Bacterial DNA Isolation and 16S rRNA Sequencing

Bacterial DNA from stool samples was isolated using a QIAamp stool DNA mini kit (QIAGEN, Hilden, Germany) by applying the method described in our previous report [39 (link)]. PCR of the V1-V3 region of the 16s rRNA gene sequences was carried out using a C1000 Touch thermal cycler with a 96-deep-well reaction module (Biorad, Hercules, CA, USA). The PCR products were purified using a LaboPass PCR purification kit (COSMO GENTECH, Seoul, Korea). The amplicons of each sample were pooled in equimolar amounts and then purified using AMPure XP beads (Agencourt Bioscience, Beverly, MA, USA) and finally quantified using a PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA, USA). The mixed amplicons were amplified on sequencing beads by undertaking emulsion PCR. The sequencing reactions were conducted on a Roche/454 GS Junior system (454 Life Sciences, Branford, CT, USA) following the manufacturer’s instructions.