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Advancements in molecular biology have revolutionized the identification and characterization of bacteria, with multiple methods leveraging DNA sequencing for enhanced precision. As sequencing technologies improve and costs decline, these approaches are increasingly used in clinical, environmental, and evolutionary studies.Multilocus Sequence Typing (MLST) examines several housekeeping genes, essential chromosomal genes encoding cellular functions, to distinguish strains. Approximately...
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Updated: Sep 9, 2025

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example
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Accurately assembling nanopore sequencing data of highly pathogenic bacteria.

Christine Thomas1,2, Hanka Brangsch1, Valentina Galeone3

  • 1Institute of Bacterial Infections and Zoonoses, Federal Research Institute for Animal Health, Friedrich-Loeffler-Institute, Naumburger Str. 96a, 07743, Jena, Germany.

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Nanopore sequencing for bacterial genomes shows variable assembly quality across species. While some pathogens yield perfect genomes, errors in core-genome MLST loci can impact outbreak analysis reliability.

Keywords:
Assembly pipelineBacteriaOutbreak analysisOxford nanopore technologiesPathogens

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Area of Science:

  • Genomics
  • Bioinformatics
  • Microbial Pathogenesis

Background:

  • Whole-genome sequencing and bioinformatics are crucial for bacterial genome exploration and outbreak analysis.
  • High sequencing accuracy and precise genome assembly are essential for reliable genotyping and genetic marker detection.

Purpose of the Study:

  • To assess the utility of Oxford Nanopore Technologies (ONT) R10.4.1 sequencing for genotyping highly pathogenic bacteria with low mutation rates.
  • To evaluate different assembly strategies and their impact on genome accuracy.

Main Methods:

  • Sequencing of six reference bacterial strains using ONT R10.4.1 chemistry and Illumina.
  • Evaluation of various assembly strategies against RefSeq assemblies as ground truth.
  • Analysis of publicly available sequencing data from key bacterial pathogens.

Main Results:

  • Assembly quality varied by species; Bacillus anthracis achieved near-perfect assembly, while Brucella spp. showed nucleotide differences.
  • Perfect genomes were obtained for Klebsiella variicola, Listeria spp., Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pyogenes.
  • Errors, particularly within coding sequences and methylation-related, were observed, though methylation-aware models showed improvement. Core-genome Multilocus Sequence Typing (cgMLST) revealed minor allele differences in some species.

Conclusions:

  • Nanopore sequencing data assembly quality for pathogenic bacteria is species and method-dependent.
  • Despite persistent errors in assemblies, including cgMLST loci, specific tool combinations can yield perfect genomes for outbreak analysis without short-read polishing.