Bacterial Gut Microbiome Analysis by 16S rDNA Sequencing

Bacterial genomic DNA was extracted from ileal digesta using NucleoSpin^® DNA Stool kit (MACHEREY-NAGEL Company, Germany). The quality of DNA samples were checked with gel electrophoresis. Bacterial 16S rDNA sequences spanning the variable regions V3–V4 were amplified according to the method described previously (Wang et al., 2019 (link)). The sequencing of the PCR products was performed on an Illumina HiSeq2500 PE250 platform (Illumina, San Diego, CA, United States). The sequencing results has been submitted to the Sequence Read Archive of the NCBI (Accession no. SAMN13610217). All the effective reads were clustered into operational taxonomic units (OTUs) based on a 97% sequence similarity. Classification of OTUs at various taxonomic levels were implemented by comparing sequences to the GreenGene database. Microbial α-diversity was analyzed using the MOTHUR v1.31.2 program (Schloss et al., 2009 (link)). The principal co-ordinates analysis (PCoA) and plot and non-metric multidimensional scaling (NMDS) were used to assess pairwise distances among samples (β-diversity). Linear discriminant analysis (LDA) combined effect size measurements (LEfSe) was applied to identify the relative richness (LDA > 2, P < 0.05) of bacteria between groups.