--- 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