Biology subjects

Bagby, S.

Publications and source records attributed to Bagby, S..

6 recordsLinked to original sources

bioRxiv2026

NAP: an open-source pipeline for cross-domain microbiome profiling using Nanopore sequencing-derived amplicon data

BackgroundNanopore sequencing offers a cost-effective and portable platform for microbiome analysis, but amplicon-based approaches remain limited by higher sequencing error rates and a lack of workflows tailored to mixed domain ribosomal RNA profiling. While short-read technologies dominate microbial community analysis, their portability and flexibility are constrained. There is therefore a need for robust pipelines designed specifically for cross-domain Nanopore amplicon data. ResultsWe introduce the Nanopore sequencing-based Amplicon Pipeline (NAP; https://github.com/Luke-B-Jones/NAP), an open-source workflow optimised for flexible mixed domain primer sets such as 515Y/926R. NAP performs adaptive quality filtering, chimera removal, centroid generation, BLAST-based taxonomic classification, hierarchical consensus correction, and domain-aware post-processing, outputting decontaminated abundance tables suitable for downstream analysis. Initial validation against two complementary commercial mock communities showed that NAP achieved strong genus-level performance across both low complexity logarithmic and more compositionally complex gut mock communities. Detection was most reliable above ca. 1% relative abundance, and replicate outputs showed strong agreement with expected composition under Bray-Curtis, Jaccard, agreement-plot, and Bland-Altman analyses. Benchmarking of NAPs internal filtering modes showed that the default adaptive setting provided the most robust balance of read quality, retained depth, and downstream taxonomic fidelity across heterogeneous inputs. Direct comparison against QIIME2 and Kraken2/Bracken further showed that NAP most accurately preserved expected community structure, with markedly fewer false positive assignments at genus level and substantially stronger species-level behaviour under the tested conditions. Species-level assignments were informative for some taxa, but remained less robust than genus-level outputs with the default V4-V5 amplicon. ConclusionsNAP provides a robust and flexible workflow for cross-domain Nanopore amplicon profiling, with strongest performance at genus level and competitive species-level behaviour for well resolved taxa. Although analysis of field-derived data was not assessed here, NAP compatibility with portable Nanopore sequencing supports accurate mixed domain microbiome profiling under the tested conditions.

bioinformatics↗

bioRxiv2026

Stress-induced DNA methylome plasticity and transcriptional re-programming in Staphylococcus aureus

Staphylococcus aureus, a major human and livestock pathogen, is the second biggest cause of antimicrobial resistance-associated mortality. Although S. aureus transcriptional regulation has been extensively characterised, the potential role of DNA methylation in S. aureus transcriptional regulation and stress response remains largely undefined. We tackled this gap by combining genome-wide nanopore sequencing-derived DNA methylation data and transcriptomic data, acquired before and during exposure of methicillin-resistant Staphylococcus aureus strain USA300 to clinically relevant oxidative, antibiotic and nitrosative stresses. Stress-induced significant DNA methylation changes were rare, with <0.1% of cytosines/adenines undergoing [≥]20% change in methylation; these changes were enriched within genomic features (protein-coding genes, predicted promoter regions, ncRNAs). Transcription changes reflected metabolic, regulatory and stress-specific pathway adjustments. Many of the stress-induced DNA methylation changes occurred alongside transcription changes, although there was no obvious overarching relationship between the directions of changes in DNA methylation and transcription. Pre-stress treatment methylation entropy tended to be elevated at sites containing a base that underwent stress-induced change in methylation level, identifying focal sites of pre-existing methylome heterogeneity relative to both local and genome-wide backgrounds; the magnitude of the site-specific methylation entropy peak, moreover, correlated with the magnitude of subsequent methylation change. Genome-wide entropy levels were consistent with a finite number of methylation patterns, distinguished by key bases, that could correspond to clinically important S. aureus subpopulations exhibiting persistence, dormancy and immune evasion. These findings support the principle that DNA methylation is an important component of the regulatory machinery underpinning S. aureus adaptability and persistence. ImportanceStaphylococcus aureus survives antibiotic treatment and host immune attack partly through phenotypic diversity, yet the regulatory processes that support this adaptability remain incompletely understood. Here, we integrate genome-wide DNA methylation and transcriptomic profiling to examine how epidemic methicillin-resistant S. aureus USA300 responds to oxidative, antibiotic, and nitrosative stresses. Stress-induced methylation changes were rare but non-random, concentrated in coding and regulatory regions linked to stress defence, metabolism, persistence, virulence, biofilm formation, and host interaction. Sites that underwent stress-induced methylation changes tended to have unusually high pre-stress methylation pattern diversity relative to local and genome-wide pattern diversity. These findings support a model in which an S. aureus population contains a pre-stress repertoire of epigenetic states that stress selectively redistributes via bases that differentiate stable methylation patterns. By revealing a layer of heterogeneity that may support pathogen resilience, this work provides a framework for investigating whether bacterial methylation dynamics can be exploited clinically.

microbiology↗

bioRxiv2023

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons

Dopa-responsive dystonia (DRD) and Parkinsons disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

neuroscience↗

bioRxiv2021

Comparative genomics of Bordetella pertussis isolates from New Zealand, a country with an uncommonly high incidence of whooping cough

2.Whooping cough, the respiratory disease caused by Bordetella pertussis, has undergone a wide-spread resurgence over the last several decades. Previously, we developed a pipeline to assemble the repetitive B. pertussis genome into closed sequences using hybrid nanopore and Illumina sequencing. Here, this sequencing pipeline was used to conduct a more high-throughput, longitudinal screen of 66 strains isolated between 1982 and 2018 in New Zealand. New Zealand has a higher incidence of whooping cough than many other countries; usually at least twice as many cases per 100,000 people as the USA and UK and often even higher, despite similar rates of vaccine uptake. To the best of our knowledge, these strains are the first New Zealand B. pertussis isolates to be sequenced. The analyses here show that, on the whole, genomic trends in New Zealand B. pertussis isolates, such as changing allelic profile in vaccine-related genes and increasing pertactin deficiency, have paralleled those seen elsewhere in the world. At the same time, phylogenetic comparisons of the New Zealand isolates with global isolates suggest that a number of strains are circulating in New Zealand which cluster separately from other global strains, but which are closely related to each other. The results of this study add to a growing body of knowledge regarding recent changes to the B. pertussis genome, and are the first genetic investigation into B. pertussis isolates from New Zealand. 3. Impact statementSince the 1990s, whooping cough has been resurgent in many countries around the world, despite the wide availability of vaccines. New Zealand has often had a higher incidence of whooping cough than other countries such as the USA, UK and Australia, both during outbreak periods and in the intervening years. One potential reason for the resurgence of whooping cough is genetic changes to the causative bacterium, Bordetella pertussis, with several recently identified, ongoing global genetic trends. No B. pertussis isolates from New Zealand have previously been sequenced, however. Here, we used hybrid sequencing to investigate the genomes of 66 New Zealand B. pertussis isolates, collected between 1982 and 2018. This revealed that genetic trends in New Zealand B. pertussis match those observed elsewhere, but over the years a number of highly similar or identical strains appear to have circulated (or are currently circulating) in the country, a phenomenon not commonly noted elsewhere. This first study of B. pertussis isolates from New Zealand contributes to the global understanding of B. pertussis genomics, as well as providing the groundwork for any future whole genome sequencing of New Zealand B. pertussis isolates. 4. Data summaryO_LINanopore and Illumina fastq sequence files for all strains have been deposited in NCBIs Sequence Read Archive, BioProject PRJNA556977. A full list of accession numbers for all sequence read files is provided in Supplementary Table S1. C_LIO_LIGenome sequences for 63 strains have been deposited in NCBIs GenBank, BioProject PRJNA556977, accession numbers in Supplementary Table S1. C_LIO_LIGenome assemblies for 3 strains assembled using only nanopore data (NZ1, NZ5 and NZ29), which had a high number of pseudogenes, were not deposited in GenBank, but are available from Figshare: https://doi.org/10.6084/m9.figshare.12640463 C_LIO_LISource code and full commands used are available from Github: https://github.com/nataliering/Comparative-genomics-of-Bordetella-pertussis-isolates-from-New-Zealand C_LI The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

genomics↗

bioRxiv2020

Duplications drive diversity in Bordetella pertussis on an underestimated scale.

Bacterial genetic diversity is often described using solely base pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplications of genomic loci are thought to be widespread among bacteria but due to their often intractable size and instability, comprehensive studies of the range and genome dynamics of these mutations are rare. We define a methodology to investigate duplications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for duplications to occur. Analysis of genome sequence data for 2430 B. pertussis isolates identified 272 putative duplications, of which 94% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of duplications, suggesting unstable and sporadic characteristics. Genome instability was further described in-vitro using long read sequencing via the Nanopore platform. Clonally derived laboratory cultures produced heterogenous populations containing multiple structural variants. Short read data was used to predict 272 duplications, whilst long reads generated on the Nanopore platform enabled the in-depth study of the genome dynamics of tandem duplications in B. pertussis. Our work reveals the unrecognised and dynamic genetic diversity of B. pertussis and, as the complexity of the B. pertussis genome is not unique, highlights the need for a holistic and fundamental understanding of bacterial genetics.

microbiology↗

bioRxiv2018

Resolving the complex Bordetella pertussis genome using barcoded nanopore sequencing

The genome of Bordetella pertussis is complex, with high GC content and many repeats, each longer than 1,000 bp. Short-read DNA sequencing is unable to resolve the structure of the genome; however, long-read sequencing offers the opportunity to produce single-contig B. pertussis assemblies using sequencing reads which are longer than the repetitive sections. We used an R9.4 MinION flow cell and barcoding to sequence five B. pertussis strains in a single sequencing run. We then trialled combinations of the many nanopore-user-community-built long-read analysis tools to establish the current optimal assembly pipeline for B. pertussis genome sequences. Our best long-read-only assemblies were produced by Canu read correction followed by assembly with Flye and polishing with Nanopolish, whilst the best hybrids (using nanopore and Illumina reads together) were produced by Canu correction followed by Unicycler. This pipeline produced closed genome sequences for four strains, revealing inter-strain genomic rearrangement. However, read mapping to the Tohama I reference genome suggests that the remaining strain contains an ultra-long duplicated region (over 100 kbp), which was not resolved by our pipeline. We have therefore demonstrated the ability to resolve the structure of several B. pertussis strains per single barcoded nanopore flow cell, but the genomes with highest complexity (e.g. very large duplicated regions) remain only partially resolved using the standard library preparation and will require an alternative library preparation method. For full strain characterisation, we recommend hybrid assembly of long and short reads together; for comparison of genome arrangement, assembly using long reads alone is sufficient.\n\nDATA SUMMARYO_LIFinal sequence read files (fastq) for all 5 strains have been deposited in the SRA, BioProject PRJNA478201, accession numbers SAMN09500966, SAMN09500967, SAMN09500968, SAMN09500969, SAMN09500970\nC_LIO_LIA full list of accession numbers for Illumina sequence reads is available in Table S1\nC_LIO_LIAssembly tests, basecalled read sets and reference materials are available from figshare: https://figshare.com/projects/Resolving_the_complex_Bordetella_pertussis_genome_using_barcoded_nanopore_sequencing/31313\nC_LIO_LIGenome sequences for B. pertussis strains UK36, UK38, UK39, UK48 and UK76 have been deposited in GenBank; accession numbers: CP031289, CP031112, CP031113, QRAX00000000, CP031114\nC_LIO_LISource code and full commands used are available from Github: https://github.com/nataliering/Resolving-the-complex-Bordetella-pertussis-genome-using-barcoded-nanopore-sequencing\nC_LI\n\nIMPACT STATEMENTOver the past two decades, whole genome sequencing has allowed us to understand microbial pathogenicity and evolution on an unprecedented level. However, repetitive regions, like those found throughout the B. pertussis genome, have confounded our ability to resolve complex genomes using short-read sequencing technologies alone. To produce closed B. pertussis genome sequences it is necessary to use a sequencing technology which can generate reads longer than these problematic genomic regions. Using barcoded nanopore sequencing, we show that multiple B. pertussis genomes can be resolved per flow cell. Use of our assembly pipeline to resolve further B. pertussis genomes will advance understanding of how genome-level differences affect the phenotypes of strains which appear monomorphic at nucleotide-level.\n\nThis work expands the recently emergent theme that even the most complex genomes can be resolved with sufficiently long sequencing reads. Additionally, we utilise a more widely accessible alternative sequencing platform to the Pacific Biosciences platform already used by large research centres such as the CDC. Our optimisation process, moreover, shows that the analysis tools favoured by the sequencing community do not necessarily produce the most accurate assemblies for all organisms; pipeline optimisation may therefore be beneficial in studies of unusually complex genomes.

microbiology↗