The importance of commensal microbes for human health is increasingly recognized [1][2][3][4][5] , yet the impacts of evolutionary changes in human diet and culture on commensal microbiota remain almost unknown. Two of the greatest dietary shifts in human evolution involved the adoption of carbohydrate-rich Neolithic (farming) diets 6,7 (beginning ~10,000 years BP 6,8 ), and the more recent advent of industrially processed flour and sugar (~1850) 9 . Here, we show that calcified dental plaque (dental calculus) on ancient teeth preserves a detailed genetic record throughout this period. Data from 34 early European skeletons indicate that the transition from hunter-gatherer to farming shifted the oral microbial community to a disease-associated configuration. The composition of oral microbiota remained surprisingly constant between Neolithic and Medieval times, after which (the now ubiquitous) cariogenic bacteria became dominant, apparently during the Industrial Revolution. Modern oral microbiota are markedly less diverse than historic Sequence data have been deposited in Genbank under accession ERP002107.The authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nat Genet. Author manuscript; available in PMC 2014 April 23. Commensal microbiota comprise the majority of cells in the body and play a key role in human health [1][2][3][4][5]10 . However, their evolution remains poorly understood, and detailed genetic records from commensal bacteria have yet to be recovered from the archaeological record. Dental calculus is ubiquitous in both present-day and ancient human populations 11 , and microscopic analysis has shown that it accurately preserves bacterial morphology over millennia [12][13][14] . Dental calculus develops when dental plaque, an extremely dense bacterial biofilm 15 , becomes mineralised with calcium phosphate 16 . Bacteria in calculus become locked in a crystalline matrix similar to bone 16 (Supplementary Figure 1), with deposits occurring both above and below the gum or gingiva (supra-and subgingivally) 17 . Calculus represents one of the few sources of preserved human and hominid microbiota, and genetic analysis has the potential to create a powerful new record of past dietary impacts, health changes, and oral pathogen genomic evolution deep into the past. In addition, oral bacteria are transferred vertically from the primary caregiver(s) in early childhood 18 and horizontally between family members later in life 18,19 , making archaeological dental calculus a potentially unique means of tracing population structure, movement and admixture between ancient cultures, as well as the spread of diseases.The increased consumption of domesticated cereals (wheat and barley in the Near East) beginning with the Neolithic was associated with a marked increase in prevalence of dental calculus and oral pathology 20 . These oral diseases include dental caries (tooth decay) 1 and periodontal disease (an infection causing damage to the supporting connective tissues of ...

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high‐throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no‐template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.

High-Throughput DNA Sequencing (HTS) technologies have changed the way in which we detect and assess DNA contamination in ancient DNA studies. Researchers use computational methods to mine the large quantity of sequencing data to detect characteristic patterns of DNA damage, and to evaluate the authenticity of the results. We argue that unless computational methods can confidently separate authentic ancient DNA sequences from contaminating DNA that displays damage patterns under independent decay processes, prevention and control of DNA contamination should remain a central and critical aspect of ancient human DNA studies. Ideally, DNA contamination can be prevented early on by following minimal guidelines during excavation, sample collection and/or subsequent handling. Contaminating DNA should also be monitored or minimised in the ancient DNA laboratory using specialised facilities and strict experimental procedures. In this paper, we update recommendations to control for DNA contamination from the field to the laboratory, in an attempt to facilitate communication between field archaeologists, anthropologists and ancient DNA researchers. We also provide updated criteria of ancient DNA authenticity for HTS-based studies. We are confident that the procedures outlined here will increase the retrieval of higher proportions of authentic genetic information from valuable archaeological human remains in the future.

No abstract