At London Calling 2024, the Nanopore Community showcases how ‘nanopore sequencing can drive new discoveries in biology’

As we round out the Innovation Days at London Calling 2024, the Nanopore Community has once again demonstrated the transformative potential of nanopore sequencing in driving new discoveries across diverse fields of biology. Here’s a recap of some of the compelling research presented at the conference.

Nanopore sequencing reveals chromosome end-specific telomere lengths conserved across 147 individuals

Delivering the opening plenary of London Calling 2024, Carol Greider (University of California Santa Cruz, USA) described the telomere length equilibrium: the observed distribution of telomere lengths, which plays a critical role in human disease. To investigate what mechanism maintains this equilibrium, Carol and her team turned to nanopore sequencing.

First, they sequenced yeast DNA using long nanopore reads, enabling identification of both the lengths and chromosomal locations of telomeres. To their surprise, this revealed that ‘we’ve been looking at the overarching equilibrium for so many years, and now we see that equilibrium is made up of individual chromosome equilibria’. To investigate whether this was the case for the human genome, the team used a MinION to perform targeted sequencing of telomeres from 147 research samples. As well as recapitulating the age-related telomere shortening observed in FISH-based assays, the data showed chromosome-end-specific — and even allele-specific — telomere lengths. These equilibria were conserved and consistent between newborns and adults, suggesting that telomere length is established at birth and shortens with age. Carol concluded that ‘new technologies — nanopore sequencing — can really drive new discoveries in biology’.

Into the unknown: portable sequencing empowers global conservation genomics

Mrinalini Watsa (San Diego Zoo Wildlife Alliance, USA) emphasised that to understand biodiversity loss in the Amazon rainforest, it is critical to first comprehensively capture its true biodiversity. However, Mrinalini highlighted the geographic, economic, and gender inequalities in who can participate in cutting-edge scientific research. In conservation genomics, ~91% of genomes are sequenced at facilities in the northern hemisphere — despite 43% originating in the Global South.

Mrinalini asked: ‘why take samples to labs when you can take labs to samples?’. She introduced the In Situ Laboratory Initiative, currently across five countries, facilitating local conservation sequencing using nanopore technology. She shared examples from the Los Amigos Wildlife Conservation Laboratory, Peru, including their use of DNA barcoding to sequence 1,200 samples on a single flow cell across a wide range of species, and DNA metabarcoding of faecal samples, producing data across 75 genera. As part of the Oxford Nanopore ORG.one initiative, they have produced reference genomes for threatened species including the short-eared dog and yellow-tailed woolly monkey on a PromethION 2 Solo. All these projects have taken place locally, including library preparation and sequencing at source, around the biodiversity the scientists are committed to conserving.

Towards personalised medicine for breast cancer in the Caribbean: a pilot study

Carika Weldon (CariGenetics & Bermuda College, Bermuda) began by highlighting the unequal burden of disease for breast cancer. Black women are more likely to die from breast cancer than white women at any age, and the ten countries with the highest death rates from the disease are all non-European. A 2021 panel-based study across seven Caribbean islands indicated that 13% of identified pathogenic variants were outside of BRCA1/2, suggesting that the targeting of these genes was insufficient for breast cancer screening in Caribbean women. In their Bermuda-based CariGenetics Caribbean breast cancer whole-genome pilot study — the first study of unique genetic markers of Caribbean women — Carika and her team investigated whether they could sequence the targets in current panels locally as well as look for novel variants.

Using a PromethION 24, they performed whole-genome sequencing of 102 breast cancer and control research samples from a Bermudian cohort. The data revealed a considerably higher rate of inherited breast cancer variants than expected, of which 60% were not in BRCA1/2, suggesting the potential for more therapeutic options than previously thought; short-read sequencing of these research samples did not capture some of the structural variants (SVs) identified in the nanopore data. Where panel testing requires samples to be sent to the USA, taking 6–14 weeks, the team’s nanopore research workflow takes just two weeks in their own lab — and ‘the cost of doing whole-genome sequencing in Bermuda on PromethION is cheaper than the panel test’. Now, the team are working to identify novel variants from their data and investigate epigenetic modifications.

Whole-genome sequencing in PulseNet foodborne molecular surveillance systems

Heather Carleton (Centers for Disease Control & Prevention, USA) started her talk by explaining the impact foodborne diseases have on public health. Each year, there are ~550 million cases globally with ~48 million people falling ill in the USA, leading to considerable knock-on effects on morbidity, mortality, and the economy. PulseNet USA is part of an international network that aims to identify foodborne disease outbreaks early, trace them back to the source, remove any affected products from the market, and prevent any further illnesses. Heather explained how whole-genome sequencing is used to monitor clusters of illnesses with the same ‘genetic fingerprints’, so that epidemiologists can be informed about potential outbreaks to investigate​.

Currently, PulseNet USA sequences ~65,000 isolates annually, and Heather described how nanopore sequencing data is being validated on historical outbreaks. With the launch of the ‘bacterial methylation-aware Dorado’ basecaller in September 2023, Heather spoke about how nanopore technology can now be used to identify isolates within outbreak clusters across a range of suspected outbreak sources. Looking to the future, she and the team at PulseNet USA want to expand validation testing to include more historical outbreaks, and the international network will roll out training on how to use nanopore sequencing for antimicrobial resistance characterisation.

Nanopore sequencing opening up access to sequencing rare genetic paediatric diseases

This panel plenary explored how nanopore sequencing is used in paediatric clinical research. Hannah Titheradge (Birmingham Women’s and Children’s NHS Foundation Trust, UK) opened the panel by introducing her study investigating the potential of nanopore whole-genome sequencing of clinical research samples from children with rare diseases. Rare diseases affect one in 17 people and 30% of affected children will not reach their fifth birthday, highlighting the importance of diagnosis. Hannah showed how nanopore sequencing could potentially be used to identify novel genetic associations with rare diseases, potentially providing the answers that affected individuals and their families are seeking.

Stephen Meyn (Centre for Human Genomics and Precision Medicine, University of Wisconsin-Madison, USA) followed up by presenting the BadgerSeq pipeline, a rapid, in-house workflow to provide potentially actionable results in 72 hours, cutting down the current sequencing timeline of 17–19 days. He ended by highlighting that ‘PromethION is a really good choice, not something you can do with your local [short-read sequencing device]’.

Next, Cate Paschal provided examples of how Seattle’s Children’s Hospital, USA, have investigated nanopore sequencing in their own labs to research rare diseases to potentially eliminate the time wasted in transporting samples to centralised laboratories for specialised assays.

Justin O’Sullivan (Liggins Institute, University of Auckland, New Zealand), the final panel speaker, further highlighted the importance of in-house sequencing. He explained that he used nanopore sequencing on a PromethION 2 Solo to study rare disorders, demonstrating that this workflow can generate 40x coverage across whole genomes in a short timeframe with comparable results to accredited short-read sequencing techniques.

Can long-read technologies transform clinical care?

In the closing plenary of this year’s conference, Professor Dame Lyn Chitty (Great Ormond Street NHS Foundation Trust & UCL Great Ormond Street Institute of Child Health, UK) presented on her lab’s exploration of the potential use of targeted long nanopore reads for rare disease diagnosis, noting their ‘potential to transform clinical care’. She shared her view that ‘a long-read sequencing assay could eliminate the need for multiple Sanger sequencing confirmatory tests’ as currently used when investigating pseudogenes, and could also allow ‘the detection of SVs that Sanger sequencing would miss’. She shared an example in which they used nanopore sequencing to rapidly characterise both SNVs and CNVs across three genes in a clinical research sample, providing ‘a much more streamlined lab flow’.

Emphasising the importance of variant phasing, Dame Chitty explained that cost-effective multiplexed nanopore sequencing has the potential to negate the need for costly and time-consuming trio sequencing. She also discussed the potential of cell-free fetal DNA sequencing to replace invasive prenatal tests that carry a risk of miscarriage. Noting the ‘dark regions’ of the genome inaccessible to short-read sequencing, including regions significant in mechanisms of disease, Dame Chitty emphasised that ‘if you can use targeted long-read sequencing, you can potentially identify those hard-to-reach areas’. She highlighted how, where current workflows can require the use of multiple technologies to identify variants in diseases such as Prader-Willi syndrome and Angelman syndrome, with nanopore sequencing there is future potential to ‘look at SNVs, CNVs, and the methylation status’ in a single assay.