2.8. Fecal Metagenomic Analysis
Extraction of microbiome DNA and metagenome library preparationThe samples were subjected to quality control according to the sample type and product requirements. Genomic DNA was randomly fragmented. The fragmented genomic DNA was selected according to a certain average size, subjected to end-repair, and then 3′ adenylated. Adaptors were then ligated to the ends of these 3′ adenylated fragments. The PCR system and program were configured and set up to amplify the product. The corresponding library quality control protocol was selected depending on the product requirements. Single-stranded PCR products were produced via denaturation. Single-stranded circular DNA molecules were produced and replicated via rolling cycle amplification, and a DNA nanoball (DNB), which contains multiple copies of DNA, was generated. DNBs of sufficient quality were then loaded into patterned nanoarrays using the high-intensity DNA nanochip technique and sequenced through combinatorial probe-anchor synthesis (cPAS).Bioinformatic AnalysisAll of the raw data were trimmed, and the host-originating reads were removed (only for samples of host origin). High-quality reads were de novo assembled, and contigs less than 300 bp in length were discarded. Genes were predicted and redundant genes were removed with an identity and coverage cut-off of 95% and 90%, respectively. Significant differences in alpha diversity between the groups were determined using estimated marginal means analysis applied to a linear mixed model, built with alpha diversity as the response variable, the different groups and time points as the predictor variables, and subject number as a random variable. To generate the annotation information, the protein sequences of the genes were aligned against the functional database KEGG with an E value cutoff of 1 × 10