Accurate age inference from biological material is an essential component of forensic investigations. DNA methylation is a particularly informative biomarker, yet most current forensic approaches rely on bisulphite conversion, which can degrade DNA and limit applicability to compromised samples. Oxford Nanopore Technologies (ONT) enables direct detection of methylated cytosines from native DNA and therefore represents a promising alternative for epigenetic age inference. In this study, whole-genome ONT sequencing was performed on buccal DNA samples from 26 individuals aged 21-77 years to identify novel age-associated methylation signatures beyond previously established CpG markers. Sequencing was conducted on MinION R9.4.1 flow cells with Remora-optimised GPU basecalling, followed by alignment to GRCh38 and read-level quality filtering (Q ≥ 10). DNA methylation was profiled using four independent tools (Nanopolish, DeepSignal, Megalodon and Remora). To maximise reliability, we applied a stringent coverage filter, retaining only sites with at least 100 reads per site read depth, and considered as candidates only those loci that showed consistent age-methylation trends across all four methods. Under these conditions, multiple genomic coordinates exhibited strong correlations with chronological age (R² > 0.95). Strikingly, a substantial proportion of these high-confidence sites clustered within the same lncRNA loci on chromosome 21 (ENSG00000280441, ENSG00000278996), indicating that DNA methylation at lncRNA genomic loci changes is reproducibly detected even after rigorous quality and coverage filtering. Additional highly correlated sites were located in unannotated genomic regions, suggesting the presence of previously unrecognised epigenetic elements. These results expand the catalogue of candidate age-associated methylation markers, highlight the robustness of lncRNA-linked signals under strict bioinformatic filtering, and underscore the value of ONT sequencing for genome-wide epigenetic discovery in forensic age inference.

A range of forensic science activities for the classroom or home, designed for children aged 7+

Background Oxford Nanopore Technologies (ONT) enables direct detection of native DNA methylation without bisulphite conversion, offering long reads and field-deployable workflows. However, multiple ONT-compatible methylation callers differ in their underlying models, default parameters and genomic target space, and most comparative studies have evaluated them at a single sequencing depth using limited performance metrics. This makes it difficult to design ONT experiments and to interpret methylation calls in applications such as epigenetic biomarker discovery and forensic age estimation. Results We systematically benchmarked four widely used ONT methylation callers—Nanopolish, DeepSignal2, Megalodon and Remora—using whole-genome nanopore sequencing data from 26 human saliva samples and a consensus reference methylation profile derived from an Illumina Infinium MethylationEPIC BeadChip array saliva dataset (GSE111631; approximately 850,000 CpG sites). Performance was assessed at five coverage thresholds (1×, 5×, 10×, 30× and 100×) under three complementary analytical strategies: individual-level comparisons, within-sample consensus across tools and pooled method-level analyses. Agreement with the reference was quantified using Pearson correlation, methylation-level deviation metrics (MAE and RMSE), Wilcoxon signed-rank tests and corresponding effect size estimates, supported by visual summaries. MAE and RMSE were interpreted as technical measures of methylation-level deviation from the reference profile, not as forensic classification error rates, false-positive/false-negative rates or age-prediction error rates. Across all tools, single-read (1×) estimates were unstable and strongly method-dependent, whereas coverage ≥5× substantially improved accuracy and inter-method agreement. Increasing coverage further to 10× often yielded the best compromise between accuracy and robustness, but also led to a marked loss of shared CpG sites, highlighting a critical trade-off between depth and site retention. Conclusions This work provides, to our knowledge, the first coverage-aware, multi-metric and multi-strategy evaluation of ONT methylation callers on human saliva. Our results show that both the choice of caller and the sequencing depth have a major impact on methylation estimates, and that very high coverage thresholds can reduce the number of evaluable CpG sites to a degree that may limit downstream analyses. The study offers practical guidance for planning ONT-based methylation experiments in epigenetic and forensic contexts, particularly for selecting coverage thresholds and callers before downstream marker validation, age-prediction modelling or tissue-specific forensic interpretation.

Cotton swabs are one of the most effective methods of retrieving biological evidence. The efficiency of swab-based DNA recovery is impacted by many factors, such as the swabbing technique, source of DNA and volume and type of wetting solution used to moisten the swab head. This study aimed to evaluate a series of different swab-moistening solutions. The types of swabbing solutions included buffers, detergent-based solutions, and chelating agents. The DNA deposits, including cell-free DNA, cellular DNA, blood, and saliva, were collected from three non-porous surfaces: plastic, glass, and metal. The difference in the performance of the swab-wetting solutions was heavily influenced by the type of biological fluid, with the chelating agents, EGTA and EDTA, being the most suitable for recovering DNA from saliva and blood samples. Conversely, water and detergent-based solutions were more appropriate for cell-free and cellular DNA material likely to be found in trace DNA deposits.

The work presented herein is the second part of a large-scale persistence project aimed at identifying trends in trace DNA persistence. This study aims to show how different environmental storage conditions and target surface characteristics influence the persistence of cellular and cell free DNA (cfDNA) over time. To eliminate variation within the experiment, we used a proxy DNA deposit consisting of a synthetic fingerprint solution, cellular DNA, and/or cfDNA. Samples were collected and analysed from eight non-metal surfaces over the course of 1 year (27 time points) under three different environmental storage conditions. The results of this experiment show that surface characteristics in conjunction with DNA type greatly influence DNA persistence. Variation in the amount of DNA recovered over time was greatly influenced by surface porosity. CfDNA persisted at significantly higher levels on non-porous surfaces, and cellular DNA persisted at higher levels on porous items. Furthermore, statistically significant differences in DNA persistence were found among the items classified as non-porous surfaces and among the items classified as porous surfaces. Additionally, this study showed that the sample storage environment had a larger impact on DNA persistence than previously observed for metal surfaces [1]. When considering DNA type, cellular DNA was shown to persist for longer than cfDNA and persistence as a whole appears to be better when DNA is deposited alone rather than in mixtures. Unsurprisingly, it was found that the amount of DNA recovered from trace deposits decreased over time. However, DNA decay is highly dependent on the surface type and exhibits higher variability at short time points and on porous surfaces. For each of the surfaces tested, DNA persisted 1 year past deposition (in some combination of DNA type and environmental condition), except for wood, on which DNA did not persist in any capacity past four months. This data is intended to add to our understanding of DNA persistence and the factors which affect it.

In this study, we compare the performance of a simple PVP extraction method with a commercially available and widely used kit for recovering DNA from adhesive tapes. This novel method shows almost 60% higher DNA recovery from blood deposits on SceneSafe Fast™ minitapes when compared to the PrepFiler™ BTA Forensic DNA Extraction Kit. We also demonstrate how a simple modification of the magnetic bead-based purification step can lead to better recovery and removal of PCR inhibitors.

Choosing an inappropriate method of sample collection can often have a detrimental impact on DNA recovery. Multiple studies highlight the importance of selecting the recovery method based on the type of surface the DNA sample is located on. This study aimed to investigate the efficacy of sample collection via the single cotton swabbing method in comparison to recovery directly from the material cut from the surface. The three types of surfaces included cotton, paper, and cardboard. DNA sources comprised cell-free and cellular DNA, as well as blood and saliva as examples of body fluids commonly encountered at crime scenes. The data analysis revealed that the cutting-out method resulted in higher DNA recovery from all but cardboard surfaces, making it the more efficient collection method. Despite its limitations, the cutting-out method should be considered as the DNA recovery method of choice when suitable.

One of the most challenging issues still present in forensic DNA analysis is identifying individuals in samples containing DNA from multiple contributors. The introduction of novel identification markers may be a useful tool in the deconvolution of such DNA mixtures. In this study, we investigated the potential of alleles from the human leukocyte antigen system (HLA) to aid in identifying individuals in complex, multiple-donor DNA samples. The most advantageous characteristic of the HLA complex is its polymorphism in the human genome. A 22-loci multiplex with HLA markers was designed and applied to two-, three-, and four-person DNA mixtures. The results of the conducted experiments demonstrated that the identification of individuals in multiple contributor samples with the help of HLA markers is possible; however, it is clear that the reliability of the method is heavily dependent on the number of unique alleles for each individual in the analysed mixture. In order to compare this novel approach against the already established process, the same group of reference and multiple-contributor samples was analysed with a commonly used set of STR markers. This proof-of-concept research shows the importance of examining alternative solutions to the current deconvolution challenge in forensic DNA profiling.

One of the most challenging issues still present in forensic DNA analysis is identifying individuals in samples containing DNA from multiple contributors. The introduction of further novel identification markers may be a useful tool in the deconvolution of such DNA mixtures. In this study, we investigated the potential of alleles from the human leukocyte antigen system (HLA) to aid in identifying individuals in complex, multiple-donor DNA samples. The most advantageous characteristic of the HLA complex is its polymorphism in the human genome. A 22-loci multiplex with HLA markers was designed and then applied to two-, three- and four-person DNA mixtures. The results of the conducted experiments demonstrated that the identification of individuals in multiple contributor samples with the help of HLA markers is possible, however, it is clear that the reliability of the method is heavily dependent on the number of unique alleles for each individual in the analysed mixture. In order to compare this novel approach against the already established method, the same group of reference and multiple-contributor samples was analysed with a commonly used set of STR markers. This proof-of-concept research shows the importance of examining alternative solutions to the current deconvolution challenge in forensic DNA profiling.

Supplementary Figure S4: Clonogenic survival of G361 cells treated with or without A23187 prior to etoposide.

A review of the literature on DNA transfer and persistence highlights many difficulties that are encountered when conducting research of this nature. One of the main problems highlighted repeatedly in the literature is the prevalence of inherent uncontrolled variation that accompany these studies, and in turn, the results obtained. This work aims to decrease the amount of intrinsic variability associated with DNA transfer and persistence experiments using a realistic proxy solution which is adaptable, of known composition, reproducible, and capable of being standardised. This proxy is composed of three parts: a synthetic fingerprint solution, cellular DNA, and cell free DNA. In this proof-of-concept study the proxy was tested with a small-scale DNA transfer and recovery experiment and the data obtained suggests that the use of a solution that mimics real fingerprint secretions, over an alternative (such as buffer or a body fluid), is important when working with non-donor provided trace DNA samples. This is because the DNA deposit solution likely impacts the transfer of DNA from fingers/hands to a surface as well as the ability to recover the biological material once deposited.

We present a novel rapid method for the recovery of cellular and free DNA from cotton swabs based on a simple elution buffer containing a high molecular weight polymer and detergent combined with a short proteinase K digestion to release cellular DNA. This method shows increased yields approaching 80% recovery of the input DNA compared to the QIAamp DNA Mini kit standard extraction protocol for swabs which has a recovery of 20-30%. The buffer components in the described method are compatible with direct PCR analysis of the isolated DNA without further purification. Recovery efficiencies were estimated by qPCR.

Supplementary Figure S1: Etoposide activates AMPK in the nucleus

Supplementary Figure S2: Low concentrations of etoposide activates nuclear AMPK in cells with DNA damage.

AMP-activated protein kinase (AMPK) coordinates energy homeostasis during metabolic and energy stress. We report that the catalytic subunit isoform AMPK-α1 (but not α2) is cleaved by caspase-3 at an early stage during induction of apoptosis. AMPK-α1 cleavage occurs following Asp529, generating an ∼58-kDa N-terminal fragment (cl-AMPK-α1) and leading to the precise excision of the nuclear export sequence (NES) from the C-terminal end. This cleavage does not affect (1) the stability of pre-formed heterotrimeric complexes, (2) the ability of cl-AMPK-α1 to become phosphorylated and activated by the upstream kinases LKB1 or CaMKK2, or (3) allosteric activation by AMP or A-769662. Importantly, cl-AMPK-α1 is only detectable in the nucleus, consistent with removal of the NES, and ectopic expression of cleavage-resistant D529A-mutant AMPK-α1 promotes cell death induced by cytotoxic agents. Thus, we have elucidated a non-canonical mechanism of AMPK activation within the nucleus, which protects cells against death induced by DNA damage.

Understanding the transfer and persistence of different types of trace evidence between different donor and receiving surfaces under specific conditions, circumstances and alleged competing defence and prosecution hypotheses is a significant need. Acquiring such a knowledge base enables hypothesis testing to be undertaken more readily and with greater confidence. A longstanding goal has been to develop a unified approach to transfer and persistence studies which are fit for purpose but also scalable. Here we propose a low cost, universal experimental protocol using a recognised and well researched proxy material for the development and aggregation of ground truth transfer and persistence data at scale. We also propose and provide the tools to enable the creation of an open source and open access data repository of experimental data to act as a resource for practitioners and researchers in addressing transfer and persistence questions.

Inhibition of PCR by metal ions can pose a serious challenge in the process of forensic DNA analysis. Samples contaminated with various types of metal ions encountered at crime scenes include swabs from metal surfaces such as bullets, cartridge casings, weapons (including guns and knives), metal wires and surfaces as well as bone samples which contain calcium. The mechanism behind the impact of metal ions on DNA recovery, extraction and subsequent amplification is not fully understood. In this study, we assessed the inhibitory effects of commonly encountered metals on DNA amplification. Of the nine tested metals, zinc, tin, iron(II) and copper were shown to have the strongest inhibitory properties having IC50 values significantly below 1 mM. In the second part of the study, three commercially available DNA polymerases were tested for their susceptibility to metal inhibition. We found that KOD polymerase was the most resistant to metal inhibition when compared with Q5 and Taq polymerase. We also demonstrate how the calcium chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) can be used as an easy and non-destructive method of reversing calcium-induced inhibition of PCR reactions.

Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1(101-152)). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by similar to 20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.