--> --- 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 --- Call SNPs and indels from aligned reads using bcftools.
Scanned 5/27/2026
Install via CLI
openskills install FreedomIntelligence/OpenClaw-Medical-Skills<!--
# COPYRIGHT NOTICE
# This file is part of the "Universal Biomedical Skills" project.
# Copyright (c) 2026 MD BABU MIA, PhD <md.babu.mia@mssm.edu>
# All Rights Reserved.
#
# This code is proprietary and confidential.
# Unauthorized copying of this file, via any medium is strictly prohibited.
#
# Provenance: Authenticated by MD BABU MIA
-->
---
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
---
# 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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