解码四种与公共卫生相关的微生物物种的细菌甲基化组:纳米孔测序可实现对DNA修饰的可重复分析。

Decoding bacterial methylomes in four public health-relevant microbial species: nanopore sequencing enables reproducible analysis of DNA modifications.

作者信息

Galeone Valentina, Dabernig-Heinz Johanna, Lohde Mara, Brandt Christian, Kohler Christian, Wagner Gabriel E, Hölzer Martin

机构信息

Bioinformatics and Translational Research, Genome Competence Center, Robert Koch Institute, Nordufer 20, 13353, Berlin, Germany.

Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Neue Stiftingtalstraße 6, Graz, 8010, Austria.

出版信息

BMC Genomics. 2025 Apr 23;26(1):394. doi: 10.1186/s12864-025-11592-z.

Abstract

Investigating bacterial methylation profiles provides essential complementary information to the native DNA sequence, significantly extending our understanding of how DNA modifications influence virulence, antibiotic resistance, and the ability of bacteria to evade the immune system. Recent advancements in real-time Nanopore sequencing and basecalling algorithms have enabled the direct detection of modified bases from raw signal data, eliminating the need for bisulfite treatment of DNA. However, decoding methylation signals remains challenging due to rapid technological and methodological progress. In this study, we focus on public health-relevant bacterial strains to analyze their methylation profiles and identify methylation motifs. Our dataset includes samples from Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecium, and Klebsiella pneumoniae, sequenced on the Nanopore GridION platform using the latest flow cell chemistry (R10.4.1) and modification basecalling models (Dorado basecalling SUP model v5). We investigated distinct methylation patterns within and between species, focusing on heavily modified genes or genomic regions. Our results reveal distinct species-specific methylation profiles, with each strain exhibiting unique modification patterns. We developed a modular pipeline using Nextflow and the Nanopore Modkit tool to streamline the detection of methylated motifs. We compared the results with outputs from MicrobeMod, a recent toolkit for exploring prokaryotic methylation and base modifications in nanopore sequencing. Our pipeline is publicly available for further use (github.com/rki-mf1/ont-methylation). We identified known methylation motifs already described in the literature and novel de novo motifs, providing deeper insights into the diversity of bacterial DNA modifications. Furthermore, we identified genomic regions that are extensively methylated, which could have implications for bacterial behavior and pathogenicity. We also assess improvements in basecalling accuracy, specifically how methylated bases can influence neighboring basecalls. Recent advances in basecalling models, particularly v5 models as part of Dorado, have reduced these issues, improving the reliability of methylation detection in bacterial genomes. In conclusion, our study highlights the potential of current nanopore sequencing tools for detecting DNA modifications in prokaryotes. By making our pipeline and results publicly available, we facilitate further research into bacterial DNA modifications and their role in microbial pathogenesis.

摘要

研究细菌甲基化谱可为天然DNA序列提供重要的补充信息,极大地扩展了我们对DNA修饰如何影响毒力、抗生素抗性以及细菌逃避免疫系统能力的理解。实时纳米孔测序和碱基识别算法的最新进展使得能够直接从原始信号数据中检测修饰碱基,无需对DNA进行亚硫酸氢盐处理。然而,由于技术和方法的快速发展,解码甲基化信号仍然具有挑战性。在本研究中,我们聚焦于与公共卫生相关的细菌菌株,以分析它们的甲基化谱并识别甲基化基序。我们的数据集包括来自金黄色葡萄球菌、单核细胞增生李斯特菌、粪肠球菌和肺炎克雷伯菌的样本,这些样本在纳米孔GridION平台上使用最新的流动池化学技术(R10.4.1)和修饰碱基识别模型(多拉多碱基识别SUP模型v5)进行测序。我们研究了物种内部和物种之间不同的甲基化模式,重点关注高度修饰的基因或基因组区域。我们的结果揭示了不同的物种特异性甲基化谱,每个菌株都表现出独特的修饰模式。我们使用Nextflow和纳米孔Modkit工具开发了一个模块化流程,以简化甲基化基序的检测。我们将结果与MicrobeMod的输出进行了比较,MicrobeMod是一个用于探索纳米孔测序中原核生物甲基化和碱基修饰的最新工具包。我们的流程可公开获取以供进一步使用(github.com/rki-mf1/ont-methylation)。我们识别出了文献中已经描述的已知甲基化基序和新的从头基序,从而更深入地了解了细菌DNA修饰的多样性。此外,我们识别出了广泛甲基化的基因组区域,这可能对细菌行为和致病性有影响。我们还评估了碱基识别准确性的提高,特别是甲基化碱基如何影响相邻碱基的识别。碱基识别模型的最新进展,特别是作为多拉多一部分的v5模型,减少了这些问题,提高了细菌基因组中甲基化检测的可靠性。总之,我们的研究突出了当前纳米孔测序工具在检测原核生物DNA修饰方面的潜力。通过公开我们的流程和结果,我们促进了对细菌DNA修饰及其在微生物发病机制中作用的进一步研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/93a5/12016153/f58e79f9827b/12864_2025_11592_Fig1_HTML.jpg

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