RAPID: Pathogen Detection with Real-Time Genetic Sequencing

早期病原体检测和疾病诊断是预防COVID-19等传染病指数传播的关键。如果不及早发现和控制，这种病原体不仅会影响当地社区，还可能跨越国家和国际边界，成为全球大流行。现有的早期病原体检测方法可能会在大流行期间延误诊断。这些识别特定于病原体类型/亚型的感兴趣区域，并在目标标本（如果存在）中扩增这些区域，使其不适用于未知病原体。另一个限制是对引物化学设计的依赖，这是一个复杂、容易出错且耗时的过程，可能会在大流行的早期阶段延迟病原体的检测。此外，追踪新突变的积累对确定疾病热点、监测病毒进化、指导免疫工作和为公共卫生计划提供信息也很重要。据估计，像asSARS-CoV-2这样的RNA冠状病毒每年可累积30次替换。该方法将有助于设计快速准确的全基因组测序（WGS）分析管道，用于菌株分型和系统发育分类。提出的实时诊断管道可以作为“数字嗅探器”，用于早期发现潜在的疫情。研究人员建议使用便携式牛津纳米孔测序仪MinION建立早期病原体检测的实时诊断管道。测序仪的read-until功能可用于对低覆盖率的基因组区域进行自适应靶向测序，并加速未知病原体的全基因组测序。该项目的最终目标是发展即时移动快速病原体诊断和分析。这将通过开发新的硬件加速技术和算法来实现，用于快速分类病毒/细菌病原体，然后进行菌株检测和流行病学分析。提出的技术包括对螺旋空间分类算法的软硬件协同设计、精细的索引、利用MinION测序设备的实时反馈特性（读直到和运行直到）以及变体调用来实现快速分析。该项目还将探索无需保留转录酶步骤的直接RNA测序，以减少文库准备时间。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Early pathogen detection and disease diagnosis is key to preventing the exponential spread of infectious diseases such as COVID-19. If not detected and contained early, the pathogen not only affects local communities but can cross national and international borders to become a global pandemic. Existing approaches for early pathogen detection can delay diagnosis during a pandemic. These identify regions of interest specific to a pathogen type/sub-type and amplify these regions in target specimens (if present), making them inapplicable to unknown pathogens. Another limitation is the dependence on the design of primer chemistry, which is a complex, error-prone, and time-consuming process that can delay pathogen detection in the early stages of a pandemic. Further, it is also important to track the accumulation of novel mutations to identify disease hotspots, monitor virus evolution, guide immunization efforts, and inform public healthcare plans. It has been estimated that RNA coronaviruses such asSARS-CoV-2 can accumulate 30 substitutions/year. The proposed approach will enable design for fast and accurate whole-genome sequencing (WGS) analysis pipelines for strain typing and phylogenetic classification. The proposed real-time diagnosis pipeline can potentially serve as a "digital sniffer" and be used to detect potential outbreaks early.The researchers propose to build real-time diagnosis pipelines for early pathogen detection using the portable Oxford Nanopore Sequencer MinION. The read-until functionality of the sequencer can be used to adaptively target sequencing towards genome regions with low-coverage and accelerate whole-genome sequencing of unknown pathogens. The ultimate goal of this project is to develop point-of-care mobile rapid pathogen diagnosis and analysis. This will be realized by developing novel hardware-acceleration techniques and algorithms for quick classification of viral/bacterial pathogens, followed by strain detection and epidemiology analysis. The proposed techniques include hardware-software co-design for squiggle-space classification algorithms, refined indexes, exploiting real-time feedback feature of MinION sequencing devices (read-until and run-until), and variant calling to achieve rapid analysis. The project will also explore direct RNA sequencing without reserve transcriptase step to reduce library-preparation time.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.