16S amplicon sequencing of Undaria pinnatifida exudate-seawater: day 7
The 16S rRNA gene V4 variable region PCR primers 515/806, with barcode on the forward primer were used in a 30-cycle PCR using the HotStarTaq Plus Master Mix Kit(Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 30-35 cycles of94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongationstep at 72C for 5 minutes was performed. After amplification, PCR products are checked in2% agarose gel to determine the success of amplification and the relative intensity ofbands. Multiple samples are pooled together (e.g., 100 samples) in equal proportions basedon their molecular weight and DNA concentrations. Pooled samples are purified usingcalibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepareillumina DNA library. Sequencing was performed at MR DNA (www.mrdnalab.com,Shallowater, TX, USA) on a MiSeq following the manufacturers guidelines.