By sequencing full-length, native RNA molecules without conversion or amplification, [direct RNA sequencing] enables simultaneous analysis of isoform structure, expression, and epitranscriptomic marks—all in a single read.
RNA isn’t just a messenger. It encodes regulatory complexity that shapes how diseases emerge, progress, and respond to therapy. But standard transcriptomics methods only tell part of the story. Traditional cDNA-based RNA-seq workflows rely on reverse transcription and amplification, steps that fragment RNA, obscure isoform structure, and erase epigenetic marks critical to understanding gene regulation.
Long-read direct RNA sequencing (dRNA) offers a clearer view. By sequencing full-length, native RNA molecules without conversion or amplification, it enables simultaneous analysis of isoform structure, expression, and epitranscriptomic marks—all in a single read. In cancer, genetic disorders, and infectious diseases, this native-resolution approach is uncovering regulatory variation and biomarkers that conventional short-read workflows miss. In this article, we explore how Oxford Nanopore’s (ONT) dRNA platform captures transcript-level complexity with high fidelity, empowering discoveries where regulatory nuance, isoform diversity, and RNA modifications matter most.
Reverse transcription (RT) distorts the true RNA landscape, undermining data quality that can lead to false signals in cancer and other disease research. These distortions arise because RT enzymes can stall or misread structured RNA and fail to replicate DNA modifications, truncating reads, misassembling isoforms, and erasing epigenetic marks1. This compromises data quality—especially in applications requiring isoform-level resolution, modification detection, or resolution of mRNA molecule ends. In diseases like cancer, where transcriptional dysregulation is central, such distortions can obscure true signals, leading to false biomarkers, missed drug targets, and flawed insights into disease mechanisms2.
For example, mRNA poly(A) tails, which are critical for regulating molecule stability, processing, and translation efficiency, are often truncated or mismeasured during reverse transcription, leading to the loss of essential information about RNA lifespan and regulatory dynamics3,4. Given that poly(A) tail length is closely tied to tumor progression, neurodegeneration, and overall transcript stability, full-resolution profiling of this region is vital for identifying new biomarkers and therapeutic targets that could reshape diagnostics and treatment approaches 3,5.
Beyond structural distortions, reverse transcription also erases native chemical modifications that play pivotal roles in RNA biology. Key RNA modifications, including N6-methyladenosine (m6A), N5-methylcytosine (m5C), pseudouridine (Ψ), and inosine (I), control RNA stability, splicing, translation efficiency, and cellular localization. These modifications are increasingly recognized as major regulators in cancer progression, immune responses to infections, and chronic disease development 6,7.
For example, m6A modifications can drive oncogenic pathways by promoting abnormal mRNA decay or enhancing translation of cancer-promoting genes. Similarly, m5C and pseudouridine influence viral replication and immune evasion strategies in infectious diseases 6,7. By converting RNA into cDNA, reverse transcription removes this layer of epitranscriptomic information, limiting opportunities to discover new biomarkers and therapeutic targets.
Taken together, these and technical biological limitations constrain the ability of traditional cDNA-based RNA sequencing to fully resolve transcriptome complexity. To overcome these challenges and reveal a more complete picture of RNA biology, researchers are turning to long-read dRNA sequencing, which preserves native RNA features and offers a higher-fidelity view of the transcriptome 4,8.
Long-read dRNA sequencing directly reads full-length, native RNA molecules without reverse transcription or amplification, preserving transcript architecture, including exon connectivity, alternative splicing, and epitranscriptomic modifications4. This enables researchers to resolve native isoform diversity and capture critical regulatory features that often go undetected.
In leukemia research, directly sequencing RNA has uncovered undetectable isoforms of the CD19 receptor, a key target in cancer immunotherapy that alters how cancer cells are recognized by the immune system10. This insight has immediate implications for improving CAR-T cell therapy design, as hidden variants potentially compromise treatment efficacy.
dRNA sequencing enables the precise measurement of poly(A) tail lengths with single-transcript resolution. This more accurately reflects how RNA stability and decay dynamics shape disease processes3. In neurodegenerative diseases, shifts in poly(A) tail length can influence neuronal transcript persistence and function, offering clues to disease progression and new potential therapeutic entry points11.
Critically, dRNA also retains native RNA modifications, such as m6A, m5C, pseudouridine, and inosine, that regulate RNA fate and are increasingly recognized as key players in cancer development, viral infection, and immune response modulation 6,7,13,14. By directly preserving and mapping these marks, dRNA provides an unprecedented window into the epitranscriptomic landscape, information that cDNA-based methods inherently lose.
The clinical potential of dRNA extends across diverse disease areas. In Alzheimer’s disease, dRNA has uncovered isoform shifts and regulatory signatures missed by standard approaches, opening new avenues for targeted intervention12. In sepsis, it has revealed pathogen-specific RNA signatures and host responses that could inform more precise diagnostics and treatments 15.
Together, long-read direct RNA sequencing delivers a comprehensive, single-molecule view of RNA biology, capturing isoform diversity, poly(A) tail dynamics, and epitranscriptomic modifications in one assay. This holistic perspective empowers researchers to uncover novel biomarkers 16, precisely design therapies, and deepen our understanding of how RNA drives disease 15. As a result, dRNA is reshaping transcriptomics from a fragmented snapshot into a dynamic, multidimensional portrait of cellular regulation and disease.
In summary, while cDNA-based RNA sequencing remains a powerful tool for many transcriptomic applications, it lacks the resolution needed to fully capture complex isoforms and RNA modifications. In contrast, long-read direct RNA sequencing offers an unparalleled view of the transcriptome by capturing native, full-length RNA molecules without the biases introduced by reverse transcription or amplification. This technology is transforming our understanding of RNA biology, uncovering the intricate regulatory layers that drive health and disease, and delivering critical insights into conditions such as neurodegenerative disorders, cancer, and infectious diseases.
