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Epigenetics in Real Time: Unlocking RNA Modifications with Nanopore Direct Sequencing

To fully understand cellular function, development, and disease progression, we must look beyond the DNA sequence itself and examine the additional layers of biological information that regulate gene activity: the epigenome and epitranscriptome.

Traditionally, detecting epigenetic and epitranscriptomic modifications, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), or N6-methyladenosine (m6A), has required chemical treatments, such as bisulfite conversion, or antibody-based enrichment approaches. These methods can degrade samples, introduce bias, lack direct single-molecule resolution, or lose important long-range sequence context.

By reading native, unamplified DNA and RNA molecules directly, Oxford Nanopore Technologies (ONT) has transformed how researchers detect and analyze nucleotide modifications in real time.

Figure 1. Direct Nanopore sequencing for detecting epigenetic and epitranscriptomic modifications.

Key Applications of Nanopore Epigenetic Sequencing

The ability to sequence long, native nucleic acid molecules has opened new opportunities in biological research.

1. Phased 5mC and 5hmC Detection in Cancer Genomics

Cancer genomes are often characterized by global hypomethylation together with hypermethylation of specific tumor suppressor genes. Because Nanopore sequencing can generate ultra-long DNA reads ranging from tens to hundreds of kilobases, it is possible to map 5mC and 5hmC modifications across repetitive elements, promoters, and enhancers.

Furthermore, long reads allow for haplotype phasing, helping determine which parental allele carries specific epigenetic modifications.

2. Direct RNA Sequencing (dRNA-seq) and the Epitranscriptome

mRNA contains more than one hundred different post-transcriptional modifications, with m6A being among the most prevalent. Traditional RNA sequencing converts RNA into cDNA, a process that removes native RNA modifications.

Nanopore Direct RNA Sequencing bypasses cDNA synthesis entirely, enabling the analysis of RNA modifications, poly(A) tail length, and alternative splicing events on the same native RNA molecule.

The Bioinformatics Pipeline: Software for Epigenetic Analysis

Translating raw ionic current signals into base calls and modification states requires sophisticated computational tools.

Basecalling and Initial Processing
Dorado / Guppy: Developed by Oxford Nanopore, these are standard basecalling tools. Current versions use deep neural networks, including RNNs and Transformer-based models, trained on datasets of modified and unmodified control nucleic acids to perform basecalling and detect common modifications, such as 5mC, 5hmC, and m6A.

Dedicated Methylation Callers

For highly accurate detection beyond standard basecalling outputs, several open-source tools are widely used:

Megapodal / Nanopolish: These tools compare observed raw ionic current signals against a reference genome to detect DNA methylation signals, including 5mC modifications.
DeepSignal / DeepMod: These tools use deep learning models, such as Bi-LSTM networks or convolutional neural networks, to extract features from raw signals and predict modification states at single-nucleotide resolution.

Direct RNA Modification Software
m6Anet: A specialized deep learning tool designed for detecting m6A modifications from Nanopore Direct RNA Sequencing data.
EpiTools / Nanocompore: These packages support comparative analysis of direct RNA current signals between different biological conditions, such as wild-type versus knockdown samples, to identify differential RNA modification sites.

Conclusion

Nanopore direct sequencing has fundamentally shifted the study of epigenetics and epitranscriptomics. By reducing the bias and sample damage associated with chemical conversion, reverse transcription, and PCR amplification, it provides a more direct view of native DNA and RNA molecules.

Are you planning an upcoming epigenomics or transcriptomics project? Contact our team at BMKGENE to learn how our ONT direct sequencing services can accelerate your biological discoveries.

References

Leger, A., Amaral, P. P., Pandolfini, L., Capitanchik, C., Capraro, F., Miano, V., Migliori, V., Toolan-Kerr, P., Sideri, T., Enright, A. J., Tzelepis, K., van Werven, F. J., Luscombe, N. M., Barbieri, I., Ule, J., Fitzgerald, T., Birney, E., Leonardi, T., & Kouzarides, T. RNA modifications detection by comparative Nanopore direct RNA sequencing. Nature Communications 12, 7198 (2021). doi:10.1038/s41467-021-27393-3

Wu, Z., Li, J., Xia, R., Dai, J., Su, J., Meng, J., & Zhang, Y. Nanopore direct RNA sequencing for RNA modification analysis: workflow assessment and computational tool benchmarking. Advanced Biotechnology 4, 9 (2026). doi:10.1007/s44307-025-00093-5