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

通过体外包装实现配对末端下一代测序的均一长度 DNA

• 批准号: 8001230
• 负责人: FREDERICK R BLATTNER
• 金额: $ 12.74万
• 依托单位: SCARAB GENOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-04 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8001230
• 关键词: Address; Affect; Age; Bacteriophage P22; Bacteriophages; Biological Process; Biology; Biopsy; Biotin; Capsid; Cell Extracts; Clinical; DNA; DNA Library; DNA Packaging; DNA Sequence; DNA Sequence Rearrangement; Diagnosis; Disease; Dreams; Effectiveness; Escherichia coli; Exposure to; Genes; Genome; Genomics; Goals; Human; Human Genome; Immune; In Vitro; Indium; Individual; Infectious Agent; Joints; Label; Length; Libraries; Life; Ligation; Malignant Neoplasms; Measures; Medicine; Methods; Organism; Patients; Persons; Preparation; Preventive Medicine; Process; Process Measure; Production; Property; Protocols documentation; Reaction; Reading; Recovery; Reliance; Research; Research Personnel; Salmonella; Sampling; Sequence Analysis; Source; Specific qualifier value; Staging; System; Techniques; Technology; Testing; Tissues; Treatment Protocols; Yeasts; base; cancer genome; clinical practice; cost; feeding; genome sequencing; human disease; improved; in vivo; innovation; insight; mutant; nanomachine; novel; particle; prototype; public health relevance; research study; terminase; tumor; tumor progression

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.

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来从珍贵样品中分离具有长且均匀的片段长度的纯DNA，用于下一代基因组测序。下一代测序的出现降低了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。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用于个体化预防医学，而在癌症进展过程中采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，包括先前暴露于感染因子的记录。该提案的目标是研究一种创新的新方法，从任何来源制备DNA文库，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应该与配对末端测序相容并且足够长以跨越人类基因组中的重复。这意味着DNA应易于连接成环状且大于10 kbp。(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。我们的方法基于为数不多的测量DNA长度的生物过程之一，即广义转导噬菌体P22的体外头部包装。P22可以无差别地包装DNA，产生具有非常紧密的大小分布的长片段，42，618 780 bp。此外，末端是酶促产生的，这意味着它们是可精确连接的，是nextgen配对末端测序的理想选择。一旦包装，噬菌体颗粒提供了用于分离大小分级的DNA的容器化货物系统。该过程需要1）通过“满头”机制包装固定长度的靶DNA，（2）纯化填充的噬菌体颗粒以去除污染物，（3）从衣壳释放均匀长度的DNA，（4）环化DNA并捕获新的接头，（5）nextgen测序这些配对末端读数，和（6）利用双端读段之间的长恒定距离组装序列以跨越重复区域并帮助正确组装。为了证明我们的方法的可行性，我们已经成功地在体外包装和回收外源添加的酵母DNA，并在一个独立的实验中，我们已经测序的43 kb包装的DNA从两个P22裂解物使用Illumina配对末端测序技术。本研究的主要目的是：1）生产一株E.大肠杆菌溶原系统体外包装细胞提取物的制备; 2）优化包装DNA产生43 kb环状物的条件。 公共卫生相关性：在下一代测序技术的巨大进步的推动下，负担得起的个人基因组学时代已经到来，即使在这个早期阶段，仅根据受影响组织的基因组序列来诊断疾病的潜力也是存在的。在这项提案中，我们描述了一种强大的新方法，可以提高基因组DNA处理的有效性，允许像活检一样小的样本被完全测序和分析。这项技术对人类疾病的诊断、分期和治疗的潜在影响是巨大的。