2.10. Fecal DNA Isolation and 16S rRNA Sequence Analysis

On day 50, feces were collected in a sterile tube filled with 1 mL PBS and then immediately frozen at −80 °C. For isolation of DNA, 100–300 mg of fecal material was ground with silica beads and extracted with a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. PCR products for the V4 region of the 16S rRNA gene were amplified with region-specific primers that included the Illumina flowcell adapter (Illumina, San Diego, CA, USA) sequences and 12-base barcodes on the reverse primer. PCR production of the bacterial DNA template was quantified using Invitrogen’s PicoGreen. Taxonomic classification of 16S rRNA targeting amplicon reads and the eight most abundant bacterial sequences was performed using Illumina 16S Metagenomics workflow in the Miseq Reporter software curated by the GreenGene taxonomic database (https://basespace.illumina.com/analyses/). Alpha diversity was calculated based on the Shannon index for richness and evenness of bacterial sequences at rarefraction depth reads of the operational taxonomic unit sample.