Metagenomic Analysis of Gut Microbiome

Total DNA was isolated from faecal samples (n = 27, caecum; n = 28, colon) by MasterPure Complete DNA&RNA Purification Kit (Epicentre, Illumina, WI, USA), with some modifications, as previously described^{50 (link)}. Isolated DNA concentrations were measured using a Qubit® 2.0 Fluorometer (Life Technology, CA, USA) and normalized to 10 ng/μL. The V3-V4 region of 16 S rDNA gene was amplified by PCR using Illumina adapter overhang nucleotide sequences following Illumina protocols. The multiplexing step was performed using Nextera XT Index Kit (Illumina, CA, USA). PCR product was checked with a Bioanalyzer DNA 1000 chip (Agilent Technologies, CA, USA) and libraries were sequenced using a 2 × 300 pb paired-end run (MiSeq Reagent kit v3) on a MiSeq-Illumina platform (FISABIO sequencing service, Valencia, Spain) according to manufacturer’s instructions (Illumina). Reagents employed for DNA extraction and PCR amplification were also sequenced as controls.