Computational protocol: Microbial Diversity and Biochemical Potential Encoded by Thermal Spring Metagenomes Derived from the Kamchatka Peninsula

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[…] To assess the prokaryotic community structure, the retrieved 16S rRNA gene sequences were analyzed using QIIME []. The obtained 16S gene sequences were edited using gap4 [] and initially checked for the presence of chimeric sequences using Mallard [], Bellerophon [], and Chimera Check []. Remaining sequences were clustered employing the UCLUST algorithm [] and the following QIIME scripts: pick_otus.py and pick_rep_set.py. The sequences were clustered in operational taxonomic units (OTUs) at 1, 3, and 20% genetic dissimilarity.The phylogenetic composition of the prokaryotic communities in both samples was determined using the QIIME assign_taxonomy.py script. A BLAST alignment [] against the most recent SILVA ARB database [] was performed. Sequences were classified with respect to the taxonomy of their best hit in the ARB database. Finally, OTU tables were generated. Rarefaction curves, Shannon indices [], and Chao1 indices [] were calculated employing QIIME. In addition, the maximal number of OTUs (n max⁡) was estimated for each sample using the Michaelis-Menten-fit alpha diversity metrics included in the QIIME software package.One sequence per OTU (1% genetic distance) was further used for the construction of phylogenetic trees. Sequences were imported into the most recent SSU Ref SILVA database of the ARB program package []. Multiple sequence alignments were checked manually and improved by employing the ARB editor tool. Phylogenetic trees were created by employing the maximum parsimony algorithm implemented in ARB. The robustness of obtained tree topologies was evaluated by bootstrap analysis with 100 resemblings. […]

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