High throughput intermediate scale sequencing for the Midlands

中部地区高通量中规模测序

• 批准号: BB/M012336/1
• 负责人: Matthew Loose
• 金额: $ 35.7万
• 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: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM012336%2F1
• 关键词: throughput; intermediate; scale; sequencing; Midlands

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.

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。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来研究细菌复制过程中基因组是如何复制的。尽管诺丁汉大学拥有一系列可用的测序技术，但该提案解决了两个层面的测序技术之间存在明显的差距。北方大学现有的技术允许在个体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。这些规模阻碍了使用 NGS 来研究具有较大基因组的多种生物体，而在这些生物体中，可以轻松应用许多为研究良好表征的基因组而开发的技术。相反，在进行靶向和随机诱变实验以设计新表型（合成生物学）时，小基因组的全基因组测序（WGS）可能非常重要。这里，补偿性或脱靶突变体的产生是一种风险，因此可以应用常规全基因组测序来筛选此类事件。在这种规模下，与文库制备相关的劳动力成本限制了这些方法的成本效益。为了解决这些问题，我们寻求购买一台 NextSeq500 测序仪 (Illumina) 以及一台 BioMEK 4000 液体处理机器人，该机器人能够同时制备 24 个独立的文库进行测序。 NextSeq 技术理论上可以在一次运行中对 96 个文库进行测序，从而能够同时对多个细菌基因组进行经济有效的高通量测序。 BioMEK 4000 还可用于制备各种适合测序的文库，从而大大降低研究人员的管理成本。所有 Illumina 测序平台之间生成的文库的交叉兼容性将使 DeepSeq 能够准备可以使用我们合作伙伴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 技术将极大地加强 UoN 在广泛领域的研究。其中一些领域包括： (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) 食品安全 (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) 探索新的工作方式。UoN 增强型 NGS 设施的开发以及区域米德兰测序联盟的建立将使 UoN 及其合作者能够解决上述领域的关键生物学问题，这将对科学、经济和社会产生重大影响。

项目成果

Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments.

• DOI: 10.1186/s13068-017-0700-9
• 发表时间: 2017
• 期刊: Biotechnology for biofuels
• 影响因子: 6.3
• 作者: Daly P;van Munster JM;Blythe MJ;Ibbett R;Kokolski M;Gaddipati S;Lindquist E;Singan VR;Barry KW;Lipzen A;Ngan CY;Petzold CJ;Chan LJG;Pullan ST;Delmas S;Waldron PR;Grigoriev IV;Tucker GA;Simmons BA;Archer DB
• 通讯作者: Archer DB

Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824.

• DOI: 10.1186/s13068-015-0410-0
• 发表时间: 2016
• 期刊: Biotechnology for biofuels
• 影响因子: 6.3
• 作者: Ehsaan M;Kuit W;Zhang Y;Cartman ST;Heap JT;Winzer K;Minton NP
• 通讯作者: Minton NP

Signatures of Selection for Environmental Adaptation and Zebu × Taurine Hybrid Fitness in East African Shorthorn Zebu.

• DOI: 10.3389/fgene.2017.00068
• 发表时间: 2017
• 期刊: Frontiers in genetics
• 影响因子: 3.7
• 作者: Bahbahani H;Tijjani A;Mukasa C;Wragg D;Almathen F;Nash O;Akpa GN;Mbole-Kariuki M;Malla S;Woolhouse M;Sonstegard T;Van Tassell C;Blythe M;Huson H;Hanotte O
• 通讯作者: Hanotte O

Transcriptomic responses of mixed cultures of ascomycete fungi to lignocellulose using dual RNA-seq reveal inter-species antagonism and limited beneficial effects on CAZyme expression.

• DOI: 10.1016/j.fgb.2016.04.005
• 发表时间: 2017-05
• 期刊: Fungal genetics and biology : FG & B
• 作者: Daly P;van Munster JM;Kokolski M;Sang F;Blythe MJ;Malla S;Velasco de Castro Oliveira J;Goldman GH;Archer DB
• 通讯作者: Archer DB