Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens

Journal of Clinical Microbiology, Год журнала: 2024, Номер 62(5)

Опубликована: Март 5, 2024

ABSTRACT Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) gained prominence may offer advantages over sequencing, particularly recent introduction R10 chemistry, which promises much lower error rates than R9 chemistry. However, limited information is available on its performance single-nucleotide polymorphism (SNP)-based investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) ( https://github.com/BioinformaticsPlatformWIV-ISP/PACU ), constructing SNP phylogenies and/or ONT R9/R10 data. The workflow was evaluated data sets Shiga toxin-producing Escherichia coli Listeria monocytogenes by comparing each technology not only separately but also integrating samples sequenced different technologies/chemistries into same phylogenomic analysis. Additionally, minimum time required to obtain accurate phylogenetic results nanopore evaluated. PACU allowed identification clusters both species all technologies/chemistries, deviated slightly more from results. showed trends very similar Illumina, we found that either or isolates analysis produced stable highly resulting these two outbreaks stabilized after ~20 hours ~8 R10. This study provides a proof concept R10, in isolation combination rapid SNP-based

Язык: Английский

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How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies

Microbial Genomics, Год журнала: 2024, Номер 10(6)

Опубликована: Июнь 4, 2024

It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing usually required for perfection. However, the effect of depth on performance not well understood. Here, we introduce Pypolca (with default and careful parameters) Polypolish v0.6.0 a new parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default Polypolish-careful commonly false-positive errors at low read depth; (2) most benefit occurs by 25× (3) almost never introduces any (4) Pypolca-careful single effective polisher. Overall, recommend following strategies: alone when very (<5×), (5–25×), sufficient (>25×).

Язык: Английский

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Do we still need Illumina sequencing data? Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and the Rapid v14 library prep kit for Gram negative bacteria whole genome assemblies

Canadian Journal of Microbiology, Год журнала: 2024, Номер 70(5), С. 178 - 189

Опубликована: Фев. 14, 2024

The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read that can bridge repetitive problematic regions. Oxford Nanopore Technologies (ONT) produce platforms they continually improving their technology to obtain higher quality read is approaching the obtained such as Illumina. As these innovations continue, we evaluated how much ONT coverage produced by Rapid Barcoding Kit v14 (SQK-RBK114) necessary generate high-quality hybrid long-read-only for panel carbapenemase-producing Enterobacterales bacterial isolates. We found 30× sufficient if Illumina available, more (at least 100× recommended assemblies. polishing still single nucleotide variants (SNVs) INDELs in also examined antimicrobial resistance genes could be accurately identified data, Flye regardless detected >96% at 100% identity length. Overall, an optimal strategy (i.e., plasmid characterization AMR detection) but finer-scale analyses SNV) benefit data.

Язык: Английский

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Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies

Microbial Genomics, Год журнала: 2024, Номер 10(5)

Опубликована: Май 8, 2024

Improvements in the accuracy and availability of long-read sequencing mean that complete bacterial genomes are now routinely reconstructed using hybrid (i.e. short- long-reads) assembly approaches. Complete allow a deeper understanding evolution genomic variation beyond single nucleotide variants. They also crucial for identifying plasmids, which often carry medically significant antimicrobial resistance genes. However, small plasmids missed or misassembled by algorithms. Here, we present Hybracter allows fast, automatic scalable recovery near-perfect first approach. can be run either as assembler only assembler. We compared to existing automated tools diverse panel samples varying levels with manually curated ground truth reference genomes. demonstrate is more accurate faster than gold standard Unicycler. show long-reads most comparable methods accurately recovering plasmids.

Язык: Английский

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Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, Klebsiella pneumoniae

Microbial Genomics, Год журнала: 2023, Номер 9(2)

Опубликована: Фев. 8, 2023

Oxford Nanopore Technologies (ONT) sequencing has rich potential for genomic epidemiology and public health investigations of bacterial pathogens, particularly in low-resource settings at the point care, due to its portability affordability. However, low base-call accuracy limited reliability ONT data critical tasks such as antimicrobial resistance (AMR) virulence gene detection typing, serotype prediction, cluster identification. Thus, Illumina remains standard surveillance despite higher capital running costs. We tested ONT-only assemblies common applied genomics (genotyping detection, implemented via Kleborate, Kaptive Pathogenwatch), using from 54 unique Klebsiella pneumoniae isolates. reads generated MinION with R9.4.1 flowcells were basecalled three alternative models [Fast, High-accuracy (HAC) Super-accuracy (SUP), available within ONT's Guppy software], assembled Flye polished Medaka. Accuracy typing was compared that Illumina-only hybrid ONT+Illumina assemblies, constructed same isolates reference standards. The most resource-intensive ONT-assembly approach (SUP basecalling, or without Medaka polishing) performed best, yielding reliable capsule (K) type calls all strains (100 % exact best matching locus), multi-locus sequence (MLST) assignment (98.3 match single-locus variants), good acquired AMR genes mutations (88-100 correct identification across various drug classes). Distance-based trees SUP+Medaka accurately reflected overall genetic relationships between definition outbreak clusters problematic inflation SNP counts by high errors. could be reliably used 'rule out' distinct lineages suspected transmission clusters. HAC basecalling + polishing similarly SUP polishing. Therefore, we recommend investing compute resources into model), wherever time allow, note is also worthwhile improved performance. Overall, our results show MLST, K determinants can identified flowcell data. challenging this technology.

Язык: Английский

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Plassembler: an automated bacterial plasmid assembly tool

Bioinformatics, Год журнала: 2023, Номер 39(7)

Опубликована: Июнь 27, 2023

With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining a long-read-first assembly approach with short-read polishing. However, existing methods for assembling plasmids from often misassemble or even miss entirely accordingly require manual curation. Plassembler was developed provide tool that automatically assembles outputs using hybrid approach. It achieves increased accuracy computational efficiency compared the gold standard Unicycler removing chromosomal reads input read sets mapping

Язык: Английский

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Genomic dissection of endemic carbapenem resistance reveals metallo-beta-lactamase dissemination through clonal, plasmid and integron transfer

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Авг. 8, 2023

Abstract Infections caused by metallo-beta-lactamase-producing organisms (MBLs) are a global health threat. Our understanding of transmission dynamics and how MBLs establish endemicity remains limited. We analysed two decades bla IMP-4 evolution in hospital using sequence data from 270 clinical environmental isolates (including 169 completed genomes) identified the gene across 7 Gram-negative genera, 68 bacterial strains distinct plasmid types. showed an initial multi-species outbreak conserved IncC plasmids (95 genomes 37 strains) allowed to be established through ability disseminate successful strain-genetic setting pairs we termed propagators, particular Serratia marcescens Enterobacter hormaechei . From this reservoir, persisted diversification genetic settings that resulted transfer between hosts integron carrying plasmids. findings provide framework for spread may have broader applicability other carbapenemase-producing organisms.

Язык: Английский

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Nanopore sequencing: flourishing in its teenage years

Journal of genetics and genomics/Journal of Genetics and Genomics, Год журнала: 2024, Номер unknown

Опубликована: Сен. 1, 2024

Язык: Английский

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How low can you go? Short-read polishing of Oxford Nanopore bacterial genome assemblies

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Март 10, 2024

Abstract It is now possible to assemble near-perfect bacterial genomes using Oxford Nanopore Technologies (ONT) long reads, but short-read polishing still required for perfection. However, the effect of depth on performance not well understood. Here, we introduce Pypolca (with default and careful parameters) Polypolish v0.6.0 a new parameter). We then show that: (1) all polishers other than Pypolca-careful, Polypolish-default Polypolish-careful commonly false-positive errors at low depth; (2) most benefit occurs by 25× (3) never introduces any (4) Pypolca-careful single effective polisher. Overall, recommend following strategies: alone when very (<5×), (5–25×), sufficient (>25×). Data Summary open-source freely available Bioconda, PyPI, GitHub ( github.com/gbouras13/pypolca ). Bioconda github.com/rrwick/Polypolish All code data reproduce analyses figures are github.com/gbouras13/depth_vs_polishing_analysis . FASTQ sequencing reads BioProject PRJNA1042815 A detailed list accessions can be found in Table S1.

Язык: Английский

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Roadmap for the integration of environmental microbiomes in risk assessments under EFSA's remit

EFSA Supporting Publications, Год журнала: 2024, Номер 21(2)

Опубликована: Фев. 1, 2024

Scientific interest in the use of environmental microbiomes for risk assessment is rapidly growing, as exemplified by various EFSA opinions. In absence official regulatory guidelines on how to integrate assessment, aims this report are therefore determine whether microbiome studies can be used such purposes, and propose a roadmap integration assessments under EFSA's remit. The identifies current gaps (in terms knowledge from technical point view) barriers that might delay implementation methods, offers recommendations standardised (multi-)omics techniques purposes. Our main findings identified five priorities: (i) defining core (what it encompasses what made of, including identification bioindicators) assess impact any type disturbance; (ii) standardising methodologies protocols, sampling interpretation, guarantee comparability analyses; (iii) developing tools facilitate interpretation; (iv) collecting microbiome-based data shared, curated maintained databases; (v) setting up European Network Microbiome Laboratories reach an agreement standardise studies, interactions between researchers access or samples, actively include multiple stakeholders discussions involving assessment. There both short- longer-term priorities, all which highlight need mobilise concurrently different agencies institutions, well research. also points out capacity building training, acceptance emerging technology, communication issues. These will hopefully contribute elaboration widely accepted framework dealing with.

Язык: Английский

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