Our study investigates the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a 3-year-long Klebsiella pneumoniae outbreak with Illumina short-read sequencing data as the point of reference. We detect considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besides K. pneumoniae. Based on these data, we explore PCR-based sequencing and a masking strategy, which both successfully address these inaccuracies and ensure accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investigating outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either using PCR-based sequencing or using our provided bioinformatic workflow. We advise that read mapping–based quality control of genomes should be provided when publishing results.

中文翻译：

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使用 Oxford Nanopore 测序进行准确的细菌爆发追踪并减少甲基化诱导的错误


我们的研究以 Illumina 短读长测序数据为参考点，对长达 3 年的肺炎克雷伯菌疫情的 33 个分离株进行重测序，调查了 Oxford Nanopore 技术准确追踪疫情的有效性。我们通过对使用 Oxford Nanopore Technologies 测序的基因组进行 cgMLST 和系统发育分析来检测相当大的碱基错误，导致一些与爆发相关的菌株被错误地排除在爆发集群之外。附近的甲基化位点会导致这些错误，也可以在肺炎克雷伯菌以外的其他物种中找到。基于这些数据，我们探索了基于 PCR 的测序和掩蔽策略，它们都成功地解决了这些不准确的问题并确保了准确的疫情追踪。我们提供掩蔽策略作为生物信息学工作流程 （MPOA），以无参考的方式识别和掩蔽有问题的基因组位置。我们的研究强调了使用 Oxford Nanopore 技术对原核生物进行测序的局限性，尤其是在研究爆发方面。对于时间紧迫的项目，如果不能等待 Oxford Nanopore Technologies 的进一步技术发展，我们的研究建议使用基于 PCR 的测序或使用我们提供的生物信息学工作流程。我们建议在发表结果时提供基于读取映射的基因组质量控制。