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Major advances over the past decade in the field of ancient DNA are providing access to past paleogenomic diversity, but the diverse functions and biosynthetic capabilities of this growing paleome remain largely elusive. We investigated the dental calculus of 12 Neanderthals and 52 anatomically modern humans ranging from 100,000 years ago to the present and reconstructed 459 bacterial metagenome-assembled genomes. We identified a biosynthetic gene cluster shared by seven Middle and Upper Paleolithic individuals that allows for the heterologous production of a class of previously unknown metabolites that we name “paleofurans.” This paleobiotechnological approach demonstrates that viable biosynthetic machinery can be produced from the preserved genetic material of ancient organisms, allowing access to natural products from the Pleistocene and providing a promising area for natural product exploration.

Dental calculus is increasingly used by researchers to study dietary patterns in past populations. The benefits of using dental calculus for this purpose have been clearly demonstrated in previous studies, with dental calculus harbouring a wealth of microremains and biomarkers for health and diet within its mineral matrix. Previous studies have demonstrated some of the limitations and biases of how methods of processing may overlook, or even remove, some of the important information contained within the mineralised matrix. However, there are many factors that are impossible to account for *in vivo* and in archaeological material, such as exact dietary intake, and individual factors such as pH and enzyme activity, leaving some limitations that may not be addressed through these types of studies and will require a different approach. We present a protocol for creating a calcifying oral biofilm model that can be used to explore the biases and limitations of dental calculus as a medium for paleodietary reconstructions. We report the microbial and mineral composition of our model in an effort to validate the model calculus as an appropriate proxy to natural dental calculus. The microbial profile and species diversity of our model was determined using metagenomic classification with the nf-core/eager pipeline and Kraken2, and compared to various reference samples from oral sites, including saliva, plaque, and dental calculus. We then assessed whether our model calculus mineralises in a manner similar to natural dental calculus using Fourier transform infrared (FTIR) spectroscopy. The metagenomic classification showed a microbial profile predominantly made up of (facultative) anaerobes, with a community structure that was somewhat distinct from other oral reference samples. The core genera of the model consisted of oral species, but clustered separately from oral reference samples, with a higher abundance of anaerobes. Mineral and organic components of our model mimic that of the modern and archaeological reference calculus that was used as a comparison. There was an overall increase in the inorganic component relative to organic over the course of the experiment, with carbonated hydroxyapatite as the principal compound, consistent with natural human-derived calculus. We conclude that oral biofilm models, such as the one presented in this study, have great potential to validate current methods used in the analysis of archaeological dental calculus, and should be used to complement, rather than replace current *in vivo* studies.

The Xiongnu established the first nomadic imperial power, controlling the Eastern Eurasian steppe from ca. 200 BCE to 100 CE. Recent archaeogenetic studies identified extreme levels of genetic diversity across the empire, corroborating historical records of the Xiongnu Empire being multiethnic. However, it has remained unknown how this diversity was structured at the local community level or by sociopolitical status. To address this, we investigated aristocratic and local elite cemeteries at the western frontier of the empire. Analyzing genome-wide data from 18 individuals, we show that genetic diversity within these communities was comparable to the empire as a whole, and that high diversity was also observed within extended families. Genetic heterogeneity was highest among the lowest-status individuals, implying diverse origins, while higher-status individuals harbored less genetic diversity, suggesting that elite status and power was concentrated within specific subsets of the broader Xiongnu population. Members of the Xiongnu Empire were genetically diverse, but elite status was concentrated within specific genetic subsets. Introduction Results - Generation of genome-wide data from Xiongnu aristocratic elites, local elites, and subordinates - Modeling Xiongnu ancestry - High genetic diversity within Xiongnu communities and across the empire - Genetic diversity and archaeological signifiers of social status - Genetic dynamics of the Xiongnu elite Discussion Materials and methods

The identity of the earliest inhabitants of Xinjiang, in the heart of Inner Asia, and the languages that they spoke have long been debated and remain contentious1. Here we present genomic data from 5 individuals dating to around 3000–2800 bc from the Dzungarian Basin and 13 individuals dating to around 2100–1700 bc from the Tarim Basin, representing the earliest yet discovered human remains from North and South Xinjiang, respectively. We find that the Early Bronze Age Dzungarian individuals exhibit a predominantly Afanasievo ancestry with an additional local contribution, and the Early–Middle Bronze Age Tarim individuals contain only a local ancestry. The Tarim individuals from the site of Xiaohe further exhibit strong evidence of milk proteins in their dental calculus, indicating a reliance on dairy pastoralism at the site since its founding. Our results do not support previous hypotheses for the origin of the Tarim mummies, who were argued to be Proto-Tocharian-speaking pastoralists descended from the Afanasievo1,2 or to have originated among the Bactria–Margiana Archaeological Complex3 or Inner Asian Mountain Corridor cultures4. Instead, although Tocharian may have been plausibly introduced to the Dzungarian Basin by Afanasievo migrants during the Early Bronze Age, we find that the earliest Tarim Basin cultures appear to have arisen from a genetically isolated local population that adopted neighbouring pastoralist and agriculturalist practices, which allowed them to settle and thrive along the shifting riverine oases of the Taklamakan Desert., A genomic analysis of human remains from the Bronze Age provides insights into the origin of the Tarim Basin mummies from the Xinjiang region.

Recent discoveries on the importance of microbes for human biology, health, and culture, the rise of antimicrobial resistance, and developing technological advancements necessitate new dialogues about human relationships with microbes. Long perceptible only through their transformations—from epidemic disease to alcoholic beverages—it is now possible to more fully perceive the diversity of ways in which we influence and are influenced by microbes and to understand that human and microbial cultures are fundamentally intertwined. In the introduction to this supplement, we outline the current state of the art of an “anthropology of microbes” in three subfields of anthropology: biological anthropology, cultural anthropology, and archaeology. Moreover, as a result of dialogues borne out of the symposium associated with this issue, and now reflected in the articles themselves, we discuss the interactions between and within the subfields of anthropology. This supplement is committed to the development of a common language for an emerging anthropology of microbes, and in order to shape genuine transdisciplinarity we argue for the continued necessity of “trading zone” points of intersection— such as the Wenner-Gren Foundation’s symposium “Cultures of Fermentation.”

The terrestrial subsurface houses a significant proportion of the Earth’s microbial biomass. Our understanding about terrestrial subsurface microbiomes is almost exclusively derived from groundwater and porous sediments. To obtain more insights about endolithic microbiomes and their metabolic status, we investigated rock samples from the vadose zone, fractured aquifers, and deep aquitards. Using methods from paleogenomics, we recovered sufficient DNA for metagenomics from rock specimens independent of porosity, lithology, and depth. We estimated between 2.81 and 4.25 × 105 cells × g−1 rock. DNA damage patterns revealed paleome signatures (genetic records of past microbial communities) for three rock specimens from the vadose zone. The taxonomy and functional potential of paleome communities revealed increased abundances of chemolithoautotrophs, and a broader metabolic potential for aromatic hydrocarbon breakdown. Our study suggests that limestones represent ideal archives for genetic records of past microbial communities, due to their specific conditions facilitating long-term DNA preservation.

Industrialization - including urbanization, participation in the global food chain, and consumption of heavily processed foods - is thought to drive substantial shifts in the human microbiome. While diet strongly influences stool microbiome composition, the influence of diet on the oral microbiome, and particularly dental plaque, is largely speculative. Here we investigated whether dental plaque microbial communities are distinctly different across populations with dissimilar subsistence strategies and degree of industrialized market integration. Using a metagenomic approach, we compared the dental plaque microbiomes of Baka foragers and Nzime subsistence agriculturalists in Cameroon (n = 46) with the dental plaque and calculus microbiomes of highly industrialized populations in North America and Europe. We found that differences in microbial taxonomic composition between populations were minimal, with high conservation of abundant microbial taxa and no significant differences in microbial diversity related to dietary practices. Instead, we find that the major source of variation in dental plaque microbial species composition is related to tooth location and oxygen availability, and may be influenced by toothbrushing or other dental hygiene measures. Our results support that dental plaque, in contrast to the stool microbiome, maintains an inherent stability against ecological perturbations in the oral environment.

Host-associated microbiomes are essential for a multitude of biological processes. Placed at the contact zone between external and internal environments, the little-studied oral microbiome has important roles in host physiology and health. Here we investigate the contribution of host evolutionary relationships and ecology in shaping the oral microbiome in three closely related gorilla subspecies (mountain, Grauer’s, and western lowland gorillas) using shotgun metagenomics of 46 museum-preserved dental calculus samples. We find that the oral microbiomes of mountain gorillas are functionally and taxonomically distinct from the other two subspecies, despite close evolutionary relationships and geographic proximity with Grauer’s gorillas. Grauer’s gorillas show intermediate bacterial taxonomic and functional, and dietary profiles. Altitudinal differences in gorilla subspecies ranges appear to explain these patterns, proposing a close connection between dental calculus microbiome and the environment, which is further supported by the presence of gorilla subspecies-specific phyllosphere/rhizosphere taxa. Mountain gorillas show high abundance of nitrate-reducing oral taxa, which may contribute to high altitude adaptation by modulating blood pressure. Our results suggest that ecology, rather than evolutionary relationships and geographic proximity, primarily shape the oral microbiome in these closely related species.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague’s formative years in terms of its early evolution and ecology.

©. This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by/4.0/ This document is the Published manuscript version of a Published Work that appeared in final form in FEMS Microbes. To access the final edited and published work see DOI: 10.1093/femsmc/xtac006 The oral cavity is a heterogeneous environment, varying in factors such as pH, oxygen levels, and salivary flow. These factors affect the microbial community composition and distribution of species in dental plaque, but it is not known how well these patterns are reflected in archaeological dental calculus. In most archaeological studies, a single sample of dental calculus is studied per individual and is assumed to represent the entire oral cavity. However, it is not known if this sampling strategy introduces biases into studies of the ancient oral microbiome. Here, we present the results of a shotgun metagenomic study of a dense sampling of dental calculus from four Chalcolithic individuals from the southeast Iberian peninsula (ca. 4500–5000 BP). Interindividual differences in microbial composition are found to be much larger than intraindividual differences, indicating that a single sample can indeed represent an individual in most cases. However, there are minor spatial patterns in species distribution within the oral cavity that should be taken into account when designing a study or interpreting results. Finally, we show that plant DNA identified in the samples is likely of postmortem origin, demonstrating the importance of including environmental controls or additional lines of biomolecular evidence in dietary interpretations.

Paleoproteomics, the study of ancient proteins, is a rapidly growing field at the intersection of molecular biology, paleontology, archaeology, paleoecology, and history. Paleoproteomics research leverages the longevity and diversity of proteins to explore fundamental questions about the past. While its origins predate the characterization of DNA, it was only with the advent of soft ionization mass spectrometry that the study of ancient proteins became truly feasible. Technological gains over the past 20 years have allowed increasing opportunities to better understand preservation, degradation, and recovery of the rich bioarchive of ancient proteins found in the archaeological and paleontological records. Growing from a handful of studies in the 1990s on individual highly abundant ancient proteins, paleoproteomics today is an expanding field with diverse applications ranging from the taxonomic identification of highly fragmented bones and shells and the phylogenetic resolution of extinct species to the exploration of past cuisines from dental calculus and pottery food crusts and the characterization of past diseases. More broadly, these studies have opened new doors in understanding past human-animal interactions, the reconstruction of past environments and environmental changes, the expansion of the hominin fossil record through large scale screening of nondiagnostic bone fragments, and the phylogenetic resolution of the vertebrate fossil record. Even with these advances, much of the ancient proteomic record still remains unexplored. Here we provide an overview of the history of the field, a summary of the major methods and applications currently in use, and a critical evaluation of current challenges. We conclude by looking to the future, for which innovative solutions and emerging technology will play an important role in enabling us to access the still unexplored "dark" proteome, allowing for a fuller understanding of the role ancient proteins can play in the interpretation of the past.

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.Research HighlightsNo associations between calculus species profiles and oral health metrics were detected in a single large populationA minority of individuals have a dental calculus species profile characterized by low levels of Streptococcus and high levels of anaerobic taxa

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

[Significance] The bacterium Yersinia pestis has caused numerous historically documented outbreaks of plague and research using ancient DNA could demonstrate that it already affected human populations during the Neolithic. However, the pathogen’s genetic diversity, geographic spread, and transmission dynamics during this early period of Y. pestis evolution are largely unexplored. Here, we describe a set of ancient plague genomes up to 5,000 y old from across Eurasia. Our data demonstrate that two genetically distinct forms of Y. pestis evolved in parallel and were both distributed across vast geographic distances, potentially occupying different ecological niches. Interpreted within the archeological context, our results suggest that the spread of plague during this period was linked to increased human mobility and intensification of animal husbandry., The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague’s formative years in terms of its early evolution and ecology., This study was funded by the Max Planck Society, Max Planck Harvard Research Center for the Archaeoscience of the Ancient Mediterranean and the European Research Council under the European Union’s Horizon 2020 research and innovation program under Grant Agreement 771234 – PALEoRIDER (to W.H.), 856453 – HistoGenes (to J.K.), and 834616 – ARCHCAUCASUS (to S.H.). The Heidelberg Academy of Science financed the genetic and archeological research on human individuals from the Augsburg region within the project WIN Kolleg: “Times of Upheaval: Changes of Society and Landscape at the Beginning of the Bronze Age. M.E. was supported by the award “Praemium Academiae” of the Czech Academy of Sciences. M.D. was supported by the project RVO 67985912 of the Institute of Archaeology of the Czech Academy of Sciences, Prague. I.O. was supported by the Ramón y Cajal grant from Ministerio de Ciencia e Innovación, Spanish Government (RYC2019-027909-I). A. H€ubner was supported by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy (EXC 2051 – Project-ID 390713860). J.F.-E. and J.A.M.-A. were supported by the Diputación Foral de Alava, IT 1223-19, Gobierno Vasco. A. Buzhilova was supported by the Center of Information Technologies and Systems (CITIS), Moscow, Russia 121041500329-0. L. M., L.B.D., and E. Khussainova were supported by the Grant AP08856654, Ministry of Education and Science of the Republic of Kazakhstan. A. Beisenov was supported by the Grant AP08857177, Ministry of Education and Science of the Republic of Kazakhstan.

We are pleased to see Ben-Dor et al.’s interest (1) in our study on the evolution and changing ecology of the hominid oral microbiome (2), which finds that starch-adapted oral bacteria are characteristic of the oral microbiota of Homo . However, in their critique, Ben-Dor et al. mischaracterize our findings by asserting claims not made in our article, and their conclusion that genetic adaptations in host microbiota have no implication for understanding hominin diets reveals misunderstandings about microbial evolution and the mechanisms of amylase-binding proteins. Ben-Dor et al. dismiss out of hand our findings of bacterial starch adaptation … [↵][1]1To whom correspondence may be addressed. Email: warinner{at}fas.harvard.edu. [1]: #xref-corresp-1-1

The oral cavity is a heterogeneous environment, varying in factors such as pH, oxygen levels, and salivary flow. These factors affect the microbial community composition and distribution of species in dental plaque, but it is not known how well these patterns are reflected in archaeological dental calculus. In most archaeological studies, a single sample of dental calculus is studied per individual and is assumed to represent the entire oral cavity. However, it is not known if this sampling strategy introduces biases into studies of the ancient oral microbiome. Here, we present the results of a shotgun metagenomic study of a dense sampling of dental calculus from four Chalcolithic individuals from the southeast Iberian peninsula (ca. 4500-5000 BP). Inter-individual differences in microbial composition are found to be much larger than intra-individual differences, indicating that a single sample can indeed represent an individual in most cases. However, there are minor spatial patterns in species distribution within the oral cavity that should be taken into account when designing a study or interpreting results. Finally, we show that plant DNA identified in the samples may be of environmental origin, showing the importance of including environmental controls or several lines of biomolecular evidence.

Incomplete documentary evidence, variable biomolecular preservation, and limited skeletal responses have hindered assessment of acute infections in the past. This study was initially developed to explore the diagnostic potential of dental calculus to identify infectious diseases, however, the breadth and depth of information gained from a particular individual, St. Louis Individual (St.LI), enabled an individualized assessment and demanded broader disciplinary introspection of ethical research conduct. Here, we document the embodiment of structural violence in a 23-year-old Black and/or African American male, who died of lobar pneumonia in 1930s St. Louis, Missouri. St.LI exhibits evidence of systemic poor health, including chronic oral infections and a probable tuberculosis infection. Metagenomic sequencing of dental calculus recovered three pre-antibiotic era pathogen genomes, which likely contributed to the lobar pneumonia cause of death (CoD): Klebsiella pneumoniae (13.8X); Acinetobacter nosocomialis (28.4X); and Acinetobacter junii (30.1X). Ante- and perimortem evidence of St.LI's lived experiences chronicle the poverty, systemic racism, and race-based structural violence experienced by marginalized communities in St. Louis, which contributed to St.LI's poor health, CoD, anatomization, and inclusion in the Robert J. Terry Anatomical Collection. These same embodied inequalities continue to manifest as health disparities affecting many contemporary communities in the United States.

DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences, but existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both on simulated aDNA data and archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, Pydamage opens up new doors to explore functional diversity in ancient metagenomic datasets.

This collection contains established ancient DNA (aDNA) specific protocols for Illumina next generation sequencing. It covers sampling of skeletal remains for regions preserving higher amounts of aDNA, DNA extraction methods designed for retrieval of very short DNA fragments, and various library construction methods (including treatments for the 'removal' of typical aDNA damage). All protocols have been adapted after published protocols (see each for the corresponding original publication), and are used primarily in the Department of Archaeogenetics at the MPI-SHH (Jena, Germany). All protocols in this collection can be used together, and in some cases swapped out with others. All protocols prior indexing are mostly performed in a dedicated ancient DNA laboratory - please see the guidelines section of each protocol for more information. The order of protocols is as follows: 1. Sampling for aDNA Sampling of Pars petrosa (os temporale, ''petrous bones') for human aDNA extraction Sampling of teeth for human and microbial (e.g. pathogen) aDNA extraction Sampling of dental calculus for oral microbiome aDNA extraction 2. Retrieval of DNA Ancient DNA Extraction from Skeletal Material (based on the Dabney et al. 2013 Proc Natl Acad Sci U S A protocol) Ancient DNA extraction from dental calculus (based on the Dabney et al. 2013 Proc Natl Acad Sci U S A with extra washing steps from Ozga et al. 2016 Am J Phys Anthropol) 3. Library Preparation Library Adapter Preparation for Dual-Index Double Stranded DNA Illumina Sequencing (based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) Non-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (requires 3.1, based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) Half-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (Variant of 3.2, with partial damage removal treatment from Rohland et al. 2015 Philos Trans R Soc Lond B Biol Sci) Full-UDG Treated double-stranded ancient DNA library preparation for Illumina sequencing (Variant of 3.2, with complete damage removal treatment from Briggs et al. 2010 Nucleic Acids Res) Dual-Indexing of aDNA libraries for Illumina Sequencing (based on Meyer and Kircher 2010 Cold Spring Harb. Protoc. protocol) 4. Preparation for Sequencing Amplification and Pooling for dual-indexed Illumina library for sequencing

Collection of protocols used for Fellows Yates et al. "The evolution and changing ecology of the hominid primate oral microbiome". Bioinformatics analysis can be found on GitHub at https://github.com/jfy133/Anthropoid_Calculus_Microbiome_Evolution/. This collection describes the laboratory procedures used for sampling, (ancient) DNA extraction, library construction and preparation for Illumina sequencing of ancient and modern dental calculus samples. Sampling - Dental calculus Field-Sampling Protocol (Warinner Version) - Dental Calculus Field-Sampling Protocol (Sabin version) Extraction - Ancient DNA Extraction from Dental Calculus with Consolidant Removal - DNA Extraction from Modern Dental Calculus Library preparation - Non-UDG treated double-stranded DNA library preparation for Illumina sequencing ofancient dental calculus - (Non-UDG treated) double-stranded modern dental calculus DNA library preparation for Illumina sequencing - Full-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing of ancient dental calculus (reduced input DNA) Indexing - Illumina double-stranded DNA dual indexing for ancient DNA Preparation for Sequencing - Amplification and Pooling

This protocol converts partially completed double-stranded DNA libraries e.g. from: Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (dx.doi.org/10.17504/protocols.io.bakricv6) into dual-indexed Illumina libraries ready for sequencing on an Illumina platform. Other variants of the above protocol can also be used. This protocol includes steps for indexing PCR, clean-up, and post-indexing qPCR. This protocol is modified after Kircher, M., Sawyer, S. & Meyer, M., 2012. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic acids research, 40(1), p.e3. http://dx.doi.org/10.1093/nar/gkr771.

The development and dispersal of agropastoralism transformed the cultural and ecological landscapes of the Old World, but little is known about when or how this process first impacted Central Asia. Here, we present archaeological and biomolecular evidence from Obishir V in southern Kyrgyzstan, establishing the presence of domesticated sheep by ca. 6,000 BCE. Zooarchaeological and collagen peptide mass fingerprinting show exploitation of Ovis and Capra, while cementum analysis of intact teeth implicates possible pastoral slaughter during the fall season. Most significantly, ancient DNA reveals these directly dated specimens as the domestic O. aries, within the genetic diversity of domesticated sheep lineages. Together, these results provide the earliest evidence for the use of livestock in the mountains of the Ferghana Valley, predating previous evidence by 3,000 years and suggesting that domestic animal economies reached the mountains of interior Central Asia far earlier than previously recognized. Results - Site excavation. - Archaeofaunal remains. - Cementum analysis and dental eruption/wear. - Animal DNA. Discussion Methods - Excavation. - Radiocarbon dating. - Thermoluminescence dating. - Zooarchaeology and ZooMS. - Cementum analysis. - DNA analysis. -- PCR amplification. -- Library construction and sequencing. -- PCA of genome-wide sequences. -- Mitochondrial genome analysis.