Human study for investigating the effects of chronic intake of Ecklonia cava (Seapolynol) on body fat level and related metabolism in obese adults. The study protocol was approved by the Institutional Review Board of Ewha Womans University (IRB No. 67-14) and registered prospectively at the WHO International Clinical Trials Registry Platform (ICTRP) via Clinical Research Information Service (CRIS) in Korea (Registration number: KCT0001074).
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Genomic DNA was extracted using the Fast DNA SPIN Kit for feces (MP Biomedicals, Santa Ana, CA, USA) and was quantified using a NanoDrop ND-2000 (Thermo Scientific, Rockford, IL, USA) at 260 nm. Quality was checked by the ratio of the absorbances at 260 and 280 nm. PCR amplification on the V1V3 region of the 16S rRNA gene was carried out with unique reverse barcoded primers, and the pyrosequencing was subsequently implemented with a 454 GS Junior Sequencing System (Roche, Branford, CT, USA). Raw sequence reads containing low-quality reads filtered by Phred quality score < 20 and reads length. Any reads shorter than 350 bp, or longer than 550 bp were discarded. Chimeric sequences introduced by PCR or amplification were identified and removed using VSEARCH with the SILVA gold database (Rognes et al., 2016). Next, the remaining sequence reads were clustered into operational taxonomic units (OTUs) using VSEARCH with de novo clustering algorithm under a threshold of 97% sequence similarity, followed by taxonomy assignment with UCLUST (parallel_assign_taxonomy_uclust.py script on QIIME 2) under default parameters (Caporaso et al., 2010). OTUs containing 1 sequence in only one subject were excluded from further analysis.