title: IRE1TFY01__D4-15__ITS3
design: Randomly selected five terminal roots (ca. 2 cm each) from each sample were used for DNA extraction. All soil was carefully removed from each root sample by immersion in 70 percent ethanol containing 1-mm-diameter zirconium balls followed by shaking for 2 min at a rate of 15 times per sec using a TissueLyser II (Qiagen, Venlo, The Netherlands) (Toju et al. 2013, Ecol Evol). Washed roots were frozen at -25 degC and then pulverized by shaking at a rate of 20 times per sec for 3 min with 4-mm-diameter zirconium balls using a TissueLyser II. Total DNA was extracted from each root sample using the cetyltrimethylammonium bromide (CTAB) method described elsewhere (Sato and Murakami 2008, Mol Phyl Evol).
library_construction_protocol: We amplified the entire ITS region and the partial ribosomal large subunit region using the fungus-specific high-coverage primer ITS1F_KYO2 and the universal primer LR3 (see Toju et al. 2012, PLOSONE and citations therein). PCR was conducted with a temperature profile of 95 degC for 10 min, followed by 20 cycles at 94 degC for 20 s, 50 degC for 30 s, 72 degC for 120 s, and a final extension at 72 degC for 7 min using an Ampdirect Plus buffer system and Nova Taq. We subjected the PCR product from each root sample to a second PCR step that targeted the ITS2 region. The second PCR was conducted using the universal primer ITS3_KYO2 (Toju et al. 2012, PLOSONE) fused with 454 Adaptor A and sample-specific molecular ID; the reverse universal primer was LR_KYO1b (Toju et al. 2013, PLOSONE) fused with 454 Adaptor B. A buffer system of Taq DNA Polymerase together with Standard Taq Buffer (New England BioLabs, Ipswich, MA, USA) was used with a temperature profile of 95 degC for 1 min, followed by 40 cycles at 94 degC for 20 s, 50 degC for 30 s, 72 degC for 60 s, and a final extension at 72 degC for 7 min.