| Construction Protocol | We sequenced fungal ITS sequences based on a tag-encoded massively-parallel pyrosequencing. The entire ITS region was amplified using the fungus-specific high-coverage primer ITS1F_KYO2 (5'-TAG AGG AAG TAA AAG TCG TAA-3') and the universal primer ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') with the buffer system of Ampdirect Plus (Shimadzu) and BIOTAQ HS DNA Polymerase (Bioline). PCR was conducted under a temperature profile of 95 degrees C for 10 min, followed by 30 cycles of 94 degrees C for 20 s, 50 degrees C for 30 s, and 72 degrees C for 30 s, and final extension at 72 degrees C for 7 min. The PCR product of each root sample was subjected to the second PCR step targeting ITS2 region using the universal primer ITS3_KYO2 (5'-GAT GAA GAA CGY AGY RAA-3') fused with the 454 Adaptor A (5'-CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG-3') and each sample-specific molecular ID, and the reverse universal primer ITS4 fused with the 454 Adaptor B (5'-CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG-3'). The second PCR was conducted with the buffer system of Taq DNA Polymerase with Standard Taq Buffer (New England BioLabs) under a temperature profile of 95 degrees C for 1 min, followed by 40 cycles of 94 degrees C for 20 s, 50 degrees C for 30 s, and 72 degrees C for 30 s, and final extension at 72 degrees C for 7 min. The ITS amplicons of the second PCR step were subjected to pyrosequencing. The first 480 and the second 480 samples were sequenced separately using a GS Junior sequencer (Roche). The ITS amplicons of the first 480 root samples were pooled and purified using ExoSAP-IT (GE Healthcare) and QIAquick PCR Purification Kit (QIAGEN). The sequencing of the first 480 samples was conducted as instructed by the manufacturer. Likewise, the amplicons of the remaining 480 samples were pooled and purified, and then sequenced in the second run. |
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