Abstract
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 5% sodium hypochlorite immersion treatments) aim to minimize the exogenous content of the outer surface of ancient calculus samples prior to DNA extraction. While these protocols are widely used, no one has systematically compared them in ancient dental calculus. Here, we compare untreated dental calculus samples to samples from the same site treated with four previously published decontamination protocols: a UV only treatment; a 5% sodium hypochlorite immersion treatment; a pre-digestion in EDTA treatment; and a combined UV irradiation and 5% sodium hypochlorite immersion treatment. We examine their efficacy in ancient oral microbiota recovery by applying 16S rRNA gene amplicon and shotgun sequencing, identifying ancient oral microbiota, as well as soil and skin contaminant species. Overall, the EDTA pre-digestion and a combined UV irradiation and 5% sodium hypochlorite immersion treatment were both effective at reducing the proportion of environmental taxa and increasing oral taxa in comparison to untreated samples. This research highlights the importance of using decontamination procedures during ancient DNA analysis of dental calculus to reduce contaminant DNA.
Highlights
Microbial communities within the human microbiota vary across different body sites
Four out of the five EDTA treated samples had no detectable soil component, while a single EDTA treated sample was comprised of 21.5% soil Operational taxonomic unit (OTU)
We summarized the percentage of environmental taxa (i.e., absent from the Human Oral Microbiome Database (HOMD)) with significant differences in frequencies (p < 0.001) relative to the no treatment controls (Fig. 4A) and the percentage of oral OTUs that increased relative to no treatment controls (Fig. 4B)
Summary
Microbial communities within the human microbiota vary across different body sites (e.g., the gut, skin, and oral cavity). Contamination, poses a significant risk for ancient dental calculus analysis[19,20,21,22,23], as it is an unwanted source of variation that obscures biological factors of interest For these reasons, strict aDNA protocols must be followed, including methods that reduce and monitor contaminant DNA contributing to a sample. Evidence suggests that sequencing extraction and non-template amplification (i.e., PCR negatives) controls can help monitor laboratory c ontamination[26], some research teams fail to include such sequencing data in their p ublications[22] Such controls cannot detect contaminant DNA that was present on the sample prior to entering the facility[15]. These direct comparisons of aDNA decontamination protocols on ancient dental calculus samples can serve as a resource for the future analysis of ancient oral microbiota
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