title: 16S sequencing of gut microbiota in healthy individuals from different regions
design: 16S sequencing of gut microbiota in healthy individuals from different regions
library_construction_protocol: Each participant provided the fresh stool sample in a collection tube filled with a DNA stabilization buffer (Canvax Biotech).  DNA microbial extraction was performed using the QIAamp DNA Stool Mini Kit (Qiagen) starting from 250 µl of each sample according to the manufacturer's protocol. After assessing DNA concentration and purity, samples were stored at -80° until processing. The V3–V4 hypervariable region of the bacterial 16S  rRNA gene was amplified from total DNA according to the Illumina 16S Metagenomic Sequencing Library Preparation instructions, as previously described in Diamanti-Picchianti A, et al (Int J Mol Med ). In summary, the V3-V4 amplicon was obtained by PCR with universal primers, followed by purification. A second PCR was then performed to barcode the libraries using the Illumina dual-index system. Following a second step of purification, libraries were diluted to 4nM and pooled.