus/Nucleocytoviricota -> route to /bio-viromics with GVClass and NCLDV marker-gene phylogeny.
• Eukaryota -> use EukCC for MAG/genome QC and lineage context; avoid CheckM/GTDB-Tk assumptions.

1. For group-appropriate marker families, run HMM searches against the relevant profile libraries (Pfam, TIGRFAM, COG/arCOG, PHROG/NCVOG for viruses, eukaryotic ribosomal/structural HMMs when applicable). Use pyhmmer (Python bindings around HMMER 3.4 with native SIMD and batch-friendly APIs) by default; fall back to the HMMER CLI (hmmsearch / hmmscan) when an upstream tool requires it. The choice of profile libraries is derived from the literature-derived playbook for the inferred group.
2. Build an annotation-wide feature inventory by genome/contig and by gene family/domain/pathway.
3. Marker-gene census: From the literature-derived playbook, list the diagnostic marker / machinery categories for the inferred group (e.g., replication, transcription, translation-related such as ribosomal proteins and translation factors, packaging, capsid/structural, chromatin/SMC/topoisomerase, host-interaction). For EACH query genome and each comparison-set genome supplied, record presence and copy number per category. Save as marker_census.tsv (columns: genome, category, family_id, family_name, copy_number, evidence_source, e_value, notes). Expected-but-absent markers are first-class rows, not silent omissions.
4. Per-family copy-number matrix: Build a Pfam/InterPro/HMM-family × genome integer matrix covering queries AND the supplied relatives. Persist as family_copy_number_matrix.parquet. Compute per-family fold change vs the relative median; flag query-specific families, missing-expected families, expansions, and contractions in family_expansion_candidates.tsv.
5. For exploratory work, read the literature-derived analysis playbook for the inferred organism or virus group before deciding what to flag.
6. Mine the inventory for discovery candidates relative to that playbook: expected features, missing expected features, rare or expanded families, unusual combinations, annotation/taxonomy conflicts, and high-value unknowns.
7. For specialized inputs such as viruses, organelles, symbionts, pathogens, or poorly characterized lineages, use the feature classes and outlier dimensions reported in the relevant literature rather than a fixed global checklist.
8. query_specific requires the family to be absent from every reference. When the reference median is 0 but at least one reference carries the family, the status is present_in_reference_minority with an empty fold_change: report it as a signal, never as a discovery.
9. Order discovery_candidates.tsv deterministically before reporting. Sort by status in the order query_specific, missing_expected, expanded, contracted, present_in_reference_minority; then by fold_change descending, with inf first and blank or non-numeric values last; then by genome and family_id ascending. Report the top rows in that order and keep the full table.

Input Requirements

Prerequisites:

• Tools declared in the project's pinned Pixi environment. See docs/README.md for expected tools.
• Reference DB root: set BIO_DB_ROOT to the project or site-local database directory.
• Input FASTA and reference DBs are readable.

Inputs:

• proteins.faa (FASTA protein sequences).
• reference_db/ (eggNOG, InterPro, DIAMOND databases + taxdump).
• For the driver: raw_annotations.tsv, genomes.tsv (at least one query and one reference), and marker_catalog.tsv with the columns listed in Instructions step 2.

Output

The driver writes every file below except the agent-authored report and logs/, and also writes run_manifest.json.

• results/bio-annotation/annotations.parquet
• results/bio-annotation/domain_routing.tsv
• results/bio-annotation/taxonomy.parquet
• results/bio-annotation/feature_inventory.parquet
• results/bio-annotation/marker_census.tsv
• results/bio-annotation/family_copy_number_matrix.parquet
• results/bio-annotation/family_expansion_candidates.tsv
• results/bio-annotation/discovery_candidates.tsv
• results/bio-annotation/annotation_report.md (agent-authored)
• results/bio-annotation/logs/
• stdout: the last line is one JSON envelope {ok, skill, out, manifest, warnings} (driver stdout contract in AGENTS.md)
• Artifact contract: schemas/artifacts.schema.json

Quality Gates

• [ ] Annotation hit rate and taxonomy rank coverage meet project thresholds.
• [ ] 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 proteins.faa is non-empty and amino acid encoded.
• [ ] Verify proteins.faa does not contain * stop symbols before InterProScan, or strip them deliberately.
• [ ] QuickClade domain routing was used when nucleotide assemblies/genomes were available, or the protein-only reason for skipping it is recorded.
• [ ] Verify InterProScan output options are valid: use -b or -d, never both together.
• [ ] Verify packaged InterProScan installs have been initialized with python3 setup.py -f interproscan.properties when required.
• [ ] Verify required InterProScan helper binaries are resolvable, especially ps_scan.pl, pfscan, and pfsearch.
• [ ] Run a short sbatch smoke test on 1-2 proteins before submitting a large cluster job; do not compute on the login node. On Dori use -M perceus-00 -A grp-org-sc-mgs -p dori --qos=jgi_normal with a short --time, never a debug partition or QOS.
• [ ] Verify required reference DBs exist under the reference root.
• [ ] Domain-specific taxonomy tools match the route: GTDB-Tk for Bacteria/Archaea, /bio-viromics plus vConTACT3/GVClass as appropriate for viruses, and EukCC for Eukaryota.
• [ ] Feature inventory summarizes all annotated and unannotated proteins, not only top hits.
• [ ] The normalized bundle is produced in a fresh directory and matches schemas/artifacts.schema.json; protein identifiers are globally unique.
• [ ] marker_census.tsv covers every literature-derived marker category for the inferred group with explicit zero rows for absent markers.
• [ ] family_copy_number_matrix.parquet includes the query AND the supplied relatives, and family_expansion_candidates.tsv flags query-specific, missing-expected, expanded, and contracted families with fold-change vs the relative median.
• [ ] Discovery candidates include evidence fields: gene/protein ID, annotation source, confidence, why notable, and recommended validation.
• [ ] Discovery candidates are justified against the literature-derived playbook and comparison baseline, not only by generic keyword matches.

Non-Goals

• No causal or phenotype claims from homology alone. An annotation transfer is a hypothesis about function, not a demonstration of it.
• No taxonomy for nucleotide assemblies, MAGs, or contigs outside the QuickClade route and its domain-specific follow-up (GTDB-Tk, EukCC, /bio-viromics).
• No reference database downloads or builds. The databases under $BIO_DB_ROOT are an input; report a missing database instead of fetching it.
• No structure-based inference. High-value unknowns go to /bio-structure-annotation.

Troubleshooting

Issue: InterProScan fails immediately with CLI or runtime setup errors Solution: Check docs/interproscan-usage.md for mutually exclusive output flags, * stripping, one-time setup.py initialization, and ProSite PATH requirements.