genomics-pipeline

Step 20: Mitochondrial Variant Calling and Heteroplasmy Detection

What This Does

Calls mitochondrial DNA variants with heteroplasmy fractions — detecting variants present in only a fraction of your mitochondrial copies. Uses GATK Mutect2 in mitochondrial mode.

Why

Step 12 (haplogrep3) assigns your mitochondrial haplogroup from chrM variants already in the main VCF. This step goes deeper:

• Heteroplasmy detection: Identifies variants present in only a fraction of mtDNA copies (clinically important for mitochondrial diseases)
• Dedicated mitochondrial calling: Mutect2’s mitochondrial mode handles the unique properties of mtDNA (high copy number, circular genome, no recombination)
• Somatic-grade sensitivity: Detects variants at allele fractions as low as 1-3%

Tool

• GATK Mutect2 (Broad Institute) in --mitochondria-mode

Note: This step was originally planned for MToolBox, but no working Docker image exists for MToolBox (see lessons-learned.md). GATK Mutect2 is the standard clinical alternative.

Docker Image

broadinstitute/gatk:4.6.1.0

Command

# Extract chrM reads
samtools view -b sorted.bam chrM > chrM.bam
samtools index chrM.bam

# Call variants in mitochondrial mode
gatk Mutect2 \
  -R reference.fasta \
  -I chrM.bam \
  -L chrM \
  --mitochondria-mode \
  --max-mnp-distance 0 \
  -O chrM_mutect2.vcf.gz

# Filter
gatk FilterMutectCalls \
  -R reference.fasta \
  -V chrM_mutect2.vcf.gz \
  --mitochondria-mode \
  -O chrM_filtered.vcf.gz

Output

• ${SAMPLE}_chrM_mutect2.vcf.gz — Raw mitochondrial variant calls
• ${SAMPLE}_chrM_filtered.vcf.gz — Filtered calls with PASS/FAIL status
• Each variant includes an AF (allele fraction) field indicating heteroplasmy level

Interpreting Heteroplasmy

| AF Level | Meaning | |—|—| | >0.95 | Homoplasmic — effectively fixed variant | | 0.10-0.95 | Heteroplasmic — mixed population, clinically significant threshold varies | | 0.03-0.10 | Low-level heteroplasmy — may be age-related somatic | | <0.03 | Near detection limit |

Runtime

~15-30 minutes per sample.

Notes

• --mitochondria-mode disables several filters inappropriate for mtDNA: no germline filtering, adjusted LOD thresholds, handles high copy number.
• --max-mnp-distance 0 prevents merging nearby variants into multi-nucleotide polymorphisms.
• The GATK Docker image is large (~2.2 GB) but well-maintained and versioned.
• For disease annotation of mitochondrial variants, cross-reference with MitoMap or the Ensembl VEP output from step 13.
• Some mitochondrial diseases require heteroplasmy above a tissue-specific threshold (e.g., m.3243A>G MELAS requires >60% in blood).

This site is open source. Improve this page.