Day 3 London Calling: range of groundbreaking research, a STEM session and further hands-on technical and informatics updates

The final day of this year’s London Calling started with a hugely popular informatics update that saw four Oxford Nanopore experts sharing their latest work. Don’t worry if you weren’t there, this will be shared through an online Knowledge Exchange next Thursday 26th May and there will be an opportunity for a Q&A following the broadcast. You can sign up here.

Following the success of Oxford Nanopore’s STEM event in 2019, 60 school children were welcomed to Old Billingsgate for a day of talks and workshops to provide an insight into what it’s like to work in a scientific technology company. During the day, the students had the opportunity to get hands-on with the tech, hear from scientists using it around the world, and talk to them about their work.

You can watch a great summary of this year’s brilliant conference here.

Welcome back!

Back on the main stage in the auditorium, Zoe McDougall welcomed the live and virtual audiences to the final day. Zoe talked about how we are working to fulfil our mission of anything, anyone, anywhere, by enabling more scientists, in more environments, to do more important research. And that Oxford Nanopore is only in the foothills of reaching beyond research as we see a pathway towards impacting health outcomes, or even food and environmental outcomes for many communities.

Zoe went on to say that accessibility has always been an absolute core value for Oxford Nanopore, that this is reflected in service design as well as product design, and we are incredibly proud to support customers in more than 120 countries. This map continues to light up with new users, particularly in the continents of Africa and South America, where access to the latest sequencing technology has historically been lower. Ground-breaking research is underway across human genetics and the important areas of conservation and food security, and of course public health, in which networks of technology have been established in countries from Brazil, to Japan, to Sri Lanka, to look ahead to the next public health threats, including the spectrum of drug resistance.

Zoe finished by reiterating that we believe that we now have a single platform that is very high performance and so versatile that it can meet all your scientific needs.

Understanding Cancer Epigenetics, Immunogenetics, and Energetics - Michael Dean

Michael Dean started by saying that cancer is a complex problem with a sizeable epigenetic component and this is currently poorly understood. For the last two years, Michael and his team have used MinION to dig deeper into cancer pathogenesis, specifically looking at human papilloma virus infection through direct cDNA and RNA sequencing. Michael and his team also used MinION to investigate the epigenetic component of cancer through methylation sequencing, and they used Pore-C to look at 3D structural changes in the genome. With Oxford Nanopore’s direct RNA sequencing, Michael and his team found that they could get bias-free transcriptomic data and identify RNA modifications in the MYC oncogene. Michael concluded by saying: “I did my Ph.D. studying the MYC oncogene, and I never thought I would be able to directly see MYC oncogene RNA transcripts”.

Panel plenary: animal conservation in the era of accessible genomics

Genomic tools for gorilla population dynamics and conservation - Ettore Fedele

• ‘We are living through one of the worst extinction events on our planet’

• We need an effective way to monitor gorilla species and DNA sequence analysis is ideal for this

• Ettore uses the MinION MK1B device, due to its  portability, low cost, and ease of use

• More specifically, Ettore uses nanopore technology to obtain whole-genome sequencing data for species identification and SNP analysis for individual identification

Educational, Field-based Sequencing Lab Facilitates Biodiversity Research in the Ecuadorian Tropical Andes - Zane Libke

• Genetic cataloguing of biodiversity is a massive task on its own. In addition, traditional sequencing technologies have not been accessible, students lack practical molecular biology experience, and funding is not readily available

• Zane is enthusiastic about establishing sustainable, field-based, education-focused nanopore sequencing labs: “Nanopore is diverse…nothing compares to it”. He discussed how he has helped to set up a 3-7 day intensive Field Genetics Course in the Ecuadorian Tropical Andes

• These courses are accessible and designed for students of all skill levels, providing theory and practical experience of molecular biology techniques and nanopore sequencing

• A lot of opportunities and collaborations have come out of the course – “conservation is a work that impacts the next 300,000 years”

• Zane also stated that “We have the tools now to study the Amazon”.

Genomics in the jungle: a field laboratory success story - Mrinalini Erkenswick Watsa

• Population sizes of mammals, birds, fish, amphibians, and reptiles have seen an alarming average decline of 68% since 1970

• The greatest advantage of using nanopore technology is that it empowers local scientists to create decentralised, community-run lab networks

• Using nanopore technology, Mrinalini is working on (i) DNA barcoding as many animals as possible, (ii) population monitoring by designing low-cost genotyping tools and nanopore-based pipelines for analysis, and (iii) disease surveillance by studying viral genomics in host-pathogen relationships

• She said that the “most exciting thing about nanopore is that it’s opening up sequencing to people who would never use it otherwise. It breaks down so many barriers”.

Endangered European sturgeon detection through non-amplified eDNA sequencing - Reindert Nijland

• Sturgeon in the Netherlands are non-indigenous; the European sturgeon was the indigenous species but it is now extinct in the lakes and ponds of the Netherlands.

• With the help of Oxford Nanopore and their ORG.one project, Reindert’s group have developed an environmental DNA (eDNA) sequencing method to help with their re-introduction

• Reindert explained how the standard for eDNA sequencing is the incorporation of a specific barcode amplification step, which adds complexity to the workflow

• Their detection workflow is based on unamplified eDNA, and they hope to extend their method to all fish species and ultimately to biodiversity as a whole.

There then followed a series of breakouts on data analysis tools, human clinical research and transcriptomics – all of these will be available to watch on demand in full on our website from next week.

Genetic dissection of structural variants in study subjects with antithrombin deficiency by nanopore sequencing - Belén de la Morena Barrio

Antithrombin deficiency is the most severe thrombophilia and is a disorder that is often explained by variants in the gene SERPINC1. Up to 20% of samples from people with the condition have no underlying variants detected, but nanopore sequencing is enabling Belén to characterise structural variants (SVs), as well as providing the sequence of the breakpoint. In her research study, she is aiming to characterise a known SV affecting the SERPINC1 gene. To date they have recruited 400 subjects with antithrombin deficiency and selected 35 clinical research samples with detected SVs in order to dissect and characterise the variants. Additionally, they will use nanopore sequencing to study new possible SVs in clinical research samples with no molecular defects detected by any other method.

Currently-used techniques are limited by their resolution, e.g. CGH arrays and MLPA, but with nanopore sequencing, all SVs could be detected, regardless of the size or the type and the exact sequences. This is allowing them to resolve conflicting results from other technologies and identify a founder defect in a recurrent variant in SERPINC1 by haplotyping with long nanopore sequencing reads. “After attempting different detection methods, nanopore sequencing is the most suitable method to fully characterise SVs associated with ATD; allowing breakpoint analysis at a nucleotide-level resolution.”

Comprehensive structural variant detection: from population to mosaic level - Fritz Sedlazeck

Fritz started by stating that structural variants (SVs) are a cause of higher diversity than single nucleotide variants (SNVs), and went on to say that long reads are providing a more comprehensive view of SVs than short reads. This gives access to repetitive regions and phasing information. With the tool Sniffles2 they have outperformed current methods in SV calling accuracy (improved F1) and improved germline SV calling, as well as resolving  population SVs and rare somatic SVs, with much improved speed for SV calling.

In brain samples with rare neurodegenerative disorders, Sniffles2 captured rare SVs missed by both short-read sequencing and optical mapping. From only 5X depth of coverage, they could perform high-sensitivity SV calling. This resulted in stable performance across different sequencing depths. Fritz also stated that Sniffles2 performs very well in genes of clinical relevance that are challenging to access. Fritz believes this could have many applications such as rapid Oxford Nanopore sequencing and single-cell whole-genome sequencing, and higher level resolution for variant detection at single-cell level. Their initial results show the ability to pinpoint both SVs and SNVs: “the boundaries are non-existent with this”.

Portable real-time sequencing to safeguard critically endangered wildlife - Marissa Le Lec and Lara Urban

Next on the main stage, we had Lara and Marissa discussing their work with the critically endangered Kākāpō parrot. These are nocturnal, flightless birds endemic to New Zealand with a current population of just 197. Lara and Marissa developed a rapid nanopore workflow using the Rapid Barcoding Kit and MinION, to study parentage and sex, aimed at conservationists who are not trained molecular biologists. Speed, portability, and cost were all vitally important. Only 1.3x coverage was required for parentage studies and they found that “any yield” gave good results for sex determination.

In 2019, an aspergillosis outbreak was identified, so they needed fast, simple in situ monitoring for Kākāpō conservationists. The pathogen Aspergillus fumigatus was detected using nanopore metagenomics on the portable MinION; this was enabled with real-time data analysis. They also used adaptive sampling to deplete host DNA and this has been successfully trialled to help speed up the detection rate.

De novo assembly of immunoglobulin loci linked to full-length single-cell transcriptome of antigen-specific plasmablasts - Christian Stevens

In their workflow, Christian’s team performed blood collection from subjects, following an MMR vaccination to induce proliferation of MMR antigen-specific plasmablasts. They used adaptive sampling to enrich for immunoglobulin (Ig) loci which were then assembled and the team also performed single-cell transcriptome sequencing with nanopore to investigate Ig transcript expression and grouped plasmablasts by isotype. Lastly, Christian explained how they could identify linked heavy and light chains in individual plasmablasts, which Christian suggested has the future potential to allow for antigen-specific antibody synthesis and testing.

Gordon Sanghera closed this year’s conference and said he’s looking forward to seeing you all in America in November for the Nanopore Community Meeting!

Finally, if you missed yesterday’s technology update from CTO Clive Brown and colleagues, you can now watch this on demand here.