Measure quantity of mitochondrial DNA in aged bones or calculate it from nuclear DNA quantitative PCR results?

Mitochondrial DNA (mtDNA) is of great value in forensics to procure information about a person when a next of kin, personal belongings, or other sources of nuclear DNA (nDNA) are unavailable, or nDNA is lacking in quality and quantity. The quality and reliability of the results depend greatly on ensuring optimal conditions for the given method, for instance, the optimal input of the copy number (CN) in next-generation sequencing (NGS) methods. The unavailability of commercial quantitative PCR (qPCR) methods to determine mtDNA CN creates the necessity to rely on recommendations to infer mtDNA CN from nDNA yield. Because nDNA yield varies between individuals, tissues, parts of the same tissue, and because mtDNA CN varies between tissues, such assumptions must be examined for a specific context, rather than be generalized. This study compares mtDNA CN calculated from nDNA yield and qPCR measured mtDNA CN. Seventy-five femurs from the Second World War victims were used as samples; they were cut below the greater trochanter, surface contaminants were removed by mechanical and chemical cleaning, samples were fully demineralized, and DNA was isolated. PowerQuant® Kit (Promega) was used to analyze DNA yield. An in-house method was used to determine mtDNA CN. Comparison of mtDNA CN from nDNA derived calculations and measured mtDNA CN highlighted vast differences. The results emphasize the need to perform qPCR to assess mtDNA CN before NGS analyses of aged bones' mitogenomes rather than estimating mtDNA CN from nDNA yield to ensure the quality and reliability of the results of NGS analysis.