Library preparation of 16S ribosomal DNA (16SrDNA) and V3-V4 sequencing

We amplified DNA (5ng/µl) extracted from feces using primers targeting 16SrDNA V3-V4. Amplification of the DNA was done using the forward 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 3′ and reverse 5′ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC 3′ primers including Illumina Overhang adapter sequences. PCR was run following those conditions 95°C for 3 min, 25 cycles of: 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, with a final extension at 72°C for 5 min on a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems), purification of amplicons was done using AMPure XP beads (A63881, Beckman Coulter). A second PCR was performed in order to attach dual indices and Illumina adapters sequences to the target 16SrDNA V3-V4 using the Nextera XT index kit. PCR was run following this program 95°C for 3 min 8 cycles of: 95°C for 30 s, 55°C for 30 s, 72°C for 30 s and final extension 72°C for 5 min. Amplicons were pooled in equimolar concentration and diluted in 10 mM Tris, pH 8.5, to obtain a final concentration of 2 nM. The pool was quantified and validated using Fragment Analyzer device (FSv2-CE2, Proteigene). Denaturation of pooled libraries was done with 0.2 N NaOH diluted with hybridization buffer, containing 10% of PhiX. Sequencing was performed on Illumina Miseq instrument (Miseq™2013) using MiSeq v3 reagent kits. A total of 11,076,396 sequencing reads were identified and 9,204,083 read pairs were generated. Analyses were performed using QIIME2 pipeline. Taxonomic assignment was achieved referring to the Human intestinal Data Base (HiTDB) version 1.0 [18]. We evaluated the alpha diversity referring to QIIME2 pipeline, statistical analysis were performed using Kruskal–Wallis test. Beta diversity was estimated using QIIME2 workflow and statistical analysis were achieved using permanova method. We evaluated the relative abundance of bacteria between groups Kruskal–Wallis test, we performed also pairwise analysis referring to Mann–Whitney test.