Ultra high throughput DNA sequencing system based on two-dimensional monolith mul

描述(申请人提供)：本项目的主要目标是开发、实施和测试一种高效的DNA测序方法和仪器，能够以100倍的成本降低100倍的成本，对哺乳动物大小的基因组进行自动重新测序和从头测序。该系统将基于二维整体柱多毛细管阵列(2D-MMCA)中凝胶基质固定的单模板分子(Polonies)菌落产生的荧光标记的末端双脱氧核苷酸DNA延伸产物的高度并行CE分离和检测。成本的降低将来自使用纳升体积反应和使用2D-MMCA，与通常使用的高通量DNA机器相比，2D-MMCA将CE分离和检测的吞吐量提高至少两个数量级。我们将进行以下工作：技术平台的开发，建立一个试验性的55x55道自动DNA测序仪，能够以450bp的Q20读取长度每秒测序1,000bp，成本为0.007美元/kBP，并展示了生产规模的重新测序和自动生成高质量的小鼠草稿基因组的基因组序列，每个哺乳动物大小的基因组不到100,000美元。进一步开发拟议的系统，并引入能够进行从头测序的技术和仪器变化：优化Polonie的培养技术，获得3-5kb DNA片段的有效扩增和测序周期；建造一台试验性的100x300通道DNA测序仪，能够以7500bp/秒的速度自动生成原始测序数据，读取长度为800bpQ20，成本为0.002美元/kBP。展示了该系统对人类大小基因组进行从头测序的潜力，覆盖范围为10倍，耗资10万至20万美元。

DESCRIPTION (provided by applicant): The main goal of the present project is a development, implementation and testing of a highly efficient method and an instrument for DNA sequencing capable of both automated re-sequencing and de-novo sequencing of mammalian size genomes with the mouse draft quality {Nature 420:520, 2002} at 100-fold reduced cost. The proposed system will be based on highly parallel CE separation and detection of fluorescently labeled dideoxynucleotide-terminated DNA extension product generated by gel matrix-immobilized colonies of single template molecules (polonies) in two dimensional monolith multi-capillary arrays (2D-MMCA). The cost reduction will come from both using nanoliter volume reactions and employing 2D-MMCAs which increase the throughput of the CE separation and detection by at least two orders of magnitude compared to commonly used high-throughput DNA machines. We will perform the following: Development of the technology platform, building of a pilot 55x55 lane automated DNA sequencer capable of sequencing 1,000 bp/second with 450bp Q20 read length at cost of $0.007/kbp, and demonstration of the production scale re-sequencing and automated generation of a genome sequence of the quality of the mouse draft genome at less than $100,000 per mammalian size genome Further development of the proposed system and introducing the technology and the instrumentation changes enabling de-novo sequencing: Optimization of polonie's cultivation technology and obtaining an efficient amplification and cycle sequencing of 3-5kb DNA fragments; Building a pilot 100x300 lane DNA sequencer capable of automated generation of raw sequencing data at 7500 bp/second with 800 bp Q20 read length at cost of $0.002/kbp. Demonstration of the system's potential for de-novo sequencing of human size genome at 10 fold coverage for $100,000 - $200,000.