Summary This source did not publish a separate summary. Review SKILL.md before using the skill.
Version Compatibility
Reference examples tested with: bcftools 1.19+
Before using code patterns, verify installed versions match. If versions differ:
CLI: <tool> --version then <tool> --help to confirm flags
If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.
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
Goal: Detect SNPs and indels from aligned reads using the bcftools pileup-and-call pipeline.
Approach: Generate per-position pileup likelihoods with mpileup, then call genotypes with the multiallelic caller.
"Call variants from my BAM file" → Generate genotype likelihoods from aligned reads and identify variant sites using a Bayesian caller.
Basic Variant Calling
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -o variants.vcf
Output Compressed VCF
bcftools mpileup -f reference.fa input.bam | bcftools call -mv -Oz -o variants.vcf.gz
bcftools index variants.vcf.gz
Call Specific Region
bcftools mpileup -f reference.fa -r chr1:1000000-2000000 input.bam | \
bcftools call -mv -o region.vcf
Call from Multiple BAMs
bcftools mpileup -f reference.fa sample1.bam sample2.bam sample3.bam | \
bcftools call -mv -o variants.vcf
BAM List File
# bams.txt: one BAM path per line
bcftools mpileup -f reference.fa -b bams.txt | bcftools call -mv -o variants.vcf
mpileup Options Goal: Control pileup generation with quality thresholds, annotations, and region restrictions.
Approach: Set minimum mapping/base quality, request specific FORMAT/INFO tags, and restrict to target regions.
Quality Filtering 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 bcftools mpileup -f reference.fa -a DP,AD input.bam | bcftools call -mv -o variants.vcf
Full Annotation Set 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) bcftools mpileup -f reference.fa -R targets.bed input.bam | \
bcftools call -mv -o variants.vcf
Max Depth bcftools mpileup -f reference.fa -d 1000 input.bam | bcftools call -mv -o variants.vcf
call Options
Calling Models Flag Model Use Case -mMultiallelic caller Default, recommended -cConsensus caller Legacy, single sample
Output Variants Only bcftools mpileup -f reference.fa input.bam | bcftools call -mv -o variants.vcf
# -v outputs variant sites only (not reference calls)
Output All Sites bcftools mpileup -f reference.fa input.bam | bcftools call -m -o all_sites.vcf
# Without -v, outputs all sites including reference
Ploidy # 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 # 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 Goal: Run production-ready variant calling workflows for single-sample and multi-sample analyses.
Approach: Chain mpileup and call with quality filters, annotations, and compressed output, optionally parallelized by chromosome.
Standard SNP/Indel Calling 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 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 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 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 DPTotal read depth ADAllelic depths MQMapping quality FSFisher strand bias SGBSegregation based metric
FORMAT Tags Tag Description GTGenotype DPRead depth per sample ADAllelic depths per sample ADFForward strand allelic depths ADRReverse strand allelic depths GQGenotype quality PLPhred-scaled likelihoods
Request Specific Annotations bcftools mpileup -f reference.fa \
-a FORMAT/DP,FORMAT/AD,FORMAT/SP,INFO/AD \
input.bam | bcftools call -mv -o variants.vcf
Performance Options Goal: Speed up variant calling for large datasets.
Approach: Use multi-threading and uncompressed BCF piping to reduce I/O overhead.
Multi-threading bcftools mpileup -f reference.fa --threads 4 input.bam | \
bcftools call -mv --threads 4 -o variants.vcf
Uncompressed BCF for Speed 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.vcfWith quality filter bcftools mpileup -f ref.fa -q 20 -Q 20 in.bam | bcftools call -mvRegion bcftools mpileup -f ref.fa -r chr1:1-1000 in.bam | bcftools call -mvMulti-sample bcftools mpileup -f ref.fa s1.bam s2.bam | bcftools call -mvWith annotations bcftools mpileup -f ref.fa -a DP,AD in.bam | bcftools call -mv
Common Errors Error Cause Solution no FASTA referenceMissing -f Add -f reference.fa reference mismatchWrong reference Use same reference as alignment no variants calledLow 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
