基于amoA基因扩增子高通量测序的氨氧化古菌多样性分析方法

Development of a method for ammonia-oxidizing archaea diversity analysis based on amoA gene amplicons with high-throughput sequencing

摘要:

【背景】对于环境样品中氨氧化古菌(Ammonia-oxidizing archaea，AOA)多样性的研究，利用amoA功能基因作为分子标记会比16S rRNA基因有更强的特异性和更高的分辨率，能更准确地反映环境样品中氨氧化古菌的种群结构和分布特征。然而，目前对amoA基因扩增子高通量测序的分析存在两大限制因素：一是缺乏相应的amoA基因参考数据库；二是AOA amoA基因在种水平上的相似性阈值未知，分析过程中没有明确的划分种水平操作分类单元(Operational taxonomic unit，OTU)的阈值。【目的】构建基于amoA功能基因序列分析氨氧化古菌多样性的方法，为基于高通量测序的功能微生物多样性分析提供参考。【方法】基于目前已通过分离纯化或富集培养获得的34株氨氧化古菌及功能基因数据库中收录的环境样品amoA基因序列，构建氨氧化古菌amoA基因参考数据库。通过菌株间两两比对获得的amoA基因相似度与16S rRNA基因相似度的相关性分析，确定amoA基因在种水平上的相似性阈值。基于MOTHUR软件平台，利用建立的参考数据库和确定的阈值对南海一个垂直水体剖面样品的amoA基因序列进行多样性分析。【结果】构建了含有26 091条序列信息的古菌amoA基因参考数据库，确定了89%作为分析过程中古菌amoA基因划分种水平OTU的阈值，对南海水体样品氨氧化古菌的多样性分析结果很好地显示了南海不同深度水层水体中氨氧化古菌的种群结构和系统发育关系，有效揭示了南海氨氧化古菌的垂直分布差异。【结论】建立了基于amoA基因高通量测序的氨氧化古菌多样性分析方法，此方法可以有效分析环境样品中氨氧化古菌的多样性。

Abstract:

[Background] Using amoA gene as marker for analyzing the diversity of ammonia-oxidizing archaea (AOA) has stronger specificity and higher resolution than 16S rRNA gene, it can more accurately reflect the community structure and distribution pattern of AOA in environmental samples. However, there are two limitations in the analysis of amoA gene sequence with high-throughput sequencing: one is the lack of corresponding amoA gene reference taxonomic database; and the other is no determined species-level cut-off value for operational taxonomic units (OTUs) clustering. [Objective] The aim of this article was to develop a method for ammonia-oxidizing archaea diversity analysis based on amoA gene sequence with high-throughput sequencing, providing a reference method to analyze the functional microbial diversity based on high-throughput sequencing. [Methods] amoA gene sequences of 34 AOA strains from pure or enrichment culture were used as seed sequences. Uncultured amoA gene sequences from environmental samples were downloaded from functional gene database. All sequences were used to construct taxonomic database for amoA gene sequences. By pairwise comparison of 16S rRNA gene and archaeal amoA identities of all recognized species of AOA, we determined the cut-off value for OTU clustering at species-level. We used the established reference database and determined cut-off value to analyze the diversity for water samples?in a vertical profile of the South China Sea with the MOTHUR software. [Results] We constructed a reference taxonomic database containing 26 091 amoA gene sequences, and determined 89% sequence identity as cut-off value for OTU clustering at species-level. The diversity analysis of AOA sufficiently showed the community structure and phylogenetic relationship, and effectively revealed the AOA vertical distribution differences in the South China Sea. [Conclusion] We developed a method for ammonia-oxidizing archaea diversity analysis based on amoA gene sequence with high-throughput sequencing. This method can effectively analyze the diversity of AOA in environmental samples.