宏基因组靶向测序分析皮肤表面微生物群落方法优化

Optimization of Method for Analyzing Microbial Community on Human Skin by Targeted Sequencing of Metagenomics

通过实验设计和数据分析,筛选出尽可能真实反映含量较低的皮肤微生物群落的高通量测序分析方法。使用多因素多水平完全随机实验设计,分析DNA提取、PCR扩增、样品采集、测序过程中各种实验因素对微生物群落组成特征分析结果的影响。结果显示,在样品采集方面,筛选出了最佳的采集拭子与采集液体;在DNA提取方面,筛选出了最佳的前处理方法及试剂盒种类;在PCR反应方面,筛选出了最佳的模板浓度及退火温度;在测序方面,筛选出了合适的测序深度。通过一系列的实验条件筛选,确定了适合分析低含量细菌群落结构组成特征的方法,并成功地应用于人类手掌面皮肤。

Based on the experimental design and data analysis, we screened a high-throughput sequencing method which may measurethe community of microorganisms in a low level on the human skin as real as possible. By completely random design of the experiments withmulti-factor multi-level, we analyzed the effects of varied factors in the DNA extraction, PCR amplification, sample collection and sequencing onthe composition characteristics of microbial community. The results showed that regarding the sample collection, the optimal collection swab andextraction liquid were screened out. In the aspect of DNA, the optimal pretreatment method and reagent box type were selected; on PCR reaction,the optimal template concentration and annealing temperature were determined; and on the sequencing aspect, the suitable sequencing depth wasalso chosen. In conclusion, through above series of experiments to screen the conditions, the appropriate method for analyzing the compositioncharacteristics of community structure of bacteria in a low level was determined, and successfully applied to human palm skin.