Genetic variants of mitochondrial genome are an important driver of mitochondrial disorder pathology. Mitochondrial disorders are heterogeneous diseases characterized by a lack of cellular energy. Using targeted nanopore sequencing in combination with CRISPR/Cas9 technology enables amplification-free sequence enrichment of mitochondrial DNA (mtDNA). Nanopore sequencing produces long reads (> 10 kb), which improves de novo assembly and detection of large sporadic deletions and tandem repeats. In this study, we demonstrated that this technology enriches the mitochondrial genome and generates full-length nanopore reads of mtDNA. We tested different bioinformatics approaches for analyzing the data and compared the results of these approaches for variant calling. We verified the ability of nanopore sequencing for the detection of low-level DNA heteroplasmy, where the cell contains a low proportion of mutated mtDNA molecules compared to wild-type mtDNA. We determined that nanopore sequencing is able to detect down to 10 % heteroplasmy. Detecting a low-heteroplasmic variance is crucial for the diagnosis of mitochondrial diseases, as the bottleneck effect during oogenesis and embryogenesis can lead to the sampling and subsequent expansion of rare mutant mtDNA. Nanopore sequencing technology is also able to detect modified bases that are important for epigenetic control of gene expression. We identified the presence of 5-methylcytosines on mtDNA and observed that their frequency is low. The application of the presented approach would provide deeper understanding of mitochondrial genetics, epigenetics and consequently, of mitochondrial biology.
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