Science unlocked: publication picks from March 2025

In this monthly series, we share a selection of recent publications in which Oxford Nanopore sequencing was used to unlock novel insights. Spanning from rare diseases, to somatic variant calling, to intraoperative tumour classification, these studies showcase the advances in scientific research made possible by Oxford Nanopore sequencing.

Featured in this edition:

1. No need for short-read polishing

2. Brain tumour biopsy-to-result in under an hour

3. Changing the game for intraoperative tumour classification

4. Cancer variant calling without matched normal samples

5. Hope is on the horizon for unsolved rare diseases

6. Oxford Nanopore direct RNA takes the transcriptomics crown

Microbiology

1. High intra-laboratory reproducibility of nanopore sequencing in bacterial species underscores advances in its accuracy (Microbial Genomics)

This research, conducted by Abdel-Glil et al., highlights the advances in Oxford Nanopore Technologies, including higher sequencing accuracy, removing the need for short-read data. Across five bacterial strains and eight replicates, the team achieved an average reproducibility accuracy of 99.9% for Oxford Nanopore-only assemblies — closely matching short-read-polished assemblies.

Key points:

• The aim of this study was to evaluate the reproducibility of nanopore sequencing under uniform conditions using a benchmark dataset of five bacterial species

• To assemble Oxford Nanopore sequencing data, the team used an open-source workflow, nanobacta

• Resistance gene detection, MLST typing, taxonomic classification, plasmid recovery, and genome completeness were all highly reproducible using Oxford Nanopore-only data

• Oxford Nanopore-only assemblies averaged 2.2 variant errors per genome, and short-read polishing only improved accuracy by 0.00005%, supporting the case for nanopore-only assemblies

Figure: Methodological workflow used in the study by Abdel-Glil et al. 2025. Redistributed under Creative Commons Attribution License CC BY.

Cancer research

2. Rapid brain tumour classification from sparse epigenomic data (Nature Medicine)

This study showcases a live brain tumour classification method combining Oxford Nanopore sequencing with MethyLYZR, an epigenomic analysis tool. By leveraging real-time genetic and epigenetic data from Oxford Nanopore sequencing, MethyLYZR achieved 94.5% accuracy within 15 minutes and required minimal computational power — highlighting its potential for future intraoperative diagnostics.

Key points:

• MethyLYZR is a naïve Bayesian framework that runs in parallel with Oxford Nanopore sequencing for live methylation-based classification

• Over 200 brain tumour samples were analysed, including 10 intraoperatively

• MethyLYZR classified tumours with 94.52% accuracy in under 15 minutes, with a 1-hour biopsy-to-result turnaround time

• It requires under 1 minute of processing time and less than 3GB of RAM, making MethyLYZR an attractive alternative to computationally intensive machine learning models

• Tumours were also classified from cerebrospinal fluid cell-free DNA, highlighting potential for non-invasive liquid biopsy diagnostics in the future

Figure: Schematic representation of the timeline for intraoperative tumour sequencing and classification in this study. The cancer class prediction was achieved within a rapid turnaround time of just 1 hour from tumour biopsy reception. The process involves genomic DNA extraction (approximately 22 min), nanopore library preparation (approximately 18 min) and loading of the library with subsequent sequencing (15–20 min). Figure redistributed from Brandl and Steiger et al. 2025 under Creative Commons Attribution 4.0 International License.

3. Prospective, multicentre validation of a platform for rapid molecular profiling of central nervous system tumours (Nature Medicine)

The Oxford Nanopore-based workflow, Rapid-CNS², together with MNP-Flex classified central nervous system tumours in under 30 minutes. The method detected key variants previously missed by short-read methods, and up to 94.6% of results matched standard diagnostic tests, highlighting the potential for this Oxford Nanopore-based approach to accelerate personalised cancer care in the future.

Key points:

• Patel et al. combined Rapid-CNS² (an adaptive sampling-based Oxford Nanopore sequencing workflow) with the methylation classifier MNP-Flex

• Methylation classification and copy number profiles were reported within a 30-minute intraoperative window, and comprehensive molecular profiling followed within 24 hours

• Rapid-CNS² achieved concordance with current diagnostic tools in 285/301 cases (94.6%)

• Oxford Nanopore sequencing detected clinically relevant mutations, fusions, and structural variants, including alterations missed by short-read methods

Watch Areeba Patel discuss this research at NCM 2021

Bioinformatics

4. ClairS-TO: a deep-learning method for long-read tumour-only somatic small variant calling (bioRxiv)

ClairS-TO is a deep learning-based tool for detecting somatic variants in tumour-only samples using long-read sequencing data. It combines dual neural networks with advanced filtering strategies to accurately distinguish somatic mutations from germline variants and sequencing noise — achieving state-of-the-art performance without requiring a matched normal sample.

Key points:

• Tumour-only somatic variant detection is challenging because it lacks a matched normal sample, making it difficult to distinguish somatic mutations from germline variants and sequencing noise

• Chen and Zheng et al. designed ClairS-TO specifically for long-read tumour-only variant calling, addressing the limitations of short-read-based tools

• It uses an ensemble of two neural networks trained on opposing tasks to boost classification accuracy

• ClairS-TO was trained on synthetic datasets derived from Genome in a Bottle HG002 and HG001 from EPI2ME labs and real tumour data from six cancer cell lines

• It outperformed DeepSomatic and short-read callers (Mutect2, Octopus, Pisces) across Oxford Nanopore, PacBio, and Illumina datasets

• Demonstrated robust performance across sequencing coverages, variant allele fractions, tumour purities, and complex genomic regions

Human genetics

5. Long read sequencing enhances pathogenic and novel variation discovery in patients with rare diseases (Nature Communications)

The authors used Oxford Nanopore sequencing and a filtration strategy to improve the detection of harmful genetic variants and abnormal methylation profiles in unsolved rare disease cases. They identified pathogenic variants in 10% of individuals with previous negative short-read testing, demonstrating their method’s potential as a rare disease diagnostic tool in the future.

Key points:

• Short-read sequencing struggles to detect complex structural variants, methylation profiles, and repeat expansions, leaving over 50% of rare disease patients without a diagnosis

• Sinha, Rabea, and Ramaswamy et al. developed a simplified funnel-down filtration strategy to enhance the identification of deleterious variants and abnormal episignature disease profiles from Oxford Nanopore sequencing data

• Oxford Nanopore sequencing captured full-length structural changes and epigenetic signatures, allowing for the identification of a novel methylation-based tag for spinal muscular atrophy

• Pathogenic variants were identified in an additional 5/51 of individuals with suspected rare diseases

Watch Ahmad Abou Tayoun discuss Oxford Nanopore sequencing as a potential diagnostic tool for genetic diseases at London Calling 2023

6. A systematic benchmark of nanopore long-read RNA sequencing for transcript-level analysis in human cell lines (Nature Methods)

Chen, Davidson, Wan, and Yao et al. present their results from the Singapore Nanopore Expression (SG-NEx) project, in which they compared five RNA sequencing protocols across a benchmark dataset from seven human cell lines. The authors report that Oxford Nanopore sequencing offers superior detection of major isoforms, novel transcripts, and RNA modifications compared to other sequencing methods, providing a valuable resource for advancing transcriptomic research.

Key points:

• Short-read RNA sequencing introduces fragmentation and biases, making it difficult to accurately identify full-length RNA transcripts — critical for understanding gene regulation and disease mechanisms

• The authors compared Oxford Nanopore direct RNA, direct cDNA and PCR-amplified cDNA kits, Illumina cDNA, and PacBio IsoSeq

• Oxford Nanopore sequencing better captured full-length transcripts and alternative isoforms compared to Illumina short-read sequencing

• The direct RNA and cDNA kits performed best at detecting novel transcripts, fusion genes, and RNA modifications

• Oxford Nanopore direct RNA sequencing was unique in its ability to sequence native RNA, avoiding biases from cDNA conversion and PCR amplification

Unlock isoform-level insights with direct RNA sequencing

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