Computational protocol: Microbiome analysis and confocal microscopy of used kitchen sponges reveal massive colonization by Acinetobacter, Moraxella and Chryseobacterium species

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[…] Pyrosequencing data were analyzed with QIIME 1.9. First, the sequences were length-filtered (480–540 nucleotides), then MID, primer, and adapter sequences were removed, and finally quality filtering was applied (quality threshold 25) with final denoising. A maximum of one primer mismatch was allowed. Operational taxonomic units (OTUs) were generated at 97% sequence similarity level (corresponding to the theoretical boundary of species) using the uclust method. After OTU taxonomic identification (via the RDP database, using 0.05 as minimum confidence-level to record an assignment), chimeric OTUs were detected using Chimera Slayer and removed. Then, singletons as well as plastidic and mitochondrial OTUs were also removed. The dataset was normalized to 357 sequences per sample, and this rarified dataset was used to assess alpha- and beta-diversity, and to identify the OTUs significantly affected by the investigated factors.Statistical comparison of alpha diversity between samples was performed with Statistica version 12 (StatSoft Inc., Tulsa, OK, USA), using ANOVA and considering top and bottom parts as replicates of the individual sponges. Beta-diversity (phylogenetic distance between samples) was computed by jackknifed-UniFrac weighted pairwise distances, and the effect of the investigated factors was statistically assessed by the Adonis test. Beta-diversity plots (Principal Component Analysis, PCoA) were visualized by Emperor. The list of specific commands used for each QIIME step is available upon request. [...] In order to identify the most closely related species, the 16S rRNA gene consensus sequences of the ten most abundant OTUs were subjected to a phylogenetic analysis together with the sequences retrieved by manual BLAST and EzTaxon alignment. Mega7 was used for the multialignment, using the ClustalW algorithm, . Phylogenetic relationships were inferred by using the neighbor joining method with the maximum composite likelihood substitution model. One-thousand bootstrap re–samplings were used to statistically evaluate the tree topology. […]

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