454 gs junior titanium system

DNA was extracted from 0.25 g soil with a Power Soil DNA Isolation Kit (Mo Bio, Carlsbad, CA, USA) according to the manufacturer's protocol, quantified using the dsDNA HS Assay Kit of the Qubit Quantification Platform (Invitrogen, Carlsbad, CA, USA), and stored at −30°C. PCR amplification of 16S rRNA genes was performed in a 50-µL reaction mixture containing 25 ng template DNA, 1 × AccuPrime PCR buffer II (Invitrogen), 200 nM forward and reverse primers, and 1 U of AccuPrime Taq polymerase (Invitrogen). Barcoded V4 forward primer (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGxxxxxxAYTGGGYDTAAAGNG-3′) and reverse primer (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCCGTCAATTCMTTTRAGT-3′) were used, where xxxxxx represents the barcode sequence designed for sample identification. PCR conditions were as follows: denaturation at 94°C for 2 min; 25 cycles of 94°C for 30 s, 52°C for 30 s, and 68°C for 45 s; and a final extension at 68°C for 3 min. PCR amplicons were purified using Agencourt AMPure reagent (Beckman Coulter, Danvers, MA, USA). The quality and quantity were checked on an Agilent Bioanalyzer (Agilent, Santa Clara, CA, USA) using an Agilent DNA 1000 kit, according to the manufacturer's protocols. Fragments were sequenced on a 454 GS Junior Titanium System (Roche, Indianapolis, IN, USA) according to the manufacturer's protocols.