Two-Sample Mendelian Randomization

Two-Sample Mendelian Randomization

When to Use This Skill

• You have GWAS summary statistics for an exposure and outcome trait
• You want to test causal direction between two traits (not just correlation)
• You need to assess whether an observed association is likely causal or confounded
• You want to use genetic variants as instrumental variables (natural experiment)
• You have OpenGWAS trait IDs or your own GWAS summary statistics files

Not suitable for: One-sample MR (individual-level data), non-linear MR, multivariable MR with >2 exposures

Installation

install.packages(c("remotes", "ggplot2", "ggprism", "dplyr", "rmarkdown"))
remotes::install_github("MRCIEU/TwoSampleMR")
# For PDF report generation (optional but recommended):
# install.packages("tinytex"); tinytex::install_tinytex()
# For MR-PRESSO outlier detection (optional but recommended):
# remotes::install_github("rondolab/MR-PRESSO")

Inputs

Option A — OpenGWAS IDs (recommended):

• Exposure ID (e.g., "ieu-a-300" for LDL cholesterol)
• Outcome ID (e.g., "ieu-a-7" for coronary heart disease)
• Browse available traits at: https://gwas.mrcieu.ac.uk/

Option B — User-provided files (CSV/TSV):

• Exposure GWAS summary statistics
• Outcome GWAS summary statistics

Outputs

Results (CSV):

• mr_results.csv — MR estimates from all 4 methods (beta, SE, p-value, nSNP, F-statistics)
• heterogeneity_results.csv — Cochran's Q test for instrument heterogeneity
• pleiotropy_results.csv — MR-Egger intercept test for directional pleiotropy
• directionality_results.csv — Steiger test confirming causal direction
• harmonized_data.csv — SNP-level harmonized exposure-outcome data
• single_snp_results.csv — Per-SNP Wald ratio estimates
• leaveoneout_results.csv — Leave-one-out robustness estimates
• MR-PRESSO outlier results (if heterogeneity significant and MRPRESSO installed)

Plots (PNG + SVG):

• mr_scatter_plot — SNP-exposure vs SNP-outcome with method regression lines
• mr_forest_plot — Individual + combined SNP effect estimates
• mr_funnel_plot — Precision vs effect size (asymmetry = pleiotropy)
• mr_leaveoneout_plot — Effect stability when removing each SNP

Report:

• mr_report.pdf — Structured analysis report (Introduction, Methods, Results, Figures, Conclusions)

Analysis objects (RDS):

• mr_object.rds — Complete analysis (results, sensitivity, harmonized data)
  • Load with: mr_obj <- readRDS("mr_results/mr_object.rds")

Clarification Questions

1. Input Data (ASK THIS FIRST):

   • Do you have specific GWAS summary statistics or OpenGWAS trait IDs?
   • If files uploaded: Are these the exposure and outcome GWAS files?
   • Expected formats: CSV/TSV with SNP, beta, se, pval, effect_allele, other_allele
   • Or use example data? LDL Cholesterol → Coronary Heart Disease demo (drug-target validation example)

2. Exposure and Outcome:

   • (If using example data) Pre-set: Exposure = LDL Cholesterol, Outcome = Coronary Heart Disease — no need to specify
   • (If using your own data) What is the exposure (potential cause)? What is the outcome (potential effect)?

3. Parameters (defaults usually fine):

   • P-value threshold for instruments? (default: 5×10⁻⁸)
   • LD clumping r²? (default: 0.001)

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/mendelian-randomization-twosamplemr'))

🚨 MANDATORY: USE SCRIPTS EXACTLY AS SHOWN — DO NOT WRITE INLINE CODE 🚨

Step 1 — Load and harmonize data:

source("scripts/load_data.R")
dat <- load_example_data()
# OR: dat <- load_from_opengwas("ieu-a-300", "ieu-a-7")
# OR: dat <- load_from_files("exposure.csv", "outcome.csv")

DO NOT write inline data loading or harmonization code. Use the functions above.

✅ VERIFICATION: You MUST see "✓ Data loaded and harmonized successfully!"

Step 2 — Run MR analysis:

source("scripts/run_mr_analysis.R")
mr_results <- run_mr(dat)
sensitivity <- run_sensitivity(dat, mr_results)

DO NOT write inline MR code. Just source the script and call the functions.

✅ VERIFICATION: You MUST see "✓ MR analysis completed successfully!" AND "✓ Sensitivity analyses completed successfully!"

Step 3 — Generate visualizations:

source("scripts/mr_plots.R")
generate_all_plots(mr_results, dat, sensitivity$singlesnp, sensitivity$leaveoneout, output_dir = "mr_results")

🚨 DO NOT write inline plotting code (ggsave, ggplot, etc.). Just use the function. 🚨

✅ VERIFICATION: You MUST see "✓ All MR plots generated successfully!"

Step 4 — Export results and generate report:

source("scripts/export_results.R")
export_all(mr_results, sensitivity, dat, output_dir = "mr_results")

DO NOT write custom export code. Use export_all(). It automatically generates the PDF report.

✅ VERIFICATION: You MUST see "✓ Report generated successfully!" AND "=== Export Complete ==="

❌ IF YOU DON'T SEE VERIFICATION MESSAGES: You wrote inline code. Stop and use the scripts.

⚠️ CRITICAL — DO NOT:

• ❌ Write inline MR analysis code → STOP: Use run_mr() and run_sensitivity()
• ❌ Write inline plotting code → STOP: Use generate_all_plots()
• ❌ Write custom export code → STOP: Use export_all()
• ❌ Write custom report code → STOP: Use generate_report()
• ❌ Try to install system dependencies → Scripts handle package installation

⚠️ IF SCRIPTS FAIL — Script Failure Hierarchy:

1. Fix and Retry (90%) — Install missing R package, re-run script
2. Modify Script (5%) — Edit the script file, document changes
3. Use as Reference (4%) — Read script, adapt approach, cite source
4. Write from Scratch (1%) — Only if genuinely impossible, explain why

Common Issues

Interpreting Results

See references/interpretation-guide.md for detailed guidance.

Quick interpretation:

• Concordant methods (IVW, Egger, WM, WMode agree on direction + significance) → stronger evidence
• Any method non-significant or discordant → must be discussed explicitly, not dismissed
• IVW significant + no heterogeneity + no pleiotropy → strongest evidence
• Egger intercept p < 0.05 → directional pleiotropy may bias IVW
• High heterogeneity (Q p < 0.05) → run MR-PRESSO, flag outlier instruments
• Steiger direction incorrect → reverse causation concern (check binary outcome R² correction)
• F-statistic < 10 → weak instrument bias toward the null

Suggested Next Steps

• Multiple exposures? → Run bidirectional MR (swap exposure/outcome)
• Pleiotropy detected? → Consider MR-PRESSO or multivariable MR
• Significant result? → Replicate with independent GWAS datasets
• Drug target validation? → Use cis-MR with variants near gene of interest
• Pathway analysis? → Combine with functional enrichment skills

Related Skills

• polygenic-risk-score — Polygenic risk score computation (LDpred2)
• polygenic-risk-score-prs-catalog — PRS from pre-computed PGS Catalog weights
• eqtl-colocalization-coloc — eQTL colocalization analysis (MR follow-up)

References

• Sanderson E, et al. (2022). Mendelian randomization. Nat Rev Methods Primers. PMC7384151
• Hemani G, et al. (2018). The MR-Base platform supports systematic causal inference across the human phenome. eLife. DOI: 10.7554/eLife.34408
• TwoSampleMR package: https://github.com/MRCIEU/TwoSampleMR
• OpenGWAS database: https://gwas.mrcieu.ac.uk/
• STROBE-MR guidelines: https://doi.org/10.1001/jama.2023.1788