DNA profiling strategies of heavily degraded human skeletal remains: Review of comparative significance of petrous bone, femur and molar teeth

Teeth are the most resistant and hardest structures of human body which are usually better preserved than other parts of skeleton and maintain their forensic odontological significance for a comparatively longer period of time. They retain their unique features even in the worst environmental conditions from taphonomic degradations to biological or chemical destructions. They can survive all sorts of natural or man-made disasters and the taphonomic destructions. Besides the unique odontological, molecular and chemical characteristics of teeth; the dental pathologies and structural anomalies also play crucial role in forensic identification of unknown human skeletal remains. The idiosyncratic features like tooth staining/coloration patterns, developmental defects, tooth wear and attritions, dental restorations/implants, cultural tooth modifications, tobacco or nut-chewing signs, occupational stigmas etc., act as valuable adjuncts to forensic examination of teeth found in forensic or bio-archaeological contexts. Dental pathologies and anomalies may reflect the oral hygiene, dietary patterns (like consumption of sweets and sugar, fats, proteins) socio-economic or socio-cultural, and the occupational status of an individual. Present review article presents a brief overview of different dental defects and their putative role in forensic anthropological identification of unknown human remains.

Ancient DNA has emerged as a powerful tool for investigating the human past and reconstructing the movements, mixtures, and adaptations that have structured genetic variation throughout human history. While the study of genome-wide ancient human DNA was initially restricted to regions with temperate climates, methodological breakthroughs have now extended the reach of ancient DNA analysis to parts of the world with hot and humid climates that are less conducive to biomolecular preservation. This includes Africa, where people harbor more genetic diversity than can be found anywhere else on the planet, reflecting deep and complex population histories. Since the first ancient African genome was published in 2015, the number of individuals with genome-wide data has increased to nearly 200, with greater coverage of diverse geographical, temporal, and cultural contexts. Ancient DNA sequences have revealed genetic variation in ancient African foragers that no longer exists in unadmixed form; illuminated how local-, regional-, and continental-scale demographic processes associated with the spread of food production and new technologies changed genetic landscapes; and discerned notable variation in interactions among people with distinct genetic ancestries, cultural practices, and, likely, languages. Despite an increasing number of studies focused on African ancient DNA, multiple regions and time periods have yet to be explored. Research to date has primarily focused on the past several thousand years in eastern and southern Africa, setting up northern, western, and central Africa, as well as deeper time periods, as key areas for future investigation. As ancient DNA research becomes increasingly integrated with anthropology and archaeology, it is advantageous to understand the basic methodological and analytical techniques, the types of questions that can be investigated, and the ways in which the discipline may continue to grow and evolve. Critically, the growth and evolution of ancient DNA research must include attention to the ethics of this work, both in African contexts and globally. In particular, it is essential that research is conducted in a way that minimizes the potential of harm to both the living and the dead. Scientists conducting ancient DNA research in Africa especially must also contend with structural challenges, including a lack of ancient DNA facilities on the continent, the extensive fragmentation of African heritage (including ancient human remains) among curating institutions worldwide, and the complexities of identifying descendant groups and other stakeholders in the wake of colonial and postcolonial disruptions and displacements. Ancient DNA research projects should be designed in a way that contributes to capacity building and the reduction of inequities between the Global North and South to ensure that the research benefits the people and communities with connections to the ancient individuals studied. While ensuring that future studies are rooted in ethical and equitable practices will require considerable collective action, ancient DNA research has already become an integral part of our understanding of African population history and will continue to shape our understanding of the African past.

Sex estimation of sub-adult human remains and determination of geographic origin: New perspectives and methodologies

Neolithic genomic data from southern France showcase intensified interactions with hunter-gatherer communities

Molecular biology has made considerable contributions to the study of human history from the earliest days of our species, when our ancestors began to walk on the savannah, to the dawn of civilization and the more recent history of mankind. The analysis of contemporary mitochondrial DNA, for example, has told us that Homo sapiens first appeared in Africa; the sequencing of the Neanderthal genome has revealed that Homo sapiens and Homo neanderthalensis lived along each other and interbred before the latter disappeared; last year, the analysis of DNA extracted from a skeleton found under a parking lot in Leicester, UK, showed that it was the remains of King Richard III. Although the advent of next‐generation sequencing and advances in bioinformatics have boosted research using ancient DNA, a major hurdle has always been the quantity and quality of the DNA itself: DNA molecules quickly degrade over time into smaller fragments, and microbial contamination makes it challenging to identify human fragments within a sample. Intriguingly, teeth have proven to be an excellent source of high‐quality ancient DNA, yielding insights into the evolution of the human diet, disease and immunity since the onset of agriculture some 10,000 years ago. Dental calculus has been the most complete source of historical sequence data, especially for probing oral microbiome populations. At the same time, dental pulp—the connective tissue at the centre of teeth—has been an important source of information about ancient diseases. Archaeologist Keith Dobney, from the University of Aberdeen in the UK, and his colleagues first identified the potential of dental calculus as a source of information about past diets and microbial populations long before the advent of ancient DNA studies [1]. Now, almost 30 years later, Dobney is among those at the forefront of the revolution in ancient DNA analysis. “It appears that dental …

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.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument

Ancient degraded remains of human origin can be identified using ancient DNA analyses. The mitochondrial DNA is a useful tool to solve unsolved cases in relation to identification of an individual where nuclear DNA is too degraded or insufficient to be used for the purpose. Comparative analysis of mtDNA content in petrous bone and the tooth-root cementum have revealed that later is best substrate for studying genomic analyses of ancient human populations The dental DNA is less prone to contamination, and the preservation of ancient DNA from dental samples is easier than bones. The selection of correct decontamination method, appropriate extraction protocol provides best results and thus increases value of bones and teeth as a forensic evidence in cases where identity establishment is problematic. In this review article, the current status of the methods used in ancient DNA research, the advantages and the limitations of this archaeogenetical method have been presented.

Community partnerships are fundamental to ethical ancient DNA research.

The petrous bone is considered the most efficient source of endogenous DNA across skeletal tissues in ancient DNA (aDNA) research as well as in forensic work. Recently, ancient DNA in auditory ossicle bones was shown to be comparably well-preserved as in the petrous, although no attempt was made to distinguish among the three ossicle bones. In this study, we compare aDNA profiles across matched ossicle- and petrous-derived sequencing libraries prepared from 29 human skeletons from Neolithic Anatolia and Medieval Iberia. We find that the stapes and incus provide higher human endogenous aDNA than the petrous bone, with >2×; higher median rates of endogenous aDNA recovery, while the malleus performs similarly to the petrous. Human aDNA fragments retrieved from the stapes were 8% longer than those from the petrous, whereas postmortem damage, clonality and contamination rates were comparable among the studied bone types. These observations are corroborated by data from non-matched ossicle or petrous libraries from 81 individuals from the same contexts, with the highest endogenous aDNA content observed in the stapes. Despite being the smallest bone in the human skeleton, the stapes, along with the incus, may be among the most optimal aDNA sources yet identified.

DNA ‘lives’ to tell the tale

The use of genetic technology in forensic science and archaeometry is applied primarily to distinguish between individials who may be the source of biological material associated with archeological remains. DNA sequences from ancient fossils have great potential for studies of phylogeny, biogeography and molecular evolution. DNA from fossils also facilitates the rigorous testing and calibration of mutation rates among related taxa, sex test and molecular divergence time (Cano et al., 1993; Burger et al., 1999). In this study, a rapid and quantitative ancient DNA extaction methods from human skeletal remains was developed for application of forensic science and archaeometry. For that reason, DNA was extracted from ancient human bones from Mugla in Turkey. Furthermore, all the bone samples which are obtained from burial place are subjected to DNA isolation and then interspecific sequence polymorphisms in the mitochondrial cytochrome b gene were analyzed by PCR to determine the species origin of Bronze Age animal and human skeletal remains. Existing techniques were refined by targeted primer design focusing on a DNA fragment shorter than 200 bp, an approach allowing us to identify up to all bone samples at the same time. For routine applications in archaeometry, food or material analyses, PCR may thus provide a simple alternative to sequencing of PCR products, allowing discrimination between species, even if the template DNA is degraded or contains traces of DNA from various species. Key words: Ancient DNA, species determination, cytochrome b gene, mtDNA.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.

High-Throughput DNA Sequencing (HTS) technologies have changed the way in which we detect and assess DNA contamination in ancient DNA studies. Researchers use computational methods to mine the large quantity of sequencing data to detect characteristic patterns of DNA damage, and to evaluate the authenticity of the results. We argue that unless computational methods can confidently separate authentic ancient DNA sequences from contaminating DNA that displays damage patterns under independent decay processes, prevention and control of DNA contamination should remain a central and critical aspect of ancient human DNA studies. Ideally, DNA contamination can be prevented early on by following minimal guidelines during excavation, sample collection and/or subsequent handling. Contaminating DNA should also be monitored or minimised in the ancient DNA laboratory using specialised facilities and strict experimental procedures. In this paper, we update recommendations to control for DNA contamination from the field to the laboratory, in an attempt to facilitate communication between field archaeologists, anthropologists and ancient DNA researchers. We also provide updated criteria of ancient DNA authenticity for HTS-based studies. We are confident that the procedures outlined here will increase the retrieval of higher proportions of authentic genetic information from valuable archaeological human remains in the future.

The identification of unknown human remains represents an important task in forensic casework. If there are no clues as to the identity of the remains, then the age, sex, and origin are the most important factors to limit the search for a matching person. Here, we present the outcome of application of so-called bomb pulse radiocarbon (14C derived from above-ground nuclear bomb tests during 1955–1963) analysis to birthdate human remains. In nine identified cases, 14C analysis of tooth crowns provided an estimate of the true date of birth with an average absolute error of 1.2 ± 0.8 years. Analysis of 14C in tooth roots also showed a good precision with an average absolute error of 2.3 ± 2.5 years. Levels of 14C in bones can determine whether a subject has lived after 1955 or not, but more precise carbon turnover data for bones would be needed to calculate date of birth and date of death. Aspartic acid racemization analysis was performed on samples from four cases; in one of these, the year of birth could be predicted with good precision, whereas the other three cases are still unidentified. The stable isotope 13C was analyzed in tooth crowns to estimate provenance. Levels of 13C indicative of Scandinavian provenance were found in known Scandinavian subjects. Teeth from four Polish subjects all showed higher 13C levels than the average for Scandinavian subjects.