Microbial Diversity Characterization of Sponge Tissue

DNA was extracted from the samples of sponge tissue (0.1–0.2 g) and primmorphs after bead beating using the TRIzol LS reagent (Invitrogen, USA) according to the manufacturer’s protocols. Total DNA from three technical replicates for each sample was suspended in 18 μl of RNase-free water and stored at −70 °C pending further analysis. The universal bacterial primers 518F and 1064R (Ghyselinck et al., 2013 (link)) were used to amplify the V4–V6 hypervariable region of the bacterial 16S rRNA gene. The following program was used to amplify 16S rRNA genes using PCR: 3 min at 96 ^∘C; 30 cycles at 94 °C for 20 s, 55 °C for 20 s and 72 °C for 1 min with a final 10-min incubation at 55 °C. PCR products were quantified using the NanoDrop device, mixed equally and sequencing using the 454 GS Junior Sequencing System (Roche, Basel, Switzerland) with GS FLX Titanium series reagents. Raw sequencing data are available in the NCBI Sequence Read Archive under accession number PRJNA480187.