Massively parallel contiguity mapping

描述（由申请人提供）：大规模并行技术已经将DNA测序的每碱基成本降低了几个数量级。然而，有限的读取长度和缺乏在甚至适度的距离上建立邻接的方法已经阻止了这些技术实现哺乳动物基因组的高质量、低成本的从头组装。即使革命性的测序技术进一步成熟，就每碱基成本而言，最好的技术产生的读段长度或质量对于大基因组的有效从头组装来说可能仍然不足。为了满足这一关键需求，我们正在利用高密度，随机，体外转座作为一种新的手段，以创造性的方式物理粉碎基因组DNA，促进在不同尺度上恢复邻接信息。我们的项目分为四个目标，其中前三个分别针对大规模并行方法的发展，以确定短距离，中距离和长距离的邻接。这些是：1）用于粉碎具有对称标签的基因组DNA的方法，所述对称标签以完全独立于一级序列含量的方式事后通知相邻片段化事件的排序; 3）用于将拉伸的DNA分子转化为衔接子侧翼文库的原位方法，使得通过大规模平行测序产生的读段将在它们起源的XY坐标方面保持线性有序。在第四个目标中，我们将整合这些方法来证明：1）小鼠基因组的高成本效益从头组装，其质量超过原始组装; 2）人类基因组的高成本效益单倍型解析重测序。

DESCRIPTION (provided by applicant): Massively parallel technologies have reduced the per-base cost of DNA sequencing by several orders of magnitude. However, limited read lengths and a lack of methods to establish contiguity over even modest distances have prevented these technologies from achieving the high-quality, low-cost de novo assembly of mammalian genomes. Even as revolutionary sequencing technologies further mature, it may continue to be the case that the best technologies in terms of cost-per-base yield reads that are of an insufficient length or quality for the effective de novo assembly of large genomes. To address this critical need, we are exploiting high density, random, in vitro transposition as a novel means of physically shattering genomic DNA in creative ways that facilitate the recovery of contiguity information at different scales. Our project is divided into four aims, the first three of which are respectively directed at the development of massively parallel methods for determining short-range, mid-range, and long-range contiguity. These are: 1) a method for shattering genomic DNA with symmetric tags that post hoc inform the ordering of adjacent fragmentation events in a way that is entirely independent of the primary sequence content; 2) a method for massively parallel, in vitro barcoding of fosmid or BAC-sized subsequences of a genome, thereby facilitating hierarchical assembly; 3) an in situ method for converting stretched DNA molecules into adaptor-flanked libraries, such that reads generated by massively parallel sequencing will remain linearly ordered in terms of the XY coordinates at which they originate. In the fourth aim, we will integrate these methods to demonstrate: 1) the highly cost-effective de novo assembly of the mouse genome with a quality that exceeds that of the original assembly; 2) the highly cost-effective haplotype resolved resequencing of a human genome.