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).