Ballast Water Metagenom-June2013
The universal 16S Eubacterial primers 515/806 with barcode on the forward primer  were used to amplify the V4 region of the 16S rRNA gene in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA). The following conditions were used: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute. A final elongation step at 72°C for 5 minutes was performed. After amplification, all samples were pooled together in equal proportions. Pooled samples were purified using calibrated Ampure XP beads. Subsequently, the DNA library was prepared by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer’s guidelines.