Gut Microbiome Analysis via 16S rRNA Sequencing

Fresh fecal samples were collected during the last week of the experiment for gut microbial characterization. Bacterial genomic DNA was extracted from frozen fecal samples stored at −80 °C. A smear approximately 5 mm² (roughly the size of a pencil eraser) was taken from a fecal sample using a sterile swab in a test tube. DNA extraction and PCR amplification of variable region 4 (V4) of the 16S rRNA gene using Illumina-adapted universal primers 515F/806R was conducted using the direct PCR protocol (Extract-N-Amp Plant PCR kit (Sigma-Aldrich, Inc., St. Louis, MO, USA)) as previously described [17 (link),18 (link)]. Briefly, PCRs were conducted in triplicate in 96-well plates (denaturation for 3 min at 94 °C; 35 cycles (98 °C, 60 s; 55 °C, 60 s; 72 °C, 60 s) followed by elongation for 10 min at 72 °C). Positive amplicons were pooled in equimolar concentrations into a composite sample that was size selected (300–500 bp) using agarose gel to reduce non-specific products from the host DNA. Sequencing was performed on the Illumina MiSeq platform (San Diego, CA, USA) with the addition of 20% PhiX (illumina cat #15017666, San Diego, CA, USA) and paired-end reads of 175 b in length were generated in each direction.