Science unlocked: publication picks from December 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 to biopharma and cancer research, these studies showcase the advances in scientific research made possible by nanopore sequencing. Read on to stay on top of what's next.

1. Optimising oncolytic virus engineering with nanopore sequencing.

2. Capturing complex HPV integration sites for insights into cervical cancer.

3. Classifying cancer using direct RNA sequencing.

4. Detecting methylation signatures to predict disease severity.

5. Large-scale study for novel insights into neurodevelopment and disease.

6. Accurate and cost-effective detection of pathogenic variants.

7. Ultra-long reads bridge the gaps in genome assembly.

Biopharma

1. Antibiotic-mediated selection of randomly mutagenised and cytokine-expressing oncolytic viruses (Nature Biomedical Engineering)

Oncolytic viruses (OVs) are engineered to selectively infect and lyse cancer cells while stimulating anti-tumour immune responses. Rezaei et al. introduce a strategy to optimise OVs by coupling antibiotic selection with mutagenesis. Insertion sites were mapped using nanopore sequencing, facilitating the creation and characterisation of stable recombinant viruses for therapeutic applications.

Key points:

• The long-read capabilities of nanopore sequencing allowed rapid analysis of mutagenised herpes simplex virus type 1 (HSV-1) and vaccinia virus (VV) genomes, producing comprehensive maps of genetic modifications.
• 988 unique transposon insertion sites in the VV genome and high-density insertion regions in the HSV-1 genome were identified.
• Nanopore technology enabled the discovery of stable intergenic regions that are ideal for transgene insertion without compromising virus replication.
• The authors reduced the time required for virus library creation from months to days by developing this streamlined method for engineering and selecting transgene-expressing recombinant viruses.
• Optimising these viruses could lead to safer and more effective cancer treatments, addressing unmet needs in oncology.

Find out more about how nanopore sequencing can provide solutions for the biopharma sector.

Cancer

2. Rearrangements of viral and human genomes at human papillomavirus integration events and their allele-specific impacts on cancer genome regulation (Genome Research)

During human papillomavirus (HPV)-driven cervical cancer, the viral DNA is integrated into the host cell genome, leading to genetic and epigenetic changes. Here Porter et al. used nanopore sequencing to study these integration sites to advance insights into cervical cancer development. This could aid diagnostics and pave the way for targeted therapies in the future.

Key points:

• The authors obtained 72 cervical cancer tumour samples previously characterised using short-read sequencing.

• In every sample, a higher number of HPV-human integration breakpoints were detected with nanopore sequencing over short-read technology.

• Complex multi-breakpoint events were the most prevalent, comprising 32% of integration events.

• Epigenetic modifications were also identified with nanopore sequencing, with consistent methylation patterns found associated with the transcription of integrants.

• Oxford Nanopore Technologies was superior to short-read sequencing for capturing the complexity of human-HPV structures.

Hear Vanessa talk about her work during a knowledge exchange (2023):

3. Epitranscriptomic rRNA fingerprinting reveals tissue-of-origin and tumour-specific signatures (Molecular Cell)

The authors enlisted Oxford Nanopore Technologies to successfully sequence native ribosomal RNA (rRNA), simultaneously revealing epigenetic modifications and isoform information. ‘Epitranscriptomic rRNA fingerprinting’ could revolutionise cancer care by using minimal samples to enable early and more precise cancer detection in the future.

Key points:

• Dynamic modifications of ribosomal (r)RNA across tissues, developmental stages, and cancer states, have the potential to act as ‘epitranscriptomic rRNA fingerprints’.

• rRNA was analysed from mouse tissues (brain, heart, liver, testis) across developmental stages, and human matched tumour-normal samples (lung, colon, testis, liver).

• This data was used to identify unannotated modification sites critical for understanding tissue-specific and tumour-related signatures.

• rRNA modifications were found to be distinct across tissues, developmental stages, and cancer states, allowing tissue-of-origin identification and tumour-normal differentiation using as few as 250 reads.

Learn more about RNA and cDNA sequencing with Oxford Nanopore Technologies.

4. Prognostic importance of direct assignment of parent-of-origin via long-read genome and epigenome sequencing in retinoblastoma (JCI Insight)

Paternal inheritance of a faulty RB1 allele leads to worse clinical outcomes for patients with retinoblastoma. Parent-of-origin of the allele can be identified via a differentially methylated region, but this is not routinely assessed during diagnosis. Here Stacey et al. used targeted nanopore sequencing to simultaneously detect genetic and epigenetic information in the RB1 allele. These genetic insights could guide personalised therapeutic decisions, leading to better patient outcomes in the future.

Key points:

• Intron 2 of RB1 is methylated on the maternal allele and unmethylated on the paternal allele.

• Nanopore sequencing enabled simultaneous phasing of genetic variants and detection of methylation, thus identifying parent-of-origin from sequencing the proband alone.

• Parent-of-origin could be identified in familial and de novo cases of retinoblastoma, including those with complex somatic mosaicism and structural variants.

• Adaptive sampling allowed targeting of RB1 gene and reduced sequencing time, offering results within 18 hours of blood sample receipt.

• Nanopore sequencing offers the potential for faster, accurate, and more comprehensive retinoblastoma classification in the future.

Hear more from Debarshi Mustafi from his talk and NCM Houston 2023:

Human Genetics

5. Long-read sequencing of hundreds of diverse brains provides insight into the impact of structural variation on gene expression and DNA methylation (bioRxiv)

Billingsley et al. utilised nanopore sequencing to create a detailed catalogue of structural variants (SVs) and methylation profiles from 351 brain samples, uncovering novel insights into gene regulation and neurodevelopment. Nanopore technology resolved complex genomic regions beyond the reach of short-read methods. This work advances understanding of the brain’s genetic architecture, paving the way for personalised medicine and neurological research.

Key points:

• SVs impact gene expression and play a crucial role in neurological and neurodegenerative diseases, but many can not be detected with short-read sequencing.

• The authors identified 234,905 SVs in 351 brain samples, including novel population-specific variants.

• Using quantitative trait locus (QTL) analyses they were able to identify SVs linked to gene expression and methylation.

• Nanopore sequencing uncovered population-specific genetic and epigenetic diversity influencing neurological traits (within European and African ancestry populations).

• Nanopore enabled the detection of both SVs and epigenetic patterns missed by short-read sequencing, providing a more comprehensive understanding of genetic contributions to brain function.

Hear more about Kimberley’s work at one of our showcase sessions at London Calling 2024:

6. Genewise detection of variants in MEFV gene using nanopore sequencing (Frontiers in Genetics)

Here Ghukasyan et al. developed a cost-effective nanopore sequencing protocol for analysing the gene associated with Familial Mediterranean Fever (FMF), MEFV. Nanopore sequencing accurately identified common variants and those outside the scope of PCR mutation panels, including variants in intronic and non-coding regions. This approach offers a scalable alternative to qPCR, and could improve accessibility to comprehensive FMF genetic testing in resource-limited settings in the future.

Key points:

• ~60 known/suspected pathogenic variants in the MEFV gene cause FMF, a genetic autoinflammatory disorder.

• Existing diagnostic methods only target a subset of pathogenic variants, or they have limited application due to their high costs.

• Oxford Nanopore multiplex amplicon sequencing enabled full-length MEFV sequencing with high accuracy.

• MEFV mutations were detected with near-complete concordance with qPCR, but nanopore sequencing also allowed the identification of intronic and regulatory variants, surpassing the scope of qPCR.

• '[Nanopore sequencing] is cost-effective, with costs comparable to those of the qPCR test, making it particularly suitable for settings with limited laboratory infrastructure.'

Discover the range of clinical research being undertaken with nanopore sequencing.

Bioinformatics

7. Verkko2: integrating proximity ligation data with long-read De Bruijn graphs for efficient telomere-to-telomere genome assembly, phasing, and scaffolding (bioRxiv)

Verkko2, a tool designed to assemble telomere-to-telomere (T2T) genomes, offers significant improvements in accuracy and runtime compared to the original Verkko tool. Antipov et al. used Verkko2 with ultra-long Oxford Nanopore reads to resolve challenging genomic regions and achieve high-resolution T2T genome assemblies. Verkko2 will help deliver high-quality assemblies, paving the way for advancements in pangenomics and personalised genomics in the future.

Key points:

• Verkko2 achieved a twofold increase in T2T scaffolds compared to its predecessor, resolving an average of 39 out of 46 chromosomes per human genome in diploid assemblies.

• Ultra-long reads closed gaps in challenging repetitive and structurally complex genomic regions such as ribosomal DNA arrays and acrocentric chromosome arms.

• Verkko2 worked seamlessly with Hi-C and Pore-C data for phasing and scaffolding.

• These advancements pave the way for large-scale pangenome studies, deeper exploration of genomic diversity, and new possibilities in personalised and comparative genomics.

Check out our telomere-to-telomere sequencing workflow.

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