Computational protocol: Spatial Variations in Microbial Community Composition in Surface Seawater from the Ultra-Oligotrophic Center to Rim of the South Pacific Gyre

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[…] Closest relatives to the 16S rRNA gene sequences were searched for in GenBank by use of the BLASTn program . 16S rRNA gene sequences from all the clones were aligned with the ClustalX program (version 2.0) , and grouped into operational taxonomic units (OTUs) based on 3% (Bacteria) and 2% (Archaea) dissimilarity cut-off values calculated by the DOTUR program . We also used DOTUR program to calculate diversity indices including coverage, species evenness (J), abundance-based coverage estimator ACE , species richness estimator Chao1 , Shannon–Wiener index (H), and Shannon index (D) . The coverage was calculated as C = [1 – (n1/N) ×100], where n1 represents the OTUs represented by only one clone (i.e. singleton) and N represents the total number of clones in a library. It presents the probability that in the given library all the unique sequences were detected at least once . UNIFRAC principal coordinate analysis (PCoA) of weighted sequence data following previously established procedures were carried out to statistically determine the relationship between the bacterial and archaeal communities at the four sampling sites. Correlation between microbial community structure and environmental factors among the four sites were analyzed by canonical correspondence analysis (CCA) using the software Canoco (version 4.5, Microcomputer Power) .Phylogenetic trees were constructed using the Kimura 2-parameters distance matrix and the neighbor-joining algorithm by use of the PHYLIP package (version 3.69). A random selection clone sequence for each OTU was included. Phylogenetic affiliation of bacterial clones was determined by use of the RDP-II classifier . […]

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