variant-calling

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name: bio-variant-calling description: Call SNPs and indels from aligned reads using bcftools mpileup and call. Use when detecting variants from BAM files or generating VCF from alignments. tool_type: cli primary_tool: bcftools measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes. allowed-tools:

• read_file
• run_shell_command

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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

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

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
-m	Multiallelic caller	Default, recommended
-c	Consensus 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

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
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

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

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.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