title: Amplicon sequencing of soil metagenome: AM day 85
design: At first PCR amplification was carried out using the 16S rRNA universal bacterial primers, 341Fbac (CCTACGGGAGGCAGCAG) (Muyzer et al., 1993) and 909R (CCCCGYCAATTCMTTTRAGT) (Tamaki et al., 2011) to amplify a 568- bp fragment of the 16S rRNA gene flanking the V3 and V4 regions. A single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was used. Following PCR, amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corpora- tion, MA, USA). Finally samples were sequenced utilizing Illumina MiSeq instruments and reagents, following manufacturers guidelines.