Mitochondrial DNA sequence data are often utilized in disease studies, conservation genetics and forensic identification. The current approaches for sequencing the full mtGenome typically require several rounds of PCR enrichment during Sanger or MPS protocols followed by fairly tedious assembly and analysis. Here we describe an efficient approach to sequencing directly from genomic DNA samples without prior enrichment or extensive library preparation steps. A comparison is made between libraries sequenced directly from native DNA and the same samples sequenced from libraries generated with nine overlapping mtDNA amplicons on the Oxford Nanopore MinION™ device. The native and amplicon library preparation methods and alternative base calling strategies were assessed to establish error rates and identify trends of discordance between the two library preparation approaches. For the complete mtGenome, 16 569 nucleotides, an overall error rate of approximately 1.00% was observed. As expected with mtDNA, the majority of error was detected in homopolymeric regions. The use of a modified basecaller that corrects for ambiguous signal in homopolymeric stretches reduced the error rate for both library preparation methods to approximately 0.30%. Our study indicates that direct mtDNA sequencing from native DNA on the MinION™ device provides comparable results to those obtained from common mtDNA sequencing methods and is a reliable alternative to approaches using PCR‐enriched libraries.

Traditional approaches for interrogating the mitochondrial genome often involve laborious extraction and enrichment protocols followed by Sanger sequencing. Although preparation techniques are still demanding, the advent of next‐generation or massively parallel sequencing has made it possible to routinely obtain nucleotide‐level data with relative ease. These short‐read sequencing platforms offer deep coverage with unparalleled read accuracy in high‐complexity genomic regions but encounter numerous difficulties in the low‐complexity homopolymeric sequences characteristic of the mitochondrial genome. The inability to discern identical units within monomeric repeats and resolve copy‐number variations for heteroplasmy detection results in suboptimal genome assemblies that ultimately complicate downstream data analysis and interpretation of biological significance. Oxford Nanopore Technologies offers the ability to generate long‐read sequencing data on a pocket‐sized device known as the MinION. Nanopore‐based sequencing is scalable, portable, and theoretically capable of sequencing the entire mitochondrial genome in a single contig. Furthermore, the recent development of a nanopore protein with dual reader heads allows for clear identification of nucleotides within homopolymeric stretches, significantly increasing resolution throughout these regions. The unrestricted read lengths, superior homopolymeric resolution, and affordability of the MinION device make it an attractive alternative to the labor‐intensive, time‐consuming, and costly mainstay deep‐sequencing platforms. This article describes three approaches to extract, prepare, and sequence mitochondrial DNA on the Oxford Nanopore MinION device. Two of the workflows include enrichment of mitochondrial DNA prior to sequencing, whereas the other relies on direct sequencing of native genomic DNA to allow for simultaneous assessment of the nuclear and mitochondrial genomes. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Enrichment‐free mitochondrial DNA sequencing Alternate Protocol 1: Mitochondrial DNA sequencing following enrichment with polymerase chain reaction (PCR) Alternate Protocol 2: Mitochondrial DNA sequencing following enrichment with PCR‐free hybridization capture Support Protocol 1: DNA quantification and quality assessment using the Agilent 4200 TapeStation System Support Protocol 2: AMPure XP bead clean‐up Support Protocol 3: Suggested data analysis pipeline