Variant Calling

Call SNPs and indels from aligned reads using bcftools.

Basic Workflow

code

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