design: bacterial 16S ribosomal DNA amplicons were prepared by amplification of the 16S rRNA gene V4 variable region with PCR primers U515F (GTGCCAGCMGCCGCGGTAA) and 805R (GGACTACHVGGGTWTCTAAT) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 C for 3 min, followed by 3035 cycles of 94 C for 30 s, 53 C for 40 s and 72 C for 1 min, after which a final elongation step at 72 C for 5 min was performed. Following amplification, PCR products were examined in 2% agarose gel to determine the success of amplification and the relative band intensity. Multiple samples are pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. The pooled and purified PCR product was used to prepare illumina DNA library.