2.4. Sample collection and gut microbiota analysis
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Fecal samples were collected twice before and after SAAT or sham treatments for participants, shipped on dry ice within half an hour after collection, and then frozen at −80°C freezer for analysis. Microbial DNA was extracted from human samples using the HiPure Soil DNA Kit B (Magen Biotech., Guangzhou, China) according to the manufacturer's protocol. The extracted DNA was quantified and the V3 and V4 hypervariable regions were amplified by polymerase chain reaction (PCR) system (ABI GeneAmp® 9700, Applied Biosystems, Waltham, MA) with the primers containing the forward sequence of “CCTACGGRRBGCASCAGKVRVGAAT” and the reverse sequence of “GGACTACNVGGGTWTCTAATCC.” The PCR reactions were conducted using the following program: 3 minutes of denaturation at 95°C, 27 cycles of 30 seconds at 95°C, 45 seconds for annealing at 55°C, and 45 seconds for elongation at 72°C, and final extension at 72°C for 10 minutes. PCR products were purified and quantified by the agarose gel electrophoresis with 1.5% agarose gel. Then the indexed adapters were added to the ends of the 16S rDNA amplicons to generate the indexed libraries ready for downstream next-generation sequencing sequencing on lllumina Miseq (Illumina, San Diego, CA). The concentrations of DNA libraries were validated by Qubit3.0 Fluorometer (Invitrogen, Waltham, MA, USA). After quantifying the library to 10 nM, the DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to the manufacturer’s instructions (TruSeq™ DNA Sample Prep Kit, Illumina, San Diego, CA). Afterward, the sequencing was performed using the paired-end approach. Further, the image analysis and base calling were conducted by the MiSeq Control Software embedded in the MiSeq instrument. The positive and negative reads were joined together to filter the results contained in the sequence of N and the sequence length larger than the 200 bp sequence was retained. The retained sequences were qualitatively filtered and the chimeric sequences were deleted, and the sequence operational taxonomic unit (OTU) cluster analysis was then performed using VSEARCH (1.9.6) with the similarity set to 97%. The reference taxonomy was SILVA release 132. The Ribosomal Database Program Classifier Bayesian Algorithm of the OTU species taxonomy was then used to analyze representative sequences and under different species classification levels, statistical community compositions of each sample were performed. Microbiota alpha diversity was assessed by using the abundance-based coverage estimator and Chao1 indices of species richness and the Shannon and Simpson indices of diversity. Next, we assessed the β diversity of gut microbiota in diverse groups using principal component analysis (PCA), unweighted principal coordinate analysis (PCoA), and weighted distance matrices (nonmetric multidimensional scaling, NMDS). Finally, Linear discriminant analysis coupled with effect size (LEfSe) was performed with LEFSE software (LEfSe 1.0) to identify potential microbial biomarkers between groups.