Science unlocked: publication picks from November 2024

In this monthly series, we share a selection of recent publications in which nanopore sequencing was used to unlock novel insights. Spanning from human genetics and clinical research to infectious disease, agrigenomics, and conservation, these studies showcase the advances in scientific research made possible by nanopore sequencing. Read on to stay on top of what's next.

Techniques

1. Nanopore sequencing of intact aminoacylated tRNAs (bioRxiv)

Transfer RNA (tRNA) plays a crucial role in translation. During aminoacylation, a tRNA binds to an amino acid and delivers it to a growing polypeptide chain, but studying this process is challenging as the amino acid binding is unstable. Here White et al. present aa-tRNA-seq, a novel nanopore-based method that stabilises the amino acid and allows direct analysis of tRNA sequence, modifications, and aminoacylation status in a single experiment.

Key points:

• The researchers trained machine learning models to translate the ionic signals from nanopore sequencing into classification of tRNAs as aminoacylated or unbound with high accuracy.
• During nutrient deprivation and heat stress in yeast they identified dynamic changes in tRNA aminoacylation levels using the new PromethION 2 integrated instrument.
• Nanopore sequencing allowed the detection of specific tRNA misaminoacylation events, offering insights into tRNA synthetase fidelity.
• By revealing comprehensive details of tRNA biology, nanopore sequencing may inform research into translation regulation and cellular stress responses.

Discover our new PromethION 2 Integrated device

Microbiology and infectious disease

2. Genomic surveillance of multidrug-resistant organisms based on long-read sequencing (Genome Medicine)

Multidrug-resistant organisms (MDRO) are a major public health threat, requiring effective genomic surveillance to manage outbreaks and track resistance mechanisms. Short-read sequencing methods struggle to detect structural variation and plasmids, which is crucial for understanding MDRO evolution and transmission. Nanopore sequencing addressed these gaps by enabling complete genome assemblies and accurate resistance gene identification.

Key points:

• Through multi-locus sequence typing, nanopore sequencing enabled detailed outbreak analysis of methicillin-resistant Staphylococcus aureus, as the researchers could differentiate and cluster isolates — essential for tracing pathogen transmission and evolution.
• With the long-read capabilities of nanopore sequencing the authors identified microbial structural variants and plasmid assemblies.
• They discriminated multi-copy antimicrobial resistance genes in mobile genetic elements such as plasmids — regions that cannot be assembled with short-read sequencing.
• Nanopore sequencing reduced costs and preparation time, making MDRO surveillance feasible for resource-limited settings.

Watch Fabian Landman present this research at ESCMID 2024:

3. Diagnostic value of nanopore sequencing of cerebrospinal fluid samples in tuberculous meningitis (Diagnostic Microbiology and Infectious Disease)

Tuberculous meningitis (TBM) accounts for 1% of global tuberculosis cases and is considered the most severe, with an 80% mortality rate if not treated within five weeks. Identifying TBM requires detection of Mycobacterium tuberculosis in cerebrospinal fluid (CSF), but the bacterial load is low. Current tools are inadequate for the job as they have low sensitivity and can take weeks to yield results, which impedes disease monitoring and delays treatment. Here Zhao and colleagues pitted nanopore sequencing against the typical methods for detecting M. tuberculosis in CSF.

Key points:

• The authors obtained 48 CSF samples (30 TBM, 18 non-TBM) and compared acid-fast staining microscopy, M. tuberculosis solid culture, DNA detection, imaging and nanopore sequencing for detecting M. tuberculosis.
• Nanopore sequencing showed higher sensitivity (43.30%) and area under the curve (AUC) (0.661) than the other methods.
• When nanopore sequencing was combined with imaging, the researchers achieved 60% sensitivity and an AUC of 0.744.
• Although currently only approved for research use, the speed, sensitivity and accuracy of nanopore sequencing suggests it may prove a valuable tool for TBM identification in the future.

Human genetics

4. Long-read sequencing for detection and subtyping of Prader-Willi and Angelman syndromes (Journal of Medical Genetics)

This study investigates the utility of nanopore sequencing to identify and subtype cases of Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS), two imprinting disorders caused by genetic or epigenetic alterations in the 15q11.2-q13 chromosomal region. By leveraging the capability of nanopore technology to simultaneously detect DNA sequence and methylation changes, the researchers accurately identified various subtypes without requiring parental samples, offering a more cost-effective and streamlined approach than other current methods.

Key points:

• Methylation-sensitive PCR, chromosomal microarray analysis, or fluorescence in situ hybridisation are common methods for obtaining a PWS or AS diagnosis, but these methods require multiple steps or confirmation using parental samples.
• Individuals with a pre-existing PWS or AS diagnosis gave blood samples for this research to understand more about the mechanism of their individual condition without needing parental samples.
• Akbari et al. performed nanopore sequencing on DNA from individuals with a pre-existing diagnosis of PWS or AS.
• They detected the molecular subtype of 18 PWS and 6 AS individuals in a single experiment, and eliminated the need for parental samples by leveraging imprinted regions like IGF1R outside the 15q11.2-q13 region.
• All cases were accurately subtyped including uniparental heterodisomy, mixed iso-/heterodisomy, type 1 and 2 deletions, microdeletion and UBE3A indels.
• A PWS case was successfully subtyped as maternal isodisomy without a sample from the mother.
• Due to its ability to detect both the nucleotide sequence and epigenetic modifications, nanopore sequencing is particularly useful for analysing PWS and AS cases, and could be applied to other imprinting disorders in the future.

Check out our epigenetics workflow

5. Parallel in-depth analysis of repeat expansions: an updated Clin-CATS workflow for nanopore R10 flow cells (bioRxiv)

Scholz et al. introduce an updated Clinical Nanopore Cas9-Targeted Sequencing (Clin-CATS) workflow using the latest Oxford Nanopore R10 chemistry to analyse repeat expansions in hereditary ataxias and related neurological disorders. The nanopore-based Clin-CATS workflow allowed for the precise quantification of repeat length, sequence composition, and methylation status, overcoming the limitations of current ‘gold standard’ tools.

Key points:

• Identifying cases of hereditary ataxia requires detection of complex repeat expansions and methylation, which is difficult even with ‘gold standard’ PCR and Southern blot.
• Nanopore sequencing overcomes the limitations of other methods due to its long-read capabilities and simultaneous detection of DNA bases and epigenetic modifications.
• By expanding the Clin-CATS ataxia panel from 10 to 27 genes, and introducing new gene panels for myopathy, neurodegeneration, and motor neuron disease, the authors enabled the simultaneous investigation of multiple repeat expansion-related genes across these conditions.
• The updated workflow provided greater coverage than what is typically achieved with adaptive sampling, so it is advantageous for applications requiring higher sequencing depth and accuracy.

Explore the targeted nanopore sequencing approach

Biopharma

6. An optimised protocol for quality control of gene therapy vectors using nanopore direct RNA sequencing (Genome Research)

Many lentiviral vectors (LVs) contain an incomplete (and thus potentially nonfunctional) RNA genome, which can undermine gene delivery and increase manufacturing costs. Here Zeglinski and colleagues compared the ability of Oxford Nanopore and another long-read sequencing platform to undertake quality control of LVs used in gene therapy. Oxford Nanopore direct RNA sequencing came out on top for the identification of incomplete transcripts caused by cryptic splicing, polyadenylation sites, and structural truncations.

Key points:

• Oxford Nanopore direct RNA sequencing directly sequences native RNA, identifying truncation sites and splicing patterns without the biases introduced by reverse transcription.
• To identify all sources of truncation in a vector, including hairpin-associated truncations that are not naturally polyadenylated, the researchers performed two nanopore direct RNA sequencing runs: one with and one without artificial polyadenylation.
• Truncations associated with RNA hairpins were identified as significant contributors to incomplete transcripts.

Watch Kathleen Zeglinski’s talk at London Calling 2024:

Zeglinski et al. 2024

Inspired? Apply nanopore sequencing to your own research questions and you'll never see sequencing the same way again.

Explore the nanopore sequencing solution

Oxford Nanopore Technologies products are not intended for use for heath assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.