**5. Ultra-sensitive next-generation sequencing techniques and mitogenomics**

Whole mitochondrial genome analysis by high-throughput next-generation sequencing (NGS) techniques enables the detection of low-level heteroplasmic mtDNA variants and completely revolutionized mitogenomics in the last few years [109]. This approach has been extensively applied to different mitochondrial disorders to carefully investigate the transmission dynamics of low-level maternal germline mtDNA variants across generations [110–112]. In a comparative analysis, it has been demonstrated that Sanger sequencing is valid for quantification of heteroplasmies with more than 10% of cells/mitochondria carrying the mutation, whereas NGS is capable of reliably detecting and quantifying heteroplasmic variants down to the 1% level [113]. Recently, a massive parallel sequencing (MPS) protocol reliably quantified low frequency, large mtDNA deletions in single cells with a lower detection limit of 0.5% [114]. mtDNA NGS has been also suggested as a useful quality check of pluripotent stem cells for drug discovery and regenerative medicine purposes [115].

Conventionally, DNA variants detected in a tumor sample but not in the germline counterpart (such as peripheral blood, buccal swab or saliva) are scored as somatic (likely pathogenic) mtDNA variants, otherwise they are considered as germinal variants (likely polymorphic/ benign). High-throughput NGS approaches may unveil low-level germinal heteroplasmies having a tumoral tissue counterpart with higher heteroplasmy simply because of increased cell replication rate or random genetic drift phenomena and, therefore, without any deleterious oncogenic effect. The ultra-sensitive detection rate of NGS methods may be used to monitor even subtle shifts in the heteroplasmy levels of the tumor during time and potentially correlate them with tumor evolution [116]. Moreover, the possibility to easily analyze the circulating cell-free mtDNA isolated from plasma/serum ("liquid biopsy") or urine [117–119], may allow non-invasive serial sampling from the same patient.
