Search Results (442)

The Chinese hamster ovary K1 cell line (CHO-K1) constitutively produces retroviral-like particles (RVLPs) containing reverse transcriptase (RT) activity, which, thus far, have not been shown to be infectious. Since infectious retroviruses have been reported in other rodent species, this study was undertaken to investigate the presence of latent, infectious, endogenous retroviruses (ERVs) in CHO-K1 cells by using chemical induction assays and detection of activated virus using the highly sensitive, product-enhanced RT (PERT) assay, with subsequent infectivity analysis in cell lines of different species, including human. The results demonstrated activation of A-type and C-type retroviral particles based on transmission electron microscopy and increased production of cell-free RT-particles after treatment of the cells with 5-iodo-2′-deoxyuridine and 5-azacytidine, which was greater with dual treatment than with each inducer alone. Induction of A- and C-type particles was confirmed in dual-drug-treated CHO-K1 cells by long-read high-throughput sequence (HTS) analysis. Infectivity studies performed by inoculating human A549, HEK-293, and MRC-5 cells; African green monkey Vero cells; Mus dunni cells; and CHO-K1 cells with supernatant containing RT-particles from dual-treated CHO-K1 cells indicated the absence of a replicating retrovirus in supernatant from extended cell culture using the PERT assay. Furthermore, short-read HTS analysis did not show evidence of integration of retroviral sequences in inoculated A549 and 293 cells. The overall results showed no evidence for latent, infectious, endogenous RVLPs in CHO-K1 cells. Full article

Background/Objectives: Plant mitogenomes display remarkable variation in size, structure, and gene content, yet their evolutionary causes remain unclear. Araceae, the most significant family within Alismatales, encompasses both aquatic and terrestrial lineages, providing an excellent system for studying how ecological shifts influence mitogenome evolution. Methods: We assembled and annotated four new mitogenomes using both short- and long-read sequencing, including three aquatic taxa (Pistia stratiotes L., Spirodela intermedia W. Koch, Wolffia australiana (Benth.) Hartog & Plas) and one terrestrial species (Amorphophallus konjac K. Koch). Along with five previously published mitogenomes, we performed comparative analyses across nine Araceae species. Results: These mitogenome sizes varied from ~178 kb to ~877 kb, consisting of one to 19 circular molecules, with aquatic species generally having smaller and simpler structures. Plastid-derived sequences (MTPTs) contributed 1.2–10.6% of genome content, peaking in Zantedeschia aethiopica (L.) Spreng. Despite significant structural heterogeneity, all species maintained core respiratory genes under strong purifying selection, while ribosomal protein-coding genes showed lineage-specific loss. RNA editing ranged from 363 to 772 sites per mitogenome, with the number of sites independent of mitogenome size. Conclusions: Overall, this study uncovers the dynamic evolutionary patterns of Araceae mitogenomes and offers a framework for understanding how habitat shifts between aquatic and terrestrial environments influence mitogenome diversity in plants. Full article

Transposable elements (TEs), once regarded as genomic “junk,” are now recognized as powerful regulators of gene expression, genome stability, and innate immunity. In the context of neurodegeneration, particularly Amyotrophic Lateral Sclerosis (ALS), accumulating evidence implicates TEs as active contributors to disease pathogenesis. ALS is a fatal motor neuron disease with both sporadic and familial forms, linked to genetic, epigenetic, and environmental factors. While coding mutations explain a subset of cases, advances in long-read sequencing and epigenomic profiling have unveiled the profound influence of non-coding regions—especially retrotransposons such as LINE-1, Alu, and SVA—on ALS onset and progression. TEs may act through multiple mechanisms: generating somatic mutations, disrupting chromatin architecture, modulating transcriptional networks, and triggering sterile inflammation via innate immune pathways like cGAS-STING. Their activity is normally repressed by epigenetic regulators, including DNA methylation, histone modifications, and RNA interference pathways; however, these controls are compromised in ALS. Taken together, these insights underscore the translational potential of targeting transposable elements in ALS, both as a source of novel biomarkers for patient stratification and disease monitoring, and as therapeutic targets whose modulation may slow neurodegeneration and inflammation. This review synthesizes the current knowledge of TE biology in ALS; integrates findings across molecular, cellular, and systems levels; and explores the therapeutic potential of targeting TEs as modulators of neurodegeneration. Full article

Listeria monocytogenes is a ubiquitous foodborne pathogen whose occurrence in food and food-processing environments raises public-health concerns, particularly when isolates carry antimicrobial-resistance determinants. Next-generation sequencing (NGS) is increasingly used to detect resistance genes and to predict phenotypic resistance. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines, PubMed, Web of Science, and Scopus were searched for original articles (2015–2024) that used second- and/or third-generation sequencing to characterize antibiotic resistance in L. monocytogenes from food and food-processing environments. After deduplication and screening, 58 studies were included from an initial 418 records. NGS reliably detected a set of recurrent resistance determinants across diverse sample types and geographies. The fosX locus (intrinsic fosfomycin-related marker) was effectively ubiquitous across studies, while acquired determinants were variably distributed: lin (35/58 studies, 60.34%), norB (33/58, 56.90%), and tetracycline genes overall in 20/58 (34.48%) with tetM as the most common (11/58, 18.97%). Reported concordance between the genotypes and phenotypes for acquired resistance was very high (>99% for most agents), with notable exceptions (e.g., ciprofloxacin and some fosfomycin cases). Common analysis pipelines and databases included ResFinder, CARD, BIGSdb-Lm, ABRicate, and ARIBA; most sequencing used Illumina short reads, with an increasing use of long-read or hybrid approaches. NGS is a powerful surveillance tool for detecting resistance determinants and for source-tracking, but its predictive value depends on integration with phenotypic testing, standardized reporting, and comprehensive, curated databases. Key gaps include inconsistent phenotype reporting, variable database coverage, and limited assessment of gene expression/regulatory effects. Full article

(1) Background: Water buffaloes (Bubalus bubalis) are important for dairy and meat production. Up to now, genomic analysis has focused on female subjects, leaving the Y chromosome essentially unknown. Advances in third-generation sequencing (TGS) made it possible to improve the study of complex genome sequences, such as buffalo and other mammalian species including humans. (2) Methods: In this study, we applied TGS-based long-read sequencing to generate, in one step, high-quality whole-genome sequences, which can take full advantage of a rapid bioinformatic pipeline, such as that described in the companion paper. (3) Results: Five male buffalo genomes have been fully sequenced at relatively high depth (20–40×) which, combined with the read length typical of TGS, provide the basis for important insights into male-specific genetic traits, including those linked to meat and milk production. (4) Conclusions: With the use of TGS technologies, we offer a complete strategy for fast, one-step genome sequencing which can also be applied to other farm animals with a comparably large genome. This approach can help in revealing genetic features characteristic of an animal individual beyond the simple assessment of a number of SNPs or other known sequence variations, thus supporting improved genetic selection for dairy productivity and future research on genetic variability in buffalo breeds. Full article

Metagenomic next-generation sequencing (mNGS) is increasingly employed for the diagnosis of lower respiratory tract infections (LRTIs). However, the relative diagnostic performance of long-read versus short-read sequencing platforms remains incompletely defined. For this systematic review, a search was conducted in PubMed, Embase, Scopus, Web of Science, and Google Scholar to identify studies directly comparing long-read (e.g., Oxford Nanopore, PacBio) and short-read (e.g., Illumina, Ion Torrent, BGISEQ) metagenomic sequencing for the diagnosis of LRTI. Eligible studies reported diagnostic accuracy or comparative performance between platforms. Risk of bias was evaluated using the QUADAS-2 tool. Thirteen studies met inclusion criteria. Reported platforms included Illumina, Oxford Nanopore, PacBio, Ion Torrent, and BGISEQ-500. A total of 13 studies met inclusion criteria. Across studies reporting sensitivity, average sensitivity was similar for Illumina (71.8%) and Nanopore (71.9%). Specificity varied substantially, ranging from 42.9 to 95% for Illumina and 28.6 to 100% for Nanopore. Concordance between platforms ranged from 56 to 100%. Illumina consistently produced superior genome coverage (approaching 100% in most reports) and higher per-base accuracy, whereas Nanopore demonstrated faster turnaround times (<24 h), greater flexibility in pathogen detection, and superior sensitivity for Mycobacterium species. Risk of bias was frequently high or unclear, particularly in patient selection (6 studies), index test interpretation (5), and flow and timing (4), limiting the robustness of pooled estimates. Long-read and short-read mNGS platforms exhibit comparable strengths in the diagnosis of LRTIs. Illumina remains optimal for applications requiring maximal accuracy and genome coverage, whereas Nanopore offers rapid, versatile pathogen detection, particularly for difficult-to-detect organisms such as Mycobacterium. However, there are certain limitations of the review, including a lack of comparable outcomes reported in all studies; therefore, further research is warranted to address this. Full article

Background/Objectives: Actinobacillus pleuropneumoniae is an important respiratory tract pathogen of swine worldwide. Insertion sequences (ISs) play a major role in the transfer of antimicrobial resistance (AMR) among various porcine respiratory tract pathogens. In this study, three A. pleuropneumoniae genomes were investigated for the presence of a novel IS. Methods: Analysis of the draft genomes of three A. pleuropneumoniae serovar 8 isolates (AP_1, AP_120, AP_123) suggested the presence of a novel IS. A closed whole-genome sequence was generated for strain AP_123 by hybrid assembly of Oxford Nanopore MinION long-reads and Illumina MiSeq short-reads, followed by sequence analysis using standard online tools. Transfer was tested by natural transformation. Antimicrobial susceptibility testing was conducted by broth microdilution following Clinical and Laboratory Standards Institute standards. Results: A novel IS, designated ISApl4, was detected in all three genomes. ISApl4 is 712 bp in size and has a transposase gene (tnp) of 654 bp. Moreover, it has perfect terminal 14-bp inverted repeats and produces 8-bp direct repeats at its integration site. This IS was found in 39 copies in the AP_123 genome, two of which formed the 5,765-bp pseudo-compound transposon Tn7560. This transposon carries four AMR genes: sul2 (sulfonamide resistance), strA-strB (streptomycin resistance), and tet(Y) (tetracycline resistance). RT-PCR confirmed tnp gene expression and horizontal transfer of Tn7560 into A. pleuropneumoniae MIDG2331. Conclusions: This study identified the novel ISApl4 in porcine A. pleuropneumoniae and its association with the novel pseudo-compound transposon Tn7560, which proved to be an active element capable of disseminating multidrug resistance amongst A. pleuropneumoniae. Full article

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) β-lactamases represent a threat for public health. Their dissemination is often mediated by mobile genetic elements (MGEs), but plasmid identification and characterization could be hindered by sequencing limitations. Hybrid assembly may overcome these barriers. Eight ESBL/pAmpC-producing E. coli isolates from broilers were sequenced using Illumina (short-read) and Oxford Nanopore MinION (long-read). Assemblies were generated individually and using a hybrid approach. Plasmids were typed, annotated, and screened for antimicrobial resistance genes (ARGs), MGEs, and virulence factors. Short-read assemblies were highly fragmented, while long reads improved contiguity but showed typing errors. Hybrid assemblies produced the most accurate and complete plasmids, including more circularized plasmids. Long and hybrid assemblies detected IS26 associated with ESBL genes and additional virulence genes not identified by short reads. ARG profiles were consistent across methods, but structural resolution and contextualization of resistance loci were superior in hybrid assembly. Hybrid assembly integrates the strengths of short- and long-read sequencing, enabling accurate plasmid reconstruction and improved detection of resistance-associated MGEs. This approach may enhance genomic surveillance of ESBL/pAmpC plasmids and support strategies to mitigate antimicrobial resistance. Full article

Advancements in genomic technologies have transformed prenatal genetic testing, offering more accurate, comprehensive, and noninvasive approaches to reproductive care. This review provides an in-depth overview of current methodologies and emerging innovations, including expanded carrier screening (ECS), cell-free DNA (cfDNA) testing, chromosomal microarray analysis (CMA), and sequencing-based diagnostics. We highlight how next-generation sequencing (NGS) technologies have revolutionized carrier screening and fetal genome analysis, enabling detection of a broad spectrum of genetic conditions. The clinical implementation of cfDNA has expanded from common aneuploidies to include copy number variants (CNVs), and single-gene disorders. Diagnostic testing has similarly evolved, with genome sequencing outperforming traditional CMA and exome sequencing through its ability to detect both sequence and structural variants in a single assay. Emerging tools such as optical genome mapping, RNA sequencing, and long-read sequencing further enhance diagnostic yield and variant interpretation. This review summarizes major technological advancements, assesses their clinical utility and limitations, and outlines future directions in prenatal genomics. Full article

The filarioid nematode Dirofilaria immitis is the causative agent of heartworm disease, a major parasitic infection of canids, felids and occasionally humans. Current prevention relies on macrocyclic lactone-based chemoprophylaxis, but the emergence of drug resistance highlights the need for new intervention strategies. Here, we applied a machine learning (ML)-based framework to predict and prioritise essential genes in D. immitis in silico, using genomic, transcriptomic and functional datasets from the model organisms Caenorhabditis elegans and Drosophila melanogaster. With a curated set of 26 predictive features, we trained and evaluated multiple ML models and, using a defined threshold, we predicted 406 ‘high-priority’ essential genes. These genes showed strong transcriptional activity across developmental stages and were inferred to be enriched in pathways related to ribosome biogenesis, translation, RNA processing and signalling, underscoring their potential as anthelmintic targets. Transcriptomic analyses suggested that these genes are associated with key reproductive and neural tissues, while chromosomal mapping revealed a relatively even genomic distribution, in contrast to patterns observed in C. elegans and Dr. melanogaster. In addition, initial evidence suggested structural variation in the X chromosome compared with a recently published D. immitis assembly, indicating the importance of integrating long-read sequencing with high-throughput chromosome conformation capture (Hi-C) mapping. Overall, this study reinforces the potential of ML-guided approaches for essential gene discovery in parasitic nematodes and provides a foundation for downstream validation and therapeutic target development. Full article

Background: Antimicrobial drugs are used to treat bacterial diseases in livestock production systems, including bovine respiratory disease (BRD) in feedlot cattle. It is recommended that therapeutic antimicrobial use (AMU) in food animals be informed by diagnostic tests to limit the emergence of antimicrobial resistance (AMR) and preserve the effectiveness of available drugs. Recent evidence demonstrates preliminary support for the pen as a prospective target for AMR testing-based interventions in higher-risk cattle. Methods: A previously reported agent-based model (ABM) was modified and then used in this study to investigate the potential for different pen-level sampling and laboratory testing-informed BRD treatment strategies to favorably impact selected antimicrobial stewardship and management outcomes in the western Canadian context. The incorporation of sample testing to guide treatment choice was hypothesized to reduce BRD relapses, subsequent AMU treatments and resultant AMR in sentinel pathogen Mannheimia haemolytica. The ABM was extended to include a discrete event simulation (DES) workflow that models the testing process, including the time at sample collection (0 or 13 days on feed) and the type of AMR diagnostic test (antimicrobial susceptibility testing or long-read metagenomic sequencing). Candidate testing scenarios were simulated for both a test-only control and testing-informed treatment (TI) setting (n = 52 total experiments). Key model outputs were generated for both the pen and feedlot levels and extracted to data repositories. Results: There was no effect of the TI strategy on the stewardship or economic outcomes of interest under baseline ecological and treatment conditions. Changes in the type and number of uses by antimicrobial class were observed when baseline AMR in M. haemolytica was assumed to be higher at feedlot arrival, but there was no corresponding impact on subsequent resistance or morbidity measures. The impacts of sample timing and diagnostic test accuracy on AMR test positivity and other outputs were subsequently explored with a theoretical “extreme” BRD treatment protocol that maximized selection pressure for AMR. Conclusions: The successful implementation of a pen-level sampling and diagnostic strategy would be critically dependent on many interrelated factors, including the BRD treatment protocol, the prevalences of resistance to the treatment classes, the accuracy of available AMR diagnostic tests, and the selected “treatment change” thresholds. This study demonstrates how the hybrid ABM-DES model can be used for future experimentation with interventions proposed to limit AMR risk in the context of BRD management. Full article

Background: Medulloblastoma is the childhood tumor with the highest morbidity and mortality worldwide. This type of cancer is characterized by a high degree of heterogeneity that gives rise to different molecular groups with disparities in the clinical presentation and prognosis. Among the molecular differences, one of the most relevant factors is alternative splicing, as it is responsible for transcriptomic diversity. EZH2 is a gene processed by alternative splicing that functions as an epigenetic regulator. In cancer, certain EZH2 mRNA variants are associated with tumorigenesis; however, in medulloblastoma, the alternative splicing pattern of EZH2 has not been studied. Currently, the best tool for identifying alternative splicing variants is long-read sequencing. Methods: We amplified the most variable region of EZH2 alternative splicing and used nanopore sequencing to obtain the transcriptional profile of the gene in patients with medulloblastoma. We verified the variants identified with Sanger sequencing and digital RT–PCR. Finally, we studied the relationship between the expression levels and the clinical–biological characteristics of the patients. Results: We identified seven mRNA variants of EZH2 expressed in medulloblastoma patients, five of which had not been reported previously. In addition, high expression of the novel variant EZH2_RetI8 was associated with patient mortality (p < 0.05). Conclusions: This is the first evidence of the EZH2 mRNA variant profile in medulloblastoma, revealing seven alternative transcripts, one of which is associated with patient mortality. This is a clear example of the complexity of the transcriptome and how long-read sequencing can resolve alternative splicing patterns. Full article

Lower respiratory tract infections (LRTIs) represent a significant global health concern, and the accurate identification of pathogens is crucial for patient care. Culture-based methods are the gold standard, but their detection abilities are limited. Next-generation sequencing (NGS) offers a promising method for comprehensive microbial detection, providing valuable information for clinical practice. In this study, 144 bronchoalveolar lavage fluid samples were collected, culture-based diagnostics were performed, and bacterial microbiome profiles were generated by short-read sequencing of the V4 region of the 16S rRNA gene using Illumina technologies and long-read sequencing with Oxford Nanopore Technologies (ONT) to determine the full-length 16S rRNA gene. The most common genera detected by NGS included Streptococcus, Staphylococcus, Veillonella, Prevotella, Rothia, Enterococcus, and Haemophilus. Short-read sequencing detected cultured bacteria at the genus level in ~85% of cases, while long-read sequencing demonstrated agreement with cultured species in ~62% of cases. In three cases, long-read sequencing identified the uncommon potential lung pathogen Tropheryma whipplei not detected with traditional culturing techniques. The NGS results showed a partial overlap with culture as the current diagnostic gold standard in LRTI. Additionally, NGS detected a broader spectrum of bacteria, revealed fastidious potential pathogens, and offered deeper insights into the complex microbial ecosystem of the lungs. Full article

Coptis trifolia (threeleaf goldthread) offers a valuable comparative system for investigating the evolution and regulation of benzylisoquinoline alkaloid (BIA) synthesis. In this study, we analyzed the leaf and root transcriptomes of C. trifolia using both long-read and short-read RNA-Sequencing. We assembled 41,926 unigenes (≥500 bp) and identified 37 genes related to BIA biosynthesis, including two transcription factors, bHLH1 and WRKY1. The number of BIA genes identified in C. trifolia was comparable to that in other Coptis species. Transcriptome analysis revealed that most of these genes were more highly expressed in roots than leaves. Consistent with previous studies, C. trifolia contained a single (S)-stylopine synthase (SPS) gene homolog, potentially multifunctional for (S)-canadine synthase (CAS), (S)-cheilanthifoline synthase (CFS), and SPS. Transcriptome and untargeted metabolomic data indicated greater variation in root samples than leaf samples, although slightly more differentially expressed transcripts and metabolites were observed in leaves. Targeted metabolite profiling showed higher BIA accumulation in roots, with epiberberine being the most abundant, followed by coptisine, berberine, and columbamine. These results provide essential genomic resources for comparative analysis of the BIA pathway across Ranunculaceae, targeted gene function studies for metabolic bioengineering, and conservation strategies for C. trifolia, a member of an early-diverging clade within the genus with limited genetic resources. Full article

The methylation of CpG sites with 5mC mark is a dynamic epigenetic modification. However, the relationship between the methylation and the surrounding genomic sequence context remains poorly explored. Investigation of the allele methylation provides an opportunity to decipher the interplay between differences in the primary DNA sequence and epigenetic variation. Here, we performed high-coverage long-read whole-genome direct DNA sequencing of one individual using Oxford Nanopore technology. We also used Illumina whole-genome sequencing of the parental genomes in order to identify allele-specific methylation sites with a trio-binning approach. We have compared the results of the haplotype-specific methylation detection and revealed that trio binning outperformed other approaches that do not take into account parental information. Also, we analysed the cis-regulatory effects of the genomic variations for influence on CpG methylation. To this end, we have used available Deep Learning models trained on the primary DNA sequence to score the cis-regulatory potential of the genomic loci. We evaluated the functional role of the allele-specific epigenetic changes with respect to gene expression using long-read Nanopore RNA sequencing. Our analysis revealed that the frequency of SNVs near allele-specific methylation positions is approximately four times higher compared to the biallelic methylation positions. In addition, we identified that allele-specific methylation sites are more conserved and enriched at the chromatin states corresponding to bivalent promoters and enhancers. Together, these findings suggest that significant impact on methylation can be encoded in the DNA sequence context. In order to elucidate the effect of the SNVs around sites of allele-specific methylation, we applied the Deep Learning model for detection of the cis-regulatory modules and estimated the impact that a genomic variant brings with respect to changes to the regulatory activity of a DNA loci. We revealed higher cis-regulatory impact variants near differentially methylated sites that we further coupled with transcriptomic long-read sequencing results. Our investigation also highlights technical aspects of allele methylation analysis and the impact of sequencing coverage on the accuracy of genomic phasing. In particular, increasing coverage above 30X does not lead to a significant improvement in allele-specific methylation discovery, and only the addition of trio binning information significantly improves phasing. We investigated genomic variation in a single human individual and coupled computational discovery of cis-regulatory modules with allele-specific methylation (ASM) profiling. In this proof-of-concept analysis, we observed that SNPs located near methylated CpG sites on the same haplotype were enriched for sequence features suggestive of high-impact regulatory potential. This finding—derived from one deeply sequenced genome—illustrates how phased genetic and epigenetic data analyses can jointly put forward a hypotheses about the involvement of regulatory protein machinery in shaping allele-specific epigenetic states. Our investigation provides a methodological framework and candidate loci for future studies of genomic imprinting and cis-mediated epigenetic regulation in humans. Full article