Lu Beifang, Guo Zhihao, Liu Xudong, Ni Ying, Xu Letong, Huang Jiadai, Li Tianmin, Feng Tongtong, Li Runsheng, Deng Xin
Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China.
Department of Infectious Diseases and Public Health, City University of Hong Kong, Hong Kong SAR, China.
Nat Commun. 2025 Apr 28;16(1):3982. doi: 10.1038/s41467-025-59187-2.
DNA N-methyladenine (6mA) serves as an intrinsic and principal epigenetic marker in prokaryotes, impacting various biological processes. To date, limited advanced sequencing technologies and analyzing tools are available for bacterial DNA 6mA. Here, we evaluate eight tools designed for the 6mA identification or de novo methylation detection. This assessment includes Nanopore (R9 and R10), Single-Molecule Real-Time (SMRT) Sequencing, and cross-reference with 6mA-IP-seq and DR-6mA-seq. Our multi-dimensional evaluation report encompasses motif discovery, site-level accuracy, single-molecule accuracy, and outlier detection across six bacteria strains. While most tools correctly identify motifs, their performance varies at single-base resolution, with SMRT and Dorado consistently delivering strong performance. Our study indicates that existing tools cannot accurately detect low-abundance methylation sites. Additionally, we introduce an optimized method for advancing 6mA prediction, which substantially improves the detection performance of Dorado. Overall, our study provides a robust and detailed examination of computational tools for bacterial 6mA profiling, highlighting insights for further tool enhancement and epigenetic research.
DNA N-甲基腺嘌呤（6mA）是原核生物中一种内在且主要的表观遗传标记，影响着各种生物学过程。迄今为止，用于细菌DNA 6mA的先进测序技术和分析工具有限。在此，我们评估了八种用于6mA鉴定或从头甲基化检测的工具。该评估包括纳米孔测序（R9和R10）、单分子实时（SMRT）测序，并与6mA-IP-seq和DR-6mA-seq进行交叉比对。我们的多维度评估报告涵盖了六种细菌菌株的基序发现、位点水平准确性、单分子准确性和异常值检测。虽然大多数工具能正确识别基序，但它们在单碱基分辨率下的表现各异，其中SMRT和Dorado始终表现出色。我们的研究表明，现有工具无法准确检测低丰度甲基化位点。此外，我们引入了一种优化方法来改进6mA预测，这显著提高了Dorado的检测性能。总体而言，我们的研究对用于细菌6mA分析的计算工具进行了全面而详细的考察，为进一步改进工具和开展表观遗传学研究提供了见解。
