Characterisation of microbial communities increasingly involves use of high throughput sequencing methods (e.g. MiSeq Illumina) that amplify relatively short sequences of 16S rRNA or functional genes, the latter including ammonia monooxygenase subunit A (amoA), a key functional gene for ammonia oxidising bacteria (AOB) and archaea (AOA). The availability of these techniques, in combination with developments in phylogenetic methodology, provides the potential for better analysis of microbial niche specialisation. This study aimed to develop an approach for sequencing of bacterial and archaeal amoA genes amplified from soil using bioinformatics pipelines developed for general analysis of functional genes and employed sequence data to reassess phylogeny and niche specialisation in terrestrial bacterial ammonia oxidisers. amoA richness and community composition differed with bioinformatics approaches used but analysis of MiSeq sequences was reliable for both archaeal and bacterial amoA genes and was used for subsequent assessment of potential niche specialisation of soil bacteria ammonia oxidisers. Prior to ecological analysis, phylogenetic analysis of Nitrosospira, which dominates soil AOB, was revisited using a phylogenetic analysis of 16S rRNA and amoA genes in available AOB genomes. This analysis supported congruence between phylogenies of the two genes and increased previous phylogenetic resolution, providing support for additional gene clusters of potential ecological significance. Analysis of environmental sequences using these new sequencing, bioinformatics and phylogenetic approaches demonstrated, for the first time, similar niche specialisation in AOB to that in AOA, indicating pH as a key ecological factor controlling the composition of soil ammonia oxidiser communities. This study presents the first bioinformatics pipeline for optimal analysis of Illumina MiSeq sequencing of a functional gene and is adaptable to any amplicon size (even genes larger than 500 bp). The pipeline was used to provide an up-to-date phylogenetic analysis of terrestrial betaproteobacterial amoA genes and to demonstrate the importance of soil pH for their niche specialisation and is broadly applicable to other ecosystems and diverse microbiomes.

中文翻译：

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高通量测序技术在陆生细菌氨氧化剂的amoA系统发育和环境生态位分析中的应用

微生物群落的表征越来越多地涉及使用高通量测序方法（例如MiSeq Illumina），该方法可扩增相对较短的16S rRNA或功能基因序列，后者包括氨单加氧酶亚基A（amoA），这是氨氧化细菌（AOB）的关键功能基因）和古细菌（AOA）。这些技术的可获得性以及系统发育方法学的发展，为更好地分析微生物生态位专门化提供了潜力。这项研究旨在开发一种方法，该方法使用为功能基因的一般分析而开发的生物信息学管道从土壤中扩增的细菌和古细菌amoA基因进行测序，并利用序列数据来重新评估陆地细菌氨氧化器的系统发育和生态位专长。amoA的丰富度和群落组成与所使用的生物信息学方法有所不同，但是MiSeq序列的分析对于古细菌和细菌amoA基因都是可靠的，并用于随后评估土壤细菌氨氧化剂的潜在生态位专长。在进行生态分析之前，使用对现有AOB基因组中16S rRNA和amoA基因的系统发育分析，重新审视了以土壤AOB为主的亚硝基螺菌的系统发育分析。该分析支持两个基因系统发育之间的一致性，并增加了先前的系统发育分辨率，为具有潜在生态意义的其他基因簇提供了支持。使用这些新的测序，生物信息学和系统发育方法进行的环境序列分析首次证明了，AOB与AOA的生态位专长相似，表明pH是控制土壤氨氧化剂社区组成的关键生态因子。这项研究提出了第一个用于对功能基因的Illumina MiSeq测序进行最佳分析的生物信息学流水线，并且适用于任何扩增子大小（甚至大于500 bp的基因）。该管道用于提供陆地β-proteobacterialamoA基因的最新系统发育分析，并证明土壤pH值对其生态位专门化的重要性，并且广泛适用于其他生态系统和各种微生物群落。这项研究提出了第一个用于对功能基因的Illumina MiSeq测序进行最佳分析的生物信息学流水线，并且适用于任何扩增子大小（甚至大于500 bp的基因）。该管道用于提供陆地β-proteobacterialamoA基因的最新系统发育分析，并证明土壤pH值对其生态位专门化的重要性，并且广泛适用于其他生态系统和各种微生物群落。这项研究提出了第一个用于对功能基因的Illumina MiSeq测序进行最佳分析的生物信息学流水线，并且适用于任何扩增子大小（甚至大于500 bp的基因）。该管道用于提供陆地β-proteobacterialamoA基因的最新系统发育分析，并证明土壤pH值对其生态位专门化的重要性，并且广泛适用于其他生态系统和各种微生物群落。