16S amplicon sequencing of soil metagenome: wheat
The 16S rDNA gene (V1-V3 region) was amplified using barcoded forward (5-AGRGTTTGATCMTGGCTCAG-3) and reverse (5- GTNTTACNGCGGCKGCTG -3) primers . Using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 30 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 were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands.  Samples were 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 illumina DNA library.