SRX4389519 041415TM2 SRP153378 The 16S rRNA gene V4 variable region PCR primers 515/806 (OR OTHER PRIMER SELECTED) with barcode on the forward primer were used in a 30 cycle PCR (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 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. Multiple samples 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 DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturers guidelines. Sequence data were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). SRS3545860 041415TM2 miRNA-Seq METAGENOMIC PCR 273 0 Application Read Forward 1 Illumina MiSeq