Approaches to Whole Mitochondrial Genome Sequencing on the Oxford Nanopore MinION

Abstract

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

Mitochondrial Genome of Fagus sylvatica L. as a Source for Taxonomic Marker Development in the Fagales

“…The assembled sequence has been successfully validated by mapping of nanopore MinION long reads ( Figure S1 ). Because short reads encounter numerous difficulties due to low-complexity homopolymeric sequence characteristics and the potential presence of large repeat regions in mitochondrial genomes [ 12 , 17 , 19 , 22 , 26 , 42 ], long read sequencing is increasingly applied (often in addition to short read sequencing) for subsequent assemblies of mitochondrial genome sequences (e.g., [ 57 , 58 , 59 ]).…”

mtDNA Heteroplasmy: Origin, Detection, Significance, and Evolutionary Consequences

“…Currently, these diseases are not curable, but there are attempts to cure or to prevent them. mtDNA modification are promising techniques for this direction [ 89 , 90 , 91 ]. In addition, a set of mitochondrial replacement techniques (MRT) have been recently developed for the prevention of mitochondrial diseases.…”

Applying genomic and transcriptomic advances to mitochondrial medicine

“…A number of protocols have been developed that enable the sequencing of entire mitochondrial genomes with nanopore technology, with or without mtDNA enrichment steps 96,97 . Nanopore approaches promise improved resolution of homopolymeric regions [G] of mtDNA and are less work-intensive than NGS approaches.…”