Illumina Sequence Sample from Hudson River Locale: Piermont Pier sampleid: HR2.108
The 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer were used in a 28 cycle PCR (5 cycle used onPCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the followingconditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 secondsand 72C for 1 minute, after which a final elongation step at 72C for 5 minutes was performed.After amplification, PCR products are checked in 2% agarose gel to determine the successof amplification and the relative intensity of bands. Multiple samples are pooled together(e.g., 100 samples) in equal proportions based on their molecular weight and DNAconcentrations. Pooled samples are purified using calibrated Ampure XP beads. Then thepooled and purified PCR product is used to prepare illumina DNA library. Sequencing wasperformed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following themanufacturers guidelines.