mtDNA Pipelines

These pipelines extract mitochondrial reads from exome data and run MToolBox to generate mtDNA variant calls, annotations, and heteroplasmy estimates.

There are two processing modes:

• Single-sample analysis: mit_single
• Cohort / family analysis: mit_cohort

Both consume WES single-sample outputs and assume this nomenclature.

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Choosing a Pipeline

Use Case	Pipeline	Description
Analyze one individual	mit_single	Fast, sample-specific mtDNA variant calling + HF/DP/GT extraction
Analyze a full family or cohort	mit_cohort	Joint variant calling across samples; useful for transmission and segregation checks

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Workflow Details

Single-Sample: mit_single

mtDNA Single-Sample Pipeline

Source: View source

Workflow Diagram

Summary

The mit_single pipeline processes one individual at a time.
It extracts mtDNA reads, runs MToolBox, and enriches the prioritized variants with:

• GT — genotype
• DP — depth
• HF — heteroplasmic fraction

Inputs

• Run inside directory: cbicall_bash_mit_single_*
• Needs WES single-sample directory: ../../cbicall_bash_wes_single*/01_bam/input.merged.filtered.realigned.fixed.bam
• env.sh provides:
  • REF
  • SAM path
  • MTOOLBOXDIR

Outputs

File	Description
VCF_file.vcf	mtDNA VCF from MToolBox
prioritized_variants.txt	Raw prioritized list
mit_prioritized_variants.txt	Final prioritized list with GT/DP/HF

Cohort: mit_cohort

mtDNA Cohort Pipeline

Source: View source

Workflow Diagram

Summary

mit_cohort processes all samples together, generating a joint mtDNA variant table useful for:

• Family mtDNA transmission studies
• Maternal-lineage analysis
• Variant segregation and consistency checks

Inputs

• Run inside: cbicall_bash_mit_cohort_*
• Expects WES single-sample directories: ../../??????????_ex/cbicall_bash_wes_single*/01_bam/
• env.sh defines REF, SAM, MTOOLBOXDIR

Outputs

File	Description
VCF_file.vcf	mtDNA VCF for full cohort
prioritized_variants.txt	MToolBox-prioritized list
mit_prioritized_variants.txt	Joint variant table with GT/DP/HF

When to Use Each Pipeline

Use mit_single when:

• You need results for one individual.
• You are adding or reprocessing a single relative.
• You want faster turnaround for a standalone case.

Use mit_cohort when:

• You want a joint variant table across multiple individuals.
• You are analyzing mtDNA inheritance within a family.
• You need a cohort-level mtDNA table for downstream review or comparison.

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Background Information

CBIcall mtDNA builds on MToolbox v1.0 and performs:

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1. Preprocessing (PicardTools)

Converts BAM → FASTQ using:

• SortSam.jar
• MarkDuplicates.jar
• SamFormatConverter.jar (PicardTools)

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2. Alignment

• Aligns reads to RSRS via mapExome.py
• Uses GSNAP (2015-12-31.v7)

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3. Variant Calling & Annotation (MToolBox)

Pipeline steps include:

• mpileup (SAMtools)
• mtVariantCaller.py
• VCFoutput.py (with PyVCF)
• mt-classifier.py (haplogroup prediction)
• variants_functional_annotation.py
• prioritization.py
• summary.py

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Reference

1. Calabrese C. et al.
   MToolBox: a highly automated pipeline for heteroplasmy annotation and prioritization analysis of human mitochondrial variants in high-throughput sequencing.
   Bioinformatics (2014).
   Read paper