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，从珍贵样品中分离出具有长而均匀片段长度的纯DNA，用于下一代基因组测序。下一代测序技术的出现降低了DNA测序的成本，因此很快就可以对个体患者的基因组进行测序以供临床使用。因此，个性化基因组医学的梦想可能很快就会实现。对患者DNA进行全基因组测序可能成为临床实践的常规部分。在一个人生命早期获得的生殖系序列可用于个体化预防医学，而在癌症进展过程中进行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深入了解，包括以前接触传染因子的记录。本提案的目标是研究一种从任何来源制备DNA文库的创新方法，该方法可用于任何当前的下一代测序技术。该方法的要求是：(1)该过程必须有效地将输入DNA转化为具有随机端点的均匀长度片段；(2)DNA必须与成对末端测序兼容，并且足够长，可以跨越人类基因组中的重复序列。这意味着DNA应该很容易连接成圆圈，并且大于10kbp。(3)该工艺必须易于集成到高通量管道中，以便在商业上可行。我们的方法是基于测量DNA长度的少数生物过程之一，通过广义转导噬菌体P22在体外头部包装。P22可以不加选择地包装DNA，产生长度片段，大小分布非常紧密，42,618 780 bp。此外，末端是酶促产生的，这意味着它们非常容易连接，是下一代配对末端测序的理想选择。一旦包装，噬菌体颗粒提供了一个集装箱货物系统，用于分离大小分离的DNA。该过程需要1)通过“头部”机制包装固定长度的目标DNA，(2)净化填充的噬菌体颗粒，使其远离污染物，(3)从衣壳中释放均匀长度的DNA，(4)对DNA进行环化并捕获新的连接，(5)对这些成对的端读序列进行下一代测序，以及(6)利用成对端读序列之间的长恒定距离来组装序列，以跨越重复区域并帮助正确组装。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，并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裂解物的43 kb包装DNA进行了测序。本文提出的研究将旨在：1)建立一个基于大肠杆菌的溶菌系统，用于生产体外包装细胞提取物；2)优化从包装DNA中生产43 kb环状物的条件。

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.