Janet Kelso

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.

N -acetyltransferase 1 (NAT1) is an enzyme that acetylates certain drugs and carcinogens and is involved in folate metabolism. Some NAT1 variants are associated with susceptibility to cancer and birth defects. We show that while nearly all present-day humans carry a valine residue at position 149 and a serine residue at position 214, some carry the ancestral amino acids isoleucine and alanine at these positions because of gene flow from Neanderthals. Neither substitution affects enzymatic activity. However, replacing the serine residue, a known phosphorylation site, with a phosphomimetic aspartate reduces NAT1 activity by ~60%. Phosphorylation at this site may allow NAT1 to be down-regulated during pregnancy to reduce birth defect risk, while maintaining high activity in adults for xenobiotic clearance. This regulatory site is a molecular feature that differs between modern humans and Neanderthals.

We present a genome sequenced to ~37-fold genomic coverage from an approximately 110,000-y-old male Neandertal from Denisova Cave in the Altai Mountains and analyze it together with previously published Neandertal genomes of high quality. We show that he belonged to a population more closely related to a ~120,000-y-old Neandertal from Denisova Cave than to Neandertals in Europe or to a ~80,000-y-old Neandertal from Chagyrskaya Cave in the Altai Mountains. Both Neandertals from Denisova Cave show evidence of gene flow from Denisovans, a pattern not seen in later Neandertals from the Altai region or from Western Europe. The extent of chromosomal regions of homozygosity in Neandertals from the Altai region between 120,000 and 80,000 y ago indicates that they lived in smaller and more isolated groups than later Neandertals in Europe (54,000 to 40,000 y ago). We estimate the extent of allele frequency differentiation among Neandertal populations and find that the older Eastern Neandertals in the Altai region and younger Western Neandertals in Europe were as differentiated as the most differentiated present-day human populations worldwide.

Synonymous single nucleotide variants (sSNVs), traditionally seen as neutral, are now recognized for their biological impact. To assess their relevance, we developed SyMetrics, a framework that integrates predictors of splicing, RNA stability, evolutionary conservation, codon usage, synonymous variation effects, sequence properties, and allele frequency. We analyzed all possible sSNVs across the human genome, and our machine-learning model achieved 97% accuracy in distinguishing deleterious from benign variants, with a ROC–AUC of 0.89, outperforming individual predictors. Our estimates indicate that about 1.98 ± 0.17% of sSNVs absent from population databases are damaging (roughly 900 000 sSNVs), with an odds ratio of 3.87 for deleteriousness compared to common sSNVs (P < 0.05). To validate predictions, we performed functional assays on selected sSNVs in the AVPR2 gene and additionally used available large scale mutagenesis screens of RAD51C and BAP1 variants. In a clinical cohort, we identified 15 predicted deleterious sSNVs in genes linked to patient phenotypes; 9 were classified as (likely) pathogenic while 6 were variants of uncertain significance (VUS) per American College of Medical Genetics guidelines. For three VUS, segregation data supported their suspected inheritance patterns (de novo, X-linked). Our findings underscore the functional importance of sSNVs. To support further research and clinical applications, we provide a Python package and web application (https://symetrics.org/) for evaluating these variants comprehensively.

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.

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 .

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.