Science unlocked: publication picks from January 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 cell-free DNA sequencing, to antiviral resistance monitoring in resource-limited settings, to real-time methylation analysis, these studies showcase the advances in scientific research made possible by Oxford Nanopore sequencing.

Featured in this edition:

1. Tracking antiretroviral resistance in low-resource settings

2. Characterising variants in cancer predisposition genes

3. Detecting tumour-specific variants in as little as 20 minutes

4. A new computational method for isoform-level transcript discovery

5. Live methylation calling during nanopore sequencing

6. Identifying new disease-associated DNA alterations in a large-scale study

Microbiology and infectious disease

1. High prevalence of reverse transcriptase inhibitors associated resistance mutations among people living with HIV on dolutegravir-based antiretroviral therapy in Francistown, Botswana (Journal of Antimicrobial Chemotherapy)

Since 2016, clinicians in Botswana have been prescribing dolutegravir as antiretroviral therapy (ART) for HIV. Before this project, the authors state, no study had characterised HIV-1 drug resistance mutations in people living with HIV on dolutegravir-based ART in the Botswana National ART program. Using Oxford Nanopore sequencing, Choga et al. achieved fast, accurate sequencing of HIV-1 genes, and uncovered key antiretroviral resistance mutations. Its cost effectiveness and rapid turnaround time made Oxford Nanopore sequencing a game changer for preventing treatment failure in resource-limited settings.

Key points:

• Choga et al. obtained plasma samples from 100 individuals with HIV

• HIV drug resistance mutations were detected in 32.8% of the sequenced samples, with prevalence not correlated to viral load

• Resistance to other antiretrovirals was prevalent, with a lower incidence of dolutegravir-associated resistance mutations (5%)

• Oxford Nanopore sequencing allowed for high-throughput, cost-effective genotyping and detection of resistance mutations, even in samples with a low viral load which proved challenging for other methods

Find out more about the GridION device used for this research.

Cancer research

2. Nanopore adaptive sampling accurately detects nucleotide variants and improves the characterisation of large-scale rearrangement for the diagnosis of cancer predisposition (Clinical and Translational Medicine)

Chevrier et al. used Oxford Nanopore adaptive sampling as an alternative method to multiplex ligation-dependent probe amplification (MLPA) for studying genes associated with cancer predisposition. They detected known and novel variants, even at low coverage, and characterised large-scale rearrangements, noting higher resolution with Oxford Nanopore than MLPA. Therefore, Oxford Nanopore sequencing could potentially be an accurate alternative method for early cancer detection in the future.

Key points:

• Adaptive sampling enriched 152 cancer predisposition genes in blood samples from individuals with hereditary solid tumour cancers

• Using Oxford Nanopore, Chevrier et al. detected known and novel variants, including a duplication of two exons and a deletion carrying over five different genes

• All large-scale rearrangements were detected in the 30 germline samples, matching MLPA results

• Oxford Nanopore sequencing characterised the exact start, stop, and size of large-scale rearrangements, offering better resolution than MLPA

• The authors identified novel intronic variants that could impact splicing

• Oxford Nanopore sequencing offered improved detection of structural variants (SVs) and single nucleotide variants (SNVs) even at low coverage

Graphical abstract. Chevrier et al. 2025. Image redistributed under Creative Commons License (Attribution 4.0 International license).

Watch lead author, Romain Boidot, discuss this research at London Calling 2023.

3. Nanopore-based consensus sequencing enables accurate multimodal tumour cell-free DNA profiling (Genome Research)

Chen et al. explored the potential of nanopore rolling circle amplification (RCA)-enhanced consensus sequencing (NanoRCS) for detecting cell-free tumour DNA. NanoRCS simultaneously identified SNVs, copy number alterations (CNAs), and fragmentomics patterns. With results in 20–110 minutes and lower error rates than short-read sequencing approaches, this non-invasive method could facilitate real-time cancer monitoring in the future.

Key points:

• NanoRCS reliably detected tumour fractions as low as 0.24%

• The authors reported low SNV error rates (0.00072 for NanoRCS consensus-called reads, 0.00674 for Oxford Nanopore non-consensus-called reads, and 0.00108 for short-read sequencing technology after error correction in overlapping regions of paired-end reads)

• NanoRCS provided real-time results, detecting tumour-specific SNVs within 20–110 minutes of sequencing

• The method demonstrated utility for oesophageal, ovarian, and granulosa cell cancers

• By leveraging Oxford Nanopore sequencing, NanoRCS offers a low-cost, portable alternative to other sequencing platforms, with faster turnaround and smaller batch compatibility

This schematic shows the process by which the authors prepared the circular cell-free DNA, aligned to the reference genome and performed consensus calling. NanoRCS then performed cell-free multimodal tumour detection through assessing SNVs, copy number profiling and fragmentomic analysis. For the full figure legend see figure 1 in Chen et al. 2025. Image redistributed under Creative Commons License (Attribution 4.0 International license).

Bioinformatics

4. Isoform-level discovery, quantification and fusion analysis from single-cell and spatial long-read RNA-seq data with Bambu-Clump (bioRxiv)

Sim et al. introduce Bambu-Clump — a new computational tool for transcript discovery and quantification in single-cell and spatial long-read RNA sequencing. Bambu-Clump can be applied to long- and short-read RNA datasets, and overcomes the challenge of low sequencing depth by leveraging information from both individual cells and cell clusters.

Key points:

• The authors generated single-cell and spatial RNA sequencing datasets for cancer cell lines and mouse brain tissue using Oxford Nanopore reads of unrestricted length and other long- and short-read sequencing technologies

• Oxford Nanopore sequencing had comparable overall performance for transcript discovery, but had a higher median read length and was faster and more cost-effective than an alternative long-read sequencing platform

• The full-length RNA sequencing capabilities of Oxford Nanopore Technologies were essential for resolving transcript isoforms and detecting fusion events

• This work could potentially benefit cancer research in the future as many cancers involve fusion genes, structural mutations, and abnormal RNA splicing

How to generate your own single-cell RNA sequencing datasets with Oxford Nanopore Technologies.

5. Realfreq: real-time base modification analysis for nanopore sequencing (bioRxiv)

Oxford Nanopore Technologies generates sequencing data in real time and annotates with base modifications within the same run. Samarasinghe et al. introduce realfreq — a new tool for instant methylation calling. Live epigenetic analysis could aid time-sensitive clinical diagnostics and forensic investigations in the future.

Key points:

• Realfreq processes raw nanopore signal data and provides immediate access to methylation patterns on DNA or RNA

• Realfreq kept up with the data output of a MinION Flow Cell on a laptop, and successfully maintained real-time processing over a 48-hour sequencing run on a PromethION Flow Cell using a desktop computer

• Base and modification calling took ~87.3% of processing time, alignment took ~8.7%, and data conversion took ~2.4%

• Realfreq is a free and open-source application on GitHub

Find out more about methylation analysis using Oxford Nanopore sequencing.

Human genetics

6. Imputation of structural variants using a multi-ancestry long-read sequencing panel enables identification of disease associations (medRxiv)

Using Oxford Nanopore sequencing, Noyvert et al. captured SVs in 888 samples from the 1000 Genomes Project, creating a reference panel that was then used for the first large-scale genome-wide SV-association study. The authors identified thousands of disease-related DNA alterations, and SV analysis highlighted potential causal genes for respiratory diseases that were missed in previous studies.

Key points:

• Previous genome-wide association studies (GWAS) focused on SNVs because SVs are often longer than the maximum read length of short-read sequencing methods

• By nanopore sequencing 888 samples from mixed ancestry backgrounds, the authors created an SV panel that was used to impute SVs in nearly 500,000 UK Biobank participants

• The authors identified 3,858 SV associations across 32 disease-relevant traits, of which 1,258 were novel

• SV analysis highlighted CFDP1, MEGF6, AAGAB, and FLI1 as potential causal genes for respiratory diseases that were missed by previous GWAS

• Studying SVs could improve genetic risk prediction and identification of potential drug targets in the future

Looking to investigate structural variants? Try our workflow.

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