16S rRNA V4 of Crassostrea virginica: mucus sample from oyster 15
The relative abundance and composition of 16S rRNA genes was determined using 16S rRNA tagged Illumina MiSeq on the 12 samples and 3 of seawater.  DNA samples were processed by MR DNA Lab (www.mrdnalab.com, Shallowater, TX, USA). The 16S rRNA gene V4 variable region PCR primers 515F/806R (Caporaso et al., 2011) with barcode on the forward primer were used in a 30 cycle PCR (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) 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. After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. All samples are pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled sample are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at MR DNA on a MiSeq following the manufacturers guidelines. Sequence data were processed using a proprietary analysis pipeline (MR DNA, Shallowater, TX, USA).