16S rRNA Gene Amplification and Sequencing

The bacterial genomic DNA was amplified with the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) primers specific for the V1-V3 hypervariable regions of the 16S rRNA gene33 (link)34 (link)45 (link). Each forward primer incorporated FLX Titanium adapters and a sample barcode at the 5′ end of the reverse primer to allow all samples to be included in the single 454 FLX sequencing run. All PCR reactions were performed in 50-μl triplicates and combined after PCR. The products were extracted with the QIAquick Gel Extraction Kit (QIAGEN) and quantified on NanoDrop ND-1000 spectrophotometer, QuantiFluor-ST Fluorometer (Promega, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Equimolar concentrations of 83 samples were pooled and sequenced on a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer’s recommendations.