Oxford Nanopore opens first live day of annual London Calling conference with a full house, as well as an additional 4000 people online

The first live day of this year’s London Calling conference saw around 600 people through the doors of Old Billingsgate and a further 4,000 people registered to attend via the virtual platform. The first hybrid day of the conference saw speakers presenting across a mix of stages including Plenary, Breakout, Showcase Stage, Mini Theatre, Poster and Spotlight Sessions. Below is a summary of the highlights of the day.

Welcome

Gordon opened this year’s live conference talking about our community of genomic explorers and how together we are creating a new crucible of innovation, with a future guided by data-driven insights. Gordon went on to discuss what’s missing matters – the community of scientists using nanopore-based sequencing has shown the biological significance of seeing more genomic insights. Over the next two days of our 9th annual London Calling conference, there’s a chance to hear from hundreds of scientists who are pushing novel discovery across all areas of science – from a nanopore journey in the footsteps of an endangered tortoise to the latest research in liquid biopsy and infectious disease.

Gordon also discussed how Oxford Nanopore Technologies was founded with a big vision: to enable the analysis of anything, by anyone, anywhere, and the incredible progress made by the Nanopore Community in the nearly two decades since. More than 8,000 customer groups used the nanopore platform in 2022 in more than 120 countries, with more than 8,200 publications of peer-reviewed research in areas spanning human health, the environment, food and agriculture. This community is just getting started and through partnerships we’ll broaden the impact of that research. To hear more from Gordon you can read his full article on this year’s conference here.

Genomics England long-read cancer whole-genome sequencing pilot - Helen Webb, Genomics England, UK

In conjunction with NHS England, Helen described how Genomics England are currently running a pilot study to investigate the use of Oxford Nanopore sequencing in large-scale, whole-genome analysis in cancer. With the aim of trying to provide more comprehensive genomic data, Helen highlighted that instead of running many tests, they would like to run just one to deliver ‘complex rearrangements, fast turnaround times and methylation data’.

Helen presented several case studies of complex variants which they were unable to detect using short-read sequencing, but were able to resolve using nanopore sequencing. Helen also presented their research on promoter methylation, describing how they got 'very clean data'. There’s still work to be done, but Helen highlighted that so far, they 'have been remarkably impressed with what we have been able to do'.

A single-cell approach to cancer mutation discovery and CRISPR phenotypic modelling - Hanlee Ji, Stanford University, USA

Hanlee described their development of a new single-cell approach to identify disease-related mutations, and highlighted their work on engineering cancer mutations and variants into cellular experimental systems for further biological characterisation. Adaptive sampling was used to help identify variants down to the single-cell level. Hanlee described how they have been using the MinION device but intend to progress onto PromethION. Hanlee summarised his talk by highlighting that they have identified cancer mutations and rearrangements and integrated single-cell transcriptomes to functionally assess cancer mutation phenotype at single-cell resolution.

Real-time genomics for One Health - Lara Urban, Helmholtz AI Institute, Helmholtz Pioneer Campus, & Technical University of Munich, Germany

The concept of ‘One Health’ is that all of the life on our planet is linked. In her talk, Lara Urban explained how she is using nanopore sequencing to better understand ‘One Health’ by leveraging the ability of the platform to sequence native DNA and detect epigenetic modifications. Lara is exploring the use of non-invasive sampling, such as water, air and soil, to better understand ecosystem health and human health.

After sampling freshwater from the River Cam in Cambridge to examine whether anthropogenic effects are altering the ecosystem, Lara and her group found enrichment of virulent pathogens downstream of a wastewater outlet. They also analysed the air microbiome in the same manner to assess anthropogenic impacts on the air microbiome, noting that bioaerosols are understudied.

Lara explained that nanopore sequencing could potentially be used in the future in clinical settings, due to its rapid turnaround times and ability to detect antimicrobial resistance. Lara concluded by explaining that real-time nanopore sequencing has the ability to contribute to equitability and inclusiveness of the One Health project, with its low upfront investment costs and accessibility.

Liquid biopsies showcase

Nanopore sequencing of cell-free DNA for methylation-based breast cancer detection in a case-control cohort - Billy Lau, Stanford University School of Medicine, USA

• Here, Billy introduced his new approach for single-molecule methylation analysis of cell-free DNA (cfDNA) from cancer research samples, using a nanopore-based sequencing method that can provide hundreds of millions of sequenced reads per cfDNA sample. This method requires only nanograms or less of input analyte without PCR, improving upon previous techniques which struggled with sensitivity, especially when studying early-stage cancers

• An analytical framework was developed for detecting breast cancer in cfDNA to identify differentially methylated CpG sites between case and control group research samples.

• The pipeline successfully distinguished breast cancer cfDNA samples from healthy controls, outperforming existing machine learning models - demonstrating the future potential as an effective and scalable tool for non-invasive detection of breast cancer from blood.

Potential application of Nanopore sequencing for liquid biopsy analysis in children with cancer - Carolin Sauer, EMBL-EBI, UK

• Carolin introduced the challenges that motivated her work: paediatric cancers are the leading cause of child death in the western world & present cancer profiling challenges exist due to limited biopsy material. Liquid biopsies using cell-free DNA (cfDNA) provide a non-invasive alternative for detecting circulating tumour-derived DNA (ctDNA).

• The approach successfully detected clinically relevant somatic aberrations in paediatric cancer research samples; the team developed novel methylation deconvolution algorithms for accurate determination of tissue-of-origin and oncotype

• Carolin described how nanopore sequencing of cfDNA from liquid biopsy clinical research samples allows simultaneous detection of fragmentation size profiles , copy number aberrations , and DNA methylation signal . She highlighted that the platform offers quick, scalable, and cost-effective ctDNA analysis, features which would make them advantageous for potential use in clinical settings.

Nanopore sequencing of ctDNA — better than short read! - Andrew Beggs, University of Birmingham, UK

• Andrew introduced his teams ‘homebrew’, ‘Birmingham method’ for ctDNA sequencing, using a PCR panel that targets around 50 genes, producing ctDNA amplicons. The process, costs around £75 for the panel and Flongle, inclusive of reagents and consumables.

• ‘We can go from DNA to result in 8 hours’, with a coverage of around 10,000x, providing high data sufficiency. Andrew mentioned that you can’t do that with short reads because of the need to allow for the whole sequencing run.

• This protocol showcases a 95% detection rate for all mutations, demonstrating the power of duplex data in detecting very low variant allele frequencies (VAF); Andrew showed the seamless detection of a 15 bp indel due to long, duplex nanopore reads, with 0.1-0.2% VAF.

Update from the Applications team - Dan Turner, SVP, Applications, Oxford Nanopore Technologies

• Dan began by describing how, though rare disease is defined as those with a frequency of ≤1:2,000 people, 3,800 such diseases have been described, meaning that they affect around one in 20 people. These are typically monogenic - caused by one inherited variant allele, which is traditionally identified by methods such as exome sequencing and microarrays. However, Dan emphasised, what you’re missing matters: where short-read sequencing technology is used, structural variants (SVs), methylation, and GC-rich areas are overlooked, whilst mismapping of reads in repetitive sequences can result in ‘dark’ regions which cannot be resolved. With nanopore sequencing, it is possible to resolve these regions and variants.

• Some diseases may involve 10s-100s of genes across the genome, making it more difficult to determine the risk of an individual developing a disease. Polygenic risk scores – calculations of the risk of developing a rare disease – are frequently calculated by taking only a ‘snapshot’ of a subset of SNPs via genotyping array. Imputation is then used to statistically predict the likelihood that other relevant variants – not captured via the chip – are present.

• Here, Dan introduced the potential of low-pass nanopore whole-genome sequencing (WGS) and imputation for the calculation of polygenic risk scores. To demonstrate this, the team sequenced the well-characterised GIAB NA12878 genome and downsampled to 0.5-5x depth of coverage. Variants were identified and imputed, then evaluated against an available truth set. Initial results revealed that shorter nanopore reads gave better results at these coverages, as more were available, so the team utilised adaptive sampling to intentionally eject all fragments after the first 500 bp had been read.

• Dan showed an example in which this novel method was tested, producing a polygenic risk score for hypertension which matched that of 30x WGS data. Examples across a range of diseases again showed imputed data to matched risk scores from higher depth WGS.

• Finally, Dan showed a dataset in which 278 pharmacogenomic targets had been enriched and sequenced to good depth of coverage at the same as generating low-coverage WGS data with adaptive sampling, enabling simultaneous imputation and enrichment of disease-associated genes.

Direct RNA sequencing update (SQK-RNA004): initial impressions of RNA004 and its application for functional profiling of cancer-associated long non-coding RNAs - Libby Snell & Martin Smith, Principal Scientist, Applications, Oxford Nanopore Technologies & Sainte-Justine University-Hospital Research Centre, Canada

Libby kicked off the talk by introducing the Direct RNA Sequencing Kit, highlighting that “it is unique; we are the only platform where you can sequence RNA directly”. In addition, you can maintain modifications as there is no conversion to cDNA. The new Direct RNA Sequencing Kit (RNA004) has improvements over the previous kit (RNA002), which include higher output due to a faster motor — 120bps (versus 70bps previously) — and a 96% single-read accuracy from the human transcriptome. The key feature of this sequencing chemistry is a new RNA pore which will have its own dedicated flow cell for RNA sequencing. The kit is currently in beta testing but will be open for early access. Before the Direct RNA Sequencing Kit was released six years ago, nobody was able to sequence native RNA. This opened new avenues of RNA research, which Martin Smith went on to explain in his talk.

Martin followed up Libby’s talk to introduce his work as a beta-tester of the new Direct RNA Sequencing Kit. His first runs with the new kit gave 14.75 million reads on the PromethION. Martin wants to use this chemistry to understand non-coding regions of the genome, as only 2% of the genome consists of protein coding genes, while non-coding RNAs constitute over 70% of the genome. Martin remarked that they are “challenging to experimentally and functionally validate” and yet they have many important roles in cells. Martin works with paediatric oncology and interrogated a list of 760 leukaemia-associated long non-coding RNAs, discovering several thousand new isoforms with excellent resolution of isoforms. Martin comments that you “can pick up all the isoforms in your samples quite cleanly”.

Oxford Nanopore technology update

As is customary the day finished with all the latest Oxford Nanopore technology updates, delivered by Clive Brown, Chief Officer, Technology, Innovation and Products. A full analysis of this year’s technology update can be found here.

There is still one full day left of our hybrid conference, so you can register for online attendance for the final day of live presentations here.