Comparing Genetic and Physical Anthropological Analyses for the Biological Profile of Unidentified and Identified Bodies in Milan.

When studying unknown human remains, the estimation of skeletal sex and ancestry is paramount to create the victim's biological profile and attempt identification. In this paper, a multidisciplinary approach to infer the sex and biogeographical ancestry of different skeletons, using physical methods and routine forensic markers, is explored. Forensic investigators, thus, encounter two main issues: (1) the use of markers such as STRs that are not the best choice in terms of inferring biogeographical ancestry but are routine forensic markers to identify a person, and (2) the concordance of the physical and molecular results. In addition, a comparison of physical/molecular and then antemortem data (of a subset of individuals that are identified during our research) was evaluated. Antemortem data was particularly beneficial to evaluate the accuracy rates of the biological profiles produced by anthropologists and classification rates obtained by molecular experts using autosomal genetic profiles and multivariate statistical approaches. Our results highlight that physical and molecular analyses are in perfect agreement for sex estimation, but some discrepancies in ancestry estimation were observed in 5 out of 24 cases.

How Physical and Molecular Anthropology Interplay in the Creation of Biological Profiles of Unidentified Migrants.

The skeletal sex and ancestry of unidentified human crania can be inferred both from physical and from molecular features. This paper depicts and discusses the experiences of physical and molecular anthropologists on a set of commingled crania from the largest Mediterranean shipwreck disaster on 18 April 2015, in order to facilitate identification of human crania. Twenty-one disarticulated crania that were recovered from the above-mentioned shipwreck were analyzed to estimate skeletal sex and ancestry, following a physical and a molecular pipeline. The physical analyses applied morphological and metric methods that provided posterior probabilities for the crania to be classified into a sex or ancestral group. The molecular analyses were performed on petrous bones via a shotgun sequencing approach that allowed us to determine the sex of each individual and to retrieve the complete mitochondrial genome, Y chromosome single nucleotide polymorphisms, up to 597573 SNPs across the human genome from each individual. The morphometric sex analyses showed that most crania belonged to male individuals, although some estimations remained uncertain or undetermined. Inconsistent results were obtained for ancestry estimation as well, since morphological methods classified the crania mostly as European/White, in contrast to the most numerous African forms determined by craniometric analyses. This quite agreed with molecular analyses that identified only African males. Overall, undetermined and contrasting results were obtained between disciplines, preventing the creation of reliable and sound biological profiles that could provide guidance on the sex and ancestral group of the victims. Therefore, the times may not be mature for a merger of physical and molecular anthropology. However, future investigations of this research avenue would pave the way to the possible development of novel tools, methods, and wider reference databases that could address the limitations of both disciplines.

Biogeographical ancestry, variable selection, and PLS-DA method: a new panel to assess ancestry in forensic samples via MPS technology.

As evidenced by the large number of articles recently published in the literature, forensic scientists are making great efforts to infer externally visible features and biogeographical ancestry (BGA) from DNA analysis. Just as phenotypic, ancestry information obtained from DNA can provide investigative leads to identify the victims (missing/unidentified persons, crime/armed conflict/mass disaster victims) or trace their perpetrators when no matches were found with the reference profile or in the database. Recently, the advent of Massively Parallel Sequencing technologies associated with the possibility of harnessing high-throughput genetic data allowed us to investigate the associations between phenotypic and genomic variations in worldwide human populations and develop new BGA forensic tools capable of simultaneously analyzing up to millions of markers if for example the ancient DNA approach of hybridization capture was adopted to target SNPs of interest. In the present study, a selection of more than 3000 SNPs was performed to create a new BGA panel and the accuracy of the new panel to infer ancestry from unknown samples was evaluated by the PLS-DA method. Subsequently, the panel created was assessed using three variable selection techniques (Backward variable elimination, Genetic Algorithm and Regularized elimination procedure), and the best SNPs in terms of inferring bio-geographical ancestry at inter- and intra-continental level were selected to obtain panels to predict BGA with a reduced number of selected markers to be applied in routine forensic cases where PCR amplification is the best choice to target SNPs.

Multivariate statistical approach and machine learning for the evaluation of biogeographical ancestry inference in the forensic field.

The biogeographical ancestry (BGA) of a trace or a person/skeleton refers to the component of ethnicity, constituted of biological and cultural elements, that is biologically determined. Nowadays, many individuals are interested in exploring their genealogy, and the capability to distinguish biogeographic information about population groups and subgroups via DNA analysis plays an essential role in several fields such as in forensics. In fact, for investigative and intelligence purposes, it is beneficial to inference the biogeographical origins of perpetrators of crimes or victims of unsolved cold cases when no reference profile from perpetrators or database hits for comparative purposes are available. Current approaches for biogeographical ancestry estimation using SNPs data are usually based on PCA and Structure software. The present study provides an alternative method that involves multivariate data analysis and machine learning strategies to evaluate BGA discriminating power of unknown samples using different commercial panels. Starting from 1000 Genomes project, Simons Genome Diversity Project and Human Genome Diversity Project datasets involving African, American, Asian, European and Oceania individuals, and moving towards further and more geographically restricted populations, powerful multivariate techniques such as Partial Least Squares-Discriminant Analysis (PLS-DA) and machine learning techniques such as XGBoost were employed, and their discriminating power was compared. PLS-DA method provided more robust classifications than XGBoost method, showing that the adopted approach might be an interesting tool for forensic experts to infer BGA information from the DNA profile of unknown individuals, but also highlighting that the commercial forensic panels could be inadequate to discriminate populations at intra-continental level.

Kinship assignment with the ForenSeq™ DNA Signature Prep Kit: Sources of error in simulated and real cases.

Kinship recognition between anonymous DNA samples is becoming a relevant issue in forensics, more so with the increasing number of DNA profiles in databanks. Also, NGS-based genotyping is being increasingly used in routine personal identification, to simultaneously type large numbers of markers of different kind. In the present work, we explored computationally and experimentally the performance of the ForenSeq™ DNA Signature Prep Kit in identifying the true relationship between two anonymous samples, distinguishing it from other possible relationships. We analyzed with Familias R series of 10,000 pairs with 9 different simulated relationships, corresponding to different degrees of autosomal sharing. For each pair we obtained likelihood ratios for five kinship hypotheses vs. unrelatedness, and used their ranking to identify the preferred relationship. We also typed 21 subjects from two pedigrees, representing from parent-child to 4th cousins relationships. As expected, the power for identifying the true relationship decays in the order of autosomal sharing. Parent-child and full siblings can be robustly identified against other relationships. For half-siblings the chance of reaching a significant conclusion is already small. For more distant relationships the proportion of cases correctly and significantly identified is 10% or less. Bidirectional errors in kinship attribution include the suggestion of relatedness when this does not exist (10-50%), and the suggestion of independence in pairs of individuals more than 4 generations apart (25-60%). The real cases revealed a relevant effect of genotype miscalling at some loci, which could only be partly avoided by modulating the analysis parameters. In conclusion, with the exception of first degree relatives, the kit can be useful to inform additional investigations, but does not usually provide probatory results.

How a Paleogenomic Approach Can Provide Details on Bioarchaeological Reconstruction: A Case Study from the Globular Amphorae Culture.

Ancient human remains have the potential to explain a great deal about the prehistory of humankind. Due to recent technological and bioinformatics advances, their study, at the palaeogenomic level, can provide important information about population dynamics, culture changes, and the lifestyles of our ancestors. In this study, mitochondrial and nuclear genome data obtained from human bone remains associated with the Neolithic Globular Amphorae culture, which were recovered in the Megalithic barrow of Kierzkowo (Poland), were reanalysed to gain insight into the social organisation and use of the archaeological site and to provide information at the individual level. We were able to successfully estimate the minimum number of individuals, sex, kin relationships, and phenotypic traits of the buried individuals, despite the low level of preservation of the bone samples and the intricate taphonomic conditions. In addition, the evaluation of damage patterns allowed us to highlight the presence of "intruders"-that is, of more recent skeletal remains that did not belong to the original burial. Due to its characteristics, the study of the Kierzkowo barrow represented a challenge for the reconstruction of the biological profile of the human community who exploited it and an excellent example of the contribution that ancient genomic analysis can provide to archaeological reconstruction.

The first evidence for Late Pleistocene dogs in Italy.

The identification of the earliest dogs is challenging because of the absence and/or mosaic pattern of morphological diagnostic features in the initial phases of the domestication process. Furthermore, the natural occurrence of some of these characters in Late Pleistocene wolf populations and the time it took from the onset of traits related to domestication to their prevalence remain indefinite. For these reasons, the spatiotemporal context of the early domestication of dogs is hotly debated. Our combined molecular and morphological analyses of fossil canid remains from the sites of Grotta Paglicci and Grotta Romanelli, in southern Italy, attest of the presence of dogs at least 14,000 calibrated years before present. This unambiguously documents one of the earliest occurrence of domesticates in the Upper Palaeolithic of Europe and in the Mediterranean. The genetic affinity between the Palaeolithic dogs from southern Italy and contemporaneous ones found in Germany also suggest that these animals were an important common adjunct during the Late Glacial, when strong cultural diversification occurred between the Mediterranean world and European areas north of the Alps. Additionally, aDNA analyses indicate that this Upper Palaeolithic dog lineage from Italy may have contributed to the genetic diversity of living dogs.

Ancient human mitochondrial genomes from Bronze Age Bulgaria: new insights into the genetic history of Thracians.

One of the best documented Indo-European civilizations that inhabited Bulgaria is the Thracians, who lasted for more than five millennia and whose origin and relationships with other past and present-day populations are debated among researchers. Here we report 25 new complete mitochondrial genomes of ancient individuals coming from three necropolises located in different regions of Bulgaria - Shekerdja mogila, Gabrova mogila and Bereketska mogila - dated to II-III millennium BC. The identified mtDNA haplogroup composition reflects the mitochondrial variability of Western Eurasia. In particular, within the ancient Eurasian genetic landscape, Thracians locate in an intermediate position between Early Neolithic farmers and Late Neolithic-Bronze Age steppe pastoralists, supporting the scenario that the Balkan region has been a link between Eastern Europe and the Mediterranean since the prehistoric time. Spatial Principal Component Analysis (sPCA) performed on Thracian and modern mtDNA sequences, confirms the pattern highlighted on ancient populations, overall indicating that the maternal gene pool of Thracians reflects their central geographical position at the gateway of Europe.

Ancestral mitochondrial N lineage from the Neolithic 'green' Sahara.

Because Africa's climate hampers DNA preservation, knowledge of its genetic variability is mainly restricted to modern samples, even though population genetics dynamics and back-migrations from Eurasia may have modified haplotype frequencies, masking ancient genetic scenarios. Thanks to improved methodologies, ancient genetic data for the African continent are now increasingly available, starting to fill in the gap. Here we present newly obtained mitochondrial genomes from two ~7000-year-old individuals from Takarkori rockshelter, Libya, representing the earliest and first genetic data for the Sahara region. These individuals carry a novel mutation motif linked to the haplogroup N root. Our result demonstrates the presence of an ancestral lineage of the N haplogroup in the Holocene "Green Sahara", associated to a Middle Pastoral (Neolithic) context.

A genetic perspective on Longobard-Era migrations.

From the first century AD, Europe has been interested by population movements, commonly known as Barbarian migrations. Among these processes, the one involving the Longobard culture interested a vast region, but its dynamics and demographic impact remains largely unknown. Here we report 87 new complete mitochondrial sequences coming from nine early-medieval cemeteries located along the area interested by the Longobard migration (Czech Republic, Hungary and Italy). From the same areas, we sampled necropoleis characterized by cultural markers associated with the Longobard culture (LC) and coeval burials where no such markers were found, or with a chronology slightly preceding the presumed arrival of the Longobards in that region (NLC). Population genetics analysis and demographic modeling highlighted a similarity between LC individuals, as reflected by the sharing of quite rare haplogroups and by the degree of genetic resemblance between Hungarian and Italian LC necropoleis estimated via a Bayesian approach, ABC. The demographic model receiving the strongest statistical support also postulates a contact between LC and NLC communities, thus indicating a complex dynamics of admixture in medieval Europe.

From unknown to known: Identification of the remains at the mausoleum of fosse Ardeatine.

During the Second World War, on 24th March 1944, 335 Italians were massacred near Rome by the occupying forces of Nazi Germany. Four months later forensic examination led to the identification of 323 out of 335 victims. After approximately 60 years, the identification of the remaining unidentified twelve victims began with anthropological and genetic analysis carried out by a team of Italian forensic experts. Anthropological analysis was performed in field in order to confirm the sex of each victim and verify the presence of only one individual in each grave for a correct sampling. Selected bone fragments for each individual were then collected and transferred to the laboratory for genetic analysis. Although the anthropological ante mortem information was limited, morphological and metrical data was collected for a possible future identification of the victims. Subsequently, the typing of autosomal loci, Y-STR and mtDNA D-loop region of all bone and available reference samples was conducted. LR and cumulative LRs obtained from autosomal STR and Y-STR results confirmed the alleged relationship between three victims and their relatives with values over 104 (one sample) and 106 (two samples). Therefore, the genetic analysis offered the families the possibility of replacing the number of the grave with the name of the victim.

Maternal DNA lineages at the gate of Europe in the 10th century AD.

Given the paucity of archaeogenetic data available for medieval European populations in comparison to other historical periods, the genetic landscape of this age appears as a puzzle of dispersed, small, known pieces. In particular, Southeastern Europe has been scarcely investigated to date. In this paper, we report the study of mitochondrial DNA in 10th century AD human samples from Capidava necropolis, located in Dobruja (Southeastern Romania, Southeastern Europe). This geographical region is particularly interesting because of the extensive population flux following diverse migration routes, and the complex interactions between distinct population groups during the medieval period. We successfully amplified and typed the mitochondrial control region of 10 individuals. For five of them, we also reconstructed the complete mitochondrial genomes using hybridization-based DNA capture combined with Next Generation Sequencing. We have portrayed the genetic structure of the Capidava medieval population, represented by 10 individuals displaying 8 haplotypes (U5a1c2a, V1a, R0a2'3, H1, U3a, N9a9, H5e1a1, and H13a1a3). Remarkable for this site is the presence of both Central Asiatic (N9a) and common European mtDNA haplotypes, establishing Capidava as a point of convergence between East and West. The distribution of mtDNA lineages in the necropolis highlighted the existence of two groups of two individuals with close maternal relationships as they share the same haplotypes. We also sketch, using comparative statistical and population genetic analyses, the genetic relationships between the investigated dataset and other medieval and modern Eurasian populations.

Neither femur nor tooth: Petrous bone for identifying archaeological bone samples via forensic approach.

One of the major challenges of molecular biology in anthropological analysis is the identification via DNA typing of bone or teeth samples that can be collected from archaeological site in order to investigate kinship relationships. Due to the difficulties of isolating and analysing DNA from such samples, several efforts have been made to solve these problems, but less work has been conducted to identify the proper type of bone samples for the DNA analysis. Therefore, following the promising results obtained from the DNA analysis of petrous bones by different groups of researchers, for the first time, here we investigated the possibility of using petrous bones as skeletal elements useful for short tandem repeat (STR) typing via capillary electrophoresis technique in ancient bone samples. In order to compare the results from petrous bone, femur and tooth samples, a total of 39 skeletal elements were collected from 13 different individuals excavated from Italian archaeological sites, dating from the sixth to seventh century C.E. The DNA was extracted, quantified, and subsequently amplified using two STR multiplex kits. The presence of a good amount of genetic material, despite high degradation, allowed us to quantify and subsequently identify STR profiles via CE analysis from ancient petrous bones that were complete for four out of thirteen samples and higher than 11 autosomal loci for all samples. Our results indicated that petrous bone is the best skeletal element with regard to DNA conservation and is a valuable element from which it is possible to obtain a complete STR profile also when analysing ancient bones. The STR results showed the possibility to use the petrous bones for identification and matching purposes in cases in which the biological material is poor and highly degraded such as in archaeological studies. Therefore, STR typing could represent a time-saving and cheap chance to verify kinship relationships in archaeological sites and evaluate sex when skeletal material is not suitable for morphometric estimate as in case of infants.

Genome diversity in the Neolithic Globular Amphorae culture and the spread of Indo-European languages.

It is unclear whether Indo-European languages in Europe spread from the Pontic steppes in the late Neolithic, or from Anatolia in the Early Neolithic. Under the former hypothesis, people of the Globular Amphorae culture (GAC) would be descended from Eastern ancestors, likely representing the Yamnaya culture. However, nuclear (six individuals typed for 597 573 SNPs) and mitochondrial (11 complete sequences) DNA from the GAC appear closer to those of earlier Neolithic groups than to the DNA of all other populations related to the Pontic steppe migration. Explicit comparisons of alternative demographic models via approximate Bayesian computation confirmed this pattern. These results are not in contrast to Late Neolithic gene flow from the Pontic steppes into Central Europe. However, they add nuance to this model, showing that the eastern affinities of the GAC in the archaeological record reflect cultural influences from other groups from the East, rather than the movement of people.

Human identification by lice: A Next Generation Sequencing challenge.

Rapid and progressive advances in molecular biology techniques and the advent of Next Generation Sequencing (NGS) have opened new possibilities for analyses also in the identification of entomological matrixes. Insects and other arthropods are widespread in nature and those found at a crime scene can provide a useful contribution to forensic investigations. Entomological evidence is used by experts to define the postmortem interval (PMI), which is essentially based on morphological recognition of the insect and an estimation of its insect life cycle stage. However, molecular genotyping methods can also provide an important support for forensic entomological investigations when the identification of species or human genetic material is required. This case study concerns a collection of insects found in the house of a woman who died from unknown causes. Initially the insects were identified morphologically as belonging to the Pediculidae family, and then, human DNA was extracted and analyzed from their gastrointestinal tract. The application of the latest generation forensic DNA assays, such as the Quantifiler(®) Trio DNA Quantification Kit and the HID-Ion AmpliSeq™ Identity Panel (Applied Biosystems(®)), individuated the presence of human DNA in the samples and determined the genetic profile.

Multimodal molecular imaging system for pathway-specific reporter gene expression.

Preclinical imaging modalities represent an essential tool to develop a modern and translational biomedical research. To date, Optical Imaging (OI) and Magnetic Resonance Imaging (MRI) are used principally in separate studies for molecular imaging studies. We decided to combine OI and MRI together through the development of a lentiviral vector to monitor the Wnt pathway response to Lithium Chloride (LiCl) treatment. The construct was stably infected in glioblastoma cells and, after intracranial transplantation in mice, serial MRI and OI imaging sessions were performed to detect human ferritin heavy chain protein (hFTH) and firefly luciferase enzyme (FLuc) respectively. The system allowed also ex vivo analysis using a constitutive fluorescence protein expression. In mice, LiCl administration has shown significantly increment of luminescence signal and a lower signal of T2 values (P<0.05), recorded noninvasively with OI and a 7 Tesla MRI scanner. This study indicates that OI and MRI can be performed in a single in vivo experiment, providing an in vivo proof-of-concept for drug discovery projects in preclinical phase.

The Neanderthal in the karst: First dating, morphometric, and paleogenetic data on the fossil skeleton from Altamura (Italy).

In 1993, a fossil hominin skeleton was discovered in the karst caves of Lamalunga, near Altamura, in southern Italy. Despite the fact that this specimen represents one of the most extraordinary hominin specimens ever found in Europe, for the last two decades our knowledge of it has been based purely on the documented on-site observations. Recently, the retrieval from the cave of a fragment of bone (part of the right scapula) allowed the first dating of the individual, the quantitative analysis of a diagnostic morphological feature, and a preliminary paleogenetic characterization of this hominin skeleton from Altamura. Overall, the results concur in indicating that it belongs to the hypodigm of Homo neanderthalensis, with some phenetic peculiarities that appear consistent with a chronology ranging from 172 ± 15 ka to 130.1 ± 1.9 ka. Thus, the skeleton from Altamura represents the most ancient Neanderthal from which endogenous DNA has ever been extracted.

Genealogical relationships between early medieval and modern inhabitants of Piedmont.

In the period between 400 to 800 AD, also known as the period of the Barbarian invasions, intense migration is documented in the historical record of Europe. However, little is known about the demographic impact of these historical movements, potentially ranging from negligible to substantial. As a pilot study in a broader project on Medieval Europe, we sampled 102 specimens from 5 burial sites in Northwestern Italy, archaeologically classified as belonging to Lombards or Longobards, a Germanic people ruling over a vast section of the Italian peninsula from 568 to 774. We successfully amplified and typed the mitochondrial hypervariable region I (HVR-I) of 28 individuals. Comparisons of genetic diversity with other ancient populations and haplotype networks did not suggest that these samples are heterogeneous, and hence allowed us to jointly compare them with three isolated contemporary populations, and with a modern sample of a large city, representing a control for the effects of recent immigration. We then generated by serial coalescent simulations 16 millions of genealogies, contrasting a model of genealogical continuity with one in which the contemporary samples are genealogically independent from the medieval sample. Analyses by Approximate Bayesian Computation showed that the latter model fits the data in most cases, with one exception, Trino Vercellese, in which the evidence was compatible with persistence up to the present time of genetic features observed among this early medieval population. We conclude that it is possible, in general, to detect evidence of genealogical ties between medieval and specific modern populations. However, only seldom did mitochondrial DNA data allow us to reject with confidence either model tested, which indicates that broader analyses, based on larger assemblages of samples and genetic markers, are needed to understand in detail the effects of medieval migration.

Pet fur or fake fur? A forensic approach.

BACKGROUND: In forensic science there are many types of crime that involve animals. Therefore, the identification of the species has become an essential investigative tool. The exhibits obtained from such offences are very often a challenge for forensic experts. Indeed, most biological materials are traces, hair or tanned fur. With hair samples, a common forensic approach should proceed from morphological and structural microscopic examination to DNA analysis. However, the microscopy of hair requires a lot of experience and a suitable comparative database to be able to recognize with a high degree of accuracy that a sample comes from a particular species and then to determine whether it is a protected one. DNA analysis offers the best opportunity to answer the question, 'What species is this?' In our work, we analyzed different samples of fur coming from China used to make hats and collars. Initially, the samples were examined under a microscope, then the mitochondrial DNA was tested for species identification. For this purpose, the genetic markers used were the 12S and 16S ribosomal RNA, while the hypervariable segment I of the control region was analyzed afterwards, to determine whether samples belonged to the same individual. RESULTS: Microscopic examination showed that the fibres were of animal origin, although it was difficult to determine with a high degree of confidence which species they belonged to and if they came from a protected species. Therefore, DNA analysis was essential to try to clarify the species of these fur samples. CONCLUSIONS: Macroscopic and microscopic analysis confirmed the hypothesis regarding the analyzed hair belonging to real animals, although it failed to prove with any kind of certainty which actual family it came from, therefore, the species remains unknown. Sequence data analysis and comparisons with the samples available in GenBank showed that the hair, in most cases, belonged to the Canidae family, and in one case only to Felidae.

Monitoring DNA contamination in handled vs. directly excavated ancient human skeletal remains.

Bones, teeth and hair are often the only physical evidence of human or animal presence at an archaeological site; they are also the most widely used sources of samples for ancient DNA (aDNA) analysis. Unfortunately, the DNA extracted from ancient samples, already scarce and highly degraded, is widely susceptible to exogenous contaminations that can affect the reliability of aDNA studies. We evaluated the molecular effects of sample handling on five human skeletons freshly excavated from a cemetery dated between the 11 to the 14(th) century. We collected specimens from several skeletal areas (teeth, ribs, femurs and ulnas) from each individual burial. We then divided the samples into two different sets: one labeled as "virgin samples" (i.e. samples that were taken by archaeologists under contamination-controlled conditions and then immediately sent to the laboratory for genetic analyses), and the second called "lab samples"(i.e. samples that were handled without any particular precautions and subject to normal washing, handling and measuring procedures in the osteological lab). Our results show that genetic profiles from "lab samples" are incomplete or ambiguous in the different skeletal areas while a different outcome is observed in the "virgin samples" set. Generally, all specimens from different skeletal areas in the exception of teeth present incongruent results between "lab" and "virgin" samples. Therefore teeth are less prone to contamination than the other skeletal areas we analyzed and may be considered a material of choice for classical aDNA studies. In addition, we showed that bones can also be a good candidate for human aDNA analysis if they come directly from the excavation site and are accompanied by a clear taphonomic history.