Reproducibility and Accuracy of Nanopore-Based Methylome Profiling of Streptococcus dysgalactiae subspecies equisimilis Strains from Cancer Patients
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Background
DNA methylation influences bacterial gene regulation, virulence, and restriction-modification (RM) systems. Advances by Oxford Nanopore Technologies (ONT) now enable direct methylome profiling from nanopore sequencing using the Dorado basecaller. However, the comparative performance of ONT-only versus hybrid-assembly reference-based methylation calling, particularly regarding genomic DNA quality and inter-operator variability, remains understudied.
Methods
Six operators independently prepared fifteen sequencing libraries each 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). MicrobeMod v1.0.3 was used to identify methylation and motif profiles using Illumina-corrected hybrid reference assemblies (HRAs) and ONT-only reference assemblies (ORAs). Reproducibility and accuracy were compared using a custom genome annotation feature-enabled modular analysis that mapped and counted methylation site calls to CDS, rRNA and tRNA coordinates.
Results
Strain UT9728 predominantly exhibited N6-methyladenine (6mA) at GATC motifs, whereas strain UT10237 displayed dual methylation patterns: C5-methylcytosine (5mC) at CCWGG motifs and 6mA at GAGNNNNNTAA motifs. Both strains contained Type I and Type II RM systems; UT10237 uniquely harbored a Type IIG RM system with combined restriction and methylation activities. Motif identification concordance using HRAs and ORAs exceeded 99.9%. Reproducibility for methylation calls was high across independent replicates for both HRA (Pearson’s r >0.989) and ORA (Pearson’s r >0.993) methylation calls in GATC and CCWGG motifs but lower in the GAGNNNNNTAA motif (Pearson’s r (HRA) = 0.80; r (ORA) = 0.78). ORA-based methylation site calls for all motifs showed excellent precision and recall compared to HRA-based calls (F1-score >99.999%).
Conclusion
Our findings support the accuracy, robustness, and utility of ONT-only data based methylome profiling for bacterial epigenetic characterization. Our analytical framework facilitates detailed evaluations of reproducibility and accuracy.
Data Summary and Availability
Unprocessed_data_files : DOI: 10.5281/zenodo.15555625
Raw paired-end FastQ Files (Illumina).
Raw FastQ Files (ONT; unmodified basecalls).
Raw uBAM Files (ONT; 6mA_5MC_modified_basecalls).
Genome_assemblies_and_annotation_files : DOI: 10.5281/zenodo.15558488
Genome_Assemblies reconstucted with ONT reads and polished with Illumina Reads (Hybrid assemblies);(strain_name_<rep_ID>_Hyb.gbk)
Genome_Assemblies reconstructed with ONT reads and polished with ONT reads (ONT-only assemblies); (strain_name_<rep_ID>_ONT.fasta)
GenBank flatfiles from Hybrid assemblies; (strain_name_<rep_ID>_Hyb.gbk)
Genbank flatfiles from ONT-only assemblies. (strain_name_<rep_ID>_ONT.gbk)
Data_Tables and Python_code for modular analysis: DOI: doi.org/10.5281/zenodo.15579791
9728_10237_ORA+HRA_all_reps_microbemod_output.zip
Methylation calls mapped to parsed genbank features & feature count matrix files.
9728_HRA_reproducibility_analysis_tables.zip
9728_ORA_reproducibility_analysis_tables.zip
10237_HRA_5mC_6mA_reproducibility_analysis_tables.zip
10237_ORA_reproducibility_analysis_tables.zip
Python code for annotation feature-based modular analysis
methylation_feature_hash_assert_FILTER_9728.py
methylation_feature_hash_NOFILTER_9728.py
methylation_feature_hash_motif_10237.py
hyb_vs_ont_per_replicate_comparison.py
hyb_vs_ont_combined_plot.py
Genome Assembly_Code Availibility : https://github.com/TSchababerle/Bacterial-Methylation
Bash script for automated Hybrid Genome Assembly
Bash Script for automated ONT-Only Assembly
Impact Statement
This study provides a focused proof-of-principle demonstrating the robustness and reproducibility of Oxford Nanopore Technologies (ONT) sequencing-based methylome profiling without short-read correction. Using independent replicates prepared by multiple operators, we show that nanopore-only methods yield consistent, accurate methylation profiles concordant with short-read corrected ONT reads. This highlights the potential of ONT-only sequencing as a broadly accessible, reliable approach for rapid bacterial epigenomic characterization across diverse clinical contexts.
