GWAS to Function via TWAS

GWAS to Function via TWAS

Identify genes whose genetically regulated expression is associated with disease risk, determine therapeutic directionality (inhibit vs. activate), and prioritize drug targets with causal genetic evidence using Transcriptome-Wide Association Study (TWAS) analysis.

Key capabilities:

• Dual TWAS tools: FUSION (comprehensive) and S-PrediXcan (fast, 10-100x faster)
• Therapeutic directionality: Determine inhibit vs. activate strategy for each gene
• Multi-tier analysis: Basic → Colocalization → Mendelian Randomization → Druggability
• Colocalization testing: Filters LD artifacts (up to 50% of TWAS hits), requires PP.H4 > 0.8
• Cross-tissue meta-analysis: S-MultiXcan across 54 GTEx v8 tissues
• Biopharma-ready reports: Excel workbooks with prioritized targets and confidence levels

When to Use This Skill

Use this skill when:

• ✅ You have genome-wide GWAS summary statistics (N > 5,000 samples)
• ✅ Need to identify effector genes for non-coding GWAS variants
• ✅ Want to determine therapeutic strategy (inhibit vs. activate gene expression)
• ✅ Prioritizing drug targets by genetic evidence strength
• ✅ Validating existing targets with causal genetic evidence

Don't use this skill when:

• ❌ Only have significant loci (p < 5×10⁻⁸) without genome-wide data
• ❌ Sample size < 5,000 (insufficient power for TWAS)
• ❌ Need functional enrichment analysis → use functional-enrichment-from-degs
• ❌ Working with individual-level genotypes → run GWAS first with gwas-genotype-qc

Data requirements: Genome-wide GWAS summary statistics with columns: SNP, CHR, BP, A1, A2, BETA/OR, SE, P, N

Installation

Required Software

Python Packages

pip install pandas numpy scipy plotnine plotnine-prism seaborn statsmodels metaxcan adjustText

For detailed installation: See references/installation-guide.md for FUSION setup, LD reference panels, and GTEx weight downloads.

License compliance: All tools permit commercial AI agent use.

Inputs

Required:

• GWAS summary statistics: Genome-wide (not just significant loci)
  • Format: Tab or comma-delimited text (gzip supported)
  • Required columns: SNP, CHR, BP, A1, A2, BETA/OR, SE, P, N
  • Minimum sample size: N > 5,000 (recommended N > 10,000)
  • Genome build: GRCh37/hg19 or GRCh38 (must specify)

Optional:

• Trait information: Type (disease/quantitative), directionality (higher = risk/protective)
• Tissue selection: LDSC heritability enrichment results (from gwas-heritability-ldsc)
• eQTL summary statistics: For colocalization (GTEx v8 available via download)

Outputs

Tier 1 (Basic): TWAS results per tissue, Manhattan/QQ plots (PNG/SVG)

Tier 2 (+ Coloc): PP.H4 scores for each gene, therapeutic recommendations (inhibit/activate)

Tier 3 (+ MR): Causal estimates with sensitivity analyses, MR summary Excel

Tier 4 (+ Drug): reports/[trait]_Target_Report.xlsx - Comprehensive Excel workbook with executive summary, TWAS results, colocalization evidence, therapeutic directionality, MR causal inference, and druggability scores

Clarification Questions

Ask these questions before starting analysis:

1. Input Files (ASK THIS FIRST):

   • Do you have specific GWAS summary statistics file(s) to analyze?
   • If uploaded: Can you confirm this is genome-wide summary statistics (not just significant loci)?
   • Expected format: SNP, CHR, BP, A1, A2, BETA/OR, SE, P, N columns
   • Or use example/demo data? Example GWAS datasets can be downloaded for testing purposes
   • Minimum requirements: N > 5,000 samples, genome-wide coverage

2. Trait Information:

   • Trait type: Disease (binary) or quantitative trait?
   • Trait directionality: Is higher value "risk" or "protective"?
   • Sample size and ancestry composition?
   • Trait category: Cardiovascular, metabolic, neurological, immune, cancer, etc.?

3. Tissue Selection Strategy:

   • Data-driven (LDSC): Run LDSC partitioned heritability first (most rigorous, see gwas-heritability-ldsc)
   • Biology-based: Use known trait-tissue relationships (see references/tissue_reference_guide.md)
   • Comprehensive: Test all 54 GTEx tissues via S-MultiXcan (discovery mode)

4. Analysis Tier (choose depth based on goals and confidence requirements):

   • Tier 1 - Basic TWAS: Association testing only, low-medium confidence
   • Tier 2 - + Colocalization (RECOMMENDED): Filters LD artifacts (PP.H4 > 0.8), medium-high confidence
   • Tier 3 - + Mendelian Randomization: Establishes causality, high confidence
   • Tier 4 - + Druggability: Full target prioritization report, very high confidence (gold standard)

5. TWAS Tool Selection:

   • FUSION (comprehensive): Multiple prediction models, built-in colocalization, requires R + >32GB RAM
   • S-PrediXcan (fast): MASHR model, 10-100x faster than FUSION, requires Python + <16GB RAM
   • See references/fusion_best_practices.md and references/spredixxcan_best_practices.md

6. Computational Resources:

   • Local machine (<16GB RAM): S-PrediXcan, analyze <5 tissues
   • Server/HPC (>32GB RAM): FUSION or S-PrediXcan, parallelize across all tissues

Standard Workflow

Note: Run from the OmicsClaw root directory and add the workflow scripts to sys.path:

import sys; import os; sys.path.insert(0, os.path.abspath('knowledge_base/scripts/gwas-to-function-twas'))

🚨 EXECUTE EXACTLY AS SHOWN - Do not modify these commands.

CRITICAL: Use relative paths (knowledge_base/scripts/gwas-to-function-twas/, references/). DO NOT construct absolute paths like /mnt/knowhow/ or /workspace/.

IMPORTANT: Call scripts via command line. DO NOT import and write inline Python code.

Phase 1: Data Preparation

Step 1: Validate and harmonize GWAS summary statistics

python scripts/validate_gwas_sumstats.py \
  --gwas gwas_sumstats.txt.gz \
  --min-n 5000 \
  --build GRCh37 \
  --output gwas_harmonized.txt

QC checks: Duplicate SNPs, allele harmonization, genomic inflation (λGC), LDSC intercept. For LDSC QC interpretation, see references/ldsc_qc_guidelines.md.

Step 2: Select tissues for TWAS analysis

Option A - Data-driven (after running gwas-heritability-ldsc):

python scripts/select_reference_panel.py \
  --ldsc-results ../gwas-heritability-ldsc/twas_tissue_recommendations.csv \
  --output selected_tissues.txt

Option B - Biology-based:

python scripts/select_reference_panel.py \
  --trait-category cardiovascular \
  --top-n 5 \
  --output selected_tissues.txt

See references/tissue_reference_guide.md for trait-tissue mappings.

Step 3: Download expression weights and LD reference panels

See references/installation-guide.md for:

• GTEx v8 weight download links (FUSION: http://gusevlab.org/projects/fusion/, S-PrediXcan: https://predictdb.org/)
• LD reference panel setup (1000 Genomes EUR or FUSION LDREF)

Phase 2: TWAS Association Testing

Step 4: Run TWAS analysis

Option A - FUSION (comprehensive):

python scripts/run_fusion.py \
  --gwas gwas_harmonized.txt \
  --weights weights/GTEx_v8/ \
  --tissues selected_tissues.txt \
  --ref-ld-panel LDREF/ \
  --output results/fusion/

Option B - S-PrediXcan (fast):

python scripts/run_spredixxcan.py \
  --gwas gwas_harmonized.txt \
  --weights weights/GTEx_v8/ \
  --tissues selected_tissues.txt \
  --output results/spredixxcan/

See references/fusion_best_practices.md and references/spredixxcan_best_practices.md for tool-specific parameters.

Step 5: Generate visualization plots

python scripts/plot_twas_results.py \
  --twas-results results/fusion/Artery_Coronary_twas.txt \
  --gwas gwas_harmonized.txt \
  --output-dir figures/

Creates Manhattan plots, QQ plots, and regional association plots (PNG + SVG formats).

Step 5b (Optional): Validate against published TWAS results

python scripts/validate_with_twas_hub.py \
  --twas-results results/fusion/Artery_Coronary_twas.txt \
  --trait coronary_artery_disease \
  --tissue Artery_Coronary

See references/twas_hub_validation_guide.md.

Phase 3: Statistical Refinement (Tier 2+)

Step 6: Colocalization analysis to filter LD artifacts

python scripts/colocalization_analysis.py \
  --twas-results results/fusion/significant_genes.txt \
  --gwas gwas_harmonized.txt \
  --eqtl-dir eqtl_data/GTEx_v8/ \
  --tissue Artery_Coronary \
  --pp4-threshold 0.8 \
  --output coloc_results/

CRITICAL: Up to 50% of TWAS hits may be LD artifacts. Only genes with PP.H4 > 0.8 have strong colocalization evidence.

Step 6b (Optional): Cross-tissue meta-analysis

python scripts/run_smultixcan.py \
  --gwas gwas_harmonized.txt \
  --weights weights/GTEx_v8/ \
  --output results/smultixcan/

Phase 4: Therapeutic Directionality

Step 7: Interpret therapeutic strategy (inhibit vs. activate)

python scripts/interpret_therapeutic_direction.py \
  --coloc-results coloc_results/high_confidence.txt \
  --trait-direction risk \
  --trait-name "Coronary Artery Disease" \
  --output therapeutic_recommendations.csv

Logic: Positive TWAS Z-score + risk trait → INHIBIT gene; Negative Z-score + risk trait → ACTIVATE gene.

See references/therapeutic_interpretation_guide.md for comprehensive directionality framework.

Step 8: Multi-layer consistency check (eQTL + TWAS directionality)

python scripts/multilayer_direction_analysis.py \
  --therapeutic-recs therapeutic_recommendations.csv \
  --gwas gwas_harmonized.txt \
  --eqtl-data eqtl_data/GTEx_v8/ \
  --twas-results results/fusion/ \
  --coloc-results coloc_results/ \
  --output multilayer_consistency.csv

Confidence levels: High (consistent + PP.H4 > 0.8), Medium (PP.H4 = 0.5-0.8), Low (inconsistent/poor coloc).

Phase 5: Causal Validation (Tier 3+)

Step 9: Mendelian Randomization for causal inference

python scripts/mendelian_randomization.py \
  --genes therapeutic_recommendations.csv \
  --exposure-data eqtl_data/GTEx_v8/ \
  --outcome-data gwas_harmonized.txt \
  --methods ivw,egger,weighted_median \
  --output mr_results/

MR establishes causal directionality (gene expression → trait). Includes sensitivity analyses for pleiotropy.

See references/mendelian_randomization_guide.md for MR principles and interpretation.

Phase 6: Druggability Assessment (Tier 4)

Step 10: Druggability scoring

python scripts/druggability_scoring.py \
  --genes multilayer_consistency.csv \
  --include-known-drugs \
  --output druggability_scores.csv

Scores based on: Protein class, known drugs, clinical precedent, assay availability.

Step 11: Generate comprehensive target report

python scripts/export_results.py \
  --twas-results results/fusion/ \
  --coloc-results coloc_results/ \
  --therapeutic-recs therapeutic_recommendations.csv \
  --mr-results mr_results/ \
  --druggability druggability_scores.csv \
  --trait "Coronary Artery Disease" \
  --output reports/CAD_Target_Report.xlsx

Creates publication-ready Excel workbook with executive summary, evidence layers, and prioritized targets.

That's it! The scripts handle everything automatically.

⚠️ CRITICAL - DO NOT:

• ❌ Write inline TWAS code → causes model mismatches, missing QC steps
• ❌ Write inline colocalization code → incorrect PP.H4 calculations, wrong priors
• ❌ Use absolute paths like /mnt/knowhow/workflows/ → use relative paths scripts/ exactly as shown
• ❌ Import script functions directly → use command-line interface for proper error handling and validation
• ❌ Skip phases based on tier → breaks dependency chain (e.g., Phase 4 requires Phase 3 colocalization results)

Phase dependencies:

• Phase 3 (Colocalization) requires Phase 2 TWAS results
• Phase 4 (Directionality) requires Phase 3 PP.H4 > 0.8 filtering
• Phase 5 (MR) requires Phase 4 directional interpretations
• Phase 6 (Druggability) requires Phase 5 causal evidence

DO NOT skip phases or run out of order.

Common Issues

For complete troubleshooting guide, see references/troubleshooting_guide.md.

Suggested Next Steps

After completing TWAS analysis:

1. Functional validation: Cross-reference with expression data, protein-protein interaction networks
2. Pathway enrichment: Use functional-enrichment-from-degs on prioritized genes
3. Variant annotation: Link causal variants to regulatory elements with genetic-variant-annotation
4. Portfolio prioritization: Rank targets by genetic evidence strength, druggability, and clinical precedent
5. Literature review: Check PubMed for supporting functional studies on top genes

Related Skills

Prerequisites:

• gwas-genotype-qc - Quality control for raw genotype data
• gwas-heritability-ldsc - Tissue enrichment analysis for tissue selection

Downstream analyses:

• functional-enrichment-from-degs - Pathway analysis for gene lists
• genetic-variant-annotation - Functional annotation of causal variants

Alternatives:

• SMR (Summary-data-based MR) - Alternative TWAS approach with built-in MR
• FOCUS (Fine-mapping Of CaUsal gene Sets) - Fine-mapping extension for TWAS

References

Internal documentation:

• references/ldsc_qc_guidelines.md - LDSC intercept interpretation
• references/tissue_reference_guide.md - Trait-tissue mappings (GTEx v8)
• references/fusion_best_practices.md - FUSION usage guidelines
• references/spredixxcan_best_practices.md - S-PrediXcan/S-MultiXcan guidelines
• references/therapeutic_interpretation_guide.md - Directionality → drug target framework
• references/mendelian_randomization_guide.md - MR principles and assumptions
• references/twas_hub_validation_guide.md - Comparison with published TWAS
• references/troubleshooting_guide.md - Common issues and solutions
• references/installation-guide.md - Detailed setup instructions

Key papers:

• FUSION: Gusev A, et al. (2016) Nat Genet 48:245-252. https://doi.org/10.1038/ng.3506
• S-PrediXcan: Barbeira AN, et al. (2018) Nat Commun 9:1825. https://doi.org/10.1038/s41467-018-03621-1
• S-MultiXcan: Barbeira AN, et al. (2019) PLoS Genet 15:e1007889
• Colocalization: Giambartolomei C, et al. (2014) PLoS Genet 10:e1004383
• MR: Hemani G, et al. (2018) eLife 7:e34408

Online resources:

• FUSION: http://gusevlab.org/projects/fusion/
• MetaXcan: https://github.com/hakyimlab/MetaXcan
• PredictDB: https://predictdb.org/ (GTEx v8 weights)
• GTEx Portal: https://gtexportal.org/
• TWAS Hub: http://twas-hub.org/ (published TWAS results)
• MR-Base: https://www.mrbase.org/