A novel Next-Generation Sequencing-based approach for concurrent detection of mitochondrial DNA copy number and mutation.

Numerous studies have identified essential contributions of altered mitochondrial DNA (mtDNA) copy number and mutations in many common disorders including cancer. To date, capture-based next-generation sequencing (NGS) has been widely applied to detect mtDNA mutations, although it lacks the ability to assess mtDNA copy number. The current strategy for quantifying mtDNA copy number relies mainly on real-time quantitative PCR, which is limited in degraded samples. A novel capture-based NGS approach was developed using both mtDNA and nuclear DNA probes to capture target fragments, enabling simultaneous detection of mtDNA mutations and copy number in different sample types. First, the impact of selecting reference genes on mtDNA copy number calculation was evaluated, and finally, 3 nuclear DNA fragments of 4000 bp were selected as an internal reference for detection. Then, the effective application of this approach was verified in DNA samples of formalin-fixed, paraffin-embedded specimens and body fluids, indicating the widespread applicability. This approach showed more accurate and stable results in detecting mtDNA copy number compared with real-time quantitative PCR in degraded DNA samples. Moreover, data indicated this approach had good reproducibility in detecting both mtDNA copy number and mutations among three sample types. Altogether, a versatile and cost-effective capture-based NGS approach has been developed for concurrent detection of mtDNA copy number and mutations, which has numerous applications in research and diagnosis.