Effectiveness of decontamination protocols when analyzing ancient DNA preserved in dental calculus

Ancient DNA analysis of human oral microbial communities within calcified dental plaque (calculus) has revealed key insights into human health, paleodemography, and cultural behaviors. However, contamination imposes a major concern for paleomicrobiological samples due to their low endogenous DNA content and exposure to environmental sources, calling into question some published results. Decontamination protocols (e.g. an ethylenediaminetetraacetic acid (EDTA) pre-digestion or ultraviolet radiation (UV) and bleach immersion treatments) aim to minimize the exogenous content of the outer surface of ancient calculus samples prior to DNA extraction. While these protocols are efficient, no one has systematically compared them. Here, we compare untreated dental calculus samples to four previously published decontamination protocols: a UV only treatment; a 5% sodium hypochlorite (bleach) immersion treatment; a pre-digestion in EDTA treatment; and a treatment with a combined UV irradiation and 5% bleach immersion. We examine their efficacy in ancient oral microbiota recovery by applying 16S rRNA gene amplicon and shotgun sequencing to ancient calculus samples from a single site. We identify ancient oral microbiota, as well as soil and skin contaminants. Overall, both the EDTA and UVB treatments are effective at reducing the proportion of environmental taxa and increasing oral taxa in relation to untreated samples.