Science unlocked: publication picks from May 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.

Bioinformatics

1. Telomere-to-telomere phased genome assembly using error-corrected Simplex nanopore reads (bioRxiv)

Researchers have demonstrated a novel approach to achieving telomere-to-telomere (T2T) phased assemblies using error-corrected Simplex nanopore reads, offering a cost-effective alternative to using multiple sequencing technologies.

Key points:

• HERRO deep learning model enhances Simplex nanopore read accuracy by up to 100-fold.
• HERRO achieves high contiguity in human genomes, reconstructing many chromosomes telomere-to-telomere, with Oxford Nanopore only.
• The model can be applied to genome assembly for various species.
• The ability to generate T2T phased genomes with Oxford Nanopore only significantly reduces the cost compared to traditional methods of genome assembly and the required genomic DNA for sequencing.
• Works with both R9.4.1 and R10.4.1 nanopore Simplex reads.

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Microbiology

2. Direct detection of 8-oxo-dG using nanopore sequencing (bioRxiv)

Genetic oxidative damage predominantly causes modification of guanine into 8-hydroxyguanine (8-oxo-dG), which has the potential to be a disease biomarker. Currently there are no methods to confidently map this modification. Here the authors trained a deep learning model to detect 8-oxo-dG from raw nanopore sequencing signals. This method allows the simultaneous measure of 5-mC and 8-oxo-dG, providing a new way to explore epigenetics.

Key Points:

• Utilised synthetic oligos to create a diverse ground truth dataset, with modifications in a range of genomic contexts - for effective training of the deep learning model.
• Demonstrated high specificity and accuracy in detecting 8-oxo-dG amidst canonical bases.
• Enabled the simultaneous assessment of 8-oxo-dG and 5-mC, revealing insights into their interaction and distribution across the genome. -Highlighted the model's potential to map other DNA modifications, advancing the field of epigenetics and biomarker discovery.

Figure 1. This figure demonstrates methylation levels of 8-oxo-dG (red) and Guanine (black) on the same DNA molecule. Reads are centred around the position of 8-oxo-dG or Guanine (position 0). The data illustrates a significant decrease in methylation levels around 8-oxo-dG compared to Guanine, highlighting the impact of 8-oxo-dG on DNA methylation.

Animal Research

3. De novo genome assembly for an endangered lemur using portable nanopore sequencing in rural Madagascar (bioRxiv)

This study highlights the successful creation of a high-quality reference genome for the endangered red-fronted brown lemur (Eulemur rufifrons) using portable nanopore sequencing at the Centre ValBio Research Station in Madagascar.

Key points:

• Conducted on-site sequencing in Madagascar, addressing disparities in sequencing capabilities and research inequalities.
• Generated over 750 Gb of sequencing data from 10 MinION flow cells, assembling a 2.21 gigabase nuclear genome and a 17,108 bp mitogenome.
• Achieved a 95.47% BUSCO completeness score for primate-specific genes.
• This genome will aid conservation efforts and demonstrates the potential to build local genomic capacity and reduce research inequalities.

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Clinical research

4. A new method using rapid nanopore metagenomic cell-free DNA sequencing to diagnose bloodstream infections (medRxiv)

This prospective observational study used metagenomic nanopore sequencing (mNGS) to identify microbial DNA in blood plasma, showcasing its future potential for rapid diagnosis of bloodstream infections (BSIs) in emergency ward patients.

Key points:

• Confirmed pathogens in positive blood cultures and detected additional pathogens in initially negative blood cultures.
• Approximately 59% of mNGS findings could have affected treatment decisions, shifting from ineffective to effective therapies.
• mNGS offers a faster (6-hour turnaround) and more sensitive diagnosis of BSIs than typical methods, potentially improving outcomes in cases of severe infections or sepsis.

Figure 2. (a) Flowchart illustrating the sample handling workflow for blood cultures and metagenomic next-generation sequencing (mNGS) samples. This includes plasma DNA extraction, nanopore sequencing, and mNGS pathogen reporting. (b) Timeline comparison for bloodstream infection (BSI) diagnostics, highlighting the significantly faster turnaround for nanopore mNGS (approximately 6 hours) compared to Illumina metagenomic sequencing (about 24 hours) and traditional blood culturing (over 48 hours). This demonstrates the efficiency and speed of nanopore sequencing in diagnosing BSIs.

5. NanoImprint: A DNA methylation tool for clinical interpretation and diagnosis of common imprinting disorders using nanopore long-read sequencing (Annals of Human Genetics​)

This study introduces the NanoImprint tool, which shows future potential to enhance the diagnosis of imprinting disorders through detailed methylation analysis using nanopore sequencing.

Key points:

• Developed for analysing methylation at 14 key imprinted regions, providing detailed and clinically relevant data.
• Outputs are easily interpretable reports and visualisation plots, which could aid in diagnosing disorders like Beckwith-Wiedemann, Angelman, and Prader-Willi syndromes.
• Demonstrated utility with phased and non-phased data: for example, in Beckwith-Wiedemann Syndrome (BWS), NanoImprint identified mosaic hypomethylation at the KCNQ1OT1 region, showing approximately 30% methylation. Phased data revealed one allele as partially methylated and the other as unmethylated, clearly indicating mosaicism.
• NanoImprint fills a critical need by providing detailed, interpretable methylation data, enhancing the diagnosis and understanding of imprinting disorders in clinical settings. For example, in Beckwith-Wiedemann Syndrome (BWS), it accurately identifies mosaic hypomethylation patterns, facilitating precise clinical diagnosis.

Human genetics

6. An atlas of expressed transcripts in the prenatal and postnatal human cortex (bioRxiv)

Here, Bamford et al. used Oxford Nanopore-based transcriptome sequencing of human cortex tissue, revealing thousands of novel transcripts and significant differences in transcript diversity between prenatal and postnatal stages. Many of these transcripts had high coding potential, with some linked to neurodevelopmental disorders. This study underscores the role of alternative splicing in gene expression and provides a valuable resource for understanding brain development and disease.

Key points:

• Oxford Nanopore-based transcriptome sequencing identified 1,895,360 unique transcripts in the human cortex, with 57.6% being novel.
• Significant differences in transcript diversity between prenatal and postnatal cortex stages were observed, highlighting the role of alternative splicing in development.
• Many novel transcripts have high coding potential, validated by proteomic data showing translation into peptides.
• Novel transcripts overlap with de novo mutations in genes associated with neurodevelopmental disorders, suggesting pathogenic potential.
• Alternative splicing significantly contributes to the complexity of gene expression in the human cortex, with implications for understanding brain function and disease.
• Novel coding sequences are highly conserved, indicating their importance in genetic studies and potential diagnostic applications for neurodevelopmental conditions.