Long-Read Sequencing of RNA Methylation — Direct Detection of m⁶A, m⁵C & Ψ at Single-Molecule Resolution by Nanopore Direct RNA Sequencing

CD Genomics provides Nanopore direct RNA sequencing for RNA methylation analysis — detecting m⁶A, m⁵C, pseudouridine (Ψ), and inosine directly from native RNA without reverse transcription, PCR amplification, or antibody enrichment. Single-molecule resolution enables isoform-resolved modification analysis, modification stoichiometry, and simultaneous poly(A) tail profiling from the same sequencing run.

RNA methylation is the most abundant and functionally diverse class of post-transcriptional modification in the epitranscriptome. Over 170 distinct RNA modifications have been characterized across all RNA biotypes — messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and non-coding RNA — where they regulate splicing, translation, RNA stability, and cellular stress responses. Among these, N⁶-methyladenosine (m⁶A) is the most prevalent internal mRNA modification, affecting over 60% of human transcripts and modulating every stage of RNA metabolism from nuclear export to decay. 5-methylcytosine (m⁵C), pseudouridine (Ψ), and inosine (I) expand the functional repertoire, yet the vast majority of modification sites remain incompletely mapped across transcriptomes, isoforms, and cell types — because the dominant detection methods have, until recently, relied on antibody enrichment or chemical conversion that obscures single-molecule resolution and isoform-level information.

Long-read direct RNA sequencing (dRNA-seq) on the Oxford Nanopore Technologies (ONT) platform eliminates these constraints entirely. By sequencing native RNA molecules without reverse transcription or PCR amplification, dRNA-seq preserves every endogenous modification in its original context — the ionic current signature generated as each RNA molecule passes through a nanopore carries a characteristic shift when a modified base is present, enabling simultaneous detection of m⁶A, m⁵C, Ψ, and inosine from the same sequencing run. Every read delivers full-length transcript sequence, isoform structure, modification status at single-nucleotide resolution, and poly(A) tail length from the same native RNA molecule. This is not a surrogate for antibody-based epitranscriptomics — it is a fundamentally different information layer that captures the complete modification landscape of each transcript isoform individually, rather than population-averaged enrichment signals.

Why Long-Read Direct RNA Sequencing for RNA Methylation — Service Highlights

• No reverse transcription, no PCR, no antibody enrichment — Native RNA is sequenced directly through the nanopore. Every endogenous modification is preserved and detected from the unaltered ionic current signal. No RT bias, no PCR duplicates, no antibody cross-reactivity or epitope occlusion
• Multi-modification detection from a single sequencing run — Nanopore dRNA-seq simultaneously detects m⁶A (DRACH motif and non-DRACH contexts using tool-dependent approaches), m⁵C, pseudouridine (Ψ), and inosine (I) from the same native RNA reads, using signal-aware basecalling and downstream modification calling algorithms. A single dRNA-seq experiment replaces separate antibody IP-seq, bisulfite-seq, and chemical labeling experiments
• Single-molecule, isoform-resolved modification analysis — Every modification call is made at single-nucleotide resolution on individual native RNA molecules, enabling isoform-level m⁶A profiling, allele-specific modification analysis, and the detection of co-occurring modifications on the same transcript — information that is structurally impossible to obtain from population-averaged short-read or antibody-based methods
• Simultaneous epitranscriptome, transcriptome, and poly(A) profiling — Every dRNA-seq read simultaneously reports transcript sequence, isoform structure, base modification status, and poly(A) tail length from the same native RNA molecule. A single experiment replaces separate RNA-seq, isoform-seq, modification-seq, and poly(A)-tail assays
• Long-read RNA is our core transcriptomics technology — Our direct RNA sequencing workflows are built around ONT PromethION native RNA sequencing from sample preparation through bioinformatics, not retrofitted from a short-read or cDNA-based service catalog
• Integrated bioinformatics for epitranscriptome analysis — Our computational team deploys validated RNA modification detection pipelines including Dorado modified-base models, m6Anet, CHEUI, Nanopolish, and Minimod, delivering per-site and per-read modification calls, differential modification analysis, isoform-level modification quantification, and integrated reports

Why Long-Read Direct RNA Sequencing for RNA Methylation — and Why the Epitranscriptome Needs Single-Molecule Resolution

RNA methylation research has been transformed over the past decade by the development of transcriptome-wide m⁶A mapping methods — MeRIP-seq (m⁶A antibody enrichment followed by short-read sequencing), m⁶A-CLIP, and m⁶A-EXO-seq — that have established m⁶A as a widespread, dynamically regulated RNA modification with essential roles in development, stress response, and disease. These methods share a fundamental limitation: they report population-averaged modification signals across thousands of cells and transcript molecules. An m⁶A peak called by MeRIP-seq tells you that a region of a transcript is enriched for m⁶A in the bulk cell population, but it cannot tell you what fraction of the transcript molecules carry the modification at that site, how m⁶A at different sites co-occurs on the same molecule, or how modification status differs across transcript isoforms originating from the same gene.

Long-read direct RNA sequencing (dRNA-seq) addresses every one of these limitations. By sequencing individual native RNA molecules end-to-end through a nanopore, dRNA-seq measures the modification status of every nucleotide on every captured molecule, producing a single-molecule modification map of the transcriptome. This single-molecule resolution unlocks four categories of biological information that no population-averaged method can provide: (1) modification stoichiometry — the fraction of transcripts carrying a modification at each site, measured directly from per-read modification calls rather than inferred from enrichment ratios; (2) modification co-occurrence — whether m⁶A sites on the same transcript are modified independently or coordinately, and how modification status at one site relates to another; (3) isoform-specific modification — whether alternative transcript isoforms from the same gene carry systematically different modification patterns, and how these patterns relate to isoform-specific functions; and (4) modification-transcript feature relationships — how m⁶A status relates to poly(A) tail length, alternative splicing, and RNA abundance on a per-molecule basis, all from the same native RNA reads. The Nanopore Direct RNA Sequencing service page provides detailed information on the core technology platform.

Our service is designed to make this single-molecule epitranscriptomics capability accessible to researchers studying RNA methylation across any biological system — from focused analysis of specific modification types in targeted transcript sets to comprehensive whole-transcriptome m⁶A, m⁵C, and Ψ profiling at isoform resolution.

Nanopore dRNA-seq Provides Single-Molecule, Multi-Modification Detection with Integrated Poly(A) Tail and Isoform Analysis

Our Service Modules Combine dRNA-seq with tRNA Modification, Poly(A) Tail, and Isoform-Level Analysis

Our long-read RNA methylation analysis extends beyond standard dRNA-seq modification calling. We offer a suite of integrated service modules that combine RNA modification detection with complementary epitranscriptomic measurements, enabling comprehensive multi-layer epitranscriptome characterization from a single service provider. Each module is available as a standalone service or as an integrated component of a broader RNA analysis project.

Nanopore Direct RNA Sequencing — Core RNA Modification Detection Service

Core service: Whole-transcriptome direct RNA sequencing on ONT PromethION (RNA004 chemistry) with multi-modification detection. Native polyA+ RNA is sequenced directly — no reverse transcription, no PCR amplification, no antibody enrichment — and base modifications (m⁶A, m⁵C, Ψ, inosine) are called from the ionic current signal using Dorado v5+ modified-base models, complemented by m6Anet for comprehensive m⁶A detection and CHEUI for simultaneous m⁶A/m⁵C analysis. Per-read modification status, single-nucleotide modification probabilities, and isoform-resolved modification profiles are delivered for every transcript detected. This core module is suitable for projects ranging from whole-transcriptome epitranscriptome discovery (baseline m⁶A landscape across tissues or conditions) to focused modification analysis of specific gene families or biological pathways. The Nanopore Direct RNA Sequencing service page provides detailed information on the core method, sample requirements, and standard deliverables.

Nano-tRNA Sequencing — Specialized tRNA Modification and Fragment Profiling

Nano-tRNA Sequencing applies Nanopore direct RNA sequencing to the most densely modified class of cellular RNA. Transfer RNAs carry the highest density and diversity of post-transcriptional modifications of any RNA biotype, with 15–30 modified residues per molecule on average — including m⁵C, pseudouridine, dihydrouridine, 1-methyladenosine (m¹A), 7-methylguanosine (m⁷G), and dozens of base- and ribose-methylations that collectively regulate tRNA folding, stability, aminoacylation, and codon decoding. Nano-tRNA-seq preserves these modifications through direct native tRNA sequencing and reports modification status at single-nucleotide resolution on individual tRNA molecules, enabling tRNA-isotype-resolved modification analysis, detection of hypo- and hyper-modified tRNA populations, and characterization of tRNA-derived fragments (tRFs) with their modification status intact.

TAIL Iso-Sequencing — Poly(A) Tail Length, 3′ UTR Modification, and APA Analysis

TAIL Iso-Sequencing combines direct RNA sequencing with specialized library preparation to capture full-length mRNA molecules with intact poly(A) tails, enabling simultaneous measurement of poly(A) tail length distribution, m⁶A modification status along the transcript body and 3′ UTR, alternative polyadenylation (APA) site usage, and transcript isoform structure from the same native RNA molecules. The per-read poly(A) tail length measurement enables direct correlation between m⁶A modification status and poly(A) tail length at single-molecule resolution — a relationship that published studies (Kim et al. 2025, Cell Genomics; see Case Study below) have shown to be more complex and context-dependent than previously understood. This integrated module replaces separate poly(A)-tail assays and m⁶A-seq experiments with a single sequencing run, preserving the molecular linkage between tail length and modification status on every captured transcript.

Full-Length Transcript Sequencing (Iso-Seq) — Isoform-Level Modification Context

Full-Length Transcript Sequencing (Iso-Seq) on the PacBio Revio platform provides complementary high-accuracy (Q30+) full-length transcript sequences that serve as reference isoform annotations for RNA modification analysis. While PacBio Iso-Seq does not directly detect RNA modifications (it requires RT and PCR), the comprehensive isoform catalog it generates — including novel isoforms not represented in reference annotations — enables more accurate assignment of m⁶A calls to specific transcript isoforms from dRNA-seq data. This service module is recommended for projects in non-model organisms or poorly annotated genomes where the isoform reference is incomplete, and for cross-platform validation studies requiring independent isoform-level confirmation of modification-associated alternative splicing events.

Our Pipelines Deliver Per-Site and Single-Molecule RNA Modification Calls with Differential and Isoform-Level Analysis

CD Genomics Provides dRNA-seq as a Core Platform with Validated Multi-Modification Bioinformatics Pipelines

Direct RNA sequencing is our core transcriptomics platform, not an add-on service.

CD Genomics has built its long-read transcriptomics service portfolio around ONT direct RNA sequencing from the ground up. Our RNA methylation analysis workflows are designed for native RNA — no RT, no PCR, no workarounds that compromise modification detection. When you work with us, your epitranscriptome project is executed by a team whose core technology platform matches the native RNA approach your project requires.

Multi-modification detection from a single sequencing run — no parallel experiments.

We detect m⁶A, m⁵C, Ψ, and inosine simultaneously from the same dRNA-seq dataset, using a tiered bioinformatics approach that combines Dorado v5+ modified-base models, m6Anet, CHEUI, and Minimod. You receive comprehensive transcriptome-wide modification profiles from a single sequencing run and a single RNA input — not from separate antibody enrichments, chemical conversions, or parallel library preparations that multiply cost and sample requirements.

Platform-appropriate bioinformatics for RNA modification data.

RNA modification calling from dRNA-seq requires fundamentally different algorithms than DNA methylation detection. Our bioinformatics team deploys pipelines specifically designed and validated for direct RNA modification detection — including m6Anet (multiple-instance learning for transcriptome-wide m⁶A), CHEUI (two-stage neural network for m⁶A + m⁵C from the same data), and Dorado modified-base models for direct basecalling-level modification identification. We do not apply DNA methylation pipelines to RNA data or use generic peak-calling approaches designed for antibody-enriched fragments.

Integrated multi-module epitranscriptome analysis from a single provider.

Beyond whole-transcriptome modification detection, our integrated service modules — Nano-tRNA Sequencing for tRNA modification profiling, TAIL Iso-Sequencing for simultaneous modification + poly(A) tail analysis, and Iso-Seq for complementary high-accuracy isoform reference annotation — enable comprehensive, multi-layered epitranscriptome characterization without coordinating across multiple service providers or reconciling data from incompatible platforms.

Validated through peer-reviewed publications.

Our direct RNA sequencing services have been used in published studies spanning cancer epitranscriptomics, plant stress RNA biology, and viral RNA modification analysis, with datasets generated on our platforms appearing in peer-reviewed journals.

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

Choose long-read RNA methylation sequencing when:

• Single-molecule and isoform-resolved modification information is required — Only dRNA-seq provides per-read m⁶A modification status linked to specific transcript isoforms, enabling detection of modification stoichiometry, co-occurrence of multiple modifications on the same molecule, and allele-specific modification that no population-averaged method (MeRIP-seq, m⁶A-CLIP) can provide
• Simultaneous modification and poly(A) tail analysis from the same molecule is valuable — Every dRNA-seq read simultaneously captures full-length transcript sequence, modification status at single-nucleotide resolution, and poly(A) tail length — enabling direct correlation between modification status and tail length at single-molecule resolution that requires separate experiments with any other approach
• Multiple modification types need to be profiled from limited RNA — From a single dRNA-seq run using standard RNA input, we simultaneously detect m⁶A, m⁵C, pseudouridine, and inosine — replacing separate MeRIP-seq (m⁶A), bisulfite-seq (m⁵C), CMC labeling (Ψ), and A-to-I editing assays that each require separate RNA input, library preparation, and sequencing
• You are working with limited or irreplaceable RNA samples — The single-assay multi-modality nature of dRNA-seq is particularly valuable for clinical biopsies, laser-capture microdissected tissue, or rare cell populations where RNA quantity is insufficient for multiple parallel modification-specific assays
• Full-length native transcript context is essential for modification interpretation — For studies requiring knowledge of which specific transcript isoform carries a modification, or how modifications at 5′ UTR, CDS, and 3′ UTR sites co-vary on the same molecule, dRNA-seq is the only method that preserves native transcript connectivity

Consider alternative methods when:

• Bulk m⁶A profiling at the lowest per-sample cost is the sole requirement — For large-cohort screening studies requiring only identification of m⁶A-enriched regions across many samples, MeRIP-seq or m⁶A-CLIP offer substantially lower per-sample cost at the cost of resolution and single-molecule information. We can advise on the most cost-effective approach for your specific study design
• Very large numbers of samples must be compared for differential modification — While dRNA-seq provides richer data per sample, its throughput (~5–20 million reads per flow cell) and RNA input requirements (~1 µg polyA+ RNA) may not be optimal for studies requiring hundreds of samples. A discovery-validation strategy (dRNA-seq on a representative subset, targeted validation on the full cohort) can optimize the cost-benefit ratio
• Only transcript expression quantification is needed — For projects focused exclusively on gene or isoform expression levels, standard short-read RNA-seq or cDNA-based long-read methods provide higher throughput and lower cost per sample than dRNA-seq, without the specialized bioinformatics required for modification analysis
• Your RNA samples are degraded or limited in quantity — dRNA-seq requires high-integrity RNA (RIN ≥ 8 recommended) and relatively high input amounts (~1 µg polyA+ RNA). For degraded RNA, low-input samples, or FFPE-derived material, short-read or cDNA-based approaches may be more practical. Contact our scientists for feasibility assessment of challenging samples

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

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