Detecting Heteroplasmy from High-Throughput Sequencing of Complete Human Mitochondrial DNA Genomes

Abstract

Heteroplasmy, the existence of multiple mtDNA types within an individual, has been previously detected by using mostly indirect methods and focusing largely on just the hypervariable segments of the control region. Next-generation sequencing technologies should enable studies of heteroplasmy across the entire mtDNA genome at much higher resolution, because many independent reads are generated for each position. However, the higher error rate associated with these technologies must be taken into consideration to avoid false detection of heteroplasmy. We used simulations and phiX174 sequence data to design criteria for accurate detection of heteroplasmy with the Illumina Genome Analyzer platform, and we used artificial mixtures and replicate data to test and refine the criteria. We then applied these criteria to mtDNA sequence reads for 131 individuals from five Eurasian populations that had been generated via a parallel tagged approach. We identified 37 heteroplasmies at 10% frequency or higher at 34 sites in 32 individuals. The mutational spectrum does not differ between heteroplasmic mutations and polymorphisms in the same individuals, but the relative mutation rate at heteroplasmic mutations is significantly higher than that estimated for all mutable sites in the human mtDNA genome. Moreover, there is also a significant excess of nonsynonymous mutations observed among heteroplasmies, compared to polymorphism data from the same individuals. Both mutation-drift and negative selection influence the fate of heteroplasmies to determine the polymorphism spectrum in humans. With appropriate criteria for avoiding false positives due to sequencing errors, next-generation technologies can provide novel insights into genome-wide aspects of mtDNA heteroplasmy.

Extensive tissue-related and allele-related mtDNA heteroplasmy suggests positive selection for somatic mutations

“…Heteroplasmies occur preferentially at positions with a high mutation rate (2,9), suggesting that mutation and drift are the primary forces influencing heteroplasmy. In addition, elevated levels of nonsynonymous (NS) heteroplasmies within individuals relative to NS polymorphisms among individuals (2) suggest that there is negative selection against heteroplasmies that involve deleterious amino acid changes. In other words, deleterious amino acid changes can be observed as heteroplasmies as long as the allele frequency stays below a certain threshold and "normal" mitochondrial function is maintained; exceeding this threshold results in impaired mitochondrial function, as is commonly observed for diseaseassociated mtDNA mutations (10).…”

“…Heteroplasmy is thought to represent an intermediate stage in the fixation of mtDNA mutations within an individual, and heteroplasmic mtDNA mutations have been implicated in various diseases, cancer, and aging (5)(6)(7)(8). Heteroplasmies occur preferentially at positions with a high mutation rate (2,9), suggesting that mutation and drift are the primary forces influencing heteroplasmy. In addition, elevated levels of nonsynonymous (NS) heteroplasmies within individuals relative to NS polymorphisms among individuals (2) suggest that there is negative selection against heteroplasmies that involve deleterious amino acid changes.…”

“…mtDNA | heteroplasmy | selection | human | tissue variation A lthough mtDNA heteroplasmy (intraindividual variability in mtDNA sequences) was initially thought to be rare in humans, studies using next-generation sequencing platforms have documented extensive heteroplasmy at low levels (<2%) (1)(2)(3)(4). Heteroplasmy is thought to represent an intermediate stage in the fixation of mtDNA mutations within an individual, and heteroplasmic mtDNA mutations have been implicated in various diseases, cancer, and aging (5)(6)(7)(8).…”

Characterization of mtDNA variation in a cohort of South African paediatric patients with mitochondrial disease

“…Several of these low-frequency, heteroplasmic variants in the muscle may indeed be due to somatic mtDNA mosaicism and not diseasecausing variants, 38,39 which highlights the importance of follow-up investigations such as cybrid studies to establish pathogenicity for these variants. We have identified a number of previously documented disease-associated variants in this cohort, of which only one (m.14484T4C LHON mutation) can be considered as a frequently occurring syndrome-associated mutation.…”

Complete mtDNA genomes of Filipino ethnolinguistic groups: a melting pot of recent and ancient lineages in the Asia-Pacific region

“…17 DNA libraries were sequenced on an Illumina GA IIx machine (Illumina Inc., San Diego, CA, USA) with post processing using Illumina software followed by the Improved Base Identification System. 18 Sequencing reads were mapped to the revised Cambridge Reference Sequence of the human mitochondrial genome (GenBank: NC012920.1) 19 using the program MIA, 20 implemented in an MPI-EVA sequence assembly-analyses pipeline for detecting mtDNA heteroplasmy 21 and low-level mutations 22 (Table 3) also does not support a discrete FEN vs FEnN grouping. The 54 haplogroups have a patchy distribution across the different FE groups ( Figure 1, Table 2).…”