Ratio of mitochondrial to nuclear DNA affects contamination estimates in ancient DNA analysis

In the last decade, ancient DNA research has grown rapidly and started to overcome several of its earlier limitations through Next-Generation-Sequencing (NGS). Among other advances, NGS allows direct estimation of sample contamination from modern DNA sources. First NGS-based approaches of estimating contamination measured heterozygosity. These measurements, however, could only be performed on haploid genomic regions, i.e. the mitochondrial genome or male X chromosomes, but provided no measures of contamination in the nuclear genome of females with their two X chromosomes. Instead, female nuclear contamination is routinely extrapolated from mitochondrial contamination estimates, but it remains unclear if this extrapolation is reliable and to what degree variation in mitochondrial to nuclear DNA ratios affects this extrapolation. We therefore analyzed ancient DNA from 317 samples of different skeletal elements from multiple sites, spanning a temporal range from 7,000 BP to 386 AD. We found that the mitochondrial to nuclear DNA (mt/nc) ratio negatively correlates with an increase in endogenous DNA content and strongly influenced mitochondrial and nuclear contamination estimates in males. The ratio of mt to nc contamination estimates remained stable for overall mt/nc ratios below 200, as found particularly often in petrous bones but less in other skeletal elements and became more variable above that ratio.