Development and applications for long read sequencing in the Midlands

自从弗雷德里克·桑格（Frederick Sanger）推动的测序技术问世以来，对生物体潜在遗传密码的了解一直是建立遗传影响表型机制的关键特征。在过去的十年中，下一代测序技术的迅速发展使我们从试图确定单个人类基因组序列的大型财团到能够以近1000美元的价格对自己的基因组进行测序的个人。除了基因组DNA的测序之外，还可以对转录组（在细胞或组织中表达的基因）进行测序，或者研究改变基因表达的DNA碱基的修饰。尽管UoN拥有一系列可用的短读段测序技术，并且一直处于MinION平台上开发长读段测序的最前沿，但在本提案所解决的高通量长读段生成水平上存在明显差距。UoN目前可用的短读技术允许对微生物群落进行测序，对人类规模的基因组进行外显子测序，以及对个体人类规模的基因组进行重新测序。对于长读段测序，诺丁汉领导了MinION测序的开发，并有能力在单个流动池上对细菌基因组进行测序。DeepSeq是诺丁汉大学的测序设施，由该提案的PI（Matt Loose博士）领导，为世界各地多个地点的多个MinION上的参考人类基因组测序做出了贡献。我们也是第一个展示使用纳米孔平台选择性测序分子的能力的人，我们希望其他人能够在未来更大规模地利用这项技术。DeepSeq鼓励其他人将MinION测序应用于他们的项目，在当地和通过全国范围内的“porecamp”系列研讨会提供培训。DeepSeq还为MinoTour等分析工具的开发做出了贡献，用于对MinION数据进行真实的实时分析。现在，我们希望将我们的知识和专业知识扩展到可通过PromethION平台的规模增加来解决的显著更高的吞吐量问题。我们寻求购买牛津Nanopore PromethION测序仪以及计算支持和重要的数据存储，能够同时对多达48个不同的样品进行测序，以极高的覆盖率对单个项目进行测序，或其任何组合。PromethION平台的灵活性是其关键优势之一，可实现大规模或大量样品的测序。我们和其他人已经表明，读长仅受输入DNA质量的限制。该平台原则上还可以通过相对简单的文库制备来检测DNA的甲基化和修饰。NGS技术适用于广泛的研究问题。这里概述的长阅读NGS技术将大大加强在UoN的研究，并通过嵌入在区域米德兰测序联盟，在整个米德兰。这些项目涵盖广泛的领域，包括：健康生物科学，食品，营养和健康，农业和粮食安全，干细胞和发育生物学，数据驱动生物学，开发新的工作方式和合成生物学。在UoN开发嵌入区域米德兰测序联盟的增强型长读NGS设施将使UoN和合作者能够解决关键的生物学问题。这些领域将对科学、经济和社会产生重大影响。

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.