Ampure xp bead purification kit

The DNeasy^® PowerSoil^® Pro Kit (Qiagen, Hilden, Germany) extracted microbial DNA from 0.5 g soil samples according to the manufacturer’s guidelines. DNA purity was checked with UV spectrophotometry (NanoDrop™ One^C spectrophotometer, Thermo Fisher Scientific) and double-checked using gel electrophoresis. DNA quantity was measured using a Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The DNA samples were kept at −80°C until used for library preparation for Illumina MiSeq sequencing.
The extracted DNA template was amplified using two-step PCR with universal primers 515F/907R for the bacterial 16S rRNA gene and ITS86F/ITS4R for the fungal internal transcribed spacer ITS (ITS1) region. Detailed descriptions of the primers and PCR conditions are provided in Table S1. The AMPure XP bead purification kit (Beckman Coulter, CA, USA) and Nextera^®XT Index Kit (Illumina, San Diego, CA, USA) were used for the cleanup library and ligation processes, respectively. The pooled library was quality checking using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). Sequencing was performed using the Illumina MiSeq platform (Illumina) at Kyungpook National University’s NGS Core Facility (Daegu, South Korea).

An aliquot of each DNA sample was normalized to 0.2 ng/μl. Once the normalization was finished the library was prepared using the Nextera XT DNA Sample Preparation Kit (Illumina Inc. San Diego, CA). Tagmentation of samples was done using 1 ng of template, as directed by manufacturer. Following tagmentation, PCR amplification was done according to manufacturer’s instructions using a unique combination of barcode primers (provided by the manufacturer) for each of the 56 samples, allowing samples to be multiplexed. Following amplification, short DNA fragments were removed using the AMPure XP bead purification kit (Beckman Coulter, Indianapolis, IN) and normalized through Library Normalization beads/additives. DNA fragments were then paired-end sequenced (250 base pair [bp] reads) in multiplexed pools on the Illumina HiSeq. 2500 platform (Illumina Inc.) at the Biotechnology Resource Center at Cornell University (Ithaca, NY).