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Includes supplementary materials for the online appendix. Human populations underwent range contractions during the Last Glacial Maximum (LGM) which had lasting and dramatic effects on their genetic variation. The genetic ancestry of individuals associated with the post-LGM Magdalenian technocomplex has been interpreted as being derived from groups associated with the pre-LGM Aurignacian. However, both these ancestries differ from that of central European individuals associated with the chronologically intermediate Gravettian. Thus, the genomic transition from pre- to post-LGM remains unclear also in western Europe, where we lack genomic data associated with the intermediate Solutrean, which spans the height of the LGM. Here we present genome-wide data from sites in Andalusia in southern Spain, including from a Solutrean-associated individual from Cueva del Malalmuerzo, directly dated to ~23,000 cal yr BP. The Malalmuerzo individual carried genetic ancestry that directly connects earlier Aurignacian-associated individuals with post-LGM Magdalenian-associated ancestry in western Europe. This scenario differs from Italy, where individuals associated with the transition from pre- and post-LGM carry different genetic ancestries. This suggests different dynamics in the proposed southern refugia of Ice Age Europe and posits Iberia as a potential refugium for western European pre-LGM ancestry. More, individuals from Cueva Ardales, which were thought to be of Palaeolithic origin, date younger than expected and, together with individuals from the Andalusian sites Caserones and Aguilillas, fall within the genetic variation of the Neolithic, Chalcolithic and Bronze Age individuals from southern Iberia. We thank all members of the Archaeogenetics Department of the Max Planck Institute for Evolutionary Anthropology, especially the Population Genetics group, and we thank Paleogenomics Lab members from Instituto de Biología Evolutiva for the data discussion. We especially thank M. Hajdinjak and I. Olalde for valuable comments and suggestions. We thank G. Brandt, A. Wissgott, S. Clayton and K. Prüfer for sequencing and handling the raw data. We thank all the members of the general research project in Cueva de Ardales and Sima de las Palomas de Teba. We thank all colleagues, archaeologists, geologists and experts in different scientific disciplines, as well as students from the Universities of Cádiz, Cologne and Tübingen, who have participated in the field and laboratory works. We are grateful for the great collaboration of the Town Councils of Ardales and Teba, within the framework of collaboration agreements with the University of Cadiz and the Neanderthal Museum. This work was supported by the Max Planck Society (V.V.-M., A.B.R., J.K. and W.H.), Margarita Salas 2022 funded by Unión Europea-Next Generation EU (V.V.-M.) and the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 771234-PALEoRIDER to W.H. and no. 803147-RESOLUTION to S.T.). The information provided on recent dating and archaeological records from Cueva de Ardales is part of the General Research Project, authorized by the Consejería de Cultura y Patrimonio Histórico de la Junta de Andalucía: entitled ‘Prehistoric societies (from the Middle Palaeolithic to the Final Neolithic) in the Cueva de Ardales and Sima de las Palomas de Teba (Málaga, Spain)’. A geoarchaeological, chronological and environmental study was conducted under the direction of J.R.-M. and G.-C.W. between 2015 and 2021. Excavations and field and laboratory studies have been carried out in Cueva de Ardales within the framework of the projects entitled ‘Analysis of prehistoric societies from the Middle Palaeolithic to the Late Neolithic on the two shores of the Strait of Gibraltar’. Relaciones y contactos—HAR2017-8734P (Ministerio de Economía, Industria y Competitividad-Agencia Estatal de Investigación, cofinanced by FEDER funds) for which J.R.-M. and Salvador Domínguez-Bella are the responsible researchers. Collaborative Research Centre—CRC 806 Our Way to Europe, funded by the Deutsche Forschungsgemeinschaft (DFG-German Research Foundation). Project no. 57444011, under the responsibility of G.-C.W.

Ludwig van Beethoven (1770–1827) remains among the most influential and popular classical music composers. Health problems significantly impacted his career as a composer and pianist, including progressive hearing loss, recurring gastrointestinal complaints, and liver disease. In 1802, Beethoven requested that following his death, his disease be described and made public. Medical biographers have since proposed numerous hypotheses, including many substantially heritable conditions. Here we attempt a genomic analysis of Beethoven in order to elucidate potential underlying genetic and infectious causes of his illnesses. We incorporated improvements in ancient DNA methods into existing protocols for ancient hair samples, enabling the sequencing of high-coverage genomes from small quantities of historical hair. We analyzed eight independently sourced locks of hair attributed to Beethoven, five of which originated from a single European male. We deemed these matching samples to be almost certainly authentic and sequenced Beethoven’s genome to 24-fold genomic coverage. Although we could not identify a genetic explanation for Beethoven's hearing disorder or gastrointestinal problems, we found that Beethoven had a genetic predisposition for liver disease. Metagenomic analyses revealed furthermore that Beethoven had a hepatitis B infection during at least the months prior to his death. Together with the genetic predisposition and his broadly accepted alcohol consumption, these present plausible explanations for Beethoven’s severe liver disease, which culminated in his death. Unexpectedly, an analysis of Y chromosomes sequenced from five living members of the Van Beethoven patrilineage revealed the occurrence of an extra-pair paternity event in Ludwig van Beethoven’s patrilineal ancestry. Introduction Results - Authentication of hair samples - Genetic variants associated with somatic disease - Hepatitis B virus DNA recovered from Beethoven’s hair Discussion - Authenticity of Beethoven’s genome - Comparison with living individuals’ genomes - Origins of Beethoven’s diseases - Hepatitis B virus DNA in Beethoven’s hair - Future directions

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.

Human expansion in the course of the Neolithic transition in western Eurasia has been one of the major topics in ancient DNA research in the last 10 years. Multiple studies have shown that the spread of agriculture and animal husbandry from the Near East across Europe was accompanied by large-scale human expansions. Moreover, changes in subsistence and migration associated with the Neolithic transition have been hypothesized to involve genetic adaptation. Here, we present high quality genome-wide data from the Linear Pottery Culture site Derenburg-Meerenstieg II (DER) (N = 32 individuals) in Central Germany. Population genetic analyses show that the DER individuals carried predominantly Anatolian Neolithic-like ancestry and a very limited degree of local hunter-gatherer admixture, similar to other early European farmers. Increasing the Linear Pottery culture cohort size to ∼100 individuals allowed us to perform various frequency- and haplotype-based analyses to investigate signatures of selection associated with changes following the adoption of the Neolithic lifestyle. In addition, we developed a new method called Admixture-informed Maximum-likelihood Estimation for Selection Scans that allowed us test for selection signatures in an admixture-aware fashion. Focusing on the intersection of results from these selection scans, we identified various loci associated with immune function (JAK1, HLA-DQB1) and metabolism (LMF1, LEPR, SORBS1), as well as skin color (SLC24A5, CD82) and folate synthesis (MTHFR, NBPF3). Our findings shed light on the evolutionary pressures, such as infectious disease and changing diet, that were faced by the early farmers of Western Eurasia. Introduction New Approaches Results and discussion - Data generation and authentication. - Genome-wide data analysis. - Uniparentally inherited markers. - Biological Relatedness. - LBK inter-site comparisons. - Phenotypic analysis. - HLA class-I and II allele analysis. - Selection Scans Conclusions Material and methods - Archaeological background/site description. - Sample preparation. - Genotyping. - F-statistics - Biological Relatedness and ROH Analyses. - Mitochondrial and Y haplotype analysis. - Shotgun data. - HLA typing. - Selection scans. - AIMLESS Admixture-Informed Maximum-Likelihood Estimation for Selection Scans. - Haplotype-based scan.

[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.

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.

Here, we present a protocol which enables the comprehensive characterization of the RNA content of single phase-separated coacervates. We adapted single-cell RNA sequencing technology in combination with fluorescence activated cell sorting (FACS) to answer the question of how one condensate differs from the other in terms of RNA composition and how it relates to condensate features such as droplet size. This approach represents a powerful addition to labor intensive and low throughput microscopy approaches which have been the state of the art approach for coacervate RNA characterization. This protocol includes droplet production, as well as a Smart-seq2 protocol adaption for lysis, reverse transcription and cDNA amplification and sequencing library preparation. This protocol ends with the library preparation. Afterwards it got sequenced on an Illumina NextSeq500 (paired end for 300 cycles). The Smart-seq2 protocol was originally published in Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc9, 171–181 (2014). https://doi.org/10.1038/nprot.2014.006

Protocol for DNA extraction from modern dental plaque dried on gauze samples for Illumina sequencing. This protocol uses the Qiagen PowerSoil DNA extraction kit, but applies modifications to improve DNA recovery. Specifically, solution C2 is not used to avoid uneccessary DNA loss during inhibitor removal.

Archaeogenetic time transect in Europe unravels genetic and social changes before and after the arrival of “steppe” ancestry., Europe’s prehistory oversaw dynamic and complex interactions of diverse societies, hitherto unexplored at detailed regional scales. Studying 271 human genomes dated ~4900 to 1600 BCE from the European heartland, Bohemia, we reveal unprecedented genetic changes and social processes. Major migrations preceded the arrival of “steppe” ancestry, and at ~2800 BCE, three genetically and culturally differentiated groups coexisted. Corded Ware appeared by 2900 BCE, were initially genetically diverse, did not derive all steppe ancestry from known Yamnaya, and assimilated females of diverse backgrounds. Both Corded Ware and Bell Beaker groups underwent dynamic changes, involving sharp reductions and complete replacements of Y-chromosomal diversity at ~2600 and ~2400 BCE, respectively, the latter accompanied by increased Neolithic-like ancestry. The Bronze Age saw new social organization emerge amid a ≥40% population turnover.

Significance The microbiome plays key roles in human health, but little is known about its evolution. We investigate the evolutionary history of the African hominid oral microbiome by analyzing dental biofilms of humans and Neanderthals spanning the past 100,000 years and comparing them with those of chimpanzees, gorillas, and howler monkeys. We identify 10 core bacterial genera that have been maintained within the human lineage and play key biofilm structural roles. However, many remain understudied and unnamed. We find major taxonomic and functional differences between the oral microbiomes of Homo and chimpanzees but a high degree of similarity between Neanderthals and modern humans, including an apparent Homo-specific acquisition of starch digestion capability in oral streptococci, suggesting microbial coadaptation with host diet., The oral microbiome plays key roles in human biology, health, and disease, but little is known about the global diversity, variation, or evolution of this microbial community. To better understand the evolution and changing ecology of the human oral microbiome, we analyzed 124 dental biofilm metagenomes from humans, including Neanderthals and Late Pleistocene to present-day modern humans, chimpanzees, and gorillas, as well as New World howler monkeys for comparison. We find that a core microbiome of primarily biofilm structural taxa has been maintained throughout African hominid evolution, and these microbial groups are also shared with howler monkeys, suggesting that they have been important oral members since before the catarrhine–platyrrhine split ca. 40 Mya. However, community structure and individual microbial phylogenies do not closely reflect host relationships, and the dental biofilms of Homo and chimpanzees are distinguished by major taxonomic and functional differences. Reconstructing oral metagenomes from up to 100 thousand years ago, we show that the microbial profiles of both Neanderthals and modern humans are highly similar, sharing functional adaptations in nutrient metabolism. These include an apparent Homo-specific acquisition of salivary amylase-binding capability by oral streptococci, suggesting microbial coadaptation with host diet. We additionally find evidence of shared genetic diversity in the oral bacteria of Neanderthal and Upper Paleolithic modern humans that is not observed in later modern human populations. Differences in the oral microbiomes of African hominids provide insights into human evolution, the ancestral state of the human microbiome, and a temporal framework for understanding microbial health and disease.

Description, Ancient human DNA from southern Iberia reveals social and genomic changes during the emergence of the Bronze Age in Europe., The emerging Bronze Age (BA) of southeastern Iberia saw marked social changes. Late Copper Age (CA) settlements were abandoned in favor of hilltop sites, and collective graves were largely replaced by single or double burials with often distinctive grave goods indirectly reflecting a hierarchical social organization, as exemplified by the BA El Argar group. We explored this transition from a genomic viewpoint by tripling the amount of data available for this period. Concomitant with the rise of El Argar starting ~2200 cal BCE, we observe a complete turnover of Y-chromosome lineages along with the arrival of steppe-related ancestry. This pattern is consistent with a founder effect in male lineages, supported by our finding that males shared more relatives at sites than females. However, simple two-source models do not find support in some El Argar groups, suggesting additional genetic contributions from the Mediterranean that could predate the BA.

Hepatitis B virus (HBV) has been infecting humans for millennia and remains a global health problem, but its past diversity and dispersal routes are largely unknown. We generated HBV genomic data from 137 Eurasians and Native Americans dated between ~10,500 and ~400 years ago. We date the most recent common ancestor of all HBV lineages to between ~20,000 and 12,000 years ago, with the virus present in European and South American hunter-gatherers during the early Holocene. After the European Neolithic transition, Mesolithic HBV strains were replaced by a lineage likely disseminated by early farmers that prevailed throughout western Eurasia for ~4000 years, declining around the end of the 2nd millennium BCE. The only remnant of this prehistoric HBV diversity is the rare genotype G, which appears to have reemerged during the HIV pandemic., The research was funded by the Max Planck Society, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (771234–PALEoRIDER, to W.H.; 805268–CoDisEASe to K. Bos; 834616–ARCHCAUCASUS to S.H.), the Slovak Academy of Sciences and the European Union’s Seventh Framework Programme and Marie Curie Actions under the Programme SASPRO (1340/03/03 to P.C.R.), the ERA.NET RUS Plus–S&T programm of the European Union’s Seventh Framework Programme (277–BIOARCCAUCASUS to S.Re. and S.H.), the Werner Siemens Stiftung (“Paleobiochemistry”, to CW), the Award Praemium Academiae of the Czech Academy of Sciences (to M.E.), the Institute of Archaeology of the Czech Academy of Sciences (RVO 67985912, to M.Dobe.), the Russian Foundation for Basic Research (19-09-00354a, to M.K.K. and V.V.K.; 19-78-10053 to SSh), the German Research Foundation (DFG-HA-5407/4-1–INTERACT to W.H. and RE2688/2 to S.Re.), the French National Research Agency (ANR-17-FRAL-0010–INTERACT, to M.F.D., M.Ri., S.Ro., S.Sai., D.Bi., and P.Le.), the Wenner-Gren Dissertation Fieldwork Grant (9558 to S.Sab.), and the Ministry of Education and Science of the Republic of Kazakhstan (AP08856654 to L.B.D., L.M., and E.Kh. and AP08857177 to A.Z.B.).

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 describes how to obtain bone powder from the pars petrosa of disarticulated ossis temporalis, specifically from the dense parts around the cochlea, in a minimally-invasive way by drilling from the outside. The pars petrosa has been shown to consistently yield high amounts of aDNA (Pinhasi et al. 2015 PLoS One, doi: 10.1371/journal.pone.0129102).

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing. This produces adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols.

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols. This protocol does not include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA) with initial USER enzyme treatment. This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream indexing protocols. The initial treatment with UDG (USER) removes molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448) and Briggs et al. (2010) Nucleic Acids Research 38 (6) (doi: 10.1093/nar/gkp1163)

Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from archaeological dental calculus, modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110) and adapted for dental calculus by Mann et al. (2018) Scientific Reports (doi: https://doi.org/10.1038/s41598-018-28091-9). This protocol includes an optional acetone wash step to remove conservation consolidants (e.g. glue) from dental calculus that can sometimes be found on samples archived in museum collections.

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA) with initial USER enzyme treatment. This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols. The initial treatment with UDG (USER) removes molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448) and Briggs et al. (2010) Nucleic Acids Research 38 (6) (doi: 10.1093/nar/gkp1163) This protocol has been optimised for reduced DNA input, to allow building of multiple libraries from a singe extract.

Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from archaeological dental calculus, modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110) and adapted for dental calculus by Mann et al. (2018) Scientific Reports (doi: https://doi.org/10.1038/s41598-018-28091-9).

This protocol describes the amplification procedure of dual-indexed double-stranded DNA libraries, for shotgun Illumina sequencing. It is typically used for libraries indexed using the following protocol: (https://dx.doi.org/10.17504/protocols.io.bakticwn)

Adapter preparation protocol for doubble-stranded ancient DNA libraries for Illumina Next-Generation-Sequencing, based on Meyer and Kircher et al. (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448). The protocol describes the production of an adapter mix for 2000 libraries. This protocol is used in conjunction with Meyer and Kircher-based ancient DNA libraries and is described accordingly (see library construction like: Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing).

Southern Italy is a key region for understanding the agricultural transition in the Mediterranean due to its central position. We present a genomic transect for 19 prehistoric Sicilians that covers the Early Mesolithic to Early Neolithic period. We find that the Early Mesolithic hunter-gatherers (HGs) are a highly drifted sister lineage to Early Holocene western European HGs, whereas a quarter of the Late Mesolithic HGs ancestry is related to HGs from eastern Europe and the Near East. This indicates substantial gene flow from (south-)eastern Europe between the Early and Late Mesolithic. The Early Neolithic farmers are genetically most similar to those from the Balkan and Greece, and carry only a maximum of ∼7% ancestry from Sicilian Mesolithic HGs. Ancestry changes match changes in dietary profile and material culture, except for two individuals who may provide tentative initial evidence that HGs adopted elements of farming in Sicily.One-sentence summaryGenome-wide and isotopic data from prehistoric Sicilians reveal a pre-farming connection to (south-) eastern Europe, and tentative initial evidence that hunter-gatherers adopted some Neolithic aspects prior to near-total replacement by early farmers.

Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols. This protocol does not include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).

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.

Protocol for DNA extraction from modern dental calculus samples for Illumina sequencing. This protocol uses the Qiagen PowerSoil DNA extraction kit.

Dental calculus, calcified oral plaque biofilm, contains microbial and host biomolecules that can be used to study historic microbiome communities and host responses. Dental calculus does not typically accumulate as much today as historically, and clinical oral microbiome research studies focus primarily on living dental plaque biofilm. However, plaque and calculus reflect different conditions of the oral biofilm, and the differences in microbial characteristics between the sample types have not yet been systematically explored. Here, we compare the microbial profiles of modern dental plaque, modern dental calculus, and historic dental calculus to establish expected differences between these substrates. - Background - Results -- Authentication of a preserved oral biofilm in calculus samples -- Dental calculus and plaque biofilm communities are distinct -- Health-associated communities of dental plaque and calculus are distinct -- Signatures of health and of disease are shared in modern and historic calculus samples -- Microbial community differences between health and disease in calculus are poorly resolved -- Absence of caries-specific microbial profiles in dental calculus -- Microbial co-exclusion patterns in plaque and calculus reflect biofilm maturity -- Microbial complexes in plaque and calculus -- Functional prediction in calculus is poorly predictive of health status -- Proteomic profiles of historic healthy site calculus -- Correlations between taxonomic, proteomic, and metabolomic profiles - Discussion - Conclusions - Materials and methods --Historic and modern calculus sample collection DNA extraction -- DNA library construction and high-throughput sequencing -- DNA sequence processing -- Genetic assessment of historic calculus sample preservation -- Genetic microbial taxonomic profiling -- Principal component analysis -- Assessment of differentially abundant taxa -- Sparse partial least squares-discriminant analysis -- Assessment of microbial co-exclusion patterns -- Gene functional categorization with SEED -- Proteomics -- Metabolomics -- Regularized canonical correlation analysis