Non-destructive extraction of DNA from preserved tissues in medical collections.

Museum specimens and histologically fixed material are valuable samples for the study of historical soft tissues and represent a possible pathogen-specific source for retrospective molecular investigations. However, current methods for molecular analysis are inherently destructive, posing a dilemma between performing a study with the available technology, thus damaging the sample, and conserving the material for future investigations. Here the authors present the first tests of a non-destructive alternative that facilitates genetic analysis of fixed wet tissues while avoiding tissue damage. The authors extracted DNA from the fixed tissues as well as their embedding fixative solution, to quantify the DNA that was transferred to the liquid component. The results show that human historical DNA can be retrieved from the fixative material of medical specimens and provide new options for sampling valuable collections.

Metagenomic analysis of Ancient Egyptian canopic jars.

Ancient Egyptian remains have been of interest for anthropological research for decades. Despite many investigations, the ritual vessels for the internal organs removed during body preparation-liver, lungs, stomach, and intestines, of Egyptian mummies are rarely used for palaeopathological or medical investigations. These artifacts, commonly referred to as canopic jars, are the perfect combination of cultural and biological material and present an untapped resource for both Egyptological and medical fields. Nevertheless, technical challenges associated with this archeological material have prevented the application of current ancient DNA techniques for both the characterization of human and pathogenic DNA. We present shotgun-sequenced metagenomic profiles and ancient DNA degradation patterns from multiple canopic jars sampled from several European museum collections and enumerate current limitations and possible solutions for the future analysis of similar material. This is the first-ever recorded evidence of ancient human DNA found in Ancient Egyptian canopic jars and the first associated metagenomic description of bacterial taxa in these funerary artifacts. OBJECTIVES: In this study, our objectives were to characterize the metagenomic profile of the Ancient Egyptian funerary vessels known as canopic jars to retrieve endogenous ancient human DNA, reconstruct ancient microbial communities, and identify possible pathogens that could shed light on disease states of individuals from the past. METHODS: We applied ancient DNA techniques on 140 canopic jars to extract DNA and generate whole-genome sequencing libraries for the analysis of both human and bacterial DNA. The samples were obtained from museum collections in Berlin (DE), Burgdorf (DE), Leiden (NE), Manchester (UK), Munich (DE), St. Gallen (CH), Turin (IT), and Zagreb (HR). RESULTS: Here we describe the first isolated DNA from the Egyptian artifacts that hold human viscera. No previous work was ever conducted on such material, which led to the first characterization of human DNA from Ancient Egyptian canopic jars and the profiling of the complex bacterial composition of this highly degraded, challenging, organic material. However, the DNA recovered was not of enough quality to confidently characterize bacterial taxa associated with infectious diseases, nor exclusive bacterial members of the human microbiome. DISCUSSION: In summary, we present the first genomic survey of the visceral content of Ancient Egyptian funerary artifacts and demonstrate the limitations of current molecular methods to analyze canopic jars, such as the incomplete history of the objects or the presence of uncharacterized compounds that can hamper the recovery of DNA. Our work highlights the main challenges and caveats when working with such complicated archeological material - and offers sampling recommendations for similarly complex future studies, such as incrementing the amount of starting material and sampling from the less exposed parts of the jar content. This is the first-ever recorded evidence of ancient human DNA found in Ancient Egyptian canopic jars, and our results open new avenues in the study of neglected archeological artifacts.

Bronze Age population dynamics and the rise of dairy pastoralism on the eastern Eurasian steppe.

Recent paleogenomic studies have shown that migrations of Western steppe herders (WSH) beginning in the Eneolithic (ca. 3300-2700 BCE) profoundly transformed the genes and cultures of Europe and central Asia. Compared with Europe, however, the eastern extent of this WSH expansion is not well defined. Here we present genomic and proteomic data from 22 directly dated Late Bronze Age burials putatively associated with early pastoralism in northern Mongolia (ca. 1380-975 BCE). Genome-wide analysis reveals that they are largely descended from a population represented by Early Bronze Age hunter-gatherers in the Baikal region, with only a limited contribution (∼7%) of WSH ancestry. At the same time, however, mass spectrometry analysis of dental calculus provides direct protein evidence of bovine, sheep, and goat milk consumption in seven of nine individuals. No individuals showed molecular evidence of lactase persistence, and only one individual exhibited evidence of >10% WSH ancestry, despite the presence of WSH populations in the nearby Altai-Sayan region for more than a millennium. Unlike the spread of Neolithic farming in Europe and the expansion of Bronze Age pastoralism on the Western steppe, our results indicate that ruminant dairy pastoralism was adopted on the Eastern steppe by local hunter-gatherers through a process of cultural transmission and minimal genetic exchange with outside groups.

Assessing Metagenomic Signals Recovered from Lyuba, a 42,000-Year-Old Permafrost-Preserved Woolly Mammoth Calf.

The reconstruction of ancient metagenomes from archaeological material, and their implication in human health and evolution, is one of the most recent advances in paleomicrobiological studies. However, as for all ancient DNA (aDNA) studies, environmental and laboratory contamination need to be specifically addressed. Here we attempted to reconstruct the tissue-specific metagenomes of a 42,000-year-old, permafrost-preserved woolly mammoth calf through shotgun high-throughput sequencing. We analyzed the taxonomic composition of all tissue samples together with environmental and non-template experimental controls and compared them to metagenomes obtained from permafrost and elephant fecal samples. Preliminary results suggested the presence of tissue-specific metagenomic signals. We identified bacterial species that were present in only one experimental sample, absent from controls, and consistent with the nature of the samples. However, we failed to further authenticate any of these signals and conclude that, even when experimental samples are distinct from environmental and laboratory controls, this does not necessarily indicate endogenous presence of ancient host-associated microbiomic signals.

Fasciola hepatica eggs in paleofaeces of the Persian onager Equus hemionus onager, a donkey from Chehrabad archaeological site, dating back to the Sassanid Empire (224-651 AD), in ancient Iran.

Fascioliasis is a highly pathogenic zoonotic disease caused by the liver trematodes Fasciola hepatica and F. gigantica. Within the multidisciplinary initiative against this disease, there is the aim of understanding how this disease reached a worldwide distribution, with important veterinary and medical repercussions, by elucidating the spreading steps followed by the two fasciolids from their paleobiogeograhical origins. Fasciola eggs were detected in paleofaeces of a donkey, probably the present-day endangered Persian onager Equus hemionus onager, found in the Chehrabad salt mine archaeological site, Zanjan province, northwestern Iran. The biological remains dated back to the Sassanid period, 224-651 AD. Egg characteristics allowed for their specific ascription to F. hepatica. The interest of this finding relies on the fact of being the first archaeological finding of Fasciola in Asia and the Near East. Moreover, it allows to reach many conclusions about historical, epidemiological and spreading aspects of the disease. The finding in Chehrabad indicates that, at that time, this fasciolid had already spread through the Zagros mountains eastward from the Fertile Crescent. In that region and in ancient Egypt, livestock domestication played a crucial role in facilitating the disease spread during the postdomestication period. Donkeys appear at present to be usually infected by fasciolids in countries of the Fertile Crescent - Ancient Egypt region or neighbouring that region, with prevalences from low to very high. The high pathogenicity and mortality induced by Fasciola in these equines should be considered as an additional potential factor among the causes of the extinctions of E. h. hemippus in Syria, E. h. hydruntinus in the Anatolia-Balkans area, E. h. onager in the Caucasus and maybe also its decline in Iran. Indeed, Eurasiatic wild asses were present in the region and neighbourhood of the Fertile Crescent when the domestication of the livestock reservoirs of Fasciola began.

Queen Nefertari, the Royal Spouse of Pharaoh Ramses II: A Multidisciplinary Investigation of the Mummified Remains Found in Her Tomb (QV66).

Queen Nefertari, the favourite Royal Consort of Pharaoh Ramses II (Ancient Egypt, New Kingdom, 19th Dynasty c. 1250 BC) is famous for her beautifully decorated tomb in the Valley of the Queens. Her burial was plundered in ancient times yet still many objects were found broken in the debris when the tomb was excavated. Amongst the found objects was a pair of mummified legs. They came to the Egyptian Museum in Turin and are henceforth regarded as the remains of this famous Queen, although they were never scientifically investigated. The following multidisciplinary investigation is the first ever performed on those remains. The results (radiocarbon dating, anthropology, paleopathology, genetics, chemistry and Egyptology) all strongly speak in favour of an identification of the remains as Nefertari's, although different explanations-albeit less likely-are considered and discussed. The legs probably belong to a lady, a fully adult individual, of about 40 years of age. The materials used for embalming are consistent with Ramesside mummification traditions and indeed all objects within the tomb robustly support the burial as of Queen Nefertari.

Genotype of a historic strain of Mycobacterium tuberculosis.

The use of ancient DNA in paleopathological studies of tuberculosis has largely been restricted to confirmation of disease identifications made by skeletal analysis; few attempts at obtaining genotype data from archaeological samples have been made because of the need to perform different PCRs for each genetic locus being studied in an ancient DNA extract. We used a next generation sequencing approach involving hybridization capture directed at specific polymorphic regions of the Mycobacterium tuberculosis genome to identify a detailed genotype for a historic strain of M. tuberculosis from an individual buried in the 19th century St. George's Crypt, Leeds, West Yorkshire, England. We obtained 664,500 sequencing by oligonucleotide ligation and detection (SOLiD) reads that mapped to the targeted regions of the M. tuberculosis genome; the coverage included 218 of 247 SNPs, 10 of 11 insertion/deletion regions, and the repeat elements IS1081 and IS6110. The accuracy of the SOLiD data was checked by conventional PCRs directed at 11 SNPs and two insertion/deletions. The data placed the historic strain of M. tuberculosis in a group that is uncommon today, but it is known to have been present in North America in the early 20th century. Our results show the use of hybridization capture followed by next generation sequencing as a means of obtaining detailed genotypes of ancient varieties of M. tuberculosis, potentially enabling meaningful comparisons between strains from different geographic locations and different periods in the past.

Brief communication: identification of the authentic ancient DNA sequence in a human bone contaminated with modern DNA.

We present a method to distinguish authentic ancient DNA from contaminating DNA in a human bone. This is achieved by taking account of the spatial distribution of the various sequence families within the bone and the extent of degradation of the template DNAs, as revealed by the error content of the sequences. To demonstrate the veracity of the method, we handled two ancient human tibiae in order to contaminate them with modern DNA, and then subjected segments of the bones to various decontaminating treatments, including removal of the outer 1-2 mm, before extracting DNA, cloning, and obtaining a total of 107 mitochondrial DNA sequences. Sequences resulting from the deliberate contamination were located exclusively in the outer 1-2 mm of the bones, and only one of these 27 sequences contained an error that could be ascribed to DNA degradation. A second, much smaller set of relatively error-free sequences, which we ascribe to contamination during excavation or curation, was also located exclusively in the outer 1-2 mm. In contrast, a family of 72 sequences, displaying extensive degradation products but identifiable as haplogroup U5a1a, was distributed throughout one of the bones and represents the authentic ancient DNA content of this specimen.