Fragment Size-Based Pathogen Enrichment in cfDNA

项目摘要 通过游离DNA片段大小选择富集病原体 血浆和无菌体液的无细胞DNA测序是一种有吸引力的方法， 广泛的病原体检测血浆cfDNA可以对多种身体部位进行采样， 允许不可知病原体检测的可预测和快速（24小时）结果， 现有的筛查方案如血液培养需要5天或更长时间才能返回 阴性鸟枪测序方法在传染病中的一个主要问题 诊断是由炎症诱导的高宿主背景可以显著地 损害了测定的分析灵敏度。人类cfDNA形成刻板印象 片段大小分布，众数为167 bp，片段少于~5% 在120 bp以下测量。病原体cfDNA通常压倒性地存在于这些 因为它们很少用核小体保护它们的DNA。在这里，我们建议 为了优化选择性测序短cfDNA片段的方法，以增加 cfDNA测序用于病原体检测的分析灵敏度。具体来说，我们将 优化血浆提取、文库制备和大小选择参数， 短片段和缺失片段> 120 bp。然后，我们将验证此方法 在一系列150个已知对覆盖DNA的病原体呈阳性的血浆样本上， 病毒、细菌、真菌/霉菌以及100份阴性血浆标本。使用此 我们的目标是将cfDNA测序的分析灵敏度扩展到超过 具体的qPCR方法。

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.