DNA methylation has been proposed as an epigenetic driver of phenotypic plasticity in social insects, yet experimental evidence remains limited. Even less is known about the role of epigenetic mechanisms underlying behavioural and social polymorphism. We quantified CpG methylation for the socially polymorphic harvester ant Pogonomyrmex californicus across larvae, pupae, workers and queens using Oxford Nanopore Technologies (ONT) sequencing. These results were compared against the current gold standard whole-genome bisulfite sequencing (WGBS). Methylation sites were highly correlated between WGBS and ONT, validating the use of ONT for high-throughput epigenomic profiling. Genome-wide methylation was low (~3%), consistent with findings in other (Hymenoptera: Formicidae), and highly clustered within gene bodies, especially exons, while introns, intergenic DNA, promoters and transposable elements were hypo-methylated. Gene body methylation (GBM) correlated positively with gene expression in queens, corroborating previous reports for other insects, suggesting a conserved regulatory role for DNA methylation in insects. A comparison between developmental stages revealed significant stage-specific differences in GBM frequencies. Workers and queens, although from different populations, shared a substantial core of methylated loci enriched for olfactory-receptor activity and biosynthetic pathways, processes that are central to caste-specific behaviour and physiology. These shared methylation signatures, coupled with stage-dependent variability, highlight DNA methylation as a possible factor in developmental stages and caste differentiation. In the future, it is essential to disentangle the effects of caste and variation between populations. Our study establishes P. californicus as a powerful model for dissecting how epigenetic modifications interface with gene expression to generate developmental and complex social organization, which is largely unexplored.

Mosquitoes transmit numerous infectious diseases, with climate change expanding their global distribution through warmer environments. Next-generation sequencing offers significant advantages for mosquito genomic surveillance and potential early warning systems. In this study, a portable metagenomic sequencing approach using Oxford Nanopore Technologies (ONT) for field-based mosquito analysis (MosquitoID protocol) was developed, enabling species and host feeding patterns identification, and pathogen detection, all coming from a single amplification-free workflow. DNA was extracted from 62 mosquito samples (Aedes albopictus, Aedes cretinus, Culex pipiens, Culiseta longiareolata) from Greece and Spain, either single-species pools (1-10 specimens) or mixed-species pools, with reverse purification method or archived samples. Additionally, 30 pooled Aedes aegypti samples from Bangladesh underwent cDNA reverse purification. All samples were sequenced using ONT rapid barcoding kits. Offline bioinformatics analysis via Geneious screened custom BLAST databases for species, host, and virus identification. MosquitoID accurately identified mosquito species in 89% of samples overall, with main discrepancies in Aedes cretinus. Virus screening detected Phasi Charoen-like virus in cDNA samples. Host DNA sequences identified multiple species including horses, cattle, and ducks. This study demonstrates metagenomic ONT sequencing's effectiveness for rapid host, species, and virus identification. After further benchmarking, the approach shows potential for real-time disease monitoring and enhanced surveillance systems. Integrating portable next-generation sequencing with offline bioinformatics tools could significantly strengthen mosquito-borne disease prevention strategies, particularly for non-bioinformaticians and in resource-limited settings.

The chloroplast (cp) genome of the tree fernCyathea delgadiiPohl ex Sternb. was assembled and annotated to investigate its structure and evolution within the Cyatheales order. The genome, sequenced using Oxford Nanopore Technologies, has a total size of 165,248bp, comprising a large single-copy (LSC) region of 94,738bp, a small single-copy (SSC) region of 22,012bp, and two inverted repeat (IR) regions of 24,251bp each. It contains 89 protein-coding genes, eight rRNAs, and 33 tRNAs. Comparative phylogenomic analyses involving 19 species of Cyatheales have revealed that the cp genome of C. delgadii shares similarities in gene content with other ferns of the Cyatheaceae family; however, it demonstrates variations in both genome size and GC content. Variations in cp genome size were observed across the Cyatheales species, ranging from 154,046bp inGymnosphaera denticulata (Baker) Copelto 168,244bp inDicksonia squarrosa (G.Forst) SW. Gene content analysis showed that most species have a conserved number of protein-coding genes, rRNAs, and tRNAs, suggesting structural stability. However,Cibotiumspp. has a reduced number of protein-coding genes (87), possibly due to gene loss or transfer to the nuclear genome. Phylogenetic analyses using both whole genome and SNP data showed comparable clustering amongAlsophilaandGymnosphaeraspecies, whileC. delgadiioccupied a basal to intermediate position. The overall guanine-cytosine (GC) content of C. delgadii was calculated to be 40.95%, with a significantly higher content of 44.03% observed in the intragenic regions. An analysis of codon usage bias indicated a preference for codons ending with adenine or thymine, which aligns with the genome's adenine-thymine (AT) richness. This study provides valuable genomic resources and insights into the evolution of Cyatheales cp genomes, emphasizing both conserved features and specific adaptations within this group of ferns.

Age-associated DNA methylation loci at lncRNA genomic regions revealed by Oxford Nanopore whole-genome sequencing using four methylation callers.

Accurate HLA allele identification is essential to ensure graft-recipient compatibility. Advances in next-generation sequencing (NGS), such as those provided by Illumina and Oxford Nanopore Technologies, have improved resolution of HLA-typing. Here, a novel HLA-C*12 allele with a silent mutation (G to A) was identified in a bone marrow donor homozygous for HLA-C*12. The allele was initially linked to HLA-C*12:03:01 by short-read NGS, but further investigation by long-read sequencing revealed the mutation to be associated with HLA-C*12:02:02. Phasing limitations of short-read NGS made accurate allele assignment difficult, but precise differentiation became possible with longer reads. The origin of the mutation was subsequently confirmed by sequencing parental samples. This case highlights the ability of long-read sequencing to resolve cis-trans ambiguities and improve allele phasing, and to enhance accuracy for new HLA allele identification.

Human leucocyte antigen (HLA) class I and human platelet antigens (HPAs) can elicit immune responses, leading to platelet transfusion refractoriness (PTRs). To support the clinical management of patients with PTR, transfusion of HLA class I and sometimes HPA-selected apheresis platelets is recommended. To facilitate this service, the HLA class I and HPA genotypes of platelet donors must be defined so that appropriate platelet units may be selected. Currently, National Health Service Blood and Transplant (NHSBT) utilises two separate assays for genotyping HLA class I and HPAs. In this study, a simple, single-well, multiplex PCR assay has been developed to genotype classical HLA class I genes (HLA-A, -B and -C) and 27/35 currently described HPAs, including all clinically relevant HPAs. Genomic DNA from 90 consented English apheresis platelet donors and 17 reference samples provided by the Australian Red Cross were amplified with the custom PCR protocol. Sequencing libraries were prepared using Oxford Nanopore Technologies (ONTs) native barcoding kit v14 (SQK-NBD114.96) and sequenced on MinION R10.4.1 flow cell. Base calling was performed live in MinKNOW v24.02.16, and sequence alignment, variant calling and genotype assignment of HPA were performed by a bespoke bioinformatics pipeline. Analysis of HLA class I genes was carried out using NGSengine v4.0.0. English apheresis platelet donors successfully genotyped (89/90) using the described assay showed 100% concordance with historic results for both HLA and clinically relevant HPAs. Further testing with reference samples from the Australian Red Cross samples showed 100% concordance with externally provided HPA results. This assay offers a simplified workflow for simultaneous HLA class I and HPA genotyping of apheresis platelet donors, permitting automation and significant expansion to include more HPA specificities. This assay provides accurate HLA class I genotyping and HPA genotyping in a single assay, requiring a minimal laboratory footprint and less maintenance than current in-use assays.

Despite the recognized impact of the gut microbiome, research on the oral microbiome is limited, particularly in pediatric patients with Crohn's disease (CD). This study aims to explore salivary microbiome signatures in pediatric patients with CD from the United Arab Emirates (UAE), compared to healthy controls (HC), by analyzing early-life, lifestyle, and disease-specific factors driving dysbiosis. Salivary samples from 52 pediatric patients with CD and HC (n = 26/group) were subjected to 16S rRNA sequencing using Oxford Nanopore technology. Bioinformatics and biostatistical analyses were employed to compare groups and identify microbiota signatures correlated with clinical data. Enrichment of several species, including Veillonella parvula, Veillonella dispar, and Prevotella denticola, with depletion of beneficial bacteria was observed in CD. Machine learning-based composite biomarker analysis identified 36 species distinguishing CD from HC, most of which are opportunistic pathogens, raising concerns about their potential impact on vulnerable pediatric patients with CD. Multifactorial analysis revealed significant oral microbiome dysbiosis in patients with CD across all 15 analyzed factors, with unique CD-specific biomarkers. The strongest associations with microbial alterations were demonstrated by disease duration, diet, exercise habits, early antibiotic exposure, and delivery method. Among the 19 species analyzed, Capnocytophaga gingivalis demonstrated multifactorial associations, emerging as an integrative biomarker of disease burden. The α-diversity was significantly lower in patients with CD, with distinctive β-diversity patterns. This is the first comprehensive multifactorial analysis of the oral microbiome in pediatric patients with CD from the Middle East, employing novel machine learning approaches for composite biomarker discovery. Core dysbiotic species in CD may serve as potential diagnostic and prognostic biomarkers requiring validation in larger-scale studies.

There is limited understanding of the replication and transmission of bandaviruses and the influence of host genotype in successful infection. An in vitro Bandavirus model, such as Lone Star virus (LSV, Bandavirus amblyommae), capable of propagating in standard cell lines, could provide some of this critical information. In this study, we sequenced the genome of LSV and profiled its relationship with a key host viral-interacting protein, Synaptogyrin-2 (SYNGR2), known to influence the replication of another Bandavirus, Bandavirus dabieense.Materials and Methods: The genome of the LSV TMA 1381 strain was sequenced and assembled using Oxford Nanopore Technology. The expression of SYNGR2 was profiled and annotated in Vero cells. SYNGR2 knockout (KO) Vero clones were obtained via CRISPR-Cas9 gene editing of the first exon, present in all SYNGR2 isoforms. Following LSV infection, expression of SYNGR2 and LSV titer was measured in SYNGR2-KO and wild-type cell lines. Sequence variation and evidence of viral heterogeneity were detected across all segments of the LSV TMA 1381 strain (4 missense substitutions out of 7 single-nucleotide polymorphisms identified, q > 16). Important amino acid sequence differences for the nonstructural protein, known to directly interact with host SYNGR2, were observed between LSV and other bandaviruses (15.5-47.4%). The change in SYNGR2 expression in wild-type Vero cells was limited following LSV infection (1.77-fold). No difference in estimated LSV titer was detected between wild-type and SYNGR2-KO Vero cells (p > 0.16). Our data illustrate key distinctions from previous Bandavirus reports and underline the need for future studies to explore the mechanisms of LSV replication and pathogenesis.

Cyclodextrin metal-organic framework engineered titanium surface: Targeted modulation of foam cell lipid homeostasis and inflammatory resolution.

Hereditary transthyretin amyloidosis (ATTRv) is a rare autosomal dominant disorder caused by pathogenic variants in the TTR gene, with marked clinical heterogeneity and frequent underdiagnosis. Genetic confirmation is essential for patient management but is traditionally based on Sanger sequencing or short-read next-generation sequencing (NGS), which are time-consuming or costly. We evaluated the clinical utility of a long-read sequencing strategy using Oxford Nanopore Technologies (ONT) for comprehensive TTR gene analysis. We developed an optimized long-range PCR (6,941 bp) followed by ONT MinION sequencing workflow covering the entire TTR gene. Analytical performance was evaluated in two independent diagnostic laboratories using a total of 89 previously characterized samples (64 from laboratory #1 and 25 from laboratory #2). The method was subsequently implemented for routine clinical testing in 445 additional patients referred for suspected ATTRv amyloidosis. Performance metrics, variant detection rates, turnaround time, and concordance with short-read NGS were assessed. All previously identified pathogenic or likely pathogenic TTR variants were correctly detected, and no false-positive calls. As expected, the most frequent variants were p.(Val142Ile) and p.(Val50Met). Results were fully concordant with short-read NGS. Flowcell washing and reuse considerably lowered reagent costs without compromising an accurate TTR genotyping. Implementation of the ONT workflow reduced turnaround time compared with Illumina-based targeted panels. Long-read ONT sequencing of the TTR gene provides a robust, rapid, and economical alternative to conventional methods for genetic diagnosis of ATTRv amyloidosis. The approach we developed is well suited for routine clinical laboratories and supports systematic screening of TTR variants in patients with suspectedATTR.

Neisseria meningitidis is a primary cause of bacterial meningitis and septicemia, which is a significant global public health concern. Here, we report Oxford Nanopore Technologies RNA sequencing for N. meningitidis serogroup B strain M0579. This strain encodes the phase-variable DNA methyltransferase ModD1, an epigenetic regulator associated with hyperinvasive meningococcal B lineages.

Genome structural variants (SVs) comprise a sizable portion of functionally important genetic variation; yet, many evade discovery using short reads. While long-read sequencing can reveal hidden SVs, their role in organismal trait variation remains largely unclear. To address this gap, we investigate the molecular basis of 50 classical phenotypes in 11 Drosophila melanogaster strains using highly contiguous de novo genome assemblies generated with Oxford Nanopore Technologies long reads. These assemblies enable construction of a pangenome graph containing nucleotide-resolution maps of SVs, including complex rearrangements such as the interchromosomal inverted duplication Dp(2;4)eyD and large tandem duplications at the Bar locus. We uncover new candidate causal mutations for 15 phenotypes and new molecular alleles for 2 mutations comprising tandem duplications, transposable element (TE) insertions, and indels. For example, the wing vein phenotype plexus (px 1) links to a 1.5 kb partial tandem gene duplication, and the century-old Curved (c1) wing phenotype links to a 7.5 kb DM412 retrotransposon disrupting the coding sequence of the muscle protein gene Strn-Mlck We also identify a candidate intergenic enhancer for Ablp eyD, a finding supported by CRISPR-Cas9. We also unveil 8 SV alleles of previously identified causal genes, including uncharacterized SVs underlying the extensively studied white and yellow phenotypes. Overall, 67.4% of genes causing phenotypic changes harbor candidate SVs >100 bp, whereas only 28% are expected based on euchromatic SVs. Together, our results indicate that SVs are strongly enriched among this class of large-effect, deleterious visible phenotypes in Drosophila.

Partial endoreplication is a prominent developmental feature and poses a significant challenge for whole genome assembly in orchids. This form of cell cycle results in highly unbalanced cell DNA content, with the highly endoreplicated (P) fraction being overrepresented in sequencing data compared to the non-endoreplicated (F) fraction. Here, we report the first genome assembly of Vanilla planifolia into 16 chromosome pairs using axillary buds enriched in non-endoreplicated 2C-nuclei (55%) as determined by flow cytometry. The assembly was generated using a hybrid approach combining PacBio HiFi sequencing and Omni-C scaffolding generated in this study, together with a GBS-SNP genetic map and Oxford Nanopore Technologies long-read data from the literature. For the first time, we identified P and F regions within reconstructed chromosomes, representing 20.57% and 79.43% of the genome, respectively based on DNA sequencing data from three tissues with varying levels of endoreplicated nuclei. P regions were gene-rich and located at chromosome ends, whereas F regions were SSR-rich and located at central parts of chromosomes. Remarkably, 97.24% of SSRs were found in F regions, predominantly comprising the trinucleotide AAG/CTT motif, which may contribute to the absence of endoreplication in these regions. Protein-encoding genes overrepresented in F regions were associated with negative regulation of flower development, mitotic cycle progression, cell division and histone modification. This accurate high-quality chromosome-scale V. planifolia genome assembly provides unprecedented insights into the structural and molecular characteristics of partial endoreplication in Orchids and represents a major step toward the characterization of this complex genome.

Pediatric obesity is the most prevalent nutritional disorder in children and adolescents and is associated with multiple comorbidities. Understanding epigenetic mechanisms, particularly DNA methylation, offers potential for early risk prediction, prevention, and personalized interventions. In this study, genome-wide DNA methylation profiles in blood from children with obesity and matched controls were compared using Oxford Nanopore Technologies. Two independent analytical tools were applied to identify differentially methylated regions. Clinical data included family history, weight, BMI, and age at first examination. Participants with monogenic obesity, identified via short-read whole-exome sequencing, were excluded. The cohort included five girls and five boys with obesity (median age 14.33, IQR 1.65; median BMI SDS 3.42, IQR 0.55) and matched controls (median age 13.94, IQR 0.52; median BMI SDS 0.26, IQR 1.71). Seven consensus differentially methylated regions were consistently identified, overlapping genes involved in metabolism and obesity-related comorbidities. Hypermethylation was observed in PM20D1, PM20D1-AS1, AC119673.2 (26.05% ± 0.78%), and GM2A (33.60% ± 1.10%) in children with obesity, primarily affecting metabolic and adipogenic pathways. Hypomethylation was detected in genes linked to obesity and its comorbidities, including S100A14, S100A16 (- 25.4% ± 0.48%), SNTG2 (- 25.55% ± 0.18%), ADARB2, LINC00200 (- 21.35% ± 0.98%), LRRC32, AP001189.1 (- 27.25%), CBLN3 and KHNYN (- 23.20% ± 0.80%). Long-read genome-wide methylation profiling can detect obesity-associated epigenetic loci in children. Blood-based markers in genes regulating metabolism and obesity comorbidities could support early risk prediction, patient stratification, and targeted prevention. Extending this work to larger and more diverse cohorts will be necessary to evaluate the robustness of these results and their potential translational relevance.

This article presents Sequenoscope: a bioinformatics pipeline for analysing Oxford Nanopore Technologies (ONT) adaptive sampling sequencing data. Sequenoscope features three main modules: filter_ONT for filtering raw reads and creating a FASTQ file with a subset of reads for further analyses, analyze for generating sequencing and read mapping statistics against the provided reference taxon sequences and plot for interactive data summarization, comparison, and visualization between two datasets/test conditions. Here, we demonstrate the ability of the pipeline to analyse ONT adaptive sampling sequence data and provide examples of the outputs users can expect using data we generated. Adaptive sampling was performed on two ZymoBIOMICS Microbial Community DNA Standards, log-distributed (Cat# D6311) and even-distributed (Cat# D6306) formulations, with targeted depletions of Listeria monocytogenes. By comparing the test and control experimental data in FASTQ files from the sequencing runs, Sequenoscope showed that depletion of L. monocytogenes was successful by providing users with parameters to compare such as taxon coverage, read length and types of pore-level decisions made during sequencing. Although Sequenoscope was designed for ONT adaptive sampling data analysis, it supports short-read data from other sequencing platforms such as Illumina, allowing for the direct comparison of any two experimental conditions or cross-platform benchmarking.

Fresh vegetables can harbor diverse bacteria, including Pseudomonas spp. In routine diagnostics, isolates are often identified by MALDI-TOF MS, but novel or unreferenced taxa can be misidentified. Here, we report the WGS of Pseudomonas sp. strain PK-RTE-24, initially assigned by MALDI-TOF MS to a known species. The aim was to generate genomic data that clarify the taxonomic assignment of PK-RTE-24 that is not linked to any described species and to highlight the discrepancy between MALDI-TOF MS identification and WGS analysis. PK-RTE-24 was isolated from ready-to-eat lettuce during screening for extended-spectrum β-lactamase (ESBL)-producing bacteria. MALDI-TOF MS reported Pseudomonas koreensis. Using Illumina and Oxford Nanopore technologies a complete 5,847,647-bp genome, with 60.04% GC content, and encoding 5,285 predicted genes, was assembled. Phylogenomic analysis and average nucleotide identity showed that PK-RTE-24 did not match any described Pseudomonas sp. and fell below the species threshold, grouping within an environment-associated clade. Antimicrobials testing revealed β-lactams non-susceptibility, consistent with a chromosomally-encoded class C β-lactamase (AmpC) gene (1,167-bp). The 388 amino-acid protein shared 96.9% identity with an AmpC from Pseudomonas sp. RG1. The PK-RTE-24's genome sequence provides valuable data for classifying environment-associated Pseudomonas spp. and elucidating β-lactam resistance in non-clinical strains.

Protocol for high-throughput processing of fecal samples for long-read metagenomic sequencing using PacBio HiFi or Oxford Nanopore Technologies.

Eupentacta fraudatrix is the only species of sea cucumbers for which transdifferentiation has been described. This type of cell type switching occurs during gut regeneration after evisceration, which makes this species a scientifically valuable model for studying regeneration mechanisms. Moreover, chromosome-level genomes for the family Sclerodactylidae have not been available until now. In this study, we employed the MGI short-read, Oxford Nanopore, and Hi-C technologies to assemble and annotate two chromosome-level, high-quality haplotypes of E. fraudatrix. The estimated heterozygosity was 5.1%, which is much higher than the known values for sea cucumber genomes. Both haplotypes are nearly equivalent, containing 23 chromosomes with a total length of approximately 1.6 gigabase pairs, and 99% of assembled bases anchored to chromosome-level scaffolds. The annotation predicted 26,352 protein-coding genes for one haplotype and 25,238 genes for the other, with BUSCO assessment revealing 98.1 and 97.9% complete metazoa_odb12 core genes. The chromosome-level assembly and annotation of E. fraudatrix genome will provide a genomic basis for further phylogenetic, comparative, and molecular biological studies of echinoderm regeneration.

Dark-field X-ray imaging visualizes structural inhomogeneities through small-angle scattering, but existing directional methods are confined to the micrometer scale. While recent advances have extended dark field capabilities to nanoscale transmission X-ray microscopy, directional scattering retrieval - critical for characterizing anisotropic nanostructures - has remained inaccessible for imaging resolutions in the sub-micrometer scale. Here, we demonstrate the first directional dark-field setup for nanoimaging, achieving orientation mapping of scattering features below the spatial resolution limit. Our method is experimentally simple to implement with existing transmission X-ray microscopy setups. We validate its performance by successfully resolving sub-resolution test structure orientations, cross-correlating orientational changes within hierarchical nanoporous materials, and mapping the directional arrangement of hydroxyapatite nanocrystals (30-70 nm) within human tooth enamel. By utilizing shadow regions in the optical configuration, we further extend the detectable scattering vector range, demonstrating a pathway toward size-selective dark-field imaging. This advancement enables the quantitative structural characterization of anisotropic nanomaterials, which are critical to biomineralization, advanced materials, and nanotechnology applications.

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) poses a major global health threat due to limited treatment options and high mortality. While the genomes of carbapenemase-positive XDR-KP strains are well-documented, there is limited data on carbapenemase-negative XDR-KP. This study characterized the genomes of two such clinical isolates, obtained from abdominal fluid and urine, that were resistant to nearly all tested antibiotics. Antimicrobial susceptibility testing was performed at the Hamilton Regional Microbiology Laboratory, ON. Carbapenemase production and the presence of carbapenemase genes were assessed using the NG-Test®CARBA-5 and a multiplex carbapenemase PCR assay, respectively. Whole-genome sequencing via Oxford Nanopore technology revealed that both strains lacked carbapenemase genes but harbored multiple other resistance genes (e.g., blaCTX-M-15, qnrB1/S1, sul1/2, dfrA17/A14, fosA) and acrR mutations. Notably, both carried ompK37 mutations, an outer membrane porin previously linked to carbapenem resistance. A number of antibiotic resistance genes were found on conjugative plasmids (IncFIB/IncFII, IncFIA, IncFIA/IncFII). Multilocus sequence typing identified different sequence types (ST307, ST45) and virulence profiling showed overlapping and distinct features, including variability in capsular and siderophore genes. These findings suggest a significant role for porin loss and efflux pump regulation in conferring resistance to broad-spectrum and last-resort antibiotics in clinical XDR-KP isolates that lack carbapenemases and other related antibiotic resistance genes.