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 genetics

1. Advancing long-read nanopore genome assembly and accurate variant calling for rare disease detection (medRxiv)

Short-read whole-genome sequencing is unable to solve 50% of suspected rare monogenic disease cases. Here, the authors performed nanopore sequencing on 98 research samples, confirming all known variants previously detected with short-read sequencing, whilst also detecting rare, functionally annotated structural variants and tandem repeats. This demonstrates the potential for the detection of clinically relevant novel variants with comprehensive nanopore sequencing.

Key points:

• The pipeline developed in this study was time- and cost-efficient, with the capacity to generate assemblies and variant, phasing, and methylation calls in a single run.
• Nanopore sequencing data covered coding exons in ~280 genes and 5 known mendelian disease genes missed by short-read methods.
• With nanopore data, 87% of protein-coding genes were completely phased.
• Out of 42 samples, causative variants were identified in 11 samples, and potential variant candidates that require further investigation were identified in 4 samples.

2. Genetic regulation of nascent RNA maturation revealed by direct RNA nanopore sequencing (bioRxiv)

The link between genetic variants and messenger RNA (mRNA) processing is not fully understood, as studies are generally performed on mature mRNA. Here the authors used nanopore sequencing of cytoplasmic and chromatin-associated RNA to identify genetic variants that directly modulate premature messenger RNA (pre-mRNA) processing.

Key points:

• To the authors knowledge, this is the first long-read subcellular RNA sequencing dataset across multiple individuals.
• Nanopore sequencing was selected to allow the detection of several introns simultaneously (to investigate splicing order), to cover highly polymorphic genes (such as HLA genes), and to enable allele-specific transcriptome analysis.
• 472 unique single nucleotide polymorphisms (SNPs) were significantly associated with allele-specific splicing order in 17 genes, including seven HLA genes.
• 37 genes had statistically significant differences in poly(A) tail length between alleles, many of which also had a skewed allelic abundance ratio.
• The identification of variants that impact this new layer of gene regulation may help to unravel the mechanisms linking variants to human traits and disease.

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3. Genomic analysis of adult thrombotic microangiopathies in less than three days: from rapid to fast genomics to treatment (Blood)

Hybrid gene formation in the CFHR genes is a rare pathological driver of thrombotic microangiopathy (TMA) that is not well-detected with current whole-exome sequencing (WES) methods. Here the authors used nanopore sequencing to explore its capacity to rapidly detect these genetic events that could be informative for patient management practice.

Key points:

• 21 patients were sequenced using targeted nanopore sequencing (adaptive sampling), alongside the standard WES approach.
• Nanopore sequencing cost between €1054.25–195.75 per sample in comparison to €2205 for WES.
• The workflow ranged from 60–105 hours with nanopore sequencing, which is a substantial reduction from the three-week timeframe that is standard for WES.
• This proof-of-concept study showed that nanopore sequencing is a reliable and cost-effective method for the analysis of variants undetectable with traditional methods, in a significantly improved timeframe.

Watch Laurent Mesnard’s talk from London Calling 2024

4. Long-read transcriptomic identification of synaptic adaptation to amyloid pathology in the App^NL-G-F knock-in mouse model of the earliest phase of Alzheimer's disease (bioRxiv)

Characterising isoform-level alterations has significant implications for the early identification and stratification of individuals with Alzheimer’s disease (AD). Previous studies have identified a network of AD risk genes, however short-read sequencing has been limited in its capacity to fully characterise isoforms and splicing patterns. Here, the authors used nanopore sequencing to confirm the significance of the risk genes previously identified, as well as identify new genes linked to AD.

Key points:

• Analysis was applied to App^NL-G-F knock-in mice to understand transcriptional changes in response to amyloid-β, the peptide that forms plaques in the brain that impairs memory and cognition.
• The authors identified significant disparities in fold-expression ranking and significance when they compared gene-level versus transcript-level analysis.
• Transcript isoform changes were seen in genes not previously linked to AD, including: Capg, Trem2, Ocln, Ctsd, Ctsa, Cd68, Gusb, Csf2ra and Ctsb.
• Tracking transcript expression over time for the genes linked to AD may provide insights into disease stage, progression, or response to intervention.
• Specific transcript isoforms may also be used to inform new therapeutic interventions.

Cancer research

5. Transcriptome profiling of paediatric extracranial solid tumours and lymphomas enables rapid low‑cost diagnostic classification (Scientific Reports)

Approximately 80% of paediatric tumours occur in low- and middle-income countries, where is it is difficult to implement the multiple, resource-intensive tests required for an accurate tumour classification. Here the authors tested the feasibility of using a MinION to classify formalin-fixed paraffin embedded (FFPE) tumour samples based on cDNA analysis. Their protocol allowed the authors to accurately determine diverse tumour subtypes in a cost-effective manner.

Key points:

• Most solid tumours are stored as FFPE specimens, however this causes the nucleic acids to crosslink and become fragmented to very short read lengths.
• Nanopore sequencing was utilised in this study as it has the capacity to reliably sequence fragments as short as 20 base pairs.
• With the nanopore data, they correctly classified 95.6% paediatric solid tumours (131/137) and 89.7% (104/116) of lymphoma specimens.
• The predictions were not impacted by tumour purity or chemotherapy treatment status.
• Capital costs were <$5000 USD, whilst cost per specimen was <$100 USD (including reagents, with 12 samples run per flow cell).

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Microbiology and infectious diseases

6. Rapid whole genome characterisation of high-risk pathogens using long-read sequencing to identify potential healthcare transmission (medRxiv)

To prevent infectious disease outbreaks, hospitals need to be able to identify and track pathogen transmission. Over a six-month period, the authors prospectively sequenced 242 bacterial isolates from 216 patients, using nanopore technology. Seventy percent of the isolates were linked to transmissions within the hospital, demonstrating the potential for this nanopore workflow to support infectious disease control measures by offering rapid, accurate results on-site.

Key points:

• Whole-genome sequencing can help identify pathogen transmission in healthcare settings, but short-read sequencing approaches are both labour- and time-intensive.
• The authors developed a pipeline utilising nanopore sequencing for whole-genome assembly, that had an average turnaround time of two-days.
• A Q score of 60 was achieved for assembled genomes, even with coverage at 40X.
• As this data could be generated using a single platform, the approach is cost-effective and more accessible to a broad variety of biomedical facilities.

Watch senior author, Samuel Shelburne, present the team’s latest research at the Nanopore Community Meeting: Boston 2024 – register here.

Animal research

7. Complete sequencing of ape genomes (bioRxiv)

Previous attempts to sequence ape genomes lacked resolution of the most dynamic regions, including lineage-specific gene families. Complete assemblies were also not achieved due to the challenging repetitive nature of ape genomes. Here the authors used a combination of techniques to assemble complete ape genomes and provide a more nuanced understanding of our human ancestral genes and how we evolved.

Key points:

• The team successfully produced haplotype-resolved reference genomes for six ape species: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan, and siamang.
• 215 gapless chromosomes were assembled, telomere-to-telomere.
• The assemblies had unparalleled accuracy (less than 1 error per 500,000 base pairs).
• Ultra-long (>100 kbp) nanopore reads were crucial for repeat resolution, local phasing, and scaffolding.
• Nanopore sequencing was the primary method for resolving larger repetitive regions such as centromeres and segmental duplications.
• The assemblies provided in-depth evolutionary insights by resolving the major histocompatibility complex and immunoglobulin loci.
• Preliminary analysis revealed many new candidate genes to account for phenotypic differences among the apes.
• These assemblies can be used as a baseline for future human and primate evolutionary studies.

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