title: PCR-seq of fungi: soil
design: The 16S rRNA gene V4 variable region PCR primers 515/806 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: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed.  PCR products were gel checked on 2% agarose gels. Samples were pooled in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using Ampure XP beads, which were used to prepare DNA libraries the following Illumina TruSeq DNA library protocol. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer's guidelines.