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Microbial Genomics logo Microbial Genomics

Volume 11, Issue 11

Reproducibility and accuracy of bacterial methylome profiling using Oxford Nanopore Technologies nanopore sequencing platform Open Access

Abstract

Oxford Nanopore Technologies (ONT) enables direct methylome profiling using the Dorado basecaller, but the comparative performance of ONT-only versus hybrid-assembly reference-based methylation calling, particularly regarding inter-operator variability, remains understudied.

Six operators independently prepared 15 sequencing libraries for nanopore (MinION R10.4.1 flow cells, Mk1D) and Illumina MiniSeq platforms for two subsp. strains (UT9728, 12 replicates; UT10237, 3 replicates). Methylation and motif profiles were identified with MicrobeMod v.1.0.3 using both Illumina-corrected hybrid reference assemblies (HRAs) and ONT-only reference assemblies (ORAs). A custom genome annotation-aware pipeline mapped methylation site calls to coding sequence, rRNA and tRNA features for reproducibility analysis. We also performed site-wise analyses to quantify concordance of methylated fractions among ORA replicates and assess the influence of sequencing coverage.

Strain UT9728 predominantly exhibited N6-methyladenine (6mA) at G6mATC motifs, whereas strain UT10237 displayed dual methylation patterns: C5-methylcytosine (5mC) at 5mCCWGG motifs and 6mA at G6mAGNNNNNTAA motifs. Motif identification concordance using HRAs and ORAs exceeded 99.9%. Reproducibility across replicates was high for G6mATC and 5mCCWGG motifs in both HRAs (Pearson’s >0.989) and ORAs (Pearson’s >0.993), but lower for the degenerate G6mAGNNNNNTAA motif (Pearson’s : HRA=0.80; ORA=0.78). ORA-based methylation site calls showed excellent precision and recall compared to HRA-based calls (F1-score>99.999%). Site-wise analysis of UT9728 G6mATC motifs revealed that discordant sites (absolute methylated fraction difference ≥0.15) were rare, with 44 of 66 pairwise replicate comparisons showing <1% discordance. Discordance was linked to low coverage (<70×), whereas sites sequenced >200× displayed complete concordance.

Although limited to a single species, three motifs and a feature-based framework that does not capture promoter-proximal events, ONT-only sequence-based methylome profiling proved accurate and reproducible across multiple operators, with sequencing coverage emerging as the principal determinant of site-level concordance.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Dorado basecaller , methylome , nanopore sequencing and short-read sequencing
Funding
This study was supported by the:
  • Fulbright U.S. Student Program (Award NA)
    • Principal Award Recipient: HananWees
  • The Dell Scholarship (Award NA)
    • Principal Award Recipient: OmarHayat
  • the McKowen Scholarship (Award NA)
    • Principal Award Recipient: MinhLe
  • the Kinder Foundation Endowed Scholarship (Award NA)
    • Principal Award Recipient: IsabellaPolic
  • Peter and Cythia Hu Cardinal Health Scholarship (Award NA)
    • Principal Award Recipient: JungJaeone
  • Peter and Cythia Hu Cardinal Health Scholarship (Award N/A)
    • Principal Award Recipient: TaylorSchababerle
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2025-11-19
2025-12-08

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Reproducibility and accuracy of bacterial methylome profiling using Oxford Nanopore Technologies nanopore sequencing platform
M Gen 11, 001564 (2025); https://doi.org/10.1099/mgen.0.001564
/content/journal/mgen/10.1099/mgen.0.001564
/content/journal/mgen/10.1099/mgen.0.001564
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