Science unlocked: publication picks from Feb 2024

Clinical microbiology

Nanopore sequencing of infectious fluid is a promising supplement for gold-standard culture in real-world clinical scenario (Frontiers in Cellular and Infection Microbiology)

Zhao et al. investigated the potential of metagenomic nanopore sequencing in diagnosing infectious diseases by analysing a variety of fluid samples suspected to be infectious, obtained from 10 Chinese clinical centres.

Key points:

• The team investigated a variety of infectious fluid samples, including cerebrospinal fluid, urine, bronchoalveolar lavage fluid (BALF), plasma, abscesses, and more, providing evidence beyond respiratory or BALF samples in real-world settings.
• Nanopore sequencing demonstrated nearly 100% positive predictive agreement.
• There was consistent performance in identifying antimicrobial resistance genes and in drug susceptibility testing.
• Rapid nanopore sequencing 'is a promising clinical tool to supplement gold-standard culture, and it has the potential improve patient prognosis and facilitate clinical treatment of infectious diseases'.

Cancer research

Assessing the efficacy of target adaptive sampling long-read sequencing through hereditary cancer patient genomes (NPJ Genomic Medicine)

The study embarked on a journey to uncover causative variants of hereditary cancers that elude existing clinical tests. By applying targeted adaptive sampling to 33 genomes, the researchers aimed to understand the intricate architecture of structural variants which are partially detected or missed by conventional methods.

Key points:

• The researchers used BED files for on-device targeted sequencing (adaptive sampling), which encompassed 147 genes associated with cancer predisposition, extending 10 kb margins around each gene.
• Showcasing high-fidelity identification of single nucleotide variants (SNVs), the data rivalled the accuracy of short-read sequencing platforms, and brought to light complex SVs, including novel SINE-R/VNTR/Alu (SVA) elements affecting the APC gene related to familial adenomatous polyposis.
• Use of off-target reads from the sequencing runs facilitated accurate genotyping of common SNVs across the entire genome, permitting the calculation of polygenic risk scores (PRS) to enhance genetic analysis — without incurring extra sequencing costs.
• Examination of methylation patterns in conjunction with phased reads led to the successful identification of allele-specific MLH1 promoter hypermethylation in a patient with Lynch syndrome.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes (medRxiv)

Here researchers from BC Cancer, Vancouver, who lead the Personalised Oncogenomics (POG) program, leveraged long nanopore reads to dissect the complex genetic landscape of solid tumours. This pivotal study showcased what had been missing with short-read sequencing technology, by using nanopore sequencing to clarify intricate SVs, somatic variants, viral integrations, methylation patterns, and allelic expression, paving the way for more personalised, precise, and effective cancer treatments, demonstrating the impact of Oxford Nanopore technology on the future of oncology.

Key points:

• The study showcased a cohort of 189 tumours and 41 matched normal samples sequenced on a PromethION 24.
• The team demonstrated the superiority of long nanopore reads compared to short-read data in revealing complex somatic structural variants, viral integrations — despite significantly lower sequencing depth than short reads.
• Variant phasing uncovered allele-specific differential methylation and expression in key cancer genes (see Figure 1).
• With the nanopore data, they could directly analyse promoter methylation in genes like MLH1 in Lynch syndrome, suggesting new avenues for precision oncology.
• BRCA1 and RAD51C promoter methylation were revealed as potential drivers in homologous recombination deficiency, even in cancers without known mutations.

Figure 1. Successful phasing of somatic variants in the PTEN gene using long nanopore sequencing reads (top), whilst many reads were unassigned to haplotypes (gray bars) in short-read data (bottom).

Watch author Kieran O'Neill's talk at London Calling 2023

Single-cell transcriptomics

Integrating short-read and long-read single-cell RNA sequencing for comprehensive transcriptome profiling in mouse retina (bioRxiv)

Here Wang et al. based at the Baylor College of Medicine in Houston, United States, presented a comprehensive comparison between short-read technologies and Oxford Nanopore for single-cell transcriptome profiling of ~30,000 mouse retinal cells. The research revealed thousands of transcript isoforms, with a significant portion previously uncharacterised by short-read sequencing, underscoring the unparalleled depth and precision of nanopore technology in capturing the transcriptome's complexity and reveal more biology.

Key Points:

• The team conducted a comprehensive assessment of single-cell RNA sequencing technologies, analysing over 30,000 cells from mouse retinas, using 1.4 billion nanopore reads.
• Around 44,325 transcript isoforms were uncovered, with 38% newly characterised and 17% uniquely expressed in distinct cellular subclasses, enhancing the known transcriptome repertoire.
• Nanopore sequencing matched short-read sequencing in gene expression and cell-type annotation/assignments, with added superiority in precise isoform identification.
• Cell-type-specific variation in isoform abundance could also be identified with nanopore data, highlighting the nuanced regulatory landscape of gene expression across different cell populations.

Human rare disease research

A dual-mode targeted Nanopore sequencing assay for comprehensive SMN1 and SMN2 variant analysis (medRxiv)

Here Hall et al. developed a PCR/nanopore sequencing assay that significantly enhanced the detection of genetic variants in the SMN1 and SMN2 genes — key genes in the diagnosis of spinal muscular atrophy (SMA). This cost-effective and scalable assay was validated with over 750 samples, exhibiting more than 98% accuracy. The authors suggest that their assay is particularly suited for broad implementation in SMA screening programs, including in resource-constrained settings.

Key points:

• A nanopore-based, dual-mode PCR/sequencing assay was developed, optimised for SMN1/SMN2 genotyping, with high precision (Figure 2).
• They used both 'short' (~800-3000 bases) and 'long' (11.2 kb) amplicons for comprehensive variant identification, including copy number variation and phased SNVs/indels.
• Over 97% accuracy was achieved in genotype agreement for both SMN1 and SMN2, indicating consistent performance across diverse sample types.
• Independent clinical sample evaluation showed 100% concordance rate for SMN1 and 97% for SMN2 copy number states, reinforcing assay reliability.
• Advantages of the assay included:
  • Time-efficient: processing was done within 48-72 hours for 24-96 sample batches, ideal for high-throughput demands.
  • Cost-effective: estimated material costs below $20 USD per sample, excluding PCR, promising affordability for large-scale screening programs.
  • Scalable assay design: capable of handling tens to thousands of samples annually, with a potential to extend to other commonly screened genetic disorders.

Figure 2. The PCR/nanopore assay combined two PCR steps to generate 2.7 kb or 11.2 kb amplicons, each barcoded and pooled into one library for MinION sequencing, followed by automated data analysis.

Plant genomics

A chromosome-scale assembly reveals chromosomal aberrations and exchanges generating genetic diversity in Coffea arabica germplasm (Nature Communications)

Coffea arabica possesses an intricate allotetraploid genome, making it a complex subject for genome assembly. Through the integrative use of nanopore sequencing and Hi-C data, Scalabrin et al. achieved a highly contiguous assembly, paving the way for improved breeding programs and in-depth evolutionary studies.

Key points:

• The completion of a chromosome-scale assembly significantly surpassed previous attempts in both contiguity and completeness, providing a vital reference for future genetic research on coffee plants.
• The researchers navigated previously elusive regions of the genome, such as the pericentromeric areas, shedding light on the chromosomal evolution within the species.
• The team uncovered evidence of both reciprocal and nonreciprocal exchanges between homoeologous chromosomes, offering insights into the ongoing generation of genetic diversity in coffee germplasm.

Discover more applications of nanopore technology in plant research

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