Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007–0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003–70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Dental calculus preserves oral microbes, enabling comparative studies of the oral microbiome and health through time. However, small sample sizes and limited dental health metadata have hindered health-focused investigations to date. Here we investigate the relationship between tobacco pipe smoking and dental calculus microbiomes. Dental calculus from 75 individuals from the 19th century Middenbeemster skeletal collection (Netherlands) were analyzed by metagenomics. Demographic and dental health parameters were systematically recorded, including the presence/number of pipe notches. Comparative data sets from European populations before and after the introduction of tobacco were also analyzed. Calculus species profiles were compared with oral pathology to examine associations between microbiome community, smoking behavior, and oral health status. The Middenbeemster individuals exhibited relatively poor oral health, with a high prevalence of periodontal disease, caries, heavy calculus deposits, and antemortem tooth loss. No associations between pipe notches and dental pathologies, or microbial species composition, were found. Calculus samples before and after the introduction of tobacco showed highly similar species profiles. Observed inter-individual microbiome differences were consistent with previously described variation in human populations from the Upper Paleolithic to the present. Dental calculus may not preserve microbial indicators of health and disease status as distinctly as dental plaque.

As data science and artificial intelligence continue to rapidly gain traction, the publication of freely available ICU datasets has become invaluable to propel data-driven clinical research. In this guide for clinicians and researchers, we aim to: 1) systematically search and identify all publicly available adult clinical ICU datasets, 2) compare their characteristics, data quality, and richness and critically appraise their strengths and weaknesses, and 3) provide researchers with suggestions, which datasets are appropriate for answering their clinical question.DATA SOURCES: A systematic search was performed in Pubmed, ArXiv, MedRxiv, and BioRxiv. STUDY SELECTION:We selected all studies that reported on publicly available adult patient-level intensive care datasets.DATA EXTRACTION: A total of four publicly available, adult, critical care, patientlevel databases were included (Amsterdam University Medical Center data base [AmsterdamUMCdb], eICU Collaborative Research Database eICU CRD], High time-resolution intensive care unit dataset [HiRID], and Medical Information Mart for Intensive Care-IV). Databases were compared using a priori defined categories, including demographics, patient characteristics, and data richness. The study protocol and search strategy were prospectively registered. DATA SYNTHESIS:Four ICU databases fulfilled all criteria for inclusion and were queried using SQL (PostgreSQL version 12; PostgreSQL Global Development Group) and analyzed using R (R Foundation for Statistical Computing, Vienna, Austria). The number of unique patient admissions varied between 23,106 (AmsterdamUMCdb) and 200,859 (eICU-CRD). Frequency of laboratory values and vital signs was highest in HiRID, for example, 5.2 (±3.4) lactate values per day and 29.7 (±10.2) systolic blood pressure values per hour. Treatment intensity varied with vasopressor and ventilatory support in 69.0% and 83.0% of patients in AmsterdamUMCdb versus 12.0% and 21.0% in eICU-CRD, respectively. ICU mortality ranged from 5.5% in eICU-CRD to 9.9% in AmsterdamUMCdb. CONCLUSIONS:We identified four publicly available adult clinical ICU datasets. Sample size, severity of illness, treatment intensity, and frequency of reported parameters differ markedly between the databases. This should guide clinicians and researchers which databases to best answer their clinical questions.

Objectives Dental calculus is among the richest known sources of ancient DNA in the archaeological record. Although most DNA within calculus is microbial, it has been shown to contain sufficient human DNA for the targeted retrieval of whole mitochondrial genomes. Here, we explore whether calculus is also a viable substrate for whole human genome recovery using targeted enrichment techniques. Materials and methods Total DNA extracted from 24 paired archaeological human dentin and calculus samples was subjected to whole human genome enrichment using in‐solution hybridization capture and high‐throughput sequencing. Results Total DNA from calculus exceeded that of dentin in all cases, and although the proportion of human DNA was generally lower in calculus, the absolute human DNA content of calculus and dentin was not significantly different. Whole genome enrichment resulted in up to four‐fold enrichment of the human endogenous DNA content for both dentin and dental calculus libraries, albeit with some loss in complexity. Recovering more on‐target reads for the same sequencing effort generally improved the quality of downstream analyses, such as sex and ancestry estimation. For nonhuman DNA, comparison of phylum‐level microbial community structure revealed few differences between precapture and postcapture libraries, indicating that off‐target sequences in human genome‐enriched calculus libraries may still be useful for oral microbiome reconstruction. Discussion While ancient human dental calculus does contain endogenous human DNA sequences, their relative proportion is low when compared with other skeletal tissues. Whole genome enrichment can help increase the proportion of recovered human reads, but in this instance enrichment efficiency was relatively low when compared with other forms of capture. We conclude that further optimization is necessary before the method can be routinely applied to archaeological samples.