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. 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.
3. For InterProScan, read docs/interproscan-usage.md and validate the exact CLI with --help or --version. Current stable is v5.77-108.0; InterProScan 6 (Nextflow-based) is a forward-looking migration target.
4. Run InterProScan for domain/family annotation.
5. Run eggNOG-mapper v2.1.13+ for orthology-based annotation.
6. 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).

• Default CPU path: DIAMOND v2.1.20+. For any search against NCBI nr, prefer a clustered nr database (e.g., a clusterednr build under $BIO_DB_ROOT) — it is dramatically faster than full nr at comparable sensitivity for most annotation tasks. Check whether a clusterednr build is available under the reference root; if not, build one with diamond makedb from a clustered FASTA (MMseqs2/CD-HIT-reduced nr) or fall back to full nr and record the choice in the run log.
• GPU node available (CUDA Turing or newer): MMseqs2-GPU as an alternative to DIAMOND. Published benchmark: 20× faster and ~71× cheaper per query versus 128-core CPU MMseqs2, and 177–199× faster than JackHMMER iterative search for profile-equivalent workflows (Kallenborn et al., Nature Methods 2025, DOI: 10.1038/s41592-025-02819-8). Use mmseqs easy-search or easy-taxonomy with the --gpu flag.

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. Rank discovery candidates by evidence strength, novelty relative to the comparison baseline, confidence, and follow-up value.

Quick Reference

Input Requirements

Prerequisites:

• Tools available in the active environment (Pixi/conda/system). 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).

Output

• 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
• results/bio-annotation/logs/

Quality Gates

• [ ] Annotation hit rate and taxonomy rank coverage meet project thresholds.
• [ ] On failure: retry with alternative parameters; if still failing, record in report and exit non-zero.
• [ ] 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 small login-node smoke test on 1-2 proteins before submitting a large cluster job.
• [ ] 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.
• [ ] 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.

Examples

Example 1: Expected input layout

proteins.faa (FASTA protein sequences).
reference_db/ (eggNOG, InterPro, DIAMOND databases + taxdump).

Troubleshooting

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

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.

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