Sanger和Pyrosequencing测序法分析健康成人口腔菌群

目的对比Sanger和Pyrosequencing测序法分析健康人口腔菌群组成。方法收集6例健康成人唾液、舌背、黏膜、龈上及龈下菌斑并构建16SrRNA基因文库,分别用Sanger和Pyrosequencing测序法分析。结果 Sanger测序所得已知的序列有5,794条(占6,535总序列数88.7%)、75个属,396个序列划分操作分类单元(operational taxonomic units,OTUs,占总OTUs的61.4%)。Pyrosequencing测序所得已知的序列有10,771条(占11,103总序列数97.0%)、66个属,322个OTUs(占总OTUs的68.0%)。Sanger和Pyrosequencing测序法所得口腔菌群在门、属的水平分布趋势基本一致,但在种的水平分布差异显著。Sanger和Pyrosequencing测序法构建的口腔菌群文库均匀度值分别为0.016和0.007,说明Pyrosequencing分析口腔菌群物种数量分布比Sanger测序方法的文库均匀性稍差,但优势种更显著。结论 Pyrosequencing测序时所构建基因文库能代表口腔菌群的多样性且经济、省时,可以应用于口腔细菌物种的分析。

The composition of oral bacteria flora in 6 healthy individuals by Sanger and Pyrosequencing

Abstract: Objective To compare Sanger vs Pyrosequencing for analysis of oral bacteria flora composition in healthy individuals. Methods The samples were collected from saliva, tongue, mucosa, as well as supragingival and subgingival plaque; 16S rRNA gene clone libraries were constructed. Results Using Sanger method, 5,794 sequences (88.7% of total 6,535 sequences) and 396 species-level operational taxonomic units (OTUs, 61.4% of total OTUs) were identified, corresponding to known and cultivated species. Using Pyrosequencing, 10,771 sequences (97.0% of total 11,103 sequences) and 322 OTUs (68.0% of total OTUs) were identified, corresponding to known and cultivated species. The values of evenness of Sanger and Pyrosequencing clone libraries were 0.016 and 0.007, respectively. The oral bacteria diversity of Sanger clone libraries was better than that of Pyrosequencing clone libraries. Conclusion Pyrosequencing is an alternative method to fully define the oral bacteria diversity of healthy subjects.