Development and applications for long read sequencing in the Midlands

中部地区长读长测序的开发和应用

• 批准号: BB/R000492/1
• 负责人: Matthew Loose
• 金额: $ 26.78万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR000492%2F1
• 关键词: Development; applications; long; read; sequencing

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.

自从弗雷德里克·桑格（Frederick Sanger）推动的测序出现以来，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。过去十年中，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，发展到能够花费近 1,000 美元对自己的基因组进行测序的个人。除了基因组 DNA 测序之外，还可以对转录组（细胞或组织中表达的基因）进行测序，或研究改变基因表达的 DNA 碱基修饰。尽管 UoN 拥有一系列可用的短读测序技术，并且一直处于 MinION 平台上开发长读测序的最前沿，但在本提案所解决的高通量长读生成水平上存在明显差距。目前 UoN 提供的短读技术允许对微生物群落进行测序、对人类规模基因组进行外显子测序以及对个体人类规模基因组进行重新测序。对于长读长测序，诺丁汉大学领导了 MinION 测序的开发，并有能力在单个流动细胞上对细菌基因组进行测序。诺丁汉大学测序机构 DeepSeq 在该提案的 PI（Matt Loose 博士）的领导下，在世界各地多个地点的多个 MinION 上对参考人类基因组进行了测序。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，我们希望其他人将来能够更大规模地利用这项技术。 DeepSeq 鼓励其他人将 MinION 测序应用到他们的项目中，在本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，用于实时分析 MinION 数据。现在，我们希望将我们的知识和专业知识扩展到可通过扩大 PromethION 平台规模来解决的显着更高的吞吐量问题。我们寻求购买一台 Oxford Nanopore PromethION 测序仪以及计算支持和重要的数据存储，该测序仪能够同时对多达 48 个不同的样本进行测序，以极高的覆盖率对单个项目进行测序，或其任意组合。 PromethION 平台的灵活性是其实现大规模测序或跨大量样本测序的关键优势之一。我们和其他人已经证明，读段长度仅受输入 DNA 质量的限制。该平台原则上还能够通过相对简单的文库制备来检测 DNA 甲基化和修饰。 NGS 技术适用于广泛的研究问题。本文概述的长篇 NGS 技术将极大地加强 UoN 的研究，并通过嵌入区域中部地区测序联盟以及整个中部地区的研究。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、探索新的工作方式和合成生物学。在北方大学开发增强型长读长NGS设施，嵌入区域中部测序联盟内，将使北方大学和合作者能够解决上述领域的关键生物学问题，这将有助于解决上述领域的关键生物学问题。 对科学、经济和社会产生重大影响。

项目成果

FindingNemo Library 1: Modified ULK001 v1

FindNemo 库 1：修改后的 ULK001 v1

• DOI: 10.17504/protocols.io.bxhhpj36
• 发表时间: 2021
• 作者: Cahyani I

FindingNemo in OneDay: Ultra-Long ONT Library Preparation from Cell to Flowcell in One Day v1

FindNemo in OneDay：一日内从 Cell 到 Flowcell 的超长 ONT 文库制备 v1

• DOI: 10.17504/protocols.io.bxhxpj7n
• 发表时间: 2021
• 作者: Cahyani I

FindingNemo Extraction 2: Phenol-free Method v2

FindNemo 提取 2：无酚方法 v2

• DOI: 10.17504/protocols.io.bxx2ppqe
• 发表时间: 2021
• 作者: Cahyani I

FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms v1

FindNemo：基于 CoHex 和玻璃珠的协议工具包，用于 ONT 平台 v1 上的超长测序

• DOI: 10.17504/protocols.io.bxwrppd6
• 发表时间: 2021
• 作者: Cahyani I

FindingNemo Library 2: Modified RAD004 v1

FindNemo 库 2：修改的 RAD004 v1

• DOI: 10.17504/protocols.io.bxv4pn8w
• 发表时间: 2021
• 作者: Cahyani I