amplicon of fungi: desertified soil
Total DNA was extracted from 0.5 g homogenized soil per sample using the Power SoilTM DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA) following the manufacturers instructions. A total of 24 extracted DNA samples were quantified with a Nanodrop-1000 spectrometer (NanoDrop Technologies, Wilmington, DE, USA) and adjusted to a final concentration of 10 ng L-1. We used the universal primers 515F 5- GTGCCAGCMGCCGCGGTAA -3 and 909R 5- CCCCGYCAATTCMTTTRAGT -3 that targets regions V4-V5 of the 16S rRNA bacterial gene, which yields accurate phylogenetic information (WEISBURG, BARNS et al. 1991; Mathews, Sabina et al. 1999). In order to allow simultaneous analyses of several samples in a single sequencing run, the 5' end of the forward primer was fused with a 12 bp different barcode sequence to a single sample (Table S2). Each 25 L PCR reaction was performed containing 10 ng extracted DNA as template, Mg2+ free PCR buffer, 3 mM MgCl2, 200 mM dNTP, 200 nM forward primer, 200 nM reverse primer, and 1 unit PrimeSTAR Max DNA Polymerase (TaKaRa). The thermal cycling protocol was 95 C for 2 min as the first step, followed by 30 cycles of PCR: at 95 C for 15 s, at 57 C for 15 s, and at 72 C for 40 s, and a final 10 min extension at 72 C. All amplicons were run in an agarose gel 1% (w/v) and bands containing exact sizes were excised from the gel and purified using the QIAquick Gel Extraction kit (QIAGEN GmbH, Hilden, Germany).