title: ITS Amplicon seq of soil fungi from southeastern pine forest
design: DNA was extracted from 3 analytical replicates per sample and then pooled to increase mass and reduce environmental heterogeneity.  Fungal sequencing was accomplished through target-based amplification of the entire fungal internal transcribed spacer (ITS) region (ITS1, 5.8s, ITS2) with primers ITS1 (5-TCCGTAGGTGAACCTGCGG-3; White et al., 1990) and ITS4 (5-TCCTCCGCTTATTGATATGC-3; White et al., 1990). These primers (ITS1 and ITS4) were selected because of sequence variability and representation in public data repositories and reference databases (Nilsson et al., 2009) Barcodes (8 bp) were attached to the forward primer (ITS1). PCR amplification was accomplished by utilizing the HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA) under the following conditions: an initial denaturation step at 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. After amplification, PCR products were verified via gel electrophoresis (2% agrose gel) to determine the success of amplification and the relative intensity of bands. Multiple samples were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified with calibrated AMPure XP beads (Agencourt Biosciences Co., Brea, CA, USA). The pooled and purified PCR products were then used to prepare an Illumina DNA library for each sample (n=36).  Synthesis-based sequencing was performed at Molecular Research DNA Laboratory (www.mrdnalab.com, Shallowater, TX, USA) on an Illumina MiSeq following the manufacturers guidelines.