Evaluation of mitochondrial DNA copy number estimation techniques

Ryan J. Longchamps, Christina A. Castellani, Stephanie Y. Yang, Charles E. Newcomb, Jason A. Sumpter, John Lane, Megan L. Grove, Eliseo Guallar, Nathan Pankratz, Kent D. Taylor, Jerome I. Rotter, Eric Boerwinkle, Dan E. Arking

Research output: Contribution to journalArticlepeer-review

72 Scopus citations


Mitochondrial DNA copy number (mtDNA-CN), a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy measure for mitochondrial function and has been associated with several aging-related diseases. Although quantitative real-time PCR (qPCR) is the current gold standard method for measuring mtDNA-CN, mtDNA-CN can also be measured from genotyping microarray probe intensities and DNA sequencing read counts. To conduct a comprehensive examination on the performance of these methods, we use known mtDNA-CN correlates (age, sex, white blood cell count, Duffy locus genotype, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence (WES) data across 1,085 participants from the Atherosclerosis Risk in Communities (ARIC) study and 3,489 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). We observe mtDNA-CN derived from WGS data is significantly more associated with known correlates compared to all other methods (p < 0.001). Additionally, mtDNA-CN measured from WGS is on average more significantly associated with traits by 5.6 orders of magnitude and has effect size estimates 5.8 times more extreme than the current gold standard of qPCR. We further investigated the role of DNA extraction method on mtDNA-CN estimate reproducibility and found mtDNA-CN estimated from cell lysate is significantly less variable than traditional phenol-chloroform-isoamyl alcohol (p = 5.44x10-4) and silica-based column selection (p = 2.82x10-7). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN, as well as re-analyze trait associations as more WGS data becomes available from larger initiatives such as TOPMed.

Original languageEnglish (US)
Article numbere0228166
JournalPloS one
Issue number1
StatePublished - Jan 1 2020

Bibliographical note

Funding Information:
This research was supported by grant R01HL131573 from the US National Institutes of Health (Longchamps, Castellani, Guallar, and Arking) and by grant P30AG021334 from the Johns Hopkins University Claude D. Pepper Older Americans Independence Center National Institute on Aging (Dr Arking). The Atherosclerosis Risk in Communities study has been funded in whole or in part with Federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, under Contract nos. (HHSN268201700001I, HHSN268201700002I, HHSN268201700003I, HHSN268201700005I, HHSN268201700004I). The Multi-Ethnic Study of Atherosclerosis is supported by contracts HHSN268201500003I, N01-HC 95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168 and N01-HC-95169 from the National Heart, Lung, and Blood Institute, and by grants UL1-TR-000040, UL1-TR-001079, and UL1-TR-001420 from the National Center for Advancing Translational Sciences (NCATS). Dr. Rotter and Dr. Taylor?s efforts were supported in part by the National Center for Advancing Translational Sciences, CTSI grant UL1TR001881, and the National Institute of Diabetes and Digestive and Kidney Disease Diabetes Research Center (DRC) grant DK063491 to the Southern California Diabetes Endocrinology Research Center. Funding for SHARe genotyping was provided by National Heart, Lung, and Blood Institute contract N02-HL-64278. The funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. We thank the staff and participants of the Atherosclerosis Risk in Communities Study, Cardiovascular Health Study, and the Multi-Ethnic Study of Atherosclerosis studies for their important contributions. A full list of participating MESA investigators and institutions can be found at http://www.mesa-nhlbi.org. Digital PCR was conducted at the Genetic Resources Core Facility, Johns Hopkins Institute of Genetic Medicine, Baltimore, MD.

Publisher Copyright:
© 2020 Longchamps et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


  • Aged
  • DNA, Mitochondrial/genetics
  • Female
  • Gene Dosage
  • Genomics/methods
  • Humans
  • Male
  • Middle Aged

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural


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