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This file is part of the "Universal Biomedical Skills" project.

Copyright (c) 2026 MD BABU MIA, PhD <md.babu.mia@mssm.edu>

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Variant Calling

Call SNPs and indels from aligned reads using bcftools.

Basic Workflow

BAM file + Reference FASTA
         |
         v
   bcftools mpileup (generate pileup)
         |
         v
   bcftools call (call variants)
         |
         v
   VCF file

bcftools mpileup + call

Basic Variant Calling

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -o variants.vcf

Output Compressed VCF

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -Oz -o variants.vcf.gz
bcftools index variants.vcf.gz

Call Specific Region

bash
bcftools mpileup -f reference.fa -r chr1:1000000-2000000 input.bam | \
    bcftools call -mv -o region.vcf

Call from Multiple BAMs

bash
bcftools mpileup -f reference.fa sample1.bam sample2.bam sample3.bam | \
    bcftools call -mv -o variants.vcf

BAM List File

bash
# bams.txt: one BAM path per line
bcftools mpileup -f reference.fa -b bams.txt | bcftools call -mv -o variants.vcf

mpileup Options

Quality Filtering

bash
bcftools mpileup -f reference.fa \
    -q 20 \           # Min mapping quality
    -Q 20 \           # Min base quality
    input.bam | bcftools call -mv -o variants.vcf

Annotate with Read Depth

bash
bcftools mpileup -f reference.fa -a DP,AD input.bam | bcftools call -mv -o variants.vcf

Full Annotation Set

bash
bcftools mpileup -f reference.fa \
    -a FORMAT/DP,FORMAT/AD,FORMAT/ADF,FORMAT/ADR,INFO/AD \
    input.bam | bcftools call -mv -o variants.vcf

Target Regions (BED)

bash
bcftools mpileup -f reference.fa -R targets.bed input.bam | \
    bcftools call -mv -o variants.vcf

Max Depth

bash
bcftools mpileup -f reference.fa -d 1000 input.bam | bcftools call -mv -o variants.vcf

call Options

Calling Models

| Flag | Model | Use Case | |------|-------|----------| | -m | Multiallelic caller | Default, recommended | | -c | Consensus caller | Legacy, single sample |

Output Variants Only

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -o variants.vcf
# -v outputs variant sites only (not reference calls)

Output All Sites

bash
bcftools mpileup -f reference.fa input.bam | bcftools call -m -o all_sites.vcf
# Without -v, outputs all sites including reference

Ploidy

bash
# Haploid calling
bcftools mpileup -f reference.fa input.bam | bcftools call -m --ploidy 1 -o variants.vcf

# Specify ploidy file
bcftools mpileup -f reference.fa input.bam | bcftools call -m --ploidy-file ploidy.txt -o variants.vcf

Prior Probability

bash
# Adjust variant prior (default 1.1e-3)
bcftools mpileup -f reference.fa input.bam | bcftools call -m -P 0.001 -o variants.vcf

Common Pipelines

Standard SNP/Indel Calling

bash
bcftools mpileup -Ou -f reference.fa \
    -q 20 -Q 20 \
    -a FORMAT/DP,FORMAT/AD \
    input.bam | \
bcftools call -mv -Oz -o variants.vcf.gz

bcftools index variants.vcf.gz

Multi-sample Calling

bash
bcftools mpileup -Ou -f reference.fa \
    -a FORMAT/DP,FORMAT/AD \
    sample1.bam sample2.bam sample3.bam | \
bcftools call -mv -Oz -o cohort.vcf.gz

bcftools index cohort.vcf.gz

Calling with Regions

bash
bcftools mpileup -Ou -f reference.fa \
    -R targets.bed \
    -a FORMAT/DP,FORMAT/AD \
    input.bam | \
bcftools call -mv -Oz -o targets.vcf.gz

Parallel by Chromosome

bash
for chr in chr1 chr2 chr3; do
    bcftools mpileup -Ou -f reference.fa -r "$chr" input.bam | \
        bcftools call -mv -Oz -o "${chr}.vcf.gz" &
done
wait

# Concatenate results
bcftools concat -Oz -o all.vcf.gz chr*.vcf.gz
bcftools index all.vcf.gz

Annotation Tags

INFO Tags

| Tag | Description | |-----|-------------| | DP | Total read depth | | AD | Allelic depths | | MQ | Mapping quality | | FS | Fisher strand bias | | SGB | Segregation based metric |

FORMAT Tags

| Tag | Description | |-----|-------------| | GT | Genotype | | DP | Read depth per sample | | AD | Allelic depths per sample | | ADF | Forward strand allelic depths | | ADR | Reverse strand allelic depths | | GQ | Genotype quality | | PL | Phred-scaled likelihoods |

Request Specific Annotations

bash
bcftools mpileup -f reference.fa \
    -a FORMAT/DP,FORMAT/AD,FORMAT/SP,INFO/AD \
    input.bam | bcftools call -mv -o variants.vcf

Performance Options

Multi-threading

bash
bcftools mpileup -f reference.fa --threads 4 input.bam | \
    bcftools call -mv --threads 4 -o variants.vcf

Uncompressed BCF for Speed

bash
bcftools mpileup -Ou -f reference.fa input.bam | bcftools call -mv -Ou | \
    bcftools filter -Oz -o filtered.vcf.gz

Quick Reference

| Task | Command | |------|---------| | Basic calling | bcftools mpileup -f ref.fa in.bam \| bcftools call -mv -o out.vcf | | With quality filter | bcftools mpileup -f ref.fa -q 20 -Q 20 in.bam \| bcftools call -mv | | Region | bcftools mpileup -f ref.fa -r chr1:1-1000 in.bam \| bcftools call -mv | | Multi-sample | bcftools mpileup -f ref.fa s1.bam s2.bam \| bcftools call -mv | | With annotations | bcftools mpileup -f ref.fa -a DP,AD in.bam \| bcftools call -mv |

Common Errors

| Error | Cause | Solution | |-------|-------|----------| | no FASTA reference | Missing -f | Add -f reference.fa | | reference mismatch | Wrong reference | Use same reference as alignment | | no variants called | Low quality/depth | Lower quality thresholds |

Related Skills

• vcf-basics - View and query resulting VCF
• filtering-best-practices - Filter variants by quality
• variant-normalization - Normalize indels
• alignment-files/pileup-generation - Alternative pileup generation

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