Nanopore sequencing: flourishing in its teenage years

Journal of genetics and genomics/Journal of Genetics and Genomics, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 1, 2024

Language: Английский

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Improving Nanopore sequencing-based core genome MLST for global infection control: a strategy for GC-rich pathogens like Burkholderia pseudomallei

Journal of Clinical Microbiology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 6, 2025

ABSTRACT Genomic surveillance of pathogens is essential to trace infections and analyze resistance markers. Core genome multilocus sequence typing (cgMLST) facilitates genomic by simplified analysis standardization. However, its application limited the poor cost-efficiency short-read (SR) sequencing. Oxford Nanopore long-read sequencing (ONT-LR), which allows fast on-site with comparatively low costs, could provide an alternative. Despite ONT-LR raw read accuracy improvement, evidence for methylation-based errors accumulates. PCR-based library preparation, suggested as a solution, presumably poses difficulties GC-rich bacteria. We challenged ONT-LR-based cgMLST using highly pathogen Burkholderia pseudomallei develop clinically applicable workflow. Our B. scheme was applied data, results were validated against SR data. Native, rapid, preparation performed combined different basecalling models (SUP@bacterial-methylation, [email protected], [email protected], [email protected]) polishing strategies (medaka_consensus, medaka_variant, r103_min_high_g360). To ensure reliability across genotypes, we included 14 types 27 genotypes. The recommended workflow at study initiation ([email protected], medaka_consensus) showed nearly 200 allele differences compared reference specific strains. resulted in missing targets up 21 alleles. Native barcoding [email protected] r103_min_high_g360 outperformed approach all parameters reducing error rate maximum two differences. optimized integrates high resolution ease implementation enhanced rapid diagnostics. developed protocol might serve guideline other pathogens. IMPORTANCE This highlights significant advancement bacterial pathogens, specifically addressing challenges posed species . widely used it combines simple improve cost efficiency thus accessibility, changed from Illumina (ONT-LR). very SR-based cgMLST, most likely due methylation-associated errors. proposed correct these errors, did not achieve required accuracy. In contrast, native advanced massively reduces allelic provides transformative solution cost-efficient, high-resolution Furthermore, this can guide similarly challenging

Language: Английский

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A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens

Journal of Clinical Microbiology, Journal Year: 2024, Volume and Issue: 62(9)

Published: Aug. 19, 2024

ABSTRACT Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared gold-standard short-read sequencing. Furthermore, although essential for widespread application, reproducibility nanopore-only remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with latest R10.4.1 flow cells V14 chemistry. Core genome MLST analysis over 500 data sets revealed highly strain-specific typing errors in all each laboratory. Investigation methylation-related consistent DNA motifs at error-prone sites across participants read level. Depending on frequency incorrect target reads, this either leads correct or typing, whereby only minimal deviations can randomly determine final result. PCR preamplification, basecalling model updates an optimized polishing strategy notably diminished non-reproducible typing. Our highlights new appear newly strain lays foundation computational approaches reduce such errors. conclusion, shows necessity a validation concept nanopore sequencing-based, standardized where single nucleotide accuracy is critical.

Language: Английский

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Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies

mSystems, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 8, 2025

ABSTRACT Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed improve the yield of low abundance sequences has not been thoroughly explored with Oxford Nanopore Technologies (ONT) use in clinical bacterial epidemiology. We guide RNAs enrich human Klebsiella pneumoniae , by targeting multi-locus type (MLST) and transfer RNA (tRNA) genes, as well common antimicrobial resistance (AMR) genes resistance-associated integron gene intI1 . validated performance 20 K isolates, finding that guides generated successful across all conserved sites except one AMR two isolates. Enrichment MLST led a correct allele call seven loci 8 out 10 isolates had depth 30× or more these regions. then enriched unenriched sequencing three fecal spiked K. at varying abundance. Enriched 56× 11.3× number reads, respectively, sequencing, required approximately one-third computational storage space. Targeting detection 10–20 proximal due long reads produced ONT sequencing. demonstrated combined enabled improved genomic outcomes over samples. This method could be used inform infection control strategies identifying patients colonized high-risk strains. IMPORTANCE Understanding bacteria complex can challenging their abundance, which results To harmful bacteria, we implemented technique aimed increasing amount target pathogens when modern technologies. Our uses specific recovery stool found our significantly outperform methods, generating far originating genes. Additionally, developed new techniques further enhance analysis, providing thorough characterizing biological

Language: Английский

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Impact of microbiological molecular methodologies on adaptive sampling using nanopore sequencing in metagenomic studies

Environmental Microbiome, Journal Year: 2025, Volume and Issue: 20(1)

Published: May 5, 2025

Abstract Introduction Metagenomics, the genomic analysis of all species present within a mixed population, is an important tool used for exploration microbiomes in clinical and environmental microbiology. Whilst development next-generation sequencing, more recently third generation long-read approaches such as nanopore have greatly advanced study metagenomics, recovery unbiased material from microbial populations remains challenging. One promising advancement sequencing Oxford Nanopore Technologies (ONT) adaptive sampling, which enables real-time enrichment or depletion target sequences. As technologies continue to develop, advances sampling become common techniques microbiological toolkit, it essential evaluate benefits advancements metagenomic studies, impact methodological choices on research outcomes. Aim methods Given rapid tools chemistry, this aimed demonstrate impacts choice DNA extraction kit chemistry downstream analyses. We first explored quality accuracy 16S rRNA amplicon extracted ZymoBIOMICS Microbial Community Standard, using range commercially available kits understand effects different biases assessment microbiome composition. next compared analyses two nanopore-based ligation with differing levels base-calling error; older error-prone (~ 97% accuracy) LSK109 newer accurate 99% LSK112 Q20 + chemistry. Finally, we assessed version output novel approach genome yeast Saccharomyces cerevisiae community. Results Firstly, methodology impacted composition yield, mechanical bead-beating methodologies providing least biased picture due efficient lysis Gram-positive microbes community standard, differences also producing variation Secondly, whilst use improved data quality, resulting assemblies were not significantly based metrics assembly statistics. Most importantly, demonstrated effective application enriching low-abundance sample. This resulted 5-7-fold increase non-adaptive despite reduction overall throughput strand-rejection processes. Interestingly, no significant efficiency observed between ONT chemistries, suggesting that performs consistently across library preparation kits. Conclusion Our findings underscore importance selecting minimises bias ensure representation diversity studies. Additionally, provided by even chemistries can achieve reliable results, enabling researchers confidently these depending their specific experimental needs. Critically, highlight potential ONT’s technology targeted genomes samples. offers broad applicability organisms genetic elements (e.g., pathogens plasmids) depleting unwanted host DNA) diverse sample types However, should carefully weigh against trade-offs throughput, particularly targets, where strand rejection lead pore blocking. These results provide valuable guidance optimising workflows objectives.

Language: Английский

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Characteristics of phage-plasmids and their impact on microbial communities

Essays in Biochemistry, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 29, 2024

Abstract Bacteria host various foreign genetic elements, most notably plasmids and bacteriophages (or phages). Historically, these two classes were seen as separate, but recent research has shown considerable interplay between them. Phage-plasmids (P-Ps) exhibit characteristics of both phages plasmids, allowing them to exist extrachromosomally within bacterial hosts also infect lyse bacteria phages. This dual functionality enables P-Ps utilize the modes transmission phage facilitating rapid dissemination material, including antibiotic resistance virulence genes, throughout populations. Additionally, have been found encode toxin-antitoxin CRISPR-Cas adaptive immune systems, which enhance survival under stress provide immunity against other elements. Despite a growing body literature on P-Ps, large gaps remain in our understanding their ecological roles environmental prevalence. review aims synthesise existing knowledge identify impacts microbial communities.

Language: Английский

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Plasmidome of Salmonella enterica serovar Infantis recovered from surface waters in a major agricultural region for leafy greens in California

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(12), P. e0316466 - e0316466

Published: Dec. 30, 2024

Non-typhoidal Salmonella enterica is a leading cause of gastrointestinal illnesses in the United States. Among 2,600 different S . serovars, Infantis has been significantly linked to human and frequently recovered from broilers chicken parts U.S. A key virulence determinant serovar presence megaplasmid pESI, conferring multidrug resistance. To further characterize potential this serovar, present study identified types plasmids harbored by strains, surface waters adjacent leafy greens farms California. Sequencing analysis showed that each examined 12 strains had large plasmid ranging size 78 kb 125 kb. In addition, second 4-kb was detected two strains. Plasmid nucleotide queries did not identify emerging pESI strains; however, similarity sequence already cataloged databases. Subsequent comparative analyses, based on gene or absence, divided into five distinct clusters, phylogram revealed these were clustered either conjugation system, IncI IncF, phage genes. Assignment putative genes functional categories contained implicated cell cycle control division, replication recombination defense mechanisms. Further mobilome, including prophages transposons, demonstrated release bactericidal peptide microcin IncF Tn10 transposon tetracycline resistance one IncI1 plasmids. These findings indicated environmental wide variety associated with adaptation, survivability antimicrobial

Language: Английский

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ReadCurrent: a VDCNN-based tool for fast and accurate nanopore selective sequencing

Briefings in Bioinformatics, Journal Year: 2024, Volume and Issue: 25(5)

Published: July 25, 2024

Abstract Nanopore selective sequencing allows the targeted of DNA interest using computational approaches rather than experimental methods such as multiplex polymerase chain reaction or hybridization capture. Compared to sequence-alignment strategies, deep learning (DL) models for classifying target and nontarget provide large speed advantages. However, relatively low accuracy these DL-based tools hinders their application in nanopore sequencing. Here, we present a tool named ReadCurrent sequencing, which takes electric currents inputs. employs modified very convolutional neural network (VDCNN) architecture, enabling significantly lower costs training quicker inference compared conventional VDCNN. We evaluated performance across 10 datasets spanning human, yeasts, bacteria, viruses. observed that achieved mean 98.57% classification, outperforming four other methods. In validation selectively sequenced microbial from human DNA, an enrichment ratio 2.85, was higher 2.7 by MinKNOW strategy. summary, can rapidly classify with high accuracy, providing alternative toolbox is available at https://github.com/Ming-Ni-Group/ReadCurrent.

Language: Английский

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