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.

Epigenetics

1. Decoding the epigenetic landscape: insights into 5mC and 5hmC patterns in mouse cortical cell types (bioRxiv)

The DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are powerful epigenetic regulators of gene expression. Yet, how these genome-wide marks determine unique transcriptional signatures across different brain cell populations is unclear. Here Wei et al. applied nanopore sequencing of native DNA to obtain a genome-wide, single-base resolution atlas of 5mC and 5hmC modifications in different cell populations in the mouse brain.

Key points:

• The approach gave genome-wide coverage (99%), across 40 million CpG sites, providing comprehensive DNA methylation data from the mouse brain.
• Sequencing of native DNA avoided the need for bisulfite conversion, whilst also preventing bias and enabling the simultaneous detection of 5mC and 5hmC in a single assay.
• Uniquely high 5hmC levels were identified in astrocytes compared to other brain cell types, which supports previous literature on methylation patterns, gene expression, and alternative splicing.
• The authors provide the data as an interactive tool (NAM-Me), to be used as a resource for exploring the methylome.
• This data bridges a gap in our understanding of the epigenetic regulation of brain function and disease, offering potential insights for numerous applications in medical research, for example biomarker discovery and identifying novel therapeutic targets.

Microbiology and infectious diseases

2.Unified metagenomic method for rapid detection of microorganisms in clinical samples (Communications Medicine)

Where infectious diseases are suspected, typical diagnostic tests such as culture, targeted multiplex PCR, and antigen detection can take days to provide comprehensive results. This leads to delays in diagnosis and appropriate management during the critical early stages of infection. Here the authors from Guy’s and St Thomas’ NHS Foundation Trust (UK) have developed a unified metagenomic approach using nanopore sequencing for simultaneous detection of pathogenic RNA and DNA in human samples, showcasing the future potential of the technology in this space.

Key points:

• The authors used a mechanical host-depletion method with zirconium-silicate beads and nonspecific endonuclease, significantly reducing human DNA and increasing microbial detection.
• Achieved a 256-fold reduction in human DNA across respiratory tract samples.
• Automated reports suitable for initial insights were generated after a mere 30 minutes of sequencing, a turnaround significantly quicker than typical methods.
• Successfully identified a wide range of pathogens, including viruses (RNA and DNA), bacteria (typical and atypical), and fungi, improving detection of pathogens compared to typical methods.
• Based on 33 respiratory samples, additional pathogens were detected in 21% of cases after 2 hours of nanopore sequencing compared to typical methods.
• This approach holds future promise for rapid, actionable results in routine microbiology laboratories, enhancing initial management of acute infections, particularly lower respiratory tract infections.

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3.Comparative analysis of 43 distinct RNA modifications by nanopore tRNA sequencing (bioRxiv)

Of all the RNA in cells, tRNA is the most abundant and diversely modified. Until now it has been challenging to identify and analyse all the different tRNA molecules and their modifications due to limitations of methods used. Here the authors sequenced tRNA from one prokaryotic and five eukaryotic species using direct RNA chemistry by Oxford Nanopore Technologies (RNA002 and RNA004) and compared the reproducibility of tRNA abundance metrics.

Key points:

• The species studied were Drosophila melanogaster, Danio rerio, Escherichia coli (E. coli), Homo sapiens, Saccharomyces cerevisiae (S. cerevisiae) and Tetrahymena thermophila.
• The authors isolated total RNA in all samples, purified the tRNA and generated matched pairs of tRNA sequencing libraries using RNA002 and RNA004.
• Mitochondrial tRNA was also isolated from S. cerevisiae to observe whether less abundant tRNA could be detected.
• They also sequenced tRNA from untreated E. coli and E. coli infected with T4 bacteriophage, allowing for both host and phage-derived tRNA to be detected.
• The protocol took five hours from the first ligation step to loading onto a flow cell.
• The new RNA004 kit achieved greater alignment accuracy and ~10-fold higher yield than its predecessor RNA002, calculated in reads per pore per minute.
• Higher tRNA sequencing yields could allow investigations into mitochondrial diseases potentially caused by tRNA gene mutations and provide insight for studying tRNA in an infection context.

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Plant and animal

4.RNA N⁶-adenine methylation dynamics impact Hyaloperonospora arabidopsidis resistance in Arabidopsis (Plant Physiology)

Studying N⁶-adenine methylation (m⁶A) changes during the biotic stress response in Arabidopsis is challenging with immunoprecipitation techniques. Direct RNA nanopore sequencing was used to find the exact m⁶A positions at single nucleotide resolution, and allowed the authors to investigate methylation changes during infection with the parasite Hyaloperonospora arabidopsidis (Hpa).

Key points:

• Direct RNA chemistry (RNA002) enabled single-nucleotide resolution analysis of m⁶A modifications.
• The authors detected and quantified m⁶A modifications from direct RNA data.
• Found global m⁶A reduction when Arabidopsis were infected with Hpa.
• Reduction corresponded to activation of basal defence genes, enhancing resistance to the parasite.
• The authors concluded that m⁶A could be a promising target to investigate for improved crop resistance against pathogens.

5.Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life (PLOS Biology)

The Drosophilidae family of flies are a pivotal model organism for many studies, but the available genomes are limited to those that are reared in labs. Here the authors performed amplification-free nanopore sequencing of single wild flies in order to expand the taxonomic diversity of known genomes in the Drosophilidae family.

Key points:

• Identified 183 new drosophilid genome assemblies for 179 species (62 from pooled lab strains, 121 from single adult flies).
• The authors used either Oxford Nanopore R9.4.1 (87 genomes) or R10.4.1 chemistry (80 genomes), combined with short-read sequencing.
• When compared to inbred lines with much greater sample volume, the single-fly diploid assemblies are comparable in contiguity, completeness and accuracy.
• Achieved a more affordable cost of US$150 per genome, and only used 35 ng genomic DNA per fly.
• Ran the nanopore assembly pipeline at different depths of coverage, and nanopore sequencing alone achieved an impressive Q score of 43.5 at 40x coverage.
• Even below 30x coverage, the genome-wide consensus accuracy of the nanopore-only datasets exceeds both the standard recommended by the Vertebrate Genomes Project (QV40) and the accuracy previously reported for a D. melanogaster R9.4.1 and hybrid assembly by the same authors.
• This study is part of a wider community-level project to build an open resource for drosophilid evolutionary genomics.

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