基于BIPES的微生物群落分析新方法研究

微生物群落结构及其动态变化分析是涉及众多学科的共性瓶颈技术。近期，该领域发展迅速，揭示了许多与人体健康以及环境科学相关的新现象。其关键原因是454测定16S rRNA基因短标签序列，进而计算微生物群落结构新方法的建立与运用。申请者原创开发BIPES法，它比454法的通量高数十倍，成本更低，并且准确性更高。前期工作已证明BIPES方法的可行性，并解决了百万条序列水平的聚类计算瓶颈。本项目针对BIPES一些潜在影响因素和不足之处，系统评估BIPES操作流程中的样品保存、DNA制备、PCR及测序等步骤的影响，探求最佳分析条件；建立内外标质控体系；建立非测通配对序列分析技术，拓展BIPES可变区的选择范围，提高序列标签的系统分类效率；从而将BIPES建成为准确、易用、高性价比的微生物群落结构分析手段。该方法可望在人体微生物组、微生物生态、环境及资源微生物、土壤及农业微生物等诸多领域获得广泛应用。

Determining microbial community structure and its dynamic change has been a bottleneck for many fields. The area has been significantly improved with many novel observations due to the development of determining 16S rRNA gene tags with 454 and calculating microbial diversity with new bioinformatic tools. Recently, we developed a method called barcoded Illumina paired-end sequenicng (BIPES), which had an order of magnitutue level higher throughput, lower cost, and higher accuracy comparing with the 454 approach. Our previous study has proved the feasibility of BIPES, and we have sucessfully resovled the bioinformatic problem of clustering millions of sequences. However, there are some shortcomings and factors potentially affecting the usage of BIPES. In this proposal, we will systematically assess the wet lab steps in the BIPES process, mainly focusing on sample preparation, DNA extraction and purification, PCR, and next generation sequencing methods, therefore to find the best experimental conditions for the process; in addition, we will develop the quality control techniques including the external and internal standards; furthermore, we will develop the gapped paired-end tag technique, which is more flexible for selecting variable gene tags and can improve the taxonomy assignment efficiency of BIPES reads. The project aims to fully develop the BIPES method to be an accurate, labor-saving, and cost-effective method for studying microbial community. The method could be used in many fields involving human health, animal health, and environmental issues.