Computational protocol: Environmental Factors Shaping the Community Structure of Ammonia-Oxidizing Bacteria and Archaea in Sugarcane Field Soil

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Protocol publication

[…] Raw sequences obtained by pyrosequencing were screened by eliminating sequences with a low quality (quality score < 25) and of insufficient length (AOB amoA < 450 nt and AOA amoA < 425 nt). Unique sequences among the trimmed sequences were obtained using the unique.seqs-function of the Mothur software (). AOB amoA sequences longer than 450 nt and AOA amoA sequences longer than 598 nt were downloaded from the NCBI database. Unique sequences from the downloaded sequence files were obtained using Mothur as described above. These sequences were used as references when a chimera check of the pyrosequencing data was performed using the chimera.uchime-function in Mothur. The non-chimeric sequences were aligned using the dpparttree algorithm in the Mafft software (http://mafft.cbrc.jp/alignment/software/tips.html). A distance matrix of the aligned sequences was calculated using the dist.seqs-function in Mothur. Clustering of the sequence data was carried out using cluster-function with the furthest neighbor algorithm in Mothur. Representative sequences of each cluster were obtained using the get.oturep-function in Mothur. The phylogenetic analysis was performed using MEGA5 software (). [...] Pairwise correlations between soil properties and the abundance or relative abundance of AOB and AOA were performed by Pearson’s correlation coefficient using SPSS (IBM, New York, NY, USA). Relationships between the composition of the AOB and AOA communities and soil properties were estimated using the Mantel test in Mothur. Dendrograms of AOB and AOA community structures were constructed using the Yue & Clayton measure of dissimilarity and unweighted pair group method with arithmetic mean (UPGMA) clustering in Mothur (). […]

Pipeline specifications

Software tools mothur, UCHIME, MAFFT, MEGA, SPSS
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Chemicals Ammonia