*2.7. Analyses of 16S rRNA Gene Sequence, Species Richness and Diversity*

Aliquots of the extracted DNA were sent to the IGA Technology Service in Udine (Italy) for metagenomic analyses; the sequencing was performed through the MiSeq Illumina system platform. QIIME software (version 1.9.1) was used to perform bacterial community analysis as fully described by Kuczynski et al. [30]. There were two amplification steps in the library workflow: an initial PCR amplification using locus specific PCR primers and a subsequent amplification that integrates relevant flow-cell binding domains and unique indices (NexteraXT Index Kit, FC-131-1001/FC-131-1002). This method was used to amplify the variable V3 and V4 regions of the 16S rRNA gene aiming to characterize bacterial community compositions. Operational taxonomic units (OTUs) were identified using a cut-off of 97% similarity. Singletons, non-bacterial OTUs were removed, and the OTU abundance levels were normalized based on the sample with the least number of sequences.

To estimate the bacterial α-diversity, CHAO1, Shannon and Simpson indices and good-coverage were calculated using the QIIME software based on 10,000 sequences per sample. Rarefaction curves endpoints and normalization of counts for diversity analysis was set to 50% of the target sequencing coverage (i.e., for 100,000 fragments a cutoff of 50,000 fragments were applied). Bacterial taxonomic assignment was conducted using the Ribosomal Database Project (RDP) Naïve Bayesian classifier and reference database with a minimum confidence threshold of 50%.
