Closing day of the Nanopore Community Meeting 2022 covers everything from pathogen surveillance to diverse genomic data

Welcome back

Rosemary Sinclair Dokos welcomed back the audience to the final day of the Nanopore Community Meeting and gave two further technology updates. The Q-line range is being refreshed to deliver ISO9001-qualified devices with locked-down, standardised format. This is available to pre-order for GridION, R9, Kit9, Kit10 with more devices in the pipeline that will integrate Q20+ chemistries. Rosemary also discussed Project TurBOT, which is a fully integrated automation that will couple sequencing with sample preparation and analysis to deliver a liquid handling solution for sample to answer sequencing.

Long-read sequencing resolves complex structural variants and identifies missing pathogenic variants in unsolved hemophilia cases - Danny Miller, University of Washington, USA

Danny began his talk by discussing the typical diagnostic odyssey and how he knows they will only resolve 50% of suspected genetic cases with current methodologies based on short-read sequencing. He went on to say that nanopore long-read sequencing resolves complex structural variants and identifies missing pathogenic variants in unsolved haemophilia cases, and that SNVs, indels, SVs, and methylation can be called and phased from a single data source. Whole genome nanopore sequencing allowed the complete reconstruction of complex genomic regions and is showing potential to shed light on unsolved haemophilia clinical cases.

Furthermore, Danny spoke about his confidence in adaptive sampling as a targeted method for resolving complex variants and said “we figured out in 24 hours of sequencing what we couldn’t figure out in months of clinical testing”. He believes in the future potential of nanopore sequencing to enter this clinical domain and stated it has real potential to shorten the time to, and increase the rate of, diagnosis.

Panel plenary: delivering the future of genomic pathogen surveillance

Identification of resistance mutations to DAA’s against HCV in infected subjects in Mexico - Kame Galan-Huerto, Autonomous University of Nuevo Leon, Mexico

• Kame highlighted that the hepatitis C virus (HCV) is a ‘big threat in Mexico’, yet a complete genome for HCV in Mexico was not available

• Using a multiplex PCR approach, combined with the Ligation Sequencing Kit, Native Barcoding and the MinION Mk1B, Kame sequenced the full 9 kb RNA virus and looked for polymorphisms associated with anti-viral resistance

• Kame’s approach enabled successful polymorphism identification, even in samples with low viral load

• Complete genome sequencing also enabled phylogenetic analysis and showed that the strains sequenced in Mexico were consistently related with samples from the USA, but they had a distinct Mexican phylogenetic clade.

Genomic investigation into yellow fever virus spread at the animal-human interface - Sarah Hill, Royal Veterinary College, UK

• Sarah began her talk by explaining that ‘emerging zoonotic pathogens are most likely to cause explosive outbreaks of severe disease’, so that it is important to understand what factors affect viral spillover from animals to humans

• Sarah and her large collaborative team wanted a deployable, distributable, cheap and easy viral screening method

• Using samples from human and non-human primates, an amplicon-based tiled PCR approach on the MinION was used to investigate viral spread. Whole virus genomes were generated, and Sarah highlighted how they “achieved really good representivity” allowing them to use phylogenetics to study outbreak dynamics

• The team generated genomes from 15% of confirmed infections of humans and non-human primates, tripling the availability of genomes

• They were able to better understand what was driving yellow fever virus spread and link climatic and landscape variables with the dynamics of yellow fever, for example, the change in outbreak size could be predicted by temperature

• Sarah highlighted that the ability to “stop the run immediately as soon as we obtain enough coverage for our genomes” enabled low-cost analyses.

Single-cell and spatial transcriptomics breakout

Single-cell transcript isoform sequencing of the activated adult mouse hippocampus with 10X Genomics and Oxford Nanopore – Sheridan Cavalier, The Johns Hopkins University School of Medicine, USA

• Sheridan explained that a transcriptional response elicited by neural cell activity is needed for learning and memory consolidation; this is known to involve thousands of genes, is cell-type specific, and also involves alternative splicing

• Sheridan’s research question is ‘what are the mRNA substrates of learning?’ which she has been exploring using single-cell isoform sequencing with 10x Genomics (Chromium) and the PromethION device, in a nanopore-only sequencing workflow

• Long nanopore reads helped to resolve transcript-level information, with 10-times the average read length produced compared to short-read sequencing technology

• Of the nanopore reads obtained, 94% were demultiplexed, and different cell types in the brain could be identified as “really beautiful cell-type communities”.

A spatiotemporal view of alternative splicing in the mouse brain – Anoushka Joglekar, Weill Cornell Medicine, USA

• Isoform diversity is more extensive in humans compared to mice – both species have ~20,000 protein-coding genes, yet humans have more isoforms per gene. This is accompanied by increased specificity of expression in humans – between tissues, regions, and cell types

• Anoushka has been investigating isoform expression in the developing mouse brain, using single-cell and spatial transcriptome sequencing, and the accompanying bioinformatic pipeline scisorseqr (available here)

• With some of her “cherry-picked” favourite examples, Anoushka demonstrated that she could obtain a single-cell and spatial view of isoform expression in the mouse brain, with “some really cool examples” of spatial isoform expression across developmental stages.

NAb-seq: an accurate, rapid, and cost-effective method for antibody long-read sequencing in hybridoma cell lines and single B cells – Kathleen Zeglinski, Walter and Eliza Hall Institute of Medical Research, Australia

• Kathleen introduced how hybridomas and B-cell cloning are two common approaches for generating antibodies from an immunised animal

• However, antibodies are a major contributor to the reproducibility crisis in science: Kathleen explained that antibodies have a lot of batch-to-batch variation and ambiguous specificity, but recombinant expression and sequence validation could help address this

• Compared to Sanger sequencing – the typical approach for sequence validation – NAb-seq with Oxford Nanopore is quick and easy (results in 2-3 days), lower cost (US$30 per antibody, with multiplexing), and has no need for optimised, specific primers, instead detecting all antibody transcripts in a cell

• Kathleen introduced the bioinformatics workflow which she stated is quick (~90 min on a PC), “super easy to use”, and freely available on GitHub; compared to previously generated Sanger sequence data, NAb-seq generated thousands of antibody reads with 100% accurate, full-length consensus sequences, including variable and constant regions.

Oxford Nanopore technology expands the research scope and increases resolution in microbiome research - Jun Wang, Institute of Microbiology, Chinese Academy of Sciences, China

• Wang and colleagues focus on human microbiome research, and how the microbiome interacts with immune function

• Regarding sequencing, they found that nanopore data improved the N50 contig length of assemblies and improved the analysis of structural variations

• The group works with DNA, RNA, and protein and is thus using both direct DNA and RNA sequencing and methylation calling with Oxford Nanopore and they are waiting for protein sequencing to be available

• “[in a] disease and health context, understanding the rules of the microbiome can be greatly sped up by nanopore sequencing”.

Using nanopore sequencing to investigate genome evolution in fungal symbioses: ploidy, repetitive elements, and reproduction – Jessica Allen, Eastern Washington University, USA

• Jessica described how lichenised fungi represent ‘an excellent’ model system: they function as ‘miniature ecosystems’, rich in bacterial communities, and feature unique secondary chemistry with potential industrial and pharmaceutical applications. Their use in evolutionary genomics research has, until recently, remained limited with the use of short-read technology
• Jessica first used nanopore sequencing to sequence the wolf lichen genome, using her office desktop computer. “The nanopore platform was the perfect solution” for sequencing primarily in an undergraduate institution with limited funding. Now, Jessica and her team routinely, rapidly generate lichen assemblies. They are able to annotate chromosome-level, mostly telomere-to-telomere assemblies
• Jessica and her team used nanopore sequencing on the MinION to investigate ploidy in wolf lichen. They generated genome assemblies for fungal and algal symbionts and the bacterial microbiome. Though most lichens are haploid, surprisingly, ~70% of individuals sampled were found to be triploid
• “Nanopore data produces highly contiguous genome sequence assemblies”: Jessica illustrated the significantly higher contig N50s for publicly-available fungi assemblies generated with long reads compared with those generated with short reads
• “What makes a lichen a lichen?” Analysis of lichenised fungi revealed lipid metabolism gene families to be expanded, suggesting major shifts in nutrient transport in the transition to lichenised fungi.
• Jessica is also training students at a regional comprehensive university in how to use nanopore technology to sequence fungal genomes, moving towards the bigger questions in fungal evolution. She noted that the workflow was scalable, economical, and without the need for expensive equipment.

A long-read nanopore sequencing platform for Indigenous genomics – Andre Reis, Garvan Institute of Medical Research, Australia

Andre highlighted the hundreds of Aboriginal nations across Australia. However, this diversity is not reflected in currently available human reference genomes. This lack of representation persists in modern global catalogues of human genomic variation, and may exacerbate existing health disparities between Indigenous and non-Indigenous people in Australia.

The National Centre for Indigenous Genomics (NCIG) was established with the goal of bridging the gap between Indigenous communities and genomics. Andre and his team are using nanopore whole-genome sequencing on the PromethION to characterise common variation in four Indigenous communities in northern Australia. They also included individuals of European ancestry for comparison. The read lengths and depths of coverage of sequenced samples enabled structural variant (SV) detection across every genome. They then characterised a non-redundant SV set and short tandem repeats (STRs).

Distinctive genetic architecture was identified between non-NCIG and NCIG communities. Andre showed how projections from plotted SV discovery curves indicate that “there is still a lot more unique Australian variation to be sampled”. The data also revealed that allelic diversity of STR expansions can be community-specific.

Summarising his talk, Andre described how there is “clear separation” in genetic architecture both between non-Indigenous and Indigenous people, and amongst different Indigenous communities; most of the variation specific to Indigenous people was also found to be specific to a given community, and there is still more diversity to be sampled.

It’s been another fantastic Nanopore Community Meeting and we’re delighted to share the dates for next year’s London Calling:17th – 19th May. Tickets will be available on 4th January!