Search Results (689)

Wastewater-based epidemiology (WBE) offers valuable insight into viral circulation at the community level. In this study, we combined digital PCR (dPCR) with molecular typing to investigate the prevalence and diversity of human adenoviruses (HAdVs) in untreated wastewater samples collected throughout Italy. HAdV genomes were detected in over 93% of the 168 samples analyzed, with concentrations up to 4.5 × 10⁶ genome copies per liter. For genotypic characterization, we used nested PCR followed by Sanger and Oxford Nanopore Technologies (ONTs) long-read sequencing. While Sanger sequencing identified three dominant genotypes (HAdV-A12, HAdV-B3, and HAdV-F41), ONT sequencing provided enhanced resolution, confirming all previously identified types and revealing seven additional genotypes: HAdV-B21, HAdV-C5, HAdV-D45, HAdV-D46, HAdV-D49, HAdV-D83, and HAdV-F40. This comprehensive approach highlights the added value of ONT long-read sequencing in uncovering the genetic complexity of adenoviruses in wastewater, particularly in detecting rare or low abundance types that conventional methods may miss. Our findings highlight the value of integrating quantitative and high-resolution molecular tools in WBE to improve surveillance and better understand the epidemiology of viral pathogens circulating in the human population. Full article

Kimchi is a fermented Korean food typically made with napa cabbage, garlic, radish, ginger, and chili pepper. It is becoming increasingly popular due to its flavor, high fiber content, and purported probiotic benefits. The microbial ecology of the fermentation community has been extensively studied, though what’s less understood is how its microbial community changes when nutrients or pathogens are introduced. To study this, we used gnotobiotic cabbage media inoculated with a kimchi starter culture as a model system. These inoculated samples were exposed to E. coli or Bacillus cereus, with or without added nutrients in the form of tryptic soy broth (TSB). We tracked pH, colony-forming units (CFUs), and community composition over time. We also used Oxford Nanopore sequencing to analyze the 16S rRNA gene (V4–V9), followed by use of the Emu algorithm for taxonomic assignments. As expected, LABs suppressed pathogens, but this effect was weaker early on in the nutrient-rich condition. Pathogen exposure changed the overall community—Lactobacillus species became more common, and Leuconostoc mesenteroides less so. Interestingly, adding nutrients alone caused similar microbial shifts to those seen with pathogen exposure. This could suggest that nutrient levels have a larger impact on the final microbiome structure than direct microbial competition. Together, these findings suggest that monitoring total microbial composition, and not just the presence of pathogens, may be important for ensuring kimchi fermentation reproducibility. Full article

Background: Sanger sequencing remains the gold standard for characterizing genetic variants in short DNA fragments (<700 bp). However, the increasing demand for short TATs and high sensitivities in variant detection, particularly in oncohematology, is driving the need for more efficient methods. Next-generation sequencing (NGS) has improved sensitivity and allows for the simultaneous analysis of multiple genes, but it is still costly and time-consuming. Consequently, Sanger sequencing continues to be widely used. In this study, we have compared Sanger sequencing with Oxford Nanopore technology (ONT), which offers enhanced sensitivity and faster sequencing, delivering diagnostic results within 24 h. Methods: This study involves 164 samples (for a total of 174 analyzed regions of interest) previously characterized using either Sanger sequencing or a next-generation sequencing (NGS) panel, categorized by their genetic alterations. Validation was conducted on 15 genes crucial for the diagnosis, prognosis, or identification of drug resistance in myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). The primary objective was to assess whether MinION could identify the same variants previously detected in these patients. Results and Conclusions: With a 99.43% concordance observed in our comparison, our results support the implementation of MinION technology in routine variant detection in MPN, MDS, AML, and CML cases due to its significant advantages over Sanger sequencing. Full article

Background: Bromus inermis is a high-quality perennial forage grass in the Poaceae family, with significant ecological and economic value. While its chloroplast genome has been sequenced, the mitochondrial genome of this species remains poorly understood due to the inherent complexity and frequent recombination of plant mitochondrial genomes. Methods: We sequenced the complete mitochondrial genome of B. inermis using both Illumina Novaseq6000 and Oxford Nanopore PromethION platforms. Subsequently, comprehensive bioinformatics analyses were performed, including genome assembly and annotation, repetitive sequence identification, codon usage analysis, RNA editing site prediction, the detection of chloroplast-derived sequences, and phylogenetic reconstruction. Results: The mitochondrial genome of B. inermis was determined to be 515,056 bp in length, with a GC content of 44.34%, similar to other Poaceae species. This genome encodes 35 protein-coding genes, 22 tRNA genes, and 10 rRNA genes. Repetitive sequences account for 16.2% of the genome, totaling 83,528 bp, including 124 simple sequence repeats, 293 dispersed repeats, and 31 tandem repeats. A total of 460 RNA editing sites were identified, among which 430 were nonsynonymous. Additionally, 110 putative chloroplast-derived sequences were detected. A phylogenetic analysis based on mitochondrial genome data clarified the species’ evolutionary position within Poaceae. Conclusions: This study provides genetic resources for evolutionary research on and the communication of organelle genomes. Meanwhile, it also lays a solid foundation for the better development and utilization of the germplasm resources of B. inermis. Full article

Antimicrobial resistance (AMR) is an emerging global threat that is expanding in many areas of the world. Wastewater-based epidemiology (WBE) is uniquely suited for use in areas of the world where clinical surveillance is limited or logistically slow to identify emerging threats, such as in Sub-Saharan Africa (SSA). Wastewater was analyzed from three urban areas of Kampala, including a local HIV research clinic and two informal settlements. Wastewater extraction was performed using a low-cost, magnetic bead-based protocol that minimizes consumable plastic consumption followed by sequencing on the Oxford Nanopore Technology MinION platform. The majority of the analysis was performed using cloud-based services to identify AMR biomarkers and bacterial pathogens. Assemblies containing AMR pathogens were isolated from all locations. As one example, clinically relevant AMR biomarkers for multiple drug classes were found within Acinetobacter baumannii genomic fragments. This work presents a metagenomic WBE workflow that is compatible with areas of the world without robust water treatment infrastructure. This study was able to identify various bacterial pathogens and AMR biomarkers without shipping water samples internationally or relying on complex concentration methods. Due to the time-dependent nature of wastewater surveillance data, this work involved cross-training researchers in Uganda to collect and analyze wastewater for future efforts in public health development. Full article

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the double homeobox 4 (DUX4) gene. In this study, an analysis of human FSHD muscle biopsies revealed differential expressions of six m6A regulators, including writers, readers and eraser proteins. In immortalized human FSHD myoblasts, we found higher levels of mRNA and protein expression of a major m6A regulator, methyltransferase-like protein 3 (METTL3), in comparison with myoblasts from unaffected siblings (UASbs). Quantification of the overall RNA m6A levels in the FSHD myoblasts revealed significant elevation compared with their UASb, which was reversed to UASb levels following treatment with an antisense oligonucleotide targeting the DUX4 mRNA. Using Oxford Nanopore direct-RNA sequencing, we mapped m6A across the transcriptome and identified genes harboring differential methylated m6A sites, including several involved in iron homeostasis. Western blot protein quantification showed that FSHD myoblasts had higher levels of ferritin-heavy chain-207 isoform and mitoferrin-1. In addition, our data showed elevation in mitochondrial ferrous iron in FSHD myoblasts. Our findings suggest that m6A RNA modifications play a pivotal role in FSHD pathophysiology and may serve as biomarker for this disease. Full article

Marssonina coronaria is the causal agent of apple blotch, which poses a significant threat to apple production worldwide. Here, Illumina and Oxford Nanopore sequencing were combined to generate a high-quality M. coronaria YL1 genome assembly (54.5 Mb, 23 contigs). Based on genome annotation, 97 candidate effector proteins (CEPs) were identified, and 61 CEPs were successfully cloned for functional analysis. Transient expression assays in Nicotiana benthamiana revealed that eight CEPs significantly suppressed BAX-induced cell death, with McCEP12, McCEP23, McCEP24, and McCEP52 concurrently inhibiting flg22-triggered reactive oxygen species bursts. Two signal peptide-dependent cell death-inducing effectors were identified: McNLP1, containing an NPP1 domain, and McCEP3. McCEP3 exhibited evolutionary conservation within Ascomycota, with its homologous gene VmMcCEP3 from Valsa mali inducing cell death in N. benthamiana. McEP03-triggered cell death was independent of BAK1/SOBIR1 receptor kinases. This study provides a high-quality genomic resource for M. coronaria and sheds light on the mechanisms by which its CEPs modulate host immunity, offering new insights into the molecular interactions between the pathogen and its host. Full article

Group A rotaviruses (RVAs) are a major cause of acute dehydrating diarrhea in infants and young animals worldwide. In rabbits, RVAs are associated with enteric disease, likely in combination with other pathogens. We report the identification and characterization of a lapine RVA strain in an Italian rabbit breeding farm. Increased mortality rates associated with enteric symptoms were reported in the facility in post-weaning rabbits around 40 days of age. By quantitative RT-PCR, an RVA strain was identified in the intestinal contents of deceased rabbits. A PCR-based enrichment protocol coupled with Nanopore sequencing allowed the reconstruction of the nearly complete genome of a rabbit RVA strain, Rabbit-wt/ITA/36-9/2022/G3P[14], with a genotype constellation (G3-P[14]-I2-R2-C2-M3-A9-N2-T6-E5-H3) conserved among lapine RVAs. Each of the 11 gene segments displayed high nucleotide identity and phylogenetic clustering with lapine rotavirus strains, as well as two Belgian human G3P[14] strains, which had been shown to have a zoonotic (lapine) origin. However, the NSP2 gene of strain 36-9 clustered closer with a group of rare human G3P[9] strains, suggesting a common path during their evolution. Gathering sequence data on animal RVAs is pivotal to reconstructing the history of homologous and heterologous RVAs in various mammals, including humans. Full article

CLN2 disease (neuronal ceroid lipofuscinosis type 2) is an ultra-rare lysosomal storage disorder caused by mutations in the TPP1/CLN2 gene, resulting in impaired tripeptidyl peptidase 1 (TPP1) activity. The timely initiation of enzyme replacement therapy is pivotal for attenuating progressive and irreversible neurodegeneration. This study aimed to benchmark the performance of Oxford Nanopore long-read sequencing (ONT-LRS) for targeted TPP1 mutation detection in a Turkish CLN2 cohort and to assess its concordance with orthogonal validation methods, including Sanger sequencing and enzymatic activity assays. Using a custom-designed primer panel, the entire TPP1 gene (6846 bp) was sequenced on the Oxford Nanopore (ONT) MinIon platform in seven clinically confirmed CLN2 index patients and sixteen unaffected family members. Detected variants were validated via Sanger sequencing and correlated with TPP1 enzyme activity in leucocytes and dried blood spots. Four pathogenic or likely pathogenic TPP1 variants were identified: c.622C>T (p.Arg208*), c.857A>G (p.Asn286Ser), c.1204G>T (p.Glu402*), and c.225A>G (p.Gln75=), along with fourteen additional benign variants. Variant allele frequencies were 50% for c.622C>T, 28.6% for c.1204G>T, 14.3% for c.857A>G, and 7.1% for c.225A>G. Notably, this is the first report to document the homozygous state of the c.857A>G variant and the compound heterozygous configuration of the c225A>G and c.622C>T variants in CLN2 patients, thereby expanding the known mutational landscape. In contrast, the globally common variant c.509-1G>C was not observed, suggesting regional variation in TPP1 mutation patterns. Consistent with the prior Turkish studies, c.622C>T (p.Arg208*) was the most prevalent variant, followed by c.1204G>T (p.Glu402*). TPP1 enzymatic activity was significantly reduced in all affected individuals (p < 0.0001), supporting the functional relevance of the identified variants. ONT-LRS offers a robust, cost-effective platform for high-resolution analysis of the TPP1 gene. Integrating molecular and biochemical data improves diagnostic precision and supports timely, targeted interventions for CLN2 disease, particularly in regions with high consanguinity and limited diagnostic infrastructure. Full article

Brucella intermedia, a gram-negative, non-lactose-fermenting, aerobic, rod-shaped bacterium, is found in environmental sources (e.g., soil and water). In 2020, Ochrobactrum was reclassified as Brucella. We conducted a genomic analysis of B. intermedia from hospital wastewater samples in western Ghana. A hybrid genome assembly was constructed integrating short-read data from DNA Nanoball sequencing with long-read sequences generated by Oxford Nanopore MinION technology. Identification and antimicrobial susceptibility profiles were determined using MicroScan autoSCAN-4 based on Clinical and Laboratory Standard Institute documents. ResFinder and CARD Resistance Gene Identifier (RGI) were used to identify antimicrobial resistance (AMR) genes, and BLAST and VFDB datasets were used to identify virulence factor genes. The complete genome had two chromosomes, no plasmid, and a high average nucleotide identity value (98.05%) with B. intermedia. Resistance to trimethoprim-sulfamethoxazole was revealed, the first report in this species. CARD RGI revealed the presence of AMR genes, including ANT(9)-Ic and adeF. Local BLAST analysis revealed Cgs, a B. melitensis virulence factor. B. intermedia is an opportunistic human pathogen clinically isolated several times, suggesting the importance of accurately identifying multidrug resistance. B. intermedia may possess virulence factors similar to those of B. melitensis. Further study is needed to fully elucidate its pathogenesis. Full article

Objective: Mitochondria drive cellular energy production and regulate key biological processes. High levels of heteroplasmic in mitochondrial DNA (mtDNA) variants can cause mitochondrial dysfunction and clinical symptoms. Third-generation sequencing overcomes the limitations of traditional mtDNA analysis methods, offering improved cost, throughput, and sensitivity. We developed an integrated approach for analyzing methylation patterns and genetic variations in mtDNA and ADME genes. Methods: We implemented Oxford Nanopore’s long-read sequencing with adaptive sampling (AS) to enrich enzymatically linearized mtDNA and absorption, distribution, metabolism, and excretion (ADME) genes without PCR amplification, enabling native sequencing in adipose-derived mesenchymal stem cells (AdMSC). Our custom algorithm preserved phase relationships between base modifications and sequence polymorphisms. Results: We identified differential methylation patterns in ADME genes correlating with specific genetic variants, suggesting epigenetic regulation of drug response. Adaptive sampling identifies a wider range of variant diversity, while whole genome sequencing (WGS) uncovers higher-frequency hotspots. Both methods offer complementary insights into mitochondrial heteroplasmy. In mtDNA, direct sequencing showed extensive hypomethylation, and low levels of non-CpG methylation were detected regardless of sequencing coverage depth. These sparse methylation patterns showed non-random distribution, correlating with functional regions and heteroplasmic sites. Conclusions: This study demonstrates the utility of adaptive sampling for the integrated analysis of mtDNA heteroplasmy and native base modifications, revealing widespread hypomethylation independent of coverage depth. The approach showcases the potential for combined pharmacoepigenomic and mitochondrial profiling in precision medicine, disease modeling, and therapeutic development. Full article

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder characterized by ataxia, sensory loss and pyramidal signs. While the majority of FRDA cases are caused by biallelic GAA trinucleotide repeat expansions in intron 1 of FXN, there is a subset of patients harboring a heterozygous pathogenic small variant compound-heterozygous with a GAA repeat expansion. We report on the diagnostic journey of a 21-year-old patient who was clinically suspected of having FRDA at the age of 12 years. Genetic testing included fragment analysis, gene panel analysis and exome sequencing, which only detected one pathogenic heterozygous missense variant (c.389 G>T,p.Gly130Val) in FXN. Although conventional repeat analyses failed to detect GAA expansions in our patient, subsequent short-read genome sequencing (GS) indicated a potential GAA repeat expansion. This finding was confirmed by long-read GS, which in addition revealed a complex pattern of interruptions. Both large and small GAA expansions with divergent interruptions containing G, A, GA, GAG and/or GAAG sequences were present within one allele, indicating mosaic sequence variations. Our findings underscore the complexity of repeat expansions which can exhibit both interruptions and somatic instability. We also highlight the utility of long-read GS in unraveling intricate genetic profiles, ultimately contributing to more accurate diagnoses in clinical practice. Full article

Kazakhstan’s rich biodiversity includes diverse apple populations, notably the wild apple tree (Malus sieversii) prized for traits like disease resistance and adaptability, potentially aiding breeding programs. Analyzing their microbiomes offers insights into bacterial diversity and how it influences apple tree development, making it a reliable method for understanding ecological interactions. In this research, 334 apple tree samples were collected from different mountain ranges in southeastern Kazakhstan. An analysis using nanopore-based 16S rRNA sequencing showed a distinct similarity in the microbiome compositions of samples from the Zhongar and Ile Alatau mountain ranges, with a predominance of Pseudomonadaceae, Enterobacteriaceae, and Microbacteriaceae. In contrast, samples from Ketmen ridge showed a higher prevalence of Enterobacteriaceae. Alongside the less represented Pseudomonadaceae family, in the Ketmen ridge region, bacteria of the Xanthomonadaceae, Alcaligenaceae, and Brucellaceae families were also present. Across all regions, beneficial plant-associated bacteria were identified, such as Pseudomonas veronii, Stenotrophomonas geniculata, and Kocuria rhizophila, potentially enhancing plant resilience. However, opportunistic phytopathogens were also detected, including Pseudomonas viridiflava and Serratia marcescens, particularly in the Ile Alatau region. These findings highlight the complex microbial interactions in M. sieversii, thus offering key insights into host—microbe relationships that can inform apple breeding and ecological preservation efforts. Full article

Transposable elements (TEs) are major drivers of plant genome plasticity, but the immediate molecular consequences of new TE insertions remain poorly understood. In this study, we generated a wild-type Arabidopsis thaliana population with novel insertions of ONSEN retrotransposon to investigate early epigenomic and transcriptomic changes using whole-genome and cDNA nanopore sequencing. We found that novel ONSEN insertions were distributed non-randomly, with a strong preference for genic regions, particularly in chromatin enriched for H2A.Z, H3K27me3, and H3K4me2. Most full-length ONSEN insertions within genes were rapidly recognized and spliced out as new introns (intronization), thereby mitigating potential deleterious effects on transcript isoforms. In some cases, ONSEN insertions provided alternative transcription start or termination sites, generating novel transcript isoforms. Genome-wide methylation analysis revealed that new ONSEN copies were efficiently and precisely targeted by DNA methylation. Independently on the location of the original ONSEN element, the euchromatic and heterochromatic insertions display distinct methylation signatures, reflecting the action of different epigenetic pathways. In conclusion, our results demonstrate that DNA methylation and alternative splicing are effective control mechanisms safeguarding the plant genome and transcriptome integrity after retrotransposition burst. Full article

The phenomenal progress in biotechnology and genomics is both inspiring and overwhelming—a classic curse of choice, particularly when it comes to selecting methods for mapping transgene DNA integration sites. Transgene localization remains a crucial task for the validation of transgenic mouse or other animal models generated by pronuclear microinjection. Due to the inherently random nature of DNA integration, reliable characterization of the insertion site is essential. Over the years, a vast number of mapping methods have been developed, and new approaches continue to emerge, making the choice of the most suitable technique increasingly complex. Factors such as cost, required reagents, and the nature of the generated data require careful consideration. In this review, we provide a structured overview of current transgene mapping techniques, which we have broadly classified into three categories: classic PCR-based methods (such as inverse PCR and TAIL-PCR), next-generation sequencing with target enrichment, and long-read sequencing platforms (PacBio and Oxford Nanopore). To aid in decision-making, we include a comparative table summarizing approximate costs for the methods. While each approach has its own advantages and limitations, we highlight our top four recommended methods, which we believe offer the best balance of cost-effectiveness, reliability, and simplicity for identifying transgene integration sites. Full article