Estimating wound age can be crucial in forensic work for establishing a causal link between the injury and death, or for better characterizing the timing and circumstances of the injury. Conventional methods for estimating wound age are not always reliable and often fail to achieve sufficient accuracy. Animal studies have shown that RNA-based methods for wound age estimation can produce reliable and reproducible results. However, the transferability of wound markers identified in animal experiments to human samples is questionable, and a model based on human tissue needs to be developed. Twenty-three paired samples including the wounded tissue and the corresponding uninjured skin tissue were collected during routine autopsies with sharp injuries inflicted between a few seconds and 28 days prior to death. The samples underwent targeted transcriptome sequencing (targeting > 20.000 human RefSeq transcripts) and were analyzed for differentially expressed genes. The samples were grouped into three categories based on wound age: acute (< 1 day), intermediate (1-3 days) and prolonged (> 3 days). A functional analysis of the differentially expressed genes revealed various biological functions that can be attributed to the individual stages of wound healing. A total of 31 potential marker candidates were identified, 21 of which have not yet been described in the context of a temporospatial association with wound healing. Results demonstrate that the analysis of human material is essential for the development of a human-specific forensic predictive model. The candidate markers identified in this study represent a first step towards an RNA-based method for estimating wound age in humans.
This research is based on a case in which the complainant alleged that the defendant had removed her underwear, while the defendant claimed they had only danced together. To assist in resolving this issue, DNA was collected from the sides of the complainant's underwear and Y-chromosome STR (Y-STR) analysis performed using the PowerPlex® Y23 System. As no published research were available on DNA recovered from the sides of underwear given the two alleged activities, 60 experiments were conducted to generate specific data on the expected results given underwear removal and given dancing. The underwear removal was simulated using a dummy, while the dancing experiments involved real participants. The paper discusses a principled approach to representing prior knowledge and combining it with the experimental observations when assigning probabilities to DNA recovery outcomes under the competing activity level propositions. To explore whether our LRs were calibrated, we applied our model to published ground truth studies. For the presence of a full Y-STR profile PP23, we show that our LRs in the order of 50 are robust. The prevalence of DNA from cohabitants was also studied. In our experiments, taking the cohabitant's Y-STR profile into account did not impact the value of the observations given activity level propositions. However, having access to the cohabitant reference sample increases the number of interpretable DNA mixtures and can provide relevant information. We therefore recommend requesting the cohabitant DNA reference samples, particularly in cases involving balanced Y-STR mixtures.
Environmental DNA (eDNA) analysis enables non-invasive detection of aquatic species, but no established framework integrates eDNA best practices with forensic validation standards. The critically endangered freshwater pearl mussel (Margaritifera margaritifera) exemplifies the need for forensically validated eDNA assays as traditional survey methods are invasive and time-consuming. This research provides a framework that bridges both ecological monitoring standards and forensic legal admissibility requirements in wildlife crime investigations. Here we describe the validation of a multiplex quantitative PCR (qPCR) assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, using the highest forensic standards and eDNA best practice.In silico and in vitro testing confirmed species specificity, with alignment against reference sequences showing 100% identity. The multiplex assay LOD and LOQ were calculated at 2.5 and 3.14 copies/µL respectively, determined by the performance of the short fragment. The assay demonstrated robust repeatability (R², 0.986–1.000), precision (coefficient of variation, 6–28%), and accuracy (recovery, 62–137%), meeting forensic thresholds. Field validation detected a previously undetected Welsh population through intelligence-led sampling, while blind proficiency testing in the Lake District achieved 95.8–100% detection rates across juvenile release sites and successfully detected distribution patterns later confirmed by stakeholders.We establish the first forensic eDNA validation of a protected species, providing a transferable methodology for conservation, regulatory enforcement, and wildlife crime investigations, ready for deployment in an operational environment. We believe that this integrated approach can be transferred to similar forensic and conservation scenarios across a broad range of species and geographical contexts.
Purtier Placenta Live Stem Cell Therapy is a food supplement by Riway Singapore Pte. Ltd., claimed to contain living deer placenta stem cells together with 11 other active ingredients. It is also promoted as maintaining health and youthfulness and as being supported by numerous patents and research trials. Individual ingredients are accompanied by many other statements implying that Purtier Placenta can alleviate or cure diseases such as diabetes, cancer, and neurodegenerative diseases. However, none of these claims is traceable in clinical trial registries, and the mechanism allowing stem cells to be alive in the capsule and be effectively delivered to the body via the digestive tract does not seem plausible. Consequently, authorities in the United Arab Emirates, the Philippines, and Singapore have issued public warnings or imposed penalties in relation to misleading claims. Nevertheless, the product has gained popularity among Czech oncological patients, who perceive it as a magic cure. This paper describes a three-stage testing process designed to verify the species origin of the declared main ingredient. Using barcoding primer mixes, melting analysis, and Sanger sequencing with species-specific primers against seven reference species, we tested whether the capsule content corresponded to the red deer (Cervus elaphus) depicted on the official website (https://www.officialpurtier.com). Our analysis identified that DNA extracted from the 6th Edition capsule was primarily Rusa unicolor/timorensis, with minor components of Dama dama and Cervus elaphus. Although the main component was confirmed to be of deer origin, the lack of specific information and the wording of the marketing materials may be misleading.
Molecular autopsy can identify candidate variants in unexplained sudden cardiac death (SUD), but functional evidence is often required to support pathogenic interpretation and family risk assessment. Here, we investigated the functional consequences of the SCN5A c.287 T > C (NaV1.5_p.Leu96Pro) variant, identified in a 29-year-old man who died of SUD. Cascade screening identified additional relatives carrying the variant, with variable clinical expression. Sodium current (INa) was analyzed in heterologously transfected human embryonic kidney (HEK) tsA201 cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from two variant carriers and two non-carrier relatives. SCN5A transcript levels, NaV1.5 membrane expression, and additional arrhythmia-associated genetic variation were also assessed. In HEK tsA201 cells, NaV1.5_p.Leu96Pro produced no measurable INa when expressed alone, whereas co-expression with wild-type SCN5A caused an approximately 50% reduction in peak INa density. Cell-surface biotinylation showed preserved total and membrane NaV1.5 expression, indicating that loss of current was not explained by impaired trafficking. hiPSC-CMs from both carriers showed reduced INa density compared with non-carrier relatives, despite no reduction in SCN5A transcript levels. Targeted sequencing did not identify variants explaining the differences between the two hiPSC-CM carriers, but revealed an additional SCN5A splice-site deletion in family members with more severe clinical manifestations. These findings show that NaV1.5_p.Leu96Pro causes severe loss of sodium channel function and support the value of combining molecular autopsy, family evaluation, and functional studies for variant interpretation in SUD.
This EDNAP (European DNA Profiling Group) collaborative study evaluated the detection and reporting of mitochondrial DNA (mtDNA) point heteroplasmy (PHP) and length heteroplasmy (LHP) across forensic laboratories using Sanger, Ion Torrent, and Illumina sequencing platforms. Standardized DNA extracts were centrally prepared and distributed to participating laboratories to assess inter-laboratory concordance and platform-specific (platform = instrument + software) effects. Raw sequence data showed high overall consistency across platforms, and observed mitotype discrepancies were due to transcription errors. PHP detection was generally concordant across laboratories and technologies. MPS data provided relatively stable mixture ratios across platforms. Low-level PHPs were often not reported in this study when they fell below laboratory-specific minor allele frequency thresholds, although raw data review confirmed their presence. In contrast, LHP interpretation showed substantially greater variation. Sanger-derived LHP patterns could be harmonized through review under current ISFG guidelines, whereas MPS-based LHP results differed both between and within sequencing technologies. Standardization of the alignment workflow improved concordance to some extent, but important discrepancies remained, particularly in Ion Torrent HVS-I (Hypervariable Segment I) poly(C)-tract data. These differences were attributable to sequencing technology, analysis software, and human interpretation.
Forensic anthropological investigations frequently involve highly degraded and skeletonized human remains in which optimal skeletal elements may be absent, compromised, or only partially preserved. In such contexts, the integration of molecular analysis within forensic anthropology becomes essential to support biological profiling and identification. Non-osseous tissues—such as hair, nails, ossified cartilage, and atherosclerotic calcifications—are often recovered alongside skeletal elements, yet their genome-wide preservation patterns remain poorly characterized. In this exploratory study, we assessed DNA preservation and sequencing performance across multiple non-skeletal tissues using low-coverage shotgun sequencing, directly comparing them with skeletal reference samples from the same individuals. Endogenous DNA content, fragment length distribution, molecular damage patterns, mitochondrial genome reconstruction, and nuclear marker recovery were evaluated. Despite low endogenous DNA proportions and extensive fragmentation, mitochondrial haplotypes and sex-informative markers were successfully retrieved from several non-osseous substrates, with concordance observed relative to skeletal references. Genome-wide nuclear SNP concordance exceeded 99.6% across all comparisons, supporting the persistence of coherent nuclear DNA signals in non-skeletal substrates despite extensive degradation. Although non-skeletal tissues cannot replace optimal skeletal sampling, these findings highlight their potential as complementary substrates within a forensic molecular anthropology framework, particularly in scenarios involving advanced decomposition, partial remains, or restricted access to preferred skeletal elements.
DNA extracted from touched surfaces is an important analyte in forensic casework, often leading to the identification of suspects. A common assumption underlying this approach is that touch DNA originates in skin cells of the touching individual. Here we test this assumption, using cell type-specific DNA methylation markers to determine the origins of touch DNA. Strikingly, hand swabs and touched objects contain only traces of skin DNA. The majority of hand swab and touch DNA originates in leukocytes (mostly neutrophils) and to a lesser extent in buccal cells, paralleling the composition of nasal and salivary DNA. Hand swab DNA from females that received bone marrow transplantation from a male donor are predominantly of a male origin, consistent with blood rather than skin origin. Finally, the amount of DNA extracted from hand swabs does not increase after prolonged glove use, further arguing against internal shedding. We conclude that touch DNA is transferred mostly from the nose or mouth, not from the skin of the touching hand. The leukocytic origins of touch DNA have implications for understanding DNA transfer and for the interpretation of forensic findings.
The identification of lineage-defining variants, frequently found in the coding region of mitochondrial DNA (mtDNA), is essential for refining haplogroup classification. Most mtDNA studies in South American populations have focused on the control region (CR), which has provided important insights into population structure and maternal lineage origins, although information needed for more robust phylogenetic resolution has been neglected. This study investigates the maternal genetic structure of Ecuadorian populations by combining CR and whole mitogenome analyses. Sequences from the mtDNA CR were obtained from 461 individuals (253 Mestizos and 208 Native Americans), while complete mitogenomes were sequenced for 127 individuals to improve phylogenetic resolution by identifying lineage-defining variants present in coding region. Most mtDNA haplogroups in the two population groups analyzed were of Native American origin (A2, B2, B4, C1, D1, D4), with significant differences in the distribution of specific lineages between them. Among Mestizos, African haplogroups (all within the L branches) and Eurasian haplogroups (H, K, R, U) were detected at low frequencies, whereas no African lineages were observed among Native Americans. The results obtained highlighted a heterogeneity within Ecuadorian populations that must be considered when developing mtDNA haplotype databases for forensic purposes. Whole mitogenome sequences enabled the identification of variants that refined haplogroup classifications, provided a more accurate reconstruction of the maternal genetic diversity, and improve the discrimination between Native American and Asian maternal lineages within haplogroup B4b.
Evaluations given activity level propositions require the expert to make a number of choices that relate to how the complexity of the world is simplified and modelled. Some of these choices relate to circumstances about the case, some to the data used and some to manner of modelling the data. In the right circumstances, the evaluation can be sensitive to any one of these categories of choice. In this paper an example is provided that demonstrates an evaluation sensitivity to the choice of how DNA transfer is modelled. Specifically, whether observations of no-DNA in a DNA transfer experiment should be considered as DNA transfer having not occurred, or having occurred at a level below the detection capability of the laboratory. An evaluation scenario is set up that demonstrates how this choice can lead to the difference between the evaluation slightly supporting one proposition to strongly supporting the other. A solution to this modelling choice is provided that shows how DNA transfer can be modelled as mixture of models, with non-DNA observations being simultaneously considered as no DNA transfer, or DNA transfer below detection capability. This is achieved using hierarchical Bayesian modelling, however could also be applied using other tools.
There is a broad consensus that forensic tests for the prediction of externally visible characteristics (EVC) and analysis of biogeographic ancestry (BGA) of an individual are technically reliable. However, interpretation of the results and population-specific genotype distribution patterns remains challenging. EVC and BGA analyses provide valuable information for population genetics studies and as investigative leads for criminal cases, as well as for historical and contemporary identification tests. However, inaccurate or incorrect predictions, for example, from subjective bias in the interpretations made, have the potential to misdirect police investigations. The legal situation regarding EVC and BGA testing varies by country: ranging from countries where it is explicitly prohibited, to those without specific regulations on biogeographic ancestry prediction, and others that have already enacted laws governing its use. The reluctance to utilize these analyses is not only due to legal restrictions and data protection concerns, but also to initial limited sets of sufficiently comprehensive forensic DNA assays. Forensic BGA marker panels typically contain up to ∼300 SNPs. This relatively small number of genetic markers, along with limited reference population data, complicates the interpretation of results from donors of unknown origin. This paper presents the results of a collaborative EDNAP study, which, for the first time, evaluated the approach to reporting EVC and BGA data between international laboratories. For the study, DNA from nine individuals with self-reported ancestry was collected and analysed using various forensic panels differing in the number and composition of ancestry-informative markers genotyped, comprising: the Precision ID mtDNA Whole Genome Panel, the VISAGE Basic Tool and the VISAGE Enhanced Tool for Appearance and Ancestry Prediction, and the Ion AmpliSeq™ PhenoTrivium Panel. To ensure full data protection, all SNP genotypes and uniparental marker haplotypes obtained were not shared with third parties. Instead, the genetic data were analysed using a range of commonly used population analysis software packages. These analysis outcomes were then distributed to twelve European forensic laboratories (both academic and law enforcement institutions), who were asked to prepare reports based on their interpretation of the phenotypes and ancestry they inferred from the analysis data. A questionnaire sent alongside the genetic information, aimed to evaluate which difficulties were encountered by the participants in processing the BGA analysis data they were given.
Unique molecular identifiers (UMI) can be used in forensic STR sequencing to reduce the level of analytical artifacts. Here, we perform an interlaboratory study across five independent sites where the previously developed UMI-based SiMSen-Seq STR assay is applied at each laboratory to both single-source and mixed samples. The assay showed consistent results between laboratories and more than 90% of the expected alleles were detected with 31 pg DNA of template. The assay tolerated ten times higher PCR inhibitor concentrations compared to an established commercial STR sequencing method. The combined results were used to determine stutter and noise thresholds, which were applied for allele calling, allowing an estimation of the sensitivity for minor contributor alleles in mixtures. We found that the SiMSen-Seq STR method is robust across laboratories and different types of PCR and sequencing equipment and that it allows for calling of alleles from contributors of smaller proportions compared to currently commercially available non-UMI STR sequencing methods.
Degraded samples are common yet challenging evidence types in forensic casework, and obtaining complete profiles from highly degraded samples has long been a major difficulty in forensic DNA analysis. However, there is a lack of effective methods for obtaining complete profiles from single-source highly degraded samples. Insertion/deletion (InDel) genetic markers, which combine the advantages of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are widely distributed throughout the human genome, exhibit low mutation rates, possess short amplicon sizes, and are compatible with capillary electrophoresis (CE) platforms. These features make InDels ideal genetic markers for the analysis of highly degraded forensic samples. To address this issue, we selected 44 InDel genetic markers with amplicon sizes of no more than 125 bp, together with an amelogenin locus, to establish a five-dye multiplex detection system for accurate genotyping of highly degraded DNA samples. Following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), the system was evaluated in terms of sensitivity, inhibitor tolerance, species specificity, and performance on highly degraded samples. Sensitivity analysis demonstrated that complete and reliable profiles could be obtained with as little as 62.5 pg of input DNA. In degradation studies, the 44-InDel panel generated complete profiles from DNA subjected to 60 min of boiling, demonstrating a clear advantage over STR-based methods in the analysis of highly degraded samples. Population analysis was conducted using 180 samples from the Southern Han Chinese population, yielding an average observed heterozygosity (Ho) of 0.4898 for all loci. The combined random match probability (CMP) and cumulative probability of exclusion (CPE) were calculated as 2.9522 × 10⁻¹ ⁹ and 0.99985, respectively. The results demonstrated that the 44-InDel panel is highly suitable for individual identification of highly degraded forensic samples and serves as a valuable complementary tool for paternity testing.
Probabilistic genotyping (PG) has become the standard framework for evaluating forensic DNA mixtures, yet most implementations were developed for STR data and PCR-based enrichment. Microhaplotypes (MHs) provide high allele diversity without stutter and are attractive for mixture deconvolution, particularly when combined with hybridization capture for degraded or limited DNA. Here, we developed a 100-locus hybridization capture MH panel and assessed its performance for mixture interpretation using two continuous PG models: (i) a MH-specific Truncated Gaussian (TG) model previously developed by our group for targeted amplification MH-MPS data, and (ii) the gamma-based model implemented in EuroForMix (EFM). Panel performance was first examined using sensitivity (0.5, 0.125, and 0.0625 ng; triplicates) and repeatability/consistency (10 individuals, 0.5 ng; duplicates) experiments. We then analyzed two- and three-person mixtures with increasing mixture imbalance (2-person: 1:1-1:40; 3-person: 1:1.5:3-1:4:20; triplicates). Across all mixtures, true contributors yielded likelihood ratios (LRs) > 1 under both models, with all tested non-contributors yielding LRs < 1. For two-person mixtures, major-contributor deconvolution accuracy approached 100%, whereas minor-contributor accuracy peaked at 83% (1:5) and declined with increasingly imbalanced mixtures. In three-person mixtures, minor-contributor accuracy depended strongly on relative proportions among contributors and showed non-monotonic trends. Overall, the TG model produced higher LRs and improved deconvolution for minor contributors compared with EFM, supporting transferability of the TG framework from targeted amplification to hybridization capture MH-MPS data. These results provide a preliminary practical foundation for probabilistic interpretation of hybridization capture microhaplotypes in forensic mixtures.
DNA methylation at CpG sites has emerged as a powerful epigenetic biomarker for predicting forensically relevant traits, including chronological age, the biological origin of forensic samples encompassing body fluid and tissue sources, and lifestyle-associated factors such as smoking. Existing models for age estimation, body fluid and tissue of origin identification, and smoking inference have demonstrated robust performance, but their reliance on separate assays limits practical application. To address this gap, we developed COSA (a Consolidated prediction panel for Origin, Smoking, and Age), an integrated methylation-based assay implemented through amplicon-based massively parallel sequencing (MPS). COSA consolidates 126 previously reported CpG markers from multiple validated models into 67 amplicons, thereby enabling the simultaneous prediction of body fluid and tissue of origin, smoking status, and chronological age from a single analysis. By unifying these established markers, COSA provides a scalable and streamlined solution for comprehensive forensic epigenetic profiling. The panel comprises three functional modules. First, body fluid identification incorporates 9 CpG markers specific to blood, semen, saliva, menstrual blood, and vaginal fluid, supporting accurate determination of sample origin in forensic framework. Additionally, body fluid and tissue of origin inference extends to internal organs through 18 CpGs targeting blood, liver, skeletal muscle, heart, brain, epidermis, dermis, kidney, and lung. Second, lifestyle inference is supported by 13 CpGs, including the well-characterized cg05575921 locus in the AHRR gene for smoking prediction. Third, age estimation is incorporated through three fluid-specific models optimized for blood, saliva, and semen, which are the fluids most frequently encountered in forensic investigations. Methodological refinements were essential to achieve balanced multiplex amplification. Multiplex PCR for bisulfite-converted DNA is challenged by issues related to primer compatibility and GC-content variation. To overcome this, we implemented a touchdown PCR strategy that improved amplification balance and coverage uniformity across multiple loci. Several primer sets were newly designed or modified to optimize amplicon length and annealing temperature, ensuring robust co-amplification within the 67-amplicon panel. Importantly, using as little as 20 ng of bisulfite-converted DNA, the COSA panel supported inference of biological origin, smoking status, and chronological age. Overall, COSA integrates three major forensic prediction modules, including origin classifiers for body fluids and organ tissues, fluid-specific age estimators, and a smoking-status predictor within a single DNA workflow. This panel represents a practical and scalable tool for forensic laboratories seeking to maximize information yield from limited DNA, advancing the application of epigenetics in human identification and investigative intelligence.
Sex-inference in forensic DNA analysis is a critical investigative parameter, commonly relying on the Amelogenin locus, along with the Y-Indel marker (rs2032678) and a limited set of Y-chromosomal markers included in autosomal STR multiplexes and quantitative PCR assays. However, structural rearrangements of the Y chromosome can result in discordant profiles that misidentify phenotypically male individuals as "false females" in standard autosomal kits. We report an exceptionally rare case from central India exhibiting three convergent genetic anomalies in a phenotypic male blood sample: the loss of primary forensic sex markers (AMELY/Y-Indel), X-chromosomal heterozygosity and an extensive Yq deletion. This "triple-failure" haplotype (AMELY-/Y-InDel-/Yq-) represents unprecedented complexity combining South Asian-pattern Yp11.2 microdeletion with massive Yq loss and X-chromosomal diploidy. Moreover, Quantification discordance occurs between Quantifiler Trio and PowerQuant confirmed selective Yq loss, while SRY positivity and NGS validation established male genetic sex. YHRD Release R69 analysis of the partial Yfiler Plus profile revealed no matches worldwide (n = 106,444; RMP <1/106,444, 95% CI up to 1/28,856) or in Eurasian-Indian (n = 1240; RMP <1/1240, 95% CI up to 1/337) and Indian national (n = 1238; RMP <1/1238, 95% CI up to 1/336) databases. The case exposes critical diagnostic limitations of single-target sex typing and Yq-biased quantification in structural variants, underscoring the need for redundant markers (SRY, Yp STRs, multi-Y qPCR targets). Integrated STR/NGS analysis with database frequency assessment remains essential for resolving such "ghost" Y-profiles in forensics.
Short tandem repeat (STR) profiling is foundational to forensic human identification, but stutter artifacts remain a persistent challenge, particularly for mixture interpretation where elevated stutter peaks can obscure minor contributor alleles or be misinterpreted as genuine contributor alleles. Here we describe the development of Reduced Stutter Polymerase (RSP), an engineered novel thermostable DNA polymerase that substantially reduces stutter artifact formation across forensic STR loci. RSP was engineered by inserting the thioredoxin-binding domain (TBD) from T3 DNA polymerase into Taq DNA polymerase and genetically fusing E. coli thioredoxin to the N-terminus via a flexible linker that enables intramolecular interaction between thioredoxin and TBD. Scanning mutagenesis across the entire TBD and thioredoxin domains identified beneficial mutations that were systematically combined with linker length refinement to yield RSP. When tested using a duplex STR assay targeting trinucleotide repeats, RSP reduced back stutter 5- to 6-fold and forward stutter ~2-fold compared to Taq DNA polymerase. Characterization across mono-, di-, tri-, tetra-, and pentanucleotide repeat structures revealed that back stutter reduction was most pronounced for tri- and tetranucleotide repeats (5- to 8-fold reduction), with dinucleotide repeats showing ~4-fold reduction, pentanucleotide repeats 3- to 4-fold reduction, and more modest improvements for mononucleotide repeats (1.4-fold). Using the Promega PowerPlex® 35GY primer set as a representative multiplex platform, RSP reduced back stutter an average of 8.1-fold across 31 loci in 56 individuals (all loci p < 0.05). RSP also substantially reduced the dependence of stutter on allele length and decreased the variability of stutter values observed at each locus. In an analysis of 22 two-person mixture samples (10:1 ratio) designed to challenge stutter-based filtering, RSP-amplified profiles showed significantly fewer stutter assignment errors (0.3 ± 0.6 per profile) compared to control amplifications (5.6 ± 3.3 per profile, p < 0.001). These results suggest that a polymerase engineered for reduced stutter can improve the discrimination between stutter artifacts and genuine contributor alleles. These findings establish the scientific foundation for future commercial forensic RSP-based STR systems.
Genotype imputation is relevant for increasing the genetic variants available from forensic or ancient samples with low quantity and quality of DNA and from targeted sequencing approaches, enabling meta-analysis to reach the required statistical power or to establish allele frequencies. Current genotype imputation tools are based on the co-inheritance of SNPs on shared haplotype segments of recombining DNA and are therefore not suited for non-recombining DNA. Imputation for non-recombining DNA, such as the human Y chromosome, would allow expansion of information from lower-cost targeted approaches to reach data quantities comparable to massively parallel sequencing-derived data. We introduce PhyloImpute, an easy-to-use software that leverage the phylogenetic nature of Y-chromosomal SNPs provided in (custom) phylogenetic trees to impute missing genetic variants. PhyloImpute characterizes samples by predicting haplogroups more accurately than state-of-the-art predictor tools, identifies deviations from the expected phylogeny, and establishes and illustrates haplotype frequencies on maps. PhyloImpute is licensed under GPL-3.0. The command line tool, extensive instructions and test data are freely available at https://github.com/ZehraKoksal/PhyloImpute. The graphical user interface tool for windows and linux with a tutorial and test data are freely available at https://zenodo.org/records/17950955.
Sequence analysis of the human mitochondrial genome (mitogenome) is of interest to the molecular anthropology, medical, and forensic communities. Quality mitogenome data is an essential component of haplotype search databases, serving as an important element of forensic investigations to ensure that weight estimates are reflective of accurate coincidental match probabilities. The European DNA Profiling group (EDNAP) Mitochondrial DNA Population database (EMPOP) is considered the gold standard for this purpose, serving as a reference database and a quality-control tool. The current study reports on the development of a sequencing pipeline for mitogenomes that is user friendly, robust, and cost effective for uploading mitogenome sequences to EMPOP. Whole blood or buffy coat samples were extracted using the Zymo Research Quick-DNA Miniprep Plus kit. Amplification of the mitogenome was performed using two overlapping long-range amplicons of approximately 8.5 kb. Batches of amplicons from 372 samples, plus eight DNA extraction reagent blanks and four amplification negative controls, were normalized and pooled using SequalPrep plates. A library of amplicons was prepared by ligation of SMRT bells (single molecule, real time adaptors), and prepared libraries were run on the PacBio Sequel IIe instrument using a high-fidelity (HiFi) approach. The total time for laboratory processing of 384 samples, prior to SMRT bell ligation, was up to 62 working hours. Total cost of reagents and supplies for all steps was approximately 20 U.S. dollars (USD) per sample. Including labor, the cost was approximately 30 USD. The success rate for 10,394 total samples tested was ∼98.2%, with only one of the two target amplicons failing to produce suitable sequence data. Therefore, on a per amplicon basis, the success rate was ∼99.2%. Concordance studies using two short-read sequencing methods confirmed the reliability of the long-read approach. The long-read pipeline can be easily adopted by laboratories and used in high-throughput studies involving quality biological samples to generate large mitogenome databases, including those for upload to EMPOP.
Touch DNA recovery from handled objects is influenced by donor, substrate, transfer, persistence, and collection-related factors; however, the role of short-term physiological activation during contact remains poorly understood. This pilot crossover study evaluated whether competitive gaming was associated with differences in Touch DNA deposition and STR profile quality on computer input devices compared with routine computer browsing. Twenty-four adult participants completed both 30-min activity conditions in randomized order, separated by a 30-min washout period. Heart rate and palmar moisture were measured before and after each activity, and Touch DNA was collected from predefined keyboard and mouse regions, generating 96 experimental samples. Samples were processed using a standardized forensic DNA workflow comprising extraction, quantification, GlobalFiler™ STR amplification, capillary electrophoresis, and profile assessment. Baseline physiological measures were comparable between conditions. Competitive gaming elicited significantly greater physiological activation than browsing, including higher post-activity heart rate (89.6 ± 10.7 bpm vs. 75.8 ± 8.4 bpm; p < 0.001) and palmar moisture scores (48.9 ± 7.4 vs. 36.2 ± 5.6; p < 0.001). Gaming was also associated with significantly greater recovered DNA quantity (p < 0.001, Cohen's d = 1.64) and detected autosomal alleles (p < 0.001, Cohen's d = 1.37) than browsing. STR profile quality was similarly improved, with higher profile completeness (p < 0.001, Cohen's d = 2.12) and mean profile RFU (p < 0.001, Cohen's d = 2.46) following gaming. At the participant-condition level, changes in palmar moisture showed strong positive associations with mean DNA recovery across devices (r = 0.89, p < 0.001), device-averaged profile completeness (r = 0.83, p < 0.001), and device-averaged mean RFU (r = 0.90, p < 0.001), whereas change in heart rate showed a more moderate association with mean DNA recovery (r = 0.49, p < 0.001). These findings indicate that activity-associated physiological and behavioural conditions, particularly local hand-surface moisture, may contribute to variation in Touch DNA deposition and STR profile quality on high-contact computer devices. However, the independent contributions of physiological activation and intensified manual interaction could not be separated. The results support a more contextual approach to Touch DNA interpretation in which activity intensity and local hand-surface conditions are considered alongside donor variability, substrate characteristics, transfer mechanisms, and case circumstances. This study provides preliminary evidence that the physiological and behavioural conditions accompanying an activity may represent underappreciated contributors to Touch DNA variability and activity-level interpretation.
