16S of Patient28
Participants were carefully instructed on the procedures for fecal sample collection. Stool samples were collected at home in a commercial sterile, dry screw-top container. Upon collection, stool samples were delivered to local hospital and immediately frozen at -80 C until further processing. Microbial DNA was extracted from fecal samples using the E.Z.N.A. Stool DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturers protocols. The V3-V4 region of the bacteria 16S ribosomal RNA gene were amplified by PCR (95 C for 2 min, followed by 25 cycles at 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s and a final extension at 72 C for 5 min) using primers 341F: 5- CCTAYGGGRBGCASCAG-3, 806R: 5- GGACTACNNGGGTATCTAAT-3, where barcode is an eight-base sequence unique to each sample. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturers instructions. Purified PCR products were quantified by Qubit3.0 (Life Invitrogen) and every twenty-four amplicons whose barcodes were different were mixed equally. The pooled DNA product was used to construct Illumina Pair-End library following Illuminas genomic DNA library preparation procedure. Then the amplicon library was paired-end sequenced (2  250) on an Illumina platform (Shanghai BIOZERON Biotech. Co., Ltd ) according to the standard protocols.