Nanopore sequencing: An enrichment‐free alternative to mitochondrial DNA sequencing

Abstract

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.

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

“…In their report on Nanopore DNA sequencing aboard the International Space Station, Castro-Wallace and colleagues found that 29 out of 83,332 reads (0.03%) from mouse DNA were derived from the mouse mtDNA [53]. In another study, Zascavage and colleagues relied on Nanopore long-read sequencing for mtDNA analysis [54]. They reported an average coverage of mtDNA reads that varied between 15 and 118 fold from a whole genome sequencing analysis.…”

Species- and strain-level assessment usingrrnlong-amplicons suggests donor’s influence on gut microbial transference via fecal transplants in metabolic syndrome subjects

“…Through the pipeline for the rrn assembly, we realised that the indels rate during the strand-wise alignments was always higher than mismatches, likely an effect of the alignment parameters (see methods). Notwithstanding, close inspections of the alignments suggest that such indels are produced in homopolymeric regions (containing single or di-nucleotide repeats), a common failure during basecalling of nanopore data [21,26]. On the other hand, the final assemblies were recovered with less than 1% indels proportions when compared to database references, thus indicating they were drastically attenuated when compared to the strand-wise evaluation, and suggesting that assemblies have largely been improved to correct typical errors of nanopore data, and likely retaining the nucleotide changes (mismatches) linked to species/strains genetic variability.…”

A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

“…In their report on Nanopore DNA sequencing aboard the International Space Station, Castro-Wallace and colleagues found that 29 out of 83,332 reads (0.03%) from mouse DNA were derived from the mouse mtDNA (44). In another study, Zascavage and colleagues relied on Nanopore long-read sequencing for mtDNA analysis (45). They reported an average coverage of mtDNA reads that varied between 15 and 118 fold from a whole genome sequencing analysis.…”