Abstract
Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both short-read NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA.