Shifts of microbial communities in the abnormal tissues of the Red Sea sponge Carteriospongia foliascens
A set of primers, designed by adding an 8-nucleotide barcode to the universal forward primer U905F (5’-TGAAACTYAAAGGAATTG-3’) and the reverse primer U1492R (5’-GGTTACCTTGTTACGACTT-3’) for amplification of bacteria and archaea, were used. A 50-µl of PCR reaction mixture was composed of 2.5 U of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), 1×Pfu reaction buffer, 0.2 mM of dNTPs (TaKaRa, Dalian, China), 0.1 µM of each pair of barcoded primers and 5-10 ng of genomic DNA template. PCR was performed using a thermal cycler (Bio-Rad, USA) under the following conditions: first denaturation at 94 ? for 5 min; 30 cycles of 40 s of denaturation at 94 ?, 40 s of annealing at 50 ? and 60 s of extension at 72 ?; and final extension at 72 ? for 5 min. PCR products were quantified with the NanoDrop device, mixed equally and pyrosequenced on the ROCHE 454 FLX Titanium platform (Roche, Basel, Switzerland) at the National Human Genome Centre of China at Shanghai, China, according to the manufacturer’s manual.