@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-target-research/SKILL.md

@@ -0,0 +1,1510 @@
+---
+name: tooluniverse-target-research
+description: Gather comprehensive biological target intelligence from 9 parallel research paths covering protein info, structure, interactions, pathways, expression, variants, drug interactions, and literature. Features collision-aware searches, evidence grading (T1-T4), explicit Open Targets coverage, and mandatory completeness auditing. Use when users ask about drug targets, proteins, genes, or need target validation, druggability assessment, or comprehensive target profiling.
+---
+
+# Comprehensive Target Intelligence Gatherer
+
+Gather complete target intelligence by exploring 9 parallel research paths. Supports targets identified by gene symbol, UniProt accession, Ensembl ID, or gene name.
+
+**KEY PRINCIPLES**:
+1. **Report-first approach** - Create report file FIRST, then populate progressively
+2. **Tool parameter verification** - Verify params via `get_tool_info` before calling unfamiliar tools
+3. **Evidence grading** - Grade all claims by evidence strength (T1-T4)
+4. **Citation requirements** - Every fact must have inline source attribution
+5. **Mandatory completeness** - All sections must exist with data minimums or explicit "No data" notes
+6. **Disambiguation first** - Resolve all identifiers before research
+7. **Negative results documented** - "No drugs found" is data; empty sections are failures
+8. **Collision-aware literature search** - Detect and filter naming collisions
+9. **English-first queries** - Always use English terms in tool calls, even if the user writes in another language. Translate gene names, disease names, and search terms to English. Only try original-language terms as a fallback if English returns no results. Respond in the user's language
+
+---
+
+## Phase 0: Tool Parameter Verification (CRITICAL)
+
+**BEFORE calling ANY tool for the first time**, verify its parameters:
+
+```python
+# Always check tool params to prevent silent failures
+tool_info = tu.tools.get_tool_info(tool_name="Reactome_map_uniprot_to_pathways")
+# Reveals: takes `id` not `uniprot_id`
+```
+
+### Known Parameter Corrections (Updated)
+
+| Tool | WRONG Parameter | CORRECT Parameter |
+|------|-----------------|-------------------|
+| `Reactome_map_uniprot_to_pathways` | `uniprot_id` | `id` |
+| `ensembl_get_xrefs` | `gene_id` | `id` |
+| `GTEx_get_median_gene_expression` | `gencode_id` only | `gencode_id` + `operation="median"` |
+| `OpenTargets_*` | `ensemblID` | `ensemblId` (camelCase) |
+
+### GTEx Versioned ID Fallback (CRITICAL)
+
+GTEx often requires versioned Ensembl IDs. If `ENSG00000123456` returns empty:
+
+```python
+# Step 1: Get gene info with version
+gene_info = tu.tools.ensembl_lookup_gene(id=ensembl_id, species="human")
+version = gene_info.get('version', 1)
+
+# Step 2: Try versioned ID
+versioned_id = f"{ensembl_id}.{version}"  # e.g., "ENSG00000123456.12"
+result = tu.tools.GTEx_get_median_gene_expression(
+    gencode_id=versioned_id,
+    operation="median"
+)
+```
+
+---
+
+## When to Use This Skill
+
+Apply when users:
+- Ask about a drug target, protein, or gene
+- Need target validation or assessment
+- Request druggability analysis
+- Want comprehensive target profiling
+- Ask "what do we know about [target]?"
+- Need target-disease associations
+- Request safety profile for a target
+
+---
+
+## Critical Workflow Requirements
+
+### 1. Report-First Approach (MANDATORY)
+
+**DO NOT** show the search process or tool outputs to the user. Instead:
+
+1. **Create the report file FIRST** - Before any data collection:
+   - File name: `[TARGET]_target_report.md`
+   - Initialize with all 14 section headers
+   - Add placeholder: `[Researching...]` in each section
+
+2. **Progressively update the report** - As you gather data:
+   - Update each section immediately after retrieving data
+   - Replace `[Researching...]` with actual content
+   - Include "No data returned" when tools return empty results
+
+3. **Methodology in appendix only** - If user requests methodology details, create separate `[TARGET]_methods_appendix.md`
+
+### 2. Evidence Grading System (MANDATORY)
+
+**CRITICAL**: Grade every claim by evidence strength.
+
+#### Evidence Tiers
+
+| Tier | Symbol | Criteria | Examples |
+|------|--------|----------|----------|
+| **T1** | ★★★ | Direct mechanistic evidence, human genetic proof | CRISPR KO, patient mutations, crystal structure with mechanism |
+| **T2** | ★★☆ | Functional studies, model organism validation | siRNA phenotype, mouse KO, biochemical assay |
+| **T3** | ★☆☆ | Association, screen hits, computational | GWAS hit, DepMap essentiality, expression correlation |
+| **T4** | ☆☆☆ | Mention, review, text-mined, predicted | Review article, database annotation, computational prediction |
+
+#### Required Evidence Grading Locations
+
+Evidence grades MUST appear in:
+1. **Executive Summary** - Key disease claims graded
+2. **Section 8.2 Disease Associations** - Every disease link graded with source type
+3. **Section 11 Literature** - Key papers table with evidence tier
+4. **Section 13 Recommendations** - Scorecard items reference evidence quality
+
+#### Per-Section Evidence Summary
+
+```markdown
+---
+**Evidence Quality for this Section**: Strong
+- Mechanistic (T1): 12 papers
+- Functional (T2): 8 papers
+- Association (T3): 15 papers
+- Mention (T4): 23 papers
+**Data Gaps**: No CRISPR data; mouse KO phenotypes limited
+---
+```
+
+### 3. Citation Requirements (MANDATORY)
+
+Every piece of information MUST include its source:
+
+```markdown
+EGFR mutations cause lung adenocarcinoma [★★★: PMID:15118125, activating mutations 
+in patients]. *Source: ClinVar, CIViC*
+```
+
+---
+
+## Core Strategy: 9 Research Paths
+
+Execute 9 research paths (Path 0 is always first):
+
+```
+Target Query (e.g., "EGFR" or "P00533")
+│
+├─ IDENTIFIER RESOLUTION (always first)
+│   └─ Check if GPCR → GPCRdb_get_protein
+│
+├─ PATH 0: Open Targets Foundation (ALWAYS FIRST - fills gaps in all other paths)
+│
+├─ PATH 1: Core Identity (names, IDs, sequence, organism)
+│   └─ InterProScan_scan_sequence for novel domain prediction (NEW)
+├─ PATH 2: Structure & Domains (3D structure, domains, binding sites)
+│   └─ If GPCR: GPCRdb_get_structures (active/inactive states)
+├─ PATH 3: Function & Pathways (GO terms, pathways, biological role)
+├─ PATH 4: Protein Interactions (PPI network, complexes)
+├─ PATH 5: Expression Profile (tissue expression, single-cell)
+├─ PATH 6: Variants & Disease (mutations, clinical significance)
+│   └─ DisGeNET_search_gene for curated gene-disease associations
+├─ PATH 7: Drug Interactions (known drugs, druggability, safety)
+│   ├─ Pharos_get_target for TDL classification (Tclin/Tchem/Tbio/Tdark)
+│   ├─ BindingDB_get_ligands_by_uniprot for known ligands (NEW)
+│   ├─ PubChem_search_assays_by_target_gene for HTS data (NEW)
+│   ├─ If GPCR: GPCRdb_get_ligands (curated agonists/antagonists)
+│   └─ DepMap_get_gene_dependencies for target essentiality
+└─ PATH 8: Literature & Research (publications, trends)
+```
+
+---
+
+## Identifier Resolution (Phase 1)
+
+**CRITICAL**: Resolve ALL identifiers before any research path.
+
+```python
+def resolve_target_ids(tu, query):
+    """
+    Resolve target query to ALL needed identifiers.
+    Returns dict with: query, uniprot, ensembl, ensembl_version, symbol, 
+    entrez, chembl_target, hgnc
+    """
+    ids = {
+        'query': query, 
+        'uniprot': None, 
+        'ensembl': None, 
+        'ensembl_versioned': None,  # For GTEx
+        'symbol': None,
+        'entrez': None,
+        'chembl_target': None,
+        'hgnc': None,
+        'full_name': None,
+        'synonyms': []
+    }
+    
+    # [Resolution logic based on input type]
+    # ... (see current implementation)
+    
+    # CRITICAL: Get versioned Ensembl ID for GTEx
+    if ids['ensembl']:
+        gene_info = tu.tools.ensembl_lookup_gene(id=ids['ensembl'], species="human")
+        if gene_info and gene_info.get('version'):
+            ids['ensembl_versioned'] = f"{ids['ensembl']}.{gene_info['version']}"
+        
+        # Also get synonyms for literature collision detection
+        ids['full_name'] = gene_info.get('description', '').split(' [')[0]
+    
+    # Get UniProt alternative names for synonyms
+    if ids['uniprot']:
+        alt_names = tu.tools.UniProt_get_alternative_names_by_accession(accession=ids['uniprot'])
+        if alt_names:
+            ids['synonyms'].extend(alt_names)
+    
+    return ids
+```
+
+### GPCR Target Detection (NEW)
+
+~35% of approved drugs target GPCRs. After identifier resolution, check if target is a GPCR:
+
+```python
+def check_gpcr_target(tu, ids):
+    """
+    Check if target is a GPCR and retrieve specialized data.
+    Call after identifier resolution.
+    """
+    symbol = ids.get('symbol', '')
+    
+    # Build GPCRdb entry name
+    entry_name = f"{symbol.lower()}_human"
+    
+    gpcr_info = tu.tools.GPCRdb_get_protein(
+        operation="get_protein",
+        protein=entry_name
+    )
+    
+    if gpcr_info.get('status') == 'success':
+        # Target is a GPCR - get specialized data
+        
+        # Get structures with receptor state
+        structures = tu.tools.GPCRdb_get_structures(
+            operation="get_structures",
+            protein=entry_name
+        )
+        
+        # Get known ligands (critical for binder projects)
+        ligands = tu.tools.GPCRdb_get_ligands(
+            operation="get_ligands",
+            protein=entry_name
+        )
+        
+        # Get mutation data
+        mutations = tu.tools.GPCRdb_get_mutations(
+            operation="get_mutations",
+            protein=entry_name
+        )
+        
+        return {
+            'is_gpcr': True,
+            'gpcr_family': gpcr_info['data'].get('family'),
+            'gpcr_class': gpcr_info['data'].get('receptor_class'),
+            'structures': structures.get('data', {}).get('structures', []),
+            'ligands': ligands.get('data', {}).get('ligands', []),
+            'mutations': mutations.get('data', {}).get('mutations', []),
+            'ballesteros_numbering': True  # GPCRdb provides this
+        }
+    
+    return {'is_gpcr': False}
+```
+
+**GPCRdb Report Section** (add to Section 2 for GPCR targets):
+
+```markdown
+### 2.x GPCR-Specific Data (GPCRdb)
+
+**Receptor Class**: Class A (Rhodopsin-like)  
+**GPCR Family**: Adrenoceptors  
+
+**Structures by State**:
+| PDB ID | State | Resolution | Ligand | Year |
+|--------|-------|------------|--------|------|
+| 3SN6 | Active | 3.2Å | Agonist (BI-167107) | 2011 |
+| 2RH1 | Inactive | 2.4Å | Antagonist (carazolol) | 2007 |
+
+**Known Ligands**: 45 agonists, 32 antagonists, 8 allosteric modulators  
+**Key Binding Site Residues** (Ballesteros-Weinstein): 3.32, 5.42, 6.48, 7.39
+```
+
+### Collision Detection for Literature Search
+
+Before literature search, detect naming collisions:
+
+```python
+def detect_collisions(tu, symbol, full_name):
+    """
+    Detect if gene symbol has naming collisions in literature.
+    Returns negative filter terms if collisions found.
+    """
+    # Search by symbol in title
+    results = tu.tools.PubMed_search_articles(
+        query=f'"{symbol}"[Title]',
+        limit=20
+    )
+    
+    # Check if >20% are off-topic
+    off_topic_terms = []
+    for paper in results.get('articles', []):
+        title = paper.get('title', '').lower()
+        # Check if title mentions biology/protein/gene context
+        bio_terms = ['protein', 'gene', 'cell', 'expression', 'mutation', 'kinase', 'receptor']
+        if not any(term in title for term in bio_terms):
+            # Extract potential collision terms
+            # e.g., "JAK" might collide with "Just Another Kinase" jokes
+            # e.g., "WDR7" might collide with other WDR family members in certain contexts
+            pass
+    
+    # Build negative filter
+    collision_filter = ""
+    if off_topic_terms:
+        collision_filter = " NOT " + " NOT ".join(off_topic_terms)
+    
+    return collision_filter
+```
+
+---
+
+## PATH 0: Open Targets Foundation (ALWAYS FIRST)
+
+**Objective**: Populate baseline data for Sections 5, 8, 9, 10, 11 before specialized queries.
+
+**CRITICAL**: Open Targets provides the most comprehensive aggregated data. Query ALL these endpoints:
+
+| Endpoint | Section | Data Type |
+|----------|---------|-----------|
+| `OpenTargets_get_diseases_phenotypes_by_target_ensemblId` | 8 | Diseases/phenotypes |
+| `OpenTargets_get_target_tractability_by_ensemblId` | 9 | Druggability assessment |
+| `OpenTargets_get_target_safety_profile_by_ensemblId` | 10 | Safety liabilities |
+| `OpenTargets_get_target_interactions_by_ensemblId` | 6 | PPI network |
+| `OpenTargets_get_target_gene_ontology_by_ensemblId` | 5 | GO annotations |
+| `OpenTargets_get_publications_by_target_ensemblId` | 11 | Literature |
+| `OpenTargets_get_biological_mouse_models_by_ensemblId` | 8/10 | Mouse KO phenotypes |
+| `OpenTargets_get_chemical_probes_by_target_ensemblId` | 9 | Chemical probes |
+| `OpenTargets_get_associated_drugs_by_target_ensemblId` | 9 | Known drugs |
+
+### Path 0 Implementation
+
+```python
+def path_0_open_targets(tu, ids):
+    """
+    Open Targets foundation data - fills gaps for sections 5, 6, 8, 9, 10, 11.
+    ALWAYS run this first.
+    """
+    ensembl_id = ids['ensembl']
+    if not ensembl_id:
+        return {'status': 'skipped', 'reason': 'No Ensembl ID'}
+    
+    results = {}
+    
+    # 1. Diseases & Phenotypes (Section 8)
+    diseases = tu.tools.OpenTargets_get_diseases_phenotypes_by_target_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['diseases'] = diseases if diseases else {'note': 'No disease associations returned'}
+    
+    # 2. Tractability (Section 9)
+    tractability = tu.tools.OpenTargets_get_target_tractability_by_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['tractability'] = tractability if tractability else {'note': 'No tractability data returned'}
+    
+    # 3. Safety Profile (Section 10)
+    safety = tu.tools.OpenTargets_get_target_safety_profile_by_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['safety'] = safety if safety else {'note': 'No safety liabilities identified'}
+    
+    # 4. Interactions (Section 6)
+    interactions = tu.tools.OpenTargets_get_target_interactions_by_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['interactions'] = interactions if interactions else {'note': 'No interactions returned'}
+    
+    # 5. GO Annotations (Section 5)
+    go_terms = tu.tools.OpenTargets_get_target_gene_ontology_by_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['go_terms'] = go_terms if go_terms else {'note': 'No GO annotations returned'}
+    
+    # 6. Publications (Section 11)
+    publications = tu.tools.OpenTargets_get_publications_by_target_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['publications'] = publications if publications else {'note': 'No publications returned'}
+    
+    # 7. Mouse Models (Section 8/10)
+    mouse_models = tu.tools.OpenTargets_get_biological_mouse_models_by_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['mouse_models'] = mouse_models if mouse_models else {'note': 'No mouse model data returned'}
+    
+    # 8. Chemical Probes (Section 9)
+    probes = tu.tools.OpenTargets_get_chemical_probes_by_target_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['chemical_probes'] = probes if probes else {'note': 'No chemical probes available'}
+    
+    # 9. Associated Drugs (Section 9)
+    drugs = tu.tools.OpenTargets_get_associated_drugs_by_target_ensemblId(
+        ensemblId=ensembl_id
+    )
+    results['drugs'] = drugs if drugs else {'note': 'No approved/trial drugs found'}
+    
+    return results
+```
+
+### Negative Results Are Data
+
+**CRITICAL**: Always document when a query returns empty:
+
+```markdown
+### 9.3 Chemical Probes
+
+**Status**: No validated chemical probes available for this target.
+*Source: OpenTargets_get_chemical_probes_by_target_ensemblId returned empty*
+
+**Implication**: Tool compound development would be needed for chemical biology studies.
+```
+
+---
+
+## PATH 2: Structure & Domains (Enhanced)
+
+**Objective**: Robust structure coverage using 3-step chain.
+
+### 3-Step Structure Search Chain
+
+**Do NOT rely solely on PDB text search.** Use this chain:
+
+```python
+def path_structure_robust(tu, ids):
+    """
+    Robust structure search using 3-step chain.
+    """
+    structures = {'pdb': [], 'alphafold': None, 'domains': [], 'method_notes': []}
+    
+    # STEP 1: UniProt PDB Cross-References (most reliable)
+    if ids['uniprot']:
+        entry = tu.tools.UniProt_get_entry_by_accession(accession=ids['uniprot'])
+        pdb_xrefs = [x for x in entry.get('uniProtKBCrossReferences', []) 
+                    if x.get('database') == 'PDB']
+        for xref in pdb_xrefs:
+            pdb_id = xref.get('id')
+            # Get details for each PDB
+            pdb_info = tu.tools.get_protein_metadata_by_pdb_id(pdb_id=pdb_id)
+            if pdb_info:
+                structures['pdb'].append(pdb_info)
+        structures['method_notes'].append(f"Step 1: {len(pdb_xrefs)} PDB cross-refs from UniProt")
+    
+    # STEP 2: Sequence-based PDB Search (catches missing annotations)
+    if ids['uniprot'] and len(structures['pdb']) < 5:
+        sequence = tu.tools.UniProt_get_sequence_by_accession(accession=ids['uniprot'])
+        if sequence and len(sequence) < 1000:  # Reasonable length for search
+            similar = tu.tools.PDB_search_similar_structures(
+                sequence=sequence[:500],  # Use first 500 AA if long
+                identity_cutoff=0.7
+            )
+            if similar:
+                for hit in similar[:10]:  # Top 10 similar
+                    if hit['pdb_id'] not in [s.get('pdb_id') for s in structures['pdb']]:
+                        structures['pdb'].append(hit)
+        structures['method_notes'].append(f"Step 2: Sequence search (identity ≥70%)")
+    
+    # STEP 3: Domain-based Search (for multi-domain proteins)
+    if ids['uniprot']:
+        domains = tu.tools.InterPro_get_protein_domains(uniprot_accession=ids['uniprot'])
+        structures['domains'] = domains if domains else []
+        
+        # For large proteins with domains, search by domain sequence windows
+        if len(structures['pdb']) < 3 and domains:
+            for domain in domains[:3]:  # Top 3 domains
+                domain_name = domain.get('name', '')
+                # Could search PDB by domain name
+                domain_hits = tu.tools.PDB_search_by_keyword(query=domain_name, limit=5)
+                if domain_hits:
+                    structures['method_notes'].append(f"Step 3: Domain '{domain_name}' search")
+    
+    # AlphaFold (always check)
+    alphafold = tu.tools.alphafold_get_prediction(uniprot_accession=ids['uniprot'])
+    structures['alphafold'] = alphafold if alphafold else {'note': 'No AlphaFold prediction'}
+    
+    # IMPORTANT: Document limitations
+    if not structures['pdb']:
+        structures['limitation'] = "No direct PDB hit does NOT mean no structure exists. Check: (1) structures under different UniProt entries, (2) homolog structures, (3) domain-only structures."
+    
+    return structures
+```
+
+### Structure Section Output Format
+
+```markdown
+### 4.1 Experimental Structures (PDB)
+
+**Total PDB Entries**: 23 structures *(Source: UniProt cross-references)*
+**Search Method**: 3-step chain (UniProt xrefs → sequence search → domain search)
+
+| PDB ID | Resolution | Method | Ligand | Coverage | Year |
+|--------|------------|--------|--------|----------|------|
+| 1M17 | 2.6Å | X-ray | Erlotinib | 672-998 | 2002 |
+| 3POZ | 2.8Å | X-ray | Gefitinib | 696-1022 | 2010 |
+
+**Note**: "No direct PDB hit" ≠ "no structure exists". Check homologs and domain structures.
+```
+
+---
+
+## PATH 5: Expression Profile (Enhanced)
+
+### GTEx with Versioned ID Fallback
+
+```python
+def path_expression(tu, ids):
+    """
+    Expression data with GTEx versioned ID fallback.
+    """
+    results = {'gtex': None, 'hpa': None, 'failed_tools': []}
+    
+    # GTEx with fallback
+    ensembl_id = ids['ensembl']
+    versioned_id = ids.get('ensembl_versioned')
+    
+    # Try unversioned first
+    gtex_result = tu.tools.GTEx_get_median_gene_expression(
+        gencode_id=ensembl_id,
+        operation="median"
+    )
+    
+    # Fallback to versioned if empty
+    if not gtex_result or gtex_result.get('data') == []:
+        if versioned_id:
+            gtex_result = tu.tools.GTEx_get_median_gene_expression(
+                gencode_id=versioned_id,
+                operation="median"
+            )
+            if gtex_result and gtex_result.get('data'):
+                results['gtex'] = gtex_result
+                results['gtex_note'] = f"Used versioned ID: {versioned_id}"
+        
+        if not results.get('gtex'):
+            results['failed_tools'].append({
+                'tool': 'GTEx_get_median_gene_expression',
+                'tried': [ensembl_id, versioned_id],
+                'fallback': 'See HPA data below'
+            })
+    else:
+        results['gtex'] = gtex_result
+    
+    # HPA (always query as backup)
+    hpa_result = tu.tools.HPA_get_rna_expression_by_source(ensembl_id=ensembl_id)
+    results['hpa'] = hpa_result if hpa_result else {'note': 'No HPA RNA data'}
+    
+    return results
+```
+
+### Human Protein Atlas - Extended Expression (NEW)
+
+HPA provides comprehensive protein expression data including tissue-level, cell-level, and cell line expression.
+
+```python
+def get_hpa_comprehensive_expression(tu, gene_symbol):
+    """
+    Get comprehensive expression data from Human Protein Atlas.
+    
+    Provides:
+    - Tissue expression (protein and RNA)
+    - Subcellular localization
+    - Cell line expression comparison
+    - Tissue specificity
+    """
+    
+    # 1. Search for gene to get IDs
+    gene_info = tu.tools.HPA_search_genes_by_query(search_query=gene_symbol)
+    
+    if not gene_info:
+        return {'error': f'Gene {gene_symbol} not found in HPA'}
+    
+    # 2. Get tissue expression with specificity
+    tissue_search = tu.tools.HPA_generic_search(
+        search_query=gene_symbol,
+        columns="g,gs,rnat,rnatsm,scml,scal",  # Gene, synonyms, tissue specificity, subcellular
+        format="json"
+    )
+    
+    # 3. Compare expression in cancer cell lines vs normal tissue
+    cell_lines = ['a549', 'mcf7', 'hela', 'hepg2', 'pc3']
+    cell_line_expression = {}
+    
+    for cell_line in cell_lines:
+        try:
+            expr = tu.tools.HPA_get_comparative_expression_by_gene_and_cellline(
+                gene_name=gene_symbol,
+                cell_line=cell_line
+            )
+            cell_line_expression[cell_line] = expr
+        except:
+            continue
+    
+    return {
+        'gene_info': gene_info,
+        'tissue_data': tissue_search,
+        'cell_line_expression': cell_line_expression,
+        'source': 'Human Protein Atlas'
+    }
+```
+
+**HPA Expression Output for Report**:
+```markdown
+### Tissue Expression Profile (Human Protein Atlas)
+
+| Tissue | Protein Level | RNA nTPM | Specificity |
+|--------|---------------|----------|-------------|
+| Brain | High | 45.2 | Enriched |
+| Liver | Medium | 23.1 | Enhanced |
+| Kidney | Low | 8.4 | Not detected |
+
+**Subcellular Localization**: Cytoplasm, Plasma membrane
+
+### Cancer Cell Line Expression
+
+| Cell Line | Cancer Type | Expression | vs Normal |
+|-----------|-------------|------------|-----------|
+| A549 | Lung | High | Elevated |
+| MCF7 | Breast | Medium | Similar |
+| HeLa | Cervical | High | Elevated |
+
+*Source: Human Protein Atlas via `HPA_search_genes_by_query`, `HPA_get_comparative_expression_by_gene_and_cellline`*
+```
+
+**Why HPA for Target Research**:
+- **Drug target validation** - Confirm expression in target tissue
+- **Safety assessment** - Expression in essential organs
+- **Biomarker potential** - Tissue-specific expression
+- **Cell line selection** - Choose appropriate models
+
+---
+
+## PATH 6: Variants & Disease (Enhanced)
+
+### 6.1 ClinVar SNV vs CNV Separation
+
+```markdown
+### 8.3 Clinical Variants (ClinVar)
+
+#### Single Nucleotide Variants (SNVs)
+| Variant | Clinical Significance | Condition | Review Status | PMID |
+|---------|----------------------|-----------|---------------|------|
+| p.L858R | Pathogenic | Lung cancer | 4 stars | 15118125 |
+| p.T790M | Pathogenic | Drug resistance | 4 stars | 15737014 |
+
+**Total Pathogenic SNVs**: 47
+
+#### Copy Number Variants (CNVs) - Reported Separately
+| Type | Region | Clinical Significance | Frequency |
+|------|--------|----------------------|-----------|
+| Amplification | 7p11.2 | Pathogenic | Common in cancer |
+
+*Note: CNV data separated as it represents different mutation mechanism*
+```
+
+### 6.2 DisGeNET Integration (NEW)
+
+DisGeNET provides curated gene-disease associations with evidence scores. **Requires**: `DISGENET_API_KEY`
+
+```python
+def get_disgenet_associations(tu, ids):
+    """
+    Get gene-disease associations from DisGeNET.
+    Complements Open Targets with curated association scores.
+    """
+    symbol = ids.get('symbol')
+    if not symbol:
+        return {'status': 'skipped', 'reason': 'No gene symbol'}
+    
+    # Get all disease associations for gene
+    gda = tu.tools.DisGeNET_search_gene(
+        operation="search_gene",
+        gene=symbol,
+        limit=50
+    )
+    
+    if gda.get('status') != 'success':
+        return {'status': 'error', 'message': 'DisGeNET query failed'}
+    
+    associations = gda.get('data', {}).get('associations', [])
+    
+    # Categorize by evidence strength
+    strong = []     # score >= 0.7
+    moderate = []   # score 0.4-0.7  
+    weak = []       # score < 0.4
+    
+    for assoc in associations:
+        score = assoc.get('score', 0)
+        disease_name = assoc.get('disease_name', '')
+        umls_cui = assoc.get('disease_id', '')
+        
+        entry = {
+            'disease': disease_name,
+            'umls_cui': umls_cui,
+            'score': score,
+            'evidence_index': assoc.get('ei'),
+            'dsi': assoc.get('dsi'),  # Disease Specificity Index
+            'dpi': assoc.get('dpi')   # Disease Pleiotropy Index
+        }
+        
+        if score >= 0.7:
+            strong.append(entry)
+        elif score >= 0.4:
+            moderate.append(entry)
+        else:
+            weak.append(entry)
+    
+    return {
+        'total_associations': len(associations),
+        'strong_associations': strong,
+        'moderate_associations': moderate,
+        'weak_associations': weak[:10],  # Limit weak
+        'disease_pleiotropy': len(associations)  # How many diseases linked
+    }
+```
+
+**DisGeNET Report Section** (add to Section 8 - Disease Associations):
+
+```markdown
+### 8.x DisGeNET Gene-Disease Associations (NEW)
+
+**Total Diseases Associated**: 47  
+**Disease Pleiotropy Index**: High (gene linked to many disease types)
+
+#### Strong Associations (Score ≥0.7)
+| Disease | UMLS CUI | Score | Evidence Index |
+|---------|----------|-------|----------------|
+| Non-small cell lung cancer | C0007131 | 0.85 | 0.92 |
+| Glioblastoma | C0017636 | 0.78 | 0.88 |
+
+#### Moderate Associations (Score 0.4-0.7)
+| Disease | UMLS CUI | Score | DSI |
+|---------|----------|-------|-----|
+| Breast cancer | C0006142 | 0.62 | 0.45 |
+
+*Note: DisGeNET score integrates curated databases, GWAS, animal models, and literature*
+```
+
+**Evidence Tier Assignment**:
+- DisGeNET Score ≥0.7 → Consider T2 evidence (multiple validated sources)
+- DisGeNET Score 0.4-0.7 → Consider T3 evidence
+- DisGeNET Score <0.4 → T4 evidence only
+
+---
+
+## PATH 7: Druggability & Target Validation (ENHANCED)
+
+### 7.1 Pharos/TCRD - Target Development Level (NEW)
+
+NIH's Illuminating the Druggable Genome (IDG) portal provides TDL classification for all human proteins:
+
+```python
+def get_pharos_target_info(tu, ids):
+    """
+    Get Pharos/TCRD target development level and druggability.
+    
+    TDL Classification:
+    - Tclin: Approved drug targets
+    - Tchem: Targets with small molecule activities (IC50 < 30nM)
+    - Tbio: Targets with biological annotations
+    - Tdark: Understudied proteins
+    """
+    gene_symbol = ids.get('symbol')
+    uniprot = ids.get('uniprot')
+    
+    # Try by gene symbol first
+    if gene_symbol:
+        result = tu.tools.Pharos_get_target(
+            gene=gene_symbol
+        )
+    elif uniprot:
+        result = tu.tools.Pharos_get_target(
+            uniprot=uniprot
+        )
+    else:
+        return {'status': 'error', 'message': 'Need gene symbol or UniProt'}
+    
+    if result.get('status') == 'success' and result.get('data'):
+        target = result['data']
+        return {
+            'name': target.get('name'),
+            'symbol': target.get('sym'),
+            'tdl': target.get('tdl'),  # Tclin/Tchem/Tbio/Tdark
+            'family': target.get('fam'),  # Kinase, GPCR, etc.
+            'novelty': target.get('novelty'),
+            'description': target.get('description'),
+            'publications': target.get('publicationCount'),
+            'interpretation': interpret_tdl(target.get('tdl'))
+        }
+    return None
+
+def interpret_tdl(tdl):
+    """Interpret Target Development Level for druggability."""
+    interpretations = {
+        'Tclin': 'Approved drug target - highest confidence for druggability',
+        'Tchem': 'Small molecule active - good chemical tractability',
+        'Tbio': 'Biologically characterized - may require novel modalities',
+        'Tdark': 'Understudied - limited data, high novelty potential'
+    }
+    return interpretations.get(tdl, 'Unknown')
+
+def search_disease_targets(tu, disease_name):
+    """Find targets associated with a disease via Pharos."""
+    
+    result = tu.tools.Pharos_get_disease_targets(
+        disease=disease_name,
+        top=50
+    )
+    
+    if result.get('status') == 'success':
+        targets = result['data'].get('targets', [])
+        # Group by TDL for prioritization
+        by_tdl = {'Tclin': [], 'Tchem': [], 'Tbio': [], 'Tdark': []}
+        for t in targets:
+            tdl = t.get('tdl', 'Unknown')
+            if tdl in by_tdl:
+                by_tdl[tdl].append(t)
+        return by_tdl
+    return None
+```
+
+**Pharos Report Section** (add to Section 9 - Druggability):
+
+```markdown
+### 9.x Pharos/TCRD Target Classification (NEW)
+
+**Target Development Level**: Tchem  
+**Protein Family**: Kinase  
+**Novelty Score**: 0.35 (moderately studied)  
+**Publication Count**: 12,456
+
+**TDL Interpretation**: Target has validated small molecule activities with IC50 < 30nM. Good chemical starting points exist.
+
+**Disease Targets Analysis** (for disease-centric queries):
+| TDL | Count | Examples |
+|-----|-------|----------|
+| Tclin | 12 | EGFR, ALK, RET |
+| Tchem | 45 | KRAS, SHP2, CDK4 |
+| Tbio | 78 | Novel kinases |
+| Tdark | 23 | Understudied |
+
+*Source: Pharos/TCRD via `Pharos_get_target`*
+```
+
+### 7.2 DepMap - Target Essentiality Validation (NEW)
+
+CRISPR knockout data from cancer cell lines to validate target essentiality:
+
+```python
+def assess_target_essentiality(tu, ids):
+    """
+    Is this target essential for cancer cell survival?
+    
+    Negative effect scores = gene is essential (cells die upon KO)
+    """
+    gene_symbol = ids.get('symbol')
+    
+    if not gene_symbol:
+        return {'status': 'error', 'message': 'Need gene symbol'}
+    
+    deps = tu.tools.DepMap_get_gene_dependencies(
+        gene_symbol=gene_symbol
+    )
+    
+    if deps.get('status') == 'success':
+        return {
+            'gene': gene_symbol,
+            'data': deps.get('data', {}),
+            'interpretation': 'Negative scores indicate gene is essential for cell survival',
+            'note': 'Score < -0.5 is strongly essential, < -1.0 is extremely essential'
+        }
+    return None
+
+def get_cancer_type_essentiality(tu, gene_symbol, cancer_type):
+    """Check if gene is essential in specific cancer type."""
+    
+    # Get cell lines for cancer type
+    cell_lines = tu.tools.DepMap_get_cell_lines(
+        cancer_type=cancer_type,
+        page_size=20
+    )
+    
+    return {
+        'gene': gene_symbol,
+        'cancer_type': cancer_type,
+        'cell_lines': cell_lines.get('data', {}).get('cell_lines', []),
+        'note': 'Query individual cell lines for dependency scores via DepMap portal'
+    }
+```
+
+**DepMap Report Section** (add to Section 9 - Druggability):
+
+```markdown
+### 9.x Target Essentiality (DepMap) (NEW)
+
+**Gene Essentiality Assessment**:
+| Context | Effect Score | Interpretation |
+|---------|--------------|----------------|
+| Pan-cancer | -0.42 | Moderately essential |
+| Lung cancer | -0.78 | Strongly essential |
+| Breast cancer | -0.21 | Weakly essential |
+
+**Selectivity**: Differential essentiality suggests cancer-type selective target
+
+**Cell Lines Tested**: 1,054 cancer cell lines from DepMap
+
+*Interpretation*: Score < -0.5 indicates strong dependency. This target is more essential in lung cancer than other cancer types - suggesting lung-selective targeting may be feasible.
+
+*Source: DepMap via `DepMap_get_gene_dependencies`*
+```
+
+### 7.3 InterProScan - Novel Domain Prediction (NEW)
+
+For uncharacterized proteins, run InterProScan to predict domains and function:
+
+```python
+def predict_protein_domains(tu, sequence, title="Query protein"):
+    """
+    Run InterProScan for de novo domain prediction.
+    
+    Use when:
+    - Protein has no InterPro annotations
+    - Novel/uncharacterized protein
+    - Custom sequence analysis
+    """
+    
+    result = tu.tools.InterProScan_scan_sequence(
+        sequence=sequence,
+        title=title,
+        go_terms=True,
+        pathways=True
+    )
+    
+    if result.get('status') == 'success':
+        data = result.get('data', {})
+        
+        # Job may still be running
+        if data.get('job_status') == 'RUNNING':
+            return {
+                'job_id': data.get('job_id'),
+                'status': 'running',
+                'note': 'Use InterProScan_get_job_results to retrieve when ready'
+            }
+        
+        # Parse completed results
+        return {
+            'domains': data.get('domains', []),
+            'domain_count': data.get('domain_count', 0),
+            'go_annotations': data.get('go_annotations', []),
+            'pathways': data.get('pathways', []),
+            'sequence_length': data.get('sequence_length')
+        }
+    return None
+
+def check_interproscan_job(tu, job_id):
+    """Check status and get results for InterProScan job."""
+    
+    status = tu.tools.InterProScan_get_job_status(job_id=job_id)
+    
+    if status.get('data', {}).get('is_finished'):
+        results = tu.tools.InterProScan_get_job_results(job_id=job_id)
+        return results.get('data', {})
+    
+    return status.get('data', {})
+```
+
+**When to use InterProScan**:
+- Novel/uncharacterized proteins (Tdark in Pharos)
+- Custom sequences (e.g., protein variants)
+- Proteins with outdated/sparse InterPro annotations
+- Validating domain predictions
+
+**InterProScan Report Section** (for novel proteins):
+
+```markdown
+### Domain Prediction (InterProScan) (NEW)
+
+*Used for uncharacterized protein analysis*
+
+**Predicted Domains**:
+| Domain | Database | Start-End | E-value | InterPro Entry |
+|--------|----------|-----------|---------|----------------|
+| Protein kinase domain | Pfam | 45-305 | 1.2e-89 | IPR000719 |
+| SH2 domain | SMART | 320-410 | 3.4e-45 | IPR000980 |
+
+**Predicted GO Terms**:
+- GO:0004672 protein kinase activity
+- GO:0005524 ATP binding
+
+**Predicted Pathways**:
+- Reactome: Signal Transduction
+
+*Source: InterProScan via `InterProScan_scan_sequence`*
+```
+
+### 7.4 BindingDB - Known Ligands & Binding Data (NEW)
+
+BindingDB provides experimental binding affinity data (Ki, IC50, Kd) for target-ligand pairs:
+
+```python
+def get_bindingdb_ligands(tu, uniprot_id, affinity_cutoff=10000):
+    """
+    Get ligands with measured binding affinities from BindingDB.
+    
+    Critical for:
+    - Identifying chemical starting points
+    - Understanding existing chemical matter
+    - Assessing tractability with small molecules
+    
+    Args:
+        uniprot_id: UniProt accession (e.g., P00533 for EGFR)
+        affinity_cutoff: Maximum affinity in nM (lower = more potent)
+    """
+    
+    # Get ligands by UniProt
+    result = tu.tools.BindingDB_get_ligands_by_uniprot(
+        uniprot=uniprot_id,
+        affinity_cutoff=affinity_cutoff
+    )
+    
+    if result:
+        ligands = []
+        for entry in result:
+            ligands.append({
+                'smiles': entry.get('smile'),
+                'affinity_type': entry.get('affinity_type'),  # Ki, IC50, Kd
+                'affinity_nM': entry.get('affinity'),
+                'monomer_id': entry.get('monomerid'),
+                'pmid': entry.get('pmid')
+            })
+        
+        # Sort by affinity (most potent first)
+        ligands.sort(key=lambda x: float(x['affinity_nM']) if x['affinity_nM'] else float('inf'))
+        
+        return {
+            'total_ligands': len(ligands),
+            'ligands': ligands[:20],  # Top 20 most potent
+            'best_affinity': ligands[0]['affinity_nM'] if ligands else None
+        }
+    
+    return {'total_ligands': 0, 'ligands': [], 'note': 'No ligands found in BindingDB'}
+
+def get_ligands_by_structure(tu, pdb_id, affinity_cutoff=10000):
+    """Get ligands for a protein by PDB structure ID."""
+    
+    result = tu.tools.BindingDB_get_ligands_by_pdb(
+        pdb_ids=pdb_id,
+        affinity_cutoff=affinity_cutoff,
+        sequence_identity=100
+    )
+    
+    return result
+
+def find_compound_targets(tu, smiles, similarity_cutoff=0.85):
+    """Find other targets for a compound (polypharmacology)."""
+    
+    result = tu.tools.BindingDB_get_targets_by_compound(
+        smiles=smiles,
+        similarity_cutoff=similarity_cutoff
+    )
+    
+    return result
+```
+
+**BindingDB Report Section** (add to Section 9 - Druggability):
+
+```markdown
+### Known Ligands (BindingDB) (NEW)
+
+**Total Ligands with Binding Data**: 156
+**Best Reported Affinity**: 0.3 nM (Ki)
+
+#### Most Potent Ligands
+
+| SMILES | Affinity Type | Value (nM) | Source PMID |
+|--------|---------------|------------|-------------|
+| CC(=O)Nc1ccc(cc1)c2... | Ki | 0.3 | 15737014 |
+| CN(C)C/C=C/C(=O)Nc1... | IC50 | 0.8 | 15896103 |
+| COc1cc2ncnc(Nc3ccc... | Kd | 2.1 | 16460808 |
+
+**Chemical Tractability Assessment**:
+- ✅ **Tchem-level target**: Multiple ligands with <30nM affinity
+- ✅ **Diverse chemotypes**: Multiple scaffolds identified
+- ✅ **Published literature**: Ligands have PMID references
+
+*Source: BindingDB via `BindingDB_get_ligands_by_uniprot`*
+```
+
+**Affinity Interpretation for Druggability**:
+| Affinity Range | Interpretation | Drug Development Potential |
+|----------------|----------------|---------------------------|
+| <1 nM | Ultra-potent | Clinical compound likely |
+| 1-10 nM | Highly potent | Drug-like |
+| 10-100 nM | Potent | Good starting point |
+| 100-1000 nM | Moderate | Needs optimization |
+| >1000 nM | Weak | Early hit only |
+
+### 7.5 PubChem BioAssay - Screening Data (NEW)
+
+PubChem BioAssay provides HTS screening data and dose-response curves:
+
+```python
+def get_pubchem_assays_for_target(tu, gene_symbol):
+    """
+    Get bioassays targeting a gene from PubChem.
+    
+    Provides:
+    - HTS screening results
+    - Dose-response data (IC50/EC50)
+    - Active compound counts
+    """
+    
+    # Search assays by target gene
+    assays = tu.tools.PubChem_search_assays_by_target_gene(
+        gene_symbol=gene_symbol
+    )
+    
+    assay_info = []
+    if assays.get('data', {}).get('aids'):
+        for aid in assays['data']['aids'][:10]:  # Top 10 assays
+            # Get assay details
+            summary = tu.tools.PubChem_get_assay_summary(aid=aid)
+            targets = tu.tools.PubChem_get_assay_targets(aid=aid)
+            
+            assay_info.append({
+                'aid': aid,
+                'summary': summary.get('data', {}),
+                'targets': targets.get('data', {})
+            })
+    
+    return {
+        'total_assays': len(assays.get('data', {}).get('aids', [])),
+        'assay_details': assay_info
+    }
+
+def get_active_compounds_from_assay(tu, aid):
+    """Get active compounds from a specific bioassay."""
+    
+    actives = tu.tools.PubChem_get_assay_active_compounds(aid=aid)
+    
+    return {
+        'aid': aid,
+        'active_cids': actives.get('data', {}).get('cids', []),
+        'count': len(actives.get('data', {}).get('cids', []))
+    }
+```
+
+**PubChem BioAssay Report Section**:
+
+```markdown
+### PubChem BioAssay Data (NEW)
+
+**Assays Targeting This Gene**: 45
+
+| AID | Assay Type | Active Compounds | Target Info |
+|-----|------------|------------------|-------------|
+| 1053104 | Dose-response | 12 | EGFR kinase |
+| 504526 | HTS | 234 | EGFR binding |
+| 651564 | Confirmatory | 8 | EGFR cellular |
+
+**Total Active Compounds Across Assays**: ~500
+
+*Source: PubChem via `PubChem_search_assays_by_target_gene`, `PubChem_get_assay_active_compounds`*
+```
+
+---
+
+## PATH 8: Literature & Research (Collision-Aware)
+
+### Collision-Aware Query Strategy
+
+```python
+def path_literature_collision_aware(tu, ids):
+    """
+    Literature search with collision detection and filtering.
+    """
+    symbol = ids['symbol']
+    full_name = ids.get('full_name', '')
+    uniprot = ids['uniprot']
+    synonyms = ids.get('synonyms', [])
+    
+    # Step 1: Detect collisions
+    collision_filter = detect_collisions(tu, symbol, full_name)
+    
+    # Step 2: Build high-precision seed queries
+    seed_queries = [
+        f'"{symbol}"[Title] AND (protein OR gene OR expression)',  # Symbol in title
+        f'"{full_name}"[Title]' if full_name else None,  # Full name in title
+        f'"UniProt:{uniprot}"' if uniprot else None,  # UniProt accession
+    ]
+    seed_queries = [q for q in seed_queries if q]
+    
+    # Add key synonyms
+    for syn in synonyms[:3]:
+        seed_queries.append(f'"{syn}"[Title]')
+    
+    # Step 3: Execute seed queries and collect PMIDs
+    seed_pmids = set()
+    for query in seed_queries:
+        if collision_filter:
+            query = f"({query}){collision_filter}"
+        results = tu.tools.PubMed_search_articles(query=query, limit=30)
+        for article in results.get('articles', []):
+            seed_pmids.add(article.get('pmid'))
+    
+    # Step 4: Expand via citation network (for sparse targets)
+    if len(seed_pmids) < 30:
+        expanded_pmids = set()
+        for pmid in list(seed_pmids)[:10]:  # Top 10 seeds
+            # Get related articles
+            related = tu.tools.PubMed_get_related(pmid=pmid, limit=20)
+            for r in related.get('articles', []):
+                expanded_pmids.add(r.get('pmid'))
+            
+            # Get citing articles
+            citing = tu.tools.EuropePMC_get_citations(pmid=pmid, limit=20)
+            for c in citing.get('citations', []):
+                expanded_pmids.add(c.get('pmid'))
+        
+        seed_pmids.update(expanded_pmids)
+    
+    # Step 5: Classify papers by evidence tier
+    papers_by_tier = {'T1': [], 'T2': [], 'T3': [], 'T4': []}
+    # ... classification logic based on title/abstract keywords
+    
+    return {
+        'total_papers': len(seed_pmids),
+        'collision_filter_applied': collision_filter if collision_filter else 'None needed',
+        'seed_queries': seed_queries,
+        'papers_by_tier': papers_by_tier
+    }
+```
+
+---
+
+## Retry Logic & Fallback Chains
+
+### Retry Policy
+
+For each critical tool, implement retry with exponential backoff:
+
+```python
+def call_with_retry(tu, tool_name, params, max_retries=3):
+    """
+    Call tool with retry logic.
+    """
+    for attempt in range(max_retries):
+        try:
+            result = getattr(tu.tools, tool_name)(**params)
+            if result and not result.get('error'):
+                return result
+        except Exception as e:
+            if attempt < max_retries - 1:
+                time.sleep(2 ** attempt)  # Exponential backoff
+            else:
+                return {'error': str(e), 'tool': tool_name, 'attempts': max_retries}
+    return None
+```
+
+### Fallback Chains (CRITICAL)
+
+| Primary Tool | Fallback 1 | Fallback 2 | Failure Action |
+|--------------|------------|------------|----------------|
+| `ChEMBL_get_target_activities` | `GtoPdb_get_target_ligands` | `OpenTargets drugs` | Note in report |
+| `intact_get_interactions` | `STRING_get_protein_interactions` | `OpenTargets interactions` | Note in report |
+| `GO_get_annotations_for_gene` | `OpenTargets GO` | `MyGene GO` | Note in report |
+| `GTEx_get_median_gene_expression` | `HPA_get_rna_expression` | Note as unavailable | Document in report |
+| `gnomad_get_gene_constraints` | `OpenTargets constraint` | - | Note in report |
+| `DGIdb_get_drug_gene_interactions` | `OpenTargets drugs` | `GtoPdb` | Note in report |
+
+### Failure Surfacing Rule
+
+**NEVER silently skip failed tools.** Always document:
+
+```markdown
+### 7.1 Tissue Expression
+
+**GTEx Data**: Unavailable (API timeout after 3 attempts)
+**Fallback Data (HPA)**:
+| Tissue | Expression Level | Specificity |
+|--------|-----------------|-------------|
+| Liver | High | Enhanced |
+| Kidney | Medium | - |
+
+*Note: For complete GTEx data, query directly at gtexportal.org*
+```
+
+---
+
+## Per-Section Data Minimums & Completeness Audit
+
+### Minimum Data Requirements (Enforced)
+
+| Section | Minimum Data | If Not Met |
+|---------|--------------|------------|
+| **6. PPIs** | ≥20 interactors | Document which tools failed + why |
+| **7. Expression** | Top 10 tissues with TPM + HPA RNA summary | Note "limited data" with specific gaps |
+| **8. Disease** | Top 10 OT diseases + gnomAD constraints + ClinVar summary | Separate SNV/CNV; note if constraint unavailable |
+| **9. Druggability** | OT tractability + probes + drugs + DGIdb + GtoPdb fallback | "No drugs/probes" is valid data |
+| **11. Literature** | Total count + 5-year trend + 3-5 key papers with evidence tiers | Note if sparse (<50 papers) |
+
+### Post-Run Completeness Audit
+
+Before finalizing the report, run this checklist:
+
+```markdown
+## Completeness Audit (REQUIRED)
+
+### Data Minimums Check
+- [ ] PPIs: ≥20 interactors OR explanation why fewer
+- [ ] Expression: Top 10 tissues with values OR explicit "unavailable"
+- [ ] Diseases: Top 10 associations with scores OR "no associations"
+- [ ] Constraints: All 4 scores (pLI, LOEUF, missense Z, pRec) OR "unavailable"
+- [ ] Druggability: All modalities assessed; probes + drugs listed OR "none"
+
+### Negative Results Documented
+- [ ] Empty tool results noted explicitly (not left blank)
+- [ ] Failed tools with fallbacks documented
+- [ ] "No data" sections have implications noted
+
+### Evidence Quality
+- [ ] T1-T4 grades in Executive Summary disease claims
+- [ ] T1-T4 grades in Disease Associations table
+- [ ] Key papers table has evidence tiers
+- [ ] Per-section evidence summaries included
+
+### Source Attribution
+- [ ] Every data point has source tool/database cited
+- [ ] Section-end source summaries present
+```
+
+### Data Gap Table (Required if minimums not met)
+
+```markdown
+## 15. Data Gaps & Limitations
+
+| Section | Expected Data | Actual | Reason | Alternative Source |
+|---------|---------------|--------|--------|-------------------|
+| 6. PPIs | ≥20 interactors | 8 | Novel target, limited studies | Literature review needed |
+| 7. Expression | GTEx TPM | None | Versioned ID not recognized | See HPA data |
+| 9. Probes | Chemical probes | None | No validated probes exist | Consider tool compound dev |
+
+**Recommendations for Data Gaps**:
+1. For PPIs: Query BioGRID with broader parameters; check yeast-2-hybrid studies
+2. For Expression: Query GEO directly for tissue-specific datasets
+```
+
+---
+
+## Report Template (Initial File)
+
+**File**: `[TARGET]_target_report.md`
+
+```markdown
+# Target Intelligence Report: [TARGET NAME]
+
+**Generated**: [Date] | **Query**: [Original query] | **Status**: In Progress
+
+---
+
+## 1. Executive Summary
+[Researching...]
+<!-- REQUIRED: 2-3 sentences, disease claims must have T1-T4 grades -->
+
+## 2. Target Identifiers
+[Researching...]
+<!-- REQUIRED: UniProt, Ensembl (versioned), Entrez, ChEMBL, HGNC, Symbol -->
+
+## 3. Basic Information
+### 3.1 Protein Description
+[Researching...]
+### 3.2 Protein Function
+[Researching...]
+### 3.3 Subcellular Localization
+[Researching...]
+
+## 4. Structural Biology
+### 4.1 Experimental Structures (PDB)
+[Researching...]
+<!-- METHOD: 3-step chain (UniProt xrefs → sequence search → domain search) -->
+### 4.2 AlphaFold Prediction
+[Researching...]
+### 4.3 Domain Architecture
+[Researching...]
+### 4.4 Key Structural Features
+[Researching...]
+
+## 5. Function & Pathways
+### 5.1 Gene Ontology Annotations
+[Researching...]
+<!-- REQUIRED: Evidence codes mapped to T1-T4 -->
+### 5.2 Pathway Involvement
+[Researching...]
+
+## 6. Protein-Protein Interactions
+[Researching...]
+<!-- MINIMUM: ≥20 interactors OR explanation -->
+
+## 7. Expression Profile
+### 7.1 Tissue Expression (GTEx/HPA)
+[Researching...]
+<!-- NOTE: Use versioned Ensembl ID for GTEx if needed -->
+### 7.2 Tissue Specificity
+[Researching...]
+<!-- MINIMUM: Top 10 tissues with TPM values -->
+
+## 8. Genetic Variation & Disease
+### 8.1 Constraint Scores
+[Researching...]
+<!-- REQUIRED: pLI, LOEUF, missense Z, pRec with interpretations -->
+### 8.2 Disease Associations
+[Researching...]
+<!-- REQUIRED: Top 10 with OT scores; T1-T4 evidence grades -->
+### 8.3 Clinical Variants (ClinVar)
+[Researching...]
+<!-- REQUIRED: Separate SNV and CNV tables -->
+### 8.4 Mouse Model Phenotypes
+[Researching...]
+
+## 9. Druggability & Pharmacology
+### 9.1 Tractability Assessment
+[Researching...]
+<!-- REQUIRED: All modalities (SM, Ab, PROTAC, other) -->
+### 9.2 Known Drugs
+[Researching...]
+### 9.3 Chemical Probes
+[Researching...]
+<!-- NOTE: "No probes" is valid data - document explicitly -->
+### 9.4 Clinical Pipeline
+[Researching...]
+### 9.5 ChEMBL Bioactivity
+[Researching...]
+
+## 10. Safety Profile
+### 10.1 Safety Liabilities
+[Researching...]
+### 10.2 Expression-Based Toxicity Risk
+[Researching...]
+### 10.3 Mouse KO Phenotypes
+[Researching...]
+
+## 11. Literature & Research Landscape
+### 11.1 Publication Metrics
+[Researching...]
+<!-- REQUIRED: Total, 5y, 1y, drug-related, clinical -->
+### 11.2 Research Trend
+[Researching...]
+### 11.3 Key Publications
+[Researching...]
+<!-- REQUIRED: Table with PMID, title, year, evidence tier -->
+### 11.4 Evidence Summary by Theme
+[Researching...]
+<!-- REQUIRED: T1-T4 breakdown per research theme -->
+
+## 12. Competitive Landscape
+[Researching...]
+
+## 13. Summary & Recommendations
+### 13.1 Target Validation Scorecard
+[Researching...]
+<!-- REQUIRED: 6 criteria, 1-5 scores, evidence quality noted -->
+### 13.2 Strengths
+[Researching...]
+### 13.3 Challenges & Risks
+[Researching...]
+### 13.4 Recommendations
+[Researching...]
+<!-- REQUIRED: ≥3 prioritized (HIGH/MEDIUM/LOW) -->
+
+## 14. Data Sources & Methodology
+[Will be populated as research progresses...]
+
+## 15. Data Gaps & Limitations
+[To be populated post-audit...]
+```
+
+---
+
+## Quick Reference: Tool Parameters
+
+| Tool | Parameter | Notes |
+|------|-----------|-------|
+| `Reactome_map_uniprot_to_pathways` | `id` | NOT `uniprot_id` |
+| `ensembl_get_xrefs` | `id` | NOT `gene_id` |
+| `GTEx_get_median_gene_expression` | `gencode_id`, `operation` | Try versioned ID if empty |
+| `OpenTargets_*` | `ensemblId` | camelCase, not `ensemblID` |
+| `STRING_get_protein_interactions` | `protein_ids`, `species` | List format for IDs |
+| `intact_get_interactions` | `identifier` | UniProt accession |
+
+---
+
+## When NOT to Use This Skill
+
+- Simple protein lookup → Use `UniProt_get_entry_by_accession` directly
+- Drug information only → Use drug-focused tools
+- Disease-centric query → Use disease-intelligence-gatherer skill
+- Sequence retrieval → Use sequence-retrieval skill
+- Structure download → Use protein-structure-retrieval skill
+
+Use this skill for comprehensive, multi-angle target analysis with guaranteed data completeness.