2.2. Microbiome DNA Extraction, PCR Amplification, and High-Throughput Sequencing

Microbiome DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions and including mechanic lysis of the samples using disruption spheres (FastPrep-24 MP). Extracted DNA was visualized in Tape Station 2200 (Agilent Technologies, Santa Clara, CA, USA) using Genomic DNA Screen Tape, according to the manufacturer’s indications and quantified by fluorescent probes (Qubit Thermo Fisher Scientific, Waltham, MA, USA).

Internal transcribed spacer (ITS) was amplified using the primer set ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’), with a barcode in the forward primer. For the amplification, the kit HotStarTaq Plus Master Mix (Qiagen, Hilden, Germany) was used with the following conditions: 94 °C 3 min, 28 cycles of 94 °C 3 s, 53 °C 4 s, and 72 °C 1 min, followed by an elongation phase of 72 °C 5 min. PCR products were examined in agarose gels (2%). Samples were purified using Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA). DNA libraries were constructed following the protocol TruSeq DNA sample preparation (Illumina, San Diego, CA, USA). Sequencing was performed by MrDNA Next Generation Sequencing Service Provider (Shallowater, TX, USA) on Illumina MiSeq platform in an overlapping 2 × 300 bp configuration to obtain a minimum throughput of 20,000 sequences (reads) per sample.