The highly polymorphic nature of mitochondrial DNA (mtDNA) poses a significant challenge for primer design in PCR-based assays, because mismatches under primer binding sites can lead to reduced amplification and thus lower sequencing read depth or even complete amplicon dropout. While manufacturers aim to prevent this by adding variant-specific combinations to the primer pools (also known as degenerate primers), it cannot be entirely avoided that some samples are still affected, particularly when rare variants appear in the primer target region. In this study, we evaluated the impact of such variants on the amplification efficiency using two widely implemented Massively Parallel Sequencing kits, the Precision ID MtDNA Control Region / Whole Genome Panels (Thermo Fisher Scientific) and the ForenSeq mtDNA Whole Genome Kit (Verogen). The samples used in this study came from routine casework applications (analysed in the Control Region) and from a study of population genetics and phylogeography (encompassing the entire mitogenome). The observed instances of drop-out or reduced read depth were examined for their phylogenetic background and for their expected abundance and relevance in forensic casework. We discuss possible solutions to mitigate these issues, including the use of overlapping amplicons and an increased number of degenerate primers.

BACKGROUND:Urothelial carcinoma (UC) is a biologically heterogeneous disease, and current molecular classifications have limited integration into clinical decision-making. To further pursue precision oncology efforts in UC, we developed a molecular classification framework applicable to transcriptomic and proteomic data from non-muscle-invasive bladder cancer (NMIBC), muscle-invasive bladder cancer (MIBC) and urothelial cancer cell lines. METHODS:Using a whole-transcriptome self-organised map and regularised semi-supervised clustering of 4439 bulk NMIBC and MIBC transcriptomes and proteomes, and 33 UC cell lines, we identified three molecular UC clusters. Making use of both in silico and in vitro approaches, we selected promising treatment approaches for each cluster. RESULTS:The three developed clusters displayed distinct signatures of mRNA, proteins, biological processes, metabolism and essential driver genes. They also differed in prognosis and machine learning-predicted treatment vulnerabilities and resistance. High-risk, stroma-rich Cluster #1 cancers were predicted to respond to selected cytotoxic drugs, ferroptosis inducers and PARP inhibitors. For the aggressive, fast-proliferating, immune-infiltrated Cluster #2 tumours with basal/squamous differentiation, cytotoxic agents and EGFR/ERBB- and MEK/ERK-targeting therapies were proposed. Cluster #3 cancers of predominantly luminal papillary phenotype with scarce stroma and immune infiltration were enriched with NMIBC and low-risk malignancies. For patients with Cluster #3 tumours, selected epigenetic drugs or EGFR/FGFR inhibitors may represent attractive treatment options. CONCLUSIONS:Our novel molecular taxonomy holds promise as a practical framework for patient risk stratification and clinical trials in UC. Our molecular classification scheme may facilitate personalised transcriptome- and proteome-based risk assessment and clinical trial design for the development of various therapeutics. KEY POINTS:We developed three UC clusters, applicable for MIBC and NMIBC, which were validated using transcriptomic- and proteomic datasets. Publically available UC cell lines were assigned to the clusters, to have in vitro models representing each cluster. The clusters differ in molecular and biological signatures, with distinct prognostic and therapeutic characteristics.

Interpretation of mitochondrial DNA (mtDNA) evidence in a forensic context faces challenges, particularly when evaluating mtDNA profiles from different tissues. In hair, for example, the segregation of mtDNA shows tight bottlenecks that can result in different mtDNA profiles between and along hair shafts, and between hair and other reference tissues of the same donor. Current forensic interpretation guidelines for mtDNA are based on conventions on the number of discrepant positions observed between the two compared profiles. Most legislations consider two discrepancies between samples as an exclusion, and one discrepancy as an inclusion or as an inconclusive result. More data are needed to understand the variation and occurrence of discrepancies in samples from the same donor, to be able to incorporate this knowledge into a mathematical approach that effectively quantifies the probability of these events. This study reports the first project of the MitoMetrics collaborative initiative. In this study, data were generated from several participating laboratories, and previously published data, along with data generated from casework. mtDNA profiles from blood/buccal reference samples were compared to those from hair shafts from the same individuals. Results report the levels of heteroplasmy detected in the different tissues, the number of differences observed between the tissue comparisons, and the number of discrepant positions observed. We suggest a preliminary model for calculating the evidential value of mtDNA-based evidence using a likelihood ratio approach, that takes into consideration the occurrence of discrepancies between profiles from the same donor. This work represents the first attempt to quantify the probability of finding discrepant events between tissues and to incorporate these when reporting mtDNA in a forensic context.

Forensic lineage markers pose a challenge in forensic genetics as their evidential value can be difficult to quantify. Lineage marker population frequencies can serve as one way to express evidential value. However, for some markers, e.g., high-quality whole mitochondrial DNA genome sequences (mitogenomes), population data remain limited. In this paper, we offer a new method, MitoFREQ, for estimating the population frequencies of mitogenomes. MitoFREQ uses the mitogenome resources HelixMTdb and gnomAD, harbouring information from 195,983 and 56,406 mitogenomes, respectively. Neither HelixMTdb nor gnomAD can be queried directly for individual mitogenome frequencies, but offers single nucleotide variant (SNV) allele frequencies for each of 30 "top-level" haplogroups (TLHG), which mainly correspond to the first letter of major mitochondrial DNA (mtDNA) haplogroups (e.g., A, B, C, D, E, etc.) except for the L0, L1, L2, L3, L4-6, HV, and R/B haplogroups. We propose using the HelixMTdb and gnomAD resources by classifying a given mitogenome within the TLHG scheme and subsequently using the frequency of its rarest SNV within that TLHG weighted by the TLHG frequency. We show that this method is guaranteed to provide a higher population frequency estimate than if a refined haplogroup and its SNV frequencies were used. Further, we show that top-level haplogrouping can be achieved by using only 227 specific positions for 99.9% of the tested mitogenomes, potentially making the method available for low-quality samples. The method was tested on two types of datasets: high-quality forensic reference datasets and a diverse collection of scrutinized mitogenomes from GenBank. This dual evaluation demonstrated that the approach is robust across both curated forensic data and broader population-level sequences. This method produced likelihood ratios in the range of 100-100,000, demonstrating its potential to strengthen the statistical evaluation of forensic mtDNA evidence. We have developed an open-source R package mitofreq that implements our method, including a Shiny app where custom TLHG frequencies can be supplied.

The discovery of glacially preserved remains offers a rare and invaluable opportunity to explore long-term tissue preservation, diagenetic alterations, and microbial colonisation. In this study, we examined a 350-year-old glacier mummy of a purple heron (Ardea purpurea) recovered from the Austrian Alps, specifically from the Gurgler Ferner glacier in Tyrol, Austria, at an altitude of 3.004 m in the area of the Hochwildehaus towards Hochwilde and the Annakegele, marking the first documented instance of an avian glacier mummy in this region. A comprehensive multimodal analytical approach was employed, integrating micro-computed tomography (micro-CT), histology with conventional and specialised stains, fluorescence microscopy, Raman spectroscopy, attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, and molecular analysis of bacterial and fungal DNA. Micro-CT and histological analyses revealed exceptional preservation of cortical and trabecular bone, intact osteocyte lacunae, and structurally well-preserved epiphyseal growth plates. Additionally, remnants of soft tissues, including muscle, skin, feather quills, and lung tissue, were identified. Raman and ATR-FTIR spectroscopy supported preservation of the bone mineral phase, characterised by carbonate apatite signatures, while indicating a reduced relative detectable organic contribution in the glacier mummy bone. Fluorescence microscopy further identified post-mortem colonisation by environmental fungi (Orbilia, Davidiella, Itersonilia) and bacteria (Sphingomonas, Paenibacillus, Alkalibacillus). These findings underscore the extraordinary preservation conditions provided by glacial environments and highlight the value of integrative, multimodal methodologies for studying ancient remains. This study not only expands our understanding of avian tissue preservation under frozen conditions but also provides critical insights into the interplay between biological and environmental factors in long-term preservation.

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.

This study evaluated the ability to produce FORensic Capture Enrichment (FORCE) genotypes using amplicon-based and capture-based enrichment assays. The FORCE panel is a standardized set of single nucleotide polymorphism (SNP) markers developed for forensic applications. Twelve DNA samples were prepared and distributed to the laboratories for testing: five control DNA samples, a dilution series ranging from 10ng to 0.03ng, two degraded DNA samples with 200bp and 150bp average fragment lengths, and one inhibited sample spiked with humic acid. Fifteen laboratories from three different continents participated in this study, choosing from one of four manufacturer-developed enrichment assays to complete the experiments, setting their own parameters for sequencing and other user-defined steps to accommodate their own preferences and expertise. A total of eighteen methods were evaluated, as three laboratories performed two methods. The results showed that all four assays were successful in producing full FORCE SNP genotypes from high quality samples. However, significant differences between and within assays and methods were observed. Read count variability and enrichment type led to significant differences in call rate. Robust SNP recovery was observed across all assays at 0.3ng DNA input, with an amplicon-based assay producing high SNP call rates at 0.03ng DNA input. Capture and single primer extension assays produced consistently high SNP call rates from degraded samples with 150-200bp fragments. Future research to optimize laboratory parameters may reduce the variation in SNP data, so that labs may equitably adopt SNP methods to make use of these powerful forensic markers.

Abstract Introduction: Urothelial carcinoma (UC) is a molecularly heterogeneous disease, and transcriptome-based classification systems have given important insights into its biology. The current consensus classifications for UC represent a major step toward biological stratification; however, its prognostic and predictive relevance remains uncertain, limiting use in clinical guidelines. Furthermore, most molecular schemes have been developed either for non-muscle invasive (NMIBC) or muscle-invasive disease (MIBC), relying only on bulk transcriptomic data. This fragmentation hinders comparability across studies and integration with proteomic or single-cell datasets. A simplified molecular framework is therefore needed to capture UC heterogeneity more comprehensively and to support personalized therapeutic approaches. Materials & Methods: Using the TCGA bulk bladder cancer transcriptome dataset, we developed three distinct molecular UC clusters, which were validated using 18 transcriptome, 3 proteome and 33 UC cell line datasets. Making use of in silico predictions, we selected promising treatment strategies, which were further screened in vitro using the IncucyteS3 live-cell imaging system and RNA-sequencing on representative cell lines. Results: Our transcriptomic and proteomic analyses revealed three UC clusters with distinct molecular, biological and clinical features. Each cluster showed specific mRNA and protein expression patterns, metabolic profiles, and driver gene alterations, translating into divergent prognoses and predicted therapeutic sensitivities. Novel approaches, like liquid biopsy-based stratification using ECM-derived urinary peptides or IHC-based profiling of the proposed distinct markers, are currently being investigated. The stroma-rich, high-risk cluster #1 was associated with responsiveness to ferroptosis inducers and PARP inhibition. Cluster #2, which is highly proliferative and immune-infiltrated with basal/squamous traits, showed predicted benefit from cytotoxic agents and inhibition of EGFR or MEK signaling pathways. Cluster #3, dominated by luminal papillary, low-risk tumors with minimal stromal and immune components, appeared susceptible to epigenetic therapies and EGFR/FGFR inhibition. Conclusion: Our new integrative molecular classification scheme provides a practical framework for patient stratification, personalized transcriptome- and proteome-based risk assessment, preclinical research and clinical trial design, including both NMIBC and MIBC. Citation Format: Nils Cornelis van Creij, Piotr Tymoszuk, Florian Handle, Andreas Seeber, Teresa Sellemond, Agnieszka Martowicz, Eva Compérat, Hamed Wafa, Steffen Ormanns, Michael Günther, Walther Parson, Maxim Noeparast, Frederic Romain Santer, Jose Daniel Subiela, Petros Grivas, Roger Li, Zoran Culig, Renate Pichler. Multi-omic profiling identified three molecular clusters in urothelial carcinoma: A path towards clinical precision [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6869.

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.

Mitochondrial DNA (mtDNA) analysis is a frequently used tool for determining the potential origin of biological traces found at crime scenes. The method typically involves comparing the genetic profile of the trace with that of a suspect. While a mismatch between the two profiles usually leads to the exclusion of the suspect, the evidential value of a match is sometimes difficult to grasp. This is particularly true in cases that are more complex than a simple trace-suspect comparison. We considered two such scenarios and developed means for appropriate statistical interpretation of the respective mtDNA results. One scenario requires the evaluation of a composite hypothesis about trace donorship in multiple cases involving an mtDNA profile match with one and the same suspect. The other scenario calls for the consideration of a second mtDNA profile found at the crime scene that matches a matrilineally unrelated contact person of the suspect. For both scenarios, we propose formally linked mathematical methods for interpreting the mtDNA data which, under certain assumptions, allow valid quantification of the evidential value of the latter for or against the suspect. Furthermore, we illustrate the application of both methods with example calculations under realistic assumptions about the required parameters.

The identification of novel molecular drivers and the development of new state-of-the-art therapies are critical challenges in ovarian cancer (OC) treatment. Cyclin-dependent kinase 12 (CDK12) is a promising target, as it’s functional activity promotes genomic stability. Here, we examined the anticancer efficacy of the dual CDK12/13-inhibitor SR-4835 in platinum-sensitive and -resistant OC cell lines, as well as its potential as a drug partner for platinum or olaparib combination therapy. SR-4835 exhibited potent anti-proliferative effects on most OC cell lines with IC50 values within the nanomolar range. A tendency for increased sensitivity of the cisplatin-resistant compared to their sensitive, parental cell lines was observed. Transcriptome analyses indicated gross changes in gene expression in numerous signaling pathways by SR-4835. Gene downregulation was in part due to alternative exon usage, which correlated with the number of intronic polyadenylation sites per gene and gene length. Furthermore, SR-4835 lead to the downregulation of key homologous recombination pathway genes rendering a BRCAness phenotype. However, the combination of SR-4835 with cisplatin or olaparib primarily exhibited an additive, not synergistic, effect. In summary, the present findings indicate that CDK12/13 inhibitor SR-4835 has potent anti-cancer effects accompanied by a BRCAness induction, but fails to achieve synergistic effects with cisplatin or olaparib in OC cells.

OBJECTIVES:The current Caribbean Colombian population is the result of migration processes and admixture that occurred throughout the country's history. The aim of this study was to investigate genetic ancestry gradients throughout the Caribbean territory, with a higher Native American ancestry expected in inland areas compared to coastal cities. Simultaneously, since the genotyping methodology used included markers of forensic relevance for predicting population of origin and pigmentary phenotypic characteristics, this study also tested the main tools commonly applied in forensic contexts. MATERIALS AND METHODS:Uniparental and biparental genetic ancestries of individuals from the Bolívar Department, in the Caribbean Colombian region, were investigated. A total of 64 samples were collected from Coast, Center, and Inland regions. Genotyping was performed with a newly developed PCR-based targeted MPS tool called COMBO, targeting over 1000 polymorphisms along mtDNA, Y chromosome, and autosomes. RESULTS AND DISCUSSION:The Coast and Center regions showed similar ancestry profiles. The Inland region displayed a contrasting pattern, being mainly composed of Native American maternal lineages and European paternal lineages. Statistically significant differences in mtDNA and AIM-SNP compositions were only found between the Coast and Inland regions, absent for the Y chromosome. The results obtained regarding the prediction of eye, hair, and skin color were as expected based on the ancestry profile of the population. Biogeographic ancestry prediction tools have presented challenges in assigning individuals to the American metapopulation, highlighting the need for better reference datasets from South America, given the great heterogeneity in the admixture patterns they present.

Background: Efficient DNA extraction from degraded skeletal remains is essential for forensic and ancient DNA analysis. The main aim of this study was to compare the performance of an automated DNA extraction system with a manual DNA extraction protocol when applied to challenging skeletal samples. Specifically, the automated Maxwell Forensic Sample Concentrator system was evaluated against a modified manual Dabney extraction protocol. Methods: DNA was extracted from skeletal material originating from twelve human individuals. Maxwell extractions using 50 mg and/or 100 mg of starting material were compared with Dabney extractions using 50 mg. DNA extracts were quantified using SD quants targeting nuclear DNA and two mitochondrial DNA fragments. Selected extracts were further analysed by mitochondrial DNA sequencing. Results: Both extraction approaches generated comparable DNA yields and sequencing results for moderately degraded samples. In the highly degraded samples analysed in this study, the Dabney protocol generally yielded higher nuclear and mitochondrial DNA quantities and was often associated with a higher sequencing performance. The Maxwell system nevertheless performed well for less degraded material and, in some cases, produced sequencing results comparable to Dabney. Maxwell extraction with 100 mg input was effective for better-preserved samples but was less consistent for highly degraded material. Conclusions: The efficiency of the extraction methods depended largely on the degree of DNA degradation. The findings of this study suggest that the Dabney protocol may be more suitable for heavily degraded skeletal remains, whereas the automated Maxwell system represents a practical and efficient option for less degraded samples. The choice of method therefore depends on the sample condition and the analytical objectives.

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.

Donor-signed informed consent is a fundamental prerequisite for ethically correct analysis and publication of genetic data in forensic population studies, including quality assessment of datasets and their inclusion into frequency databases. While considerations on the requirement and content of informed consent have been published, little information is available with regard to the actual nature of the documents currently in use. This study investigated 50 recent informed consent forms submitted to EMPOP and STRidER from a broad range of contributors across worldwide legislations, irrespective of the quality of the associated genetic data. The common ground of the informed consent forms, their specific content and differences, and the extent to which they contain suggested components are outlined. This evaluation of authentic informed consent form diversity adds to the discussion on formal aspects to be covered at the time of sampling and may expedite future harmonization of informed consent in forensic population studies, assuring ethical principles in the application of precious sample sets for a broad range of investigations across genetic disciplines.

For decades, there has been scientific interest in the variation and geographic distribution of paternal lineages associated with the human Y chromosome. However, the relevant data have been dispersed across numerous publications, making it difficult to consolidate. Additionally, understanding the relationships between different variants, and the tools used to analyze them, have evolved over time, further complicating efforts to harmonize this information. The Universal Y-SNP Database (UYSD) marks a substantial advancement by providing a comprehensive and accessible platform for Y-SNP and haplogroup data from populations around the world. UYSD harmonizes diverse datasets into a unified repository, facilitating the exploration of global Y-chromosomal variation. The platform handles data generated with both high- and low-throughput technology and is compatible with the automated analysis software tool, Yleaf v3. Key functionalities include the ability to: i) visualize haplogroup distributions on an interactive world map, ii) estimate haplogroup frequencies in geographic regions with sparse data through interpolation, and iii) display detailed phylogenetic trees of Y-chromosomal haplogroups. Currently, UYSD encompasses data from over 6,600 males across 27 populations. This dataset largely aligns with known global Y-haplogroup patterns, but also reveals unexplored finer-scale geographic variations. While the present dataset is largely European-centered, UYSD is designed for ongoing expansion by the scientific community, aiming to include more global data and higher-resolution population sequencing data. The platform thus offers valuable insights into human genetic diversity and migration patterns, serving several fields of research such as: human population genetics, genetic anthropology, ancient DNA analysis and forensic genetics.