PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.
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.
Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, United States of America.
Meetha P
MP
Gould
Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, United States of America.
Colleen M
CM
Bosworth
Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, United States of America.
Sarah
S
McMahon
Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, United States of America.
Sneha
S
Grandhi
Center for Global Health and Disease, Case Western Reserve University, Cleveland, Ohio, United States of America.
Brian T
BT
Grimberg
Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, Ohio, United States of America; Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio, United States of America; Department of Electrical Engineering and Computer Science, Case Western Reserve University, Cleveland, Ohio, United States of America; Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, United States of America.
Thomas
T
LaFramboise
D002478:Cells, Cultured; D003106:Colon / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification*; D014644:Genetic Variation / Q000235:genetics; D054629:Genome, Mitochondrial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004272:DNA, Mitochondrial