Our study investigated the effectiveness of Oxford Nanopore Technology for accurate outbreak tracing by resequencing a three-year-long Klebsiella pneumoniae outbreak with Illumina short-read sequencing data as the point of reference. We detected considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Nanopore compared to the Illumina data, 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 this data, we explored PCR-based sequencing and a masking strategy, which both successfully addressed these inaccuracies and ensured accurate outbreak tracing. Additionally, we offer a bioinformatic workflow to identify and mask problematic genome positions in a reference-free manner. Without further technological developments, our study recommends PCR-based sequencing for outbreak tracing to avoid spurious base calls from Nanopore data.

中文翻译：

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纳米孔测序可准确追踪细菌爆发

我们的研究以 Illumina 短读长测序数据为参考点，对长达三年的肺炎克雷伯菌疫情进行了重新测序，调查了牛津纳米孔技术在准确疫情追踪方面的有效性。与 Illumina 数据相比，我们通过 cgMLST 和对使用 Nanopore 测序的基因组进行系统发育分析，检测到相当大的碱基错误，导致从爆发集群中错误地排除了一些与爆发相关的菌株。附近的甲基化位点会导致这些错误，并且也可以在肺炎克雷伯菌以外的其他物种中发现。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性并确保了准确的疫情追踪。此外，我们提供生物信息学工作流程，以无参考的方式识别和掩盖有问题的基因组位置。如果没有进一步的技术发展，我们的研究建议使用基于 PCR 的测序来进行疫情追踪，以避免 Nanopore 数据中的虚假碱基识别。