Uniform length DNA for paired end nextgen sequencing via in vitro packaging

描述(由申请人提供)：我们已经认识到迫切需要一种商业上适用的高通量方法，从珍贵的样本中分离出具有长而统一的片段长度的纯DNA，用于NextGen基因组测序。NextGen测序的出现降低了DNA测序的成本，因此很快就可以将单个患者的基因组测序用于临床。因此，个性化基因组医学的梦想可能很快就会实现。患者DNA的全基因组测序可能成为临床实践的常规部分。在人的生命早期获得的生殖系序列可以用于个性化的预防医学，而在癌症进展过程中获取的活检序列将为个性化治疗方案提供指导。此外，免疫组织的体细胞序列将提供对患者免疫状态的洞察，包括以前接触感染性物质的记录。这项提议的目标是研究一种创新的新方法，从任何来源准备DNA文库，该来源可以被任何当前的NextGen测序技术使用。这种方法的要求是：(1)该过程必须有效地将输入的DNA转换成具有随机终点的均匀长度的片段，(2)DNA应该与成对末端测序兼容，并且足够长以跨越人类基因组中的重复序列。这意味着DNA应该可以很容易地连接成圈，并且大于10kbp。(3)该工艺必须容易地集成到大输量管道中，以使其在商业上可行。我们的方法是基于少数几个测量DNA长度的生物学过程之一，即由通用转导噬菌体P22进行体外头部包装。P22可以不分青红皂白地包装DNA，产生大小分布极其紧密的长片段，大小为42,618 780个碱基。此外，末端是酶生成的，这意味着它们可以精致地连接，是NextGen配对末端测序的理想选择。一旦包装好，噬菌体颗粒就提供了一个集装箱运输系统，用于分离大小不同的DNA。该过程包括：1)通过“Headful”机制包装固定长度的靶DNA，(2)从污染物中提纯填充的噬菌体颗粒，(3)从衣壳中释放均匀长度的DNA，(4)环化DNA并捕获新的接头，(5)NextGen对这些成对的末端读出进行测序，以及(6)利用成对末端读出之间的长的恒定距离来组装序列以跨越重复区域并帮助正确的组装。为了证明我们方法的可行性，我们成功地在体外包装和回收了外源添加的酵母DNA，在一个独立的实验中，我们使用Illumina配对末端测序技术对两个P22裂解物中43kb的包装DNA进行了测序。本研究的目的是：1)生产一种基于大肠杆菌的溶原菌系统，用于生产体外包装细胞提取物；2)优化包装DNA生产43kb环的条件。 公共卫生相关性：在下一代测序技术的戏剧性进步的推动下，负担得起的个人基因组学的时代已经到来，即使在这个早期阶段，也存在仅基于受影响组织的基因组序列来诊断疾病的可能性。在这项提案中，我们描述了一种强大的新方法，它可以提高处理基因组DNA的效率，允许对小到活检的样本进行完全测序和分析。这项技术将对人类疾病的诊断、分期和治疗产生巨大的潜在影响。

DESCRIPTION (provided by applicant): We have recognized an urgent need for a commercially applicable high throughput way to isolate pure DNA with long and uniform fragment length for nextgen genomic sequencing from precious samples. The advent of nextgen sequencing has driven the cost of DNA sequencing down so that soon it will be feasible to sequence the genomes of individual patients for clinical use. Thus the dream of personalized genomic medicine may soon be realized. Whole genome sequencing of patients' DNA could become a routine part of clinical practice. Germline sequences obtained early in the life of a person can be used for individualized preventive medicine, whereas, biopsy sequences taken over the course of a cancer progression will provide a guide for personalized treatment regimens. Additionally, somatic sequences of immune tissue will provide insight into the immune status of patients, including a record of previous exposure to infectious agents. The goal of this proposal is to investigate an innovative new method of preparing DNA libraries from any source that could be used by any of the current nextgen sequencing technologies. The requirements for this method are: (1) The process must efficiently convert input DNA into uniform length fragments with random endpoints, (2) The DNA should be compatible with paired end sequencing and long enough to span repeats in the human genome. This means that the DNA should be readily ligatable into circles and larger than 10 kbp. (3) The process must be readily integrated into the high throughput pipelines so that it is commercially feasible. Our method is based on one of the few biological processes that measure the length of DNA, in vitro headful packaging by the generalized transducing bacteriophage, P22. P22 can package DNA indiscriminately, producing long fragments with an extremely tight size distribution, 42,618 780 bp. Moreover, the ends are enzymatically generated meaning they are exquisitely ligatable, ideal for nextgen paired end sequencing. Once packaged, the phage particle provides a containerized cargo system for isolating the size fractionated DNA. The process entails 1) packaging a fixed length of the target DNA by the "headful" mechanism, (2) purifying the filled phage particles away from contaminants, (3) releasing the homogeneous length DNA from the capsids, (4) cyclizing the DNA and capturing the novel joint, (5) nextgen sequencing these paired end reads, and (6) assembling the sequence taking advantage of the long constant distance between paired end reads to span repeat regions and aid in correct assembly. To demonstrate the feasibility of our method we have successfully in vitro packaged and recovered exogenously added yeast DNA and in an independent experiment we have sequenced the 43 kb packaged DNA from two P22 lysates using Illumina paired end sequencing technology. Research proposed here will be aimed at: 1) produce an E. coli based lysogen system for production of in vitro packaging cell extracts, 2) optimize conditions for the production of 43 kb circles from packaged DNA. PUBLIC HEALTH RELEVANCE: The age of affordable personal genomics is upon us, driven by dramatic advances in nextgen sequencing technologies, and even at this early stage, the potential exists to diagnose an illness based solely on the affected tissue's genome sequence. In this proposal, we describe a powerful new method that increases the effectiveness with which genomic DNA can be processed, allowing samples as small as a biopsy to be fully sequenced and analyzed. The potential impact this technology will have on diagnosis, staging and treatment of human disease is immense.