Ethical considerations when co-analyzing ancient DNA and data from private genetic databases.

Ancient DNA studies have begun to explore the possibility of identifying identical DNA segments shared between historical and living people. This research requires access to large genetic datasets to maximize the likelihood of identifying previously unknown, close genetic connections. Direct-to-consumer genetic testing companies, such as 23andMe, Inc., manage by far the largest and most diverse genetic databases that can be used for this purpose. It is therefore important to think carefully about guidelines for carrying out collaborations between researchers and such companies. Such collaborations require consideration of ethical issues, including policies for sharing ancient DNA datasets, and ensuring reproducibility of research findings when access to privately controlled genetic datasets is limited. At the same time, they introduce unique possibilities for returning results to the research participants whose data are analyzed, including those who are identified as close genetic relatives of historical individuals, thereby enabling ancient DNA research to contribute to the restoration of information about ancestral connections that were lost over time, which can be particularly meaningful for families and groups where such history has not been well documented. We explore these issues by describing our experience designing and carrying out a study searching for genetic connections between 18th- and 19th-century enslaved and free African Americans who labored at Catoctin Furnace, Maryland, and 23andMe research participants. We share our experience in the hope of helping future researchers navigate similar ethical considerations, recognizing that our perspective is part of a larger conversation about best ethical practices.

The genetic legacy of African Americans from Catoctin Furnace.

Few African Americans have been able to trace family lineages back to ancestors who died before the 1870 United States Census, the first in which all Black people were listed by name. We analyzed 27 individuals from Maryland's Catoctin Furnace African American Cemetery (1774-1850), identifying 41,799 genetic relatives among consenting research participants in 23andMe, Inc.'s genetic database. One of the highest concentrations of close relatives is in Maryland, suggesting that descendants of the Catoctin individuals remain in the area. We find that many of the Catoctin individuals derived African ancestry from the Wolof or Kongo groups and European ancestry from Great Britain and Ireland. This study demonstrates the power of joint analysis of historical DNA and large datasets generated through direct-to-consumer ancestry testing.

Genome-wide data from medieval German Jews show that the Ashkenazi founder event pre-dated the 14th century.

We report genome-wide data from 33 Ashkenazi Jews (AJ), dated to the 14th century, obtained following a salvage excavation at the medieval Jewish cemetery of Erfurt, Germany. The Erfurt individuals are genetically similar to modern AJ, but they show more variability in Eastern European-related ancestry than modern AJ. A third of the Erfurt individuals carried a mitochondrial lineage common in modern AJ and eight carried pathogenic variants known to affect AJ today. These observations, together with high levels of runs of homozygosity, suggest that the Erfurt community had already experienced the major reduction in size that affected modern AJ. The Erfurt bottleneck was more severe, implying substructure in medieval AJ. Overall, our results suggest that the AJ founder event and the acquisition of the main sources of ancestry pre-dated the 14th century and highlight late medieval genetic heterogeneity no longer present in modern AJ.

The Anglo-Saxon migration and the formation of the early English gene pool.

The history of the British Isles and Ireland is characterized by multiple periods of major cultural change, including the influential transformation after the end of Roman rule, which precipitated shifts in language, settlement patterns and material culture1. The extent to which migration from continental Europe mediated these transitions is a matter of long-standing debate2-4. Here we study genome-wide ancient DNA from 460 medieval northwestern Europeans-including 278 individuals from England-alongside archaeological data, to infer contemporary population dynamics. We identify a substantial increase of continental northern European ancestry in early medieval England, which is closely related to the early medieval and present-day inhabitants of Germany and Denmark, implying large-scale substantial migration across the North Sea into Britain during the Early Middle Ages. As a result, the individuals who we analysed from eastern England derived up to 76% of their ancestry from the continental North Sea zone, albeit with substantial regional variation and heterogeneity within sites. We show that women with immigrant ancestry were more often furnished with grave goods than women with local ancestry, whereas men with weapons were as likely not to be of immigrant ancestry. A comparison with present-day Britain indicates that subsequent demographic events reduced the fraction of continental northern European ancestry while introducing further ancestry components into the English gene pool, including substantial southwestern European ancestry most closely related to that seen in Iron Age France5,6.

Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

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.

Social stratification without genetic differentiation at the site of Kulubnarti in Christian Period Nubia.

Relatively little is known about Nubia's genetic landscape prior to the influence of the Islamic migrations that began in the late 1st millennium CE. Here, we increase the number of ancient individuals with genome-level data from the Nile Valley from three to 69, reporting data for 66 individuals from two cemeteries at the Christian Period (~650-1000 CE) site of Kulubnarti, where multiple lines of evidence suggest social stratification. The Kulubnarti Nubians had ~43% Nilotic-related ancestry (individual variation between ~36-54%) with the remaining ancestry consistent with being  introduced through Egypt and ultimately deriving from an ancestry pool like that found in the Bronze and Iron Age Levant. The Kulubnarti gene pool - shaped over a millennium - harbors disproportionately female-associated West Eurasian-related ancestry. Genetic similarity among individuals from the two cemeteries supports a hypothesis of social division without genetic distinction. Seven pairs of inter-cemetery relatives suggest fluidity between cemetery groups. Present-day Nubians are not directly descended from the Kulubnarti Nubians, attesting to additional genetic input since the Christian Period.

COMBINING ANCIENT DNA AND RADIOCARBON DATING DATA TO INCREASE CHRONOLOGICAL ACCURACY.

This paper examines how ancient DNA data can enhance radiocarbon dating. Because there is a limit to the number of years that can separate the dates of death of related individuals, the ability to identify relatives through ancient DNA analysis can serve as a constraint on radiocarbon date range estimates. To determine the number of years that can separate related individuals, we modeled maximums derived from biological extremes of human reproduction and death ages and compiled data from historic and genealogical death records. We used these data to jointly study the date ranges of a global dataset of individuals that have been radiocarbon dated and for which ancient DNA analysis identified at least one relative. We found that many of these individuals could have their date uncertainties reduced by building in date of death separation constraints. We examined possible reasons for date discrepancies of related individuals, such as dating of different skeletal elements or wiggles in the radiocarbon curve. We also developed a program, refinedate, which researchers can download and use to help refine the radiocarbon date distributions of related individuals. Our research demonstrates that when combined, radiocarbon dating and ancient DNA analysis can provide a refined and richer view of the past.

Assessing the performance of qpAdm: a statistical tool for studying population admixture.

qpAdm is a statistical tool for studying the ancestry of populations with histories that involve admixture between two or more source populations. Using qpAdm, it is possible to identify plausible models of admixture that fit the population history of a group of interest and to calculate the relative proportion of ancestry that can be ascribed to each source population in the model. Although qpAdm is widely used in studies of population history of human (and nonhuman) groups, relatively little has been done to assess its performance. We performed a simulation study to assess the behavior of qpAdm under various scenarios in order to identify areas of potential weakness and establish recommended best practices for use. We find that qpAdm is a robust tool that yields accurate results in many cases, including when data coverage is low, there are high rates of missing data or ancient DNA damage, or when diploid calls cannot be made. However, we caution against co-analyzing ancient and present-day data, the inclusion of an extremely large number of reference populations in a single model, and analyzing population histories involving extended periods of gene flow. We provide a user guide suggesting best practices for the use of qpAdm.

A minimally destructive protocol for DNA extraction from ancient teeth.

Ancient DNA sampling methods-although optimized for efficient DNA extraction-are destructive, relying on drilling or cutting and powdering (parts of) bones and teeth. As the field of ancient DNA has grown, so have concerns about the impact of destructive sampling of the skeletal remains from which ancient DNA is obtained. Due to a particularly high concentration of endogenous DNA, the cementum of tooth roots is often targeted for ancient DNA sampling, but destructive sampling methods of the cementum often result in the loss of at least one entire root. Here, we present a minimally destructive method for extracting ancient DNA from dental cementum present on the surface of tooth roots. This method does not require destructive drilling or grinding, and, following extraction, the tooth remains safe to handle and suitable for most morphological studies, as well as other biochemical studies, such as radiocarbon dating. We extracted and sequenced ancient DNA from 30 teeth (and nine corresponding petrous bones) using this minimally destructive extraction method in addition to a typical tooth sampling method. We find that the minimally destructive method can provide ancient DNA that is of comparable quality to extracts produced from teeth that have undergone destructive sampling processes. Further, we find that a rigorous cleaning of the tooth surface combining diluted bleach and UV light irradiation seems sufficient to minimize external contaminants usually removed through the physical removal of a superficial layer when sampling through regular powdering methods.

ContamLD: estimation of ancient nuclear DNA contamination using breakdown of linkage disequilibrium.

We report a method called ContamLD for estimating autosomal ancient DNA (aDNA) contamination by measuring the breakdown of linkage disequilibrium in a sequenced individual due to the introduction of contaminant DNA. ContamLD leverages the idea that contaminants should have haplotypes uncorrelated to those of the studied individual. Using simulated data, we confirm that ContamLD accurately infers contamination rates with low standard errors: for example, less than 1.5% standard error in cases with less than 10% contamination and 500,000 sequences covering SNPs. This method is optimized for application to aDNA, taking advantage of characteristic aDNA damage patterns to provide calibrated contamination estimates, and is available at https://github.com/nathan-nakatsuka/ContamLD .

A Paleogenomic Reconstruction of the Deep Population History of the Andes.

There are many unanswered questions about the population history of the Central and South Central Andes, particularly regarding the impact of large-scale societies, such as the Moche, Wari, Tiwanaku, and Inca. We assembled genome-wide data on 89 individuals dating from ∼9,000-500 years ago (BP), with a particular focus on the period of the rise and fall of state societies. Today's genetic structure began to develop by 5,800 BP, followed by bi-directional gene flow between the North and South Highlands, and between the Highlands and Coast. We detect minimal admixture among neighboring groups between ∼2,000-500 BP, although we do detect cosmopolitanism (people of diverse ancestries living side-by-side) in the heartlands of the Tiwanaku and Inca polities. We also highlight cases of long-range mobility connecting the Andes to Argentina and the Northwest Andes to the Amazon Basin. VIDEO ABSTRACT.

Human auditory ossicles as an alternative optimal source of ancient DNA.

DNA recovery from ancient human remains has revolutionized our ability to reconstruct the genetic landscape of the past. Ancient DNA research has benefited from the identification of skeletal elements, such as the cochlear part of the osseous inner ear, that provides optimal contexts for DNA preservation; however, the rich genetic information obtained from the cochlea must be counterbalanced against the loss of morphological information caused by its sampling. Motivated by similarities in developmental processes and histological properties between the cochlea and auditory ossicles, we evaluate the ossicles as an alternative source of ancient DNA. We show that ossicles perform comparably to the cochlea in terms of DNA recovery, finding no substantial reduction in data quantity and minimal differences in data quality across preservation conditions. Ossicles can be sampled from intact skulls or disarticulated petrous bones without damage to surrounding bone, and we argue that they should be used when available to reduce damage to human remains. Our results identify another optimal skeletal element for ancient DNA analysis and add to a growing toolkit of sampling methods that help to better preserve skeletal remains for future research while maximizing the likelihood that ancient DNA analysis will produce useable results.

The formation of human populations in South and Central Asia.

By sequencing 523 ancient humans, we show that the primary source of ancestry in modern South Asians is a prehistoric genetic gradient between people related to early hunter-gatherers of Iran and Southeast Asia. After the Indus Valley Civilization's decline, its people mixed with individuals in the southeast to form one of the two main ancestral populations of South Asia, whose direct descendants live in southern India. Simultaneously, they mixed with descendants of Steppe pastoralists who, starting around 4000 years ago, spread via Central Asia to form the other main ancestral population. The Steppe ancestry in South Asia has the same profile as that in Bronze Age Eastern Europe, tracking a movement of people that affected both regions and that likely spread the distinctive features shared between Indo-Iranian and Balto-Slavic languages.

Ancient DNA from the skeletons of Roopkund Lake reveals Mediterranean migrants in India.

Situated at over 5,000 meters above sea level in the Himalayan Mountains, Roopkund Lake is home to the scattered skeletal remains of several hundred individuals of unknown origin. We report genome-wide ancient DNA for 38 skeletons from Roopkund Lake, and find that they cluster into three distinct groups. A group of 23 individuals have ancestry that falls within the range of variation of present-day South Asians. A further 14 have ancestry typical of the eastern Mediterranean. We also identify one individual with Southeast Asian-related ancestry. Radiocarbon dating indicates that these remains were not deposited simultaneously. Instead, all of the individuals with South Asian-related ancestry date to ~800 CE (but with evidence of being deposited in more than one event), while all other individuals date to ~1800 CE. These differences are also reflected in stable isotope measurements, which reveal a distinct dietary profile for the two main groups.

Ancient human genome-wide data from a 3000-year interval in the Caucasus corresponds with eco-geographic regions.

Archaeogenetic studies have described the formation of Eurasian 'steppe ancestry' as a mixture of Eastern and Caucasus hunter-gatherers. However, it remains unclear when and where this ancestry arose and whether it was related to a horizon of cultural innovations in the 4th millennium BCE that subsequently facilitated the advance of pastoral societies in Eurasia. Here we generated genome-wide SNP data from 45 prehistoric individuals along a 3000-year temporal transect in the North Caucasus. We observe a genetic separation between the groups of the Caucasus and those of the adjacent steppe. The northern Caucasus groups are genetically similar to contemporaneous populations south of it, suggesting human movement across the mountain range during the Bronze Age. The steppe groups from Yamnaya and subsequent pastoralist cultures show evidence for previously undetected farmer-related ancestry from different contact zones, while Steppe Maykop individuals harbour additional Upper Palaeolithic Siberian and Native American related ancestry.

Publisher Correction: Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation.

In the original version of this Article, references in the format 'First author et al.' were inappropriately deleted. These errors have been corrected in the PDF and HTML versions of the Article.

Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation.

The material culture of the Late Chalcolithic period in the southern Levant (4500-3900/3800 BCE) is qualitatively distinct from previous and subsequent periods. Here, to test the hypothesis that the advent and decline of this culture was influenced by movements of people, we generated genome-wide ancient DNA from 22 individuals from Peqi'in Cave, Israel. These individuals were part of a homogeneous population that can be modeled as deriving ~57% of its ancestry from groups related to those of the local Levant Neolithic, ~17% from groups related to those of the Iran Chalcolithic, and ~26% from groups related to those of the Anatolian Neolithic. The Peqi'in population also appears to have contributed differently to later Bronze Age groups, one of which we show cannot plausibly have descended from the same population as that of Peqi'in Cave. These results provide an example of how population movements propelled cultural changes in the deep past.

Ancient genomes document multiple waves of migration in Southeast Asian prehistory.

Southeast Asia is home to rich human genetic and linguistic diversity, but the details of past population movements in the region are not well known. Here, we report genome-wide ancient DNA data from 18 Southeast Asian individuals spanning from the Neolithic period through the Iron Age (4100 to 1700 years ago). Early farmers from Man Bac in Vietnam exhibit a mixture of East Asian (southern Chinese agriculturalist) and deeply diverged eastern Eurasian (hunter-gatherer) ancestry characteristic of Austroasiatic speakers, with similar ancestry as far south as Indonesia providing evidence for an expansive initial spread of Austroasiatic languages. By the Bronze Age, in a parallel pattern to Europe, sites in Vietnam and Myanmar show close connections to present-day majority groups, reflecting substantial additional influxes of migrants.

Erratum: The Beaker phenomenon and the genomic transformation of northwest Europe.

This corrects the article DOI: 10.1038/nature25738.