title: Seawater2
design: The Tridacna specimens were cut open with a scalpel, and tissue samples of three different compartment were collected: gills, mantle, and the digestive system. All samples were weighed to the nearest 0.001 mg. The clam tissue was then homogenized using an Ultra Turrax T18 homogenizer (IKA-Werke GmbH and Co. KG, Staufen, Germany) with 15 mL MilliQ water (sterilized under UV light for one hour, and filtered through a 0.2 m syringe filter). The homogenate was frozen at -80C until further processing. In total, we extracted DNA from 62 samples, including 54 T. maxima tissue samples, four seawater samples and four negative controls (to assess for kit and lab contaminations; except for one negative control, which originated from the subsequent PCRs). DNA from water and tissue samples were extracted with the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions with minor modifications. Briefly, 90 L of the tissue homogenate was added to 1.5 ml Eppendorf tubes containing 90 L of ATL buffer and 20 L of Proteinase K and incubated at 56 C for 1 hr. DNA extractions were then conducted according to manufacturers instructions. Water filters were thawed and placed in 1.5 mL Eppendorf tubes, 360 L of ATL buffer and 40 L Proteniase K buffer were added, and the tubes were incubated at 56 C for 20 minutes. DNA extractions were then conducted according to manufacturers instructions. DNA concentrations were measured using a Qubit 2.0 fluorometer (ThermoFisher Scientific), and samples were adjusted to 10 ng/L. To amplify the hypervariable regions v5 and v6 of the bacterial 16S rRNA gene, the primers 784F [5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGGATTAGATACCCTGGTA-3] and 1061R [5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCRRCACGAGCTGACGAC-3] (Illumina adapter sequences underlined) were used (Andersson et al., 2008). For all samples, triplicate PCRs were performed using 10 ng of DNA using the Qiagen Multiplex PCR kit and a final primer concentration of 0.3 M in a reaction volume of 10 l. Thermal cycling conditions were: 95C for 15 min, followed by 27 cycles of 95C for 30 s, 55C for 90 s, 72C for 30 s, and a final extension cycle of 72C at 10 min. Five L of the PCR were run on a 1% agarose gel to visualize amplification. Triplicated PCRs from each sample were pooled, cleaned with ExoProStar 1-step (GE Healthcare, UK), and subsequently underwent an indexing PCR step (according to the Illumina 16S metagenomic sequencing library preparation protocol). Successful addition of indexes was checked by size comparison on a 1% agarose gel. Samples were purified and normalized using the SequalPrep Normalization Plate Kit (Invitrogen, Carlsbad, USA). Next, from each pooled and normalized sample, 4 L were transferred into a 1.5 mL Eppendorf tube, and concentrated using the CentriVap Benchtop Vacuum Concentrator (LABNOCO, USA). The final library was assessed using a Bioanalyzer Agilent Technologies, USA) and quantified using the Qubit dsDNA HS assay with the Qubit 2.0 Fluorometer (ThermoFisher Scientific, USA). The 16S rRNA gene amplicon library was sequenced at the KAUST sequencing facility on the Illumina MiSeq platform. Sequencing was performed at 6 pM with 20% PhiX on the Illumina MiSeq platform at 2 x 301 bp paired-end V3 chemistry according to the manufacturers specifications.