Accurate bacterial outbreak tracing with Oxford Nanopore sequencing and reduction of methylation-induced errors

Lohde, Mara; Wagner, Gabriel E.; Dabernig-Heinz, Johanna; Viehweger, Adrian; Braun, Sascha D.; Monecke, Stefan; Diezel, Celia; Stein, Claudia; Marquet, Mike; Ehricht, Ralf; Pletz, Mathias W.; Brandt, Christian

doi:10.1101/2023.09.15.556300

Cited by 6 publications

(1 citation statement)

References 49 publications

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“…For example, in direct-RNA sequencing, EpiNano has demonstrated that alignment features can accurately predict m6A modifications ( 18 ). And the DNA modification in bacterial DNA is creating G to A mismatches in Nanopore basecalls ( 19 ). Exploring the alignment features would facilitate the development of DNA/RNA modification finding programs and help to validate the potential modification sites.…”

Section: Introductionmentioning

confidence: 99%

Nanopore Current Events Magnifier (nanoCEM): a novel tool for visualizing current events at modification sites of nanopore sequencing

Guo,

Ni,

Tan

et al. 2024

NAR Genomics and Bioinformatics

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Summary: Nanopore sequencing technologies have enabled the direct detection of base modifications in DNA or RNA molecules. Despite these advancements, the tools for visualizing electrical current, essential for analyzing base modifications, are often lacking in clarity and compatibility with diverse nanopore pipelines. Here, we present Nanopore Current Events Magnifier (nanoCEM, https://github.com/lrslab/nanoCEM), a Python command-line tool designed to facilitate the identification of DNA/RNA modification sites through enhanced visualization and statistical analysis. Compatible with the four preprocessing methods including ‘f5c resquiggle’, ‘f5c eventalign’, ‘Tombo’ and ‘move table’, nanoCEM is applicable to RNA and DNA analysis across multiple flow cell types. By utilizing rescaling techniques and calculating various statistical features, nanoCEM provides more accurate and comparable visualization of current events, allowing researchers to effectively observe differences between samples and showcase the modified sites.

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Section: Introductionmentioning

confidence: 99%

Nanopore Current Events Magnifier (nanoCEM): a novel tool for visualizing current events at modification sites of nanopore sequencing

Guo,

Ni,

Tan

et al. 2024

NAR Genomics and Bioinformatics

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Oxford Nanopore’s 2024 sequencing technology forListeria monocytogenesoutbreak detection and source attribution: progress and clone-specific challenges

Biggel,

Cernela,

Horlbog

et al. 2024

Preprint

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Whole genome sequencing is an essential cornerstone of pathogen surveillance and outbreak detection. Established sequencing technologies are currently challenged by Oxford Nanopore Technologies (ONT), which offers an accessible and cost-effective alternative enabling gap-free assemblies of chromosomes and plasmids. Limited accuracy has hindered its use for investigating pathogen transmission, but recent technology updates have brought significant improvements. To evaluate its readiness for outbreak detection, we selected 78Listeria monocytogenesisolates from diverse lineages or known epidemiological clusters for sequencing with ONT’s V14 Rapid Barcoding Kit and R10.4.1 flow cells. The most accurate of several tested workflows generated assemblies with a median of one error (SNP or indel) per assembly. For 66 isolates, cgMLST profiles from ONT-only assemblies were identical to those generated from Illumina data. Eight assemblies were of lower quality with more than 20 erroneous sites each, primarily caused by methylations at the GAAGAC motif (5′-GAAG6mAC-3 / 3′-GT4mCTTC-5′). This led to inaccurate clustering, failing to group isolates from a persistence-associated clone that carried the responsible restriction-modification system. Out of 50 methylation motifs detected among the 78 isolates, only the GAAGAC motif was linked to substantially increased error rates. Our study shows that mostL. monocytogenesgenomes assembled from ONT-only data are suitable for high-resolution genotyping, but further improvements of chemistries or basecallers are required for reliable routine use in outbreak and food safety investigations.

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

Dabernig-Heinz,

Lohde,

Hölzer

et al. 2024

J Clin Microbiol

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

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Accurate bacterial outbreak tracing with Oxford Nanopore sequencing and reduction of methylation-induced errors

Cited by 6 publications

References 49 publications

Nanopore Current Events Magnifier (nanoCEM): a novel tool for visualizing current events at modification sites of nanopore sequencing

Nanopore Current Events Magnifier (nanoCEM): a novel tool for visualizing current events at modification sites of nanopore sequencing

Oxford Nanopore’s 2024 sequencing technology forListeria monocytogenesoutbreak detection and source attribution: progress and clone-specific challenges

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

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