Abstract
Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.
Highlights
Mitochondria are involved in fundamental cellular processes including generating ATP for cellular energy, storing calcium for cell signaling, and mediating cell growth and death
Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest
Since MT-ND1 is present at hundreds to thousands of copies per cell, using a two-copy gene as a comparator limits precision in measuring relative abundances of mitochondrial DNA (mtDNA) across samples. 18S is present at some 300–400 copies per cell [13], closer to mitochondrial genome abundance
Summary
Mitochondria are involved in fundamental cellular processes including generating ATP for cellular energy, storing calcium for cell signaling, and mediating cell growth and death. Dysregulation is often the result of DNA-level mutations affecting mitochondrial proteins [1], which may be encoded in either the nuclear genome or the mitochondrial genome. Detecting DNA-level variants in the mitochondrial genome presents unique challenges. A mutation may be present in a very low percentage of mtDNA copies, in contrast to nuclear variants which are normally present in 0%, 50%, or 100% of the cell’s nuclear genome (nDNA) copies. Variant calling can be confounded by the presence of nuclear mitochondrial DNA sequences (numts). Numts are tracts of nDNA that are near or perfect matches to the mtDNA sequence. These numts can give rise to false positive variant calls when mistaken for mtDNA, as their small deviations from the mitochondrial reference sequence are misidentified as mitochondrial variants. To avoid numt-induced false positives, it is important to enrich the DNA sample for mtDNA, either through mitochondrial isolation or mtDNA amplification
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