Methods for the identification of mitochondrial DNA variants

Abstract Detection of human mitochondrial DNA variants and quantification of heteroplasmy have quickly evolved since the discovery of the mitochondrial genome in the early 1960s. Traditionally, mitochondrial variants were studied through a combination of molecular biology techniques, often limited to the analysis of single variants. The development of Sanger sequencing led to a paradigm shift in mitochondrial studies, which enabled to perform a comprehensive screening of the entire genome. Progress in robotics and nanotechnologies led to the development of high-throughput detection techniques, like modern DNA microarrays and next-generation sequencing. In this chapter, we provide a compendium of the most widely used techniques to identify mitochondrial variants, from PCR-based methods to third-generation sequencing, and also address the challenges posed by detection and heteroplasmy quantification. Likewise, we provide a description of the most up-to-date bioinformatic tools available to the scientific community to perform mitochondrial genome analysis as well as guidelines for variant detection in high-throughput sequencing data