PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

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.

Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue

“…A method (II) based on alkaline extraction–commonly used to extract plasmid DNA and thus designed to extract circular DNA 28 , 30 , 32 , 33 –yielded median 144 ng (IQR 140 ng) dsDNA with 0.5% (IQR 0.6%) mtDNA. Extracting DNA from isolated mitochondria (III) 34 yielded median 825 ng (IQR 529 ng) dsDNA with 0.2% (IQR 0.1%) mtDNA. A selective lysis method (IV) that starts with the disruption of the plasma membrane to release the cellular components 29 , 37 followed by sedimentation of cell nuclei, and DNA extracted from the remaining cytosol fraction yielded median 403 ng (IQR 321 ng) dsDNA with 1.0% (IQR 0.8%) mtDNA.…”

“…In the study described here, we aimed to develop a sensitive procedure to detect low-frequent single-nucleotide mtDNA variants in frozen tumor tissue. Multiple efforts in developing methods for extraction of pure mtDNA exist 27 – 34 . These include methods using commercial kits or (laborious) ultracentrifugation to obtain pure mitochondria, and techniques to enrich for mtDNA by either the isolation technique or enzymatic degradation of nDNA.…”

Detection and quantification of mitochondrial DNA deletions from next-generation sequence data

“…When designing NGS experiments for this purpose, researchers also must take into account the high numbers of nuclear genome reads present in the sequencing data, which will decrease the average number of reads per mitochondrial base position. Indeed, unless an mtDNA enrichment procedure is applied in the laboratory prior to DNA sequencing, only approximately 0.2% of DNA is expected to be mitochondrial [ 22 ]. Even with enrichment, the sensitivity of our computational procedure clearly depends on the number of mtDNA reads, which is a function of the enrichment protocol’s efficiency.…”

“…Even with enrichment, the sensitivity of our computational procedure clearly depends on the number of mtDNA reads, which is a function of the enrichment protocol’s efficiency. We and others have performed studies developing and comparing various mtDNA enrichment protocols [ 22 , 23 ], with varying results depending on the tissue type and other factors. Theoretical sensitivity for a sequencing experiment therefore here takes into account various enrichment levels.…”

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

“…Other methods that, conversely, reduce the level of the nuclear genome by using exonuclease V to digest the linear nuclear DNA (24), or by isolating mitochondria with differential centrifugation or magnetic beads for immunoprecipitation (25) require a large amount of starting materials, which is not suited for large-scale population studies with limited DNA sources or frozen tissue biospecimens. Importantly, by processing only mtDNA, the mtDNA-targeted sequencing methods lose valuable information on mtDNA levels in relation to nuclear DNA in the sample, making them unable to quantify mtDNA content in the same assay.…”