Human Mitochondrial DNA Sequencing Service — Full-Length, PacBio Revio and ONT PromethION Platform

Human mitochondrial DNA (mtDNA) is a 16,569 bp circular molecule present in hundreds to thousands of copies per cell, and its variant landscape—single nucleotide variants, small indels, single large-scale deletions (SLSMD), and multiple deletions (MMD)—is central to diagnosing and studying primary mitochondrial disease. CD Genomics' human mitochondrial DNA sequencing service uses long-read, amplification-free sequencing on the PacBio Revio system, with ONT PromethION available as an alternative or complementary platform, to read native full-length mtDNA molecules directly from total genomic DNA.

By skipping long-range PCR, our workflow avoids the amplification bias that limits conventional short-read mtDNA testing, capturing single nucleotide variants, small indels, large-scale deletions, and deletion heteroplasmy from the same dataset—without a second orthogonal assay such as ddPCR. This gives research teams a single, comprehensive readout of the mitochondrial genome for translational and non-clinical research programs.

Why researchers choose our mtDNA sequencing service

• Amplification-free, full-length mtDNA sequencing—no long-range PCR bias
• Simultaneous SNV, indel, and large-scale deletion detection in one workflow
• Deletion heteroplasmy quantification without a secondary ddPCR assay
• Precise deletion breakpoint mapping, including microhomology analysis
• Choice of PacBio Revio or ONT PromethION depending on project needs

Introduction

Mitochondrial DNA instability and heteroplasmy—the coexistence of wild-type and variant mtDNA copies within a cell—underlie a wide range of research areas, from primary mitochondrial disease to aging and neurodegeneration. Conventional workflows rely on long-range PCR (LR-PCR) to enrich full-length mtDNA before short-read sequencing (SRS). This amplification step introduces bias, obscures deletion breakpoints in samples with multiple large-scale deletions (MMD), and cannot on its own quantify deletion heteroplasmy, typically requiring a follow-up Droplet Digital PCR (ddPCR) assay. Long-read sequencing removes this bottleneck by reading native mtDNA molecules end to end, directly from total genomic DNA.

Sequencing Platforms We Use

CD Genomics runs human mtDNA sequencing on two current-generation long-read platforms, selected based on project scale, sample type, and analysis goals.

Both platforms are offered as amplification-free, full-length mtDNA workflows. Our team will help you choose the platform—or combination—that best matches your sample type, cohort size, and heteroplasmy sensitivity requirements. Structural variants outside the mitochondrial genome can also be assessed through our human genome structural variation detection service.

Applications of mtDNA Sequencing

Primary Mitochondrial Disease Research

• Detect and differentiate single large-scale deletions (SLSMD) from multiple mtDNA deletions (MMD) in muscle and blood samples.
• Quantify deletion heteroplasmy directly from long reads, including low-level deletions difficult to resolve with ddPCR.
• Map precise deletion breakpoints and assess microhomology to inform deletion-formation mechanisms.

SNV and Small Indel Profiling

• Identify mtDNA single nucleotide variants and small indels across the full mitochondrial genome in a single run.
• Support variant-of-uncertain-significance follow-up with orthogonal long-read evidence.

Aging, Neurodegeneration, and Tissue-Specific Studies

• Track accumulation of common deletions (such as the well-characterized 4977 bp deletion) across tissues or age groups.
• Compare mtDNA content and variant burden between tissue types with differing mitochondrial density.

Method Development and Cohort Studies

• Benchmark amplification-free long-read approaches against existing LR-PCR/SRS/ddPCR pipelines.
• Scale to multi-sample cohorts on PromethION for population-level mtDNA variant surveys.

Bioinformatics Analysis

Sample Requirements

Case Study: PacBio Long-Read Sequencing for mtDNA Deletion and Heteroplasmy

Jadhav, T., Aruta, M., Diaz-Miranda, M.A. et al. Evaluation of PacBio Long-Read and PCR-Based Short-Read Sequencing for Mitochondrial DNA (mtDNA) Variant Detection, with an Emphasis on Detection and Quantification of mtDNA Deletion. International Journal of Molecular Sciences 27(8), 3562 (2026).

1. Background

Conventional clinical mtDNA testing relies on long-range PCR followed by short-read sequencing (SRS), which can detect deletions but cannot accurately quantify deletion heteroplasmy without a secondary Droplet Digital PCR (ddPCR) assay, and struggles to resolve breakpoints in samples carrying multiple co-occurring deletions.

This study evaluated whether amplification-free PacBio long-read sequencing (LRS), applied directly to total genomic DNA, could deliver SNV detection, large-scale deletion detection, and heteroplasmy quantification in a single assay.

2. Methods

Seventeen samples—4 single large-scale mtDNA deletion (SLSMD) cases, 9 multiple mtDNA deletion (MMD) cases, and 4 deletion-negative controls, from blood and muscle—were sequenced on a PacBio Sequel IIe system without PCR amplification. The workflow included:

• Total genomic DNA extraction and shearing, without mtDNA-targeted amplification
• SMRTbell library preparation and HiFi long-read sequencing
• DBSCAN-based breakpoint clustering to classify samples as SLSMD or MMD
• LoFreq and Mutect2 variant calling for SNVs and small indels
• Deletion heteroplasmy quantification from the proportion of deletion-containing reads, benchmarked against ddPCR

Results were compared against each sample's existing clinical LR-PCR/SRS and ddPCR data.

3. Results

PacBio LRS detected large-scale deletions in all four SLSMD samples and in seven of nine MMD samples, with breakpoints consistent with prior SRS-based findings. Deletion heteroplasmy computed from LRS correlated strongly with ddPCR estimates, with a Pearson correlation coefficient of 0.94 across all deletion samples—0.98 for SLSMD samples and 0.83 for MMD samples.

Coverage across the mitogenome and circos-style deletion breakpoint maps for control, SLSMD, and MMD samples, with the common 4977 bp deletion highlighted.

Key Findings

• High-confidence SNV detection:
• SNV calling using LoFreq achieved 100% sensitivity, specificity, precision, and recall for variants above 10% heteroplasmy across all four control samples.
• Reliable low-heteroplasmy detection:
• Raw long-read data captured SNVs at approximately 5% heteroplasmy, comparable to a prior LRS/SRS comparison study.
• SLSMD vs. MMD differentiation:
• Breakpoint clustering correctly classified deletion patterns, distinguishing single recurrent deletions from heterogeneous multi-deletion profiles.
• Precise breakpoint microhomology mapping:
• On average, 74.4% of deletions in MMD samples showed ≥1 bp of breakpoint microhomology, with homology lengths comparable to those reported in the MITOMAP and MitoBreak reference databases.
• Single-assay heteroplasmy quantification:
• Deletion heteroplasmy was calculated directly from LRS read counts without requiring a follow-up ddPCR assay.

4. Conclusions

This study demonstrates that amplification-free PacBio long-read sequencing provides a comprehensive, single-assay approach to human mtDNA analysis. Importantly:

• SNV and small indel detection above 10% heteroplasmy matched clinical SRS results with high accuracy
• Deletion heteroplasmy from long reads closely tracked ddPCR quantification
• SLSMD and MMD samples were reliably distinguished from breakpoint clustering patterns
• Deletion breakpoints were mapped with base-level precision, enabling microhomology analysis
• Sequencing depth of at least 300–500× across the mitogenome supported stable heteroplasmy estimates

FAQs

For Research Use Only. Not for use in diagnostic procedures.