Search Results (91)

Background/Objectives: Interpretation of mixture profiles generated from crime scene samples is an important element in forensic genetics. Here, a workflow for mixture deconvolution of sequenced microhaplotypes (MHs) and STRs using the probabilistic genotyping software MPSproto v0.9.7 was developed, and the performance of the two types of loci was compared. Methods: Sequencing data from a custom panel of 74 MHs (the MH-74 plex) and a commercial kit with 26 autosomal STRs (the ForenSeq™ DNA Signature Prep Kit) were used. Single-source profiles were computationally combined to create 360 two-person and 336 three-person mixtures using the Python script MixtureSimulator v1.0. Additionally, 72 real mixtures typed with the MH-74 plex and 18 real mixtures typed with the ForenSeq Kit from a previous study were deconvoluted using MPSproto. Results: The deconvoluted MH profiles were more complete and had fewer wrong genotype calls than the deconvoluted STR profiles. The contributor proportion estimates were more accurate for MH profiles than for STR profiles. Wrong genotype calls were mostly caused by locus and heterozygous imbalances, noise reads, or an inaccurate contributor proportion estimation. The latter was especially problematic in STR sequencing data, when two contributors contributed equally to the mixture. A total of 34,800 deconvolutions of the simulated mixtures were performed with two defined hypotheses: H_p, “The sample consists of DNA from one/two unknown contributor(s) and the suspect” and H_d, “The sample consists of DNA from two/three unknown individuals”. All true contributors were identified (LR > 10¹⁵ for MHs and LR > 10⁹ for STRs) and all non-contributors excluded (LR < 10⁻⁶ for MHs and LR < 0.2 for STRs). Conclusions: In simulated and real mixtures, the MHs performed better than STRs. Full article

Background/Objectives: Obtaining reliable DNA profiles from archival tissue preserved as formalin-fixed, paraffin-embedded (FFPE) samples remains a major challenge in both forensic and medical evaluations. The quality of DNA isolated from FFPE material is frequently compromised due to formalin-induced fragmentation and chemical modifications. These limitations are particularly relevant in cases of suspected medical malpractice related to cancer diagnosis or treatment, where retrospective molecular analyses may provide critical evidence. The aim of this study was to evaluate the performance of the Maxwell^® RSC Xcelerate DNA FFPE Kit (Promega) in generating DNA profiles from archival FFPE tissue blocks of endometrial cancer and to identify the limitations associated with this approach. Methods: Archival FFPE blocks of endometrial cancer were analyzed using the Maxwell^® RSC Xcelerate DNA FFPE Kit. DNA yield, purity, and degradation indices were assessed using standard real-time PCR-based quantification methods. Short tandem repeat (STR) profiling was performed with forensic genotyping kits, and the completeness, allele balance, and reliability of obtained profiles were evaluated. The obtained results were compared with reference quality thresholds commonly used in forensic practice. Results: The Maxwell^® RSC Xcelerate Kit allowed for recovery of relatively high DNA yields with consistently low degradation indices, confirming good extraction efficiency from FFPE samples. Nevertheless, despite favorable quantitative values, the generation of complete STR profiles was often unsuccessful. Partial or incomplete profiles were frequent, characterized by allele dropout and imbalance, which substantially reduced their evidentiary value. These findings suggest that DNA fragmentation and fixation-related artifacts impair amplification efficiency and limit the usefulness of STR analysis. Conclusions: This study emphasizes the persistent challenges of DNA profiling from FFPE tissue in forensic-medical contexts. Although the Maxwell^® RSC Xcelerate Kit demonstrated effective DNA recovery, the ability to generate complete and interpretable STR profiles remained limited. Further refinement of extraction protocols, as well as improved interpretative strategies, are required to enhance the reliability and evidentiary significance of molecular analyses based on archival FFPE material. Full article

The acid phosphatase (AP) test is widely utilised in forensic biology laboratories to examine for the presence of semen on crime evidence. If semen is present, the AP-positive areas are marked on the exhibit to indicate the precise location of the semen stain. However, documenting AP-positive areas with a crayon is time-consuming and laborious. In this proof-of-concept study, we evaluated the use of Saral Wax-Free Transfer Taper (TP) as an alternative tool for tracing the boundaries of AP-positive areas. We demonstrated that the TP pigment did not inhibit PCR amplification, as indicated by consistent internal PCR control (IPC) C_T values during real-time DNA quantification. While a reduction in DNA yield was observed under stress-test conditions, where TP pigment was intentionally included in the samples, complete STR profiles were still recovered with no allele dropout. Importantly, the documenting time for AP mapping was reduced by approximately five-fold with TP compared to crayon, underscoring its potential to enhance efficiency in forensic laboratory workflows. Full article

Background: Probabilistic genotyping software has become an essential tool in forensic genetics, particularly for interpreting complex DNA mixtures. Previous studies measured the impact of considering widely divergent statistical approaches in quantifying evidence, both inter- and intra-software. At a much smaller scale, this data-driven study shows how different models implemented on distinct versions of the same tool may affect the results. Among the available tools, EuroForMix stands out as a quantitative, open-source software that models various aspects of the DNA profile, including artefacts like stutter peaks. Its freeware nature allowed the use of both versions 1.9.3. and 3.4.0, between which several updates were made, including the possibility to model both back and forward stutter, compared to only modeling back stutters inputted by the expert in the earlier version. Methods: A total of 156 real casework sample pairs (comprising mixtures with two or three estimated contributors and associated reference) from the Portuguese Scientific Police Laboratory were analyzed using both software versions. The same input data, containing alleles and artefactual peaks, were used to reflect operational conditions. Statistical measurements were compared and further investigated. Results: Most Likelihood Ratio values differed in less than one order of magnitude across versions. However, exceptions were found in more complex samples, such as those with more contributors, unbalanced contributions, or greater degradation. Conclusions: This work emphasizes the relevance of model selection in forensic evidence quantification, even when considering different versions of the same tool. The impact of different models in statistical evaluation depends on several factors, such as sample technical conditions, genotypic profiles, and population distribution. Full article

Background/Objectives: Advancements in DNA profiling have made it possible to retrieve intact DNA profiles from increasingly minute biological samples. This increased sensitivity in DNA detection has highlighted crucial considerations to be made when handling and packing items from the crime scene to minimize potential contamination from either direct or indirect transfer of DNA. To investigate potential DNA transfer between items stored within the same evidence package, we conducted a simulation study with items commonly encountered during forensic casework. Methods: Participants were grouped in pairs, each of them handling the same type of item to simulate the activity conducted at the crime scene. The items were then collected from each pair of participants and stored in the same evidence package for 4 to 5 days. To evaluate the basal DNA transfer between items within the same package, the packed items were not subjected to friction, force, or long-distance movement in this study. Results: We have observed the occurrence of DNA transfer on 39% of the studied items inside the package, which changed the source attribution of the DNA profiles for 10% of the recovered samples. Our results showed that the types of items were associated with the number of transferred alleles and the amount of DNA recovered, while no association was found between the number of transferred alleles and the amount of DNA on the studied items. Conclusions: Taken together, the results from this study reiterate the importance of packing each item from the crime scene separately, especially when packing items together may impact the interpretation of source attribution. Full article

This review presents an in-depth analysis of the synergistic role of molecular biology in advancing forensic entomology. The study discusses how insects associated with decomposing bodies provide critical data for estimating the post-mortem interval (PMI), and how molecular techniques improve species identification and trace analysis. The manuscript examines DNA-based methods such as RAPD, RFLP, and mitochondrial sequencing, along with innovative applications like gene expression profiling and entomotoxicology analysis. Additionally, it presents real case studies illustrating how molecular data from insects can be used not only to estimate PMI but also to identify victims or suspects through human DNA retrieved from insect tissues. These advances confirm the fundamental role of molecular biology in strengthening the reliability and applicability of forensic entomology in legal contexts. Full article

Background: The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. For example, the genetic casework unit of the forensic institute of the French gendarmerie analyzes more than 70,000 casework samples per year mainly from swabs that are fully consumed during DNA extraction. The only way to process further analyses is to preserve DNA. Currently, the most common technique used for the long-term preservation of DNA is to freeze the extracted DNA at −20 °C or −80 °C. However, this preservation method involves significant constraints (large equipment), risks (equipment failure), and is not ecologically sustainable due to its high energy consumption. Many solutions for DNA preservation at room temperature exist based either on fibrous supports or on anhydrobiosis. However, few studies have examined the efficiency of these systems in preserving very-low DNA amounts, such as those in forensic samples (≤1 ng), while ensuring full recovery and the ability to retest the samples many years later. Methods: We choose to evaluate the ability of the anhydrobiosis technology from GenTegra^® LLC to preserve DNA extracts from one month to one accelerated year from different DNA quantities (from 1 ng to 0.2 ng) and sources (NIST, mocked samples, and true casework mixtures). We studied the quantity, integrity of DNA, and also the quality of the STR genetic profiles obtained. Results and Conclusions: Our results prove the high potential of this technology to preserve and to allow an effective recovery of the DNA extracts for forensic purposes. Full article

Background/Objectives: DNA methylation profiles have emerged as robust biomarkers of ageing, leading to the development of “epigenetic clocks” that estimate biological age. Most established clocks (e.g., Horvath’s 353-CpG pan-tissue clock and Hannum’s 71-CpG blood clock) require dozens to hundreds of CpG sites. This study presents a novel saliva-specific epigenetic clock built on 10 sites identified from Illumina MethylationEPIC (850 k) array data. Methods: Saliva DNA methylation was analysed from 3408 individuals (age range 15–89 years, 68% male, 32% female, no diagnosed disease) from the Muhdo Health Ltd. dataset (2022–2024), and 10 CpG sites were selected where methylation levels showed the strongest positive correlations with chronological age (Pearson r = 0.48–0.66, p < 1 × 10−20). These CpGs map to genes involved in developmental and metabolic pathways (including ELOVL2, CHGA, OTUD7A, PRLHR, ZYG11A, and GPR158). A linear combination of the 10 methylation sites was used to calculate a “DNA methylation age”. Results: The 10-CpG clock’s predictions were highly correlated with chronological age (r = 0.80, R2 = 0.64), with a mean absolute error of ~5.5 years. Its performance, while slightly less precise than Horvath’s or Hannum’s multi-CpG clocks, is notable given the minimal marker set. It was observed that all 10 clock CpGs undergo age-related hypermethylation. The biological significance of these loci is discussed, along with the potential health and forensic applications of a saliva-based epigenetic age predictor. Conclusions: This study demonstrates that a saliva-specific epigenetic clock using only 10 CpG sites can capture a substantial portion of age-related DNA methylation changes, providing a cost-effective tool for age estimation. Full article

Forensic genetics has experienced remarkable advancements over the past decades, evolving from the analysis of a limited number of DNA segments to comprehensive genome-wide investigations. This progression has significantly improved the ability to establish genetic profiles under diverse conditions and scenarios. Beyond individual identification, forensic genetics now enables the inference of physical traits (e.g., eye, hair, and skin color, as well as body composition), biogeographic ancestry, lifestyle habits such as alcohol and tobacco use, and even the transfer of genital microbiomes post-coitus, among other characteristics. Emerging trends point to a future shaped by the integration of cutting-edge technologies, including CRISPR-Cas systems, artificial intelligence, and machine learning, which promise to further revolutionize the field. This review provides a thorough exploration of forensic genetics, tracing its evolution from its foundational methods (past) to its diverse modern applications (present) and offering insights into its potential future directions. Full article

Background/Objectives: Identification of human remains is of utmost importance for criminal investigations and providing closure to the families. The reconstruction of a biological profile of the individual will narrow down the list of candidates for identification. From another perspective, facial approximations performed by a forensic artist can provide investigative leads, with the identity being confirmed by primary or secondary methods of identification. In recent years, DNA analysis has evolved, trying to create a portrait of the perpetrator/victim based on External Visible Characteristics (EVCs), the color of the eyes, hair, and skin and Biogeographical ancestry (BGA), called DNA phenotyping. Despite these advances, currently, there are no studies integrating the biological profile performed by forensic anthropologists, the facial approximation created by forensic artists and EVCs determined by DNA. The goal of this work was to integrate these three investigative leads to enhance the possibility of human identification. Methods: Five donated remains from Mercyhurst were studied through these approaches: reconstruction of biological profile, facial approximation and estimation of EVCs based on previous studies. Results: Our results indicated the feasibility of integrating this biological profile and EVCs data into the facial approximation developed by the forensic artist, aiming to an enhance portrait of the remains. In a second phase of this project, the accuracy of the integrated facial approximation will be assessed. Conclusions: This study pointed out the importance of an interdisciplinary approach towards the identification of human remains, as well as the combination of current methods with new technologies. Full article

Background/Objectives: The Y chromosome is a crucial tool in forensic genetics due to its unique characteristics, such as its haploid inheritance and lack of recombination. Y-STRs (short tandem repeats on the Y chromosome) are widely used for identifying male genetic profiles in DNA mixtures, especially in sexual assault cases where high levels of female DNA hinder autosomal analysis. This study evaluates the applicability of Y-STRs in forensic investigations, addressing their limitations and the impact of advanced technologies, such as rapidly mutating Y-STRs (RM Y-STRs). Methods: A comprehensive literature review was conducted to analyze existing knowledge on the application of Y-STRs in sexual crimes. The study also examines the role of population databases, such as YHRD, in estimating haplotype frequencies and enhancing forensic reliability. Results: Y-STR analysis proves essential for male DNA identification in complex mixtures, with RM Y-STRs enhancing discriminatory power. However, limitations persist, particularly in cases involving closely related male lineages. The population database coverage remains insufficient in regions like Cape Verde, affecting forensic reliability. Case studies demonstrate Y-STR effectiveness in solving cold cases and sexual crimes, reinforcing the need for expanded databases and methodological advancements. Conclusions: Y-STRs play a fundamental role in forensic genetics, particularly in sexual assault investigations. Their integration with advanced sequencing technologies and expanded databases is critical for improving forensic accuracy. Ethical considerations regarding genetic data privacy and potential discrimination must be addressed through clear regulations and forensic best practices. Full article

Forensic DNA phenotyping (FDP) has emerged as an essential tool in criminal investigations, enabling the prediction of physical traits based on genetic information. This review explores the genetic factors influencing skin pigmentation, particularly within Asian populations, with a focus on Thailand. Key genes such as Oculocutaneous Albinism II (OCA2), Dopachrome Tautomerase (DCT), KIT Ligand (KITLG), and Solute Carrier Family 24 Member 2 (SLC24A2) are examined for their roles in melanin production and variations that lead to different skin tones. The OCA2 gene is highlighted for its role in transporting ions that help stabilize melanosomes, while specific variants in the DCT gene, including single nucleotide polymorphisms (SNPs) rs2031526 and rs3782974, are discussed for their potential effects on pigmentation in Asian groups. The KITLG gene, crucial for developing melanocytes, includes the SNP rs642742, which is linked to lighter skin in East Asians. Additionally, recent findings on the SLC24A2 gene are presented, emphasizing its connection to pigmentation through calcium regulation in melanin production. Finally, the review addresses the ethical considerations of using FDP in Thailand, where advances in genetic profiling raise concerns about privacy, consent, and discrimination. Establishing clear guidelines is vital to balancing the benefits of forensic DNA applications with the protection of individual rights. Full article

Molecular diversity, which reflects variation in species abundance and genetic structure, plays a pivotal role in forensic entomology by enabling the accurate identification of insect evidence through tools such as DNA barcoding. In Pakistan, the absence of trained forensic entomologists and limited research on insect biodiversity hinder the effective use of entomological evidence in criminal investigations. Traditional morphological identification methods are insufficient for resolving complex forensic cases, particularly when dealing with immature insect stages. This highlights the urgent need for molecular approaches, such as DNA barcoding, to enhance species identification and genetic analysis of forensically relevant insects. This study uniquely focuses on evaluating the utility of a 658 bp fragment of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene for identifying dipteran species collected from cadavers in Lahore, Pakistan. The primary goal was to identify forensically relevant insect species, assess their genetic diversity and population structure, and compare these findings with global data to contextualize the results within forensic entomology. Three blow fly species were identified: Chrysomya megacephala (Fabricius, 1794), Chrysomya saffranea (Bigot, 1877), and Chrysomya rufifacies (Macquart, 1843). Low genetic diversity was observed within populations, while significant genetic differentiation among populations was indicated by a high fixation index (FST = 0.83992). These findings suggest unique genetic signatures for blow fly populations in Lahore. This study underscores the importance of molecular tools like DNA barcoding for species identification and highlights the need for further research to establish a comprehensive database of forensically relevant insects in Pakistan, given the limited species diversity and unique genetic profiles observed. By laying the groundwork for future research, this study contributes to advancing forensic entomology in Pakistan by improving species identification, which, when combined with future thermobiological data, can enhance postmortem interval (PMI) estimation and forensic investigations. Full article

Background/Objectives: On 22 February 2021, a coastal landslide in Italy caused the collapse of an old cemetery, displacing approximately 370 coffins, with over 200 plunging into the sea. This disaster necessitated the recovery and identification of human remains under challenging conditions to provide closure to families and uphold the dignity of the deceased. Methods: Recovery operations involved firefighters and scuba divers, followed by forensic analysis conducted by the Medical Staff of Legal and Forensic Medicine. A post-mortem team utilized forms adapted from Interpol’s Disaster Victim Identification (DVI) standards to document remains, which included 140 decomposed bodies and 193 bags of commingled skeletal remains. DNA samples were collected from 147 bone fragments, primarily long bones and teeth, and compared with ante-mortem data gathered from relatives. Results: Of the 77 eligible relatives, 66 consented to DNA sample collection for genetic profiling, and 28 bodies were identified. Personal effects, clothing, medical devices, and a strong match between non-genetic AM and PM data led to an attribution of identity of other 19 individuals. Advanced post-mortem phenomena were observed in remains spanning from the late 19th century to 2017. An identification area at the cemetery facilitated streamlined operations, emphasizing environmental preservation and forensic accuracy. Conclusions: The cemetery collapse highlights the necessity for tailored forensic approaches in disaster scenarios. Accurate identification methods, combining genetic analysis and secondary means, are crucial for ensuring dignified burials and providing closure to affected families. Full article

Background/Objectives: The study of DNA transfer and persistence has become increasingly significant, driven by advancements in DNA detection sensitivity and the need for reliable forensic evidence. In forensic investigations, saliva and saliva-stained materials are recognised as valuable DNA sources, particularly in cases of homicide, sexual assault, and burglary, where saliva can be transferred between individuals during the criminal act. The time between the crime and sample collection is a critical factor that can influence the success of the analysis. The value of the specimens collected from the victim’s skin or mouth (perilabial and labial sites, teeth and tongue) after the crime has not been investigated with currently used highly sensitive and specific molecular methods. Methods: On the assumption that a significant loss of DNA occurred, in our study, 10 voluntary pairs were tested at different time points after intense kissing and samples were taken from the above-mentioned sites to assess the presence of the donor’s DNA. Extracted DNA was quantified using the Plexor HY System kit (Promega), and both autosomal STRs and Y-STRs were analysed. Results: The results reveal a greater persistence of male DNA on the female partner, particularly in the labial and perilabial regions, even up to 120 min after contact, in terms of both concentration and duration. Conclusions: This study emphasises the forensic importance of salivary DNA as a solid source of evidence, particularly in investigations involving mixed DNA profiles. Full article