Ileum Cirrhosis 69_S69 sample
16S libraries were carried out through a 2-step PCR-based protocol, following the Il-lumina 16S Metagenomic Sequencing Library Preparation guidelines (Illumina, San Diego, CA). The first PCR reaction allowed the amplification of a custom 548 bp amplicon covering the V4-V6 hypervariable regions of the 16S rRNA gene, as we previously de-scribed. Primers used in the first round of PCR also contained the overhang sequences with Illumina adapters; forward primer, 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCAGCAGCCGCGGTAAT-3; reverse primer, 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGTTGCGCTCGTTGC- 3. In detail, the PCR reactions were incubated at 95 C for 10 minutes; followed by 30 cycles of 95 C for 30 seconds, 59 C for 30 seconds, and 72 C for 1 minute. Amplicons obtained were purified using AMPure XP magnetic beads (Beckman Coulter, Milan, Italy). A second round of PCR was used to add the Illumina index barcodes (unique sequences required for multiple samples pooling and for clusters generation and sequencing, re-spectively) to the amplicons according to the Nextera XT protocol (Illumina, San Diego, CA). PCR conditions, this time, were 95 C for 3 minutes; 8 cycles of 95 C for 30 seconds, 55 C for 30 seconds, and 72 for 30 seconds. A second step of purification was set up and the quality of each library was assessed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Each library was diluted at 10 nM and pooled together to obtain a final 10 nM pool. Then two sequencing runs were performed using the Illumina MiSeq in-strument PE 600 cycles.