Nanopore Community Meeting 2020 Online

Examination of gene expression with single cell or spatial resolution led to important advances in our understanding of physiology and disease. The most widely used single cell sequencing workflows use co-encapsulation of cells with barcoded beads into emulsion droplets. A recently developed spatial transcriptomics approach synthesizes cDNA on slides that contain grids of spatial barcodes. When Illumina sequencing is used in either of those approaches, only 3’ terminal sequence information is obtained and most information on splicing and SNVs is lost. We demonstrated previously that nanpore sequencing combined with Illumina data guided barcode and UMI assignment, allowing profiling of transcript isoforms and identification of SNVs with single cell or spatial resolution. Recent improvements in nanopore read accuracy and a novel barcode and UMI assignment strategy now allow skipping of Illumina sequencing to perform accurate nanopore single cell and spatial transcript isoform sequencing without any guidance by Illumina data.

The North Sea ecosystem is degraded – compared to a century ago, complete trophic levels are missing and predatory fish like bluefin tuna, halibut and large sharks are rare or absent. The current construction of large offshore wind farms can help restore the North Sea ecosystem, as these are closed for fishing and provide novel habitats. Fish presence in wind parks can be monitored through environmental DNA analysis – using the MinION enables time and cost-effective biodiversity monitoring ‘in the field’ in the North Sea. Upcoming are autonomous eDNA sampling and analysis, allowing for high resolution, non-invasive monitoring strategies.

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For many countries with a high burden of canine rabies, control and eradication efforts are hampered by lack of information about the distribution and spread of the rabies virus. Rapid, cost-effective sequencing could provide critical information for control and vaccination strategies. PCR amplification was used to enrich rabies virus RNA from over 100 clinical and field samples for sequencing on the MinION in a veterinary laboratory lacking the resources and expertise for sequencing in Goa, India. Phylogenetic analysis provided valuable insight into rabies distribution in Goa, including evidence of rabies importation after extensive vaccination and surveillance efforts. Disclaimer: Use of trade names and commercial sources is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services.

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Frontotemporal dementia is a subtype of dementia predominantly characterized by language and behavioral impairment. We use PromethION genome sequencing on DNA extracted from the frontal cortex from patients and controls to identify structural and epigenetic variants underlying the disease. Nanopore sequencing enables the detection of modified nucleotides, such as methylcytosine, and phasing of reads in parental haplotypes, and based on this we can detect allele-specific epigenetic marks. These marks will include loci for which allele-specific modifications is a biological feature, such as parental imprinting. More interestingly, this approach can also provide epigenetic evidence of disease-associated variants.

Improvements in sequencing technology and computational assembly methods have led to remarkable milestones in human genome assembly. Recently, the Telomere-to-Telomere (T2T) consortium generated the first telomere-to-telomere assembly of a complete human genome, due in part to the use of ultralong nanopore sequencing reads to stretch across these difficult regions. For the first time, we have a complete human reference spanning multi-megabase satellite arrays and rDNA arrays in the peri/centromeric regions and acrocentric short arms, as well as regions enriched in segmental duplications. While these large repetitive regions of the genome remain largely unexplored, there is evidence that epigenetic control of repeats contributes strongly to genome stability and disease. Previous attempts to study epigenetics in centromeric, pericentromeric, telomeric, and macrosatellite regions have been precluded by the difficulty in mapping conventional NGS data to large repetitive arrays. Ultra-long (>100kb) reads allow us to accurately map nanopore reads to these regions and investigate the epigenome of previously unassembled and unannotated sequences. We investigated the methylome of the centromeric higher order repeat (HOR) arrays, and observed a distinctive pattern of hyper and hypomethylation. Furthermore, we can use allele-specific methylation alone to phase microsatellite repeats, which we have demonstrated on the DXZ4 region of the X chromosome. There we found two clusters of reads which we attribute to either the active or inactive X allele. Our demonstrated ability to probe the epigenome of these repetitive areas will allow us to gain a greater understanding of their regulation and significance.