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. Targeted long-read sequencing enriches disease-relevant genomic regions of interest to provide complete Mendelian disease diagnostics (JCI Insight)

Identifying causative variants in Mendelian diseases is a challenge. Here the authors present Targeted Long-read Sequencing of Mendelian Disease genes (TaLon-SeqMD) which uses low-cost, real-time adaptive nanopore sequencing with integrated methylation analysis, and show its capabilities to validate prior clinical testing.

Key points:

• The participants of this study were 18 individuals who had previously undergone targeted genetic testing for 373 inherited retinal disease (IRD) genes.
• TaLon-SeqMD confirmed the previous molecular diagnosis in all participants, and identified non-coding and structural variants in two IRD cases where prior clinical testing was inconclusive.
• Drastically improved turnaround time — TaLon-SeqMD identified and phased two disease variants within 12 hours, compared to a standard clinical exome panel taking seven weeks to yield results (without the phasing data).
• Accessible to resource-limited settings — due to the targeted nature of TaLon-SeqMD only a single MinION flow cell and minimal computational resources were required.
• The ability to provide phased genetic and epigenetic information from a single sequencing run has the potential to improve the diagnostic rate of Mendelian conditions in the future.

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2. Multi-pass, single-molecule nanopore reading of long protein strands (Nature)

Proteins play a vital role in our biological processes. We have many more proteins than genes due to differences in transcription, translation and post-translational modifications. However, our understanding of proteome diversity is incomplete as other technologies can't sequence single protein molecules in their native, full-length form. This information would enable a more comprehensive understanding of health and disease states, and aid the development of therapeutics. Here the authors present a streamlined two-step process using nanopore sequencing to sequence native polypeptides.

Key points:

• In the first step, the protein substrate was threaded into the nanopore by electrophoretic force (cis-to-trans). Then, ClpX was added to the cis solution to steadily pull the substrate protein back out of the pore (trans-to-cis).
• Nanopore sequencing allowed the examination of full-length, folded protein domains for complete end-to-end analysis.
• The authors were able to re-read individual protein molecules multiple times.
• Nanopore technology enabled the sequencing of single-amino-acid substitutions and post-translational modifications.
• ‘These results provide proof of concept for a platform that has the potential to identify and characterise full-length proteoforms at single-molecule resolution’.

3. Targeted long-read sequencing as a single assay improves diagnosis of spastic-ataxia disorders (medRxiv)

Genetic testing for spastic-ataxia disorders is complex, often requiring multiple assays due to the diversity of underlying genetic variants. Limitations of other assays (e.g. short-read sequencing, repeat-primed PCR, and Southern blog) leave 71% of individuals with hereditary cerebellar ataxia and 45-50% of individuals with a hereditary spastic paraplegia phenotype currently without a genetic diagnosis. Here the authors characterised genetic variation within 469 disease-associated genes in a single nanopore-based assay.

Key points:

• The research samples came from the peripheral blood of 34 undiagnosed individuals and 5 positive controls.
• Nanopore sequencing identified potential causative pathogenic variants in 14/34 (41%) of the unsolved participants.
• The authors found that short tandem repeat expansions in FGF14 were the most common cause, present in 7/34 (21%) of the participants.
• The availability of a single comprehensive assay has the potential to streamline the diagnostic process in future, allowing earlier access to therapeutics and potentially improving patient outcomes.

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4. Long-read whole-genome sequencing-based concurrent haplotyping and aneuploidy profiling of single cells (bioRxiv)

Genome-wide haplotyping methods for single cells that use genotyping arrays or short-read sequencing require DNA from parents and first-degree relatives (who may not be willing or available) for genetic phasing. Nanopore sequencing could be a future method of preimplantation genetic testing (PGT) as it could allow direct haplotype phasing of both parents and the embryo, including parental de novo mutations, without requiring relatives. Here the authors present a proof-of-concept study using single-cell nanopore data for single nucleotide variant (SNV) and insertion-deletion (indel) calling, and haplotyping using a Genome in a Bottle trio of Ashkenazi Jewish heritage.

Key points:

• In single-cell and multi-cell samples, 92% and 98% of heterozygous SNVs, and 74% and 78% of heterozygous indels were accurately haplotyped respectively.
• A previous study phased the autosomes of the same child into 19,215 blocks with a switch error rate of 0.37% for SNVs and indels using an alternative long-read sequencing technology. In this study the authors obtained 1,964 phased blocks with a lower switch error rate of 0.22% using ~24x nanopore sequencing reads.
• The approach was then tested on five embryos from two different couples, achieving 100% concordance with SNP array-based PGT. Obtained 21-31x coverage of the parents and embryos, covering 93-95% of the human genome.
• For profiling single cells, nanopore sequencing required fewer family members and offered a more comprehensive genomic analysis (direct variant detection, haplotyping and aneuploidy assessment).
• Concurrent haplotyping and aneuploidy profiling provides a potential alternative to current PGT methods, and could allow cell-based prenatal diagnosis in animal and plant breeding in the future.
• Nanopore sequencing also holds promise for being able to perform non-invasive prenatal diagnosis by analysing single foetal cells in maternal blood in the future.

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Cancer research

5. Long-read DNA and cDNA sequencing identify cancer-predisposing deep intronic variation in tumour-suppressor genes (Genome Research)

Some deep intronic variants lead to exonification of intronic sequences which can result in cancer. Currently these are underreported as testing does not include full intronic sequences – they often lie in complex repetitive regions, and in silico tools have historically not accurately predicted the consequences of these variants. Accurate identification of pathogenic variants can inform treatment and empower patients and their families, but a high proportion of familial risk for cancers remains unexplained. Here the authors use targeted nanopore sequencing to identify causative variants in unsolved families.

Key points:

• 120 unsolved families affected by breast, ovarian, or metastatic prostate cancer were investigated for the presence of deep intronic variants.
• They used adaptive sampling targeted at BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51C, RAD51D, and TP53.
• 92 rare or private deep intronic variants were identified in 88/120 unsolved families.
• In silico tools identified 7/92 variants that might create cryptic donor or acceptor sites within introns, these were then evaluated by cDNA sequencing.
• All seven of these variants yielded transcripts including pseudoexons.
• Eight of the 120 previously unsolved families (6%) were found to have a rare deep intronic variant, leading to a stop codon and loss of gene function.
• The ability of nanopore technology to sequence complex genomic regions offers insights into the causative variants behind these cancers, which could inform personalised cancer treatments in the future.

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6. ROBIN: a unified nanopore-based sequencing assay integrating real-time, intraoperative methylome classification and next-day comprehensive molecular brain tumour profiling for ultra-rapid tumour diagnostics (medRxiv)

Brain tumours are routinely classified based on their epigenetic signatures. Current testing relies on microarrays which have high capital costs and require multiplex batch processing. The average turnaround time takes days to weeks. ROBIN (Rapid nanpOre Brain intraoperatIve classificatioN) is a nanopore-based tool for providing real-time, intraoperative methylome classification and next-day comprehensive molecular profiling within a single assay. The tool uniquely integrates three methylation classifiers to improve performance.

Key points:

• ROBIN achieved a turnaround time of two hours, meaning surgeons could receive the results mid-operation and tailor their approach accordingly.
• 38 cases (76%) within the prospective cohort were classified intraoperatively.
• The initial classifier identified 90% of tumours correctly according to the fully integrated diagnosis available the next day.
• 'Implementing nanopore-based sequencing for routine practice would [in the future] democratise access to rapid results, with immediate benefits to patients, medical professionals and the wider health system. Nanopore-based intraoperative classification enables characterisation of the tumour subtype in far greater detail than smear or frozen section alone'.

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7. Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumour-normal pair for somatic benchmarks (bioRxiv)

In the past, tumour cell lines were created from patient samples without informed consent for their tissue to be used for research or commercial gain (a famous example is HeLa cells). Researchers therefore need new tumour-normal pairs of cell lines from individuals who give informed consent for the broad dissemination of any genomic information derived from the cells. After giving informed consent, a woman with European heritage donated tissue samples which were used for sequencing and genomic analysis to offer insights into somatic variation, mosaicism and epigenetics. The resulting data will be used by the National Institute of Standards and Technology (NIST) Genome in a Bottle Consortium to develop matched tumour-normal benchmarks for future cancer research.

Key points:

• The authors obtained pancreatic ductal adenocarcinoma tissue which was used to make a new immortal cell line plus paired normal pancreatic and duodenal tissue.
• They used 13 technologies to collect whole-genome information, including short- and long-read sequencing, optical mapping, chromatin conformation capture, G-banded karyotyping, directional genomic hybridisation, and single-cell sequencing.
• This approach was chosen to produce a comprehensive dataset that should be suitable for all future benchmarking efforts, with the aim of improving the field of cancer research.
• The resulting genomic data will be available via NIST Genome in a Bottle Consortium, and the cell line will be deposited into a public repository.
• Projects like this are important to raise awareness of the unethical practises that happened in the past, whilst providing a new benchmark resource for future studies so that we can move away from using immortal cell lines that were created without patient consent.

Microbiology and infectious diseases

8. Implementing portable, real-time 16S rRNA sequencing in the healthcare sector enhances antimicrobial stewardship (medRxiv)

Antimicrobial resistance poses a significant global health burden, so healthcare settings need rapid, accurate identification of bacterial infections in order to prescribe the correct treatment. Here the authors assessed whether nanopore sequencing could be the answer.

Key points:

• The authors sequenced 16S ribosomal RNA (rRNA) in patient research samples using nanopore technology.
• Their findings showed the potential to impact antibiotic treatment in 34.2% of cases.
• Nanopore sequencing identified additional bacterial organisms missed by reference labs, and confirmed non-infectious conditions in 5.4% of cases.
• Using nanopore sequencing reduced patient stays due to the fast turnaround time (24-72 hours). Previous methods, such as 16S PCR from culture or mass-spectrometry, took approximately seven days.
• '[16S rRNA nanopore sequencing offers] a faster, more sensitive, and accurate bacterial identification method. Earlier use of this assay in cases where routine cultures are likely to fail could enhance patient outcomes further by enabling timely, targeted antibiotic therapies, reducing hospital stays, and curbing unnecessary antibiotic use [in the future]'.

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