Splicing errors drive an estimated 15% of inherited diseases and cancers, yet many of these events go undetected with standard RNA-seq1. As the transcriptome emerges as a vital layer in understanding and targeting disease biology, short-read sequencing methods are increasingly proving inadequate.
Long-read sequencing, however, captures full-length isoforms and complex splicing patterns in single reads, offering a clearer path to discovering disease-relevant variants, novel transcripts, and functional insights that fragmented methods often miss. By sequencing full transcripts, long-read cDNA sequencing provides a clearer, more complete view of the transcriptome, enabling researchers to detect clinically relevant splice variants and structural features with greater confidence. This article explores how long-read cDNA sequencing is advancing transcriptomic biomarker discovery and cancer research with a focus on the advantages of Oxford Nanopore’s platform over short-read technologies.
By fragmenting cDNA transcripts into short reads, standard RNA-seq makes it difficult to reconstruct full-length transcripts, obscuring which coding regions are connected, how splicing patterns vary, and how these changes influence biological function2. Significant computational resources are required to accurately reassemble these short reads into full-length transcripts, slowing analysis, increasing error potential, and reducing confidence in transcript-level insights that inform discovery and development decisions3. Indeed, a study determining the full structure of RNA transcripts from fragmented reads was proven to be difficult as tests using 25 different lab protocols and 14 software tools showed major differences when the results were compared4.
Short reads also struggle to resolve transcript start and poly(A) sites, regions that define where transcripts begin and end 5. These boundaries often carry regulatory significance. Missing them can obscure alternative transcript usage that influences gene expression, protein output, or therapeutic relevance. Taken together, these limitations—fragmentation, incomplete transcript reconstruction, and unresolved transcript boundaries—undermine the accuracy of short-read RNA-seq and restrict researchers’ ability to confidently interpret transcript-level data critical to discovery and development.
Capturing full-length transcripts in a single read enables more accurate isoform detection, transcript quantification, and gene regulation analysis, revealing features often missed by short-read methods. Long-read cDNA sequencing spans entire transcripts, capturing key regions that shape transcript structure and function, like exons, introns, and untranslated regions. By resolving full exon junctions, it allows researchers to directly observe splice events and alternative isoform usage without relying on fragmented reconstruction6. In one study, Oxford Nanopore sequencing uncovered an unexpected level of transcriptomic complexity in B cells, revealing thousands of novel transcription start and end sites and hundreds of alternative splicing events, highlighting the full extent of transcript isoform diversity and the resulting biology7.
Further, capturing both transcript start and end sites helps identify alternative initiation and termination points, which can affect protein output, regulatory dynamics, or disease relevance. Tang et al. identified numerous novel alternative transcription start and polyadenylation sites in cancer, revealing disease-specific isoforms that arise from splicing dysregulation and are linked to distinct functional consequences in tumor biology8. Thus, long-read cDNA sequencing simplifies analysis and reveals biologically meaningful isoforms by capturing complete transcripts, including exon junctions and transcript boundaries, in a single read.
Improved accuracy and full transcript coverage deepen insight into gene function, fueling advances in health, disease research, and drug development through more robust datasets. This comprehensive view is especially critical for detecting gene fusions, abnormal transcripts formed by the joining of two separate genes, which are common drivers in many cancers but often missed by fragmented RNA-seq data. In adenocarcinoma cell lines, long-read cDNA sequencing have uncovered full-length fusion transcripts and alternative fusion breakpoints in key oncogenes, delivering unprecedented resolution of tumor-driving rearrangements with direct implications for targeted therapy and clinical prognosis9.
Long-read cDNA sequencing helps uncover cryptic, disease-driving mutations, such as deep intronic variants, that often go undetected by short-read methods, offering new opportunities for early detection and genetic risk assessment in familial cancers. These variants occur in introns, non-coding regions that are typically removed during RNA processing, and can introduce premature stop signals that disrupt protein production10. Because they fall outside of normal splice sites, these variants frequently go undetected by traditional sequencing workflows. Long-read cDNA sequencing can detect how these intronic mutations alter splicing, sometimes causing intronic sequences to be misread as exons, and revealing mechanisms that silence tumor suppressor genes. These long-read insights are especially valuable for improving genetic screening protocols and stratifying patients by inherited cancer risk.
Long-read cDNA sequencing also provides high-resolution immune profiling,revealing functional biomarkers that inform disease monitoring and therapeutic responses. Using Oxford Nanopore long-read cDNA sequencing, researchers sequenced the transcript expression of T and B cell receptors from thousands of individual cells from primary tumors and nearby lymph nodes. This transcript-level view of receptor diversity and structural variation allowed them to map immune responses with single-cell resolution11. In cancers like prostate and metastatic melanoma, patients who maintained key T cell clonotypes before and after treatment consistently displayed better clinical outcomes. These findings highlight the prognostic value of immune repertoire monitoring and the role of long-read cDNA sequencing in capturing these dynamic, clinically relevant changes12.
These examples illustrate how long-read cDNA sequencing goes beyond transcript quantification, uncovering functionally relevant isoforms, regulatory anomalies, and immune features that drive disease biology and open new paths for discovery and clinical development.
Oxford Nanopore’s long-read cDNA sequencing delivers a more complete and confident view of the transcriptome, capturing full-length isoforms, splice variants, and regulatory features in a single read. By reducing reliance on computational reconstruction, it improves isoform-level resolution and enhances the discovery of transcriptomic changes that drive disease biology, advancing applications in biomarker development, oncology, and therapeutic research.
Because the process relies on reverse transcription, convertingRNA to cDNA, it does not preserve native RNA modifications, making it less suited for studying RNA methylation or epitranscriptome regulation. For researchers focused on those aspects of transcriptomic biology, direct RNA sequence may offer a better alternative. Still, for applications centered on transcript structure and gene expression, long-read cDNA sequencing provides a powerful and scalable tool for unlocking new layers of insight.
