DNA extraction, amplification and sequencing
Fecal DNA was individually extracted from sub-samples at more than 3 different regions of the original sample, using the QIAGEN QIAamp fast stool mini kit as per the manufacturer’s instructions. DNA was amplified targeting the V3-V4 region of bacterial 16S rRNA gene using primers, 341F (5’-CCTAGGGGNGGCWGCAG-3’) and 805R (5’GACTACHVGGGTATCTAATCC-3’) (Klindworth et al., 2013), and amplification was done using the following protocol: one denaturation step of 94°C for 3 min; 5 cycles of denaturation at 94°C for 15s and extension at 65°C for 60s, 20 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 20 s and extension at 72°C for 30 s, and a final extension at 72°C for 5 min. Sequencing library construction and amplicon sequencing were performed at Macrogen (Seoul, South Korea) using a 2×300bp Illumina MiSeq sequencing system (Illumina, USA).