high-throughput sequence of environmental bacterial 16S rRNA gene: arctic soil
The V4 region of the bacterial 16S rRNA gene was amplified using the primers 533F (5'- TGCCAGCAGCCGCGGTAA-3') and Bact 806R (5'-GGACTACCAGGGTATCTAATCCTGTT-3')) with a unique 8-bp barcode at the 5' end of 533F. PCR was carried out in triplicate reactions using 10-50 ng of DNA. Thermal cycling conditions consisted of initial denaturation at 94 oC for 5 min followed by 25 cycles of denaturation at 94 oC for 30 s, annealing at 58 oC for 40 s, and extension at 72 oC for 30 s with a final extension at 72 oC for 10 min. PCR products were gel-purified using an EZNA Gel Extraction Kit (Omega Bio-Tek, Inc., USA). The purified DNA was sequenced on the Illumina MiSeq platform. The primer sequence were included in the reads.