454 Life Sciences Massively Parallel Sys.:DNA Sequencing

454 生命科学大规模并行系统：DNA 测序

• 批准号: 6954120
• 负责人: MARCEL MARGULIES
• 金额: $ 148.91万
• 依托单位: 454 LIFE SCIENCES CORPORATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-29 至 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954120
• 关键词: bioengineering /biomedical engineering; biomedical equipment development; biotechnology; clinical research; high throughput technology; human subject; nucleic acid sequence

DESCRIPTION (provided by applicant): 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 characterize individual susceptibilities to many chronic and acute diseases or disorders. Additionally, genomic data will lead to advances in agriculture, environmental sciences and further our understanding of evolution and ecological systems. However, the cost of sequencing mammalian-sized genomes is currently too high and we remain too far away from being able to afford the use of comprehensive genomic sequence information on a routine basis, in part because such large-scale sequencing requires a great deal of equipment and is labor intensive. Of equal importance with a significant decrease in cost, is the need to develop a complete platform that brings to any research laboratory the capability to perform sequencing of sizable organisms without a large and expensive infrastructure. Background. 454 Life Sciences has developed a massively parallel, high-throughput sequencing system, designed to simplify, parallelize and speed up all aspects of sequencing viral and bacterial genomes, from sample preparation, through amplification and sequencing, to data processing and assembly. There is one sample preparation and one amplification process for a whole genome, done without need for robotics, cloning or colony picking, by one individual, in one laboratory. That same individual can do whole genome sequencing on a single high throughput instrument that simultaneously sequences all fragments in hundreds of thousands to millions of picoliter-scale reaction wells, and performs basecalling and scaffolding in real time, with consensus accuracies of > 99.99%. Currently, sequencing of viruses and bacteria is performed at a throughput of 5 Mbp/hour. Under a recently funded NIH grant, 454 will be scaling up this system to perform paired-end sequencing of whole genomes up to the size of small fungi: Projects. We will further expand the existing platform to handle resequencing and de novo sequencing of mammalian genomes at very low cost and high accuracy in 3 projects: (i) Scaling the 454 hardware to achieve two orders of magnitude reduction in the cost per base, at a throughput of up to 50 Mb/hour, and at an accuracy of > 99.99%. (ii) Extending the 454 molecular biology to very small beads and to combined read lengths of 400 basepairs with paired-end sequencing of very long fragments, (iii) Extending the modular assembler algorithms to allow the use of large-span paired-end reads, leading to resequencing and de novo assembly of mammalian-sized genomes. 454 relies on a very talented, multi-disciplinary team that encompasses engineering, molecular biology, chemistry, software and bioinformatics groups. The hardware, fluidics, optics, software and bioinformatics efforts will be led by PI Dr. Marcel Margulies. The molecular biology, chemistry and sequencing efforts will be led by co-Pi, Dr. Michael Egholm. This facility will cover less than 3,000 sq. ft and be staffed by less than 10 personnel. We will be ready to deploy such a facility commercially at other sites at the end of year 3.

描述（由申请人提供）：相关性。研究、制药和临床领域对低成本、高通量的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表征个体对许多慢性和急性疾病或病症的易感性的巨大潜力。此外，基因组数据将导致农业，环境科学的进步，并进一步加深我们对进化和生态系统的理解。然而，目前测序大规模基因组的成本太高，并且我们仍然离能够负担得起在常规基础上使用全面的基因组序列信息太远，部分原因是这种大规模测序需要大量的设备并且是劳动密集型的。与成本显著降低同等重要的是，需要开发一个完整的平台，该平台为任何研究实验室带来在没有大型和昂贵的基础设施的情况下对相当大的生物体进行测序的能力。 背景454 Life Sciences开发了一种大规模并行、高通量测序系统，旨在简化、并行化和加速病毒和细菌基因组测序的各个方面，从样品制备、扩增和测序，到数据处理和组装。在一个实验室里，一个人可以完成一个样品制备和一个完整基因组的扩增过程，而不需要机器人、克隆或菌落挑选。同一个人可以在单个高通量仪器上进行全基因组测序，该仪器同时对数十万至数百万皮升规模的反应威尔斯孔中的所有片段进行测序，并在真实的时间内进行碱基判定和支架构建，一致性准确度> 99.99%。目前，病毒和细菌的测序以5 Mbp/小时的通量进行。在最近资助的NIH拨款下，454将扩大该系统的规模，对整个基因组进行配对末端测序，直到小型真菌的大小： 项目我们将进一步扩展现有的平台，以极低的成本和高精度在3个项目中处理哺乳动物基因组的重新测序和从头测序：（i）扩展454硬件，以实现每个碱基成本降低两个数量级，吞吐量高达50 Mb/小时，准确度> 99.99%。(ii)将454分子生物学扩展到非常小的珠粒，并将400个碱基对的读取长度与非常长片段的配对末端测序相结合，（iii）扩展模块化组装器算法，以允许使用大跨度配对末端读取，从而导致重新测序和重新组装拟南芥大小的基因组。 454依靠一个非常有才华的多学科团队，包括工程，分子生物学，化学，软件和生物信息学小组。硬件，流体，光学，软件和生物信息学工作将由PI Marcel Margulies博士领导。分子生物学，化学和测序工作将由co-Pi，Michael Egholm博士领导。 该设施占地面积不到3,000平方米。并配备少于10名员工。我们将准备在第三年年底在其他地点部署这种设施。