4.6. Taxonomic and Phylogenetic Analyses

The obtained Illumina MiSeq reads were analyzed by using a bioinformatic workflow relying on the ASVs (Amplicon Sequence Variants) inference and their taxonomic classification. In particular, the Nextera adaptors and PCR (Polymerase Chain Reaction) primers were trimmed by using cutadapt [152] and avoiding any quality trimming in order to not influence the following denoising procedure. The obtained ASVs were taxonomically annotated in BioMaS by using the release 11.5 of the RDP database [153,154] and the NCBI taxonomy, as 16S rRNA reference collection and taxonomy, respectively. In particular, the query sequences were aligned to the reference collection by using bowtie2 [155] and the resulting alignments were filtered according to query coverage (≥70%) and identity percentage (≥90%).

The phylogenetic inference was achieved by using the align-to-tree-mafft-fasttree plugin: A multiple sequence alignment of ASVs sequences was obtained by using MAFFT [156] and the phylogenetic tree was inferred by applying the maximum-likelihood procedure implemented in Fasttree 2 [157]. Statistical comparisons between jellyfish compartments and sampling periods (T1 and T2) were performed by using DESeq2 [158]. Alpha (Shannon index (H’) [159] and Faith Phylogenetic index (PD) [160]) and Beta diversity (based on Bray–Curtis Dissimilarity matrix [161]) analysis were performed by using the phyloseq [162] and vegan package R packages [163]. PERMANOVA and SIMPER analysis (both with 999 permutations) were used to test differences between groups and infer the ASVs contribution in dissimilarity between groups, respectively.