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.

Human Genomics

1.Human telomere length is chromosome end–specific and conserved across individuals (Science)

Oxford Nanopore Technologies has played a pivotal role in a groundbreaking study led by Nobel laureate Professor Carol Greider, which offers new insights into measuring human telomere length and its implications for age-related diseases and cancer predisposition. This research utilises nanopore sequencing to explore the critical role of telomeres in human health, providing a foundation for future preventative health strategies and drug discovery.

Key points:

• The study introduces a novel method using MinION to examine telomere length, revealing significant variations in telomere length among individuals and even within the same chromosomes of individuals.
• Findings suggest that specific telomeres may trigger stem-cell failure, highlighting new potential targets for clinical research and drug development.
• Nanopore sequencing's ability to produce precise native DNA reads enabled detailed analysis of telomere length regulation.
• Identified telomeric regions and binding proteins that may serve as new targets for drugs aimed at preventing degenerative diseases.

If you would be interested in hearing more, Carol Greider will be giving a keynote at our annual customer event, London Calling. To find out more and register, click here: https://nanoporetech.com/about/events/conferences/lc24/agenda

Microbiology

2.Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As (Nature Microbiology)

Researchers, led by Alice Baek, have unveiled new insights into the role of specific RNA modifications, known as N6-methyladenosine (m6A), in the replication of human immunodeficiency virus (HIV-1). By employing direct RNA sequencing with MinION, the team has discovered three predominant m6A modifications at the 3′ end of the HIV-1 RNA genome. These modifications play a critical role in maintaining normal levels of HIV-1 RNA splicing and translation.

Key Points:

• Nanopore direct RNA sequencing (DRS) allowed for the analysis of HIV-1 RNAs at a single-molecule level, providing unprecedented detail about RNA modifications.
• The study identified three significant m6A modifications near the 3′ end of the HIV-1 RNA, that were found to be crucial for the normal functioning of the virus.
• These m6A sites are more densely installed in spliced viral messenger RNAs than in genomic RNAs, suggesting a sophisticated mechanism that enhances the virus's ability to replicate effectively.
• Understanding the specific roles of these m6A modifications opens new avenues for the development of therapeutic strategies aimed at interfering with these critical viral processes.

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Agrigenomics

3.Evaluation of NGS DNA barcoding for biosecurity diagnostic applications: case study from banana freckle incursion in Australia (Australasian Plant Pathology)

In a crucial study of rapid diagnostic solutions for biosecurity threats, researchers have addressed a significant banana freckle outbreak in Australia, caused by the fungal pathogen Phyllosticta cavendishii (P. cavendishii). The study compares traditional Sanger sequencing with nanopore sequencing on the MinION, assessing their ability to quickly detect this harmful pathogen. With MinION, the researchers not only effectively identified P. cavendishii but attained faster turnaround times with the added benefit of portability. This advancement establishes MinION sequencing as a potentially transformative tool for immediate, on-site biosecurity responses, crucial for combating agricultural and ecological threat.

Key points:

• A biosecurity incursion occurs when a harmful biological agent such as a pathogen or pest enters a new area, posing threats to agriculture, ecosystems, or public health. Rapid and accurate identification of these threats is crucial.
• Traditional sequencing methods like Sanger sequencing can take 4–5 days to identify species, which is impractical for rapid biosecurity responses.
• Researchers utilised 95 banana samples infected with this fungal pathogen P. cavendishii for their analysis.
• The internal transcribed spacer (ITS) gene barcode was used for species identification on both Sanger and Oxford Nanopore MinION platforms.
• MinION sequencing was performed on now legacy Flow Cells (R9.4.1) (newer Flow Cells have significant improvements in accuracies)
• MinION sequencing provided quicker results and is portable, making it ideal for use directly at the point of incursion.

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Clinical research

4.Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing (Journal of Assisted Reproduction and Genetics)

Here Madritsch et al., showcase the application of nanopore sequencing for rapid genetic screening of polar bodies, highlighting its future potential to reduce miscarriages and implantation failures in IVF treatments.

Key points:

• Nanopore sequencing demonstrated high concordance (96%) in ploidy classification when compared with traditional array-based comparative genomic hybridisation (aCGH), ensuring reliable results.
• “The ONT workflow can sequence and analyse 300 k reads in approximately 12 h, which is approximately half the time of a standard aCGH workflow” allowing for the prompt detection of genetic abnormalities that could affect pregnancy outcomes.
• Compared to other genetic screening methods, nanopore sequencing offers a more affordable solution, reducing the financial burden on prospective parents.
• Nanopore sequencing was advantageous as it could detect ploidy classification per chromosome, whereas aCGH does not consider the presence of three chromatids in the pooled polar bodies.

5.Concordance of whole-genome long-read sequencing with standard clinical testing for Prader-Willi and Angelman syndromes (medRxiv​)

Here, the authors highlight recent advancements in clinical genetics showcasing the future potential of whole-genome sequencing (WGS) using the PromethION platform to detect imprinting disorders like Prader-Willi and Angelman syndrome. This study underscores the precise and effective use of our technology in identifying a range of genetic and epigenetic variation across 25 individuals, achieving an impressive accuracy.

Key points:

• The research focused on identifying copy number variants (CNVs), single nucleotide variants (SNVs), structural variants (SVs), and methylation differences specifically in Prader-Willi or Angelman syndrome cases.
• Utilised the PromethION platform to perform deep sequencing with an average depth of 36x.
• Developed a custom report for assessing critical variants in the genetic region 15q11.2-q13.1.
• The custom report (see figure) included detailed analyses of CNVs, SVs, and methylation patterns at specific CpG sites.
• Three evaluators, not specialised in nanopore data but experienced with sequencing technologies, achieved 100% concordance in 22 blinded cases with the user-friendly report, highlighting its reliability and future potential to simplify complex genetic diagnostics.

Figure 1. This figure presents a report used to assess imprinting disorders in the 15q11-q13.3 region, using a case of Angelman Syndrome caused by a BP2–BP3 deletion. Panel A shows the depth of coverage in this region, showing misalignments and a deletion between breakpoint 2 and 3 (BP2 and BP3), with genes highlighted in different colours. Panel B illustrates the SNP frequency, confirming reduced heterozygosity in the deleted, now hemizygous, area. Panel C compares methylation levels at specific CpG sites between the case (red) and controls (grey), notably showing methylation loss at the SNURF-SNRPN CpG island, important for clinical evaluations.

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References

• Karimian, K., Groot, A., Huso, V. et al. Human telomere length is chromosome end–specific and conserved across individuals. Science (2024). https://doi.org/10.1126/science.ado0431
• Baek, A., Lee, G.-E., Golconda, S. et al. Single-molecule epitranscriptomic analysis of full-length HIV-1 RNAs reveals functional roles of site-specific m6As. Nature Microbiology (2024). https://doi.org/10.1038/s41564-024-01638-5
• Galaihalage, K., Patel, S., Yadav, S. Evaluation of NGS DNA barcoding for biosecurity diagnostic applications: case study from banana freckle incursion in Australia. Australasian Plant Pathology (2024). https://doi.org/10.1007/s13313-024-00978-4
• Madritsch, S., Arnold, V., Haider, M. et al. Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing. Journal of Assisted Reproduction and Genetics (2024). https://doi.org/10.1007/s10815-024-03108-7
• Paschal, C.R., Zalusky, M.P.G., Beck, A.E. et al. Concordance of whole-genome long-read sequencing with standard clinical testing for Prader-Willi and Angelman syndromes. medRxiv (2024). https://doi.org/10.1101/2024.04.02.24305233