Current and new Next-Generation Sequencing approaches to study mitochondrial DNA.

Abstract

Mitochondria harbor multiple copies of a maternally inherited non-nuclear genome; point mutations, deletions, or depletion of the mitochondrial DNA have been associated with various human diseases. Different approaches have been used to investigate mitochondrial DNA defects: Sanger sequencing, Southern blot, long and quantitative PCR. All these technologies are inherently hampered by limitations in speed, throughput, sensitivity, and associated costs. Recently, Next Generation Sequencing has been introduced in this field and all its potential applications still need to be fully validated. Analysis of mitochondrial DNA from 16 control samples and 33 affected samples, previously investigated by traditional techniques, was performed. Different Next Generation Sequencing approaches were tested, using classical library preparation based on PCR amplifications and an innovative PCR-free protocol, defining their suitability and utility for: (i) generating full accurate mtDNA sequence, (ii) assessing heteroplasmy for single point mutations with high accuracy, (iii) detecting break positions and heteroplasmy of single large deletions. This study confirmed that PCR-based library preparations are appropriate for the first two points while showed that a new PCR-free method gave the best results for the third aim.