Nanomito enables amplification-free full-length analysis of mitochondrial DNA variants and deletions

Abstract Accurate analysis of mitochondrial DNA (mtDNA) requires reliable quantification of heteroplasmy and robust detection of large-scale deletions. However, routine PCR-based short-read workflows can introduce amplification bias and have limited structural resolution. Here, we developed Nanomito, an amplification-free long-read workflow for full-length sequencing of native mtDNA using Oxford Nanopore Technologies, and compared it with the routine long-range PCR-based Illumina approach in 15 retrospectively selected clinical samples previously characterized in mitochondrial diagnostics. The long-read workflow detected all clinically confirmed causal point variants and identified one low-level m.3243 A > G variant, present at 3.8% heteroplasmy by digital PCR, that was detected only among quality-filtered short-read calls. Relative to digital PCR, long-read heteroplasmy estimates showed a more stable quantitative relationship than the PCR-based short-read workflow. In deletion-positive samples, long-read sequencing confirmed the major deletions detected by routine testing with closely matching breakpoint coordinates and resolved a complex deletion spectrum in one sample. These findings support amplification-free full-length nanopore sequencing as a promising framework for integrated mtDNA variant and deletion analysis.