Clair3 Variant Calling

Basic Usage

# ONT variant calling
run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --threads=32 \
    --platform=ont \
    --model_path=${CONDA_PREFIX}/bin/models/ont \
    --output=clair3_output

# PacBio HiFi variant calling
run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --threads=32 \
    --platform=hifi \
    --model_path=${CONDA_PREFIX}/bin/models/hifi \
    --output=clair3_output

# Output: clair3_output/merge_output.vcf.gz

Platform-Specific Models

Platform	Model	Recommended Coverage
ONT R10	r1041_e82_400bps_sup_v430	30-60x
ONT R9	r941_prom_sup_g5014	30-60x
PacBio HiFi	hifi	20-40x
PacBio CLR	-	Use PEPPER-Margin-DeepVariant

# List available models
ls ${CONDA_PREFIX}/bin/models/

# Specify exact model
run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --model_path=${CONDA_PREFIX}/bin/models/r1041_e82_400bps_sup_v430 \
    --output=clair3_out \
    --threads=32

Key Parameters

Parameter	Description
--platform	ont, hifi, or ilmn
--model_path	Path to trained model
--bed_fn	Restrict calling to regions
--include_all_ctgs	Call on all contigs (not just chr1-22,X,Y)
--no_phasing_for_fa	Disable phasing
--gvcf	Output gVCF format
--qual	Minimum variant quality (default: 2)

Region-Specific Calling

# Call variants in specific regions
run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --bed_fn=target_regions.bed \
    --threads=32 \
    --platform=ont \
    --model_path=${CONDA_PREFIX}/bin/models/ont \
    --output=clair3_targeted

# Call on non-human genomes (all contigs)
run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --include_all_ctgs \
    --threads=32 \
    --platform=hifi \
    --model_path=${CONDA_PREFIX}/bin/models/hifi \
    --output=clair3_all_contigs

gVCF Output

# Generate gVCF for joint calling
run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --gvcf \
    --threads=32 \
    --platform=ont \
    --model_path=${CONDA_PREFIX}/bin/models/ont \
    --output=clair3_gvcf

# Joint genotyping multiple samples
bcftools merge sample1.g.vcf.gz sample2.g.vcf.gz -o cohort.vcf.gz

Phased Variant Calling

# With phasing information (requires haplotagged BAM)
run_clair3.sh \
    --bam_fn=haplotagged.bam \
    --ref_fn=reference.fasta \
    --enable_phasing \
    --longphase_for_phasing \
    --threads=32 \
    --platform=ont \
    --model_path=${CONDA_PREFIX}/bin/models/ont \
    --output=clair3_phased

Quality Filtering

# Filter by quality score
bcftools view -i 'QUAL>20' clair3_output/merge_output.vcf.gz -Oz -o filtered.vcf.gz

# Filter by genotype quality
bcftools view -i 'GQ>30' clair3_output/merge_output.vcf.gz -Oz -o high_gq.vcf.gz

# SNPs only
bcftools view -v snps clair3_output/merge_output.vcf.gz -Oz -o snps.vcf.gz

# Indels only
bcftools view -v indels clair3_output/merge_output.vcf.gz -Oz -o indels.vcf.gz

Python Wrapper

import subprocess
from pathlib import Path

def run_clair3(bam, reference, output_dir, platform='ont', model_path=None,
               threads=32, bed=None, gvcf=False, include_all_ctgs=False):
    if model_path is None:
        import os
        conda_prefix = os.environ.get('CONDA_PREFIX', '')
        model_path = f'{conda_prefix}/bin/models/{platform}'

    cmd = [
        'run_clair3.sh',
        f'--bam_fn={bam}',
        f'--ref_fn={reference}',
        f'--threads={threads}',
        f'--platform={platform}',
        f'--model_path={model_path}',
        f'--output={output_dir}'
    ]

    if bed:
        cmd.append(f'--bed_fn={bed}')
    if gvcf:
        cmd.append('--gvcf')
    if include_all_ctgs:
        cmd.append('--include_all_ctgs')

    subprocess.run(cmd, check=True)
    return Path(output_dir) / 'merge_output.vcf.gz'

def filter_variants(vcf, output, min_qual=20, variant_type=None):
    cmd = ['bcftools', 'view', '-i', f'QUAL>{min_qual}']
    if variant_type:
        cmd.extend(['-v', variant_type])
    cmd.extend([vcf, '-Oz', '-o', output])
    subprocess.run(cmd, check=True)
    subprocess.run(['bcftools', 'index', '-t', output], check=True)
    return output

# Example
vcf = run_clair3('sample.bam', 'ref.fa', 'clair3_out', platform='hifi', threads=48)
snps = filter_variants(str(vcf), 'snps_q20.vcf.gz', min_qual=20, variant_type='snps')

Comparison with Other Callers

Caller	Best For	Speed	Accuracy
Clair3	ONT/HiFi germline	Fast	High
DeepVariant	HiFi, Illumina	Medium	Very high
PEPPER-DV	ONT (integrated)	Slow	Very high
Longshot	ONT SNPs	Fast	Good

Troubleshooting

Issue	Solution
Missing model	Download from Clair3 releases or use conda models
Low call rate	Check coverage; increase --qual threshold
Slow performance	Reduce --threads or use --bed_fn for targeted calling
Wrong variants on non-human	Use --include_all_ctgs

Docker Usage

# Using Docker
docker run -v /data:/data \
    hkubal/clair3:latest \
    /opt/bin/run_clair3.sh \
    --bam_fn=/data/sample.bam \
    --ref_fn=/data/reference.fasta \
    --threads=32 \
    --platform=ont \
    --model_path=/opt/models/ont \
    --output=/data/clair3_output

# Singularity
singularity exec clair3.sif run_clair3.sh \
    --bam_fn=sample.bam \
    --ref_fn=reference.fasta \
    --threads=32 \
    --platform=ont \
    --model_path=/opt/models/ont \
    --output=clair3_output

Related Skills

• variant-calling/bcftools-basics - VCF manipulation
• variant-calling/filtering-best-practices - Quality filtering
• long-read-sequencing/long-read-qc - Input quality control
• long-read-sequencing/long-read-alignment - Mapping with minimap2