Soil Fungal Diversity Analysis Using ITS2 Sequencing

Three replicate sub-samples were obtained from each soil sample under strict control conditions to avoid external contamination, as described by Rosa et al. [11 (link)]. Total DNA was extracted from these using the FastDNA Spin Kit for Soil (MPBIO, Solon, OH, USA). DNA quality was analyzed using agarose gel electrophoresis (1% agarose in 1× Trisborate-EDTA) and then quantified using the Quanti-iT™ Pico Green dsDNA Assay (Invitrogen). The internal transcribed spacer 2 (ITS2) of the rDNA was used as a DNA barcode for fungal identification [14 (link),15 (link)], using the primers ITS3 and ITS4 [16 ]. The Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 was used for library construction and DNA amplification followed the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Part #15044223, Rev. B). Paired-end sequencing (2 × 300 bp) was performed on a MiSeq platform (Illumina) by Macrogen Inc. (Seoul, South Korea).