Massively Parallel High Throughput, Low Cost Sequencing

描述：(申请人提供)背景：大规模基因组测序目前需要高成本的设备，并且是劳动密集型的。传统测序的吞吐量已经不足以满足不断增长的基因组序列需求。要了解人类基因组织的错综复杂及其与人类健康和遗传的关系，需要进行基因组水平的比较分析，但由于缺乏序列信息，目前无法进行这种分析。 454公司开发了一种大规模并行的高通量测序仪，它将数十万皮升规模的反应井中的同时测序与高性能生物信息学相结合。尽管基因组大小呈指数级增长，但该方法不需要在工作量或成本上呈指数级增长。传统测序的工作量和成本与基因组的大小成比例地扩大。454方法的成本将很低，并使各种实验室都可以对大型基因组进行测序。 具体目标：在本计划中，我们将(I)构建一种从每个单独片段的两端生成短序列的健壮的双端测序方法(Ii)开发一种适用于双端测序的健壮的序列组装工具(Iii)将我们的放大系统用于经济、快速的哺乳动物单倍型的双端测序。 我们的研究设计融合了454公司分子生物学、化学、工程、软件和生物信息学小组的多学科努力。分子生物学和测序工作将由PI Kenton L.Lohman博士领导。硬件、流体、光学、软件和生物信息学工作将由联席PI马塞尔·马古利斯博士领导。我们将利用现有的454基础设施和关键人员。 相关性：研究、制药和临床领域对低成本、高通量基因组测序的需求日益增长。比较基因组学、SNP和单倍型分析已经显示出巨大的潜力来快速表征个体对许多类别的慢性和急性疾病和障碍的易感性。目前全基因组测序的成本只能由大型机构承担。自动样品准备和测序的成本与正在测序的基因组的大小成比例地扩大。454测序系统同时分析哺乳动物生物体大规模并行测序中的数百万个片段。大规模并行测序和生物信息学分析的使用创造了一种低成本、高通量的测序系统。

DESCRIPTION: (provided by applicant) Background: Large-scale genomic sequencing currently requires high cost equipment and is labor intensive. The throughput of conventional sequencing has grown inadequate in fulfilling the escalating demands for genomic sequence. Understanding the intricacies of human genetic organization and how it relates to human health and inheritance, requires genomic-level comparative analyses that cannot currently be performed due to the lack of sequence information. The 454 Corporation has developed a massively parallel, high-throughput sequencing instrument that combines simultaneous sequencing in hundreds of thousands of picoliter-scale reaction wells, with high-powered bioinformatics. The method does not require an exponential scale up in effort or cost, despite exponential increases in genome size. The effort and cost of conventional sequencing scales up proportionately with the size of the genome. The 454 approach will be low cost, and make sequencing large genomes available to a wide variety of laboratories. Specific Aims: In this Program we will (i) Construct a robust double-ended sequencing method that generates short sequences from both ends of each individual fragment (ii) Develop a robust sequence assembly tool appropriate for double-ended sequencing (iii) Prototype the use of our scaled up system, with double-ended sequencing for economical, rapid haplotyping of mammalian organisms. Our Study design incorporates a multi-disciplinary effort across molecular biology, chemistry, engineering, software and bioinformatics groups at 454 Corporation. The molecular biology and sequencing efforts will be lead by the PI, Dr. Kenton L. Lohman. The hardware, fluidics, optics, software and bioinformatics efforts will be lead by co-PI, Dr. Marcel Margulies. We will be taking advantage of current 454 infrastructure, and key personnel. Relevance: There is a growing need across the research, pharmaceutical and clinical communities for low-cost, high throughput genomic sequencing. Comparative genomics, SNP and haplotype analyses have shown tremendous potential to rapidly characterize individual susceptibilities to many classes of chronic and acute diseases and disorders. Current costs of whole genome sequencing can only be borne by large institutions. The cost of automated sample preparation and sequencing, scales up proportionately to the size of the genome being sequenced. The 454 Sequencing system simultaneously analyzes millions of fragments in massively parallel sequencing of mammalian organisms. The use of massively parallel sequencing and bioinformatics analysis creates a low cost, high throughput sequencing system.