GWAS Pipeline

Complete workflow for genome-wide association studies from genotype data to significant associations.

Workflow Overview

VCF/PLINK files
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    v
[1. QC Filtering] ------> Sample and variant QC
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    v
[2. LD Pruning] --------> Independent variants for PCA
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    v
[3. Population Structure] --> PCA for covariates
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    v
[4. Association Testing] --> Logistic/linear regression
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    v
[5. Results] -----------> Manhattan plot, QQ plot
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    v
Significant associations

Step 1: Data Import and QC

Convert VCF to PLINK

# VCF to PLINK binary format
plink2 --vcf input.vcf.gz \
    --make-bed \
    --out study

# Or with phenotype/covariate files
plink2 --vcf input.vcf.gz \
    --pheno phenotypes.txt \
    --make-bed \
    --out study

Sample QC

# Calculate sample statistics
plink2 --bfile study \
    --missing \
    --out study_stats

# Remove samples with high missing rate (>5%)
plink2 --bfile study \
    --mind 0.05 \
    --make-bed \
    --out study_sample_qc

# Check for sex discrepancies (if sex chromosome data available)
plink2 --bfile study_sample_qc \
    --check-sex \
    --out study_sex_check

# Remove related individuals (optional, requires IBD)
plink2 --bfile study_sample_qc \
    --king-cutoff 0.0884 \
    --make-bed \
    --out study_unrelated

Variant QC

# Apply standard variant filters
plink2 --bfile study_sample_qc \
    --geno 0.05 \
    --maf 0.01 \
    --hwe 1e-6 \
    --make-bed \
    --out study_qc

# Summary
plink2 --bfile study_qc --freq --out study_qc

QC Checkpoint:

• Sample call rate >95%
• Variant call rate >95%
• MAF >1%
• HWE p-value >1e-6 (controls only for case-control)

Step 2: LD Pruning for PCA

# Identify independent variants
plink2 --bfile study_qc \
    --indep-pairwise 50 5 0.2 \
    --out pruned

# Extract pruned variants
plink2 --bfile study_qc \
    --extract pruned.prune.in \
    --make-bed \
    --out study_pruned

Step 3: Population Structure (PCA)

# Calculate principal components
plink2 --bfile study_pruned \
    --pca 10 \
    --out study_pca

# The eigenvec file contains PCs for use as covariates

Visualize PCA

library(ggplot2)

# Load PCA results
pca <- read.table('study_pca.eigenvec', header = FALSE)
colnames(pca) <- c('FID', 'IID', paste0('PC', 1:10))

# Load phenotype for coloring
pheno <- read.table('phenotypes.txt', header = TRUE)
pca <- merge(pca, pheno, by = c('FID', 'IID'))

# Plot
ggplot(pca, aes(x = PC1, y = PC2, color = as.factor(PHENO))) +
    geom_point(alpha = 0.5) +
    labs(title = 'PCA of Study Samples', color = 'Phenotype') +
    theme_minimal()
ggsave('pca_plot.pdf', width = 8, height = 6)

Step 4: Association Testing

Case-Control (Binary Trait)

# Logistic regression with PCA covariates
plink2 --bfile study_qc \
    --pheno phenotypes.txt \
    --covar study_pca.eigenvec \
    --covar-col-nums 3-12 \
    --glm hide-covar \
    --out gwas_results

# Results in gwas_results.PHENO.glm.logistic

Quantitative Trait

# Linear regression
plink2 --bfile study_qc \
    --pheno phenotypes.txt \
    --pheno-name BMI \
    --covar study_pca.eigenvec \
    --covar-col-nums 3-12 \
    --glm hide-covar \
    --out gwas_bmi

# Results in gwas_bmi.BMI.glm.linear

With Additional Covariates

# Include age, sex, and PCs
plink2 --bfile study_qc \
    --pheno phenotypes.txt \
    --covar covariates.txt \
    --covar-name AGE,SEX,PC1-PC10 \
    --glm hide-covar \
    --out gwas_adjusted

Step 5: Results Visualization

Manhattan Plot

library(qqman)

# Load results
results <- read.table('gwas_results.PHENO.glm.logistic', header = TRUE)
results <- results[!is.na(results$P),]

# Manhattan plot
png('manhattan.png', width = 1200, height = 600)
manhattan(results, chr = 'X.CHROM', bp = 'POS', snp = 'ID', p = 'P',
          suggestiveline = -log10(1e-5), genomewideline = -log10(5e-8))
dev.off()

# QQ plot
png('qq_plot.png', width = 600, height = 600)
qq(results$P)
dev.off()

Calculate Genomic Inflation

# Lambda (genomic inflation factor)
chisq <- qchisq(1 - results$P, 1)
lambda <- median(chisq) / qchisq(0.5, 1)
cat('Lambda:', round(lambda, 3), '\n')
# Lambda should be close to 1.0 (1.0-1.1 acceptable)

Extract Significant Hits

# Genome-wide significant (p < 5e-8)
awk '$12 < 5e-8' gwas_results.PHENO.glm.logistic > significant_hits.txt

# Suggestive (p < 1e-5)
awk '$12 < 1e-5' gwas_results.PHENO.glm.logistic > suggestive_hits.txt

Parameter Recommendations

Step	Parameter	Value
Sample QC	--mind	0.05
Variant QC	--geno	0.05
Variant QC	--maf	0.01
Variant QC	--hwe	1e-6
LD pruning	--indep-pairwise	50 5 0.2
PCA	--pca	10
Significance	p-value	5e-8

Troubleshooting

Issue	Likely Cause	Solution
High lambda (>1.1)	Population stratification	Add more PCs, check ancestry
No significant hits	Low power	Increase sample size, meta-analysis
Deflated lambda (<1)	Over-correction	Reduce PC covariates
QQ deviation at low end	Batch effects	Check for technical artifacts

Complete Pipeline Script

#!/bin/bash
set -e

INPUT_VCF="genotypes.vcf.gz"
PHENO="phenotypes.txt"
OUTDIR="gwas_results"

mkdir -p ${OUTDIR}

# Step 1: Convert and QC
plink2 --vcf ${INPUT_VCF} --make-bed --out ${OUTDIR}/raw
plink2 --bfile ${OUTDIR}/raw --mind 0.05 --geno 0.05 --maf 0.01 --hwe 1e-6 \
    --make-bed --out ${OUTDIR}/qc

# Step 2: LD pruning
plink2 --bfile ${OUTDIR}/qc --indep-pairwise 50 5 0.2 --out ${OUTDIR}/pruned
plink2 --bfile ${OUTDIR}/qc --extract ${OUTDIR}/pruned.prune.in \
    --make-bed --out ${OUTDIR}/pruned

# Step 3: PCA
plink2 --bfile ${OUTDIR}/pruned --pca 10 --out ${OUTDIR}/pca

# Step 4: Association
plink2 --bfile ${OUTDIR}/qc --pheno ${PHENO} \
    --covar ${OUTDIR}/pca.eigenvec --covar-col-nums 3-12 \
    --glm hide-covar --out ${OUTDIR}/gwas

echo "=== GWAS Complete ==="
echo "Results: ${OUTDIR}/gwas.*.glm.*"

Related Skills

• population-genetics/plink-basics - PLINK file formats and commands
• population-genetics/population-structure - PCA and admixture
• population-genetics/association-testing - Statistical models
• population-genetics/linkage-disequilibrium - LD concepts