Biology subjects

Hope, R.

Publications and source records attributed to Hope, R..

6 recordsLinked to original sources

bioRxiv2025

Nanopore long-read only genome assembly of clinical Enterobacterales isolates is complete and accurate

Whole bacterial genome sequence reconstruction using Oxford Nanopore Technologies ("Nanopore") long-read only sequencing may offer a lower-cost, higher-throughput alternative for pathogen surveillance to hybrid assembly with recent improvements in Nanopore sequencing accuracy. We evaluated the accuracy, including plasmid reconstruction, of Nanopore long-read only genome assemblies of Enterobacterales. We sequenced 92 genomes from clinical Enterobacterales isolates, collected in England under a national surveillance program, with long-read Nanopore (R10.4.1, Dorado v5.0.0 super-high-accuracy basecalled) and short-read Illumina (NovaSeq) sequencing approaches. Genomes were assembled using three long-read only (Flye; Hybracter long; Autocycler), and three hybrid assemblers (Hybracter hybrid; Unicycler normal; bold). Three polishing modalities (Medaka v2 with subsampled or un-subsampled long-reads; Polypolish + Pypolca with short-reads) were investigated. Autocycler circularised the most chromosomes (87/92 [95%]). Plasmid sequence reconstruction was comparable between all assemblers except Flye, all recovering 90-96% of plasmids, although the ground truth was uncertain. Flye performed worse than other assemblers on almost all metrics. Autocycler + Medaka (un-subsampled long-reads) was the most accurate long-read only assembler/polisher combination, comparable to hybrid assemblies (median 0 [IQR:0-0] SNPs and 0 [IQR:0-1] indels per genome; quality value/Q score, 100 [IQR: 64-100]), with only 4/92 genome sequences having >10 SNPs/indels. Medaka polishing with un-subsampled long-reads resulted in small improvements in indels but not SNPs for both Flye and Autocycler assemblies. Seven-locus MLST, antimicrobial resistance, virulence, and stress gene annotation was equivalent across assembler/polisher combinations. Nanopore long-read only bacterial genome assembly with Autocycler combined with Medaka polishing (using un-subsampled reads) is similarly accurate and possibly more complete than hybrid assemblies, representing a viable alternative for incorporating high-quality genomic data, including plasmids, into Enterobacterales surveillance. Data SummaryNanopore long-reads and Illumina short-reads from the 92 Enterobacterales isolates from this study have been uploaded to ENA (BioProject accession: PRJEB93885). Code for the Nextflow assembly pipeline, downstream analysis scripts, and R statistical analysis scripts are available on GitHub (https://github.com/oxfordmmm/NEKSUS_ont_hybrid_assembly_comparison). The following supplementary data tables are available on FigShare (https://figshare.com/account/home#/projects/253775): O_LIENA Sample accessions and sample metadata (accessions_and_metadata.csv) C_LIO_LISeqkit stats summaries of the Illumina and Nanopore reads (raw_qc_sup.cav) C_LIO_LISummary of assembly contig features (contigs_summary_sup_cleaned.csv) C_LIO_LIPairwise mash distances between contigs (mash_cleaned.csv) C_LIO_LIPlasmids matching across different assemblers compared to the Hybracter (hybrid) and manually-curated reference sets (plasmids_match_hybracter_mash.csv; plasmids_match_manual_mash.csv, respectively) C_LIO_LISeven-locus multi-locus sequence type annotation (mlst_cleaned.csv) C_LIO_LICheckM2 summaries of assemblies (checkm2_cleaned.csv) C_LIO_LINucleotide-level accuracy of assemblies (SNP, Indels, and Quality value compared to short-read mapping; assembly_nucleotide_accuracy_cleaned.csv) C_LIO_LIBakta annotation (bakta_by_contig_cleaned.csv) C_LIO_LIAMRFinderPlus annotations of contigs (amrfinder_plus_cleaned.csv) C_LIO_LIMOB-suite annotation summaries of contigs (mobsuite_cleaned.csv) C_LI Impact StatementNanopore long-reads have historically been too error-prone to use alone for accurate bacterial genome assembly, necessitating additional Illumina short-reads to achieve structurally complete and accurate hybrid genome assemblies for public health surveillance. This increases cost and complexity. Previous studies have shown that recent improvements in Nanopore chemistry (R10.4.1 flowcell) and basecalling (super-high accuracy) allow high-quality long-read only assemblies on a small number of laboratory reference strains. This is the first evaluation, to our knowledge, to assess Nanopore long-read only genome assembly compared with hybrid assembly on a large number of clinical isolates. In addition, this is the first large-scale evaluation of the recently released automated consensus long-read assembly tool, Autocycler. We show that Autocycler long-read only assemblies are more structurally complete for chromosomal sequences, while reconstructing a similar number of plasmids to other long-read and hybrid assemblers. Most long-read polished, Autocycler-assembled genome sequences have 0 errors (median: 0 SNPs/indels) relative to a short-read polished (hybrid) Autocycler assemblies, enabling accurate annotation of key genes.

bioinformatics↗

bioRxiv2025

Phylogenetic clustering of microbial communities as a biomarker for chemical pollution

Microbial communities play a critical role in ecosystem functioning and offer promising potential as bioindicators of chemical pollution in aquatic environments. Here we examine the responses of both bacterial isolates and microbial communities to a range of pollutants, focusing on the phylogenetic predictability of their responses. We found that bacterial isolates exhibited a strong phylogenetic signal in their growth responses, with closely related taxa responding similarly to chemical stress. In microbial communities, pollutants that significantly impacted isolates also reduced community diversity and growth, causing shifts in community structure toward increased phylogenetic clustering, suggesting environmental filtering. The net relatedness index (NRI) effectively captured these shifts, indicating its potential as a simple metric for monitoring pollution. Our findings highlight the predictability of microbial responses to pollution and suggest that microbial-based bioindicators, coupled with rapid sequencing technologies, could transform environmental monitoring.

microbiology↗

bioRxiv2022

IGHV allele similarity clustering improves genotype inference from adaptive immune receptor repertoire sequencing data

In adaptive immune receptor repertoire analysis, determining the germline variable (V) allele associated with each T- and B-cell receptor sequence is a crucial step. This process is highly impacted by allele annotations. Aligning sequences, assigning them to specific germline alleles, and inferring individual genotypes are challenging when the repertoire is highly mutated, or sequence reads do not cover the whole V region. Here, we propose an alternative naming scheme for the V alleles as well as a novel method to infer individual genotypes. We demonstrate the strength of the two by comparing their outcomes to other genotype inference methods and validated the genotype approach with independent genomic long read data. The naming scheme is compatible with current annotation tools and pipelines. Analysis results can be converted from the proposed naming scheme to the nomenclature determined by the International Union of Immunological Societies (IUIS). Both the naming scheme and the genotype procedure are implemented in a freely available R package (PIgLET). To allow researchers to explore further the approach on real data and to adapt it for their future uses, we also created an interactive website (https://yaarilab.github.io/IGHV_reference_book).

immunology↗

bioRxiv2021

The spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei is induced by perturbations of endoplasmic reticulum, Golgi, or mitochondrial proteins factors and functional analysis of SLS inducing kinase, PK3

In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5 exon derived from the spliced leader RNA (SL RNA). Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA binding protein TRF4, leading to the shut-off of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi-localized quiescin sulfhydryl oxidase (QSOX1). Most strikingly, silencing of Rhomboid-like 1(TIMRHOM1) involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structure of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.

molecular biology↗

bioRxiv2019

Using hospital network-based surveillance for antimicrobial resistance as a more robust alternative to self-reporting

Hospital performance is often measured using self-reported statistics, such as the incidence of hospital-transmitted micro-organisms or those exhibiting antimicrobial resistance (AMR), encouraging hospitals with high levels to improve their performance. However, hospitals that increase screening efforts will appear to have a higher incidence and perform poorly, undermining comparison between hospitals and disincentivising testing, thus hampering infection control. We propose a surveillance system in which hospitals test patients previously discharged from other hospitals and report observed cases. Using NHS Hospital Episode Statistics data, we analysed patient movements across England and assessed the number of hospitals required to participate in such a reporting scheme to deliver robust estimates of incidence. With over 1.2 million admissions to English hospitals previously discharged from other hospitals annually, even when only a fraction of hospitals (41/155) participate (each screening at least 1000 of these admissions), the proposed surveillance system can estimate incidence across all hospitals. By reporting on other hospitals, the reporting of incidence is separated from the task of improving own performance. Therefore the incentives for increasing performance can be aligned to increase (rather than decrease) screening efforts, thus delivering both more comparable figures on the AMR problems across hospitals and improving infection control efforts.

epidemiology↗

bioRxiv2017

A National Estimate of the Health and Cost Burden of Escherichia coli Bacteraemia in the Hospital Setting: The Importance of Antibiotic Resistance.

BackgroundAntibiotic resistance poses a threat to public health and a burden to healthcare systems. Escherichia coli causes more bacteraemia cases in England than any other bacterial species, these infections, in part due to their high incidence, also pose a significant antibiotic resistance burden. The main aim of this study was to estimate the impact of E. coli bacteraemia on patient in-hospital mortality and length of stay. Secondarily, this study also aimed to estimate the effect of antibiotic resistance on these outcomes.\n\nMethods and FindingsCase patients were adult E. coli bacteraemia patients infected between July 2011 and June 2012, as reported in an English national mandatory surveillance database, with susceptibility data taken from a national laboratory surveillance database. Control patients were all non-case, adult patients with an English hospital admission record. Case and control patient characteristics and admission information were taken from NHS Digital Datasets. Resistance was defined as non-susceptible and intermediate isolates, whilst susceptible was defined as susceptible and non-tested isolates. Time to in-hospital mortality and discharge was investigated through Cox proportional hazards models. To acquire estimates of excess length of stay, multistate models were constructed, with a unit bed day cost applied to estimate cost burden. The total number of case and control hospital spells was 14,051 and 8,919,275 respectively. Acquisition of E. coli bacteraemia was associated with a statistically significant increased daily risk of in-hospital mortality, especially for the first eight days of someones hospital admission [Hazard Ratio = 2.77 (95% confidence interval; 2.61-2.94)]. Antibiotic resistance did not seem to significantly increase this risk further, though did significantly reduce risk of experiencing a discharge event (dead or alive). E.coli bacteraemia was estimated to cost {pound}14,340,900 over the study period (rounded to the nearest {pound}100), with resistance associated with excess costs per infection of {pound}220 - {pound}420 dependent on resistance type, for those where a significant impact was found (rounded to the nearest {pound}10).\n\nConclusionsE. coli bacteraemia places a significant burden on patient health and on the hospital sector in England. Resistance is an important factor on length of stay with regards to such infections.

epidemiology↗