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Comprehensive comparison of the third-generation sequencing tools for bacterial 6mA profiling

Author

Listed:
  • Beifang Lu

    (City University of Hong Kong)

  • Zhihao Guo

    (City University of Hong Kong)

  • Xudong Liu

    (City University of Hong Kong)

  • Ying Ni

    (City University of Hong Kong)

  • Letong Xu

    (City University of Hong Kong)

  • Jiadai Huang

    (City University of Hong Kong)

  • Tianmin Li

    (City University of Hong Kong)

  • Tongtong Feng

    (City University of Hong Kong)

  • Runsheng Li

    (City University of Hong Kong
    City University of Hong Kong)

  • Xin Deng

    (City University of Hong Kong
    City University of Hong Kong
    City University of Hong Kong)

Abstract

DNA N6-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.

Suggested Citation

  • Beifang Lu & Zhihao Guo & Xudong Liu & Ying Ni & Letong Xu & Jiadai Huang & Tianmin Li & Tongtong Feng & Runsheng Li & Xin Deng, 2025. "Comprehensive comparison of the third-generation sequencing tools for bacterial 6mA profiling," Nature Communications, Nature, vol. 16(1), pages 1-18, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-59187-2
    DOI: 10.1038/s41467-025-59187-2
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    2. Zaka Wing-Sze Yuen & Akanksha Srivastava & Runa Daniel & Dennis McNevin & Cameron Jack & Eduardo Eyras, 2021. "Systematic benchmarking of tools for CpG methylation detection from nanopore sequencing," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    3. Zhen-Dong Zhong & Ying-Yuan Xie & Hong-Xuan Chen & Ye-Lin Lan & Xue-Hong Liu & Jing-Yun Ji & Fu Wu & Lingmei Jin & Jiekai Chen & Daniel W. Mak & Zhang Zhang & Guan-Zheng Luo, 2023. "Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
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