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.

对钙化牙菌斑（calculus，即牙结石）内人类口腔微生物群落的古DNA分析，已为人类健康、古人口统计学（paleodemography）及文化行为研究提供了关键洞见。然而，由于古微生物样本的内源DNA含量极低且易暴露于环境污染源，污染问题始终是古微生物学研究的重大隐患，这也对部分已发表的研究结果提出了质疑。去污方案（例如乙二胺四乙酸（EDTA）预消化法、紫外线（UV）照射结合漂白剂浸泡处理法）旨在于DNA提取前，尽可能去除古牙结石样本表面的外源DNA成分。尽管上述方案均具备一定效果，但目前尚无研究对这些方案开展系统性对比。本研究将未处理的牙结石样本与四种已发表的去污方案进行对比：仅紫外线（UV）照射处理法、5%次氯酸钠（漂白剂）浸泡处理法、EDTA预消化处理法，以及紫外线（UV）照射结合5%漂白剂浸泡的联合处理法。我们通过对单一样本来源的古牙结石样本实施16S rRNA基因扩增子测序与鸟枪法（shotgun）测序，评估各方案在恢复古口腔微生物组方面的有效性。研究成功鉴定出古口腔微生物组，以及土壤与皮肤来源的污染物。总体而言，与未处理样本相比，EDTA处理法与紫外线B（UVB）照射处理法均可有效降低环境微生物类群的占比，并提升口腔微生物类群的占比。