16S of fecal samples
Briefly, 16S metagenomic sequencing library protocol (Illumina) was used to amplify the designated variable regions using region of interest-specific primers with overhang adapters attached. Forward primer 5TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG Reverse primer 5GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. Each PCR reaction consisted of the 2.5 l of 5 ng/l DNA template, 5 l 1 M reverse primer, 5 l 1 M forward primer and 12.5 l of 2x KAPA HiFi HotStart ReadyMix. PCR was performed using the following program: initial denaturation at 95C for 3 minutes, followed by 25 cycles of: 95C for 30 seconds, 55C for 30 seconds, 72C for 30 seconds, 72C for 5 minutes and held at 4C. The size of the PCR product was verified on a Bioanalyzer DNA 1000 chip and followed by purifying the V3-V4 regions using AMPure XP beads. A second round of PCR reaction was performed using Nextera XT Index Kit, followed by purification and size verification preparing samples for library quantification, normalization and pooling using Illumina guidelines. Samples were sequenced on the MiSeq sequencing platform, using a 2 300 cycle kit, following standard Illumina sequencing protocols. Bioinformatic analysis was performed by merging the pair-end sequences using FLASH. The sequences were then filtered and trimmed, and the error-free reads were picked for OUT clustering using CD-HIT-OUT.