Development and applications for long read sequencing in the Midlands

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Since the advent of sequencing, driven by Frederick Sanger, knowledge of the underlying genetic code of an organism has been a key feature in establishing the mechanisms by which inheritance influences phenotype. The rapid explosion in next generation sequencing technology in the past decade has taken us from huge consortia trying to determine the sequence of a single human genome to individuals being able to sequence their own genomes for close to $1,000. Alongside the sequencing of the genomic DNA it is also possible to sequence the transcriptome (genes expressed in a cell or tissue) or investigate modifications to the bases of DNA that alter the expression of genes. Although the UoN has a range of short read sequencing technologies available and has been at the forefront of developing long read sequencing on the MinION platform, there is a clear gap at the level of high throughput long read generation that is addressed by this proposal. Current short read technologies available at UoN allow sequencing microbial communities, exon sequencing of human scale genomes and resequencing of individual human scale genomes. For long read sequencing, Nottingham has led the development of MinION sequencing, and has the capacity to sequence bacterial genomes on single flow cells. DeepSeq, the University of Nottingham sequencing facility, led by this proposal's PI (Dr Matt Loose), contributed to the sequencing of a reference human genome on multiple MinIONs at a number of locations around the world. We were also the first to demonstrate the ability to selectively sequence molecules using the nanopore platform, a technique which we hope others will be able to exploit on a larger scale in the future. DeepSeq has encouraged others to apply MinION sequencing to their projects, providing training both locally and via the 'porecamp' series of workshops nationally. DeepSeq has also contributed to the development of analysis tools such as minoTour for real time analysis of MinION data. We now wish to extend our knowledge and expertise to significantly higher throughput problems solvable with the increased scale of the PromethION platform. We seek to purchase an Oxford Nanopore PromethION sequencer alongside compute support and significant data storage that will be capable of sequencing up to 48 different samples simultaneously, sequencing single projects at extremely high coverage, or any combination thereof. The flexibility of the PromethION platform is one of its key advantages enabling sequencing at scale or across wide numbers of samples. We, and others, have shown that read length is limited only by the quality of the input DNA. The platform also enables in principle the detection of methylation and modifications to DNA with relatively simple library preparations. NGS technologies are applicable to a wide range of research questions. The long read NGS technologies outlined here will greatly enhance research at the UoN and, by embedding within the regional Midlands Sequencing Consortium, in the Midlands as a whole. The projects cover a broad range of fields including: Bioscience for Health, Food, Nutrition and Health, Agriculture and Food Security, Stem Cell and Developmental Biology, Data Driven Biology, Exploiting New Ways of Working and Synthetic Biology.The development of an enhanced long read NGS facility at the UoN embedded within the regional Midlands Sequencing Consortium will enable the UoN and collaborators to address key biological questions in the above areas which will have a significant impact in science, the economy and society.