454 sequencing of soil sample: subtropical forest
The nuclear ribosomal internal transcribed spacer region (ITS rDNA gene) was amplified by polymerase chain reactions (PCRs) using the fungal primer set of ITS1 (5ĄŻ-NNNNNNNNNNTCCGTAGGTGAACCTGCGG-3ĄŻ) and ITS4 (5ĄŻ-TCCTCCGCTTATTGATATGC-3ĄŻ) (White et al. 1990; Arnold & Lutzoni 2007), where NNNNNNNNNN represents the 10 base-pair (bp) barcode designed for sample identification. The PCRs were performed in a 20 ŚĚL reaction mixture containing 0.8 ŚĚL of 5 ŚĚM of each primer and 10ng of template DNA, 4 ŚĚL 5ĄÁ FastPfu Buffer, 0.4 ŚĚL FastPfu polymerase, 2 ŚĚL of 2.5-mM dNTP mix (all from Beijing TransGen Biotech Co., Ltd. Beijing, China). The PCR amplification program was set as follows: samples were initially denatured at 95ĄăC for 2 min, then amplified by 35 cycles of denaturation at 95ĄăC for 45 s, primer annealing at 50?C for 30 s, extension at 72?C for 30 s. A final extension step of 5 min at 72ĄăC was added to ensure complete amplification of the target region. We conducted PCR reactions in triplicate for each sample. Then PCR products were purified with AcyPrepTM DNA gel extraction kit (Axygen Biosciences, CA, USA) as directed by manufacturer. Equimolar ratios of amplification products from individual soil samples were combined to generate the composite sample for 454 sequencing as previously described (Fierer et al. 2008). The amplicon library was sequenced on a Genome Sequencer 454 FLX System (Roche 454 Life Sciences, CT, USA).