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It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3′ ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5′ adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30−35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired. Here the authors describe an updated protocol for single-stranded sequencing library preparation suitable for highly degraded DNA from ancient remains or other sources. The procedure can be performed manually or in an automated fashion.

Neanderthals and Denisovans are extinct groups of hominins that separated from each other more than 390,000 years ago 1 , 2 . Here we present the genome of ‘Denisova 11’, a bone fragment from Denisova Cave (Russia) 3 and show that it comes from an individual who had a Neanderthal mother and a Denisovan father. The father, whose genome bears traces of Neanderthal ancestry, came from a population related to a later Denisovan found in the cave 4 – 6 . The mother came from a population more closely related to Neanderthals who lived later in Europe 2 , 7 than to an earlier Neanderthal found in Denisova Cave 8 , suggesting that migrations of Neanderthals between eastern and western Eurasia occurred sometime after 120,000 years ago. The finding of a first-generation Neanderthal–Denisovan offspring among the small number of archaic specimens sequenced to date suggests that mixing between Late Pleistocene hominin groups was common when they met. Genomic evidence of the offspring of a Neanderthal mother and a Denisovan father suggests that mixing among different hominin groups may have more been frequent than previously appreciated.

To date, the only Neandertal genome that has been sequenced to high quality is from an individual found in Southern Siberia. We sequenced the genome of a female Neandertal from ~50,000 years ago from Vindija Cave, Croatia, to ~30-fold genomic coverage. She carried 1.6 differences per 10,000 base pairs between the two copies of her genome, fewer than present-day humans, suggesting that Neandertal populations were of small size. Our analyses indicate that she was more closely related to the Neandertals that mixed with the ancestors of present-day humans living outside of sub-Saharan Africa than the previously sequenced Neandertal from Siberia, allowing 10 to 20% more Neandertal DNA to be identified in present-day humans, including variants involved in low-density lipoprotein cholesterol concentrations, schizophrenia, and other diseases.

Denisova Cave in southern Siberia is the type locality of the Denisovans, an archaic hominin group who were related to Neanderthals 1 – 4 . The dozen hominin remains recovered from the deposits also include Neanderthals 5 , 6 and the child of a Neanderthal and a Denisovan 7 , which suggests that Denisova Cave was a contact zone between these archaic hominins. However, uncertainties persist about the order in which these groups appeared at the site, the timing and environmental context of hominin occupation, and the association of particular hominin groups with archaeological assemblages 5 , 8 – 11 . Here we report the analysis of DNA from 728 sediment samples that were collected in a grid-like manner from layers dating to the Pleistocene epoch. We retrieved ancient faunal and hominin mitochondrial (mt)DNA from 685 and 175 samples, respectively. The earliest evidence for hominin mtDNA is of Denisovans, and is associated with early Middle Palaeolithic stone tools that were deposited approximately 250,000 to 170,000 years ago; Neanderthal mtDNA first appears towards the end of this period. We detect a turnover in the mtDNA of Denisovans that coincides with changes in the composition of faunal mtDNA, and evidence that Denisovans and Neanderthals occupied the site repeatedly—possibly until, or after, the onset of the Initial Upper Palaeolithic at least 45,000 years ago, when modern human mtDNA is first recorded in the sediments. Ancient mitochondrial DNA from sediments reveals the sequence of Denisovan, Neanderthal and faunal occupation of Denisova Cave, and evidence for the appearance of modern humans at least 45,000 years ago.

Ancient DNA (aDNA) analyses necessitate the destructive sampling of archaeological material. Currently, the cochlea, part of the osseous inner ear located inside the petrous pyramid, is the most sought after skeletal element for molecular analyses of ancient humans as it has been shown to yield high amounts of endogenous DNA. However, destructive sampling of the petrous pyramid may not always be possible, particularly in cases where preservation of skeletal morphology is of top priority. To investigate alternatives, we present a survey of human aDNA preservation for each of ten skeletal elements in a skeletal collection from Medieval Germany. Through comparison of human DNA content and quality we confirm best performance of the petrous pyramid and identify seven additional sampling locations across four skeletal elements that yield adequate aDNA for most applications in human palaeogenetics. Our study provides a better perspective on DNA preservation across the human skeleton and takes a further step toward the more responsible use of ancient materials in human aDNA studies.

Ancient DNA recovered from Pleistocene sediments represents a rich resource for the study of past hominin and environmental diversity. However, little is known about how DNA is preserved in sediments and the extent to which it may be translocated between archaeological strata. Here, we investigate DNA preservation in 47 blocks of resin-impregnated archaeological sediment collected over the last four decades for micromorphological analyses at 13 prehistoric sites in Europe, Asia, Africa, and North America and show that such blocks can preserve DNA of hominins and other mammals. Extensive microsampling of sediment blocks from Denisova Cave in the Altai Mountains reveals that the taxonomic composition of mammalian DNA differs drastically at the millimeter-scale and that DNA is concentrated in small particles, especially in fragments of bone and feces (coprolites), suggesting that these are substantial sources of DNA in sediments. Three microsamples taken in close proximity in one of the blocks yielded Neanderthal DNA from at least two male individuals closely related to Denisova 5, a Neanderthal toe bone previously recovered from the same layer. Our work indicates that DNA can remain stably localized in sediments over time and provides a means of linking genetic information to the archaeological and ecological records on a microstratigraphic scale.

The general view is that Eurasians mostly descend from a single group of humans that dispersed outside of sub-Saharan Africa around 50,000 to 100,000 years ago. Present-day North Africans share a majority of their ancestry with present-day Near Easterners, but not with sub-Saharan Africans. To investigate this conundrum, Van de Loosdrecht et al. sequenced high-quality DNA obtained from bone samples of seven individuals from Taforalt in eastern Morocco dating from the Later Stone Age, about 15,000 years ago. The Taforalt individuals were found to be most closely related to populations from the Near East (Natufians), with a third of their ancestry from sub-Saharan Africa. No evidence was found for introgression with western Europeans, despite attribution to the Iberomaurusian culture. None of the present-day or ancient Holocene African groups are a good proxy for the sub-Saharan genetic component. Science , this issue p. 548 Ancient human genomes suggest dynamic interactions among Pleistocene African populations. North Africa is a key region for understanding human history, but the genetic history of its people is largely unknown. We present genomic data from seven 15,000-year-old modern humans, attributed to the Iberomaurusian culture, from Morocco. We find a genetic affinity with early Holocene Near Easterners, best represented by Levantine Natufians, suggesting a pre-agricultural connection between Africa and the Near East. We do not find evidence for gene flow from Paleolithic Europeans to Late Pleistocene North Africans. The Taforalt individuals derive one-third of their ancestry from sub-Saharan Africans, best approximated by a mixture of genetic components preserved in present-day West and East Africans. Thus, we provide direct evidence for genetic interactions between modern humans across Africa and Eurasia in the Pleistocene.

The straight-tusked elephants Palaeoloxodon spp. were widespread across Eurasia during the Pleistocene. Phylogenetic reconstructions using morphological traits have grouped them with Asian elephants (Elephas maximus), and many paleontologists place Palaeoloxodon within Elephas. Here, we report the recovery of full mitochondrial genomes from four and partial nuclear genomes from two P. antiquus fossils. These fossils were collected at two sites in Germany, Neumark-Nord and Weimar-Ehringsdorf, and likely date to interglacial periods ~120 and ~244 thousand years ago, respectively. Unexpectedly, nuclear and mitochondrial DNA analyses suggest that P. antiquus was a close relative of extant African forest elephants (Loxodonta cyclotis). Species previously referred to Palaeoloxodon are thus most parsimoniously explained as having diverged from the lineage of Loxodonta, indicating that Loxodonta has not been constrained to Africa. Our results demonstrate that the current picture of elephant evolution is in need of substantial revision. Understanding how extinct species are related to each other or to their living relatives is often a difficult task. Many extinct species have been identified only from incomplete fragments of some of their bones. However, even if complete skeletons have been found, determining the relationships between species can be tricky because researchers often have to rely solely on the shapes of the bones. It is sometimes possible to retrieve DNA sequences from fossil bones. This is easier with younger fossils and those that have been recovered from cold environments. Ancient DNA sequences have been retrieved from only a few fossils older than 100,000 years, but such DNA sequences can be tremendously useful in determining how different species are related to each other. Today there are three living elephant species: the African forest elephant, the African savanna elephant and the Asian elephant. However, there are many extinct elephant species. For example, the European straight-tusked elephant went extinct at least 30,000 years ago, although most of the fossils that have been discovered are at least 100,000 years old. Straight-tusked elephants are generally assumed to be closely related to the Asian elephant, but this conclusion had been based solely on reconstructing skeletons. Meyer et al. have now obtained DNA sequences from fossils of four straight-tusked elephants ranging from around 120,000 to 240,000 years in age. These sequences were analysed to determine how straight-tusked elephants are related to the three living elephant species and the extinct mammoth, the DNA sequences for which can be found in public databases. The analyses revealed that straight-tusked elephants are in fact most closely related to the African forest elephant, not the Asian elephant as previously thought. This result completely changes our picture of elephant evolution and suggests that it is extremely difficult to determine elephant relationships based on the shape of their skeleton alone. It also shows that the African elephant lineage was not restricted to the African continent (the place where all elephant lineages originated), but that it also left Africa. Overall, the results presented by Meyer et al. confirm that DNA sequences are of critical importance for understanding the evolution of animals. Future research should include obtaining DNA sequences from additional extinct elephant species as well as careful re-evaluation of skeletal measurements for reconstructing elephant evolution.

Although measurement data from the civil engineering sector are an important basis for scientific analyses in the field of non-destructive testing (NDT), there is still no uniform representation of these data. An analysis of data sets across different test objects or test types is therefore associated with a high manual effort. Ontologies and the semantic web are technologies already used in numerous intelligent systems such as material cyberinfrastructures or research databases. This contribution demonstrates the application of these technologies to the case of the 1H nuclear magnetic resonance relaxometry, which is commonly used to characterize water content and porosity distribution in solids. The methodology implemented for this purpose was developed specifically to be applied to materials science (MS) tests. The aim of this paper is to analyze such a methodology from the perspective of data interoperability using ontologies. Three benefits are expected from this approach to the study of the implementation of interoperability in the NDT domain: First, expanding knowledge of how the intrinsic characteristics of the NDT domain determine the application of semantic technologies. Second, to determine which aspects of such an implementation can be improved and in what ways. Finally, the baselines of future research in the field of data integration for NDT are drawn.

Nuclear DNA sequences from Middle Pleistocene Sima de los Huesos hominins show they were more closely related to Neanderthals than to Denisovans, and indicate a population divergence between Neanderthals and Denisovans that predates 430,000 years ago. Neanderthal-like hominins in Middle Pleistocene Spain This genomic analysis of Middle Pleistocene hominins from Sima de los Huesos in the Sierra de Atapuerca in Spain shows that they were more closely related to Neanderthals than to Denisovans, and indicates a divergence between Neanderthals and Denisovans that predates 430,000 years ago. A previous report based on analyses of mitochondrial genomes from these specimens had suggested close relationship to Denisovans, which was in contrast to other archaeological evidence including morphological features shared with Late Pleistocene Neanderthals. A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago 1 . An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals 2 . However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history.