Library preparation was performed according to Illumina 16 S Metagenomic Sequencing Library Preparation protocol (Illumina, San Diego, CA, USA). The V3-V4 hypervariable region of the 16 S rRNA gene was amplified by PCR in 50 µL final volume, containing 25 ng of microbial DNA, 2X KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), and 200 nmol/L forward 314 and reverse 785 primers carrying Illumina overhang adapter sequences [21 (link)]. The PCR thermocycle consisted of 3 min at 95 °C, then 30 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and a final elongation step at 72 °C for 5 min [20 , 22 (link)]. Amplified products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Indexed libraries were prepared by limited-cycle PCR using Nextera technology (Illumina) and purified again as described above. The libraries were then quantified using a Qubit 3.0 fluorimeter (Invitrogen, Waltham, MA, USA), normalized to 4 nM, and pooled. Finally, the library pool was denatured with 0.2 N NaOH and diluted to 4.5 pM with a 20% PhiX control. Sequencing was performed on an Illumina MiSeq platform using a 2 × 250-bp paired-end protocol, according to the manufacturer’s instructions (Illumina).
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