Abstract
Massively parallel resequencing of mitochondrial DNA (mtDNA) has led to significant advances in the study of heteroplasmic mtDNA variants in health and disease, but confident resolution of very low-level variants (<2% heteroplasmy) remains challenging due to the difficulty in distinguishing signal from noise at this depth. However, it is likely that such variants are precisely those of greatest interest in the study of somatic (acquired) mtDNA mutations. Previous approaches to this issue have included the use of controls such as phage DNA and mtDNA clones, both of which may not accurately recapitulate natural mtDNA. We have therefore explored a novel approach, taking advantage of mtDNA with a known stereotyped mutational motif (nAT>C, from patient with MNGIE, mitochondrial neurogastrointestinal encephalomyopathy) and comparing mutational pattern distribution with healthy mtDNA by ligation-mediated deep resequencing (Applied Biosystems SOLiD). We empirically derived mtDNA-mutant heteroplasmy detection limits, demonstrating that the presence of stereotypical mutational motif could be statistically validated for heteroplasmy thresholds ≥0.22% (P=0.034). We therefore provide empirical evidence from biological samples that very low-level mtDNA mutants can be meaningfully resolved by massively parallel resequencing, confirming the utility of the approach for studying somatic mtDNA mutation in health and disease. Our approach could also usefully be employed in other settings to derive platform-specific deep resequencing resolution limits.
Highlights
Parallel (‘ generation’) resequencing is potentially a powerful tool with which to study heteroplasmic mitochondrial DNA mutations because of the possibility of the high breadth and depth of coverage
Sequencing was performed using the AB SOLiD platform with a 50-bp paired-end fragment library. mitochondrial DNA (mtDNA) was enriched from total DNA extract by using three overlapping long-range PCR (LR-PCR) fragments, designed to avoid the amplification of nuclear mitochondrial pseudogenes
In MNGIE mtDNA, the total number of mutations detected with this stereotyped pattern increased as the variant detection threshold was lowered; the proportion of total variants corresponding to the stereotypical pattern progressively increased with increasing detection threshold
Summary
Parallel (‘ generation’) resequencing is potentially a powerful tool with which to study heteroplasmic mitochondrial DNA (mtDNA) mutations because of the possibility of the high breadth and depth of coverage. Recent studies using amplicon-based resequencing (Roche 454 GS-FLX pyro-sequencing, Branford, CT, USA) have indicated (using mtDNA clones) that mtDNA mutations at B0.2% heteroplasmy level or greater can be successfully resolved, but only after very stringent data cleaning, including the exclusion of homopolymeric tracts that are known hot spots for technical artefact on this platform This approach is not well suited for analysing low-level mutation across whole mtDNA genomes.[1,2] In contrast, methods using fragment resequencing (Illumina GA platform, San Diego, CA, USA) have tended to use more conservative limits of resolution (for example of 41.5% heteroplasmy level), which potentially excludes much of the variants of interest.[3,4]. We explored the lower heteroplasmy limit to which this stereotypical mutational pattern could be resolved
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