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.

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.

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.

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.

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.

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.

Prediction of autosomal STR typing success in ancient and Second World War bone samples.

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.