A targeted RNA nanopore sequencing workflow developed by Kylie Montgomery and colleagues (University College London, UK*) is helping researchers investigate some of the most challenging rare disease cases. In a recent publication, the team developed and introduced RAPID (RNA analysis pipeline for integrated diagnostics).
RAPID is a sample-to-answer workflow that uses cDNA nanopore sequencing to capture near-full-length transcripts and reveal splicing abnormalities that can be difficult to resolve using legacy approaches. Applied retrospectively to six previously unsolved rare disease cases, RAPID generated findings in every investigation, helping to confirm splice-disrupting variants, identify candidate regions for follow-up DNA analysis, or exclude genes that were unlikely to be driving disease.
We spoke to Kylie about her journey to develop RAPID, the challenges of investigating unresolved rare disease cases, and why she wanted to make her workflow accessible to all.
*Research conducted at University College London, UK. Some of the team have since moved to UK Dementia Research Institute at Cambridge, UK.
Why rare disease research?
Before starting her PhD, Kylie worked in molecular pathology diagnostic laboratories, first in New Zealand and later in the UK. She then became a clinical scientist, and found herself increasingly drawn to variant interpretation and the challenge of delivering answers for families affected by rare disease. That work included involvement in the 100,000 Genomes Project at Great Ormond Street Hospital, where she saw both the strengths and limitations of modern genomic approaches.
‘I started getting quite frustrated over time, when you're not able to give the time and the energy and the resources to the cases that aren't getting their diagnosis’
‘I started getting quite frustrated over time, when you're not able to give the time and the energy and the resources to the cases that aren't getting their diagnosis’
For Kylie, the unresolved cases became impossible to ignore. In 2022 she moved into academia, joining the sequencing service at University College London, UK, where exposure to a huge variety of samples and research questions helped shape her thinking about how Oxford Nanopore sequencing might support rare disease investigations.
Her PhD, supervised by Mina Ryten, Emil Gustavsson, and Henry Houlden, focused on understanding whether rare diseases could be investigated differently by integrating functional genomic information and transcript-level evidence. That work eventually evolved into RAPID.
Why is RAPID needed?
Many of the patients Kylie encountered had already undergone extensive genetic testing. Whole-exome or whole-genome short-read sequencing had only narrowed the search to a likely candidate gene, identified a variant of uncertain significance, or revealed a single candidate variant in what appeared to be a recessive disorder. The missing piece was often functional evidence.
‘We just needed that last piece of evidence to get it over the line. That's where nanopore RNA sequencing was introduced as the next step’
‘We just needed that last piece of evidence to get it over the line. That's where nanopore RNA sequencing was introduced as the next step’
The RAPID workflow emerged from a simple question: if DNA sequencing had already taken researchers as far as it could, could RNA provide the next clue? Up to 62% of pathogenic single nucleotide variants are estimated to affect splicing, yet the pathogenicity of many newly identified or unannotated variants remains uncertain.
Functional validation is often needed to determine whether these variants alter gene function, but legacy short-read sequencing cannot accurately reconstruct full-length transcripts to provide that evidence. RAPID was designed as a sample-to-answer targeted sequencing workflow that leverages transcript-level information to reveal the functional consequences of genetic variants, without relying on large reference cohorts or whole-transcriptome sequencing approaches.
Why nanopore sequencing?
For Kylie, the choice of nanopore sequencing was driven as much by practicality as by performance. Rather than designing a workflow that required significant investment in new infrastructure or specialist bioinformatics expertise, Kylie wanted to create something that could realistically fit within public healthcare and translational research settings.
‘It needs to be accessible, and it needs to be targeted. By doing a targeted approach, you can multiplex your samples, and that brings the costs down further’
‘It needs to be accessible, and it needs to be targeted. By doing a targeted approach, you can multiplex your samples, and that brings the costs down further’
The RAPID workflow combines targeted cDNA amplification with Oxford Nanopore sequencing to capture near-full-length transcripts from genes of interest. By characterising full-length isoforms, researchers can directly observe exon skipping events, transcript imbalance, nonsense-mediated decay signatures, and other transcript-level effects that go beyond what is visible from DNA sequence alone.
A key motivation was overcoming the limitations of legacy short-read RNA sequencing. As the authors explain in the preprint, short reads provide only a fragmented view of transcript structure, making it difficult to resolve exon connectivity, alternative splicing events, and complex transcript architectures. Oxford Nanopore any-length reads offer a more complete picture.
Building the sample-to-answer workflow
Kylie’s goal was to create an end-to-end workflow that could take researchers from sample to interpretable result as simply as possible. The resulting workflow combines targeted cDNA amplification, Oxford Nanopore sequencing, and a modular computational pipeline designed to generate clinician-facing outputs and support interpretation of transcript-level changes. The protocol was developed to support single-sample interpretation and to integrate with workflows already familiar to diagnostic laboratories.
Although Oxford Nanopore technology is for research use only, Kylie emphasised that translation into a clinical setting follows a familiar validation pathway. Before findings are used to support clinical decision-making, laboratories undertake their own validation studies using previously characterised samples and orthogonal methods to ensure the workflow performs to the required standard.
Figure 1. The end-to-end RAPID workflow. (A) Preparation of cDNA sequencing libraries from blood or fibroblasts. B) Data analysis pipeline, including quality control, alignment to the human reference genome, transcript assembly, and quantification. Figure redistributed from Montgomery et al. 2026 under Creative Commons Attribution License CC BY 4.0.
One of the study's most notable achievements was speed. The workflow can generate informative results within two working days under suitable conditions.
‘By the end of the next day, you could have your data if you're familiar with the process and the region you're targeting is not too long, meaning the PCR process can be relatively quick’
‘By the end of the next day, you could have your data if you're familiar with the process and the region you're targeting is not too long, meaning the PCR process can be relatively quick’
In comparison, Kylie said it could take months or even years to gather sufficient control samples for short-read analysis.
What has RAPID revealed so far?
Rather than focusing on a single type of variant, the six-case retrospective pilot study demonstrated several ways that Oxford Nanopore RNA analysis has the potential to contribute to rare disease investigations.
In some research samples, RAPID identified previously unseen splice events, enabling Kylie to confirm that variants were disrupting normal splicing and providing functional evidence that supported variant reclassification. In others, transcript-level changes pointed her towards regions requiring further DNA investigation, helping narrow the search for deep intronic or regulatory variants that had escaped earlier analyses.
The workflow also demonstrated value when normal splicing was observed. For one research sample, transcript analysis enabled the researchers to confidently exclude a candidate gene and redirect their efforts elsewhere.
One particularly memorable example for Kylie involved using nanopore sequencing on both the DNA and RNA sides of an investigation. After DNA sequencing identified a deep intronic candidate variant, targeted RNA sequencing revealed a transcript change not seen in public databases. The resulting evidence helped explain the variant's biological consequences and ultimately contributed information that could be assessed by a clinician.
Here’s Kylie presenting her research at London Calling 2026.
Making RAPID available to everyone
From the beginning, Kylie wanted RAPID to be more than a one-off research project. The laboratory protocol has been made publicly available through protocols.io, while the computational pipeline is available on GitHub.
‘I’d love others to use it if they want to. Particularly for the NHS where whole-genome or whole-transcriptome sequencing isn’t the best use of resources. The RAPID protocol is already targeted and more straightforward to analyse’
‘I’d love others to use it if they want to. Particularly for the NHS where whole-genome or whole-transcriptome sequencing isn’t the best use of resources. The RAPID protocol is already targeted and more straightforward to analyse’
That openness reflects one of the core ideas behind the project: enabling other laboratories to explore transcript-level analysis without needing to reinvent the workflow themselves. Kylie believes that targeted approaches such as RAPID may be particularly valuable for resource-limited settings, and she hopes the workflow can help others investigate difficult cases more efficiently.
What’s next?
Kylie is continuing to explore how transcript-level analysis of research samples with RAPID can support rare disease research. She also has a Direct RNA Sequencing Kit ready to try. Since many of Kylie’s transcripts of interest are longer than average, cDNA conversion can introduce 3' bias. By sequencing native RNA fragments instead, she hopes to improve 5' coverage and learn more about the untranslated region of each transcript.
What excites Kylie most is the opportunity to turn difficult genomic questions into actionable biological insights. By generating near-full-length isoforms from a practical sample-to-answer workflow, RAPID demonstrates how RNA nanopore sequencing could help researchers investigate unresolved rare disease cases more efficiently in the future.
For more rare disease research using nanopore technology, read our blog: ending the diagnostic odyssey starts here.
Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.
- Montgomery, K.-A. et al. RAPID: a targeted long-read RNA workflow for functional resolution of splicing variants in rare disease. Genome Med (2026). DOI: https://doi.org/10.1186/s13073-026-01754-3
