design: A total of 4 ng of DNA extracted from each sample were used for 16S rRNA gene amplicon sequencing. The amplicon library was prepared to target the V1V3 variable regions, as previously described (Matturro et al., 2021b). PCR reactions were performed in 25 L total volume containing Phusion Master Mix High Fidelity (Thermo Fisher Scientific, Italy) and 0.5 M final concentration of the library adaptors with V1V3 primers (Bacteria, 27F: 5-AGAGTTTGATCCTGGCTCAG- 3 and 534R: 5-ATTACCGCGGCTGCTGG-3). Libraries were purified using the Agencourt AMpureXP-beads protocol (Beckmann Coulter, Italy) and the concentration was measured with Qubit 3.0 fluorometer (Thermo Fisher Scientific, Italy). Purified libraries were pooled in equimolar concentrations and diluted to 4 nM. Phix control (15%) was added in the pooled libraries.