Gut Microbiota Analysis in Mice

Feces (0.25 g per mouse) of different cages were used in each experimental group for microbiota analysis following a previous protocol [56 (link)]. In summary, metagenomic DNA was extracted from 0.25 g of feces using the DNeasy PowerSoil Kit (Qiagen, MD, USA) using the Miseq300 platform (Illumina, San Diego, CA, USA) at the Omic Sciences and Bioinformatics Centre and Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University. The raw sequences were quality processed and classified into operational taxonomic units (OTU) following Mothur’s standard operating platform procedures [57 (link),58 (link)]. Bioinformatic analyses included good coverage, alpha diversity (e.g., Chao), and beta diversity. Linear discriminant effect size analysis (LEfSe) and meta-stats were also performed to determine the species marker and the unique representative species of the interested group, respectively [57 (link),59 (link)].