Prediction of mitochondrial genome-wide variation through sequencing of mitochondrion-enriched extracts

Kelsey E. Fisher,Brad S. Coates,Steven P. Bradbury

doi:10.1038/s41598-020-76088-0

Kelsey E. Fisher, Brad S. Coates + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-76088-0

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Abstract

Although mitochondrial DNA (mtDNA) haplotype variation is often applied for estimating population dynamics and phylogenetic relationships, economical and generalized methods for entire mtDNA genome enrichment prior to high-throughput sequencing are not readily available. This study demonstrates the utility of differential centrifugation to enrich for mitochondrion within cell extracts prior to DNA extraction, short-read sequencing, and assembly using exemplars from eight maternal lineages of the insect species, Ostrinia nubilalis. Compared to controls, enriched extracts showed a significant mean increase of 48.2- and 86.1-fold in mtDNA based on quantitative PCR, and proportion of subsequent short sequence reads that aligned to the O. nubilalis reference mitochondrial genome, respectively. Compared to the reference genome, our de novo assembled O. nubilalis mitochondrial genomes contained 82 intraspecific substitution and insertion/deletion mutations, and provided evidence for correction of mis-annotated 28 C-terminal residues within the NADH dehydrogenase subunit 4. Comparison to a more recent O. nubilalis mtDNA assembly from unenriched short-read data analogously showed 77 variant sites. Twenty-eight variant positions, and a triplet ATT codon (Ile) insertion within ATP synthase subunit 8, were unique within our assemblies. This study provides a generalizable pipeline for whole mitochondrial genome sequence acquisition adaptable to applications across a range of taxa.

Highlights

Mitochondrial DNA haplotype variation is often applied for estimating population dynamics and phylogenetic relationships, economical and generalized methods for entire mitochondrial DNA (mtDNA) genome enrichment prior to high-throughput sequencing are not readily available
Enrichment was demonstrated by the ratio of amplified fragment intensities by semi-quantitative PCR of mitochondrial compared to the apn[1] nuclear target gene (Fig. 1b, S1)
Mitochondrial c oxidase subunit I (coxI) amplification was inconsistent or non-existent within DNA extracts obtained from fractions pelleted at 2000 and 4000 rcf

Summary

Introduction

Mitochondrial DNA (mtDNA) haplotype variation is often applied for estimating population dynamics and phylogenetic relationships, economical and generalized methods for entire mtDNA genome enrichment prior to high-throughput sequencing are not readily available. The number of sequenced whole mitochondrial genomes has greatly increased in recent y ears[28] This increase has facilitated mitochondrial genome assemblies directly from high-throughput short-read sequencing of shotgun total genomic libraries[8], or libraries enriched for mtDNA using commercial miniprep-29, rolling circle-30, or probe-based methods[31]. Regardless, many of these methods co-enrich nuclear-fragments with integrated mitochondrial DNA fragments (NUMTs)[32,33,34,35,36], or arguably require expensive commercial reagents. This study demonstrates the utility of mitochondrion enrichments within a full mtDNA genome sequencing protocol, and how detected variation can be used in population and phylogenetic studies

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Results

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