Computational protocol: Community shift of ammonia-oxidizing bacteria along an anthropogenic pollution gradient from the Pearl River Delta to the South China Sea

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Protocol publication

[…] The bacterial amoA gene sequences from this study were firstly evaluated by the rarefaction analysis, and richness estimators (Chao1 and Shannon) and diversity index (Simpson) were simultaneously calculated using DOTUR (Schloss and Handelsman ) prior to other analyses. To keep the congruence between different studies, the distance cut-off was set at 5% for operational taxonomic units (OTUs) definition in DOTUR (Schloss and Handelsman ).Bacterial amoA gene sequences recovered from the present study and the relevant ones retrieved from GenBank including those from the Mai Po Nature Reserve marshes and the Pearl River Estuary were transferred into MEGA 5.0 (Tamura et al. ) and merged for editing and further analyses (Cao et al. ; Jin et al. ; Li et al. ). All sequences were aligned by Clustal X 1.81 (Higgins et al. ) and the phylogenetic analysis was conducted in MEGA 5.0 (Tamura et al. ). Phylogenetic tree was constructed based on the neighbor-joining criterion and credible values for every node were evaluated with bootstrap method via 1,000 replicates in MEGA 5.0 (Tamura et al. ).Principal coordinates analyses (PCoA), Jackknife Environment Clusters analyses were conducted in an online software UniFrac ( (Lozupone et al. ) which employs the genetic distances to evaluate the community similarity based on the gene sequences data. The bacterial amoA gene sequence communities from the SCS surface sediments and other former studies were classified on the basis of the environmental variables. The environment clusters tree was projected in MEGA 5.0 (Tamura et al. ). Canonical correspondence analysis (CCA) was executed in CANOCO 4.5 to determine the correlations between bacterial amoA gene sequence community compositions from northern SCS and physicochemical parameters (ter Braak and Smilauer ). In addition, Pearson moment correlations were also evaluated with Microsoft Excel to relate the physicochemical factors with the diversity and richness indices of bacterial amoA genes. […]

Pipeline specifications

Software tools DOTUR, Clustal W, UniFrac
Applications Phylogenetics, 16S rRNA-seq analysis
Chemicals Ammonia