Archaeal 16S rRNA gene amplicon sequencing
Archaeal 16S rRNA genes were PCR-amplified using the barcoded primer pairs 524F-10-ext (5-TGYCAGCCGCCGCGGTAA-3) / Arch958R-mod (5-YCCGGCGTTGAVTCCAATT-3). PCR mixtures (20 L) were prepared in triplicate including 4 L of 5FastPfu Buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L of bovine serum albumin, and 10 ng of template DNA. PCR reactions contained an initial denaturation at 95 C for 3 min, followed by 37 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, elongation at 72 C for 45 s, and a final extension at 72 C for 10 min. Amplicon products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA) and quantified using QuantiFluorTM-ST (Promega, USA). Adaptor sequences were ligated onto the amplicons for library construction. The libraries were mixed in equimolar amounts, and sequenced using an Illumina MiSeq platform with a paired-end strategy (Illumina, USA).