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.



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