Why use direct RNA sequencing for transcriptomics?

Multiomic data is not optional for answering complex biological questions — it is essential. To link genotype to phenotype, researchers need comprehensive datasets that cover genetics, transcriptomics, epigenetics, epitranscriptomics, and proteomics. Legacy multiomic workflows remain complex and time consuming, requiring multiple different assays, platforms, and analytical approaches. As a result, many studies focus on one omic layer, limiting insight into the links between different regulatory elements due to the challenges of integrating these datasets¹.

However, we provide a range of workflows for multiomic sequencing, covering an assortment of applications. These end-to-end techniques include library preparation kits to prepare DNA, cDNA, or native RNA for nanopore sequencing. You can sequence libraries prepared with these kits on any of our devices, such as our PromethION 24. With our direct RNA sequencing kits, you can capture full-length transcripts and RNA modifications — in one go.

What does this mean? You can now generate richer datasets and explore entirely new biological questions. In this Nanopore Know-How blog, find out what insights you can gain with direct RNA analysis, and how researchers are using it to better understand translational regulation, neurological development, cancer, and diabetes.

Detect isoform-specific modifications

As discussed in our transcriptomics Nanopore Know-How blog, cDNA sequencing is currently the main method for investigating the transcriptome. When undertaken with legacy short-read sequencing, most full-length transcripts cannot be fully sequenced in a single read, so isoforms and alternative splicing cannot be identified with certainty. In addition, reverse transcription of RNA for cDNA sequencing means that base modifications are lost, obscuring an important regulatory layer.

Modifications are key to an organism’s development, and epigenetic dysregulation is increasingly recognised for its role in human diseases so it could be a target for treatment development². This is apparent in pathologies such as acute myeloid leukaemia (AML), where disease outcomes have not drastically improved despite a broad understanding of the genetic anomalies underpinning this cancer. In light of this, Chen et al. (2024) used direct RNA nanopore sequencing to investigate the biological impact of demethylation therapy, a novel therapeutic approach for AML³.

In their study, they identified two genes that were highly expressed in AML patients who were in complete remission following demethylation treatment, even though these genes are typically associated with poor prognosis according to the Cancer Genome Atlas (TCGA) database³. By identifying modifications at the isoform level, they could see that the isoforms that were being expressed in these patients were differentially methylated³. This finding provides a mechanistic explanation for the therapeutic benefit of demethylation in AML that would have been invisible without drilling down to the isoform level³.

Detect multiple RNA modification types

Many multiomic studies still focus on a single modification type. By using Oxford Nanopore sequencing, researchers are moving beyond isolated signals and assessing multiple RNA base-modifications in one go. Read our methylation Nanopore Know How blog to learn how nanopore sequencing captures modifications without bisulfite treatment or enzymatic conversion.

So, what are the benefits of studying multiple RNA modifications at once? Bansal et al. (2026) assessed the interplay between pseudouridine (pseU), N6-methyladenosine (m⁶A), and 5-methylcytosine (m⁵C) to understand human genomic regulation⁴. They observed that reduced pseU levels correlated with increased m⁶A and m⁵C levels⁴. This suggests that pseudouridylation is the key function that modulates messenger RNA (mRNA) translation and protein synthesis⁴.

Functional insights such as this can also help us to understand mechanisms of disease. In their study on diabetes, Mulroney et al. (2025) applied a similar multi-modification approach to study pancreatic beta-cell responses to glucose stimulation⁵. By integrating gene and transcript expression analysis with modification profiling (pseU, m⁶A, m⁵C, and inosine patterns), they found differentially modified sites were enriched in type 2 diabetes genes (except for pseU), independently of gene and transcript expression changes⁵. This indicates that all three types of modifications could potentially play a role in this disease⁵.

Incorporate polyadenylation analysis

Isoform expression and RNA modification profiling provide valuable insight, but they represent only part of the regulatory landscape. Poly(A) tails are necessary to export mature mRNAs out of the nucleus, and the length is suspected to influence the stability and translation of these molecules⁶. As direct RNA nanopore sequencing can capture full-length molecules, researchers are also incorporating poly(A) tail length analysis into their multiomic studies.

Gleeson et al. (2025) combined isoform expression, m⁶A modification profiling, and poly(A) tail length analysis to better understand the human brain transcriptome⁷. Differential isoform expression is of established importance in the human brain, making Oxford Nanopore isoform-level analysis essential to this study. Immunoprecipitation methods cannot resolve the exact nucleotide position and stoichiometry of m⁶A sites, whereas chemical and enzyme-based methods cannot provide methylation patterns at isoform resolution⁷. By using direct RNA sequencing, Gleeson et al. (2025) found clear differences in regulatory patterns at the isoform level in distinct cell types across different brain regions⁷. Meanwhile Kim et al. (2025) used direct RNA sequencing in MOLM13 leukaemia cells to better understand the role of m⁶A in cancer development⁸. Following METTL3 knockdown, they observed global reductions in m⁶A methylation that in turn influenced gene expression, poly(A) tail length, and alternative splicing⁸.

By linking these features within a single experiment, researchers can more clearly define RNA regulation pathways, how they drive cellular behaviour, and identify key intervention points for disease.

What’s next? Scaling up.

Direct RNA sequencing is already reshaping how researchers study transcript regulation, opening new approaches to tackle disease. With our new direct RNA barcoding kits, a scaled-up approach to transcriptomics, without sacrificing deep multiomic insights, is now within reach.

Want to be part of the action? Find out more in our direct RNA sequencing kit flyer.

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