Abstract
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 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, incident cardiovascular disease) to evaluate mtDNA-CN calculated from qPCR, two microarray platforms, as well as whole genome (WGS) and whole exome sequence 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 on average is more significantly associated with traits by 5.6 orders of magnitude and is 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 × 5.44×10-4) and silica-based column selection (p × 2.82×10-7). In conclusion, we recommend the field moves towards more accurate methods for mtDNA-CN estimation such as microarray and WGS, as well as re-analyzing trait associations as more WGS data becomes available from larger initiatives such as TOPMed.