Fragment Size-Based Pathogen Enrichment in cfDNA

基于片段大小的 cfDNA 病原体富集

• 批准号: 10039854
• 负责人: Alexander L Greninger
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-22 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10039854
• 关键词: Antimicrobial Resistance; Applications Grants; Area; Bacteria; Bioinformatics; Biological Assay; Blood; Blood Plasma Volume; Body Fluids; CLIA certified; Cells; Cerebrospinal Fluid; Clinical; Communicable Diseases; Cytomegalovirus; DNA; DNA Viruses; DNA sequencing; Data; Detection; Diagnosis; Diagnostic; Digit structure; Genomics; Hour; Human; Human Genome; Infection; Infectious Agent; Inflammation; Laboratories; Length; Leukocytes; Libraries; Liquid substance; Measures; Metagenomics; Methods; Methylation; Microbiology; Molds; Nucleic Acids; Nucleosomes; Oncology; Parasites; Pathogen detection; Plasma; Predictive Value; Preparation; Protocols documentation; Recovery; Regimen; Sampling; Series; Serologic tests; Shotgun Sequencing; Shotguns; Signal Transduction; Site; Specimen; Stereotyping; Sterility; System; Taxonomy; Technology; Testing; Translating; Work; base; bioinformatics pipeline; cell free DNA; cost; cost effective; experimental study; fungus; human DNA; improved; pathogen; pathogen genome; prenatal testing; resistance gene; screening; synergism; tumor DNA

Project Summary Pathogen enrichment by fragment size selection of cell-free DNA Cell-free DNA sequencing of plasma and sterile body fluids is an attractive approach for broad-based pathogen detection. Plasma cfDNA can sample a variety of body sites and allows for predictable and rapid (24-hour) results for agnostic pathogen detection, while existing screening regimens such as blood culture require 5 days or more to return negative. A major issue with shotgun sequencing approaches to infectious disease diagnostics is the high host background induced by inflammation can significantly compromise the analytical sensitivity of the assay. Human cfDNA forms a stereotyped fragment size distribution with a mode of 167 bp with fewer than ~5% of fragments measuring under 120 bp. Pathogen cfDNA is often overwhelmingly found in these shorter fragments as they rarely protect their DNA with nucleosomes. Here, we propose to optimize methods to selectively sequence short cfDNA fragments to increase the analytical sensitivity of cfDNA sequencing for pathogen detection. Specifically, we will optimize plasma extraction, library preparation, and size selection parameters to recover shorter fragments and deplete fragments > 120bp. We will then validate this approach on a series of 150 plasma specimens known to be positive for pathogens covering DNA viruses, bacteria, fungi/molds as well as 100 negative plasma specimens. Using this approach, our aim is to extend the analytical sensitivity of cfDNA sequencing to surpass that of specific qPCR approaches.

项目摘要 通过无细胞DNA的片段选择的病原体富集 血浆和无菌体液的无细胞DNA测序是一种吸引人的方法 基于广泛的病原体检测。等离子体CFDNA可以采样各种身体部位，并且 允许可预测的快速（24小时）进行不可知病原体检测，而 现有的筛查方案（例如血液培养）需要5天或更长时间才能返回 消极的。传染病的shot枪测序方法的主要问题 诊断是由炎症引起的高宿主背景 损害分析的分析灵敏度。人类CFDNA形成了刻板印象 片段尺寸分布，模式为167 bp，片段不足〜5％ 测量120 bp。病原体CFDNA通常在这些中被绝大多数发现 较短的片段很少用核小体保护其DNA。在这里，我们建议 优化方法以选择性序列短CfDNA片段以增加 CFDNA测序对病原体检测的分析灵敏度。具体来说，我们会的 优化等离子体提取，库准备和尺寸选择参数以恢复 较短的碎片和耗尽片段> 120bp。然后，我们将验证这种方法 在一系列150个血浆标本中，已知对覆盖DNA的病原体阳性 病毒，细菌，真菌/霉菌以及100个阴性血浆标本。使用此 方法，我们的目的是扩展CFDNA测序的分析灵敏度至超过 特定QPCR方法的方法。