title: Osyter microbiome (eDNA 16S)
design: DNA was extracted from 1 L of seawater and 10 mL of microalgal culture filtered through 47 mm diameter, 0.45 m pore-size, mixed cellulose ester filters (Advantec) under 45 mm Hg negative pressure, and extracted from the filters using a DNeasy Power Soil DNA extraction kit (Qiagen, Hilden, Germany) following the manufacturers protocol. DNA was also extracted from 250 L of suspended fertilized eggs or XX oyster larvae using the same approach. The bead-beating step for the Power Soil extraction was carried out on a 1600 MiniG automated tissue homogeniser (SPEX Sample Prep, Metuchen, USA) at 1500 rpm for two minutes. Two blocking primers were designed to target Chaetoceros (5'-TCT AAT CCC ATT TGC TAC CCT A-C3-3') and Tisochrysis lutea (5'- TCT AAT CCC TTT TGC TAC CCT A-C3-3') species, the species of microalgae used as feed during this experiment. The blocking primers contained at least five mismatches with bacterial 16S rRNA gene sequences and annealed to the microalgal template at the 3' end of the 805-R reverse primer. The blocking primers also included a C3 spacer at the 3' end so that extension of the PCR product did not occur. The blocking primers were included in the PCRs at 10 times the concentration of universal 16S rRNA gene primers. For the final PCR amplifications, the universal 16S rRNA gene primers were modified to include IlluminaTM overhang adaptors (forward: 5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-3' and reverse: 5'- GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G-3'), as described in Kozich et al. (2013).