gut microbiota stool sample: healthy control
Fecal DNA extraction was performed using the Spin stool DNA kit (Stratec Molecular, Berlin, Germany), according to manufacturers instructions. For each sample, 25 ng of extracted DNA was used to construct the sequencing library. The V3V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with a two-step barcoding approach according to the Illumina 16S Metagenomic Sequencing Library Preparation (Illumina, San Diego, CA, USA). Briefly, DNA samples were amplified with dual-index primers using a Nextera XT DNA Library Preparation Kit (Illumina) and library concentration and quantification were determined using a KAPA Library Quantification Kit (Kapa Biosystems, Woburn, MA, USA) and Agilent 2100 Bioanalyzer System (Agilent, Santa Clara, CA, USA), respectively. The libraries were pooled and sequenced with a MiSeq platform (Illumina) for 2  250 base paired-end reads and a total of 2.21 Gbases raw reads were obtained.