ITS1 rDNA amplicon sequencing of the terrestrial fungal community, sample NTJ
The fungal ITS1 rDNA was amplified by nested PCR using  the fungal-specific forward primer ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3) and the universal reverse primer ITS4 (5-TCC TCC GCT TAT TGA TAT GC-3) in the first step, and barcoded ITS1_NGS_Cladonia_forward primer (5-TGCGGAAGGATCATTAATGAG-3) and ITS1_NGS_Cladonia_reverse primer (5-AGATCCGTTGAAAGTTTT-3) in the second step. The later primer pair contained three fully degenerated positions (Ns) at 5 end and sample-specific barcodes. The PCRs were performed using the ExTaq DNA polymerase (Takara Bio), they were run in 27 and 22 cycles in the first and second step, respectively and the conditions were: initial denaturation at 94 C for 2 min, 94 C denaturation for 30 s, 56 C amplification for 45 s and 72 C elongation for 1 min, with a final 72 C extension for 5 min. Four PCR negative controls (with water used instead of sample DNA) and twelve multiplexing controls (i.e., unused label combinations) were included. The PCR products were purified with SPRI AMPure XP paramagnetic beads (Beckman Coulter), pooled equimolarly and sent for library preparation and sequencing to Fasteris (Plan-les-Ouates, Switzerland). Sequencing was performed on the Illumina MiSeq platform with SINGLE end mode (2  300 bp). Illumina MiSeq SINGLE-end raw reads were filtered for average read quality threshold 26 using the script Reads_Quality_Length_distribution.pl (Bilint et al., 2014) and reads shorter than 150 bp were discarded. Sequencing adapters were removed from  both ends of the sequences using the Fastx Toolkit. SINGLE-end assembly was performed using PANDAseq, followed by removing primer artifacts using the script python remove_multiprimer.py (Bilint et al., 2014). SINGLE-end reads were then reoriented in 5'-3' direction using fqgrep. Demultiplexing was carried out using the demultiplex.sh script (Bilint et al., 2014) modified to output fastq files.