mtDNA pipelines

The bundled cbicall-core mtDNA workflows use mtDNA-only BAMs exported by native GATK 4.6 WES/WGS single-sample runs. MToolBox then calls, annotates, and prioritizes mtDNA variants.

Mode	Use
single	Analyze one individual.
cohort	Analyze all eligible samples in one project together.

Architecture

The bundled MToolBox workflow supports x86_64 Linux only.

Required input

Run WES/WGS single-sample processing with export_mtdna_bam: true first. mtDNA workflows require exports/mtdna/<sample>-DNA_MIT.bam and its index; they do not extract reads from a full BAM.

The exported BAM inherits mitochondrial read placement from the preceding BWA-MEM alignment. CBIcall uses a fixed BWA input batch size so changing the workflow thread count does not change which reads are passed to MToolBox. See the WES/WGS single-sample pipeline for details.

Single sample

Single-sample workflow

Set input_dir to a sample directory containing a completed native WES/WGS single-sample run with an exported mtDNA BAM. CBIcall copies that BAM into the MToolBox workspace and appends genotype (GT), depth (DP), and heteroplasmy fraction values to the prioritized report.

mode:             single
pipeline:         mit
workflow_backend: bash
software_stack:   gatk-3.5
input_dir:        CNAG999_exome/CNAG99901P_ex

Cohort

Cohort workflow

Set input_dir to a project directory containing the sample directories and their completed native WES/WGS single-sample runs. Each run must contain its exported mtDNA BAM and index. CBIcall collects these files and runs MToolBox jointly.

mode:             cohort
pipeline:         mit
workflow_backend: bash
software_stack:   gatk-3.5
input_dir:        CNAG999_exome

Use cohort mode for a family, maternal-lineage analysis, or a project-level mtDNA table. A WES/WGS cohort run is not required beforehand.

Outputs

Both modes produce the same principal artifacts:

File	Description
01_mtoolbox/mit_prioritized_variants.txt	Annotated variants with CBIcall-added GT, DP, and heteroplasmy values.
01_mtoolbox/VCF_file.vcf	mtDNA VCF from MToolBox.
01_mtoolbox/mt_classification_best_results.csv	Predicted mitochondrial haplogroups.
01_mtoolbox/mit.filtered.json	Canonical filtered records used to generate the browser.
02_browser/<run-id>.html	Standalone interactive mtDNA report.

Haplogroup assignments

When the available mtDNA sequence does not distinguish a single haplogroup, MToolBox may report multiple candidate assignments. CBIcall preserves these assignments and does not select among them.

See the mtDNA end-to-end example for the complete run procedure and browser screenshots.

Implementation details and source files

The workflow builds on MToolBox v1.0. It converts and prepares the input BAM, aligns mitochondrial reads to RSRS, calls variants, predicts haplogroups, and performs functional annotation and prioritization. CBIcall then creates the canonical filtered JSON and standalone browser report.

• Single-sample Bash workflow
• Cohort Bash workflow
• 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). Article