16S rDNA of fecal microbiome
Total genome DNA from fecal samples was extracted using TIANamp Bacteria DNA Kit (TIANGEN Biotechnology, China) according to the manufacturer protocol. Extracted DNA was further processed on an Illumina Miseq platform17. The V3-V4 region of the 16S ribosomal RNA gene was targeted for amplicon sequencing using sequence-specific primers (341F: CCTAYGGGRBGCASCAG, 806R: GGACTACNNGGGTATCTAAT) with attached Illumina adapter overhang sequences. All PCR reactions were carried out in 30 L reactions with 15 L of PhusionHigh-Fidelity PCR Master Mix (New England Biolabs). Then PCR products was mixed in equidensity ratios and mixture PCR products was purified with AxyPrepDNA Gel Extraction Kit (AXYGEN). Subsequently, sequencing libraries were generated using NEB NextUltraDNA Library Prep Kit for Illumina (NEB, USA) following manufacturers recommendations. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina Miseq platform and 250/300 bp paired-end reads were generated.