Results

1. K.T. Ziesemer Abstract Materials & Methods Results The ancient oral microbiome consists of a complex, mineralized bacterial biofilm, which forms on the surfaces of teeth. Research by Warinner and co-authors (2014) has shown that biomolecules become entrapped in and are relatively well preserved in ancient dental calculus, although the effects of different environments and climates on preservation is not yet well known1. Targeted ampliconmetagenomics (16S rRNA V3) on dental calculus of a select group of post-medieval Dutch and pre-colonial Caribbean human skeletal remains has therefore been used to assess the DNA preservation. Overall, DNA yields are significantly higher in DNA that is extracted from dental calculus rather than dentine or bone. Furthermore, preliminary results of this study indicate that endogenous DNA preservation is better in the Dutch post-medieval rural site than in the pre-colonial Caribbean site, which shows signs of environmental contamination. • Samples and Sites • Dental calculus (Fig. 8) from a select group of postmedival Dutch rural (MB fig. 6; n=76) and pre-colonial Anse à la Gourde (Fig. 7; n=4) and pre-colonialLavoutte (Fig. 7; n=5) human skeletalremains. • Procedure • Decontamination, digestionanddecalcification(Fig. 9) • Phenol-chloroform extraction • V3 16S rRNAtargetedamplicon • IlluminaMiSeqsequencing Assessment of DNA preservation in dental calculus from post-medieval Dutch and pre-colonial Caribbean human skeletal remainsKirsten A. Ziesemer1, Menno L.P. Hoogland1, Christina G. Warinner2 & Corinne L. Hofman11. Leiden University, The Netherlands 2. University of Oklahoma, U.S.A. Fig.6 map of the Beemster, adaptedfromCentraal Bureau voor de Statistiek / Topografische Dienst Kadaster (2006) Fig.7 map of the Caribbean, by M.L.P. Hoogland Fig.8 dental calculus on MB and Caribbean samples Fig.13 Genus-level taxonomic analysis of bacteria present in MB and Caribbean dental calculus Conclusion Fig. 9 Digestionanddecalcification of dental calculus DNA yields are >10x higher for dental calculus than dentine or bone, even in tropical samples. Preliminary results indicate better DNA preservation in the Dutch post-medieval samples compared to the pre-colonial Caribbean samples. Nearly all (>98%) of Dutch samples contain >90% endogenous bacterial DNA, while only one third of Caribbean dental calculus samples contain at least 50% endogenous bacterial DNA. Although post-mortem soil contamination is high in the Caribbean samples, oral taxa are nevertheless present at >1% in all samples. Tropical environments pose challenges for endogenous DNA preservation. 16S rRNA V3 analysis is an effective screening tool for identifying well-preserved dental calculus samples for further analysis. Introduction Results The human microbiome is defined as the ecological community of commensal, symbioticandpathogenicmicro-organismsthat co- existwith human beings2. Research byWarinner et al. (2014) demonstrated the presence of oral microbiome biomolecules in dental calculus of human skeletalremains (Fig. 2, Hoechststainindicative of DNA). It is composed of mineralizedlayers of plaque (Fig.3), whichmayentrapmicroorganisms, food particlesand human biomolecules (Fig. 4). In samples testedto date, these biomolecules appeartobeminimallycontaminated (Fig. 5)3,4,5. This is the first large scalestudytoassess the preservation of DNA in human dental calculus. The human skeletalremainsoriginatedfromthreegeographicallocationsandtwo time periods. We targeted the variableregion 3 of the 16S gene of ribosomes. Thisregion is composed of a short (65 bp) fragment, fromwhich genus level information maybe deducted6. Moreover, as ribosomes have stayedfairlysimilarthroughouthistory, mutationsmightbeindicative of evolution6. Table 1 | DNA extraction yields References Warinner, C., Rodrigues, J. F. M., Vyas, R., Trachsel, C., Shved, N., Grossmann, J., ... & Cappellini, E. (2014). Pathogensand host immunity in the ancient human oralcavity. Nature genetics, 46(4), 336-344. Lederberg, J., & Mccray, A. (2001). The Scientist:'OmeSweet'Omics--A Genealogical Treasury of Words. The Scientist, 17(7). Hilson, S. (2008). Dental Pathology. In M. Katzenberg, & S. Saunders, Biological Anthropology of the Human Skeleton (pp. 301-340). Hoboken: John Wiley & Sons, Inc. Jin, Y., & Yip, H. (2002). Supragingival Calculus: Formation and Control. Critical Reviews in Oral Biology & Medicine, 915-922. Preus, H., Marvik, O., Selvig, K., & Bennike, P. (2011). Ancient bacterial DNA (aDNA) in dental calculus from archaeological human remains. Journal of Archaeological Science, 38, 1827-1831. Chakravorty, S., Helb, D., Burday, M., Connell, N., & Alland, D. (2007). A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. Journal of microbiological methods, 69(2), 330-339. Metcalf, J. L., Ursell, L. K., & Knight, R. (2014). Ancient human oral plaque preserves a wealth of biological data. Nature Genetics, 46(4), 321-323. Fig.10 Visualization of MB 16S rRNA V3 PCR products on 2% agarose gel Fig.11 Visualization of Caribbean 16S rRNA V3 PCR products on 2% agarose gel Acknowledgements This research has receivedfundingfrom the European Research Council under the European Union'sSeventh Framework Programme (FP7/2007-2013) / ERC grant agreement n° 319209 underthe direction of Prof. Dr. C.L. Hofman. Contact information: Kirsten A. Ziesemer k.a.ziesemer@arch.leidenuniv.nl Calculus DNA Calculus DNA Calculus DNA Dimer Dimer Dimer Fig.2 Adaptedfrom Warinner et al. (2014). HistologicalsectionwithHoechststain Fig.3 Adaptedfrom Warinner et al. (2014). Histologicalsectionwith Gram staining Fig.4 AdaptedfromMetcalf et al. (2014). Visualization of dental calculus content Fig.5 Adaptedfrom Warinner et al. (2014). EDS visualization of Ca (red) andsilicon (green) bp bp bp 0 0 0 180 180 180 280 280 280 480 480 480 880 880 880 2880 2880 2880 80 80 80 Fig.12 Sizedistribution of dental calculus shotgun DNA libraries (Bioanalyzer 2100) . Estimatedlibrarysize without adapters. Caribbean 1948 MB 454 MB 108