Development of a high throughput technology for detecting virus infection and immunity in the natural environment

本研究的目的是验证这一概念，并建立一种高通量的方法来筛选野生植物和昆虫群落中的病毒感染和免疫。我们建议使用Solexa高通量测序获得植物和昆虫群落的小RNA图谱。将筛选宿主基因沉默系统针对病毒RNA产生的抗病毒小干扰（avsi）RNA的病毒来源。我们预计在该研究中心检测到针对已知流行病毒的avsiRNA，并将使用这些病毒作为阳性对照，以优化样品制备、测序和生物信息学的条件。我们还希望发现以前未经证实和未知病毒的流行，我们计划通过使用常规方法（例如，RTPCR、克隆和测序、北方印迹等）以确定高通量方法的灵敏度和准确度。为了使传统的方法评估，我们计划通过使用序列标签来标记每个采样物种的样品。采样制度的目的是实现5%的植物种群感染率的鸟枪检测的灵敏度。将标记的样品合并在一起进行高通量测序运行，以实现成本效益。将所得序列分类回其原始样品身份并进行分析。将使用常规方法对分选后的特定样本进行结果验证。还将进行大规模测序后分析，而不将样品分选至其原始身份。将比较特定分析和质量分析的结果。不需要对样本进行分类的质量分析是为了检验从生态系统中进行遗传随机抽样的能力而设计的，不受抽样制度的限制。该技术将提供广泛的应用，从环境变化期间自然条件下的大规模随机抽样，到农业和其他管理条件下的定义调查。

This proposal is to prove the concept and develop a high throughput methodology for screening virus infections and immunities in wild plant and insect communities. We propose to obtain small RNA profiles of the plant and insect communities from the Wytham Wood, Oxfordshire, by using Solexa high throughput sequencing. The anti-virus small interfering (avsi)RNAs that are produced by the host gene silencing systems against the virus RNAs will be screened for viral origins. We anticipate the detection of the avsiRNAs against the known prevalent viruses at the site and will use these viruses as positive controls to optimize conditions of sample preparation, sequencing, and bioinformatics. We also expect to discover the prevalence of previously unconfirmed and unknown viruses, and we plan to validate these newly detected infections by using the conventional methods (e.g., RTPCR, cloning and sequencing, Northern Blotting, etc.) to determine the sensitivity and accuracy of the high throughput methodology. To enable the conventional method assessment, we plan to label samples for each sampled species by using sequence tags. The sampling regime is designed for achieving a sensitivity of shotgun detection of 5% infection rate for plant populations. The tagged samples will be pooled together for high throughput sequencing runs to achieve cost effectiveness. The resulting sequences will be sorted back to their original sample identities and analyzed. Results will be validated by using the conventional methods with the sorted specific samples. Mass post-sequencing analyses will also be performed without sorting the samples to their original identities. Results from the specific analyses and mass analyses will be compared. The mass analyses without the requirement of sorting samples are designed for testing a capacity of genetic random sampling from an ecosystem without restriction of sampling regimes. The technology will offer a broad range of applications from large scale random sampling in natural conditions during the environment change, to defined survey in agricultural and the other managed conditions.