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

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.

Key words:Functional gene, Taxonomic database, Cut-off value, amoA gene, AOA