design: The 16S rRNA gene V4 variable region PCR primers 515/806 (OR whichever PRIMER set the investigator has SELECTED) were used in a 30-35 cycle (depends on primers and DNA. Most studies use 30 cycles. Send inquiries to MR DNA as needed) PCR  (5 cycles used on PCR products if you sent PCR products instead of gDNA) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 95C for 5 minutes, followed by 30-35 cycles of 95C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 10 minutes was performed.  After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Samples are multiplexed using unique dual indices and are pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare an Illumina DNA library.