High throughput intermediate scale sequencing for the Midlands

自从弗雷德里克·桑格（Frederick Sanger）推动测序技术问世以来，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。诺丁汉大学的研究人员已经使用NGS来研究基因组内序列拷贝数的变化，甚至使用NGS来研究基因组在细菌复制过程中是如何复制的。尽管UoN有一系列可用的测序技术，但在本提案所解决的两个测序水平上存在明显的差距。UoN现有的技术允许在单个微生物基因组水平上进行测序，或对人类基因组的小区域进行靶向测序。这些规模排除了使用NGS来研究具有较大基因组的多种生物，其中许多用于研究特征良好的基因组的技术可以很容易地应用。在相反的规模上，小基因组的全基因组测序（WGS）在进行靶向和随机诱变实验以设计新表型（合成生物学）时可能非常重要。在这里，补偿性或脱靶突变体的产生是一种风险，因此可以应用常规WGS来筛选此类事件。为了解决这些问题，我们寻求购买NextSeq500测序仪（Illumina）以及BioMEK 4000液体处理机器人，该机器人将能够在任何时间制备24个独立的文库用于测序。NextSeq技术理论上可以在单次运行中对96个文库进行测序，从而能够同时对多个细菌基因组进行具有成本效益的高通量测序。BioMEK 4000还可用于制备适用于测序的各种文库，大大降低了研究人员的间接成本。所有Illumina测序平台之间生成的文库的交叉兼容性将允许DeepSeq准备可以使用我们合作机构的能力进行测序的文库。NGS技术有广泛的研究问题。这里概述的NGS技术将极大地增强UoN在广泛领域的研究。其中一些字段包括：（i）合成生物学（ii）分子微生物学（ii）工业生物技术和生物能源（iv）粮食安全（v）再生医学（vi）干细胞和发育生物学（vii）健康老龄化（viii）衰老和疾病的表观遗传基础（ix）健康生物科学（x）系统生物学和生物信息学（Xi）开发新的工作方式。在建立区域米德兰测序联盟的同时，在UoN开发增强的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, the 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. Researchers at the University of Nottingham have used NGS to investigate changes in the copy number of sequences within the genome and even used NGS to investigate how genomes are copied during bacterial replication.Although the UoN has a range of sequencing technologies available, there is a clear gap at two levels of sequencing that are addressed by this proposal. Current technologies available at UoN allow sequencing at the level of individual microbial genomes or targeted sequencing of small regions of the human genome. These scales preclude the use of NGS to study diverse organisms with larger genomes where many of the techniques developed for studying well-characterised genomes can be readily applied. At the opposite scale, whole genome sequencing (WGS) of small genomes can be of enormous importance when carrying out both targeted and random mutagenesis experiments to engineer new phenotypes (synthetic biology). Here the generation of compensatory or off target mutants is a risk and so routine WGS can be applied to screen against such events. At this scale, the labour costs associated with library preparation limit the cost effectiveness of these approaches.To address these issues we seek to purchase a NextSeq500 sequencer (Illumina) alongside a BioMEK 4000 liquid handling robot that will be capable of preparing 24 independent libraries for sequencing at any one time. The NextSeq technology can theoretically sequence 96 libraries in a single run enabling cost effective high throughput sequencing of multiple bacterial genomes simultaneously. The BioMEK 4000 can also be used to prepare a wide range of libraries suitable for sequencing drastically reducing overhead costs to researchers. The cross compatibility of libraries generated between all the Illumina sequencing platforms will allow DeepSeq to prepare libraries which can be sequenced using capacity at our partner institutions.NGS technologies have a wide range of research questions. The NGS technologies outlined here will greatly enhance research at the UoN in a broad range of fields. Some of these fields include: (i) Synthetic Biology (ii) Molecular Microbiology (ii) Industrial Biotechnology and Bioenergy (iv) Food Security (v) Regenerative Medicine (vi) Stem Cell and Developmental Biology (vii) Healthy Aging (viii) Epigenetic Basis of Aging and Disease (ix) Bioscience for Health (x) Systems Biology and Bioinformatics (xi) Exploiting New Ways of Working.The development of an enhanced NGS facility at the UoN alongside the establishment of a 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.