Long-Read Sequencing of DNA Methylation — Direct Detection of 5mC, 5hmC & 6mA at Single-Nucleotide Resolution Using PacBio Revio & ONT PromethION

CD Genomics provides long-read sequencing-based DNA methylation analysis on PacBio Revio and ONT PromethION platforms — detecting 5mC, 5hmC, 6mA, and 4mC directly from native DNA without bisulfite conversion, PCR amplification, or fragmentation. Single-molecule resolution enables haplotype-phased methylation calls and simultaneous genetic-epigenetic analysis from the same sequencing run.

Bisulfite conversion — the methodological cornerstone of DNA methylation analysis for over three decades — introduces a fundamental trade-off: it enables single-nucleotide resolution of 5-methylcytosine (5mC) at the cost of degrading 90–99% of the input DNA, fragmenting genomes into short pieces, and eliminating all sequence context beyond the methylation call itself. This trade-off is baked into every short-read bisulfite sequencing experiment, and it means that researchers studying DNA methylation in repetitive regions, GC-rich promoters, or large structural variant-associated differentially methylated regions have been working with incomplete or entirely missing data. Long-read sequencing of DNA methylation eliminates this trade-off entirely.

CD Genomics provides long-read sequencing-based DNA methylation analysis on both PacBio Revio (HiFi kinetic-based detection) and ONT PromethION (direct current-based detection) platforms. Both approaches detect native DNA modifications directly from unamplified, unmodified DNA — no bisulfite conversion, no PCR bias, no fragmentation. Single-molecule resolution means every methylation call is phased to its haplotype, localized within its repetitive genomic context, and directly comparable across alleles. This is not a supplement to bisulfite sequencing — it is a fundamentally different information layer that captures genetic sequence, epigenetic modification, and haplotype phase from the same native DNA molecule in a single experiment.

Why Long-Read Sequencing for DNA Methylation Analysis — Service Highlights

• Bisulfite-free, PCR-free, fragmentation-free — Native DNA is sequenced directly without chemical conversion or amplification. Methylation patterns are preserved exactly as they exist in the original sample, eliminating the GC bias and DNA loss inherent to bisulfite-based methods
• Single-nucleotide, single-molecule resolution — Both PacBio Revio and ONT PromethION detect base modifications on every individual DNA molecule. Every 5mC, 5hmC, 6mA, and 4mC call is made at single-nucleotide resolution on a per-read basis, enabling allele-specific and haplotype-phased methylation analysis that is structurally impossible from bulk bisulfite sequencing
• Dual-platform flexibility — PacBio Revio HiFi uses kinetic signatures from polymerase incorporation dynamics to detect modifications with single-molecule accuracy (optimal for 4mC/6mA in bacterial epigenomics and CpG-context 5mC in eukaryotic genomes). ONT PromethION detects modifications directly from ionic current shifts, providing comprehensive 5mC/5hmC detection across all sequence contexts in large genomes at lower per-Gb cost
• Simultaneous genetic and epigenetic analysis — Every long-read methylation dataset also produces complete genome sequence information. Variant calling, structural variant detection, and methylation analysis are performed on the same native DNA molecules, enabling direct correlation of genetic and epigenetic variation without data integration across separate experiments
• Long-read is our core technology, not an add-on — We specialize in third-generation sequencing. Our DNA methylation workflows are built around PacBio Revio and ONT PromethION from sample preparation through bioinformatics, not retrofitted onto an NGS-centric service catalog
• Integrated downstream analysis — Our bioinformatics team delivers fully processed methylation calls (per-site and per-read), differentially methylated region (DMR) analysis, haplotype-phased methylation tables, and integration with chromatin conformation data from related service modules

Why Long-Read Sequencing for DNA Methylation — and Why Direct Detection Changes the Question

DNA methylation is the most extensively characterized epigenetic modification in eukaryotic genomes, with 5-methylcytosine (5mC) at CpG dinucleotides serving as the primary regulatory mark in mammals. Additional modifications — 5-hydroxymethylcytosine (5hmC) in active demethylation intermediates, N6-methyladenine (6mA) in bacterial and emerging eukaryotic epigenomes, and N4-methylcytosine (4mC) in prokaryotes — expand the functional repertoire of DNA methylation across the tree of life. Profiling these modifications at single-nucleotide resolution across entire genomes has been the domain of whole-genome bisulfite sequencing (WGBS) for the past 15 years.

WGBS works, but it works within constraints that limit biological discovery. Bisulfite conversion under acidic conditions deaminates unmethylated cytosines to uracil while leaving 5mC intact — but the reaction simultaneously fragments DNA, reduces sequence complexity, and destroys the original methylation pattern's haplotype context. Short-read WGBS cannot determine whether two adjacent CpG sites are methylated on the same DNA molecule (cis) or on different molecules (trans), cannot read through repetitive elements where methylation plays critical regulatory roles, and cannot simultaneously detect sequence variation and methylation status on the same read. Every one of these constraints is eliminated by long-read direct methylation detection.

Long-read sequencing platforms detect DNA base modifications natively — PacBio through polymerase kinetics (the polymerase pauses at modified bases, creating measurable inter-pulse duration differences) and ONT through ionic current shifts (modified bases alter the electrical signal as DNA passes through the nanopore). Both approaches read the original DNA molecule without chemical transformation, preserving every modification in its native context and linking it to the underlying sequence on the same molecule. This direct detection unlocks methylation analysis in repetitive regions, haplotype-resolved epigenetic profiling, and simultaneous genetic-epigenetic analysis from a single sequencing run — capabilities that no short-read bisulfite method can provide. Our epigenetics and methylation analysis hub provides additional context on how long-read approaches compare with established methodologies.

Long-Read Sequencing Provides Haplotype-Resolved, Multi-Modification Methylation Detection from Native DNA

Our Service Modules Combine Methylation Detection with Chromatin Accessibility and 3D Genome Analysis

Our long-read DNA methylation analysis extends beyond single-platform methylation calling. We offer a suite of integrated service modules that combine methylation detection with complementary epigenetic measurements, enabling multi-layered epigenome characterization from a single service provider. Each module is available as a standalone service or as an integrated component of a comprehensive epigenetic analysis project.

Standalone DNA Methylation Analysis

Core service: Direct detection of 5mC, 5hmC, and 6mA from native genomic DNA using either PacBio Revio kinetic detection or ONT PromethION current-based detection. Available as whole-genome methylation profiling (whole-genome shotgun libraries) or targeted methylation analysis (long-range PCR or adaptive sampling enrichment of specific loci). Per-site and per-read methylation calls are delivered in standard BEDmethyl and modified-base SAM tag (MM/ML) formats, enabling direct integration with established analysis workflows. This service is suitable for projects ranging from bacterial methylome analysis (PacBio preferred, 4mC/6mA focus) to whole-genome human 5mC profiling (ONT preferred for cost-effective genome-wide coverage) and targeted methylation analysis of imprinted regions, differentially methylated loci, or candidate gene promoters. See our epigenetics and methylation analysis resource for detailed method comparisons.

Fiber-seq — Simultaneous Chromatin Accessibility and DNA Methylation

Fiber-seq combines m6A methyltransferase labeling of open chromatin with PacBio HiFi sequencing to detect both chromatin accessibility and endogenous 5mC DNA methylation from the same native DNA molecules. A single Fiber-seq library produces three data layers simultaneously: (1) high-accuracy genome sequence with variant calls, (2) single-molecule chromatin accessibility profiles (nucleosome positioning, transcription factor footprinting), and (3) endogenous 5mC methylation status at single-nucleotide resolution. This multi-omics-per-molecule approach eliminates the need for separate ATAC-seq and bisulfite-seq experiments, reducing sample input requirements and cross-experiment technical variation. Fiber-seq is particularly valuable for projects requiring integrated maps of chromatin state and DNA methylation in the same cells — including developmental epigenetics, cancer epigenome characterization, and regulatory element discovery.

Pore-C — Combined 3D Genome Architecture and DNA Methylation

Pore-C is an ONT-based chromatin conformation capture method that simultaneously detects high-order (3+-way) chromatin contacts and native 5mC DNA methylation from the same long-read dataset. Unlike conventional Hi-C, which captures pairwise contacts from short reads and requires a separate bisulfite experiment for methylation analysis, Pore-C provides multi-way contact information and single-molecule methylation status in a single sequencing run. The ultra-long reads span multiple restriction fragments, revealing multi-locus chromatin interactions — promoter–enhancer hub structures, polycomb-repressed domain contacts, and CTCF-mediated loop architectures — while simultaneously reporting the methylation status of each contacted locus. This integrated approach is ideal for projects investigating the relationship between 3D genome organization and epigenetic regulation, including imprinting mechanisms, X-chromosome inactivation, and cancer genome rewiring. Pore-C requires 25–50× coverage for comprehensive contact and methylation analysis.

HiFi-C — High-Accuracy 3D Genome Architecture with Methylation Phasing

HiFi-C combines chromosome conformation capture with PacBio HiFi sequencing to deliver ultra-high-accuracy (Q40, 99.99%) multi-contact chromatin interaction maps with haplotype-resolved methylation phasing. HiFi-C achieves 77–94% valid contact rates (compared to 38–76% for Pore-C), making it the platform of choice for projects requiring the highest-confidence contact calls — T2T genome scaffolding, structural variant breakpoint mapping in repetitive regions, and polyploid haplotype resolution. Methylation status is reported on phased haplotypes, enabling allele-resolved analysis of methylation–chromatin interactions in diploid and polyploid genomes. HiFi-C can be performed with as few as 60,000 cells, opening applications in rare cell populations, clinical biopsies, and developmental time-series datasets where sample material is limiting.

Our Pipelines Deliver Per-Site and Single-Molecule Methylation Calls with Integrated Multi-Omics Analysis

CD Genomics Provides Dual-Platform Methylation Analysis with Platform-Agnostic Expert Guidance

Long-read is our specialty, not a side service.

CD Genomics is a long-read sequencing-focused service provider. Our DNA methylation analysis workflows are built from the ground up around PacBio Revio and ONT PromethION platforms — we do not default to short-read bisulfite sequencing and offer long-read as an afterthought. When you work with us, your methylation project is executed by a team whose core technology platform matches the direct-detection approach your project requires.

True dual-platform independence with platform-agnostic recommendations.

We operate both PacBio Revio and ONT PromethION in-house, with dedicated expertise in each platform's methylation detection chemistry and bioinformatics. Our project scientists provide platform-agnostic guidance based on your modification targets, genome size, and resolution requirements — not based on platform availability or margin considerations.

Platform-appropriate bioinformatics for methylation data.

PacBio and ONT methylation data require fundamentally different detection algorithms and downstream analysis tools. We deploy platform-optimized pipelines: Primrose/ccsmeth for PacBio kinetic data, Dorado modified-base models/Megalodon for ONT current-based data, with per-site and per-read methylation calls delivered in standard formats. We do not apply a one-size-fits-all pipeline to data from different platforms.

Integrated multi-omics epigenetic analysis from a single provider.

Beyond standalone methylation detection, our integrated service modules — Fiber-seq for simultaneous methylation + chromatin accessibility, Pore-C for methylation + 3D genome contacts, and HiFi-C for ultra-high-accuracy methylation + chromatin interaction mapping — enable comprehensive, multi-layered epigenetic characterization without coordinating across multiple service providers or reconciling data from incompatible platforms.

Published validation across diverse applications.

Our customer publication library includes studies spanning cancer epigenomics, bacterial methylome characterization, imprinting disorder research, and plant epigenetic diversity analysis, all generated using long-read sequencing-based methylation detection on CD Genomics platforms.

When to Choose Long-Read DNA Methylation Sequencing — and When Alternative Methods May Be More Suitable

Choose long-read DNA methylation sequencing when:

• Single-molecule and haplotype-resolved methylation information is required — Only long-read direct detection provides per-read methylation status phased to individual haplotypes, enabling allele-specific methylation (ASM) analysis, imprinting studies, and single-molecule methylation heterogeneity assessment that no bulk method can provide
• Your regions of interest include repetitive elements, GC-rich promoters, or centromeric/telomeric chromatin — Long reads spanning complete repetitive elements generate methylation calls in genomic compartments where short-read bisulfite sequencing maps less than 60% of reads, directly validating the key advantage of long-read technology for comprehensive epigenome coverage
• Simultaneous genetic and epigenetic analysis from a single experiment is valuable — Every long-read methylation dataset simultaneously produces complete genome sequence data. SNV, indel, and structural variant calls are generated from the same native DNA reads as the methylation data, enabling direct genetic-epigenetic correlation without cross-experiment integration
• You need to distinguish 5hmC from 5mC, or detect 6mA and 4mC — Bisulfite sequencing cannot discriminate 5mC from 5hmC. Only direct long-read detection provides modification type resolution through distinct kinetic (PacBio) or current (ONT) signatures
• Multi-layered epigenetic analysis (methylation + chromatin accessibility + 3D contacts) is planned — Our integrated service modules — Fiber-seq, Pore-C, and HiFi-C — capture methylation as an integral part of broader epigenetic characterization from a single library preparation

Consider alternative methods when:

• Bulk CpG methylation at the lowest per-sample cost is the sole requirement — For projects requiring only gene-level or regional average methylation across many samples with large existing reference datasets, WGBS or methylation arrays may be more cost-effective per sample at high throughput
• Your project requires comparison with large legacy WGBS or array-based datasets — While inter-method correlation is high (0.76–0.99), cross-platform differences exist. Projects that must integrate directly with existing short-read bisulfite datasets should consider generating matching data on the same platform or budgeting for cross-platform normalization analysis
• You have a very limited budget for a large number of samples — For large-cohort screening studies where relative methylation differences (not single-molecule resolution) are sufficient, targeted bisulfite methods or methylation arrays provide more samples per dollar, and our project scientists can advise on the optimal cost-benefit trade-off for your specific study design

CD Genomics provides free project consultation to help determine the optimal DNA methylation analysis strategy for your specific research question. Contact our scientists to discuss your project requirements.

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