Reproducibility and accuracy of bacterial methylome profiling using Oxford Nanopore Technologies nanopore sequencing...

Background. 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.Methods. Six operators independently prepared 15 sequencing libraries for nanopore (MinION R10.4.1 flow cells, Mk1D) and Illumina MiniSeq platforms for two Streptococcus dysgalactiae subsp. equisimilis 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.Results. 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 r>0.989) and ORAs (Pearson's r>0.993), but lower for the degenerate G6mAGNNNNNTAA motif (Pearson's r: 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.Conclusion. 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.