Use of domesticated pigs by Mesolithic hunter-gatherers in northwestern Europe

Acknowledgements We thank the Archaeological State Museum Schleswig-Holstein, the Archaeological State Offices of Brandenburg, Lower Saxony and Saxony and the following individuals who provided sample material: Betty Arndt, Jo¨rg Ewersen, Frederick Feulner, Susanne Hanik, Ru¨diger Krause, Jochen Reinhard, Uwe Reuter, Karl-Heinz Ro¨hrig, Maguerita Scha¨fer, Jo¨rg Schibler, Reinhold Schoon, Regina Smolnik, Thomas Terberger and Ingrid Ulbricht. We are grateful to Ulrich Schmo¨lcke, Michael Forster, Peter Forster and Aikaterini Glykou for their support and comments on the manuscript. We also thank many institutions and individuals that provided sample material and access to collections, especially the curators of the Museum fu¨r Naturkunde, Berlin; Muse´um National d0 Histoire Naturelle, Paris; Smithsonian Institution, National Museum of Natural History, Washington D.C.; Zoologische Staatssammlung, Mu¨nchen; Museum fu¨r Haustierkunde, Halle; the American Museum of Natural History, New-York. This work was funded by the Graduate School ‘Human Development in Landscapes’ at Kiel University (CAU) and supported by NERC project Grant NE/F003382/1. Radiocarbon dating was carried out at the Leibniz Laboratory, CAU. This work is licensed under a Creative Commons AttributionNonCommercial-NoDerivs 3.0 Unported License.Peer reviewedPublisher PD

Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

BACKGROUND: Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. METHODOLOGY/PRINCIPAL FINDINGS: We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3'-end complementary to the template bacterial sequence and a 5'-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10-100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. CONCLUSIONS/SIGNIFICANCE: Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories

Scrapheap Challenge: A novel bulk-bone metabarcoding method to investigate ancient DNA in faunal assemblages

Highly fragmented and morphologically indistinct fossil bone is common in archaeological and paleontological deposits but unfortunately it is of little use in compiling faunal assemblages. The development of a cost-effective methodology to taxonomically identify bulk bone is therefore a key challenge. Here, an ancient DNA methodology using high-throughput sequencing is developed to survey and analyse thousands of archaeological bones from southwest Australia. Fossils were collectively ground together depending on which of fifteen stratigraphical layers they were excavated from. By generating fifteen synthetic blends of bulk bone powder, each corresponding to a chronologically distinct layer, samples could be collectively analysed in an efficient manner. A diverse range of taxa, including endemic, extirpated and hitherto unrecorded taxa, dating back to c.46,000 years BP was characterized. The method is a novel, cost-effective use for unidentifiable bone fragments and a powerful molecular tool for surveying fossils that otherwise end up on the taxonomic “scrapheap”

Phylogéographie au pléistocène et à l'holocène et domestication des équidés

Certains aspects de la phylogéographie des Equidés au Pléistocène et à l'Holocène présentent des zones d'ombres malgré de nombreuses études archéozoologiques et des analyses génétiques sur les populations modernes. L'analyse génétique de fossiles provenant d'Asie du sud-ouest et d'Europe nous a permis d'aborder cette question sous un angle différent. Afin de repousser les limites des analyses paléogénétiques, des méthodes de traitement de fossiles post-fouille, d'extraction et d'amplification de l'AD ont d'abord été optimisées, permettant d'améliorer le rendement et la qualité des résultats paléogénétiques. En utilisant ces méthodes, nous avons ainsi mis en évidence une structure phylogéographique marquée des hémiones en Asie du sud-ouest à l'Holocène. La caractérisation de ces dernières peut avoir une influence sur les plans de sauvegarde de cette espèce en voie de disparition. Nous avons également remis en question le statut d'espèce de l'hydrontin basé sur critères morphologiques, l'ADN mitochondrial des os de cette espèce paléontologique correspondant soit à l'hémione, soit au cheval selon l'origine géographique et la période des os analysés. Ces données suggèrent une forte plasticité morphologique des Équidés, leur permettant des adaptations à des conditions environnementales diverses en créant des écomorphotypes sans événement de spéciation. Finalement, nous avons pour la première fois mis en évidence la présence d'hybrides hémione x âne dans un contexte archéologique.Parts of the phylogeography of Equids during the Pleistocene and the Holocene are still unresolved despite a great number of archeozoological studies and genetic analyses of modern populations. We performed a genetic analysis of Equid archaeological bones from South-west Asia and Europe. To push back the limits of ancient DNA analyses and increase the reliability of paleogenetic studies, we optimized post-excavation treatments of fossil bones, and DNA extraction and amplification methods. Our data reveals a strong phylogeographic structure of the past Asiatic wild ass populations in South-west Asia. This result should be taken into consideration in the design of conservation projects for this endangered species. Our data also question the species status of the hydruntine, a palaeontological species, since on mitochondrial level the bones appear to correspond to either hemiones or horses depending on their geographic origin and the time period they lived in. These results suggest that the Equus species have a significant morphological plasticity allowing morphological adaptations to various environmental conditions and forming ecomorphotypes without speciation events. Finally, we show for the first time in an archaeological context the presence of hemione x donkey hybrids.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications.

BACKGROUND: PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. METHODOLOGY/PRINCIPAL FINDINGS: Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. CONCLUSIONS/SIGNIFICANCE: There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA

DNA diagenesis and palaeogenetic analysis : Critical assessment and methodological progress

International audiencePalaeogenetic data obtained from fossilizing or fossil bones and teeth are of great importance to studies of vertebrate evolution, human biological and cultural evolution, plant and animal domestication and reconstructions of palaeoenvironment and palaeoecology. These studies are based on the retrieval of previous termDNAnext term preserved in fossilizing bones and teeth. previous termDNAnext term is present in fossils, if at all, in only very small amounts, which makes its amplification with PCR necessary for detailed sequence analysis. Erroneous nucleotides can be incorporated during in vitro amplification either because of post-mortem base damage of the original previous termDNAnext term template or simply because the fidelity of previous termDNAnext term polymerases is not absolute and can be decreased by suboptimal buffer conditions or possibly by compounds in the fossil extracts. These erroneously introduced nucleotides can be mistaken for authentic mutations of the ancient sequence compared to the closest extant sequence. Moreover, contamination by modern previous termDNA,next term which is not chemically modified and therefore a better substrate for the Taq polymerase, can also lead to erroneous results. Here, we will present the procedures that we have developed in order to (i) ensure negligible mutagenicity of the PCR reaction, (ii) eliminate contamination by previous termDNAnext term molecules originating from previous PCR reactions and cloning procedures, (iii) prevent contamination with modern previous termDNAnext term of fossil bones and teeth during and after their excavation, and (iv) prevent degradation of ancient previous termDNAnext term after excavation. Finally, we will discuss our results on previous termDNAnext term preservation as a function of the taphonomy of the skeletal part that is analyzed and of the depositional context of preservation

The genetic identity of the earliest human-made hybrid animals, the kungas of Syro-Mesopotamia

International audienceBefore the introduction of domestic horses in Mesopotamia in the late third millennium BCE, contemporary cuneiform tablets and seals document intentional breeding of highly valued equids called kungas, for use in diplomacy, ceremony and warfare. Their precise zoological classification, however, has never been conclusively determined. Morphometric analysis of equids uncovered in rich Early Bronze Age burials at Umm el-Marra, Syria, placed them beyond the ranges reported for other known equid species. We sequenced the genomes of one of these ~4,500-year-old equids, together with an ~11,000-year-old Syrian wild ass (hemippe) from Göbekli Tepe and two of the last surviving hemippes. We conclude that kungas were F1 hybrids between female domestic donkeys and male hemippes, thus documenting the earliest evidence of hybrid animal breeding