MitoRS, a method for high throughput, sensitive, and accurate detection of mitochondrial DNA heteroplasmy

Abstract

BackgroundMitochondrial dysfunction is linked to numerous pathological states, in particular related to metabolism, brain health and ageing. Nuclear encoded gene polymorphisms implicated in mitochondrial functions can be analyzed in the context of classical genome wide association studies. By contrast, mitochondrial DNA (mtDNA) variants are more challenging to identify and analyze for several reasons. First, contrary to the diploid nuclear genome, each cell carries several hundred copies of the circular mitochondrial genome. Mutations can therefore be present in only a subset of the mtDNA molecules, resulting in a heterogeneous pool of mtDNA, a situation referred to as heteroplasmy. Consequently, detection and quantification of variants requires extremely accurate tools, especially when this proportion is small. Additionally, the mitochondrial genome has pseudogenized into numerous copies within the nuclear genome over the course of evolution. These nuclear pseudogenes, named NUMTs, must be distinguished from genuine mtDNA sequences and excluded from the analysis.ResultsHere we describe a novel method, named MitoRS, in which the entire mitochondrial genome is amplified in a single reaction using rolling circle amplification. This approach is easier to setup and of higher throughput when compared to classical PCR amplification. Sequencing libraries are generated at high throughput exploiting a tagmentation-based method. Fine-tuned parameters are finally applied in the analysis to allow detection of variants even of low frequency heteroplasmy. The method was thoroughly benchmarked in a set of experiments designed to demonstrate its robustness, accuracy and sensitivity. The MitoRS method requires 5 ng total DNA as starting material. More than 96 samples can be processed in less than a day of laboratory work and sequenced in a single lane of an Illumina HiSeq flow cell. The lower limit for accurate quantification of single nucleotide variants has been measured at 1% frequency.ConclusionsThe MitoRS method enables the robust, accurate, and sensitive analysis of a large number of samples. Because it is cost effective and simple to setup, we anticipate this method will promote the analysis of mtDNA variants in large cohorts, and may help assessing the impact of mtDNA heteroplasmy on metabolic health, brain function, cancer progression, or ageing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3695-5) contains supplementary material, which is available to authorized users.

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

“…Such an enrichment was of interest, but it came at the cost of a limited amount of DNA which is inadequate for sequencing without an amplification step. We decided to amplify the mtDNA using a Whole Mitochondrial Genome Amplification (WMGA) method based on multiple displacement amplification (34). We optimized primer design and concentration in the MDA reaction to avoid template-independent amplification, a technical bias usually observed with MDA.…”

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

“…The mitochondrial genome is a maternally inherited small circular dsDNA found inside mitochondria that accounts for only a small portion of the total DNA in eukaryotic cells (Ashley, Harris, & Poulton, 2005; Marquis et al, 2017). Human mtDNA contains only approximately 16.6‐kilo base pairs and encodes multiple tRNAs, rRNAs, and proteins necessary for the respiratory chain (Marquis et al, 2017).…”

“…The mitochondrial genome is a maternally inherited small circular dsDNA found inside mitochondria that accounts for only a small portion of the total DNA in eukaryotic cells (Ashley, Harris, & Poulton, 2005; Marquis et al, 2017). Human mtDNA contains only approximately 16.6‐kilo base pairs and encodes multiple tRNAs, rRNAs, and proteins necessary for the respiratory chain (Marquis et al, 2017). Of note, mtDNAs suffer from a high mutation rate, and their mutations are strongly correlated with a broad range of debilitating and fatal diseases (Hyslop et al, 2016); thus, detection of mutant mtDNAs is of great importance for both basic research and genetic diagnosis.…”

“…Of note, mtDNAs suffer from a high mutation rate, and their mutations are strongly correlated with a broad range of debilitating and fatal diseases (Hyslop et al, 2016); thus, detection of mutant mtDNAs is of great importance for both basic research and genetic diagnosis. However, it is complicated by heteroplasmy, a situation in which cells often contain mtDNAs with different genotypes as mutations are acquired as each cell carries several hundred copies of mtDNA and the number of present mtDNA molecules within each cell varies between cell types (Marquis et al, 2017; Mechta, Ingerslev, & Barres, 2018; Weerts et al, 2018). Therefore, detecting mutations in mtDNA in single cells is in great demand.…”

STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

“…mtDNA-targeted sequencing is an economical alternative to genome-wide methods. The main strategy is to isolate and enrich mtDNA from the total genomic background, and thus focus the sequencing capacity on mtDNA reads (16)(17)(18). These methods normally start with amplification of mtDNA from total genomic DNA (16,17).…”