Evaluation of whole-genome enrichment and sequencing of T. pallidum from FFPE samples after 75 years.

The recent developments in genomic sequencing have permitted the publication of many new complete genome sequences of Treponema pallidum pallidum, the bacterium responsible for syphilis, which has led to a new understanding of its phylogeny and diversity. However, few archived samples are available, because of the degradability of the bacterium and the difficulties in preservation. We present a complete genome obtained from a Formalin-Fixed Paraffin-Embedded (FFPE) organ sample from 1947, kept at the Strasbourg Faculty of Medicine. This is the preliminary, proof-of concept study of this collection/biobank of more than 1.5 million FFPE samples and the evaluation of the feasibility of genomic analyses. We demonstrate here that even degraded DNA from fragile bacteria can be recovered from 75-year-old FFPE samples and therefore propose that such collections as this one can function as sources of biological material for genetic studies of pathogens, cancer, or even the historical human population itself.

At the Origins of Tobacco-Smoking and Tea Consumption in a Virgin Population (Yakutia, 1650-1900 A.D.): Comparison of Pharmacological, Histological, Economic and Cultural Data.

(1) Background: The way tobacco and tea spread among virgin populations is of major interest our understanding of how ancient economic and cultural practices could have influenced current habits. (2) Methods: hair concentrations of theobromine, theophylline, caffeine, nicotine, and cotinine were measured in hair samples from 47 frozen bodies of people from eastern Siberia, dated from the contact with Europeans to the assimilation of people into Russian society. (3) Results: hair concentration of theobromine, theophylline, and caffeine vary with the type of beverage consumed: green, black, or local herbal teas. Shortly after the first contacts, a few heavy consumers of tobacco were found among light or passive consumers. Tobacco-related co-morbidities began to be recorded one century after and heavy tea users were only found from the 19th century (4) Conclusions: Economic factors and social and family contacts seem to have played a decisive role in tobacco consumption very early on. Behavioral evolution governed the process of substance integration into Siberian culture and was a determinant for the continuity of its use across long periods of time. Analyzing the respective contributions of social and economic processes in the use of these substances opens avenues of investigation for today's public health.

Sucrose Is Not the Whole Story: Risk Factors and Oral Health at the Contact (Yakutia, Siberia-16th/19th).

(1) Background: contact between indigenous and European populations has often resulted in changes in oral health attributed to the introduction of sucrose. Most studies are per tooth over considerable periods and with few ethnological references. (2) Aim: dental epidemiology of 96 autochthonous frozen bodies from Yakutia between the early 17th century and the late 19th century; comparisons with historical texts and ethnographic data. (3) Material and methods: we use descriptive statistics and discriminant factorial analyses to identify dominant variables in the dataset and compare periods and subjects, considering all variables. (4) Results: the peculiarities of the population are the rarity of cavities and the relative frequency of dental pathologies leading to death. Assimilation into the Russian Orthodox culture has led to decreased tooth wear and an increase in tooth loss. Dental health evolves only two centuries after the contact. (5) Conclusions: the confrontation with historical data suggests that changes are not related to the growing importance of sucrose but to a combined action: the substitution of dendrophagy by cereal flour; the decrease in immunity linked to the development of chronic infectious diseases; tobacco addiction and the mandibular torus: a risk factor promoting apical cysts.

Genetic evidence suggests a sense of family, parity and conquest in the Xiongnu Iron Age nomads of Mongolia.

In an effort to characterize the people who composed the groups known as the Xiongnu, nuclear and whole mitochondrial DNA data were generated from the skeletal remains of 52 individuals excavated from the Tamir Ulaan Khoshuu (TUK) cemetery in Central Mongolia. This burial site, attributed to the Xiongnu period, was used from the first century BC to the first century AD. Kinship analyses were conducted using autosomal and Y-chromosomal DNA markers along with complete sequences of the mitochondrial genome. These analyses suggested close kin relationships between many individuals. Nineteen such individuals composed a large family spanning five generations. Within this family, we determined that a woman was of especially high status; this is a novel insight into the structure and hierarchy of societies from the Xiongnu period. Moreover, our findings confirmed that the Xiongnu had a strongly admixed mitochondrial and Y-chromosome gene pools and revealed a significant western component in the Xiongnu group studied. Using a fine-scale approach (haplotype instead of haplogroup-level information), we propose Scytho-Siberians as ancestors of the Xiongnu and Huns as their descendants.

The genetic legacy of legendary and historical Siberian chieftains.

Seventeen years of archaeological and anthropological expeditions in North-Eastern Siberia (in the Sakha Republic, Yakutia) have permitted the genetic analysis of 150 ancient (15th-19th century) and 510 modern individuals. Almost all males were successfully analysed (Y-STR) and this allowed us to identify paternal lineages and their geographical expansion through time. This genetic data was confronted with mythological, historical and material evidence to establish the sequence of events that built the modern Yakut genetic diversity. We show that the ancient Yakuts recovered from this large collection of graves are not representative of an ancient population. Uncommonly, we were also able to demonstrate that the funerary preference observed here involved three specific male lineages, especially in the 18th century. Moreover, this dominance was likely caused by the Russian conquest of Siberia which allowed some male clans to rise to new levels of power. Finally, we give indications that some mythical and historical figures might have been the actors of those genetic changes. These results help us reconsider the genetic dynamics of colonization in some regions, question the distinction between fact and myth in national histories and provide a rare insight into a funerary ensemble by revealing the biased process of its composition.

The petrous bone: Ideal substrate in legal medicine?

Over the last few years, palaeogenomic studies of the petrous bone (the densest part of the temporal bone) have shown that it is a source of DNA in both larger quantities and of better quality than other bones. This dense bone around the otic capsule has therefore been called the choice substrate in palaeogenomics. Because the practice of forensic genetics responds to different imperatives, we implemented a study aimed at (i) understanding how and why the petrous bone is an advantageous substrate in ancient DNA studies and (ii) establishing whether it is advantageous in forensic STR typing. We selected 50 individual skeletal remains and extracted DNA from one tooth and one petrous bone from each. We then amplified 24 STR markers commonly used in forensic identification and compared the quality of that amplification using the RFU intensities of the signal as read on the STR profiles. We also performed histological analyses to compare (i) the microscopic structure of a petrous bone and of a tooth and (ii) the microscopic structure of fresh petrous bone and of an archaeological or forensic sample. We show that the RFU intensities read on STR profiles are systematically higher in experiments using DNA extracted from petrous bones rather than teeth. For this reason, we were more likely to obtain a complete STR profile from petrous bone material, increasing the chance of identification in a forensic setting. Histological analyses revealed peculiar microstructural characteristics (tissue organization), unique to the petrous bone, that might explain the good preservation of DNA in that substrate. Therefore, it appears that despite the necessity of analysing longer fragments in forensic STR typing compared to NGS palaeogenomics, the use of petrous bones in forensic genetics could prove valuable, especially in cases involving infants, toothless individuals or very degraded skeletal remains.

The limitations of kinship determinations using STR data in ill-defined populations.

The likelihood ratio (LR) method is commonly used to determine kinship in civil, criminal, or forensic cases. For the past 15 years, our research group has also applied LR to ancient STR data and obtained kinship results for collections of graves or necropolises. Although we were able to reconstruct large genealogies, some pairs of individuals showed ambiguous results. Second-degree relationships, half-sibling pairs for example, were often inconsistent with detected first-degree relationships, such as parent/child or brother/sister pairs. We therefore set about providing empirical estimations of the error rates for the LR method in living populations with STR allelic diversities comparable to that of the ancient populations we had previously studied. We collected biological samples in the field in North-Eastern Siberia and West Africa and studied more than 800 pairs of STR profiles from individuals with known relationships. Because commercial STR panels were constructed for specific regions (namely Europe and North America), their allelic makeup showed a significant deficit in diversity when compared to European populations, replicating a situation often faced in ancient DNA studies. We assessed the capacity of the LR method to confirm known relationships (effectiveness) and its capacity to detect those relationships (reliability). Concerns over the effectiveness of LR determinations are mostly an issue in forensic studies, while the reliability of the detection of kinship is an issue for the study of necropolises or other large gatherings of unidentified individuals, such as disaster victims or mass graves. We show that the application of LR to both test populations highlights specific issues (both false positives and false negatives) that prevent the confirmation of second-degree kinship or even full siblingship in small populations. Up to 29% of detected full sibling relationships were either overestimated half-sibling relationships or underestimated parent-offspring relationships. The error rate for detected half-sibling relationships was even higher, reaching 41%. Only parent-offspring pairs were reliably detected or confirmed. This implies that, in populations that are small, ill-defined, or for which the STR loci analyzed are inappropriate, an examiner might not be able to distinguish a pair of full siblings from a pair of half-siblings. Furthermore, half-sibling pairs might be overlooked altogether, an issue that is exacerbated by the common confusion, in many languages and cultures, between half-siblings and full siblings. Consequently, in the study of ancient populations, human remains of unknown origins, or poorly surveyed modern populations, we recommend a conservative approach to kinship determined by LR. Next-generation sequencing data should be used when possible, but the costs and technology involved might be prohibitive. Therefore, in potentially contentious situations or cases lacking sufficient external information, uniparental markers should be analyzed: ideally, complete mitochondrial genomes and Y-chromosome haplotypes (STR, SNP, and/or sequencing).

Genetic kinship and admixture in Iron Age Scytho-Siberians.

Scythians are known from written sources as horse-riding nomadic peoples who dominated the Eurasian steppe throughout the Iron Age. However, their origins and the exact nature of their social organization remain debated. Three hypotheses prevail regarding their origins that can be summarized as a "western origin", an "eastern origin" and a "multi-regional origin". In this work, we first aimed to address the question of the familial and social organization of some Scythian groups (Scytho-Siberians) by testing genetic kinship and, second, to add new elements on their origins through phylogeographical analyses. Twenty-eight Scythian individuals from 5 archeological sites in the Tuva Republic (Russia) were analyzed using autosomal Short Tandem Repeats (STR), Y-STR and Y-SNP typing as well as whole mitochondrial (mtDNA) genome sequencing. Familial relationships were assessed using the Likelihood Ratio (LR) method. Thirteen of the 28 individuals tested were linked by first-degree relationships. When related, the individuals were buried together, except for one adult woman, buried separately from her mother and young sister. Y-chromosome analysis revealed a burial pattern linked to paternal lineages, with men bearing closely related Y-haplotypes buried on the same sites. Inversely, various mtDNA lineages can be found on each site. Y-chromosomal and mtDNA haplogroups were almost equally distributed between Western and Eastern Eurasian haplogroups. These results suggest that Siberian Scythians were organized in patrilocal and patrilineal societies with burial practices linked to both kinship and paternal lineages. It also appears that the group analyzed shared a greater genetic link with Asian populations than Western Scythians did.

New genetic evidence of affinities and discontinuities between bronze age Siberian populations.

OBJECTIVES: This work focuses on the populations of South Siberia during the Eneolithic and Bronze Age and specifically on the contribution of uniparental lineage and phenotypical data to the question of the genetic affinities and discontinuities between western and eastern populations. MATERIALS AND METHODS: We performed molecular analyses on the remains of 28 ancient humans (10 Afanasievo (3600-2500 BC) and 18 Okunevo (2500-1800 BC) individuals). For each sample, two uniparentally inherited systems (mitochondrial DNA and Y-chromosome DNA) were studied, in order to trace back maternal and paternal lineages. Phenotype-informative SNPs (Single Nucleotide Polymorphisms) were also analyzed, along with autosomal STRs (Short Tandem Repeats). RESULTS: Most of the Afanasievo men submitted to analysis belonged to a single sub-haplogroup, R1b1a1a, which reveals the predominance of this haplogroup in these early Bronze Age populations. Conversely, Okunevo individuals carried more diverse paternal lineages that mostly belonged to Asian/Siberian haplogroups. These differences are also apparent, although less strongly, in mitochondrial lineage composition and phenotype marker variant frequencies. DISCUSSION: This study provides new elements that contribute to our understanding of the genetic interactions between populations in Eneolithic and Bronze Age southern Siberia. Our results support the hypothesis of a genetic link between Afanasievo and Yamnaya (in western Eurasia), as suggested by previous studies of other markers. However, we found no Y-chromosome lineage evidence of a possible Afanasievo migration to the Tarim Basin. Moreover, the presence of Y-haplogroup Q in Okunevo individuals links them to Native American populations, as was suggested by whole-genome sequencing.

The genetics of kinship in remote human groups.

For fifteen years, part of the work of our research team has been focused on the study of parental links between individuals living hundreds or thousands of years ago, whose remains have been found in single graves or large funerary complexes. These studies have been undertaken using methods developed by forensic genetics to identify individuals, mainly based on the genotyping of autosomal STR (Short Tandem Repeats). Issues arose from this work, namely the limits of studying small numbers of subjects, originating from groups of finite sizes where kinships cannot be inferred a priori and for which reference allelic frequencies do not exist. Although ideal human populations are rare when undertaking such studies, the Yakuts of Eastern Siberia constitute a very advantageous model, with large numbers of small pastoral communities and well-preserved archaeological material. The study of kinship in the ancient Yakuts allowed us to highlight the difficulties in analysing genetic data from small ancient human groups and to develop a strategy to improve the accuracy of statistical computations. This work describes this strategy and possible solutions to the study of populations outside of the frame of reference of global meta-populations, due either to isolation, remoteness or antiquity.