Sequencing and Bioinformatics

测序和生物信息学

• 批准号: 10650879
• 负责人: Andrew Laurence Routh
• 金额: $ 26.38万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-22 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10650879
• 关键词: Area; Behavior; Bioinformatics; Biological; Biology; Capsid; Cell Culture Techniques; Cells; Chromatin Structure; Chromosome Structures; Clinical; Collaborations; Communities; Complementary DNA; Computer Analysis; Computer software; Core Facility; DNA Sequencing Facility; Data; Databases; Development; Drug resistance; Environment; Event; Evolution; Experimental Designs; Exposure to; Gene Expression; Genetic Recombination; Genome; Genomic approach; HIV; HIV Genome; Human Resources; In Situ; In Vitro; Individual; Integrase; Length; Life Cycle Stages; Location; Measures; Messenger RNA; Minority; Molecular; Molecular Biology; Molecular Virology; Mutation; Nucleic Acids; Nucleotides; Nuts; Output; Pattern; Pharmacotherapy; Polymerase; Process; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA Viruses; RNA-Protein Interaction; Research; Research Personnel; Research Project Grants; Resolution; Resources; Role; Sampling; Services; Single Nucleotide Polymorphism; Site; Specimen; Structure; System; Techniques; Technology; Training; Universities; Variant; Viral; Viral Genome; Virus; Virus Replication; Work; antiretroviral therapy; chromosomal location; crosslink; experience; experimental study; genome sequencing; innovation; insight; integration site; molecular sequence database; nanopore; new technology; next generation; next generation sequencing; novel; novel strategies; particle; pressure; resistance mutation; response; sequencing platform; single cell sequencing; single cell technology; single molecule; skills; small molecule inhibitor; technology platform; tool; transcriptome sequencing; transcriptomics; viral genomics; virology

ABSTRACT, CORE 4 Next-Generation Sequencing is an essential and fundamental approach in molecular virology. In vitro, it can capture and characterize protein-nucleic interactions and RNA structure. In cell culture and in clinical settings, it can measure rates of viral polymerase error, viral response to small molecule inhibitors or changes in host gene expression. NGS can chart viral evolution, patterns of genome conservation, characterize origins of drug resistance and discover new viruses. As such, NGS has been transformative in our ability to study virus diversity and evolution and is still a burgeoning field. The Sequencing Core comprises a group of experts with experience with the implementation of the wide range of approaches in terms of molecular biology, experimental design, and downstream computational analyses. We will provide expert support and resources to provide Next- Generation Sequencing and downstream computational analyses to the B-HIVE Research Projects. Importantly, a key focus of the core will be to develop and apply approaches that go beyond what is accessible via commercial and university core facilities. We will provide and develop state-of-the-art technologies in close collaboration with the Research Projects that will help provide unique insights into HIV biology and evolution, whilst also providing novel platforms for deployments in other viral systems. A central focus of the B-HIVE is to understand the molecular mechanisms governing HIV entry, integration, particle assembly, and egress. Each of these processes involve profiling the viral and host nucleic acids during the viral lifecycle. The sequencing core will support the entire B-HIVE reflecting stages throughout the viral replication cycle. We will develop single-cell sequencing approaches, leveraging our extensive expertise in this area, to characterize the effects of integration site location, chromatin structure, HIV mutations, and exposure to latency reversing agents (LRAs) on proviral expression. We will apply and advance long-read sequencing technologies using the Oxford Nanopore Technologies MinION platform for both host transcriptomics and full- length HIV genome sequencing to characterize host and virus splicing. We will develop and implement novel approaches in virus genomics to characterize virus sequence diversity in both clinical and experimental settings in support of the main Research Projects. The Sequencing Core has extensive experience developing, deploying and maintaining new computational software and sequencing databases. These will provide an important resource to the B-HIVE centers, as well as allow the rapid dissemination and sharing of capabilities with the broader HIV and virus research community. Finally, we will provide training in each of our respective platforms and technologies in view of disseminating these skills throughout the B-HIVE and to support the development and progression of trainees working with the B-HIVE and its collaborators. 1

摘要，核心 4 下一代测序是分子病毒学中必不可少的基本方法。在体外，它可以 捕获并表征蛋白质-核相互作用和 RNA 结构。在细胞培养和临床环境中，它 可以测量病毒聚合酶错误率、病毒对小分子抑制剂的反应或宿主基因的变化 表达。 NGS 可以绘制病毒进化图、基因组保存模式、表征药物起源 抵抗力并发现新病毒。因此，NGS 改变了我们研究病毒多样性的能力 和进化论，并且仍然是一个新兴领域。测序核心由一群经验丰富的专家组成 随着分子生物学、实验设计等方面广泛方法的实施， 和下游计算分析。我们将提供专家支持和资源，以提供下一步- B-HIVE 研究项目的生成测序和下游计算分析。 重要的是，核心的重点将是开发和应用超出可访问范围的方法 通过商业和大学核心设施。我们将密切提供和开发最先进的技术 与研究项目的合作将有助于提供对艾滋病毒生物学和进化的独特见解， 同时还为其他病毒系统中的部署提供新颖的平台。 B-HIVE 的一个中心重点是了解控制 HIV 进入、整合、 粒子组装和出口。这些过程中的每一个都涉及在过程中对病毒和宿主核酸进行分析。 病毒生命周期。测序核心将支持整个病毒传播过程中的整个 B-HIVE 反映阶段 复制周期。我们将利用我们在这方面的丰富专业知识开发单细胞测序方法 区域，以表征整合位点位置、染色质结构、HIV 突变和暴露的影响 潜伏期逆转剂（LRA）对原病毒表达的影响。我们将应用和推进长读长测序 使用 Oxford Nanopore Technologies MinION 平台进行宿主转录组学和全转录组学的技术 长度 HIV 基因组测序来表征宿主和病毒剪接。我们将开发并实施新颖的 病毒基因组学方法在临床和实验环境中表征病毒序列多样性 支持主要研究项目。测序核心拥有丰富的开发、部署经验 维护新的计算软件和测序数据库。这些将提供一个重要的 向 B-HIVE 中心提供资源，并允许与 更广泛的艾滋病毒和病毒研究界。最后，我们将在各自的平台上提供培训 和技术，以在整个 B-HIVE 中传播这些技能并支持发展 以及与 B-HIVE 及其合作者一起工作的学员的进步。 1