bio-variant-calling-joint-calling

name	bio-variant-calling-joint-calling
description	Joint genotype calling across multiple samples using GATK CombineGVCFs and GenotypeGVCFs. Essential for cohort studies, population genetics, and leveraging VQSR. Use when performing joint genotyping across multiple samples.
tool_type	cli
primary_tool	GATK

Joint Calling

Call variants jointly across multiple samples for improved accuracy and consistent genotyping.

Why Joint Calling?

• Improved sensitivity - Leverage information across samples
• Consistent genotyping - Same sites called across all samples
• VQSR eligible - Requires cohort for machine learning filtering
• Population analysis - Allele frequencies across cohort

Workflow Overview

Sample BAMs
    │
    ├── HaplotypeCaller (per-sample, -ERC GVCF)
    │   └── sample1.g.vcf.gz, sample2.g.vcf.gz, ...
    │
    ├── CombineGVCFs or GenomicsDBImport
    │   └── Combine into cohort database
    │
    ├── GenotypeGVCFs
    │   └── Joint genotyping
    │
    └── VQSR or Hard Filtering
        └── Final VCF

Step 1: Per-Sample gVCF Generation

# Generate gVCF for each sample
gatk HaplotypeCaller \
    -R reference.fa \
    -I sample1.bam \
    -O sample1.g.vcf.gz \
    -ERC GVCF

# With intervals (faster)
gatk HaplotypeCaller \
    -R reference.fa \
    -I sample1.bam \
    -O sample1.g.vcf.gz \
    -ERC GVCF \
    -L intervals.bed

Batch Processing

# Process all samples
for bam in *.bam; do
    sample=$(basename $bam .bam)
    gatk HaplotypeCaller \
        -R reference.fa \
        -I $bam \
        -O ${sample}.g.vcf.gz \
        -ERC GVCF &
done
wait

Step 2a: CombineGVCFs (Small Cohorts)

For <100 samples:

gatk CombineGVCFs \
    -R reference.fa \
    -V sample1.g.vcf.gz \
    -V sample2.g.vcf.gz \
    -V sample3.g.vcf.gz \
    -O cohort.g.vcf.gz

From Sample Map

# Create sample map file
# sample1    /path/to/sample1.g.vcf.gz
# sample2    /path/to/sample2.g.vcf.gz

ls *.g.vcf.gz | while read f; do
    echo -e "$(basename $f .g.vcf.gz)\t$f"
done > sample_map.txt

# Combine with -V for each
gatk CombineGVCFs \
    -R reference.fa \
    $(cat sample_map.txt | cut -f2 | sed 's/^/-V /') \
    -O cohort.g.vcf.gz

Step 2b: GenomicsDBImport (Large Cohorts)

For >100 samples, use GenomicsDB:

# Create sample map
ls *.g.vcf.gz | while read f; do
    echo -e "$(basename $f .g.vcf.gz)\t$f"
done > sample_map.txt

# Import to GenomicsDB (per chromosome for parallelism)
gatk GenomicsDBImport \
    --sample-name-map sample_map.txt \
    --genomicsdb-workspace-path genomicsdb_chr1 \
    -L chr1 \
    --reader-threads 4

# Or all chromosomes
for chr in {1..22} X Y; do
    gatk GenomicsDBImport \
        --sample-name-map sample_map.txt \
        --genomicsdb-workspace-path genomicsdb_chr${chr} \
        -L chr${chr} &
done
wait

Update GenomicsDB with New Samples

gatk GenomicsDBImport \
    --genomicsdb-update-workspace-path genomicsdb_chr1 \
    --sample-name-map new_samples.txt \
    -L chr1

Step 3: GenotypeGVCFs

From Combined gVCF

gatk GenotypeGVCFs \
    -R reference.fa \
    -V cohort.g.vcf.gz \
    -O cohort.vcf.gz

From GenomicsDB

gatk GenotypeGVCFs \
    -R reference.fa \
    -V gendb://genomicsdb_chr1 \
    -O chr1.vcf.gz

# All chromosomes
for chr in {1..22} X Y; do
    gatk GenotypeGVCFs \
        -R reference.fa \
        -V gendb://genomicsdb_chr${chr} \
        -O chr${chr}.vcf.gz &
done
wait

# Merge chromosomes
bcftools concat chr{1..22}.vcf.gz chrX.vcf.gz chrY.vcf.gz \
    -Oz -o cohort.vcf.gz

With Allele-Specific Annotations

gatk GenotypeGVCFs \
    -R reference.fa \
    -V gendb://genomicsdb \
    -O cohort.vcf.gz \
    -G StandardAnnotation \
    -G AS_StandardAnnotation

Step 4: Filtering

VQSR (Recommended for >30 Samples)

# SNPs
gatk VariantRecalibrator \
    -R reference.fa \
    -V cohort.vcf.gz \
    --resource:hapmap,known=false,training=true,truth=true,prior=15.0 hapmap.vcf.gz \
    --resource:omni,known=false,training=true,truth=false,prior=12.0 omni.vcf.gz \
    --resource:1000G,known=false,training=true,truth=false,prior=10.0 1000G.vcf.gz \
    --resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp.vcf.gz \
    -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR \
    -mode SNP \
    -O snps.recal \
    --tranches-file snps.tranches

gatk ApplyVQSR \
    -R reference.fa \
    -V cohort.vcf.gz \
    --recal-file snps.recal \
    --tranches-file snps.tranches \
    -mode SNP \
    --truth-sensitivity-filter-level 99.5 \
    -O cohort.snps.vcf.gz

# Indels
gatk VariantRecalibrator \
    -R reference.fa \
    -V cohort.snps.vcf.gz \
    --resource:mills,known=false,training=true,truth=true,prior=12.0 mills.vcf.gz \
    --resource:dbsnp,known=true,training=false,truth=false,prior=2.0 dbsnp.vcf.gz \
    -an QD -an MQRankSum -an ReadPosRankSum -an FS -an SOR \
    -mode INDEL \
    -O indels.recal \
    --tranches-file indels.tranches

gatk ApplyVQSR \
    -R reference.fa \
    -V cohort.snps.vcf.gz \
    --recal-file indels.recal \
    --tranches-file indels.tranches \
    -mode INDEL \
    --truth-sensitivity-filter-level 99.0 \
    -O cohort.filtered.vcf.gz

Hard Filtering (Small Cohorts)

# See filtering-best-practices skill
gatk VariantFiltration \
    -R reference.fa \
    -V cohort.vcf.gz \
    --filter-expression "QD < 2.0" --filter-name "QD2" \
    --filter-expression "FS > 60.0" --filter-name "FS60" \
    --filter-expression "MQ < 40.0" --filter-name "MQ40" \
    -O cohort.filtered.vcf.gz

Complete Pipeline Script

#!/bin/bash
set -euo pipefail

REFERENCE=$1
OUTPUT_DIR=$2
THREADS=16

mkdir -p $OUTPUT_DIR/{gvcfs,genomicsdb,vcfs}

echo "=== Step 1: Generate gVCFs ==="
for bam in data/*.bam; do
    sample=$(basename $bam .bam)
    gatk HaplotypeCaller \
        -R $REFERENCE \
        -I $bam \
        -O $OUTPUT_DIR/gvcfs/${sample}.g.vcf.gz \
        -ERC GVCF &

    # Limit parallelism
    while [ $(jobs -r | wc -l) -ge $THREADS ]; do sleep 1; done
done
wait

echo "=== Step 2: Create sample map ==="
ls $OUTPUT_DIR/gvcfs/*.g.vcf.gz | while read f; do
    echo -e "$(basename $f .g.vcf.gz)\t$(realpath $f)"
done > $OUTPUT_DIR/sample_map.txt

echo "=== Step 3: GenomicsDBImport ==="
gatk GenomicsDBImport \
    --sample-name-map $OUTPUT_DIR/sample_map.txt \
    --genomicsdb-workspace-path $OUTPUT_DIR/genomicsdb \
    -L intervals.bed \
    --reader-threads 4

echo "=== Step 4: Joint genotyping ==="
gatk GenotypeGVCFs \
    -R $REFERENCE \
    -V gendb:///genomicsdb \
    -O /vcfs/cohort.vcf.gz

 
bcftools index -t /vcfs/cohort.vcf.gz

 
bcftools stats /vcfs/cohort.vcf.gz > /vcfs/cohort_stats.txt

 
 

Tips

Memory for Large Cohorts

# Increase Java heap
gatk --java-options "-Xmx64g" GenotypeGVCFs ...

# Batch size for GenomicsDBImport
gatk GenomicsDBImport --batch-size 50 ...

Incremental Updates

# Add new samples to existing database
gatk GenomicsDBImport \
    --genomicsdb-update-workspace-path existing_db \
    --sample-name-map new_samples.txt

Related Skills

• variant-calling/gatk-variant-calling - Single-sample calling
• variant-calling/filtering-best-practices - VQSR and hard filtering
• population-genetics/plink-basics - Population analysis of joint calls
• workflows/fastq-to-variants - End-to-end germline pipeline