454 Life Sciences Massively Parallel Sys.:DNA Sequencing

描述（由申请人提供）：相关性。研究、制药和临床界对低成本、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巨大的潜力，可以表征个体对许多慢性和急性疾病或失调的易感性。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，而且我们还远远不能负担得起在常规基础上使用全面的基因组序列信息，部分原因是这种大规模测序需要大量的设备，而且是劳动密集型的。与大幅降低成本同样重要的是，需要开发一个完整的平台，使任何研究实验室都有能力在没有大型和昂贵的基础设施的情况下对大型生物体进行测序。

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.