Metataxonomic analysis of fungi: Phragmites australis roots
DNA was extracted from 0.5 g of roots using the PowerPlant DNA Isolation Kit (Mo Bio, Carlsbad, California, USA). The library of ITS2 amplicons was prepared by PCR with primers ITS3_KYO2 and ITS4_KYO3 (Toju et. al., 2012) containing Illumina overhanging adaptors. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System and used for index PCR to attach dual indices and sequencing adapters using the Nextera XT Index Kit (Illumina). The products were purified using the Wizard SV Gel and PCR Clean-Up System. The libraries were analyzed with Agilent 2200 TapeStation System with D1000 ScreenTape kit (Agilent Technologies) and quantified using the Invitrogen QubitTM fluorometer with the QubitTM dsDNA HS Assay kit (Thermo Fisher Scientific). The libraries were normalized, pooled and pair-end sequenced (2 x 350 bp) using MiSeq (Illumina) with the output of ~100 000 reads per library.