Science unlocked: publication picks from October 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.

Human genetics

1. Revolutionising high-resolution HLA genotyping for transplant assessment: validation, implementation and challenges of Oxford Nanopore Technologies' Q20+ sequencing (HLA)

Human leukocyte antigen (HLA) matching is a crucial determinant for the clinical outcome of haematopoietic cell transplantation (HCT). Here the authors aimed to validate the Native Barcoding Kit V14 for HLA genotyping in combination with a full gene HLA PCR assay for target enrichment.

Key points:

• They validated the kit using an internal reference panel of 42 samples representing common HLA allele groups. Almost all alleles (736/737) were resolved to at least three fields of resolution (with the last allele resolved to two fields).
• Following validation, complete HLA genotype was assigned in 858/924 samples using the nanopore workflow during routine testing. Nearly all alleles were resolved to minimum two-field resolution (16047/16062; 99.9%).
• This study identified 517 novel alleles, of which 24 were in exon regions and 493 were in introns and untranslated regions.
• Nanopore sequencing provided uniform coverage across the target region and resolved some ambiguous alleles, such as cis-trans ambiguities, missed by short-read sequencing platforms.
• The consensus read accuracy was comparable between R10.4.1 (99.98%) and short-read sequencing (99.99%).
• There was a three-day turnaround time from DNA extraction to analysis, which the authors claim is significantly faster than short-read sequencing workflows.

Get your hands on the latest Native Barcoding Kit V14

2. scNanoSeq-CUT&Tag: a single-cell long-read CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells (Nature Methods)

Chromatin modifications are epigenetic marks that regulate genome functions. Short-read methods struggle to profile chromatin modifications in repetitive or complex genome regions, but repetitive elements account for 52% of the human genome. Here Fuchou Tang and colleagues introduce scNanoSeq-CUT&Tag, which enables efficient profiling of histone modifications and DNA-binding proteins within individual cells.

Key points:

• The authors applied scNanoSeq-CUT&Tag to accurately detect chromatin modifications on individual copies of repetitive elements in both human and mouse genomes.
• The method profiled histone marks and transcription factor occupancy patterns at single-cell resolution and distinguished different cell types.
• The merged genomic tracks from as few as 146 cells were highly comparable with ‘gold-standard’ bulk ChIP–seq data from millions of cells.
• ScNanoSeq-CUT&Tag captured cell type-specific chromatin modifications from archival tissues, eliminating the need for prior isolation of pure cell populations.
• The minimum sequencing depth per cell for accurate cell type identification was 1000 reads, which could cost as little as 0.1 dollar per cell on a PromethION 48 with a throughput of 10,000 cells per sequencing run.

Read the interview with Fuchou Tang – the pioneer of single-cell sequencing.

Bioinformatics

3. Benchmarking reveals superiority of deep learning variant callers on bacterial nanopore sequence data (eLife)

Deep learning variant callers have mostly been applied to human data so far. We need a benchmarking assessment of variant callers for bacteria, as variant calling is an essential step in tracking the emergence and spread of disease, identifying new strains of bacteria, and examining their evolution. Here Hall et al. investigate the accuracy of different variant callers on nanopore datasets compared to short-read sequencing.

Key points:

• They compared four deep learning-based and three traditional variant callers on nanopore datasets from 14 bacterial species, along with variants called by Snippy on short-read data.
• Using Clair3 or DeepVariant on nanopore data at 10x depth provided F1 scores consistent with, or better than, full-depth [average > 200x] short-read data for both SNPs and indels.
• The authors attributed the superior performance of nanopore technology to its ability to align reads in repetitive and variant-dense genomic regions, which is often a shortcoming with short-read methods.
• 'These results underscore the potential of [nanopore] sequencing, combined with advanced variant calling algorithms, to replace traditional short-read sequencing methods in bacterial genomics, particularly in resource-limited settings'.

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4. Parallel molecular data storage by printing epigenetic bits on DNA (Nature)

We urgently need new methods for large-scale data storage due to the rapidly expanding global data-sphere. Using DNA has potential, but writing large-scale data directly into DNA sequences by de novo synthesis is uneconomical in time and cost. Here Cheng Zhang and colleagues share a method of writing data onto premade nucleic acids (DNA) using epigenetic modification.

Key points:

• The methyltransferase DNMT1 specifically recognises hemimethylated sites in DNA and transfers a methyl group from S-adenosylmethionine to the opposite cytosine in the DNA template, achieving the writing of an epigenetic information bit (epi-bit).
• As nanopore sequencing detects both genetic and epigenetic data, it was able to read the complex methylation pattern, even when methylated bases were close together.
• Methylation calling reliably detected all methylated sites, even when only one 5-methylcytosine was present in that segment.
• Algorithms were developed to resolve 240 modification patterns per sequencing reaction.
• Storage density could be increased further by incorporating a variety of DNA modifications matched with accurate detection methodology.
• This DNA data storage method is programmable, stable and scalable. It could be useful for dual-mode data functions in biomolecular systems in the future.

Detect DNA and RNA base modifications at single nucleotide resolution

Cancer research

5. Real-time genomic characterisation of paediatric acute leukaemia using adaptive sampling (bioRxiv)

Most cancer treatment centres globally are in low and middle-income countries (LMICs), often without access to reliable cytogenetic testing approaches. Geyer et al. trialled on-device targeted nanopore sequencing (adaptive sampling) as an accessible, scalable approach to improve genomic classification of acute leukaemia.

Key points:

• Peripheral blood or bone marrow from 54 paediatric acute leukaemia cases with diverse genomic subtypes were analysed.
• Adaptive sampling was used to enrich 59 genes commonly involved in translocations/fusions in B-cell acute lymphoblastic leukaemia (B-ALL) and acute myeloid leukaemia (AML).
• Nanopore sequencing provided all genomic information currently offered by traditional diagnostic testing (karyotype, FISH, and occasional microarray) for paediatric acute leukaemia, but was also able to characterise aneuploidy and structural variants in challenging repetitive regions.
• There was no loss in sensitivity or throughput between freshly collected and cryopreserved samples, whereas karyotype and FISH are reliant on high-quality viable cells.
• The researchers suggested that nanopore sequencing could be a low-cost future alternative to current diagnostic tools, increasing accessibility to LMICs.

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Microbiology and infectious disease

6. The rapid detection of a neonatal unit outbreak of a wild-type Klebsiella variicola using decentralised Oxford Nanopore sequencing (Research Square)

During routine weekly nanopore sequencing of patient-derived isolates, a lab in New Zealand noticed an increased incidence of Klebsiella pneumoniae (K. pneumoniae) bloodstream infections at a neonatal intensive care unit. The rapid turnaround time of nanopore sequencing allowed infection control measures to be implemented quickly.

Key points:

• Within 48 hours, nanopore sequencing revealed that 3/4 of the cases were actually Klebsiella variicola (K. variicola), and two were sequence type ST6385 which had not been reported in clinical settings previously.
• Reanalysis of recent K. pneumoniae cases found a further two were K. variicola ST6385, suggesting transmission between individuals.
• Using nanopore sequencing to analyse environmental samples uncovered that K. variicola ST6385 persisted in two sinks that were close to the beds of the affected individuals. This led to the review and improvement of cleaning protocols.
• Nanopore sequencing offered accurate isolate identification in a timely manner that allowed the outbreak to be controlled.

Watch Rhys White’s talk on real-time genomic surveillance at London Calling 2024

Plant

7. Nanopore ultra-long sequencing and adaptive sampling spur plant complete telomere-to-telomere genome assembly (Molecular Plant)

Assembling complete telomere-to-telomere (T2T) genomes is crucial for plant functional genomics and molecular breeding. However, due to repetitive regions and structural variation, T2T plant genome assembly is challenging with the restricted read lengths of short-read sequencing. Nanopore sequencing offers ultra-long reads, and here Dongdong Lu and colleagues evaluate how these can be used in addition to adaptive sampling to target challenging areas of plant genomes.

Key points:

• They developed an optimised extraction and library prep workflow, focused on isolating ultra-long DNA fragments.
• This achieved DNA lengths exceeding 485 Kb (average N50: 80.57 Kb, max N50: 440 Kb).
• They also demonstrated that adaptive sampling can be used to target common gaps in genome assemblies such as segmental duplications, highly repetitive centromeres and telomeres, and tandem repeats.
• The complete T2T genome of Sorghum bicolor (717 Mb) was sequenced on a PromethION using two flow cells and cost $2,200.

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