design: V3&V4 hyper variable regions of 16S gene was amplified using KAPA HiFi HotStart ReadyMix PCR Kit (Catalog: 0370, KAPA BIOSYSTEMS).Upon the confirmation on the amplification, the PCR products were cleaned up, using 0.9X AMPure XP beads. Further unique P7 and P5 barcoding was performed and the final PCR products were again purified using 0.9X AMPure XP beads and the final library was eluted in 15uL of 0.1X TE buffer.