Matthias Meyer

Archaeological, osteological and genetic evidence suggests that Neanderthals lived in small groups ^1,2 ; however, less is known about whether these groups were part of isolated communities or belonged to larger, well-connected populations ³ . The dense concentration of broadly contemporaneous Neanderthal sites in the Meuse Basin, Belgium ⁴ , provides a rare opportunity to study regional populations at high resolution. Here we generated genetic data from 27 Neanderthals who lived less than approximately 52,500 years ago from ten archaeological sites in Belgium and France, including a high-coverage genome from a 45,000-year-old individual from Goyet, Belgium. We show that most of these individuals are more closely related to one another than to other contemporaneous late Neanderthals in Europe. Further, some of these individuals carry DNA from a Neanderthal lineage predating the split of late Neanderthals. Although these Neanderthals overlapped temporally with early modern humans in northwestern Europe from around 47,000 years ago, we find no evidence of recent gene flow from modern humans. They also do not show the genetic signatures of mating among close relatives found in Altai Neanderthals, suggesting that they lived in larger or better-connected groups. Moreover, genetic load did not accumulate over time, arguing against progressive genetic deterioration as a driver of Neanderthal extinction.

Previous efforts to link Palaeolithic cultural records to specific populations through DNA analysis have focused on materials from archaeological floor deposits such as bones, sediments, and artefacts. In this study, we explore whether rock art, a spatially distinct expression of human activity, can also preserve DNA traces from its creators. We analyse DNA preservation in pigment samples collected in and around 24 rock art panels from 11 caves across Spain and Portugal, including simple marks (from nine sites), hand stencils (Maltravieso Cave, Extremadura, Spain), and figurative paintings (Cave of Altamira, Cantabria, Spain). We recover traces of ancient human mitochondrial and nuclear DNA, unaccompanied by faunal DNA, from a pigmented calcite crust at Escoural Cave (Portugal), as well as from an unpigmented cave wall sample from the same site. The absence of faunal DNA in both samples suggests direct DNA deposition through human contact. In contrast, three additional unpigmented samples, from Escoural and Covarón Cave (Asturias, Spain), yielded mixtures of human and faunal DNA, suggesting indirect deposition. Although our results do not conclusively link ancient human DNA preservation to the generation of cave art, we show that traces of human DNA can persist on cave walls for thousands of years.

We here provide an implementation of the ssDNA2.0 single-stranded library preparation method (Gansauge et al. 2017, Gansauge et al. 2020) for automated liquid handling on the Hamilton NGS STAR workstation in 96-well format. The protocol derives from a previous implementation of the ssDNA2.0 method on the Bravo workstation (Nagel et al. 2024). The ssDNA2.0 method was developed primarily for ancient DNA research but is also suitable for library preparation from other sources of degraded or single-stranded DNA, such as cell-free DNA, formalin-fixed samples or synthetic oligonucleotides. To use the protocol, a Hamilton NGS STAR workstation is required. For a fully walk-away solution that does not require adding pipette tips during the run, the addition of a Cytomat (Thermo Fisher Scientific) is required. Calibration of the instrument for this protocol has to be performed by the user and requires significant expertise in using the platform. Electronic protocol files for the Hamilton STAR workstation are provided together with this protocol and a brief description of the steps performed by the liquid handling system. Some of the instructions, for example regarding the documentation and location of files, are specific to the environment and workflows of the Ancient DNA Core Unit of the MPI-EVA and have to be amended in other environments. We strongly advise against manual execution of the protocol, as manual handling lacks the precision of automated liquid handling and may lead to cross-contamination of samples or non-optimal results. For manual ssDNA 2.0 library preparation, please refer to Gansauge et al. 2020. Note that the adapter sequences added with this method differ from standard Illumina adapters by a 5-bp deletion in the P5-adapter, requiring a custom sequencing primer (see Gansauge et al. 2020). This protocol consists of two parts: (i) library preparation, with or without Uracil-DNA-glycosylase treatment for the removal of internal uracils from ancient DNA molecules, and (ii) library quantification by quantitative real-time PCR. A protocol for library amplification and indexing is provided separately. Two control oligonucleotides are added during library preparation. CL304 (60 million molecules) is added at the beginning of the workflow to assess the efficiency of DNA conversion into sequencing libraries (Glocke & Meyer 2017) and to identify inhibition or saturation in individual reactions. TL250 (1 million molecules) is added during the elution step for additional quality control. References Gansauge, M.-T., Gerber, T., Glocke, I., Korlevic, P., Lippik, L., Nagel, S., Riehl, L. M., Schmidt, A., & Meyer, M. (2017). Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. Nucleic Acids Research, 45(10): e79. Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300. Glocke, I., & Meyer, M. (2017). Extending the spectrum of DNA sequences retrieved from ancient bones and teeth. Genome Research, 27, 1230-1237. Nagel, S., Schmidt, A., Aximu-Petri, A., Patova, A., Szymanski, M., Essel, E. & Meyer, M. (2026). Bravo workstation: automated single-stranded DNA library preparation (ssDNA2.0). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32bdpv25/v2

Ancient DNA extracted from the sediments of archaeological sites (sedaDNA) can provide fine-grained information about the composition of past ecosystems and human site use, even in the absence of visible remains. However, the growing amount of available sequencing data and the nature of the data obtained from archaeological sediments pose several computational challenges; among these, the rapid and accurate taxonomic classification of sequences. While alignment-based taxonomic classifiers remain the standard in sedaDNA analysis pipelines, they are too computationally expensive for the processing of large numbers of sedaDNA sequences. In contrast, alignment-free methods offer fast classification but suffer from higher false-positive rates. To address these limits, we developed quicksand, an open-source Nextflow pipeline designed for rapid and accurate taxonomic classification of mammalian mitochondrial DNA in sedaDNA samples. quicksand combines fast alignment-free classification using KrakenUniq with post-classification mapping, filtering, and ancient DNA authentication. Based on simulations and reanalyses of published datasets, we demonstrate that quicksand achieves accuracy and sensitivity comparable to or better than existing methods, while significantly reducing runtime. quicksand offers an easy workflow for large-scale screening of sedaDNA samples for archaeological research and is freely available at https://github.com/mpieva/quicksand.

Denisovans, an extinct sister group of Neandertals who lived in Eastern Eurasia during the Middle and Late Pleistocene, are known only from a handful of skeletal remains and limited genetic data, including the high-coverage genome of a woman who lived ∼65,000 years ago. Here, we present a second high-quality Denisovan genome, reconstructed from a molar found at Denisova Cave. It belonged to a man who lived ∼200,000 years ago in a small Denisovan group. This group mixed with early Neandertals and was then replaced by Denisovans who had mixed with later Neandertals. We show that in addition Denisovans received gene flow from hominins that diverged before the split of the ancestors of Denisovans and modern humans. The two Denisovan genomes allow us to disentangle Denisovan ancestry in present-day humans revealing contributions from at least three distinct Denisovan groups. In particular, Oceanians and South Asians independently inherited DNA from a deeply diverged Denisovan population which was likely isolated in South Asia. This supports an early migration of the ancestors of Oceanians through South Asia followed by the later arrival of the ancestors of present-day South Asians. East Asians do not share this Denisovan component in their genomes, suggesting that their ancestors arrived independently, perhaps by a northerly route. Finally, the two high-quality Denisovan genomes allow us to refine the catalogue of genetic changes that arose on the Denisovan lineage, some of which were contributed to present-day humans.

We here provide a protocol for the first step of ancient DNA extraction - lysate preparation - from bones, teeth and sediments, building on the silica-based extraction method described by Dabney et al. 2013 and refined by Rohland et al. 2018. The protocol includes specific adaptations to the clean room environment and workflows of the Ancient DNA Core Unit at the MPI-EVA, as well as preparatory steps for downstream automated DNA purification using the Bravo NGS (Agilent Technologies) or NGS Star (Hamilton) workstations. Sample material (drilled subsamples of bones/teeth, subsamples of sediment) should be provided in 2.0 ml LoBind Eppendorf tubes. Lysates are prepared in volumes ranging from 300 to 2000µl, depending on the sample weight. A standard volume of 150µl, aliquoted to screw-cap tubes (option A), is subsequently used for DNA purification (see separate protocol), allowing two or more extractions from a single lysate. Alternatively, aliquots with different volume (option B) may be removed for manual DNA extraction or other purposes. References Dabney, J., Knapp, M., Glocke, I., Gansauge, M.-T., Weihmann, A., Nickel, B., Valdiosera, C., García, N., Pääbo, S., Arsuaga, J.-L., & Meyer, M. (2013). Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences of the United States of America, 110(39), 15758-15763. Rohland N, Hofreiter M. Ancient DNA extraction from bones and teeth. Nat Protoc. 2007;2(7):1756-62. doi: 10.1038/nprot.2007.247. PMID: 17641642.

Ancient DNA extracted from the sediments of archaeological sites (sedaDNA) can provide fine-grained information about the composition of past ecosystems and human site use, even in the absence of skeletal remains. However, the growing amount of available sequencing data and the nature of the data obtained from archaeological sediments pose several computational challenges; among these, the rapid and accurate taxonomic classification of sequences. While alignment-based taxonomic classifiers remain the standard in sedaDNA analysis pipelines, they are too computationally expensive for the processing of large numbers of sedaDNA sequences. In contrast, alignment-free methods offer fast classification but suffer from higher false-positive rates. To address these limits, we developed quicksand, an open-source Nextflow pipeline designed for rapid and accurate taxonomic classification of mammalian mitochondrial DNA (mtDNA) in sedaDNA samples. quicksand combines fast alignment-free classification using KrakenUniq with post-classification mapping, filtering, and ancient DNA authentication. Based on simulations and reanalyses of published datasets, we demonstrate that quicksand achieves accuracy and sensitivity comparable to or better than existing methods, while significantly reducing runtime. quicksand offers an easy workflow for large-scale screening of sedaDNA samples for archaeological research and is freely available at https://github.com/mpieva/quicksand .

Following the preparation of lysates (see Nagel et al. 2025), this protocol describes the second step of ancient DNA extraction - DNA purification - from bones, teeth and sediments, building on the silica-based extraction method described by Dabney et al. 2013 and refined by Rohland et al. 2018. Purification is performed using binding buffer 'D' (Rohland et al. 2018). The protocol includes specific adaptations to the clean room environment and workflows of the Ancient DNA Core Unit at the MPI-EVA. Lysate aliquots of 150 µl each should be provided in screw-cap tubes, as detailed in Nagel et al. 2025. After purification, DNA extracts will be delivered in screw-cap tubes in 30 µl volumes for downstream library preparation (e.g., see Nagel et al. 2026). References Dabney, J., Knapp, M., Glocke, I., Gansauge, M.-T., Weihmann, A., Nickel, B., Valdiosera, C., García, N., Pääbo, S., Arsuaga, J.-L., & Meyer, M. (2013). Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences of the United States of America, 110(39), 15758-15763. Nagel, S., Schmidt, A., Meyer, M. & Essel, E. (2025). Ancient DNA extraction part I: lysate preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l212oqg1y/v1 Nagel, S., Schmidt, A., Aximu-Petri, A., Patova, A., Szymanski, M., Essel, E. & Meyer, M. (2026). Bravo workstation: automated single-stranded DNA library preparation (ssDNA2.0). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32bdpv25/v2 Rohland N, Hofreiter M. Ancient DNA extraction from bones and teeth. Nat Protoc. 2007;2(7):1756-62. doi: 10.1038/nprot.2007.247. PMID: 17641642.

Denisova Cave in southern Siberia is the only site known to have been occupied by Denisovans, Neanderthals and modern humans. The cave consists of three chambers (Main, East and South), with the archaeological assemblages and remains of hominins, fauna and flora recovered from Main and East Chambers being the most thoroughly investigated to date. Here we report the results of analyses of the Palaeolithic artefacts, faunal remains and hominin and mammalian mitochondrial (mt) DNA recovered from renewed excavations in South Chamber. We construct a calendar-year time scale for the stratified Pleistocene deposits from optical dating of the sediments. The timing of hominin occupation and major turnovers in the mtDNA of Denisovans and large mammals largely accords with the patterns detected in Main and East Chambers. Time gaps in those sequences are partly filled by the South Chamber data and the sediment DNA record of Denisovans after 80,000 years ago is more than doubled in size. We combine the sediment dating and DNA records for all three chambers to reveal the whole-of-cave history of this unique site and the climatic conditions experienced by hominins and fauna over the past 300,000 years, including potential changes in habitat suitability for Denisovans and Neanderthals.

We here provide a protocol for the simultaneous amplification and dual-indexing of up to 384 DNA libraries through automated liquid handling using the Hamilton NGS Star workstation. The benefits of dual-indexing for preventing and detecting cross-contamination have been documented elsewhere (Kircher et al. 2012, Zavala et al. 2022). This protocol is optimized specifically for ancient and degraded DNA (i.e., libraries with short inserts) and is compatible with both double- and single-stranded methods for preparing Illumina-type libraries (e.g., Meyer and Kircher 2010, Gansauge et al. 2020). Amplification is carried out to PCR plateau, ensuring approximately equal yields across all samples. The use of AccuPrime Pfx polymerase minimizes length and GC biases during amplification (Dabney et al. 2012). Additionally, we provide sequences for 384 unique 8-bp indices, incorporated into the interior of both the P5 and P7 indexing primers. The protocol also includes steps for purifying indexed libraries using Solid Phase Reversible Immobilization (SPRI) technology (deAngelis et al. 1995) and for determining the concentration of purified indexed libraries using a NanoDrop photospectrometer. Some of the instructions, for example regarding the documentation and location of files, are specific to the environment and workflows of the Ancient DNA Core Unit of the MPI-EVA and have to be amended in other environments. Notes Default input volume of library into indexing PCR is 49 µl (the final library volume in library preparation with the ssDNA2.0 method), for a total reaction volume of 100 µl. However, lower input volumes are possible. Input files are provided containing unique index combinations for column-wise (or half column-wise) pipetting of 576 indexing PCR plates, each with 96 reactions, making for a total of 55,296 reactions. There is no overlap in P5 or P7 indices among four consecutive plates (labeled A, B, C and D), which together form one set of plates. Thus, four PCR plates can be set up consecutively using only a single P5 and P7 index primer plate each. In addition to the purified, indexed libraries, the protocol produces plates with 2.5-fold and 10-fold dilutions intended for Nanodrop measurements and templates for reamplification, as well as a backup of unpurified library. References Dabney, J., & Meyer, M. (2012). Length and GC-biases during sequencing library amplification: A comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. BioTechniques, 52(2), 87-94. DeAngelis, M. M., Wang, D. G., Hawkins, T. L. (1995) Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 23(22):4742-3. doi: 10.1093/nar/23.22.4742. Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300. Kircher, M., Sawyer, S. & Meyer, M. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res., 40(1):e3, doi: 10.1093/nar/gkr771. Meyer, M., & Kircher, M. (2010). Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harbor Protocols, 2010(6): prot5448. Zavala, E. I., Ayinuer-Petri, A., Richter, J., Nickel, B., Vernot, B., & Meyer, M. (2022). Quantifying and reducing cross‐contamination in single‐ and multiplex hybridization capture of ancient DNA. Molecular Ecology Resources, 22(6), 2196-2207.