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PubMed2026

MHASS: Microbiome HiFi Amplicon Sequencing Simulator.

SUMMARY: Microbiome HiFi Amplicon Sequence Simulator (MHASS) creates realistic synthetic PacBio HiFi amplicon sequencing datasets for microbiome studies, by integrating genome-aware abundance modeling, realistic dual-barcoding strategies, and empirically derived pass-number distributions from actual sequencing runs. MHASS generates datasets tailored for rigorous benchmarking and validation of long-read microbiome analysis workflows, including ASV clustering and taxonomic assignment. AVAILABILITY AND IMPLEMENTATION: Implemented in Python with automated dependency management, the source code for MHASS is freely available at https://github.com/rhowardstone/MHASS along with installation instructions. Our code is also published on Zenodo at https://doi.org/10.5281/zenodo.17486364. The data underlying this article are available on GitHub at https://github.com/rhowardstone/MHASS_evaluation/.

Software↗

PubMed2025

Comparison of a long-read amplicon sequencing approach to short-read amplicons for microbiome analysis.

Most microbiome studies to date rely on sequencing short amplicons of the 16S rRNA gene on Illumina's platforms. Because of the short read length, sequences often can be identified reliably only to the family or genus levels. Long read sequencing with whole-length 16S rRNA sequencing can improve taxonomic resolution, but often only to the species level. StrainID is an alternative approach that amplifies a large segment of the ribosomal operon, including the entire 16S rRNA gene, internal transcribed spacer, and a portion of the 23S rRNA gene. This longer amplicon is designed to allow ribotype-level classification. Although studies have demonstrated the utility of StrainID for several sample types, it has not yet been validated for saliva. Here, we compared the performance of StrainID to short read amplicons with saliva samples as well as a synthetic mock DNA community and human and mouse fecal samples. Short reads were amplified with primer pairs appropriate for the corresponding sample type, and were classified with two different taxonomic databases. For both saliva and fecal samples, we found that StrainID performed similarly to short reads overall and demonstrated a key benefit with phylogenetic-based beta diversity tests and taxonomic classification. Our results further build on establishing StrainID as a valid method and specifically validate its use with saliva samples.

Journal Article↗

PubMed2026

Upscaling Genotyping by Amplicon Sequencing With GBAS-GUI.

Genotyping by amplicon sequencing (GBAS) is a relatively low-cost approach for generating genotypic data compared with established genomic methods, making it highly scalable and particularly suitable for large-scale genetic monitoring projects. However, most existing analytical pipelines are either marker-specific, insufficiently scalable, or lacking efficient data management systems for the long-term integration of genotypic information, limiting the full potential of GBAS. Here, we address this gap by introducing GBAS-GUI (https://github.com/sonnenbe-dot/GBAS-GUI), a pipeline capable of generating GBAS-based genotypic data for a wide variety of loci at scale. GBAS-GUI integrates a graphical user interface with multiple checkpoints to improve accessibility and robustness. It implements multiprocessing architecture and a relational database that links genotypic data with associated sample metadata to enhance scalability and data management. The pipeline further enables marker screening through automated calculation of polymorphism information content (PIC) and implements a strategy to recover homologous genotypic information from paralogous loci with non-overlapping amplicon length ranges. Using multiple empirical datasets, we demonstrate substantial improvements in processing speed, database management and handling artefacts related to co-amplification of unspecific regions and duplicates of the same genomic region. We further show that incorporating the full sequence information captured by an amplicon increases marker information content beyond what is achievable with length-based genotyping alone and expands the analytical versatility of GBAS. Overall, GBAS-GUI provides a robust, scalable and versatile framework that unlocks the potential of GBAS for large-scale population genetic and phylogeographic studies.

Genotyping Techniques↗

PubMed2025

Determining genotype and antimicrobial resistance of Salmonella Typhi in environmental samples by amplicon sequencing.

BACKGROUND: Estimates of the burden of typhoid fever due to Salmonella enterica serovar Typhi (S. Typhi) rely on data from clinical surveillance, which is rarely done in low income settings and is also limited by the poor sensitivity of the assays used and the reliance on health seeking by patients. Environmental surveillance for S. Typhi shed by symptomatic and asymptomatic individuals in wastewater offers a sensitive surveillance tool that could help to inform burden estimates. Sequencing S. Typhi direct from wastewater concentrates has the potential to identify circulating genotypes and associated antimicrobial resistance (AMR) genes, supporting public health interventions such as vaccination and antimicrobial usage. METHODOLOGY AND PRINCIPAL FINDINGS: We designed a multiplex targeted amplicon sequencing protocol for genotyping and determining AMR in S. Typhi from wastewater samples, targeting SNPs that identify genotypes of interest and both chromosomal and plasmid-borne AMR. PCR products were sequenced using the Oxford Nanopore Technologies (ONT) MinION, and genotypes and AMR identified using the GenoTyphi program. We tested this approach on samples from south India from both hospital outflow and wastewater collected from the community. All samples tested were suspected to be positive for S. Typhi following quantitative PCR for ttr, tviB, and staG gene targets. Out of 110 samples tested we were able to determine a genotype and/or AMR for 8. All samples that gave a genotype call suggested a genotype consistent with those found in clinical cases in India during the same time period and produced consensus sequences that clustered with S. Typhi when included in a phylogenetic tree. CONCLUSIONS: In this study, we provide proof of concept data for amplicon sequencing of S. Typhi in wastewater which with further optimisation could be used to complement clinical surveillance data or provide data on S. Typhi presence in the absence of clinical surveillance. This information can inform public health interventions, and the concept could be applied to other pathogens of interest for genotyping from environmental surveillance samples.

Salmonella typhi↗

PubMed2026

Genomic characterization of avian metapneumovirus subtypes A and B in United States poultry by targeted amplicon sequencing.

Avian metapneumovirus (aMPV) subtypes A and B emerged in United States poultry in late 2023 and early 2024, prompting genome-scale surveillance from clinical samples. Here, we developed and optimized targeted amplicon sequencing (TAS) assays for both subtypes and applied them to 104 subtype-positive clinical samples collected from chicken and turkey farms across nine US states between early 2024 and early 2026. TAS recovered 91 genomes suitable for comparative analysis, including 44 aMPV-A and 47 aMPV-B sequences, with successful recovery extending to Ct values of 34.6 for aMPV-A and 31.2 for aMPV-B. Recovered genomes showed near-complete breadth and high mapping efficiency. Phylogenetic analyses of both G-gene and whole-genome datasets showed that US aMPV-A field strains formed a distinct monophyletic lineage within group IV and resolved into three closely related clusters. Cluster 2, first recognized in North Carolina and later detected in Ohio, spread across 30 turkey and chicken farms. Cluster 2 genomes were defined by a concentrated G-protein hotspot within residues 209-275, and most North Carolina Cluster 2 genomes carried 10-11 nonsynonymous substitutions in this region, including multiple proline substitutions suggestive of local structural change. Missouri Cluster 3 remained cohesive but distinct from both Cluster 1 and Cluster 2 in the G-gene and whole-genome trees. In contrast, US aMPV-B field strains remained highly homogeneous across hosts and states, with more than 99% nucleotide identity by both G-gene and WGS analyses. We also identified 12 vaccine-derived genomes on both vaccinated and nonvaccinated farms. These included six genomes related to aMPV-A vaccine and six related to aMPV-B vaccines (VCO3/50 and 1062), all of which retained vaccine-defining markers together with additional substitutions consistent with continued circulation after vaccine use in the field. Selection analyses showed that the G gene had the highest gene-wise dN/dS ratio in both subtypes. Additional elevated signal was observed in SH and M2, and candidate positively or episodically selected codons were concentrated in the subtype A Cluster 2 G-gene hotspot. These findings show that TAS supports direct-from-sample aMPV genomic surveillance and provides genomic context for field clusters, vaccine-derived lineages, and continued adaptive change in aMPV in US poultry.

Animals↗

PubMed2026

ALPINE: a scalable pipeline for comprehensive classification of gene-editing outcomes from long-read amplicon sequencing.

SUMMARY: CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing supporting both PacBio HiFi and Oxford Nanopore platforms. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Dual-target benchmarking on simulated datasets demonstrated high accuracy for transgene integration events. Independent validation on public crosslinked-HDR dataset confirmed ALPINE's integration detection capabilities, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling. AVAILABILITY: ALPINE is available under MIT license at https://github.com/Maggi-Chen/ALPINE and https://doi.org/10.5281/zenodo.20272510. All analysis scripts and visualization code used in this manuscript are available at https://github.com/Maggi-Chen/ALPINE-manuscript-analysis. Simulated datasets are deposited at Zenodo (https://doi.org/10.5281/zenodo.20260865). Public dataset PRJNA913199 is available through NCBI SRA.

Gene Editing↗

PubMed2027

Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.

This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.

Chikungunya virus↗

PubMed2026

Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus.

Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0-5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.IMPORTANCERSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.

bioinformatics pipeline↗

PubMed2026

Nanopore-based, long-range Parvovirus B19 amplicon sequencing for near-whole genome characterization.

BACKGROUND: Whole-Genome Sequencing (WGS) enables monitoring of genomic variation and evaluation of diagnostic PCR assays. However, WGS data for Parvovirus B19 (B19V) remains limited despite its relevance for clinical care and transfusion safety. To increase the availability of high-quality B19V genomic data, a near-WGS protocol was developed and validated. METHOD: The protocol combines long-range PCR to generate a 4.6-kb amplicon, covering ∼82% of the B19V genome, with Oxford Nanopore sequencing. Validation was performed using six reference samples and nineteen B19V-positive donor plasma samples. RESULTS: After quality control, samples achieved a median sequencing depth of 152x. Sequences generated from the six reference samples showed 100% concordance with previously published data. Genomic analysis of donor samples explained atypical amplification profiles observed during routine PCR screening. CONCLUSION: The newly developed protocol provides a scalable method for B19V genome characterization, enabling assessment of oligonucleotide-binding regions for PCR assay monitoring and facilitating the generation of genomic data for future epidemiological investigations.

PCR assay monitoring↗

PubMed2025

Survival and safety evaluation of Bifidobacterium longum subsp. longum ZS-8 in healthy adults, determined using PMAxx-qPCR and amplicon sequencing.

UNLABELLED: Species-level quantitative PCR (qPCR) provides in-depth knowledge of oral probiotics in the human gastrointestinal tract (GIT). However, it lacks the capability to differentiate exogenous strains from native microbiota, nor can it distinguish between live and dead bacteria. In this study, we employed improved propidium monoazide (PMAxx)-qPCR to evaluate the survival and colonization of Bifidobacterium longum subsp. longum ZS-8 (designated ZS-8) on the strain level in the GIT and its impact on human gut microbiota. By spiking in live and dead ZS-8, we demonstrated that strain-level PMAxx-qPCR could identify and quantify the viable ZS-8 in fecal samples accurately. Using this method, we found that, in healthy humans, oral administration of ZS-8 can transiently survive in the GIT, and multi-layer seamless capsules (MLSC) significantly improve the gastrointestinal tolerance and survivability of ZS-8 compared to its powder form. Furthermore, through selective cultivation and PMAxx-microbiome sequencing, we investigated the response of gut viable microbiome to ZS-8. Results showed that, while the microbiota diversity and total viable counts of Bifidobacterium and Lactobacillus remained stable, certain indigenous species of Bifidobacterium and Lactobacillus increased in abundance, confirming ZS-8's probiotic potential in healthy individuals. Overall, our study demonstrates the effectiveness of combining strain-specific comparative genomics with PMAxx-qPCR for evaluating probiotic survival and colonization in the human gut and highlights the safety of ZS-8 oral administration in healthy individuals. IMPORTANCE: The survival and colonization of probiotics in the gut are critical for their functional efficacy, yet conventional species-level quantitative PCR (qPCR) fails to distinguish exogenous strains from native microbiota or differentiate live from dead bacteria. By integrating strain-specific comparative genomics with propidium monoazide (PMAxx)-qPCR, we precisely quantified the viability of Bifidobacterium longum ZS-8 at the strain level in the human gut after its oral administration. Our study demonstrated that 1.53-6.90% of cells surviving transit and multi-layer seamless capsules (MLSC) significantly enhanced the gastrointestinal tolerance of ZS-8. While ZS-8 administration did not alter gut microbiota diversity or total viable counts of Bifidobacterium and Lactobacillus, it selectively increased the abundance of specific indigenous beneficial species. This method overcomes the dual limitations of traditional techniques (strain-level specificity and viability discrimination), providing a robust tool for probiotic research. Furthermore, our findings confirm the safety of ZS-8 in healthy individuals and its potential to modulate gut ecology, offering a scientific foundation for personalized probiotic development and clinical translation.

Humans↗

PubMed2025

AmpSeqR: an R package for amplicon deep sequencing data analysis.

Amplicon sequencing (AmpSeq) is a methodology that targets specific genomic regions of interest for polymerase chain reaction (PCR) amplification so that they can be sequenced to a high depth of coverage. Amplicons are typically chosen to be highly polymorphic, usually with several highly informative, high frequency single nucleotide polymorphisms (SNPs) segregating in an amplicon of 100-200 base pair (bp). This allows high sensitivity detection and quantification of the frequency of each sequence within each sample making it suitable for applications such as low frequency somatic mosaicism detection or minor clone detection in mixed samples. AmpSeq is being increasingly applied to both biological and medical studies, in applications such as cancer, infectious diseases and brain mosaicism studies. Current bioinformatics pipelines for AmpSeq data processing lack downstream analysis, have difficulty distinguishing between true sequences and PCR sequencing errors and artifacts, and often require bioinformatic expertise. We present a new R package: AmpSeqR, designed for the processing of deep short-read amplicon sequencing data, with a focus on infectious diseases. The pipeline integrates several existing R packages combining them with newly developed functions to perform optimal filtering of reads to remove noise and improve the accuracy of the detected sequences data, permitting detection of very low frequency clones in mixed samples. The package provides useful functions including data pre-processing, amplicon sequence variants (ASVs) estimation, data post-processing, data visualization, and automatically generates a comprehensive Rmarkdown report that contains all essential results facilitating easy inclusion into reports and publications. AmpSeqR is publicly available at https://github.com/bahlolab/AmpSeqR.

High-Throughput Nucleotide Sequencing↗

PubMed2026

VisPan: real-time visualisation of multiplex amplicon-based sequencing panels for rapid syndromic surveillance and pathogen detection.

MOTIVATION: Infectious diseases persist as a major global public health challenge. Diverse factors, including climate change, globalization, deforestation, human-animal interactions, lifestyle choices, and various biological factors, can contribute to their emergence and reemergence. Rapid detection and characterization of (re)emerging pathogens are therefore critical for effective outbreak management and for enhancing our understanding of epidemics by monitoring the transmission, spread, evolution, and genomics of pathogens. In this context, next-generation sequencing technologies (NGS), particularly long-read platforms such as Oxford Nanopore Technologies (ONT), have opened new avenues for real-time pathogen monitoring. However, the bioinformatics bottleneck remains a challenge, emphasizing the need for efficient, accessible, and user-friendly analysis tools. RESULTS: Here, we present a tool adapted from the RAMPART software that enables real-time data visualisation of multiplex PCR syndromic panels combined with Oxford Nanopore sequencing. This real-time analysis enables rapid pathogen detection, from raw data acquisition to taxonomic assignment, within minutes. The interface offers dynamic visual tracking of the sequencing run and amplicon coverage, facilitating immediate insights during diagnostic workflows. Validation experiments confirmed the system's reliability, accurately identifying all pathogens present in complex clinical or environmental samples. This tool provides an integrated, user-friendly solution for genomic pathogen surveillance in field or clinical settings.

Software↗

PubMed2026

Mulberry-derived endophytic Bacillus velezensis suppresses gray mold and promotes mulberry growth via reshaping the root metabolism and microbiome.

INTRODUCTION: Gray mold is an important fungal disease caused by Botrytis cinerea which threatens global agriculture. As chemical control faces limitations, biological control using Bacillus has gained attention for its environmental friendliness and growth promotion. However, their ecological basis and application potential in mulberry gray mold control remain insufficiently understood. OBJECTIVE: This study aimed to evaluate the biocontrol efficacy of the mulberry derived endophytic strain Bacillus velezensis ZJU_268 and to investigate its associated effects on plant growth, root-associated microbiomes, and metabolic profiles. METHODS: Greenhouse assays were combined with genomic and comparative genomic analyses, amplicon sequencing, non-targeted metabolomics, and functional validation of isolated microbes and metabolites to assess the effects of ZJU_268 and its cell free supernatant (CFS) on mulberry seedlings. RESULTS: This study isolated a mulberry derived endophytic bacterium, B. velezensis ZJU_268, which exhibits strong antifungal activity and reduces the incidence of gray mold in mulberry seedlings. Whole-genome sequencing and comparative genomic analyses revealed strain-specific regions and genes associated with root colonization, stress adaptation, and antimicrobial biosynthesis. Both live cells and CFS significantly promoted seed germination, seedling growth, and biomass accumulation in a dose dependent manner. Amplicon sequencing showed that ZJU_268 and its supernatant reshaped the mulberry root microbiome, enriching beneficial bacterial and fungal taxa while reducing potentially pathogenic members. Cultivable members of the enriched microbiota displayed strong antifungal activity against B. cinerea and promoted mulberry growth. Metabolomic profiling further showed that ZJU_268 and its supernatant were associated with marked metabolic shifts in mulberry roots, accompanied by the accumulation of selected metabolites that supported the growth of representative enriched isolates. CONCLUSIONS: This study demonstrates that ZJU_268 suppresses gray mold and promotes mulberry growth in association with direct antagonistic activity, microbiome restructuring, and holobiont-level metabolic shifts, providing a promising biological strategy for sustainable mulberry disease management.

Bacillusvelezensis↗

PubMed2026

An Amplicon Panel for High-Throughput and Low-Cost Genotyping of Yesso Scallop Mizuhopecten yessoensis.

The Yesso scallop Mizuhopecten yessoensis was imported from Japan to western Canada in the late 1980s to establish an economically viable scallop aquaculture industry. Since this time, the industry in Canada has operated with existing genetic diversity within the broodstock, which is considerably limited relative to wild populations. The sector has not been able to realise its full potential in part due to idiopathic hatchery failures and farm stock collapses due to disease outbreaks associated with the intracellular bacterial pathogen Francisella halioticida. To support Yesso scallop production and breeding, here we generate a low-density, genotyping-by-sequencing amplicon panel using single nucleotide polymorphism (SNP) markers that are evenly spaced across the M. yessoensis genome and that show high heterozygosity in Canada and Japan. The panel can also exploit the high genetic polymorphism of the M. yessoensis genome, with de novo SNP calling identifying over 2,500 high quality SNPs within the 579 sequenced amplicons. We demonstrate the utility and versatility of this new genotyping tool for breeding applications including parentage assignment, low density family-based genome-wide association study, trait heritability evaluation to determine potential for genomic selection, and species differentiation (against the weathervane scallop Patinopecten caurinus). We did not find any genomic regions significantly associated with F. halioticida resistance but did identify potential for genomic selection. We could separate the two species based on genotypes, and did not see evidence of a past M. yessoensis x P. caurinus hybridization event within the M. yessoensis breeding population at Vancouver Island University. This low-cost genotyping panel is expected to accelerate selective breeding improvements for M. yessoensis in Canada and elsewhere.

Animals↗

PubMed2026

Diversity analysis of indoor and outdoor fungal bioaerosols in UK households: a prospective, observational, longitudinal study.

BACKGROUND: Long-term exposure to indoor fungal bioaerosols is a recognised risk factor for respiratory illness, particularly in damp and poorly ventilated housing. However, the diversity and seasonal variability of these fungal communities are poorly understood. As part of the West London Healthy Home and Environment Study (WellHome), this study aimed to characterise the composition, diversity, and temporal dynamics of indoor fungal bioaerosols in urban UK homes, as compared with outdoor air, to inform future exposure baselines and policy development. METHODS: In this prospective, community-based observational study, 118 households were recruited across West London, UK, via community networks and partner organisations, prioritising families with children aged 5-17 years with asthma or allergies, from diverse socioeconomic backgrounds. Sampling occurred between Oct 3, 2022, and June 14, 2024. Participant data were collected via questionnaires completed by household members, capturing demographics, building characteristics, and respiratory health. Passive-air samplers were used in living rooms for 28 days during two seasonal campaigns, with concurrent outdoor sampling at four fixed community sites. Fungal bioaerosols were identified by ITS2 amplicon sequencing and quantified using broad-range quantitative PCR targeting the 18S rRNA gene. Diversity indexes and temporal dynamics were analysed using ecological statistics and generalised additive models. FINDINGS: 118 households were enrolled, comprising 504 residents (263 women, 237 men, and four not reported). Among 504 participants who self-identified, the largest groups comprised individuals identifying as Black African (n=47), Somali (n=46), White British (n=42), and African (n=38), with additional representation from mixed race ethnic backgrounds (n=29), Black British (n=27), White (n=22), and Black Caribbean (n=18), alongside several other ethnicities each represented at lower frequencies. Of 118 households, 104 completed both seasonal campaigns and 14 completed one, yielding 262 air samples (222 indoor and 40 outdoor). DNA was successfully recovered from all samples, identifying 2027 fungal genera. Indoor environments showed significantly higher richness (mean 646 vs 495 amplicon sequence variants; p<0·0001) and Shannon diversity (4·21 vs 3·53; p<0·0001) than outdoors. Community composition differed markedly (permutational multivariate ANOVA p<0·0001), with Penicillium, Aspergillus, and Wallemia enriched indoors. Indoor fungal communities presented stronger seasonal cycling (R2=0·203) than outdoor communities (R2=0·012). Fungal burden across all homes had a median 11 043 genomic equivalence (GE); IQR 4598-20 579 GE. The highest levels were observed in homes with visible mould; one household showed elevated Aspergillus exposure linked to repeated asthma hospitalisations in a sensitised resident. INTERPRETATION: Indoor fungal bioaerosols are more diverse and dynamic than outdoor communities in urban UK homes. These findings establish foundational exposure data and highlight the need for incorporating fungal bioaerosol monitoring into public health policy to mitigate mould-related health risks. FUNDING: UK Research and Innovation (UKRI) Strategic Priorities Fund (SPF) Clean Air Programme.

Humans↗

PubMed2026

XXYLT1 and Mendelian Retinal Dystrophy.

IMPORTANCE: Substantial unexplained heritability remains for pathogenic inherited retinal disease (IRD) variants. Application of genome-wide association studies (GWAS) could help identify causal genes in rare diseases. OBJECTIVE: To leverage a GWAS for the discovery of IRD-associated genes. DESIGN, SETTING, AND PARTICIPANTS: This GWAS analysis was combined with replication of findings in 2 independent IRD cohorts. The study was conducted from January 2024 to December 2025 in a multicenter setting through FinnGen, 100 000 Genomes Project, and the National Health Service Genomic Medicine Service combined with clinical cohort from the Oulu University Hospital. Using IRD criteria from the International Classification of Diseases, 9th and 10th Revisions, 540 individuals with IRD and 473 945 control individuals were identified in the FinnGen study. For validation of FinnGen results, 49 patients were recruited from Oulu University Hospital. Results were further validated in 2 individuals identified from the UK cohort. MAIN OUTCOMES AND MEASURES: The GWAS and proteomics analysis were performed in the FinnGen cohort. Sanger and whole-genome sequencing and RNA approaches were used in a clinical IRD cohort to validate pathogenicity of the identified XXYLT1 variant. RESULTS: This GWAS identified 13 recessive loci reaching genome-wide significance (defined as P < 5 × 10-8). Of these, 4 (near or within XXYLT1, ANKRD10, DYM, and CBLN4) had not been associated with IRD, including the XXYLT1 c.505-1G>C founder variant. This variant was further genotyped in the clinical replication cohort, leading to identification of 5 more homozygous individuals from 4 families. The phenotype was consistent with a cone-rod or macular dystrophy, with visual deterioration, cystoid macular edema and/or schisislike macular abnormalities. The effect of the XXYLT1 c.505-1G>C variant was further investigated using RNA sequencing and complementary DNA amplicon sequencing, demonstrating exon 2 skipping and a loss-of-function effect. These findings were replicated in an independent population identifying 2 patients from the UK harboring a homozygous XXYLT1 c.766G>A, p.(Glu256Lys) missense variant. CONCLUSIONS AND RELEVANCE: This GWAS identified an association between XXYLT1 and IRD. These results affirm that GWAS in a founder population can be used as a potential tool for the discovery of rare mendelian disease genes and that XXYLT1 should be considered in clinical IRD gene panels.

Humans↗

PubMed2025

Intratumoral fungus Neurospora crassa is associated with worsened prognosis in ovarian cancer via modulation of extracellular matrix.

Landmark studies on intratumoral fungi (ITF) have raised concerns due to irreproducible results and data-analysis errors. We aimed to determine whether ITF exist in ovarian cancer (OvCa) and, if so, whether they play a role in disease biology. Formalin-fixed, paraffin-embedded OvCa samples and multiple controls underwent operational decontamination, qPCR, internal transcribed spacer sequencing, and post-hoc data decontamination. We also leveraged updated fungal reads from The Cancer Genome Atlas generated by the TCMbio group, which addressed human-read contamination and artificial inflation, to validate findings and assess prognostic associations. A murine syngeneic model established using mouse ovarian cancer cell line (OVHM) with intratumoral Neurospora crassa injection was established. Transcriptomic and metabolomic analyses were performed to explore mechanisms. Tumor-containing blocks harbored significantly higher fungal loads than environmental controls but had loads comparable to paraffin controls. Applying a two-pass decontamination filter reduced raw sequence features from 9289 amplicon sequence variants (ASVs) to 659 ASVs. We focused on high-abundance features present in human tissues but absent from xenografts and paraffin controls and identified one candidate, N. crassa, associated with unfavorable prognosis in OvCa. Integrating human and murine data, we found Neurospora correlated with eosinophils, whereas N. crassa itself was not immune-related. Neurospora crassa promoted OvCa progression with downregulation of integrin-linked kinase and decreased extracellular matrix-receptor interaction. Most ITF signals are likely contaminants. We identified N. crassa as associated with unfavorable prognosis in OvCa, potentially via modulation of the extracellular matrix.

Neurospora crassa↗

PubMed2025

Integrated 16 S rRNA and transcriptome analysis reveal molecular and microbial mechanisms of cold-tolerant germination in hulless barley.

BACKGROUND: Elucidating the mechanisms underlying cold-tolerant germination is crucial for enhancing crop resilience to low temperatures. Hulless barley (Hordeum vulgare var. coeleste L.), with remarkable natural cold adaptation, serves as an ideal model to study cold stress tolerance mechanisms in gramineous crops. In this study, cold-tolerant variety 37 and cold-sensitive variety 44 were screened and used to investigate the molecular mechanisms of cold-tolerant germination, via seed germination assays, combined with phytohormone determination, transcriptome sequencing and 16 S rRNA amplicon sequencing. RESULTS: Low temperature significantly inhibited hulless barley seed germination: the germination rate of cold-sensitive variety 44 decreased by 69%, while that of cold-tolerant variety 37 only decreased by 2%. Transcriptome analysis identified 2,647 and 2,392 differentially expressed genes (DEGs) in variety 37 and 44, respectively. Weighted gene co-expression network analysis (WGCNA) revealed a green module significantly positively correlated with gibberellic acid (GA) content, containing 10 core genes such as late embryogenesis abundant protein (LEA) and Homeobox genes. 16 S rRNA sequencing showed that the cold-tolerant variety 37 had enriched abundances of dominant endophytes including Sphingomonas and Pelomonas, with correlation coefficients of 0.70 and 0.87 with GA content, respectively. Additionally, exogenous GA treatment significantly increased germination rates under cold stress by 176.67% in cold-sensitive variety 44. CONCLUSIONS: This study confirms that the enhanced cold tolerance of hulless barley during seed germination originates from the synergistic interaction between beneficial endophytes (Sphingomonas, Pelomonas), GA, and core genes (e.g., LEA, Homeobox). Exogenous GA application can significantly restore the germination ability of cold-sensitive varieties. These findings provide a critical theoretical basis for improving cold tolerance in hulless barley germplasm.

Hordeum↗