Illumina MiSeq of 16S rRNA gene amplicon: on-site wastewater treatment
Primers A519F (3-CAGCMGCCGCGGTAA-5) and 802R (3-TACNVGGGTATCTAATCC-5) were used to amplify the 16S V4 variable region. Samples were amplified with a barcoded forward primer and HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany) under the following conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 minutes was performed. PCR products from multiple samples were pooled together in equal proportions and purified using calibrated Ampure XP beads. Then the pooled and purified PCR product was used to prepare a DNA library by following Illumina TruSeq DNA library preparation protocol and sequenced as a paired-end set of reads.