Abstract
This protocol describes the methodology to characterize mitochondrial DNA (mtDNA) heteroplasmy by parallel sequencing. Mitochondria play an important role in essential cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondria genomes. Mutant and wild-type mtDNA may co-exist as heteroplasmy, and cause human disease. The purpose of this protocol is to simultaneously determine mtDNA sequence and quantify the heteroplasmic level. This protocol includes a two-fragment mitochondrial genome DNA PCR amplification. The PCR product is then mixed at an equimolar ratio. The samples are then barcoded and sequenced with high-throughput, next-generation sequencing technology. This technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the level of heteroplasmy.