Rapid virus detection by primer-independent linear rolling circle amplification combined with DNA-templated silver nanoclusters.

BackgroundUpper tract urothelial carcinoma (UTUC), including renal pelvic urothelial carcinoma and ureter urothelial carcinoma, accounts for 10% of urothelial carcinoma (UC). Poorer outcomes and different genetic characteristics of UTUC were reported compared to urothelial carcinoma of the bladder (UCB), which accounts for most cases of UC. Therefore, there is an urgent need for the development of molecular characterization and precision therapies tailored specifically for UTUC.MethodsTo elucidate the genetic landscape of UTUC, we included 4 UTUC samples in this study and also perform next-generation sequencing (NGS) of whole exome sequencing. After that, long-read sequencing (LRS) was employed to conduct Whole genome sequencing (WGS) analyses on tumor samples obtained from the four UTUC patients, utilizing the Pacific Biosciences (PacBio) REVIO platform.ResultsThe clinical phenotypes of four UTUC patients including tumor stage, location and response to treatment, etc. were collected. NGS of four patients yielded negative results. The WGS mapped the mutation landscape in the tumor tissues of four UTUC patients, and screened the sequencing results according to UTUC and solid tumor related genes. Seven pathogenic or likely pathogenic single nucleotide variant (SNV) were obtained. Among the detected structural variations (SVs), four patients shared multiple segments of SVs with close positions. The 12q24.31-p11.1 inversion was shared by four patients. In the detection of STR and DNA methylation, comparing the results of patients and normal controls, many different fragments were obtained. It shows that LRS has important advantages over NGS for accurate tumor detection and treatment.ConclusionThe analysis revealed multiple genetic variants potentially associated with UTUC carcinogenesis or development, and indicated advantages of TGS over next-generation sequencing (NGS) in cancer genetic variant detection, especially in SV and Short Tandem Repeats (STRs). This study may lay the groundwork for molecular classification and offer valuable insights into the development of precision therapies for UTUC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-15254-x.

Appendix-like morphology in primary ovarian mucinous tumors lacking a concurrent mature teratoma: A series of 4 cases illustrating the utility of STR analysis.

Nondestructive automated DNA extraction method from the tooth root surface.

BackgroundMedicago is an economically important forage genus widely distributed across China, yet its mitochondrial genomes remain poorly understood. Comprehensive characterization of mitochondrial genome structure, function, and evolution is crucial for elucidating plant biological mechanisms, improving germplasm utilization, and advancing molecular breeding.ResultsWe assembled and compared the mitochondrial genomes of eight Medicago species, including six newly sequenced genomes. Five species contained typical single circular mitochondrial genomes, whereas M. falcata, M. platycarpos, and M. sativa displayed complex multipartite circular conformations. Genome sizes ranged from 281,240 to 356,577 bp, with 55–74 functional genes. Repeat analysis identified 141 simple sequence repeats (SSRs) and 76 tandem repeats (TSRs), largely composed of A/T-rich mononucleotide motifs, while dispersed repeats were mainly 30–49 bp in length. Codon usage showed a strong A/T bias and a preference for leucine, serine, and isoleucine. RNA editing events were predominantly C-to-U substitutions, primarily occurring at the first and second codon positions. Phylogenetic reconstruction based on 31 shared mitochondrial protein-coding genes (PCGs) strongly supported the monophyly of Medicago and recovered interspecific relationships consistent with previous chloroplast and nuclear genome studies. Most PCGs were under purifying selection, while a few, such as matR, exhibited signatures of positive selection, suggesting lineage-specific adaptive evolution.ConclusionsThis study expands the mitochondrial genomic resources of Medicago and provides new insights into its structural evolution and phylogenetic relationships, offering a valuable foundation for evolutionary and functional studies in Fabaceae.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07650-z.

Patient-derived xenograft models are crucial in cancer research, although authentication methods remain limited. This study developed a comprehensive DNA profiling strategy for patient-derived xenograft quality control in the J-PDX Library by comparing short tandem repeat and single nucleotide polymorphism analyses. Overall, 325 patient-derived xenograft models were analyzed using both short tandem repeat and single nucleotide polymorphism profiling. Concordance rates between patient-derived samples and patient-derived xenograft models were assessed. Single nucleotide polymorphism-based analysis cutoffs classified models as matched, undeterminable, or unmatched. Additionally, the impact of microsatellite instability and loss of heterozygosity on concordance were evaluated. Short tandem repeat analysis showed greater variability than single nucleotide polymorphism analysis. Among the 325 models, 296 (91.1%) were matched, 24 (7.4%) were undeterminable, and five (1.5%) were unmatched. Notably, undeterminable models showed significantly higher microsatellite instability, with single nucleotide polymorphism analysis providing reliable authentication, even when short tandem repeat analysis failed. This study established an efficient patient-derived xenograft model quality control workflow using short tandem repeat analysis for primary screening and confirmatory single nucleotide polymorphism analysis for challenging cases, particularly microsatellite instability tumors. This workflow enables robust quality control of the J-PDX Library, enhancing its reliability for drug development and preclinical research.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21299-6.

Background/Objectives: Formalin-fixed paraffin-embedded (FFPE) tissues are sometimes the only DNA source for forensic applications. The quantity and integrity of the DNA extracted from these samples depend on multiple factors. In this work, we analyzed, for the first time, whether Masson’s trichrome (MT) staining alters the results of genetic profiles obtained from DNA extracted from FFPE tissue sections. Methods: Three pairs of sections from the year 2024 and three pairs from the year 2001 were analyzed. Each pair consisted of serial sections, one stained with hematoxylin and eosin and the other with MT. DNA was extracted using the PrepFiler Express BTA™ Forensic DNA Extraction Kit and quantified by real-time PCR using the Quantifiler™ HP DNA Quantification Kit. DNA samples were processed for short tandem repeat (STR) profiling using the GlobalFiler™ PCR Amplification Kit. The amplified alleles were separated and analyzed using an ABI PRISM® 3500 genetic analyzer. Results: All MT-stained samples showed deficiency in most or all of the parameters assessed: DNA yield, degradation index, number of alleles detected, random match probability value, and intensity of the electropherogram peaks. In fact, DNA could not even be quantified in the samples processed in 2001. Conclusions: These results could be due to the large number of acids used in MT staining, which cause chemical modification and hydrolysis of DNA, affecting the success of PCR-based methods used subsequently. In conclusion, DNA obtained from MT-stained FFPE tissue sections may be highly degraded and should therefore be used with great caution in forensic settings.

Dendrobium officinale, a perennial epiphytic herb of the orchid family renowned for its ornamental value and diverse medicinal properties, has attracted considerable global attention due to its rarity and increasing market demand. However, large-scale cultivation has led to confusion regarding its germplasm resources and genetic backgrounds, posing significant challenges for the effective conservation, management, and utilization of D. officinale germplasm. In this study, we systematically analyzed the abundance and characteristics of simple sequence repeats (SSRs) and developed highly polymorphic genomic SSR markers using whole-genome resequencing (WGRS) data from 15 D. officinale genotypes. A total of 494,783 SSRs were identified in the “Niu2020” reference genome. Mononucleotide repeats were the most abundant motifs, followed by dinucleotide repeats, with A/T and AT/TA being the predominant types, respectively. Of the SSRs with unique flanking sequences (unique SSRs), 121,544 out of 388,553 (31.28%) were polymorphic across the 15 genotypes. The polymorphism information content (PIC) values of these SSRs ranged from 0.13 to 0.92, with an average of 0.59. Subsequently, 10,364 pairs of SSR primers were successfully designed from polymorphic SSR loci with PIC values ≥ 0.5 and major allele size differences ≥ 3 bp. Ultimately, 20 primer pairs were randomly selected for experimental validation. Of these, 18 successfully amplified the target fragments and exhibited high levels of polymorphism (PIC values ranged from 0.31 to 0.74), confirming the effectiveness and practical utility of the newly developed markers. The SSR fingerprints and polymorphic markers developed in this study provide a valuable resource and establish a robust technical foundation for enhancing the efficiency of cultivar identification, genetic research, and molecular breeding in D. officinale.

The genus Takydromus (grass lizards) represents a diverse and ecologically significant group of lacertid lizards widely distributed across East and Southeast Asia. However, phylogenetic relationships within the genus remain contentious, primarily due to limited molecular data and inconsistent results from previous studies based on single or few mitochondrial genes. This study aimed to (1) sequence and characterize the complete mitogenome of T. intermedius; (2) perform a comparative analysis of mitogenomic features across the genus; and (3) reconstruct a robust phylogeny to clarify intra-generic evolutionary relationships. The mitogenome of T. intermedius was 18,770 bp in size and contained the typical set of 37 genes. Comparative analyses revealed characteristic features including AT-richness, strand asymmetry, and considerable length variation in the control region attributable to tandem repeats. The ATP8 gene showed the highest nucleotide diversity, and all protein-coding genes were found to be under strong purifying selection. Phylogenetic trees were reconstructed from a concatenated dataset of 13 protein-coding genes and two rRNA genes using both maximum likelihood and Bayesian inference methods. The resulting phylogeny strongly supported the monophyly of Takydromus and resolved several species relationships; however, it did not support the recognition of Platyplacopus as a distinct subgenus. Moreover, our mitogenomic analysis strongly validates the forest-grassland ecological speciation hypothesis and the southern–northern lineage division in Takydromus. Our study provides valuable mitogenomic resources and underscores the utility of complete mitochondrial genomes in elucidating phylogenetic relationships within Takydromus. These findings lay a solid foundation for future taxonomic and evolutionary studies, although expanded species sampling is needed to fully understand the genus’s diversification history.

Transposable elements (TEs) are dynamic components of eukaryotic genomes, playing a crucial role in genome evolution and plasticity, particularly in unstable regions such as chromosome ends. In the globally significant fungal pathogen Fusarium oxysporum, we identified and characterized a novel family of non-LTR retrotransposons named FoTeRs (F. oxysporum Telomeric Retrotransposons). These elements are consistently and uniquely localized at chromosome ends, representing a rare example of site-specific TE integration. Phylogenetic analysis confirmed that FoTeRs form a distinct clade with other telomere-targeting retrotransposons, suggesting a shared evolutionary history and a convergent mechanism for telomeric integration. We found that individual FoTeR elements exhibit a duality in their evolutionary status. Putatively functional elements are under strong purifying selection, indicating that their protein-coding regions are highly conserved. This contrasts with the presence of other, non-functional copies that exhibit signs of mutational decay, a process accelerated by Repeat-Induced Point (RIP) mutations -a fungal-specific defense mechanism. The high density of upstream variable number tandem repeats (VNTRs) also contributes to their genomic plasticity. Furthermore, FoTeRs frequently co-localize with host Telomere-Linked Helicases (TLHs), suggesting a potential functional link in telomere maintenance. This study provides crucial insights into the role of TEs in shaping the genome architecture and adaptive potential of this important fungal pathogen.

Short tandem repeat (STR) DNA, one of which is the FGA locus, can be used as a profiling tool for personal identification analysis in the forensic field because it has many allelic combinations based on the complex repetitive pattern of DNA sequences. The FGA locus was reported to have the highest discrimination power in the Indonesian population. The aim of this study was to understand the ability of personal discrimination from the FGA locus sequences in a DNA forensics testing scenario of Indonesian subjects. The specimens were used towels (T), toothbrushes (TB), and hair ties (HT) from certain people and buccal swabs (BS) from the same people and two others as controls whose identities were analyzed. DNA extraction using the Phenol CIAA ( chloroform isoamyl-alcohol ) method followed amplification of the DNA of FGA loci that targeted repetitive sequences, agarose gel electrophoresis, and Sanger sequencing of FGA loci. The amplification showed the DNA bands from the FGA loci were around 350 bp. The sequencing results showed that the number of repetitions of the FGA allele from samples T, TB, and HT was identical to BS1, which was 19 repetitions, while BS2 and BS3 were 22 and 21 repetitions, respectively. Thus, the sample items T, TB, and HT are owned by individual BS1. The FGA gene sequenced by the Sanger method can be used in a limited way, only detecting repetitive sequences on the longest allele as a differentiator between one individual and another.

Evaluation of a rapid flow cytometry assay to assess functional engraftment in CGD patients post-transplant and comparison with molecular chimerism.

Tandemly repeated non-coding sequences, widely known as satellite DNAs (satDNAs), are extremely diverse and highly variable components of eukaryotic genomes. In recent years, advances in high-throughput sequencing and new bioinformatics platforms have enabled in-depth studies of all (or nearly all) tandem repeats in any genome (the satellitome), while a growing number of telomere-to-telomere assemblies facilitates their detailed mapping. Research performed on a large number of non-model plant and animal species changed significantly the “classical” view on these sequences, both in an organizational and functional sense, from ballast compacted in the form of heterochromatin to elements that are important for structuring the entire genome, as well as for its functions and evolution. The diversity of repeat families, and the complexity of their intraspecies and interspecies distribution patterns, posed new questions, urging for species-by-species comparative analyses. Here we integrate some basic features of different forms of sequences repeated in tandem and rapidly growing data evidencing extensive dispersal of satDNA sequences in euchromatin, their putative roles and evolutionary significance. Importantly, we also present and discuss various issues brought on by the use of new methodological approaches and point out potential threats to the analysis of satDNAs and satellitomes.

Root-knot nematodes (Meloidogyne spp.) are among the most destructive agricultural pests that cause significant yield losses across a wide range of crops. Meloidogyne hapla is a valuable model for studying root-knot nematodes due to its parasitic diversity, small diploid genome, and a reproductive strategy that facilitates genetic analysis. Here, we report the most contiguous genome assembly to date for any plant-parasitic nematode built using PacBio HiFi, Oxford Nanopore, Illumina, and Hi-C sequencing. Genetic linkage analysis of F2 populations derived from crosses between M. hapla strains validated the assembly but also revealed anomalies indicating chromosome structure differences between parental isolates such as fissions, fusions, and rearrangements. Strikingly, we identified sharply delimited zones with extraordinarily high recombination on most chromosomes. Notably, several of these high recombination zones were significantly enriched for genes encoding secreted proteins, many of which contribute to parasitism. These findings suggest that meiotic recombination facilitates effector diversification and offer insight into how these parasites diversify their effector protein repertoire to change or expand their extraordinary host range. We further report the discovery of a novel 16-nucleotide tandem repeat and lack of canonical telomere repeats at chromosome ends. The localization of this 16-nt repeat at chromosome ends highlights a potentially divergent mechanism of chromosome-end maintenance in this nematode group. Overall, our study integrates high-resolution structural genomics, genetic mapping, and functional inference to uncover links between genome architecture, recombination landscapes, and host-parasite interactions.

Root-knot nematodes (Meloidogyne spp.) are among the most destructive agricultural pests that cause significant yield losses across a wide range of crops. Meloidogyne hapla is a valuable model for studying root-knot nematodes due to its parasitic diversity, small diploid genome, and a reproductive strategy that facilitates genetic analysis. Here, we report the most contiguous genome assembly to date for any plant-parasitic nematode built using PacBio HiFi, Oxford Nanopore, Illumina, and Hi-C sequencing. Genetic linkage analysis of F2 populations derived from crosses between M. hapla strains validated the assembly but also revealed anomalies indicating chromosome structure differences between parental isolates such as fissions, fusions, and rearrangements. Strikingly, we identified sharply delimited zones with extraordinarily high recombination on most chromosomes. Notably, several of these high recombination zones were significantly enriched for genes encoding secreted proteins, many of which contribute to parasitism. These findings suggest that meiotic recombination facilitates effector diversification and offer insight into how these parasites diversify their effector protein repertoire to change or expand their extraordinary host range. We further report the discovery of a novel 16-nucleotide tandem repeat and lack of canonical telomere repeats at chromosome ends. The localization of this 16-nt repeat at chromosome ends highlights a potentially divergent mechanism of chromosome-end maintenance in this nematode group. Overall, our study integrates high-resolution structural genomics, genetic mapping, and functional inference to uncover links between genome architecture, recombination landscapes, and host–parasite interactions.

Epstein-Barr virus (EBV) infects over 95% of the world's population and is tightly associated with multiple human malignant diseases. As the first discovered human oncovirus, EBV is known to induce genomic instability by promoting various types of genomic modifications in host chromosomes. However, the mechanisms through which EBV interacts with the host genome and regulates cellular gene expression in genomic modifications are not yet fully elucidated. In this study, we conducted primary EBV infection in B cells and performed the analyses of copy number variants using whole genome sequencing. The results showed genomic regions susceptible to EBV-induced mutations and unveiled MUC19 to be a critical host factor in EBV latency, which was distinctively activated transcriptionally upon EBV infection. Finally, we identified the intrinsic tandem repeats in MUC19 to be its functional domain, which promotes cell survival and cell cycle through activating mechanistic target of rapamycin (mTOR) signaling in EBV-positive cells. Further results indicate that EBV nuclear antigen 1 binds to the promoter of the MUC19 gene and enhances its expression. In conclusion, these results provide novel insights into the roles of MUC19 in EBV latency, highlighting its potential as a promising therapeutic target for the treatment of EBV-associated lymphomas.IMPORTANCEGenomic instability is a hallmark of cancer. EBV contributes to host genomic instability after primary infection. This study maps the EBV-induced genomic variations using deep whole genome sequencing and identifies the critical factor MUC19, which is one of the most understudied genes, with a genomic sequence exceeding 177 kbp that encodes a protein over 800 kD. In this study, we revealed that EBV induced the duplicated copy number variants of the MUC19 gene and enhanced its expression, which further promotes cell survival and cell cycle via mTOR signaling. Overall, this study maps the genomic perturbations induced by EBV primary infection and offers new insights into the critical role of MUC19 in EBV latency.

Εffects of MTR and AS3MT variants on antipsychotic response: prospective evidence from a naturalistic study in Greece.

On-site forensic DNA analysis has been hampered by inadequate automation and the limited scope of genetic information profiling. Here, a modular fully integrated microfluidic system is presented paired with a portable trolley-case analyzer to enable truly automated, on-site forensic DNA profiling. The system consists of a disposable plastic chip for DNA extraction/amplification (DEA) and a universal glass capillary electrophoresis (CAE) chip. The sample-in-answer-out platform generates complete DNA profiling within 124min without any manual intervention in field environments. The automated system is successfully validated to directly process six types of forensic samples. Modular microfluidic cartridge interfaces are designed to support parallel analyses, including human identification via short tandem repeats (STRs), ancestry inference using insertion/deletion polymorphism (InDels), and paternity testing based on Y-chromosomal STRs (Y-STRs) and single nucleotide polymorphisms (Y-SNPs). Validation demonstrated a sensitivity of 0.5ng for 21-locus STR profiling, enabling non-expert users to obtain complete profiles with just 1min of hands-on operation. The DIP38 ancestry panel exhibited complete concordance with self-reported ethnicities. Crucially, the system allows for simultaneous Y-STR/Y-SNP detection in outdoor settings, facilitating assignment of the O-M122 haplogroup (ISOGG 2023). The development of this microsystem offer a fully automated solution for rapid on-site forensic DNA detection.

The Iberian (Calé) Roma constitute one of the largest Roma communities in Europe, yet their internal genetic structure and connections to other Roma groups remain understudied. This study explores the microgeographical structure of the Iberian Roma and their relationships with other Roma groups by analysing paternal lineages using 17 Y-chromosome short tandem repeat markers in a geographically stratified sample of 173 Spanish Roma individuals. The haplogroup distribution patterns indicate that the paternal genetic profile of the Spanish Roma is shaped by founder effects, population bottlenecks, and multiple admixture events with non-Roma groups. Haplogroups H and J2a1b dominate the genetic landscape, reflecting their South Asian origin and subsequent dispersal patterns through West Asia into Europe. A distinctive feature of the Spanish Roma is the high frequency of haplogroup R1b, indicating significant gene flow from non-Roma Iberian populations. The absence of North African or Jewish genetic influences rules out the possibility of a North African migration route for the Calé Roma into the Iberian Peninsula. Microgeographical analyses (AMOVA) reveal substantial genetic substructure among Calé Roma across Spanish regions, consistent with historical isolation and localised gene flow. Additionally, a striking sex-biased admixture is observed when comparing the current results with previous mitochondrial DNA (mtDNA) data, with paternal South Asian ancestry being twice as high as maternal contributions, suggesting that Roma communities have historically been more inclined to integrate non-Roma women. The genetic landscape of the Iberian Roma is shaped by a complex history of founder effects, admixture, and isolation. The observed genetic substructure and sex-biased admixture reflect historical social dynamics. These results contribute to the broader understanding of Roma genetic diversity and demography in Spain and underscore the importance of integrating Y chromosome, autosomal, and mtDNA data in future studies.

CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.