Bones are an important source of DNA for identification in forensic medicine, especially when the remains are skeletonized, which is the case when dealing with victims of the Second World War. Often ...the amount of bone available for sampling is limited, and therefore it is crucial to sample the bone segment with the highest adequate DNA quantity for identification. Studies performed on all representative skeletal element types of the human body showed that the amount of DNA obtained from different skeletal elements of different body regions varies greatly. When bones from torso were analyzed, thoracic vertebrae outperformed other vertebrae (cervical and lumbar) and, alongside the first ribs, were among the most appropriate bone elements for identification purposes. It was also shown that the quantity of DNA varies significantly within a single bone type. This study focused on exploring intra-bone DNA variability between five parts of 12th thoracic vertebrae (laminae + spinous process, pedicles + transverse processes, and corpus right, left, and middle). The research was based on the theory that the distribution of body weight and consequently bone remodeling, as well as the ratio between cancellous and cortical bone, contribute to different quantities of DNA in different parts of vertebra sampled. The vertebrae were cleaned and cut into five parts, and each part was completely ground to obtain homogenous bone powder. Half a gram of powder from each part was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 machine (Qiagen). DNA quantity and quality were determined using the PowerQuant System (Promega) and autosomal STR typing success using the GlobalFiler Amplification Kit (Applied Biosystems). Thirty-five 12th thoracic vertebrae were sampled from a single Second World War mass grave. The best results with the highest DNA quantity were found in laminae and the spinous process, and among them all vertebrae analyzed yielded full STR profiles except three, where only a few dropouts occurred. The second-ranked bone part was the pedicles and transverse processes. The comparison of DNA degradation in the vertebral segments analyzed does not show statistically significant differences. Considering our research, when only the torso is available for identification, the 12th thoracic vertebra should be collected and the vertebral arch should be sampled for genetic analyses.
•Higher DNA yield is present in areas with the highest strain or weight bearing due to a higher degree of bone remodeling.•More DNA was obtained from the vertebral arch than the vertebral body.•DNA quality (Auto/Deg ratio) did not vary across the 12th vertebra.•In all lamina + spinous process segments, full or almost full genetic profiles were obtained.•When only the torso is present, the vertebral arch of the 12th vertebra should be considered for DNA identification.
In forensic kinship testing and missing person identification, it is a fundamental question to choose the most informative reference relatives, select appropriate genotyping systems, and evaluate the ...weight of evidence comprehensively. Despite that several useful tools have been developed, they have not addressed these questions satisfactorily. In this paper, we develop a flexible and user-friendly online tool,
Easykin
, to address the aforementioned issues. It has some promising features: (i) Pedigrees can be constructed easily and presented intuitively with just a few mouse clicks. (ii) System power can be estimated before testing based on certain set of markers and reference relatives. (iii) The pruning function of
EasyKin
enables users to choose appropriate subsets of available references. (iv) Parameters at a specific LR for a single case may ease evidence interpretation. (v) The user interface (UI) is an HTML-based dashboard, which is friendly to both professional and non-professional users and can be used anytime and anywhere. Here, we presented three common cases as examples to demonstrate how kinship testing and missing person identification can be improved with
EasyKin
. In conclusion, this tool provides a one-stop solution for forensic use, that is, instructing users to choose appropriate kits and reference relatives before testing, calculating LR in the testing, and providing parameters for data interpretation after testing.
EasyKin
is freely available at
https://forensicsysu.shinyapps.io/EasyKin/
.
The present work proposes a general strategy for dealing with missing person identification cases through DNA-database search. Our main example is the identification of abducted children in the last ...civic-dictatorship of Argentina, known as the “Missing Grandchildren of Argentina”. Particularly we focus on those pedigrees where few, or only distant relatives of the missing person are available, resulting in low statistical power. For such complex cases we provide a statistical method for selecting a likelihood ratio (LR) threshold for each pedigree based on error rates. Furthermore, we provide an open-source user friendly software for computing LR thresholds and error rates. The strategy described in the paper could be applied to other large-scale cases of DNA-based identification hampered by low statistical power.
•General strategy for dealing with low statistical power in missing person and disaster victim identification cases.•Method for selecting a likelihood ratio (LR) threshold for identifications through DNA-database search.•Error rates estimated by conditional simulation.•Examples based on unsolved cases of ‘Missing Grandchildren of Argentina’.•Freely available software.
In forensic applications, there is an increasing demand for the analysis of DNA profiles arising from missing person identification (MPI) cases. A specific DNA profile may originate from a single ...source or more than one contributor (i.e., a DNA mixture). When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. As a novel kind of multiallelic marker, microhaplotypes have proven promising for relatedness determination and mixture deconvolution. Herein, we developed a large panel of 185 microhaplotype markers and demonstrated its application in different scenarios of relationship inference through a simulation study and real pedigree analysis, combined with probabilistic genotyping models for data interpretation. Based on single-source profiles, it was shown that the present microhaplotype panel was sufficient for pairwise close relative testing (parent/child, full-sibling and 2nd-degree relative). For more distant relatives (3rd-degree relatives), there was a clear improvement when data from one well-chosen extra relative were available. We further sought to evaluate the theoretical systematic effectiveness and actual performance of microhaplotype markers in identifying the contribution of a missing pedigree member to a two-person mixture (as a minor donor). It was observed that 100% correct assignments were made in the balanced mixtures (with no dropout) when referenced to close relatives. When the mixture profiles suffered from dropout, incorrect assignments of minor donors were markedly associated with relatedness and the dropout level. Meanwhile, the studied scenarios generally exhibited zero or very low false-positive rates, indicating a low probability of incorrectly assigning an unrelated contributor as a close relative of the reference. Our results indicate that microhaplotype data can be reliably interpreted for identifying missing persons through kinship analysis based on DNA profiles of single-source samples or two-person mixtures. Furthermore, this study could be extended to more complex scenarios, such as determining the relatedness of contributors in (or among) mixed DNA profiles, if combined with different statistical frameworks.
•A large panel of 185 microhaplotype markers was developed.•Microhaplotype sequencing can be utilized for identification of missing persons through kinship analysis.•Microhaplotype data can be reliably interpreted when a two-person mixture was involved.
•Second World War victims were genetically tested by combining STRs and phenotypic HIrisPlex SNPs.•Badly preserved and incomplete skeletons were disinterred, and four tooth samples and two bone ...samples were analyzed.•A combination of autosomal and Y-STRs allowed identification of the male with a PP of more than 99.9%.•For the female victim, eye and hair color were predicted using NGS technology.•The phenotypic prediction agreed with the eye and hair color in a painting.
Genetic identification of a Slovenian prewar elite couple killed in 1944 was performed by typing autosomal and Y-chromosomal STRs, and phenotypic HIrisPlex SNPs for hair and eye color prediction were analyzed for the female skeleton using next-generation sequencing (NGS) technology. The clandestine grave containing the couple’s skeletal remains was found in 2015 and only the partial remains were found. Living distant relatives could be found only for the male victim. Because of a lack of comparative reference samples, it was not possible to identify the female victim through autosomal and mitochondrial DNA typing. However, the possibility of comparison of eye and hair color with a painting exhibited in the City Museum of Ljubljana by the prominent Slovenian painter Ivana Kobilca existed. Nuclear DNA obtained from the samples was quantified using the PowerQuant System, and then STR typing was carried out with different autosomal and Y-STR kits. From 0.09–9.36 ng DNA/g of powder was obtained from teeth and bones analyzed. Complete autosomal and Y-STR profiles made it possible to identify the male skeleton via comparison with two nephews. For the female victim, predicted eye and hair color was compared to colors on the painting. Kobilca’s painting confirms the genetically predicted eye and hair color. After more than seventy years, the skeletal remains of the couple were handed over to their relatives, who buried the victims with dignity in a family grave.
Often bones are the only biological material left for the identification of human remains. As situations may occur where samples need to be stored for an extended period without access to cooling, ...appropriate storage of the bone samples is necessary for maintaining the integrity of DNA for profiling. To simulate DNA preservation under field conditions, pig rib bones were used to evaluate the effects of bone cleaning, buffer composition, storage temperature, and time on DNA recovery from bone samples. Bones were stored in three different buffers: TENT, solid sodium chloride, and ethanol-EDTA, at 20 °C and 35 °C for 10, 20, and 30 days. Bones were subsequently dried and ground to powder. DNA was extracted and quantified. Results show that temperature and storage time have no significant influence on DNA yield. DNA recovery from bones stored in solid sodium chloride or ethanol-EDTA was significantly higher compared to bones stored in TENT, and grinding of bones was facilitated by the extent of dehydration in solid sodium chloride and ethanol-EDTA compared to TENT. Overall, solid sodium chloride was found to be superior over ethanol-EDTA; when it comes to transportation, dry material such as salt eliminates the risk of leaking; it is non-toxic and in contrast to ethanol not classified as dangerous goods. Based on this study’s results, we recommend NaCl as a storage substrate for forensic samples in cases where no cooling/freezing conditions are available.
In this study, we present a correlation between δ18OC values of carbonate in tooth enamel samples from the modern Brazilian population and the available δ18ODW data for the meteoric water from the ...Global Network of Isotopes in Precipitation (GNIP). Tooth enamel from 119 Brazilian individuals from five different regions of the country were analyzed. The δ18OC isoscape obtained is in good agreement with the isoscape based on regional meteoric and drinking water. The regression matrix obtained for the δ18O values of the carbonate tooth enamel and meteoric water was used to build an isoscape using the regression-kriging approach. Our data show that Brazil can be divided in two main regions with respect to the δ18O values of the carbonate tooth enamel: (1) the most easterly part of the northeast region, which is characterized by a warm and dry climate and (2) the remainder of the country, stretching from the Amazon rain forest to the more southernly regions. The data herein reported can be used for forensic purposes related to human identification.
•Isoscape based on tooth enamel for the Brazilian population•δ18O isoscape based on tooth enamel for human identification•Isotopic database based on tooth enamel for the modern population in Brazil•Isoscape built using the regression-kriging approach
•Genetic identification of the Second World War victims of the largest family massacre in Slovenia was performed.•Incomplete, poorly preserved skeletons were exhumed and 12 bone and tooth samples ...were genetically typed.•By combining autosomal and Y-STRs three male victims were identified with PP greater than 99.9 %.•The aunt was identified using the Precision ID GlobalFiler NGS STR Panel.•Identification was successful for four out of seven victims exhumed from the hidden mass grave.
The killings during the Second World War, with nearly one hundred thousand victims, is one of the greatest losses of life in Slovenia’s modern history. This article presents the genetic identification of the victims of the largest family massacre that occurred in Slovenia, in which 10 members of the same family were killed. Seven of them were buried in a hidden mass grave and only two children survived. In 2015 and 2016, two graves were found and three incomplete female skeletons and at least three incomplete male skeletons were exhumed. A total of 12 bones and teeth were analysed and compared to two living relatives. Extracted DNA was quantified using the PowerQuant kit, and various autosomal and Y-STR kits were used for STR typing. Up to 2.7 ng DNA/g of powder was acquired from the samples analysed. We managed to obtain nuclear DNA for successful STR typing from seven bones and one molar. From the female grave, autosomal profiles were obtained only from one skeleton, and from the male grave from five out of six femurs. The relationships between the males were additionally confirmed by analyses of Y-STRs. STR profiles made possible the identification of four family members; one of the aunts from the female grave, and two uncles and the father of the surviving children, who were used as family references, from the male grave. The product rule was used to calculate a combined likelihood ratio for autosomal and Y-STRs, and statistical analyses showed high confidence of correct identification with posterior probability (PP) greater than 99.9 % for three out of four victims identified. For identifying the aunt, the PP obtained after ESI-17 and NGM STR typing was too low. To increase the PP, the next-generation sequencing Precision ID GlobalFiler NGS STR Panel was used and, after the analysis of additional STR loci, the statistical analysis showed a PP greater than 99.9 %, indicating that a sufficient number of genetic markers had been investigated in identifying the skeletal remains of the aunt. An elimination database containing the genetic profiles of all individuals that had been in contact with the bones was created to ensure traceability in case of contamination, and no matches were found. After more than 70 years, the skeletal remains were returned to the surviving children, who buried their relatives in a family grave.
DNA sampling and typing are used for identifying missing persons or war victims. In recent forensic studies, little focus has been placed on determining intra-bone variability within a single ...skeletal element. When dealing with aged human bones, complete skeletal remains are rarely present. In cases in which only the torso is available, studies have shown that ribs are one of the most appropriate samples, but intra-bone variability has not yet been studied. A higher degree of remodeling was found to contribute to higher DNA yield in the parts of the skeletal element where the most strain is concentrated. This study explores intra-bone variability in proximal, middle, and distal parts of the first human rib by determining the quantity and quality of DNA using the PowerQuant System (Promega) and autosomal STR typing success using the PowerPlex ESI 17 Fast System (Promega). Thirty first ribs from a single Second World War mass grave were sampled. No variation in DNA degradation was observed across the individual rib. The highest quantity of DNA was measured in the proximal part of the first rib, and in all ribs except three, full or almost full genetic profiles were obtained. Thus, when only the torso is present in archaeological or medico-legal cases, first ribs are recommended to be collected if possible, and the proximal or vertebral ends should be sampled for genetic analysis.
Abstract We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal ...remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation – ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories.
