Biology subjects

Carr, P. G.

Publications and source records attributed to Carr, P. G..

4 recordsLinked to original sources

bioRxiv2026

Long read and short read whole genome sequencing are equivalent for genomic characterisation of bacteriophage: considerations for high throughput analysis

BackgroundAntimicrobial resistance (AMR) is a global health crisis, necessitating alternative antibacterial strategies. Bacteriophages (phages) offer a promising solution, and their use as a therapeutic agent relies on stringent bioinformatic characterisation using whole genome sequencing (WGS) technologies. However, phages are highly diverse, with no clear consensus on best practices concerning phage DNA extraction or sequencing platform. Efficient and repeatable DNA extraction, sequencing, and bioinformatics processes are critical for safety assessments but remain poorly defined. Additionally, the impact of sequencing platform choice and DNA extraction methods on downstream genomic analyses is not well understood. MethodsWe evaluated multiple DNA extraction, library preparation, and sequencing approaches using a diverse collection of Pseudomonas phages from the PhageWA biobank. Column-based and precipitation-based DNA extraction methods were compared for DNA yield and recovery efficiency. Genome sequencing was performed using short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms, incorporating multiple library preparation kits and Nanopore basecalling models. Assemblies were assessed for completeness, quality, and sequence concordance using standardised bioinformatics pipelines, with hybrid Illumina-Nanopore assemblies used as references for comparison. ResultsDNA extraction efficiency varied substantially between protocols, with the Puregene precipitation-based method yielding significantly higher DNA recovery than column-based approaches when normalised to phage titre. Illumina sequencing consistently generated complete genome assemblies, although assembly fragmentation was observed for several jumbo phages when using the SeqWell ExpressPlex 2.0 library preparation method. For Nanopore sequencing, ligation-based native barcoding libraries produced longer reads than rapid barcoding libraries, while selection of the Dorado v5.0.0 basecalling model significantly improved read quality. Genome assembly success was dependent on phage genus; native Nanopore sequencing failed to assemble several Pbunavirus genomes, likely due to modified DNA bases, but an amplification-based library preparation successfully resolved these genomes. Across successfully assembled samples, Illumina and Nanopore platforms produced highly concordant genomes with comparable completeness scores, and hybrid polishing identified only minor sequence differences. ConclusionsDNA extraction methodology, sequencing chemistry, and basecalling model selection significantly influence phage WGS outcomes. Precipitation-based DNA extraction improved DNA recovery, while both Illumina and Nanopore sequencing generated high-quality phage genomes suitable for therapeutic characterisation. Nanopore sequencing provided assemblies comparable to Illumina with minimal benefit from hybrid polishing, supporting its routine use for phage genomics. These findings provide practical guidance for phage genome characterisation workflows and contribute to the development of standardised, regulatory-grade approaches for therapeutic phage assessment.

microbiology↗

bioRxiv2026

Animal-free peptones do not alter bacteriophages propagated for therapeutic use

AimsBacteriophage (phage) propagation has traditionally relied on bacterial culture media containing animal-derived ingredients; however, safety concerns with animal-derived materials for production of phages for therapeutic use limit their acceptability. We compared animal-free and traditional media formulations, and evaluated their effects on phage yield, bactericidal activity, and genomic characteristics, hypothesizing no significant differences would be observed. Methods and ResultsPhages targeting Pseudomonas aeruginosa (n=8) and Staphylococcus aureus (n=1) were propagated in solid and liquid media containing animal-free (AF) or animal-derived (LB) peptones. Kinetic assays were used to assess phage suppression of host bacterial growth. In a mock therapeutic phage screen, spot tests, Efficiency of Plating (EOP) and kinetic assays were performed against novel bacterial targets. Whole genome sequencing of phages and their bacterial hosts propagated in AF or LB broth was used to observe genomic differences between formulations. Animal-free peptone did not impact phage yield, with both AF and LB phage stocks growing to high titers ([≥]108 PFU/mL). Kinetic assay results showed similar suppression indices for AF and LB-grown phages. Likewise, phage screen spot test, EOP, and kinetic assay results were similar between AF and LB phages. Comparisons of phage and bacterial genome annotations showed no major differences arising from media formulation. ConclusionsFindings suggest animal-free peptones do not significantly alter phage yield, bactericidal activity, or genomic characteristics, supporting use of animal-free medium for medicinal phage manufacture. This is one of the first studies to systematically combine phenotypic and genomic assessment of phages and hosts across animal-free and traditional media. Impact StatementPhage therapy is increasingly used to treat antimicrobial resistance infections. Emerging guidelines and regulations for the manufacture of phage therapeutics will impact laboratory processes and materials used for phage production. Here, we explored the use of an animal-free medium for medicinal phage propagation, providing data on phage yield and metrics of phage activity.

microbiology↗

bioRxiv2026

Genomic Determinants of Phage Activity Against Pseudomonas aeruginosa: Roles of Receptors, Defence Systems, and Anti-Defences

Abstract/SummaryPseudomonas aeruginosa is a priority pathogen in chronic and multidrug-resistant infections, yet therapeutic phages targeting this organism often exhibit variable and unpredictable efficacy. A mechanistic understanding of the genomic determinants governing phage-host interactions is therefore critical for the rational design of robust phage therapeutics. Here, we systematically dissected the genetic featrures underlying lytic outcomes across a diverse panel of P. aeruginosa-infecting phages. We comprehensively annotated receptor-binding proteins (RBPs), bacterial defence systems and phage-encoded anti-defence genes, and experimentally defined host receptor usage for each phage, linking receptor specificity to cognate RBPs. We identified multiple anti-defence systems--including vcrx089, acrIIA24, atd1, gnarl1, klcA, darA and nmna (NARP2-associated)--and RBPs targeting lipopolysaccharide, type IV pili and flagella that are associated with enhanced lytic activity and represent tractable engineering targets. Across 174 genomic features analysed, 110 significantly influenced phage activity, with heterogeneous and context-dependent effects. Leveraging these features, we trained a machine-learning classifier that accurately predicted phage-host outcomes (AUC-ROC = 0.875), demonstrating that interaction phenotypes are encoded in definable genomic signatures. Together, our findings reveal the quantitative contribution of phage anti-defence systems to infectivity in P. aeruginosa and define a genomic framework for predicting and engineering lytic success. These results establish a foundation for the rational design of synthetic phages with enhanced host ranges.

microbiology↗

bioRxiv2024

Optimisation of DNA extraction from nasal lining fluid to assess the nasal microbiome using third-generation sequencing

BackgroundSampling nasal lining fluid (NLF) via nasosorption is minimally invasive and well tolerated, but the feasibility of assessing the nasal microbiome using this technique is unknown. However, low biomass makes airway samples particularly susceptible to issues related to contaminant DNA. In this study, we evaluated the suitability of DNA isolated using methodologies for low-biomass respiratory samples and assessed how well lining fluid collected via nasosorption captures the nasal microbial diversity and composition compared to the traditional swab sampling approach. MethodsNasal swabs and NLF were collected from adult volunteers. DNA was extracted from a mock microbial community and NLF using a column-based kit (ZymoBIOMICS), a precipitation-based kit (Qiagen), or a previously published precipitation-based method. Quality and quantity of DNA was assessed and short-read 16S rRNA sequencing performed to assess feasibility and extraction bias. An optimised extraction methodology was then used to extract DNA from NLF and nasal swabs, and full-length 16S rRNA sequencing performed to compare microbial profiles between NLF and nasal swabs. Taxonomy was assigned using the nf-core/ampliseq pipeline, the PacificBiosciences/pb-16S-nf pipeline, or the software Emu, and downstream analyses were performed using R packages phyloseq and mixOmics. ResultsAll extraction methods recovered DNA from the mock community, but only precipitation-based methods yielded sufficient DNA from NLF. Extraction methodologies significantly affected microbial profiles, with mechanical lysis needed to minimize bias against specific genera. Profiles obtained from NLF and swabs were comparable with long-read sequencing. ConclusionsOur findings demonstrate the feasibility of profiling the nasal microbiome using NLF collected via nasosorption and validated two extraction methodologies as suitable for full-length 16S rRNA sequencing of low-biomass respiratory samples. Our data demonstrate the importance of unbiased DNA extraction methodologies in low-biomass respiratory samples, and the subsequent impact of DNA extraction on observed microbial profiles. Additionally, we demonstrated NLF may be an appropriate surrogate samples for nasal swabs to assess the nasal microbiome using 16S rRNA sequencing.

microbiology↗