454 Life Sciences Massively Parallel Sys.:DNA Sequencing

描述(申请人提供)：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表征个体对许多慢性和急性疾病或障碍的易感性。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，目前对哺乳动物大小的基因组进行测序的成本太高，我们距离能够在常规基础上使用全面的基因组序列信息还太远，部分原因是这种大规模的测序需要大量的设备，而且是劳动密集型的。在显著降低成本的同时，同样重要的是需要开发一个完整的平台，使任何研究实验室都能够在不需要大型且昂贵的基础设施的情况下对大规模生物进行测序。 背景资料。454生命科学公司开发了一种大规模并行的高通量测序系统，旨在简化、并行化和加快病毒和细菌基因组测序的各个方面，从样本准备到扩增和测序，再到数据处理和组装。整个基因组只需一次样本准备和一次扩增过程，不需要机器人、克隆或菌落挑选，由一个实验室的一个人完成。同一个人可以在一台高通量仪器上进行全基因组测序，该仪器可以同时对数十万到数百万皮升规模的反应井中的所有片段进行测序，并实时执行基本操作和脚手架，普遍的准确率为99.99%。目前，病毒和细菌的测序速度为5 MBP/小时。根据美国国立卫生研究院最近资助的一项拨款，454将扩大这一系统的规模，对小真菌大小的整个基因组进行配对末端测序： 项目。我们将进一步扩展现有平台，在3个项目中以极低的成本和高精度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50Mb/小时，准确率为99.99%。(Ii)将454分子生物学扩展到非常小的珠子，并将400个碱基对的读取长度与超长片段的配对末端测序相结合；(Iii)扩展模块化汇编算法，以允许使用大跨度的配对末端读取，从而导致哺乳动物大小的基因组的重新测序和从头组装。 454依靠一支非常有才华的多学科团队，其中包括工程、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圆周率博士马塞尔·马古利斯领导。分子生物学、化学和测序工作将由联席PI迈克尔·埃格霍尔姆博士领导。这个设施的面积将不到3,000平方米。英国《金融时报》，员工人数不到10人。我们将准备在第三年年底在其他地点以商业形式部署这种设施。

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.