Microbial Community DNA Extraction and Sequencing

DNA was extracted in duplicate from membrane filters using a PowerWater DNA Isolation Kit (MO BIO, USA) according to the manufacturer’s instructions. The DNA was quantified and then stored at −20 °C until further use.
Polymerase chain reaction (PCR) was used to amplify the V3–V4 hypervariable region of the bacterial 16S rRNA gene and the V3–V6 hypervariable region of the archaeal 16S rRNA gene. The primers used for bacterial 16S rRNA gene PCR amplification were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), and the primers for archaeal 16S rRNA gene PCR amplification were Arch344F (5′-ACGGGGYGCAGCAGGCGCGA-3′) and Arch915R (5′-GTGCTCCCCCGCCAATTCCT-3′). All primers contained barcodes for each sample43 (link). The products from the three replicate amplifications of the bacterial or archaeal 16S rRNA gene were pooled and evaluated on 2% agarose gels (TBE buffer). Amplicons were purified with a DNA gel extraction kit (Axygen, China), quantified with a QuantiFluorTM-ST fluorometer (Promega, Madison, WI, USA), pooled at equimolar concentrations, and finally sequenced on an Illumina MiSeq PE300 platform at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).