A beetle for prosecution: exogenous DNA detection from larval and adult gut of a Neotropical carrion beetle.

The use of recovered DNA ingested by necrophagous or hematophagous insects has increased in forensic sciences, mainly with representatives of flies. However, some beetles are also important for medico-legal forensic entomology because they feed on carcasses until advanced decomposition. This study evaluated whether the Neotropical carrion beetle Oxelytrum discicolle (Silphidae) has the potential for the detection of exogenous DNA into the gut. The whole gut or the gut contents were extracted from O. discicolle larvae and adult previously fed on pig carcass. The pig DNA recovery rate was 33.3% in larvae and 25% in adults, indicating that the carrion beetle's gut may be useful for DNA identification of ingested food. Samples with the whole gut or only gut contents showed the same DNA recovery rate. Exogenous DNA from the whole gut was recovered from samples stored in ethanol at -20 ºC for 11 days, showing that samples of O. discicolle can be stored in the forensic laboratory without loss in DNA recovery rate.

Cross-Platform Application of DNA Methylation Age Estimation Model in Eastern Chinese Han Population.

OBJECTIVES: To evaluate the forensic application value of an age estimation model based on DNA methylation in eastern Chinese Han population, and to provide a theoretical basis for exploring age estimation models suitable for different detection platforms. METHODS: According to the 6 age-related methylation sites in the published blood DNA methylation age estimation models of Chinese Han population, the DNA methylation level of 48 samples was detected by pyrosequencing and next-generation sequencing (NGS). After submitting DNA methylation levels to the age estimation model, the DNA methylation ages were predicted and compared with their real ages. RESULTS: The 6 DNA methylation sites in both detection techniques were age-related, with an R2 of 0.85 and a median absolute deviation (MAD) of 4.81 years when using pyrosequencing；with an R2 of 0.84 and MAD of 4.41 years when using NGS. CONCLUSIONS: The blood DNA methylation age estimation model can be used under pyrosequencing and multi-purpose regional methylation enrichment sequencing technology based on NGS and it can accurately estimate the age.

[Establishment of DNA Genetic Marker Identification System for Plant Evidence].

OBJECTIVES: To establish a species identification system based on DNA genetic markers for plant evidence. METHODS: Two hundred common plants in Shanghai were collected and identified by morphological characteristics. The primers of gene segments rbcL, matK, and ITS were designed and amplified. The PCR amplicon was detected by agarose gel electrophoresis. After the sequencing, the universality and the identification capacity of the three markers were evaluated. RESULTS: The success rate of amplification was in order of rbcL （99.5%） > matK （92.5%） > ITS （86.0%）. The identification capacity of the combination of rbcL and matK was better than that of rbcL or matK, by which most plant species could be identified to the genus or higher. ITS was not suitable to be a unique marker because of its unstable result, but it still could be a powerful supplement. The identification capacity of the combination of rbcL, matK and ITS was higher than that of rbcL and matK, by which most plant species could be identified to the genus or lower. CONCLUSIONS: The identification system with the combination of rbcL, matK and ITS as markers has excellent universality for plant evidence, which can distinguish most plant species to the genus or lower.

Evaluation of forensic genetic parameters of 12 STR loci in the Korean population using the InvestigatorⓇ HDplex kit.

We genotyped and calculated the forensic parameters of 10 non-CODIS loci and 2 CODIS loci of 990 Korean individuals using the InvestigatorⓇ HDplex kit. No significant deviations from Hardy-Weinberg equilibrium (after Bonferroni correction for multiple testing) or genetic linkage disequilibrium were observed. The calculated matching probability and power of discrimination ranged from 0.0080 to 0.2014, and 0.7986 to 0.9920, respectively. We conclude that the markers of the kit are highly informative corroborative tools for forensic DNA analysis.

Law enforcement use of genetic genealogy databases in criminal investigations: Nomenclature, definition and scope.

Although law enforcement use of commercial genetic genealogy databases has gained prominence since the arrest of the Golden State Killer in 2018, and it has been used in hundreds of cases in the United States and more recently in Europe and Australia, it does not have a standard nomenclature and scope. We analyzed the more common terms currently being used and propose a common nomenclature: investigative forensic genetic genealogy (iFGG). We define iFGG as the use by law enforcement of genetic genealogy combined with traditional genealogy to generate suspect investigational leads from forensic samples in criminal investigations. We describe iFGG as a proper subset of forensic genetic genealogy, that is, FGG as applied by law enforcement to criminal investigations; hence, investigative FGG or iFGG. We delineate its steps, compare and contrast it with other investigative techniques involving genetic evidence, and contextualize its use within criminal investigations. This characterization is a critical input to future studies regarding the legal status of iFGG and its implications on the right to genetic privacy.

Dense SNP-based analyses complement forensic anthropology biogeographical ancestry assessments.

Identification of unidentified human remains (UHRs) is crucial yet challenging, especially with traditional forensic techniques. Forensic anthropological examinations can yield ancestry estimations; however, the utility of these estimates is limited by the data points that can be collected from partial remains, complexities of admixture, and variation of phenotypic expression due to environmental effects. While it is generally known that anthropological estimates can be imprecise, the performance of these methods has not been studied at scale. Genome-wide SNP testing is an orthogonal approach for estimating ancestry and offers a unique opportunity to measure the magnitude of anthropological ancestry misattribution. Genomic ancestry inference leverages principal component analysis (PCA) and model-based clustering approaches. This study compares anthropologically determined ancestry with those estimated using genome-wide SNP markers. A dataset of 611 UHR samples with publicly available ancestry assessments from National Missing and Unidentified Persons System (NamUs) was analyzed. The genetic ancestry approach, validated against reference population samples, offers robust ancestry calculations for major population groups. Inconsistency between anthropological and genomic ancestry assignments were observed, particularly for admixed populations. Although forensic anthropological examinations remain valuable, their limitations emphasize the need for refinement and enhancement through the augmentation of SNP-based analyses. Further validation studies are crucial to define the uncertainty associated with both anthropological and genome-based ancestry estimates to resolve cases and aid law enforcement investigations. Additionally, current policy and practices for reporting ancestry for UHRs should be revisited to reduce potential misinformation.

Optimizing Analytical Thresholds for Low-Template DNA Analysis: Insights from Multi-Laboratory Negative Controls.

When analyzing challenging samples, such as low-template DNA, analysts aim to maximize information while minimizing noise, often by adjusting the analytical threshold (AT) for optimal results. A potential approach involves calculating the AT based on the baseline signal distribution in electrophoresis results. This study investigates the impact of reagent kits, testing quarters, environmental conditions, and amplification cycles on baseline signals using historical records and experimental data on low-template DNA. Variations in these aspects contribute to differences in baseline signal patterns. Analysts should remain vigilant regarding routine instrument maintenance and reagent replacement, as these may affect baseline signals. Prompt analysis of baseline status and tailored adjustments to ATs under specific laboratory conditions are advised. A comparative analysis of published methods for calculating the optimal AT from a negative signal distribution highlighted the efficiency of utilizing baseline signals to enhance forensic genetic analysis, with the exception of extremely low-template samples and high-amplification cycles. Moreover, a user-friendly program for real-time analysis was developed, enabling prompt adjustments to ATs based on negative control profiles. In conclusion, this study provides insights into baseline signals, aiming to enhance genetic analysis accuracy across diverse laboratories. Practical recommendations are offered for optimizing ATs in forensic DNA analysis.

Moving from the unknown to the known: a multidisciplinary approach to the identification of skeletal remains from Sandy Point, Australia.

When human remains are inadvertently located, case-related circumstantial information is used to generate an identification hypothesis, and the preservation of the remains typically informs which identification methods may then be used to validate that hypothesis. What happens, however, when there is no contextual information to generate an identification hypothesis? This paper presents the case of a near-complete human skeleton discovered at Sandy Point in Victoria, Australia. The circumstances of the case did not facilitate an identification hypothesis, and with no hypothesis to triage the identification process, all possible identification methods were employed. Preservation of the individual meant neither a visual nor a fingerprint identification was possible, and the lack of an identification hypothesis meant there was no antemortem reference data to compare with the postmortem DNA or dental information. Consequently, in addition to historical research, novel methods, such as radiocarbon dating and genetic intelligence, were utilized to complement information provided by the forensic anthropology and odontology analyses, which ultimately resulted in the identification. This example highlights the complexity of cases of unidentified skeletal remains and emphasizes the fact that identification is a process that necessarily requires a multidisciplinary and collaborative approach. Key points: Human skeletal remains were recovered from Sandy Point, Victoria.The absence of circumstantial information and the poor preservation (i.e. skeletonized) of the remains precluded the formation of an identification hypothesis, rendering the identification process complex.Only through the integration of anthropology, odontology, molecular biology, radiocarbon dating, historical research, and genealogy were the remains able to be identified as Mr. Christopher Luke Moore, who drowned in 1928.Human identification is a process that necessarily requires a multidisciplinary and collaborative approach.

Prioritizing privacy and presentation of supportable hypothesis testing in forensic genetic genealogy investigations.

Investigative leads are not generated by traditional forensic DNA testing, if the source of the forensic evidence or a 1st degree relative of unidentified human remains is not in the DNA database. In such cases, forensic genetic genealogy (FGG) can provide valuable leads. However, FGG generated genetic data contain private and sensitive information. Therefore, it is essential to deploy approaches that minimize unnecessary disclosure of these data to mitigate potential risks to individual privacy. We recommend protective practices that need not impact effective reporting of relationship identifications. Examples include performing one-to-one comparisons of DNA profiles of third-party samples and evidence samples offline with an "air gap" to the internet and shielding the specific shared single nucleotide polymorphisms (SNP) states and locations by binning adjacent SNPs in forensic reports. Such approaches reduce risk of unwanted access to or reverse engineering of third-party individuals' genetic data and can give these donors greater confidence to support use of their DNA profiles in FGG investigation.

[Box: see text].

Analysis of the privacy-performance tradeoff of reference testing in Forensic Investigative Genetic Genealogy.

During an investigation using Forensic Investigative Genetic Genealogy, which is a novel approach for solving violent crimes and identifying human remains, reference testing-when law enforcement requests a DNA sample from a person in a partially constructed family tree-is sometimes used when an investigation has stalled. Because the people considered for a reference test have not opted in to allow law enforcement to use their DNA profile in this way, reference testing is viewed by many as an invasion of privacy and by some as unethical. We generalize an existing mathematical optimization model of the genealogy process by incorporating the option of reference testing. Using simulated versions of 17 DNA Doe Project cases, we find that reference testing can solve cases more quickly (although many reference tests are required to substantially hasten the investigative process), but only rarely (<1%) solves cases that cannot otherwise be solved. Through a mixture of mathematical and computational analysis, we find that the most desirable people to test are at the bottom of a path descending from an ancestral couple that is most likely to be related to the target. We also characterize the rare cases where reference testing is necessary for solving the case: when there is only one descending path from an ancestral couple, which precludes the possibility of identifying an intersection (e.g., marriage) between two descendants of two different ancestral couples.

Ethical considerations for Forensic Genetic Frequency databases: First Report conception and development.

The Forensic Databases Advisory Board (FDAB), an independent board that assists the International Society for Forensic Genetics (ISFG), has presented a First Report on ethical aspects of the following Forensic Genetic Frequency Databases (FGFD): EMPOP, STRidER and YHRD. The FDAB designed an ethical framework to evaluate the content of these FGFD, and the factors to be considered for retention and acceptance of submissions. The FDAB framework proposes to categorize submissions according to the risk of having contravened the universal ethical principles outlined by international organizations, and the guidelines adopted by the ISFG. The report has been open to discussion by the scientific community since 2023. Herein we present the conception and development of the First Report along with a summary of its content, with consideration of the feedback received.

Investigative genetic genealogy for human remains identification.

Investigative genetic genealogy (IGG) has emerged as a highly effective tool for tying a forensic DNA sample to an identity. While much of the attention paid to IGG has focused on cases where the DNA is from an unknown suspect, IGG has also been used to help close hundreds of unidentified human remains (UHR) cases. Genome-wide single-nucleotide polymorphism (SNP) genotype data can be obtained from forensic samples using microarray genotyping or whole-genome sequencing (WGS) with protocols optimized for degraded DNA. After bioinformatic processing, the SNP data can be uploaded to public GG databases that allow law enforcement usage, where it can be compared with other users' data to find distant relatives. A genetic genealogist can then build the family trees of the relatives to narrow down the identity of the source of the forensic DNA sample. To date, 367 UHR identifications using IGG have been publicly announced. The same IGG techniques developed and refined for UHR cases have significant potential for disaster victim identification, where DNA is often extremely compromised, and close family references may not be available. This paper reviews the laboratory, bioinformatic, and genealogical techniques used in IGG for UHR cases and presents three case studies that demonstrate how IGG is assisting with remains identification.

Forensic Characterization and Genetic Portrait of the Gannan Tibetan Ethnic Group via 165 AI-SNP Loci.

BACKGROUND: The Tibetan group is one of the oldest Sino-Tibetan ethnic groups. The origin, migration as well as the genetic background of Tibetans have become the research hotspots in the field of forensic genetics. The use of ancestry informative markers (AIMs) allows the investigation of the genetic background of the Gannan Tibetan group. METHODS: In this study, the 165 ancestry informative single nucleotide polymorphism (AI-SNP) loci included in the Precision ID Ancestry Panel were used to genotype 101 Gannan Tibetans using the Ion S5 XL system. The forensic statistical parameters of 165 AI-SNP in the Gannan Tibetan group were calculated. Population genetic analyses including Nei's genetic distances, phylogenetic analyses, pairwise fixation index, principal component analyses and population ancestry composition analyses were also conducted to evaluate the genetic relationships between the Gannan Tibetan group and other reference populations. RESULTS: Forensic parameters of the 165 AI-SNP loci indicated that not all of the SNPs showed high genetic polymorphisms in the Gannan Tibetan group. Population genetic analyses indicated that the Gannan Tibetan group had close genetic affinities with East Asian populations, especially with the groups residing in its neighboring geographical regions. CONCLUSIONS: The 165 AI-SNP loci in the Precision ID Ancestry Panel showed high ancestral prediction powers for different continental populations. When trying to predict the ancestral information of East Asian subpopulations using this panel, the prediction results are not particularly accurate. The 165 AI-SNP loci showed varying degrees of genetic polymorphisms in the Gannan Tibetan group, and the combined use of these loci could be an effective tool in the forensic individual identification and parentage testing of this group. The Gannan Tibetan group has close genetic affinities with East Asian populations compared with other reference populations, especially tighter genetic relationships with the groups residing in its neighboring geographical regions.

Application of Y-STR, DIP-STR and SNP-STR Markers in Interpretation of Forensic Genetic Profiling: A Narrative Review.

Y-STR, DIP-STR, and SNP-STR are useful alternatives for testing the low quantity of DNA in solving the challenges in interpreting forensic genetic profiling. In an unbalanced mixed DNA, partial DNA is often not detected due to the effect of masking by the dominant DNA. Therefore, in such cases interpretation of the results is limited. Furthermore, profiling of these specimens cannot be performed using conventional forensic genetic methods. Biomarkers including Y-STR, DIP-STR and SNP-STR perform well in detecting DNA contributes in the mixed sample. In the present research, the performance of each is evaluated separately.

Evaluating the Impact of Dropout and Genotyping Error on SNP-Based Kinship Analysis With Forensic Samples.

Technological advances in sequencing and single nucleotide polymorphism (SNP) genotyping microarray technology have facilitated advances in forensic analysis beyond short tandem repeat (STR) profiling, enabling the identification of unknown DNA samples and distant relationships. Forensic genetic genealogy (FGG) has facilitated the identification of distant relatives of both unidentified remains and unknown donors of crime scene DNA, invigorating the use of biological samples to resolve open cases. Forensic samples are often degraded or contain only trace amounts of DNA. In this study, the accuracy of genome-wide relatedness methods and identity by descent (IBD) segment approaches was evaluated in the presence of challenges commonly encountered with forensic data: missing data and genotyping error. Pedigree whole-genome simulations were used to estimate the genotypes of thousands of individuals with known relationships using multiple populations with different biogeographic ancestral origins. Simulations were also performed with varying error rates and types. Using these data, the performance of different methods for quantifying relatedness was benchmarked across these scenarios. When the genotyping error was low (<1%), IBD segment methods outperformed genome-wide relatedness methods for close relationships and are more accurate at distant relationship inference. However, with an increasing genotyping error (1-5%), methods that do not rely on IBD segment detection are more robust and outperform IBD segment methods. The reduced call rate had little impact on either class of methods. These results have implications for the use of dense SNP data in forensic genomics for distant kinship analysis and FGG, especially when the sample quality is low.

Forensic and genetic landscape explorations of Chinese Kyrgyz group based on autosomal SNPs, Y-chromosomal SNPs and STRs.

To assess the effect of population genetic polymorphism on forensic research, we investigated the genetic polymorphisms of Chinese Kyrgyz group (n = 98) and evaluated forensic application values in Chinese Kyrgyz group and other 26 reference populations at 90 autosomal SNPs, and then combined with 34 SNPs and 37 STRs on Y chromosome to reveal the genetic background of Kyrgyz group in autosomal and Y-chromosomal inheritances, respectively. The 90 autosomal SNPs and 34 Y-chromosomal SNPs were sequenced base on next generation sequencing technology, and 37 Y-chromosomal STRs were analyzed by capillary electrophoresis platform. The results showed that cumulative power of discrimination and cumulative power of exclusion of 90 autosomal SNPs in the panel met the application need of forensic genetics in Kyrgyz group. The forensic effectivenesses of the panel were high in all 27 populations, although there were genetic differences among these populations. The forensic effectiveness of the panel was relatively higher in the European populations, but relatively lower in the African populations. The population genetic results indicated that the Kyrgyz group had the relatively closer genetic relationships with the reference East Asian populations at autosomal SNPs, and there were gene exchanges between the Kyrgyz group and East Asian, European populations based on the analytical results of autosomal SNPs, Y-chromosomal SNPs and STRs.

Bridging Disciplines to Form a New One: The Emergence of Forensic Genetic Genealogy.

Forensic Genetic Genealogy (FGG) has fast become a popular tool in criminal investigations since it first emerged in 2018. FGG is a novel investigatory tool that has been applied to hundreds of unresolved cold cases in the United States to generate investigative leads and identify unknown individuals. Consumer DNA testing and the public's increased curiosity about their own DNA and genetic ancestry, have greatly contributed to the availability of human genetic data. Genetic genealogy has been a field of study/interest for many years as both amateur and professional genetic genealogists use consumer DNA data to explore genetic connections in family trees. FGG encompasses this knowledge by applying advanced sequencing technologies to forensic DNA evidence samples and by performing genetic genealogy methods and genealogical research, to produce possible identities of unknown perpetrators of violent crimes and unidentified human remains. This combination of forensic genetics, genetic genealogy, and genealogical research has formed a new subdiscipline within the forensic sciences. This paper will summarize the individual disciplines that led to the emergence of FGG, its practice in forensic investigations, and current/future considerations for its use.

Analysis of the genealogy process in forensic genetic genealogy.

The genealogy process is typically the most time-consuming part of-and a limiting factor in the success of-forensic genetic genealogy, which is a new approach to solving violent crimes and identifying human remains. We formulate a stochastic dynamic program that-given the list of matches and their genetic distances to the unknown target-chooses the best decision at each point in time: which match to investigate (i.e., find its ancestors and look for most recent common ancestors between the match and the target), which set of potential most recent common ancestors to descend from (i.e., find its descendants, with the goal of identifying a marriage between the maternal and paternal sides of the target's family tree), or whether to terminate the investigation. The objective is to maximize the probability of finding the target minus a cost associated with the expected size of the final family tree. We estimate the parameters of our model using data from 17 cases (eight solved, nine unsolved) from the DNA Doe Project. We assess the Proposed Strategy using simulated versions of the 17 DNA Doe Project cases, and compare it to a Benchmark Strategy that ranks matches by their genetic distance to the target and only descends from known common ancestors between a pair of matches. The Proposed Strategy solves cases ≈10 - fold faster than the Benchmark Strategy, and does so by aggressively descending from a set of potential most recent common ancestors between the target and a match even when this set has a low probability of containing the correct most recent common ancestor. Our analysis provides a mathematical foundation for improving the genealogy process in forensic genetic genealogy.

Identification of a decedent in a 103-year-old homicide case using forensic anthropology and genetic genealogy.

Anthropologists are often the custodians of long-term unidentified human remains though their positions as curators of university or museum skeletal collections. Various factors decrease the solvability of these legacy cases including the passage of time, the loss of provenience for specific cases, and lack of documentation or case records. While anthropologists can contribute important information toward identification, it is often necessary to explore novel and cross-disciplinary strategies to resolve difficult cold cases. In long cold cases, the postmortem interval, in particular, may be difficult to estimate leading to further challenges in achieving identification. Modern advances in radiocarbon bomb pulse dating, isotope analysis, and actualistic studies have contributed to positive identification of unidentified human remains in some legacy cases, but may not be available to all forensic practitioners and law enforcement from resource-poor agencies. Pooling resources, as well as collaborating with professionals outside of forensic anthropology, is a useful strategy to pursue when anthropological methods are exhausted.The case study presented here demonstrates a collaborative approach between forensic anthropologists, forensic genetic genealogists, and law enforcement in a century-old homicide. The dismembered and mummified parts of a male body were recovered in a remote cave in 1979 and again in 1991. Despite forensic anthropologists creating and updating the biological profile over the decades from recovery to present, no identification was made until the application of forensic genetic genealogy (FGG) to the case in 2019. New interpretations of bone microstructure and trauma analysis are presented for the case, alongside the historical documentation and "proof of life" evidence used by the genealogy team. A review of the FGG methods underscores the challenges in this case (e.g. significant endogamy, multiple aliases used by the victim) and the steps taken toward resolution. Ultimately, a combined anthropology and genealogy approach resulted in a confirmed identity for a man who was murdered in 1916.Key pointsForensic scientists should leverage a collaborative, interdisciplinary approach toward human identification.When combined with forensic anthropology methods, forensic genetic genealogy is a valuable tool linking biological and cultural-historical aspects of identity.Forensic anthropologists should review challenging cases in their labs as new methods are introduced and new resources become available.

Fast and accurate kinship estimation using sparse SNPs in relatively large database searches.

Forensic genetic genealogy (FGG) has primarily relied upon dense single nucleotide polymorphism (SNP) profiles from forensic samples or unidentified human remains queried against online genealogy database(s) of known profiles generated with SNP microarrays or from whole genome sequencing (WGS). In these queries, SNPs are compared to database samples by locating contiguous stretches of shared SNP alleles that allow for detection of genomic segments that are identical by descent (IBD) among biological relatives (kinship). This segment-based approach, while robust for detecting distant relationships, generally requires DNA quantity and/or quality that are sometimes not available in forensic casework samples. By focusing on SNPs with maximal discriminatory power and using an algorithm designed for a sparser SNP set than those from microarray typing, performance similar to segment matching was reached even in difficult casework samples. This algorithm locates shared segments using kinship coefficients in "windows" across the genome. The windowed kinship algorithm is a modification of the PC-AiR and PC-Relate tools for genetic relatedness inference, referred to here as the "whole genome kinship" approach, that control for the presence of unknown or unspecified population substructure. Simulated and empirical data in this study, using DNA profiles comprised of 10,230 SNPs (10K multiplex) targeted by the ForenSeq™ Kintelligence Kit demonstrate that the windowed kinship approach performs comparably to segment matching for identifying first, second and third degree relationships, reasonably well for fourth degree relationships, and with fewer false kinship associations. Selection criteria for the 10K SNP PCR-based multiplex and functionality of the windowed kinship algorithm are described.