Making structurally complex genomic regions accessible

使结构复杂的基因组区域变得可访问

• 批准号: 9249078
• 负责人: David C. Page
• 金额: $ 90.11万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-24 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9249078
• 关键词: Academia; Animal Model; Autistic Disorder; Bacterial Artificial Chromosomes; Basic Science; Biological; Biomedical Research; Blood capillaries; ChIP-seq; Chickens; Chromosomes; Chromosomes, Human, X; Communities; Complex; Computer software; DNA Fragmentation; DNA Resequencing; Discipline; Disease; Ensure; Eye; Failure; Foundations; Frequencies; Generations; Genes; Genetic; Genetic Polymorphism; Genome; Genomic Segment; Goals; Haplotypes; Human; Human Genetics; Human Genome; Individual; Industry; Intellectual functioning disability; Kinetics; Laboratories; Length; Libraries; Link; Macaca mulatta; Malignant Neoplasms; Maps; Mediating; Medical; Methodology; Mus; Nature; Nucleotides; Pan Genus; Plasmids; Predisposition; Preparation; Process; Production; Reagent; Repetitive Sequence; Reproducibility; Research; Research Personnel; Resources; Rest; Role; Shotguns; Technology; Time; Translational Research; Turner' s Syndrome; Validation; Variant; X Chromosome; Y Chromosome; base; capillary; cost; design; exome; experimental study; flexibility; genome sequencing; human reference genome; improved; indexing; insight; interest; mammalian genome; new technology; public health relevance; reference genome; sex; tool; transcriptome sequencing; whole genome

 DESCRIPTION (provided by applicant): The most structurally complex regions in the genome are comprised of ampliconic sequences, which are defined as repeats that display >99% identity and are >10 kb in length. Ampliconic regions are of immensely disproportionate biomedical significance and interest. However, these regions are inaccessible by standard genome sequencing strategies, so are grossly misrepresented in or entirely missing from reference genome assemblies. Biomedical researchers cannot extract insights from parts of the genome to which they have no access, so our understanding of the frequency and mechanism of amplicon-mediated rearrangements and their role in disease is far from complete. Furthermore, ampliconic sequences are systematically excluded from all experiments based on mapping to the reference sequence (e.g. exome re-sequencing, RNA-seq, ChIP-seq), severely limiting the insights to be gained from such studies. The chief obstacle to accessing entire genomes is not a lack of interest on the part of the biomedical research community, but the lack of a practical, affordable, and distributable technology with which to generate reference-quality sequence of ampliconic regions. Single Haplotype Iterative Mapping and Sequencing (SHIMS) is the only proven strategy to assemble such regions. SHIMS relies on the use of mapped large-insert clones (usually BACs) derived from a single haplotype so that polymorphisms do not confound the assembly of ampliconic repeats. The major bottleneck and cost associated with the traditional SHIMS approach - SHIMS 1.0 - is the sequencing of individual BACs. Using standard capillary-based sequencing, this endeavor is expensive in terms of both reagents and highly skilled labor. Here we propose to dramatically restructure the SHIMS operational paradigm, so that ultra-high-quality reference sequence can be generated by a small research team at modest cost. We will achieve this by setting up an efficient SHIMS 2.0 pipeline encompassing all steps in generating finished BAC sequence using the Illumina MiSeq platform. We will sequence pools of 192 indexed BACs, generating deep sequence coverage that will dramatically reduce if not eliminate the need for directed finishing. We will optimize all components of the process, from high-throughput plasmid preparation and DNA fragmentation to de novo sequence assembly and quality assessment, with an eye toward quality of product, cost, efficiency, and reproducibility. We will ensure that this new technology and software is distributable and actively promote and support the application of the SHIMS 2.0 pipeline by other researchers to complex genomic regions. For example, it will be possible to use SHIMS 2.0 to assemble multiple human genomes, providing an invaluable resource for studies in human genetics. The SHIMS 2.0 strategy can be applied in other species, enabling insight into the evolutionary dynamics of ampliconic regions. In addition, applying SHIMS 2.0 to improve the genomes of model organisms will be of tremendous benefit to researchers in multiple biomedical disciplines.

 描述（由适用提供）：基因组中最结构上复杂的区域的扩增序列完成，该序列定义为重复序列，显示> 99％的身份，长度> 10 kb。扩增区域具有极大的生物医学意义和兴趣。但是，这些区域是由标准基因组测序策略无法访问的，因此在参考基因组组件中严重少了或完全缺少。生物医学研究人员无法从无法进入的部分基因组中提取见解，因此我们对扩增子介导的重排的频率和机制的理解及其在疾病中的作用远非完整。此外，基于对参考序列的映射（例如，外部重新序列，RNA-Seq，chip-seq）的所有实验中，扩增序列被系统地排除在外，严重限制了从此类研究中获得的见解。获取整个基因组的主要障碍并不是生物医学研究界缺乏兴趣，而是缺乏实用，负担得起且可分布的技术来产生扩增区域的参考质量序列。单个单倍型迭代映射和测序（SHIMS）是组装此类区域的唯一经过验证的策略。 Shims依赖于使用源自单个单倍型的映射的大插入克隆（通常是BAC），因此多态性不会混淆扩展重复序列的组装。与传统垫片方法相关的主要瓶颈和成本 - 垫片1.0-是单个BAC的测序。使用基于标准的毛细管测序，就试剂和高技能劳动而言，这项工作都是昂贵的。在这里，我们建议大幅度恢复Shims操作范式，以便以适度的成本以适度的成本生成超高质量的参考顺序。我们将通过设置有效的Shims 2.0管道来实现这一目标，其中包含使用Illumina Miseq平台生成完成的BAC序列的所有步骤。我们将对192个索引BAC的池进行序列库，从而产生深层序列覆盖范围，如果不消除对定向饰面的需求，将大大减少。我们将优化该过程的所有组成部分，从高通量质粒制备和DNA碎片到从头序列组装和质量评估，并着眼于产品质量，成本，效率和可重复性。我们将确保该新技术和软件是可以分发的，并积极促进和支持其他研究人员在复杂的基因组区域中应用Shims 2.0管道。例如，可以使用Shims 2.0组装多个人类基因组，为人类遗传学研究提供了宝贵的资源。 SHIMS 2.0策略可以应用于其他物种，从而深入了解扩增区域的进化动力学。此外，应用SHIMS 2.0改善模型生物的基因组将对多个生物医学学科的研究人员带来巨大的好处。

项目成果

Sequence analysis in Bos taurus reveals pervasiveness of X-Y arms races in mammalian lineages.

• DOI: 10.1101/gr.269902.120
• 发表时间: 2020-12
• 期刊: Genome research
• 影响因子: 7
• 作者: Hughes JF;Skaletsky H;Pyntikova T;Koutseva N;Raudsepp T;Brown LG;Bellott DW;Cho TJ;Dugan-Rocha S;Khan Z;Kremitzki C;Fronick C;Graves-Lindsay TA;Fulton L;Warren WC;Wilson RK;Owens E;Womack JE;Murphy WJ;Muzny DM;Worley KC;Chowdhary BP;Gibbs RA;Page DC
• 通讯作者: Page DC