Nanopore sequencing technology uniquely delivers high outputs of long reads that span full-length RNA transcripts, supporting quantification and complete transcriptome characterisation at the isoform level. Differential isoform expression and allele-specific effects are known to be important in the susceptibility to and progression of diseases (e.g. cancer), and individual drug responses. Nanopore sequencing allows the generation of novel insights into transcript isoform expression and usage across different experimental conditions and cells, including single-cell analysis. Furthermore, with direct RNA sequencing, it is possible to assess modified bases in native RNA transcripts.

Without direct RNA sequencing, we would have to perform a series of different experiments to obtain this data… now we can get all this information from a single experiment

Josie Gleeson, The University of Melbourne, Australia

Technology comparison

Oxford Nanopore sequencing

Traditional short-read technologies

Unrestricted read length

  • Generate full-length transcripts — >20 kb single-read transcripts demonstrated
  • Unambiguously identify all splice variants
  • Perform accurate allele-specific, isoform-level gene expression analysis — even at single-cell resolution
  • Easily identify antisense transcripts and lncRNA isoforms
  • Approximately 50-fold fewer reads required to cover the same number of transcripts

Read length typically 50–100 bp

The short reads generated by traditional RNA-Seq techniques only partially cover the length of a transcript, making it challenging to accurately assemble and quantify transcript isoforms. Short reads also exhibit high rates of multimapping, leading to data loss and reduced utility for genome or transcript annotation.

Direct, amplification-free protocols

Amplification required

The amplification requirement of traditional RNA-Seq approaches can introduce bias, reducing the complexity of the total RNA pool and potentially causing increased abundance or drop-out of some RNA species.

Accessible and scalable

  • Scalable devices to suit your needs — portable MinION starting at just $1,999, including sequencing reagents
  • Flexible throughput with modular GridION and PromethION — run extensive time-course or treatment studies simultaneously
  • Develop targeted assays using low-cost Flongle
  • Sample multiplexing — reduce costs and maximise experimental efficiency

Less accessible

Platform costs and infrastructure requirements can limit global accessibility. Sample batching may be required for optimal efficiency, potentially delaying results.

Streamlined workflows

  • Sample prep in ~210 mins + PCR (cDNA-PCR) or ~135 mins (Direct RNA)
  • Choice of two unique kits: cDNA-PCR and direct RNA — minimising bias and providing flexibility in speed, yield, and identification of epigenetic modifications

Laborious workflows

Typical sample prep times of ~7 hours with ~3 hours of hands-on time.

Real-time data streaming

  • Stop sequencing when sufficient data generated — wash and reuse flow cell
  • Immediate access to results

Fixed run time with bulk data delivery

Increased time to result and inability to identify workflow errors until it’s too late, plus additional complexities of handling large volumes of bulk data all at once.

White papers

The value of full-length transcripts without bias

Discover more about how long nanopore RNA sequencing reads enable unambiguous isoform-level gene expression studies and, with direct RNA sequencing, supports simultaneous detection of base modifications. Read case studies covering a wide variety of research areas, including how nanopore RNA sequencing revealed nine of the top ten expressed isoforms of the CACNA1C gene (a therapeutic target for neuropsychiatric disease) to be novel transcripts.

Get more transcriptomics content, including getting started guides, workflows, and videos, in our Resource centre.

Case study

Potential real-time molecular classification of leukaemia with nanopore transcriptome sequencing

Acute lymphoblastic leukaemia (ALL) can present with life-threatening symptoms at the point of diagnosis; however, currently used clinical workflows are costly, take 2–14 days, and leave ~40% of cases unclassified. Sagniez et al. demonstrated the potential clinical utility of nanopore transcriptome sequencing to characterise ALL. Using low-cost Flongle Flow Cells on MinION, they were able to correctly assign all subtypes from clinical research samples — in less than five minutes of sequencing.

In addition to its rapid turnaround time, the accuracy, cost and portability of nanopore RNA sequencing provides exciting new opportunities for molecular medicine in the post-genomics era

Sagniez et al., MedRxiv (2022)

Case study

Direct RNA sequencing of mouse brain samples from the RIKEN Aging project

At the Nanopore Community Meeting 2022, Callum Parr (RIKEN Institute, Japan) presented his ongoing work investigating the role of RNA splicing on the brain during aging. He described how he will leverage long-read direct RNA nanopore sequencing to overcome current sequencing limitations and map the dynamic nature of RNA splicing in different brain cell types. The aims of this work are to discover isoforms and RNA modifications involved in the ageing process and age-associated diseases, such as Alzheimer’s disease.

Get started

Scalable transcriptome sequencing

From powerful, portable Flongle and MinION devices to the high-throughput, high-output benchtop GridION and PromethION platforms — scale your RNA sequencing to match your specific research requirements.

Recommended for transcriptome sequencing


Running up to five independent MinION or Flongle Flow Cells with powerful, integrated compute, GridION provides the flexibility to run multiple RNA and DNA sequencing experiments, on demand — ideal for differential gene expression studies and busy labs.


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