MMKhulais_P.SS
The recently proposed guidelines for standardizing the description of next-generation sequencing datasets in publications as described by Nilsson et al. (2008) were followed. Genomic DNA was extracted from 8 g of soil subsample after mixing the various samples of each site using the Mo Bio Power soil kit following the manufacturers instructions. High-throughput tag-encoded GS-FLX amplicon sequencing was used to assess fungal diversity in the selected sites. Amplicon libraries were performed using a combination of tagged primers designed for the variable ITS region, as recommended for the tag-encoded pyrosequencing method (Schloss et al. 2011). Genomic DNA samples were amplified using the fungal-specific primer pair ITS1F (59-XCTTGGTCATTTAGAGGAAGTAA) and ITS4 (59-YxxxxxTCCTCCGCTTATTGATATGC), where X and Y represent the two pyrosequencing primers (CCTATCCCCTGTGTGCCTTGGCAGTCTCAGT and CCATCTCATCCCTGCGTGTCTCCGACTCAGA) and xxxxx represents the barcodes designed for sample identification. Barcodes were unique and differed from each other by at least two nucleotides. Four PCR reactions were performed per sample using the following temperature program: 95şC/2 min, 25 cycles of 95şC/30 s, 54şC/1 min, 72şC/2 min, and 72şC/10 min. One ml of DNA template was used for the 40 ml PCR reaction containing 25.6 ml of MQ water, 4 ml of 10x buffer, 1.5 ml dNTPs (2.5 mM), 1.5 ml of reverse and forward primers (10 mM), 4 ml MgCl2 (50 mM), 0.5 ml BSA (10 mg/ml) and 0.4 ml BIOTAQ polymerase (5 U/ml). PCR products were pooled for each sample, purified and sequencing-adapters/tags were integrated via MegaPrimers (PCR). The amplicon length and concentration were estimated, and an equimolar mix of all amplicon libraries was used for pyrosequencing (with the ITS4 primer). Tagged samples were pooled and sequenced using the Genome Sequencer GS FLX titanium System (454 Life Sciences/Roche Applied Biosystems, Nutley, NJ, USA) at Macrogen Inc. (Seoul, Korea).