EAGER: Viral tagging: Combining flow cytometry and genomics to explore virus-host interactions

研究人员将开发一种高通量的病毒标记方法，以调查自然海洋群落中病毒与宿主的相互作用。在这种方法中，培养的或野生的病毒通过其核酸的荧光标记并允许吸附到细胞上，从而标记细胞，以便通过流式细胞术对标记病毒的细胞进行区分分类。使用这种方法检测、分类和识别带有病毒的海洋微生物(即带有病毒标记的细胞)的初步实验已经准确和特异，显示了带有病毒标记的目标宿主细胞群体的明显荧光位移，并极大地减少了对非宿主细胞的标记。该项目将开发并严格评估这种病毒标记方法，用于野生病毒和微生物种群。首先，在应用于野生种群之前，需要对培养的病毒-宿主对的病毒标记方法进行额外的系统表征。研究人员将使用具有生态重要性宿主细胞谱系的三种主要类型的海洋DNA病毒的代表来评估不同的细胞生理状态、实验条件和宿主特异性对病毒标记效率和信号的影响。此外，冷冻保存对于在远程站点使用该方法至关重要。研究人员将测试各种冷冻保存技术对病毒标签签名和下游分子生物学的影响。单细胞基因组研究人员面临着平行的挑战，这使得智能交叉受精变得可取和可能。最后，在用培养宿主作为“钩子”捕获野生病毒多样性的实验中，人们必须在对带有病毒标记的细胞进行分类后进行病毒DNA的浓缩。根据稳定的同位素探测方案，研究人员将使用生长在13C标记底物上的培养的“诱饵”微生物进行测试，以产生沉重的宿主DNA，从而允许密度梯度分离宿主和病毒DNA。轻型病毒DNA然后被接头介导的扩增和焦解测序，以揭示宿主特有的病毒元基因组。更广泛的影响：一种经过实验验证的病毒标签方法缓解了理解病毒-微生物相互作用的三个瓶颈。具体地说，研究人员预计，该方法将允许研究人员进行高通量实验，这些实验可以(1)绘制病毒的原位宿主范围，(2)定义感染给定宿主的病毒的自然范围，以及(3)实现有针对性的元基因组测序，以进一步揭示病毒影响野外微生物新陈代谢的无数方式。这些进展将对病毒和微生物生态学领域以及寻求种群规模数据集的生物地球化学家和生态模型师有用。博士后研究人员将接受先进的流式细胞术和分子生物学方法方面的大量方法学培训，并有机会通过与富布赖特学者和系统管理员的合作，在拨款和论文写作、教学和跨学科和跨文化合作方面获得经验，以建立高通量数据分析管道。

The investigator will develop a high-throughput viral-tagging method to investigate virus-host interactions in natural marine communities. In this approach, cultured or wild viruses are tagged by fluorescent labeling of their nucleic acid and allowed to adsorb to cells, thereby labeling the cells for differential sorting of viral-tagged cells by flow cytometry. Preliminary experiments using this method to detect, sort, and identify ocean microbes with attached viruses (i.e., viral-tagged cells) have been accurate and specific, showing distinct fluorescent shifts for viral-tagged target host cell populations and greatly reduced tagging of non-host cells. This project will develop and rigorously evaluate this viral-tagging method for use with wild populations of viruses and microbes. First, additional systematic characterization of viral-tagging methodology with cultured virus-host pairs will be needed before application to wild populations. The investigator will use representatives of the three major types of marine DNA viruses with ecologically important host cell lineages to evaluate the effects of variable cell physiological states, experimental conditions, and host specificity on viral-tagging efficiency and signal. In addition, cryopreservation is critical for use of this method at remote sites. The investigator will test various cryopreservation techniques for impact on viral-tagging signatures and on downstream molecular biology. Parallel challenges face single-cell genomic researchers, making intellectual cross-fertilization desirable and likely. Finally, in experiments using cultured hosts as "hooks" to capture wild viral diversity, one must enrich for viral DNA after sorting viral-tagged cells. Based on stable isotope probing protocols, the investigator will test using cultured "bait" microbes grown on a 13C-labeled substrate to create heavy host DNA, thus allowing density-gradient separation of host from viral DNA. Light viral DNA is then linker-mediated amplified and pyrosequenced to reveal the host-specific viral metagenome. Broader impacts: An experimentally validated viral-tagging method offers to alleviate three bottlenecks in understanding virus-microbe interactions. Specifically, the investigator anticipates that the method would allow a researcher to perform high-throughput experiments that could (1) map the in situ host range of a virus, (2) define the natural range of viruses that infect a given host, and (3) enable targeted metagenomic sequencing to further uncover the myriad ways that viruses impact microbial metabolism in the wild. Such advances would be useful across fields of viral and microbial ecology, as well as to biogeochemists and ecological modelers seeking population-scale datasets. A post-doctoral researcher will receive significant methodological training in advanced flow cytometry and molecular biological methods, as well as the opportunity to gain experience in grant and paper writing, teaching and cross-discipline and cross-cultural collaboration through collaboration with a Fulbright scholar and a systems administrator to enable high throughput data analysis pipelines to be established.