The Future of DNA Methylation Detection: How PacBio and Nanopore Change the Game
In our last post, we explored how long-read sequencing is transforming DNA methylation research by enabling direct analysis of native DNA while preserving long-range genomic context. One major question remains: How do these platforms detect methylation without chemical conversion?
The answer lies in two very different approaches used by the leading long-read sequencing platforms: Oxford Nanopore Technologies (ONT) and PacBio HiFi sequencing.
Nanopore (ONT): Detecting Methylation Through Electrical Signals
Oxford Nanopore sequencing measures changes in the ionic electrical current as DNA strands pass through nanoscale protein pores embedded in a membrane. Methyl groups alter the physical size and charge of the nucleotide, resulting in a distinct electrical signal shift (relative to unmodified bases) as the DNA strand is being sequenced.
This architecture provides several major advantages:
- Real-time methylation detection
- Native DNA analysis without conversion chemistry
- Flexible read lengths and scalable workflows
Because methylation is encoded in the raw electrical signal, improvements in machine learning continue to expand the range of detectable DNA modifications without changing the sequencing hardware. This flexibility has made ONT especially attractive for exploratory epigenetics and rapid clinical workflows.
PacBio: Detecting Methylation Through Polymerase Kinetics
PacBio detects methylation by monitoring DNA polymerase activity during sequencing. When the polymerase encounters a methylated base, the addition of a methyl group has fundamentally changed the structure of the DNA. This causes the DNA polymerase to hesitate or slow down while incorporating the complementary base, resulting in a statistically significant change in two kinetic measurements.
The two primary kinetic measurements are:
- Inter-Pulse Duration (IPD): The time between nucleotide incorporations
- Pulse Width (PW): The duration of the fluorescent signal during incorporation
Rather than detecting the methyl group directly, PacBio technology infers methylation from these kinetic signatures.
Combined with highly accurate HiFi reads (sequencing reads generated through multiple passes over the same DNA molecule, i.e. CCS sequencing) this approach delivers:
- High-resolution methylation profiling
- Exceptional consensus accuracy
- Strong performance in repetitive and structurally complex genomic regions
Two Different Technologies, One Major Advantage
Although ONT and PacBio rely on different detection mechanisms, they share several important advantages over traditional methylation methods:
- Direct analysis of native DNA without chemical conversion
- Simultaneous DNA sequencing and methylation detection
- Single-base methylation resolution
- Long reads that preserve genomic context across thousands to millions of bases
- Improved characterization of repetitive regions, structural variants, and phased methylation patterns
These capabilities are helping researchers move beyond isolated methylation measurements toward a more complete understanding of how genetic and epigenetic information work together.
In our next post, we’ll compare the strengths of ONT and PacBio for different methylation applications and discuss how researchers choose the right platform for their specific biological questions.
Follow us on social to see part four of this series in August.
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