Fragment Size-Based Pathogen Enrichment in cfDNA

基于片段大小的 cfDNA 病原体富集

• 批准号: 10199981
• 负责人: Alexander L Greninger
• 金额: $ 23.33万
• 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/10199981
• 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; Serology; 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.

项目概要 通过无细胞 DNA 片段大小选择进行病原体富集 对血浆和无菌体液进行无细胞 DNA 测序是一种有吸引力的方法 广泛的病原体检测。血浆 cfDNA 可以对身体的各个部位进行采样 可以为不可知的病原体检测提供可预测的快速（24 小时）结果，同时 现有的筛查方案（例如血培养）需要 5 天或更长时间才能返回 消极的。传染病鸟枪法测序方法的一个主要问题 诊断是由炎症引起的高宿主背景可以显着 损害测定的分析灵敏度。人类cfDNA形成定型 片段大小分布模式为 167 bp，片段少于约 5% 测量低于 120 bp。病原体 cfDNA 通常绝大多数存在于这些物质中 较短的片段，因为它们很少用核小体保护 DNA。在此，我们建议 优化选择性测序短 cfDNA 片段的方法，以提高 用于病原体检测的 cfDNA 测序的分析灵敏度。具体来说，我们将 优化血浆提取、文库制备和大小选择参数以恢复 较短的片段和耗尽片段 > 120bp。然后我们将验证这个方法 对一系列 150 个血浆样本进行检测，这些样本已知对覆盖 DNA 的病原体呈阳性 病毒、细菌、真菌/霉菌以及 100 份阴性血浆样本。使用这个 方法，我们的目标是将 cfDNA 测序的分析灵敏度扩展到超越 特定 qPCR 方法的方法。

项目成果

In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

• DOI: 10.1128/jvi.00250-21
• 发表时间: 2021-05-24
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Addetia A;Phung Q;Bradley BT;Lin MJ;Zhu H;Xie H;Huang ML;Greninger AL
• 通讯作者: Greninger AL