Metagenomic Analysis of Gut Microbiome

DNA was extracted from fecal samples and 16S rRNA analyses were performed as previously described [57 (link)] by MyMetagenome Co., Ltd. (Tokyo, Japan). Briefly, PCR was performed using 27Fmod 5′-AGRGTTTGATYM TGGCTCAG-3′ and 338R 5′-TGCTGCCTCCCGTAGG AGT-3′ primers to amplify the V1–V2 region of the bacterial 16S rRNA gene. The amplified DNA (~330bp) was purified using AMPure XP (Beckman Coulter) and quantified using a Quant-iT Picogreen dsDNA assay kit (Invitrogen) and a TBS-380 Mini-Fluorometer (Turner Biosystems). The 16S amplicons were then sequenced using a MiSeq according to the Illumina protocol. The paired-end reads were merged using the fastq-join program based on overlapping sequences. Reads with an average quality value of <25 and inexact matches to both universal primers were filtered out. Filter-passed reads were analyzed further after trimming off both primer sequences. For each sample, 3,000 high-quality filter-passed reads were rearranged in descending order according to quality value and then clustered into operational taxonomic units (OTUs) with a 97% pairwise-identity cutoff using the UCLUST program version 5.2.32 (https://www.drive5.com). Taxonomic assignments of OTUs were performed based on similarity searches against the Ribosomal Database Project and the National Center for Biotechnology Information genome database using the GLSEARCH program [58 (link)].