Does methylation matter? Maximising insights with epigenomics

Our genomes are blueprints, the set of instructions that code for all living things. But if all our cells have the same set of instructions, why do some fire neurological signals, others carry oxygen, and some form the tough outer layer of skin? The answer lies in epigenetics, tags which help the body find and use the right instructions as needed. When we explore the epigenome, we start to untangle how our genes switch on and off, unlocking new ways to understand biology, health, and disease.

In this Nanopore Know-How blog, we explore the most common epigenomic modification: methylation. What exactly does it do? Why does it matter? How can we use it to push the frontiers of our scientific knowledge?

What is methylation?

Methylation is a process where a methyl group attaches to a molecule. In mammalian DNA, 5-methylcytosine (5mC), where a methyl group attaches to a cytosine, is a methylation type that is frequently studied. It regularly occurs on cytosines, which are followed by a guanine (CpG sites). These sites are dispersed throughout the genome, typically at high concentration in regions known as CpG islands, which are often located in promoter and enhancer regions of genes. Therefore, due to their location, the methylation of CpG islands is crucial for understanding gene expression.

However, cytosine is not the only base that can be methylated. Another common modification is N⁶-methyladenine (or N⁶-methyladenosine), where a methyl group attaches to an adenine in both DNA (6mA) and RNA (m⁶A)¹.

Figure 1. The chemical structure of unmodified bases: adenine (top left) and cytosine (top right), and the methylated bases: 6mA (bottom left) and 5mC (bottom right).

Image created using Marvin JS by Chemaxon.

Besides 5mC, 6mA, and m⁶A modifications, there are other variants that make up the complete epigenomic picture. For example, in 2009, scientists discovered that 5mC can be converted to 5-hydroxymethylcytosine (5hmC), revealing a new epigenetic mark^2,3. Further investigation revealed that 5hmC could hold promise as a biomarker for the early detection of cancer⁴. This unexpected finding is indicative of the breadth of methylation types underexplored in genomic research.

There is also much more to be investigated in transcriptomics research, as methylation patterns on RNA are not widely understood but could shine a light on post-transcriptional control of gene expression⁵. We will discuss RNA modifications in more detail in a future Nanopore Know-How blog.

So next let's address the question: what does methylation actually do?

What does methylation do?

Methylation changes how DNA and RNA interact and bind with proteins, such as transcription factors, which in turn shape gene expression. The type, location, and concentration of these chemical modifications can influence genome stability, chromatin structure, embryonic development, and cell differentiation. Furthermore, we can map the full methylome to understand which genes are switched on or off, enabling us to connect the modifications to the traits we observe. For example, the colouration and markings on female cats’ coats link to random X chromosome inactivation, which is controlled by methylation.

An unusual gain, change, or loss of methylation can signal underlying conditions, such as metabolic disorders, cancer, or neurological disease^6,7. To understand how methylation links to health, researchers need precise tools that can detect and map methylation down to single-base-pair resolution.

How is methylation sequenced?

Traditional short-read sequencing cannot directly detect methylation. To adapt it to epigenetic analysis, researchers add extra laborious workflow steps that fall into two categories: bisulfite treatment or enzymatic conversion. These methods convert unmethylated cytosine to uracil (read as thymine during sequencing), while methylated cytosines remain unchanged^7,8. Researchers can then align the converted reads to a reference genome to identify which regions are methylated based on which cytosines were converted.

Scientists have successfully used these methods across many studies to analyse 5mC modifications, but the additional workflow steps are time-consuming and can introduce bias or result in DNA degradation^7,9. An alternative method is PacBio HiFi sequencing, which generates reads through a circular consensus sequencing approach and detects methylation via enzymatic kinetics. However, it only picks up a few types of methylation modification: 6mA, N4-methylcytosine (4mC), and 5mC¹⁰.

Both short-read and HiFi sequencing are limited by which methylation modifications they can analyse. So, how can we sequence methylation completely and capture the full epigenomic picture?

Why use Oxford Nanopore sequencing for methylation analysis?

Oxford Nanopore sequencing directly analyses native DNA or RNA molecules. As each strand passes through a pore, the bases and epigenomic modifications disrupt the electrical current flowing through the membrane. Our basecaller Dorado decodes these disruptions to detect 5mC, 5hmC, 4mC, 6mA, m⁶A, and more. We are continuously expanding the list of modifications we can analyse — check out the full list of modified basecalling models, along with our best-practice and benchmarking blogs for DNA and RNA modifications.

So, what does an Oxford Nanopore approach to epigenomics mean in practice? With one run, both genomic and epigenomic data are sequenced without any additional damaging treatment required. Our technology analyses reads of any length, letting researchers phase both genomic and epigenomic variants to decode even the most complex regions. With only a single click in MinKNOW, you can enable modification detection to unlock multiomic insights.

Across the Nanopore Community, researchers are embracing this deep, comprehensive data to reveal biology that would have been missed by looking at genetics alone.

Real-world discoveries from the Nanopore Community

The ability to capture both genetic and epigenetic data in a single sequencing run unlocks large-scale multiomic studies. For example, Kimberley Billingsley et al. analysed 359 brain samples, uncovering the link between structural variants, methylation, and gene expression to decode complex regulatory mechanisms that impact neurodevelopment and neurodegenerative diseases such as Alzheimer’s¹¹. Dig into the details by watching Kimberley’s talk.

Methylation is also transforming cancer research. With the help of nanopore sequencing, scientists are investigating how to track cancer progression non-invasively by profiling methylation in cell-free DNA¹² and are classifying brain tumours intraoperatively to inform surgical decisions within a couple of hours¹³. Furthermore, during our flagship 2025 conference, London Calling, Dr Salvatore Benfatto and Dr L. A. Lennart Kester proved that methylation patterns could be used to classify acute leukaemia and paediatric solid tumours in record time.

Finally, researchers are using epigenetic analysis to investigate its potential for molecular diagnosis in complex and rare diseases. In X-linked disorders, this is already proving valuable, as illustrated by Wojcik et al., who used methylation analysis to determine X-inactivation patterns in patients with incontinentia pigmenti, providing explanations for disease severity and atypical presentations¹⁴. Researchers are also finding success in genomic imprinting disorders, shedding new light on Prader-Willi syndrome, Angelman syndrome, and fragile X syndrome.

This is just the beginning. Ready to dive deeper than ever before into the epigenome? Start your journey with our flyer on methylation detection.

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*Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.