The mysteries of DNA preservation in bone: A comparative study of petrous bones and metacarpal epiphyses using ATR-FTIR spectroscopy.

A comparative analysis of 26 petrous bones and epiphyses of metacarpals from the Second World War era revealed no significant differences in DNA yield or success in STR typing. This unexpected parity in DNA preservation between the petrous bone, a renowned source of endogenous DNA in skeletal remains, and the epiphyses of metacarpals, which are porous and susceptible to taphonomic changes, is surprising. In this study, we introduced ATR-FTIR spectroscopy as an approach to unravel the correlation between bone molecular structure and DNA preservation. Metacarpals and petrous bones with same taphonomic history were sampled and prepared for DNA analyses. While one portion of the sample was used for DNA analysis, the other underwent ATR-FTIR spectroscopic examination. The normalized spectra and FTIR indices between the epiphyses of metacarpals and petrous bones were compared. Because the taphonomic history of the remains used is relatively short and stable, the ATR-FTIR spectroscopy unveiled subtle structural differences between the two bone types. Petrous bones exhibited higher mineralization, whereas epiphyses contained more organic matter. The unexpected preservation of DNA in the epiphyses of metacarpals can likely be attributed to the presence of soft tissue remnants within the trabeculae. Here observed differences in the molecular structure of bones indicate there are different mechanisms enabling DNA preservation in skeletal tissues.

Genetic sexing of subadult skeletal remains.

When subadult skeletons need to be identified, biological sex diagnosis is one of the first steps in the identification process. Sex assessment of subadults using morphological features is unreliable, and molecular genetic methods were applied in this study. Eighty-three ancient skeletons were used as models for poorly preserved DNA. Three sex-informative markers on the Y and X chromosome were used for sex identification: a qPCR test using the PowerQuant Y target included in PowerQuant System (Promega), the amelogenin test included in ESI 17 Fast STR kit (Promega), and a Y-STR amplification test using the PowerPlex Y-23 kit (Promega). Sex was successfully determined in all but five skeletons. Successful PowerQuant Y-target, Y-amelogenin, and Y-chromosomal STR amplifications proved the presence of male DNA in 35 skeletons, and in 43 subadults female sex was established. No match was found between the genetic profiles of subadult skeletons, and the elimination database and negative control samples produced no profiles, indicating no contamination issue. Our study shows that genetic sex identification is a very successful approach for biological sexing of subadult skeletons whose sex cannot be assessed by anthropological methods. The results of this study are applicable for badly preserved subadult skeletons from routine forensic casework.

Comparative analysis of DNA preservation in permanent and deciduous teeth of adults and non-adults: Implications for archaeological and forensic research.

This study investigates the preservation of DNA in different categories of teeth, including permanent and deciduous, fully developed and not fully developed, in both adults and non-adults. Teeth were sampled from a modern-era cemetery in Ljubljana, Slovenia. DNA extraction was performed using a full demineralisation protocol. DNA quantity and quality were assessed using qPCR analyses, and autosomal STR typing was conducted to verify genetic profiles. Results revealed significant differences in DNA preservation among various tooth categories. Fully developed permanent teeth of adults exhibited the highest DNA yields, attributed to their fully developed roots and thicker cementum, which is rich in DNA. Deciduous teeth, with thinner enamel and cementum, showed lower DNA preservation regardless of developmental stage. Non-adult teeth generally yielded less DNA compared to adults, even when considering only fully developed permanent teeth, indicating factors beyond developmental stage. These findings suggest that, in archaeological and forensic contexts, researchers should prioritize fully developed permanent teeth for DNA analysis due to their superior preservation. Additionally, this study underscores the importance of considering tooth type and developmental stage when selecting samples for genetic analysis in cases where petrous bone is unavailable, expanding our understanding of DNA preservation in human remains.

Comparison of DNA preservation and ATR-FTIR spectroscopy indices of cortical and trabecular bone of metacarpals and metatarsals.

Shape, size, composition, and function of the bones in the human body vary on the macro, micro and nanoscale. This can influence changes caused by taphonomy and post-mortem preservation, including DNA. Highly mineralised compact bone is less susceptible to taphonomic factors than porous trabecular bone. Some studies imply that DNA can be better preserved in trabecular bone, due to remnants of the soft tissue or bacteria better digesting organic matter while not digesting DNA. The aim of this study was to understand the differences between compact (diaphyses) and trabecular (epiphyses) bone on a molecular level and thus the reasons for the better preservation of the DNA in the trabecular bone. The powder obtained from epiphyses and diaphyses of metacarpals and metatarsals was analysed using ATR-FTIR spectroscopy and compared. Samples with poorest DNA preservation originated from diaphyses, predominantly of metatarsals. They were characterised by higher concentrations of phosphates and crystallinity, while lower collagen quality in comparison to samples with the best DNA preservation. Epiphyses presented higher concentrations of better-preserved collagen while diaphyses had higher concentrations of carbonates and phosphates and higher crystallinity. Due to better-preserved collagen in the epiphyses, the soft tissue remnants hypothesis seems more likely than the bacteria hypothesis.

Comparison of Morphological Sex Assessment and Genetic Sex Determination on Adult and Sub-Adult 17th-19th Century Skeletal Remains.

The first step in the analysis of human skeletal remains is the establishment of the biological profile of an individual. This includes sex assessment, which depends highly on the age of the individual and on the completeness and preservation state of the remains. Macroscopic methods only provide the assessment of sex, while for sex determination, molecular methods need to be included. However, poor preservation of the remains can make molecular methods impossible and only assessment can be performed. Presented research compares DNA-determined and morphologically assessed sex of adult and non-adult individuals buried in a modern-age cemetery (17th to late 19th century) in Ljubljana, Slovenia. The aim of the study was to assess the accuracy of commonly used macroscopic methods for sex assessment on a Slovenian post-medieval population. Results demonstrate that for adults, macroscopic methods employed are highly reliable and pelvic morphology, even the sciatic notch alone, is more reliable than skull. In non-adults, macroscopic methods are not as reliable as in adults, which agrees with previous research. This study shows how morphological and molecular methods can go hand in hand when building a biological profile of an individual. On their own, each methodology presented some individuals with undetermined sex, while together, sex of all the individuals was provided. Results confirm suitability of sex assessment based on skull and especially pelvic morphology in Slovenian post-medieval adults, while in the non-adult population molecular methods are advised.

Eye and hair color prediction of an early medieval adult and subadult skeleton using massive parallel sequencing technology.

Phenotypic trait prediction in ancient DNA analysis can provide information about the external appearance of individuals from past human populations. Some studies predicting eye and hair color in ancient adult skeletons have been published, but not for ancient subadult skeletons, which are more prone to decay. In this study, eye and hair color were predicted for an early medieval adult skeleton and a subadult skeleton that was anthropologically characterized as a middle-aged man and a subadult of unknown sex about 6 years old. When processing the petrous bones, precautions were taken to prevent contamination with modern DNA. The MillMix tissue homogenizer was used for grinding, 0.5 g of bone powder was decalcified, and DNA was purified in Biorobot EZ1. The PowerQuant System was used for quantification and a customized version of the HIrisPlex panel for massive parallel sequencing (MPS) analysis. Library preparation and templating were performed on the HID Ion Chef Instrument and sequencing on the Ion GeneStudio S5 System. Up to 21 ng DNA/g of powder was obtained from ancient petrous bones. Clean negative controls and no matches with elimination database profiles confirmed no contamination issue. Brown eyes and dark brown or black hair were predicted for the adult skeleton and blue eyes and brown or dark brown hair for the subadult skeleton. The MPS analysis results obtained proved that it is possible to predict hair and eye color not only for an adult from the Early Middle Ages, but also for a subadult skeleton dating to this period.

Kinship analysis of 5th- to 6th-century skeletons of Romanized indigenous people from the Bled-Pristava archaeological site.

The familial relationship between skeletons buried together in a shared grave is important for understanding the burial practices of past human populations. Four skeletons were excavated from the Late Antiquity part of the Bled-Pristava burial site in Slovenia, dated to the 5th to 6th century. They were anthropologically characterized as two adults (a middle-aged man and a young woman) and two non-adults (of unknown sex). Based on stratigraphy, the skeletons were considered to be buried simultaneously in one grave. Our aim was to determine whether the skeletons were related. Petrous bones and teeth were used for genetic analysis. Specific precautions were followed to prevent contamination of ancient DNA with contemporary DNA, and an elimination database was established. Bone powder was obtained using a MillMix tissue homogenizer. Prior to extracting the DNA using Biorobot EZ1, 0.5 g of powder was decalcified. The PowerQuant System was used for quantification, various autosomal kits for autosomal short tandem repeat (STR) typing, and the PowerPlex Y23 kit for Y-STR typing. All analyses were performed in duplicate. Up to 28 ng DNA/g of powder was extracted from the samples analyzed. Almost full autosomal STR profiles obtained from all four skeletons and almost full Y-STR haplotypes obtained from two male skeletons were compared, and the possibility of a familial relationship was evaluated. No amplification was obtained in the negative controls, and no match was found in the elimination database. Autosomal STR statistical calculations confirmed that the adult male was the father of two non-adult individuals and one young adult individual from the grave. The relationship between the males (father and son) was additionally confirmed by an identical Y-STR haplotype that belonged to the E1b1b haplogroup, and a combined likelihood ratio for autosomal and Y-STRs was calculated. Kinship analysis confirmed with high confidence (kinship probability greater than 99.9% was calculated for all three children) that all four skeletons belonged to the same family (a father, two daughters, and a son). Through genetic analysis, the burial of members of the same family in a shared grave was confirmed as a burial practice of the population living in the Bled area in Late Antiquity.

Improving kinship probability in analysis of ancient skeletons using identity SNPs and MPS technology.

In forensic kinship analysis and human identification cases, analysis of STRs is the gold standard. When badly preserved ancient DNA is used for kinship analysis, short identity SNPs are more promising for successful amplification. In this work, kinship analysis was performed on two skeletons from the Early Middle Ages. The surface contaminants of petrous bones were removed by chemical cleaning and UV irradiation; DNA was isolated through full demineralization and purified in an EZ1 Advanced XL machine. The PowerQuant kit was used to analyze DNA yield and degradation, and on average, 17 ng DNA/g of petrous bone was obtained. Both skeletons were typed in duplicate for STR markers using the Investigator EssplexPlus SE QS kit, and comparison of partial consensus genotypes showed shared allelic variants at most loci amplified, indicating close kinship. After statistical calculation, the full-sibling kinship probability was too low for kinship confirmation, and additional analyses were performed with PCR-MPS using the Precision ID Identity Panel. The HID Ion Chef Instrument was used to prepare the libraries and for templating and the Ion GeneStudio S5 System for sequencing. Analysis of identity SNPs produced full genetic profiles from both skeletons. For combined likelihood ratio (LR) calculation, the product rule was used, combining LR for STRs and LR for SNPs, and a combined LR of 3.3 × 107 (corresponding to a full-sibling probability of 99.999997%) was calculated. Through the SNP PCR-MPS that followed the STR analysis, full-sibling kinship between the ancient skeletons excavated from an early medieval grave was confirmed.

A fast and highly efficient automated DNA extraction method from small quantities of bone powder from aged bone samples.

An efficient extraction method is important for obtaining high-quality DNA from degraded aged bone samples. An automated full-demineralization method using the EDTA and DNA Investigator Kit (Qiagen) combined with Qiagen's biorobots was optimized in our laboratory in the past to extract the DNA from 500 mg of aged bone samples. The purpose of this research was to further improve the method with the aim of reducing the required sample material, shortening the extraction time, and achieving higher throughput. To process extremely small samples, the amount of bone powder was reduced to 75 mg, EDTA was replaced with reagents from the Bone DNA Extraction Kit (Promega), and decalcification was shortened from overnight to 2.5 h. Instead of 50 ml tubes, 2 ml tubes were used, which allows higher throughput. The DNA Investigator Kit (Qiagen) and EZ1 Advanced XL biorobot (Qiagen) was used for DNA purification. A comparison between both extraction methods was made on 29 Second World War bones and 22 archaeological bone samples. The differences between both methods were explored by measuring nuclear DNA yield and STR typing success. After cleaning the samples, 500 mg of bone powder was processed using EDTA, and 75 mg of powder from the same bone was processed using the Bone DNA Extraction Kit (Promega). DNA content and DNA degradation were determined using PowerQuant (Promega), and the PowerPlex ESI 17 Fast System (Promega) was used for STR typing. The results showed that the full-demineralization protocol using 500 mg of bone was efficient for Second World War and archaeological samples, and the partial-demineralization protocol using 75 mg of bone powder was only efficient for the Second World War bones. The improved extraction method-for which significantly lower amounts of bone powder can be used, the extraction process is faster, and higher throughput of bone samples is possible-is applicable for genetic identification of relatively well-preserved aged bone samples in routine forensic analyses.

Comparison of DNA preservation between adult and non-adult ancient skeletons.

Studies evaluating DNA preservation in non-adults, or comparing preservation in adults and non-adults, are very rare. This study compares the preservation of DNA in the skeletal remains of adults and non-adults. It compares the quality and quantity of DNA recovered from different skeletal elements of adults and non-adults, and from non-adults of different age classes. In addition, the preservation of DNA in males and females is compared. Bone DNA preservation was estimated by measuring nuclear DNA concentration and its degradation, and through STR typing success. The study analyzed 29 adult skeletons and 23 non-adult skeletons from the Ljubljana-Polje archeological site, dating from the seventeenth to nineteenth century, and up to four skeletal elements (petrous bone, femur, calcaneus, and talus) were included. After full demineralization extraction, the PowerQuant System and the PowerPlex ESI 17 Fast System (Promega) were used for qPCR and STR typing, respectively. The results showed that, among the four bone types analyzed, only the petrous bone proved to be a suitable source of DNA for STR typing of non-adult skeletal remains, and DNA yield is even higher than in the adult petrous bone, which can be attributed to the higher DNA degradation observed in the adult petrous bone. In adult skeletons, petrous bones and tali produced high STR amplification success and low DNA yield was observed in adult femurs. The results of this study are applicable for the sampling strategy in routine forensic genetics cases for solving identification cases, including badly preserved non-adult and also adult skeletons.

Eye and Hair Color Prediction of Ancient and Second World War Skeletal Remains Using a Forensic PCR-MPS Approach.

To test the usefulness of the forensic PCR-MPS approach to eye and hair color prediction for aged skeletons, a customized version of the PCR-MPS HIrisPlex panel was used on two sets of samples. The first set contained 11 skeletons dated from the 3rd to the 18th centuries AD, and for each of them at least four bone types were analyzed (for a total of 47 samples). In the second set, 24 skeletons from the Second World War were analyzed, and only petrous bones from the skulls were tested. Good-quality libraries were achieved in 83.3% of the cases for the ancient skeletons and in all Second World War petrous bones, with 94.7% and 100% of the markers, respectively, suitable for SNP typing. Consensus typing was achieved for about 91.7% of the markers in 10 out of 11 ancient skeletons, and the HIrisPlex-S webtool was then used to generate phenotypic predictions. Full predictions were achieved for 3 (27.3%) ancient skeletons and 12 (50%) Second World War petrous bones. In the remaining cases, different levels of AUC (area under the receiver operating curve) loss were computed because of no available data (NA) for 8.3% of markers in ancient skeletons and 4.2% of markers in Second World War petrous bones. Although the PCR-based approach has been replaced with new techniques in ancient DNA studies, the results show that customized forensic technologies can be successfully applied to aged bone remains, highlighting the role of the template in the success of PCR-MPS analysis. However, because several typical errors of ancient DNA sequencing were scored, replicate tests and accurate evaluation by an expert remain indispensable tools.

Bone fragment or bone powder? ATR-FTIR spectroscopy-based comparison of chemical composition and DNA preservation of bones after 10 years in a freezer.

Freezing bone samples to preserve their biomolecular properties for various analyses at a later time is a common practice. Storage temperature and freeze-thaw cycles are well-known factors affecting degradation of molecules in the bone, whereas less is known about the form in which the tissue is most stable. In general, as little intervention as possible is advised before storage. In the case of DNA analyses, homogenization of the bone shortly before DNA extraction is recommended. Because recent research on the DNA yield from frozen bone fragments and frozen bone powder indicates better DNA preservation in the latter, the aim of the study presented here was to investigate and compare the chemical composition of both types of samples (fragments versus powder) using ATR-FTIR spectroscopy. Pairs of bone fragments and bone powder originating from the same femur of 57 individuals from a Second World War mass grave, stored in a freezer at - 20 °C for 10 years, were analyzed. Prior to analysis, the stored fragments were ground into powder, whereas the stored powder was analyzed without any further preparation. Spectroscopic analysis was performed using ATR-FTIR spectroscopy. The spectra obtained were processed and analyzed to determine and compare the chemical composition of both types of samples. The results show that frozen powdered samples have significantly better-preserved organic matter and lower concentrations of B-type carbonates, but higher concentrations of A-type carbonates and stoichiometric apatite. In addition, there are more differences in the samples with a low DNA degradation index and less in the samples with a high DNA degradation index. Because the results are inconsistent with the current understanding of bone preservation, additional research into optimal preparation and long-term storage of bone samples is necessary.

Separating forensic, WWII, and archaeological human skeletal remains using ATR-FTIR spectra.

ATR-FTIR spectroscopy is a fast and accessible, minimally or non-destructive technique which provides information on physiochemical characteristics of analyzed materials. In forensic and archaeological sciences, it is commonly used for answering numerous questions, including the archaeological or forensic context of the human skeletal remains. In this research, the accuracy of ATR-FTIR-obtained spectra for separation between forensic, WWII, and archaeological human skeletal remains was investigated. Building from the previously proposed methodological procedures, various ratio-based and whole spectra separation procedures were applied, carefully analyzed, and evaluated. Results showed that employing whole spectral domains works best for the separation of archaeological, WWII, and forensic samples, even with samples of highly variable origin. Principal component analysis (PCA) further highlighted the necessity of acknowledging all the major components in the remains: amides, phosphates, and carbonates for the separation. Most influential proved to be amide I, namely its secondary structure, which presented well-preserved and organized collagen structure in forensic and WWII samples, while highly degraded in archaeological samples. Using the whole spectral domain for separation between samples from different contexts proved to be fast and simple, with no manipulation beyond baseline correction and normalization of spectra necessary. However, a dataset with samples of known origin is required for the learning model and predictions. A less accurate alternative is separation based on combining ratios of peaks correlating to organics and minerals in the bone, which eliminated overlapping and managed to classify the majority of the samples correctly as archaeological, WWII, or forensic.

ATR-FTIR spectroscopy combined with data manipulation as a pre-screening method to assess DNA preservation in skeletal remains.

Skeletal remains are commonly subjected to various analyses, including DNA. As the remains are exposed to taphonomic processes after the death of the organism, their physicochemical structure undergoes alterations. The success and integrity of a DNA analysis is thus conditioned by the preservation state of the sample. In this study, ATR-FTIR spectroscopy with further data exploration was employed to characterize the physicochemical structure of the samples and its correlation with the preservation state of the DNA. The aim was to test the hypothesis that ATR-FTIR-obtained spectra contain enough information to allow classification of the samples based on the preservation of the DNA in the remains. In the study, 138 human bones and teeth originating from the 16th century BC to the 21 st century AD were used. The samples were cleaned and powdered following the established methodological procedures for DNA extraction. DNA was extracted and quantified. The samples were separated into four categories based on the amount of quantified DNA. The remaining powder was analyzed with ATR-FTIR spectroscopy and the spectra obtained were explored to extract physicochemical information. Before the exploration of the acquired data, samples were divided into groups A (n = 107) and B (n = 31). Statistical analyses and machine learning were performed on the group A samples. The protocol was then validated on the group B samples, which served to make predictions on the preservation of the DNA in the remains. The best results were achieved using a random forest learning algorithm employing either normalized spectra, second-derivative spectra, or five highest-ranked ratios. Even though overlapping remained, these findings indicate that ATR-FTIR spectroscopy with further exploration of the data has good potential as a pre-screening method for evaluating DNA preservation in skeletal remains.

Auditory ossicles: a potential biomarker for maternal and infant health in utero.

Background: Carbon (δ13C) and nitrogen (δ15N) isotope ratios of collagen from teeth and bone are used to study human nutrition and health. As bones are constantly remodelling throughout life, isotopic values of bone collagen represent an average of several years. In contrast, human teeth do not remodel and their primary dentine contains only the isotopic data from the time of formation. In contrast to all other bones, human auditory ossicles also appear not to remodel. As they develop in utero and finish formation in the first 2 years of life, their collagen should also represent isotopic values of these two relatively short periods.Aim: By comparing δ13C and δ15N data from ossicles and incremental dentine, this study aims to investigate how two developmental periods of the ossicles, in utero and the first 2 years of life, reflect in collagen obtained from the ossicles.Subject and methods: Ossicle and tooth samples of 12 individuals aged 0.5 ± 0.4 years to 13 ± 1 years from the nineteenth century St. Peter's burial ground in Blackburn were collected and processed to obtain bulk bone and incremental dentine collagen which was measured for δ13C and δ15N.Results: Averaged δ13C and δ15N of ossicles are lower when compared to every age group except after 3 years of age. Average offset between ossicles and dentine of different groups ranges from 0.4-0.9‰ for δ13C and from 0.3-0.9‰ for δ15N, with highest counterbalance at birth and after the first 5 months after birth.Conclusions: There appears to be a systematic offset between the dentine and ossicle data. It seems that the second phase of development does not influence the isotopic values of collagen significantly and the data we are obtaining from ossicles represents the in utero period.