Abstract
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.
Avoid common mistakes on your manuscript.
Introduction
Improvements in forensic genetic analysis using massive parallel sequencing (MPS) technology [4, 8, 25] have introduced a powerful tool for phenotypic trait prediction of skeletal remains. This can be used not only in a forensic context [6, 16, 40, 44, 48, 74, 78] but also in ancient DNA (aDNA) analysis to obtain information about externally visible characteristics of individuals from past human populations. In forensics, phenotypic traits associated with pigmentation, such as eye, hair, and skin color, were introduced in the past [38, 39, 77], and the HIrisPlex and HIrisPlex-S systems were developed and are used together with genotype and phenotype databases established for pigmentation prediction [79]. Even if aDNA is poorly preserved, and if it shows low DNA quantity and quality, high susceptibility for contamination, and the presence of inhibitors [41], several studies have successfully predicted pigmentation of ancient skeletons. With comparison of predicted colors and historical documentation, a Polish general was positively verified [16], and some other historical questions have been solved [29, 58]. Pigmentation traits were predicted for the remains of King Richard III of England [42], and even for skeletons several hundred years old, it has been possible to obtain information about what people’s ancestors looked like [83]. However, in the literature reviewed, eye and hair color prediction was established on ancient adult skeletons, but it was not possible to find data on eye and hair color prediction for ancient subadults. It is known that extrinsic factors affect subadult skeletons more than adult ones [3, 31, 52, 66], and, in comparison to adult skeletons, subadult skeletons are more susceptible to loss and decay [14, 27, 50, 75, 76, 80], resulting in low DNA preservation. Some studies have shown that DNA is better preserved in ancient adult skeletons than in subadult skeletons, and differences have been identified in the bone characteristics of adults and subadults [73]. We therefore wondered whether it is possible to determine hair and eye color not only for ancient adult skeletons, but also for subadult skeletons using MPS technology. To verify this, an adult skeleton and a subadult skeleton excavated from the same grave from the Early Middle Ages were selected for analysis, ensuring that DNA preservation was influenced by similar environmental factors for the same amount of time.
For successful aDNA analysis, the most suitable type of bone and its intra-bone part has to be selected because of high differences in preservation of DNA in different skeletal element types [1, 17, 18, 23, 32, 34, 49, 51, 53, 82]. Various studies have shown that the petrous bone performs best in aDNA analyses because of its hard and dense tissue (the petrous bone is considered the hardest and densest of all the bones in the body), resulting in better protection and higher resistance of DNA to difficult environmental conditions [11, 21, 22, 28, 30, 56, 59, 60, 68]. Accordingly, petrous bones were used for eye and hair color prediction of the adult and subadult early medieval skeletons in this study.
Approval from the Slovenian Medical Ethics Committee was obtained for this study (145/06/13, 0120–412/2021/3), and the persons included in the elimination database signed an informed consent statement.
Material and methods
Selection of bone samples
In 1974, excavation of skeletal remains from the Bled–Pristava archaeological site was performed under the leadership of Timotej Knific from the Ljubljana National Museum, and the remains were stored in the museum warehouse. The Bled–Pristava archaeological site is located in northern Slovenia, near Bled. It is a multiperiod necropolis that was active from the third to ninth centuries AD. The individuals buried there are partially indigenous people (third to sixth century) and partially Slavs that migrated to the area. A unique feature of this necropolis is a multiple burial grave, which was separate from the other graves, located on the edge of the necropolis. Four individuals were excavated from this multiple grave and, based on stratigraphy, they were all placed in the grave at the same time, indicating their simultaneous deaths. Because of the uniqueness of the finding, kinship analysis of the four skeletons has been performed, and a father–children kinship relationship has been proven for the excavated skeletons [88]. Because genetic studies of younger subadults (especially below age 3) are the most challenging [27] due to higher susceptibility to decay, as a result of low tensile and compressive strength of immature bones [14], the youngest subadult skeleton from the multiple burial grave was selected for a phenotyping study. In addition to the youngest subadult skeleton, the oldest adult skeleton was selected for eye and hair color prediction. Both skeletons (the father and the youngest child) were dated to the fifth to sixth century AD. Based on anthropological analysis, the adult skeleton was characterized as a middle-aged man (between 35 and 45 years old). For the subadult skeleton, it was not possible to determine sex anthropologically, and the age was estimated to be around 6 years. The right petrous bone was sampled from each skeleton. To consider intra-bone variability in DNA content, the densest portion—the cochlea, in which DNA is preserved the best—was used for genetic analysis. The cochlea was detached from the rest of the petrous bone following a detailed protocol constructed by Pinhasi et al. [61]. To build an elimination database, we collected buccal swabs from every individual that had had recent contact with the skeletons studied and from every individual that took part in the anthropological and genetic investigations.
Preventing contamination
The very high risk of contamination with modern DNA limits the success of aDNA analysis [65], and so a number of precautions were followed to prevent contamination and to verify ancient DNA authenticity [12, 20, 55, 64]. Extraction of DNA from ancient petrous bones was performed in a special room designed for processing ancient skeletons only, and all the work was done in a closed MC 3 microbiological safety cabinet (Iskra Pio, Šentjernej, Slovenia) outfitted with UV light and a HEPA filter [84]. All the tools used for drilling, cutting, and grinding were cleaned with bleach, water, and ethanol, sterilized in a Europa B xp sterilizer (Tecno-Gaz, Parma, Italy), and UV irradiated for 30 min in a BLX-Multichannel BioLink DNA Crosslinker (Vilber, Collégien, France). The laboratory plastics and reagents (except those labeled DNase-free or DNA-free) were also sterilized and UV irradiated before use. For contamination monitoring, extraction-negative controls (ENCs) were processed together with the bone samples [57], and all individuals involved in the analyses were typed, and their genotypes were entered into the elimination database. Various purification machines were used for purifying low-DNA-template bone samples (the EZ1 Advanced XL biorobot,Qiagen, Hilden, Germany) and high-DNA-template elimination database samples (the BioRobot EZ1 machine; Qiagen).
DNA extraction
All samples were mechanically and chemically cleaned in order to eliminate surface contamination [84]. Chemical cleaning consisted of 5% Alconox (Sigma-Aldrich, St. Louis, MO, USA), 80% ethanol (Merck, Kenilworth, NJ, USA), and sterile bi-distilled water (Sartorius-Stedim Biotech, Göttingen, Germany). After processing, the bones were dried and then cooled with liquid nitrogen. Cooled bones were cut with a sterilized diamond saw (Schick, Schemmerhofen, Germany). We exposed the bones to UV light for 30 min with a BLX-Multichannel BioLink DNA Crosslinker (Vilber) before grinding them. A Bead Beater MillMix 20 homogenizer (Tehtnica, Domel, Železniki, Slovenia) was used for grinding, which produced a fine homogenous powder. Petrous bones were used to extract DNA from 500 mg of powder following the extraction protocol reported by Zupanič Pajnič [84], with decalcification with 0.5 M of ethylenediaminetetraacetic acid, or EDTA (Sigma-Aldrich), and purification was carried out with the EZ1 DNA Investigator Kit (Qiagen) and EZ1 Advanced XL machine (Qiagen), selecting trace protocol, TE buffer elution, and 50 µl of final volume option as reported by Zupanič Pajnič [84]. We performed duplicated DNA extractions from petrous bones, obtaining extract 1 and extract 2 from each petrous bone, and ENC samples were added to each extraction batch to monitor the purity of the reagents and plastics used for extraction of DNA.
DNA quantification
Determination of the concentration of human nuclear DNA (short 85 bp autosomal fragment, Auto target), male DNA (Y target), and long 294 bp autosomal fragment (Deg target) was achieved using the PowerQuant System (Promega), according to the technical manual [62]. The results calculated for the Auto and Deg targets were used to calculate the degradation index (the Auto/Deg ratio), and we detected the PCR inhibitors using an internal PCR control (IPC shift). Quantification was performed using the QuantStudio 5 Real-Time PCR system, Quant-Studio Design and Analysis Software 1. 5. 1 (Applied Biosystems, AB, Foster City, CA, USA), and the PowerQuant Analysis Tool software (https://worldwide.promega.com/resources/tools/powerquant-analysis-tool/).
To lower the variability of results, real-time quantification was carried out in duplicate [62]. The efficiency of extraction was displayed in DNA quantity obtained from 1 g of bone and expressed in ng DNA per gram of bone powder; calculation was performed from the PowerQuant Auto target results.
STR typing
Short tandem repeat (STR) typing was performed for authentication of ancient samples and comparison with the elimination database. Together with the positive control (9948-Qiagen), the petrous bone and elimination database samples, PCR negative control, and ENCs were typed to exclude any possibility of contamination with modern DNA during DNA isolation. Two extracts were obtained from each petrous bone, and both of them were typed for autosomal STR markers. The Investigator EssplexPlus SE QS kit was used for typing, using 30 amplification cycles and a 25-μl reaction volume following the manufacturer’s protocols [63]. One nanogram of bone DNA (calculated from the PowerQuant Auto target) and positive control DNA were used as a template, and the maximum volume of DNA extract was used for amplification of ENCs. PCR amplification was run in the Nexus Master Cycler (Eppendorf, Hamburg, Germany, EU). In addition, positive and negative PCR controls were amplified. The SeqStudio Genetic Analyzer for HID (Thermo Fisher Scientific, TFS, Carlsbad, CA, USA) [71], SeqStudio Data Collection Software v 1.2.1 (TFS), and GeneMapper ID-X Software v 1.6 (TFS) were used for generation of genetic profiles. Together with monitoring the cleanliness of ENC samples, differentiation of the skeletons’ profiles and the profiles obtained from the elimination database makes it possible to authenticate aDNA.
For calculating the frequency of the adult’s and subadult’s STR genotype, composite profiles were constructed from the duplicated analysis [7], and DNA VIEW software version 37.37 [5] was used, employing allele frequencies of the modern Slovenian population [85,86,87].
SNP typing with the HIrisPlex panel
A customized HIrisPlex panel for amplification of 24 SNPs using 17 pairs of PCR primers [6, 44] was used for pigmentation prediction with the MPS technology for the extracts obtained in the second round of extraction (extract 2) from the adult and subadult petrous bone. Analysis was performed on the Ion S5 System (TFS). One nanogram of DNA (calculated from the PowerQuant Auto target) was used for library preparation for the adult and subadult skeleton extracts, following the manufacturer’s recommendations [71]. For target amplification, 25 cycles were used. Library preparation and templating were fully automated using the Ion Chef System (TFS). The Precision ID DL8 Kit for Ion Chef was used for library preparation following the manufacturer’s instructions [71]. The concentration of the combined library pools was determined in duplicate along with standards and negative controls with the Ion Library TaqMan Quantification Kit (TFS) according to the manufacturer’s recommendations [72]. The library pool, which included amplicons from the adult and subadult skeleton from the fifth to sixth century, was diluted to 30 pM. The template was loaded onto an Ion 530 sequencing chip (TFS) in a single chip loading workflow. For sequencing, Ion S5 Precision ID sequencing reagents (TFS) and Ion S5 Precision ID sequencing solutions (TFS) were used. Ion Torrent™ Suit Software 5.6 (TFS) was used to align reads against the Homo sapiens reference genome (hg19). The Coverage Analysis v 5.6.0.1. plugin was used to obtain data on the sequence coverage level in targeted regions. The default settings for genotyping were applied according to the manufacturer’s recommendations [72] using Converge™ software version 2.0 (TFS).
For the HIrisPlex eye and hair color prediction of the adult and subadult skeletons from the fifth to sixth century, the R-script program (https://walshlab.sitehost.iu.edu) was used, and the skeletons’ genotypes were converted into the corresponding input codes, and these were uploaded into the HIrisPlex web tool (accessible at https://hirisplex.erasmusmc.nl). The most likely eye and hair color prediction based on the p-value and AUC (area under the receiver operating curve) loss was used to predict eye and hair color.
Results
DNA quantification
Real-time PowerQuant results (Auto, Deg, and Y targets (calculated as ng DNA/µl of extract), IPC shift, and Auto/Deg ratio) for all of the extracts acquired from petrous bones and ENCs processed in combination with the samples analyzed are presented in Supplementary material, SM 1. Because amplifications were performed in duplicate, average measurements are reported here. For each petrous bone, we present the quantity of DNA per 1 g of powder and express it in ng DNA/g of powder. The petrous bone extracts yielded a high amount of DNA (on average, 17.4 ng DNA/g of the adult petrous bone and 16.6 ng DNA/g of the subadult petrous bone). Amplifying the Y PowerQuant targets confirmed the presence of male DNA in both petrous bones, and male sex was additionally confirmed with amplification of the Y chromosome amelogenin locus included in the STR typing amplification kit (see SM 2).
We observed a high degradation index in both skeletons; the average value was 276 for the adult petrous bone and 53 for the subadult petrous bone (see SM 1). The values of the IPC shift were less than 0.30 (threshold recommended by the manufacturer; [62]) in all extracts obtained from the adult and subadult petrous bone, indicating efficient DNA purification (see SM 1). Considering the ENC employed, no PowerQuant targets were detected, except for ENC 2, where Auto target amplification resulted in a measurement of 0.0002 ng DNA/µl, as can be seen in SM 1. When the PowerQuant System validation study was performed, it was shown that a concentration of DNA as small as 0.0005 ng DNA/µl can be detected with certainty [19], and the Auto target measurement of ENC 2 was lower. However, as expected, no STR genetic profiles were obtained from any ENCs, indicating no presence of contamination originating from the extraction process.
STR typing
For authentication of ancient samples, autosomal STRs were typed and compared with elimination database profiles. Out of 16 STR loci and the amelogenin gene, 13 STR loci and the amelogenin locus were successfully amplified in the adult skeleton, 15 STR loci and the amelogenin gene were successfully typed in the subadult skeleton, and no match was found with elimination database profiles. No profiles were obtained from the ENC samples and the PCR negative control sample. STR profile of PCR-positive control matched the profile of 9948 DNA [63]. The autosomal STR profiles of the adult and subadult skeleton from the fifth to sixth century acquired using the Investigator Essplex SE QS kit (Qiagen) are presented in SM 2. Although an optimal quantity of DNA (1 ng) was amplified in the petrous bones, there were allelic drop-out events. The explanation for this lies in the severe degradation of DNA that is isolated from fifth- to sixth-century skeletons. Specifically, allele drop-outs were identified at high-molecular-weight alleles at the D8S1179, D21S11, and D2S1338 loci in the adult skeleton and at D2S1338 in the subadult skeleton. All dropped-out loci were the longest STR loci in the Investigator EssplexPlus SE QS kit (Qiagen). Allelic drop-out events frequently occur with ancient samples, and high degradation has an effect on the allelic drop-out phenomenon, in particular at loci with a high molecular weight, which are more susceptible to drop-outs due to less efficient PCR, more frequent stuttering, and more evident degradation effects [7]. The genotype frequencies of the adult and subadult skeleton calculated for composite profiles employing allele frequencies of the modern Slovenian population [85,86,87] were 2.2 × 10−17 for the adult skeleton and 1.04 × 10–20 for the subadult skeleton. Because allele frequencies of ancient populations that inhabited Slovenia in the past are not available, comparison of genotype frequencies with the modern Slovenian population was not possible.
HIrisPlex SNP typing and prediction of eye and hair color
Quantification results obtained using the Ion Library TaqMan Quantification Kit (TFS) indicated that the library pool including extracts obtained from the adult and subadult ancient skeleton attained over 30 pM of DNA, and after dilution, 30 pM of DNA was used in MPS according to the manufacturer’s recommendations [71].
The main sequencing parameters of the bone samples from the adult and subadult ancient skeleton showed that the libraries yielded from 55.2% (adult skeleton) to 91.6% (subadult skeleton) on-target reads, the uniformity of sequencing was 100% for both skeletons, the average mapped reads values were 513,387 for the adult skeleton and 515,047 for the subadult skeleton, and the mean depth values were 14,574 for the adult skeleton and 25,636 for the subadult skeleton. When using the settings from Converge software (TFS), all the reads exceeded the threshold of 20 × . Coverage values for each HIrisPlex SNP marker and for each nucleotide variant detected by MPS in each of the markers, together with the genotypes for both skeletons analyzed, are shown in SM 3. From the adult skeleton, a HIrisPlex genotype with one missing SNP marker was obtained (NN was attributed to SNP marker rs201326893), and the subadult skeleton generated a complete HIrisPlex genotype (see SM 3). The HIrisPlex-S panel validation study [6] sets the recommended coverage value thresholds for each SNP marker for samples above and below 100 pg DNA input. In the adult and subadult ancient skeletons, the MPS coverage values significantly exceeded the recommended thresholds for > 100 pg DNA input, as expected for samples in which the optimal template DNA amount of 1 ng was used for amplification in HIrisPlex PCR-MPS analysis. For the ancient adult and subadult skeleton samples, no heterozygous or homozygous genotypes were flagged by the Converge software (TFS), showing no unambiguous results, except for rs201326893 missing data for the adult skeleton.
Eye and hair color were predicted from genotypes obtained from the adult and subadult skeleton using the HIrisPlex web tool and the user manual for HIrisPlex (https://hirisplex.erasmusmc.nl/). Table 1 presents the p-values for various eye colors, hair colors, and hair shades, and AUC loss values and the most probable phenotype predicted for both skeletons studied are shown.
The AUC loss in the subadult sample extract was 0 because there were no allelic drop-outs, and a complete HIrisPlex genotype was generated. Because of missing data for amplification of SNP marker rs201326893 in the adult skeleton, AUC loss was calculated for blond hair and red hair, and the highest was for red hair color prediction (0.003).
In the adult skeleton, the highest p-value was 0.859 for brown eyes, and in the subadult skeleton, it was 0.876 for blue eyes. For hair color prediction, the p-value for brown hair was 0.59 and for light hair value, it was 0.146, resulting in a dark brown/black hair color prediction for the adult skeleton. For the subadult skeleton, the p-value for brown hair was 0.571, and for light hair value, it was 0.817, resulting in a brown/dark brown hair color prediction. A phenotype of brown eyes and dark brown or black hair was predicted for the adult ancient skeleton, whereas the most probable hair color for the subadult ancient skeleton was brown or dark brown, and the eye color was blue (see Table 1).
Discussion
Partial or incomplete HIrisPlex profile generation is expected for ancient samples because low amounts of poor-quality DNA are usually obtained from them. Surprisingly, full or almost full profiles were obtained not only for the adult skeleton but also for the subadult skeleton from the Early Middle Ages, and hair and eye colors were successfully predicted even though some AUC losses were calculated for prediction of the hair color of the adult skeleton. We have to be aware that Table 1 shows the most probable phenotypes predicted using the HIrisPlex tool, and developmental validation study of the HIrisPlex System showed overall prediction accuracies for eye color at 0.94 for blue, 0.74 for intermediate, and 0.95 for brown, and for hair color at 0.81 for blond, 0.75 for brown, 0.92 for red, and 0.85 for black hair with a hair color shade at 0.90 [78]. When the HIrisPlex prediction tool was used on a set of 119 individuals from the Polish population, eye color was predicted with 84% accuracy, and hair color with 73% accuracy on average, meaning that in three out of four individuals, prediction was accurate [78]. None of the p values for eye and hair color prediction was above the accuracy threshold values in the adult and subadult skeleton analyzed, showing that regardless of whether we are testing recent individuals or ancient skeletons, DNA phenotyping is a probability-based prediction, and the results are based on knowledge from a combination of genotypes from thousands of individuals included in a model used in the HIrisPlex prediction tool. An anomaly with the subadult skeleton having a high probability for light hair, but a conclusion of brown or dark brown hair shown in Table 1, is presumably also related to lower probability values that do not reach the accuracy thresholds. However, it would be interesting to compare eye and hair color frequencies in the contemporary Slovenian population with the predicted phenotypes of the ancient adult and subadult skeleton from the fifth- to sixth-century Bled–Pristava archaeological site. In 2012, eye and hair color frequencies of the contemporary Slovenians were published, and for eye color, 44.7% blue, 25.7% intermediate, and 29.6% brown color were observed, whereas hair color was defined as blond in 5.7% of Slovenians, dark blond or light brown in 41%, and dark brown or black in 52.4% [37]. Unfortunately, no information on the combination of both phenotypes is provided for the contemporary Slovenian population, and information on how common phenotypes are with a combination of brown eyes and dark brown or black hair (predicted for the adult skeleton) and blue eyes and brown or dark brown hair (predicted for the subadult skeleton) is not available. The combination of blue eyes and brown or dark brown hair seems to be a less common phenotype in the Slovenian population. However, when several ancient and Second World War skeletons from Slovenia were used for eye and hair color prediction, in two skeletons, dark hair was predicted using the HIrisPlex tool despite the predicted blue color phenotype for the eyes. The ancient skeleton from the sixteenth century had a high probability for brown or dark brown hair and the Second World War skeleton even for dark brown or black hair [83]. Genetic similarities of skeletons excavated from the multiple burial grave from the Bled–Pristava archaeological site to contemporary Slovenians were confirmed in our previous study through Y chromosomal STR markers, and E1b1b haplogroup was predicted [88]. The same haplogroup was detected with a frequency of 7.3% in the current Slovenian population [81]. As expected (because of the very high variability of autosomal STR markers), the STR genotypes of the adult and subadult skeleton showed low frequencies in the contemporary Slovenian population. As it was shown, modern forensic genetics methods can interact and enhance modern archeology, not only through confirmation of kinship between individuals from the same archaeological site [88] and through searching for genetic similarities to the contemporary human populations, but also through prediction of phenotypic traits to obtain information about the external appearance of adult and subadult individuals from past human populations.
A search for pigmentation trait prediction in ancient subadult skeletons did not yield any studies on genetic prediction of eye and hair color for subadults. The reasons for this lie in the small number of subadult skeletons represented in archaeological osteological collections and in the poor preservation of DNA in subadult skeletons compared to adult skeletons. Namely, in archaeology, subadults are very often underrepresented in collections of excavated skeletons due to their bones’ suboptimal survival rate [10, 27, 36, 45, 66, 67]. Compared to adults, subadults have greater susceptibility to environmental factors affecting DNA preservation [3, 31, 52, 66], and, because of their smaller, more porous, and less dense bones, subadult skeletons are more prone to decay [14, 27, 50, 75, 76, 80]. In addition, subadult bones have lower inorganic and higher organic content, resulting in less preserved DNA in comparison to adult skeletons [73].
The results of a study performed by Šuligoj et al. [70], which analyzed adult and subadult ancient skeletons dating to the seventeenth to nineteenth century, showed that among the various skeletal element types analyzed (femurs, tali, calcanei, and petrous bones were studied), only the petrous bones yielded amplifiable amounts of DNA in subadult skeletons. In contrast, in adult skeletons, amplifiable DNA yields were obtained not only from petrous bones but also from tali and calcanei, whereas femurs showed low DNA yields and low STR typing success. Petrous bones proved to be the only suitable source of DNA for aDNA analysis of subadult skeletons (when compared to tali, calcanei, and femurs), and, interestingly, DNA yields were even higher than in the adult petrous bones, which the authors attributed to the higher degradation of DNA measured in adult petrous bones [70]. In the case of the adult and subadult early medieval skeletons analyzed in this study, similar DNA yields were observed from both skeletons, but nonetheless, amplification of HIrisPlex SNPs was more successful in the subadult than adult skeleton, obtaining a full HIrisPlex genotype with higher coverage values in all SNPs typed in the subadult skeleton and partial genotype—with one HIrisPlex SNP missing—and lower coverage values in the adult skeleton in spite of the fact that 1 ng of DNA was used for HIrisPlex PCR-MPS analysis for both petrous bones. In addition, in the adult skeleton, the percentage of on-target reads was lower than in the subadult skeleton (55% versus 91%). The difference in sequencing parameters and typing performance is probably the result of differences in DNA degradation because the degradation index in the adult skeleton was higher than in the subadult skeleton. A similar difference in DNA degradation rate was observed in the study performed by Šuligoj et al. [70], in which the DNA quantity and quality of 20 ancient adult and 20 ancient subadult petrous bone extracts were compared, and greater degradation of DNA was observed in adult petrous bones than in subadult ones. The reasons for the less-degraded DNA observed in the ancient subadult petrous bone in comparison to the ancient adult petrous bone, even though they were both exposed to similar environmental factors for the same amount of time, are unknown. The petrous bone is characterized by unique properties and completes its development in the embryonic stage. In the 16th week of gestation, when the cartilage labyrinth reaches its final size, ossification begins and is completed by birth [46]. The petrous bone has an atypical structure and unique tissue organization, with very dense, thick, compact bone, high cellularity, and low trabecular bone content [13, 15, 35, 43]. Because of a lack of remodeling, the petrous bone is characterized by its immature nature, and its limited vascularization inhibits contamination by exogenous microorganisms [24]. The petrous bone’s high density improves its resistance and decreases bacterial DNA decomposition, damage, and degradation [59]. The protective shell that surrounds the inner ear additionally promotes the preservation of endogenous DNA in the petrous bone [24]. As shown previously by various aDNA studies, it was also proved in the study of eye and hair color prediction for the early medieval adult and subadult skeleton presented here that petrous bones generate high-quality HIrisPlex genotypes in adult and subadult skeletons.
Because not only HIrisPlex SNPs were typed, but STR typing was also performed on the ancient adult and subadult skeletons, it was possible to determine the sex of the skeletons analyzed. Morphological methods to assess subadults’ sex are not sufficiently accurate and are not advisable at this time [9, 69]. Some research has acknowledged the strong potential of DNA testing for increasing the precision of morphological sex assessment [2, 26, 33, 47, 54], which was also clear in the study of the adult and subadult skeletons from the Early Middle Ages, for which anthropological analysis was only able to assess the sex of the adult skeleton, whereas determination of sex could not be made for the subadult skeleton. Molecular genetic methods corroborated the adult skeleton’s anthropologically defined male sex, and the sex of the subadult skeleton was also successfully established through successful qPCR PowerQuant Y target amplification and Y chromosome amelogenin amplification. Male sex was determined for the adult and subadult skeletons.
Because this study was carried out on ancient skeletons, measures to prevent contamination were necessary [55, 84]. Both skeletons were typed for autosomal STRs in duplicate (from different extracts obtained from the same bone) to verify the genetic typing results. We carried out STR typing of the personnel in the elimination database, and STR analysis was used to monitor the purity of ENCs [57]. ENCs were examined only for STR markers because of the expense of the MPS reagents. When the adult and subadult petrous bones were analyzed, there was no contamination of ENCs, which yielded no qPCR or STR typing results. Moreover, no match occurred between the STR profiles of the bones analyzed and the profiles in the elimination database. In addition, autosomal STR matches of duplicated STR analysis of both extracts obtained from the petrous bones also confirmed the absence of contamination events. Degradation rate is utilized as a criterion for the authenticity of ancient DNA [55], and in both skeletons analyzed, high degradation was proved (with much lower copy numbers of Deg than Auto target, as determined through PowerQuant quantification).
Conclusions
The results of this study have confirmed successful HIrisPlex-based PCR-MPS eye and hair color prediction for early medieval skeletons. As already shown in various aDNA studies, it was proved again that petrous bones are an excellent source of DNA for various genetic analyses. Exceptional preservation of DNA in petrous bones resulted in generation of high-quality HIrisPlex genotypes not only in an adult skeleton but also in a subadult skeleton dated to the fifth to sixth century. Moreover, sequencing parameters and genotyping performance were even higher in the subadult skeleton than in the adult skeleton. Studies on pigmentation trait prediction performed on subadult ancient skeletons are very rare, and so the results of this study may be helpful to forensic experts when it is necessary to predict eye and hair color in missing person identification cases when aged subadult skeletal remains are found and must be identified, but there are no clues regarding who the missing child might be. By predicting the hair and eye color, the circle of missing children can be narrowed, and investigation can be directed to successful identification. In the case of forensic subadult skeletal remains, better DNA preservation can be expected than in ancient skeletons due to shorter exposure of the remains to harmful environmental influences, and petrous bone sampling is recommended for phenotyping.
Data availability
The authors declare that all the data are available.
References
Antinick TC, Foran DR (2019) Intra- and inter-element variability in mitochondrial and nuclear DNA from fresh and environmentally exposed skeletal remains. J Forensic Sci 64:88–97
Bauer CM, Niederstätter H, McGlynn G, Stadler H, Parson W (2013) Comparison of morphological and molecular genetic sex-typing on mediaeval human skeletal remains. Forensic Sci Int Genet 7:581–586
Bello S, Andrews P (2006) The intrinsic pattern of preservation of human skeletons and its influence on the interpretation of funerary behaviours. In: Gowland R, Knüsel C (eds) Social archaeology of funerary remains. Oxbow Books Oxford, pp 1–13
Børsting C, Morling N (2015) Next generation sequencing and its applications in forensic genetics. Forensic Sci Int Genet 18:78–89
Brenner CH (2007) DNA-VIEW 2007 User Guide, Oakland (CA)
Breslin K, Wills B, Ralf A, Ventayol Garcia M, Kukla-Bartoszek M, Pospiech E et al (2019) HIrisPlex-S system for eye, hair, and skin color prediction from DNA: massively parallel sequencing solutions for two common forensically used platforms. Forensic Sci Int Genet 43:102152. https://doi.org/10.1016/j.fsigen.2019.102152
Bright JA, Gill P, Buckleton J (2012) Composite profiles in DNA analysis. Forensic Sci Int Genet 6:317–321
Bruijns B, Tiggelaar R, Gardeniers H (2018) Massively parallel sequencing techniques for forensics: a review. Electrophoresis 39:2642–2654
Buckberry J (2018) Techniques for identifying the age and sex of children at death. Crawford S, Hadley D, Shepherds G. (2018) The Oxford Handbook of the Archaeology of Childhood. Oxford Handbooks Collection. Oxford: Oup, pp 55–70
Buckberry J (2000) Missing, presumed buried? Bone diagenesis and the under-representation of Anglo-Saxon children. Assemblage. Issue 5. Available at. http://archaeologydataservice.ac.uk/archives/view/assemblage/html/5/buckberr.html. Accessed 15 Feb 2023
Campos PF, Craig OE, Turner-Walker G, Peacock E, Willerslev E, Gilbert MTP (2012) DNA in ancient bone - where is it located and how should we extract it? Ann Anat 194:7–16
Cooper A, Poinar HN (2000) Ancient DNA: do it right or not at all. Science 289:1139
Cummaudo M, Cappella A, Biraghi M et al (2018) Histomorphological analysis of the variability of the human skeleton: forensic implications. Int J Legal Med 132:1493–1503
Djurić M, Djukić K, Milovanović P et al (2011) Representing children in excavated cemeteries: the intrinsic preservation factors. Antiquity 85:250–262
Doden E, Halves R (1984) On the functional morphology of the human petrous bone. Am J Anat 169:451–462
Draus-Barini J, Walsh S, Pośpiech E, Kupiec T, Głąb H, Branicki W et al (2013) Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains. Investig Genet 4:3
Edson SM, Ross JP, Coble MD, Parson TJ, Barritt SM (2004) Naming the dead: confronting the realities of rapid identification of degraded skeletal remains. Forensic Sci Rev 16:63–90
Emmons AL, Davoren J, DeBruyn JM, Mundorff AZ (2020) Inter and intra-individual variation in skeletal DNA preservation in buried remains. Forensic Sci Int Genet 44:102193
Ewing MM, Thompson JM, McLaren RS, Purpero VM, Thomas KJ, Dobrowski PA et al (2016) Human DNA quantification and sample assessment: developmental validation of the PowerQuant system. Forensic Sci Int Genet 23:166–177
Furtwängler A, Reiter E, Neumann GU, Siebke I, Steuri N, Hafner A et al (2018) Ratio of mitochondrial to nuclear DNA affects contamination estimates in ancient DNA analysis. Sci Rep 8:14075
Gamba C, Jones ER, Teasdale MD, McLaughlin RL, Gonzalez-Fortes G, Mattiangeli V et al (2014) Genome flux and stasis in a five millennium transect of European prehistory. Nat Commun 5:5257
Geigl EM, Grange T (2018) Ancient DNA: the quest for the best. Mol Ecol Resour 18:1185–1187
Geršak ŽM, Zupanič Pajnič I, Črešnar M, Zupanc T (2019) Determination of DNA yield rates in six different skeletal elements in ancient bones. Forensic Sci Int Genet Suppl Ser 7:120–122
Gonzalez A, Cannet C, Zvénigorosky V, Geraut A, Koch G et al (2020) The petrous bone: ideal substrate in legal medicine? Forensic Sci Int Genet 47:102305
Goodwin S, McPherson JD, McCombie WR (2016) Coming of age: ten years of nextgeneration sequencing technologies. Nat Rev Genet 17:333–351
Götherström A, Liden K, Ahlström T, Källersjö M, Brown TA (1997) Osteology, DNA and sex identification: morphological and molecular sex identifications of five neolithic individuals from Ajvide, Gotland. Int J Osteoarchaeol 7:71–81
Guy H, Masset C, Baud CA (1997) Infant taphonomy. Int J Osteoarchaeol 7:221–229. https://doi.org/10.1002/(SICI)1099-1212(199705)7:3%3c221::AID-OA338%3e3.0.CO;2-Z
Haber M, Doumet-Serhal C, Scheib C, Xue Y, Danecek P, Mezzavilla M et al (2017) Continuity and admixture in the last five millennia of Levantine history from ancient Canaanite. Am J Human Gen 101:274–282
Haeusler M, Haas C, Lösch S, Moghaddam N, Villa LM, Walsch S et al (2016) Multidisciplinary identification of the controversial freedom fighter Jörg Jenatsch, assassinated 1639 in Chur, Switzerland. PLoS One 11:0168014
Hansen HB, Damgaard PB, Margaryan A, Stenderup J, Lynnerup N, Willerslev E et al (2017) Comparing ancient DNA preservation in petrous bone and tooth cementum. PLoS One 12:e0170940
Henderson J (1987) Factors determining the state of preservation of human remains. In: Boddington A, Garland AN, Janaway RC (eds) Death, Decay, and Reconstruction: Approaches to Archaeology and Forensic Science. Manchester University Press, Manchester, UK, p 43–54
Hines DZC, Vennemeyer M, Amory S, Huel R, Hanson I, Katzmarzyk C et al (2014) Prioritizing sampling of bone and teeth for DNA analysis in commingled cases. In: Adams BJ, Byrd JE (eds) Commingled Human Remains: Methods in Recovery, Analysis, and Identification, Elsevier Inc. pp 275–305
Hummel S, Bramanti B, Finke T, Herrmann B (2000) Evaluation of morphological sex determinations by molecular analyses. Anthropol Anz 58:9–13
Inkret J, Podovšovnik E, Zupanc T, Haring G, Zupanič PI (2021) Intra-bone nuclear DNA variability in Second World War metatarsal and metacarpal bones. Int J Legal Med 135:1245–1256
Jeffery N, Spoor F (2004) Prenatal growth and development of the modern human labyrinth. J Anat 204:71–92
Kamp KA (2001) Where have all the children gone?: the archaeology of childhood. J Archaeol Method Th 8:1–34
Kastelic V, Drobnič K (2012) A single-nucleotide polymorphism (SNP) multiplex system: the association of five SNPs with human eye and hair color in the Slovenian population and comparison using a Bayesian network and logistic regression model. Croat Med J 53:401–408
Kayser M, de Knijff P (2011) Improving human forensics through advances in genetics, genomics and molecular biology. Nat Rev Genet 12:179–192
Kayser M, Schneider PM (2009) DNA-based prediction of human externally visible characteristics in forensics: motivations, scientific challenges, and ethical considerations. Forensic Sci Int Genet 3:154–161
Kayser M (2015) Forensic DNA phenotyping: predicting human appearance from crime scene material for investigative purposes. Forensic Sci Int Genet 18:33–48
Keyser-Tracqui C, Ludes B (2005) Methods for the study of ancient DNA. In: Carracedo A (ed) Forensic DNA typing protocols. Humana Press Inc, New York, pp 253–264
King TE, Fortes GG, Balaresque P, Thomas MG, Balding D, Delser PM et al (2014) Identification of the remains of King Richard III. Nat Commun 5:5631
Kontopoulos I, Penkman K, McAllister GD, Lynnerup N, Damgaard PB, Hansen HB et al (2019) Petrous bone diagenesis: a multi-analytical approach. Palaeogeogr Palaeoclimatol Palaeoecol 518:143–154
Kukla-Bartoszek M, Szargut M, Pośpiech E, Diepenbroek M, Zielińska G, Jarosz A et al (2020) The challenge of predicting human pigmentation traits in degraded bone samples with the MPS-based HIrisPlex-S system. Forensic Sci Int Genet 47:102301
Lewis M (2007) The bioarchaeology of children: perspectives from biological and forensic anthropology. Cambridge University Press, Cambridge
Mansour S, Magnan J, Haidar H et al (2013) The Temporal Bone. In: Mansour S, Magnan J, Haidar H (eds) Comprehensive and Clinical Anatomy of the Middle Ear. Springer, Berlin, Heidelberg, pp 1–17
Matheson CD, Loy TH (2001) Genetic sex identification of 9400-year-old human skull samples from Çayönü Tepesi, Turkey. J Archaeol Sci 28:569–575
M’charek A (2008) Silent witness, articulate collective: DNA evidence and the inference of visible traits. Bioethics 22:519–528
Montelius K, Lindblom B (2012) DNA analysis in disaster victim identification. Forensic Sci Med Pathol 8:140–147
Morton RJ, Lord WD (2006) Taphonomy of child-sized remains: a study of scattering and scavenging in Virginia, USA. J Forensic Sci 51:475–479
Mundorff A, Davoren JM (2014) Examination of DNA yield rates for different skeletal elemenets at increasing post mortem intervals. Forensic Sci Int Genet 8:55–63
Nawrocki SP (1995) Taphonomic processes in historical cemeteries. In: Grauer AL (ed) Bodies of evidence: reconstructing history through skeletal analysis. Wiley-Liss, New York, pp 49–66
Obal M, Zupanič Pajnič I, Gornjak Pogorelc B, Zupanc T (2019) Different skeletal elements as a source of DNA for genetic identification of Second World War victims. Forensic Sci Int Genet Suppl Ser 7:27–29
Ovchinnikov IV, Ovtchinnikova OI, Druzina EB, Buzhilova AP, Makarov NA (1998) Molecular genetic sex determination of Medieval human remains from North Russia: comparison with archaeological and anthropological criteria. Anthropol Anz 56:7–15
Pääbo S, Poinar H, Serre D, Jaenicke-Després V, Hebler J, Rohland N et al (2004) Genetic analyses from ancient DNA. Annu Rev Genet 38:645–679
Parker C, Rohrlach AB, Friederich S, Nagel S, Meyer M, Krause J et al (2020) A systematic investigation of human DNA preservation in medieval skeletons. Sci Rep 10:18225. https://doi.org/10.1038/s41598-020-75163-w
Parson W, Gusmão L, Hares DR, Irwin JA, Mayr WR, Morling N et al (2014) DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing. Forensic Sci Int Genet 13:134–142
Peltzer A, Mittnik A, Wang CC, Begg T, Posth C, Nieselt K et al (2018) Inferring genetic origins and phenotyping traits of George Bähr, the architect of the Dresden Frauenkirche. Sci Rep 8:2115
Pilli E, Vai S, Caruso MG, D’Errico G, Berti A, Caramelli D (2018) Neither femur nor tooth: petrous bone for identifying archaeological bone samples via forensic approach. Forensic Sci Int 283:144–149
Pinhasi R, Fernandes D, Sirak K, Novak M, Connell S, Alpaslan-Roodenberg S et al (2015) Optimal ancient DNA yields from the inner ear part of the human petrous bone. PLoS One 10:e0129102
Pinhasi R, Fernandes DM, Sirak K, Cheronet O (2019) Isolating the human cochlea to generate bone powder for ancient DNA analysis. Nat Protoc 14:1194–1205
Promega Corporation (2022) PowerQuant System Technical Manual, Madison, WI
Qiagen Companies (2021) Investigator ESSplex SE QS handbook, Hilden
Rohland N, Hofreiter M (2007) Ancient DNA extraction from bones and teeth. Nat Protoc 2:1756
Sampietro ML, Gilbert MTP, Lao O, Caramelli D, Lari M, Bertranpetit J et al (2006) Tracking down human contamination in ancient human teeth. Mol Biol Evol 23:1801–1807
Saunders SR, Barrans L (1999) What can be done about the infant category in skeletal samples? In: Hoppa RD, Firzgerald CM (eds) Cambridge Studies in Biological and Evolutionary Anthropology. Cambridge University Press, Cambridge, pp 183–209
Shea JJ (2006) Child’s play: reflections on the invisibility of children in the Paleolithic record. Evol Anthropol Issues News Rev: Issues News Rev 15:212–216
Sirak K, Fernandes D, Cheronet O, Harney E, Mah M, Mallick S et al (2020) Human auditory ossicles as an alternative optimal source of ancient DNA. Genome Res 30:427–436
Stull KE, Cirillo LE, Cole SJ, Hulse CN (2020) Chapter 14 - subadult sex estimation and KidStats. In Klales AR. (Ed.), Sex Estimation of the Human Skeleton (pp 219–242). Academic Press. https://doi.org/10.1016/B978-0-12-815767-1.00014-6
Šuligoj A, Mesesnel S, Leskovar T, Podovšovnik E, Zupanič PI (2022) Comparison of DNA preservation between adult and non-adult ancient skeletons. Int J Legal Med 136:1521–1539
Thermo Fisher Scientific (2019) Precision ID SNP Panel with the HID Ion S5TM/HID Ion Gene StudioTM S5 System, Application Guide, Thermo Fisher Scientific, Carlsbad. CA
Thermo Fisher Scientific (2018) Torrent SuiteTM software 5.10, User Guide, Thermo Fisher Scientific, Carlsbad. CA
Tierney SN, Bird JM (2015) Molecular sex identification of juvenile skeletal remains from an Irish medieval population using ancient DNA analysis. J Archaeol Sci 62:27–38
Tozzo P, Politi C, Delicati A, Gabbin A, Caenazzo L (2021) External visible characteristics prediction through SNPs analysis in the forensic setting: a review. Front Biosci 26:828–850
von Endt DW, Ortner DJ (1984) Experimental effects of bone size and temperature on bone diagenesis. J Archaeol Sci 11:247–253
Waldron T (1987) The relative survival of the human skeleton: implications for palaeopathology. In: Boddington A, Garland AN, Janaway RC (eds) Death, Decay, and Reconstruction: Approaches to Archaeology and Forensic Science. Manchester University Press, Manchester, pp 55–64
Walsh S, Chaitanya L, Breslin K, Muralidharan C, Bronikowska A, Pospiech E et al (2017) Global skin colour prediction from DNA. Hum Genet 136:847–863
Walsh S, Chaitanya L, Clarisse L, Wirken L, Draus-Barini J, Kovatsi L et al (2014) Developmental validation of the HIrisPlex system: DNA-based eye and hair colour prediction for forensic and anthropological usage. Forensic Sci Int Genet 9:150–161
Walsh S, Liu F, Wollstein A, Kovatsi L, Ralf A, Kosiniak-Kamysz A et al (2013) The HIrisPlex system for simultaneous prediction of hair and eye colour from DNA. Forensic Sci Int Genet 7:98–115
Zapata J, Pérez-Sirvent C, Martínez-Sánchez MJ, Tovar P (2006) Diagenesis, not biogenesis: two late Roman skeletal examples. Sci Total Environ 369:357–368
Zupan A, Vrabec K, Glavač D (2013) The paternal perspective of the Slovenian population and its relationship with other populations. Ann Hum Biol 40:515–526
Zupanc T, ZupaničPajnič I, Podovšovnik E, Obal M (2021) High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains. Forensic Sci Int Genet 51:102426. https://doi.org/10.1016/j.fsigen.2020.102426
Zupanič Pajnič I, Zupanc T, Leskovar T, Črešnar M, Fattorini P (2022) Eye and hair color prediction of ancient and Second World War skeletal remains using a forensic PCR-MPS approach. Genes 13:1432. https://doi.org/10.3390/genes13081432
ZupaničPajnič I (2016) Extraction of DNA from human skeletal material. In: Goodwin W (ed) Forensic DNA Typing Protocols, Methods in Molecular Biology, vol 1420, Springer Science&Business Media, LLC, New York. pp 89–108
Zupanič I, Balažic J, Komel R (1998) Analysis of nine short tandem repeat (STR) loci in the Slovenian population. Int J Legal Med 11:248–250
Zupanič Pajnič I, Šterlinko H, Balažic J et al (2001) Parentage testing with 14 STR loci and population data for 5 STRs in the Slovenian population. Int J Legal Med 114:178–180
Zupanič Pajnič I, Podovšovnik Axelsson E, Balažic J (2014) Slovenian population data for five new European Standard Set short tandem repeat loci and SE33 locus. Croat Med J 55:14–18
Zupanič Pajnič I, Geršak ŽM, Leskovar T, Črešnar M (2023) Kinship analysis of 5th- to 6th-century skeletons of Romanized indigenous people from the Bled-Pristava archaeological site. Forensic Sci Int Genet, p 102886. https://doi.org/10.1016/j.fsigen.2023.102886
Acknowledgements
The authors would like to thank the Ljubljana National Museum, especially Dr. Timotej Knific, for including the archaeological human remains from the museum into our study. We also thank Prof. Paolo Fattorini for analyzing BAM files of the adult skeleton.
Funding
This study was financially supported by the Slovenian Research Agency (the project “Inferring ancestry from DNA for human identification” J3-3080). Javna agencija za raziskovalno dejavnost Republike Slovenije: project J3-3080.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical standards
This research project was approved by the Medical Ethics Committee of the Republic of Slovenia (145/06/13, 0120–412/2021/3).
Research involving human participants and/or animals
Research involvs aged skeletons and genetic profiles of persons included in elimination database, and from them, informed consents were obtained and submitted to the Medical Ethics Committee of the Republic of Slovenia. After submission, the Medical Ethics Committee of the Republic of Slovenia approved the research (number of approval is 145/06/13 and 0120–412/2021/3).
Informed consent
This research project was approved by the Medical Ethics Committee of the Republic of Slovenia (145/06/13, 0120–412/2021/3), and informed consents of persons included in elimination database were submitted to the Medical Ethics Committee of the Republic of Slovenia.
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Highlights
- MPS technology allows prediction of the external appearance of individuals from past human populations.
- Studies predicting eye and hair color in ancient subadults are very rare.
- Prediction of eye and hair color based on PCR-MPS HIrisPlex was carried out on fifth- to sixth-century adult and subadult skeletons.
- Petrous bones were used for genetic analysis.
- The HIrisPlex genotypes obtained allowed successful phenotypic predictions for both skeletons.
- Exceptional preservation of DNA in petrous bones resulted in successful HIrisPlex SNP typing even for the subadult skeleton.
Supplementary information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Zupanič Pajnič, I., Leskovar, T. & Črešnar, M. Eye and hair color prediction of an early medieval adult and subadult skeleton using massive parallel sequencing technology. Int J Legal Med 137, 1629–1638 (2023). https://doi.org/10.1007/s00414-023-03032-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00414-023-03032-y