| Construction Protocol | After frozen sponge specimens were defrosted, sponge tissues were washed briefly with TNE buffer (10 mM Tris-HCl [pH 8.0], 3.5 % [w/v] NaCl, and 50 mM EDTA [pH 7.5]) to remove temporally attached debris. Ten g of washed sponge tissues were cut into small pieces (<1 cm3) and homogenized with twice volume of TNE buffer using a food processing device for 1 min. Genomic DNAs of sponge and bacterial residents were extracted from the homogenate by using a DNA extraction kit (ISOPLANT, Nippon gene, Ohtsu, Japan) according to manufacturer???s protocol. The extracted DNA was further purified with a Genomic-tip (QIAGEN) to eliminate inhibitory elements for PCR. The quality and the quantity of the extracted DNA were checked with a NanoDrop spectrophotometer (Thermo Fisher Scientific) and a Qubit fluorometer (Life technologies), respectively. Sediment soils collected near sponge sampling sites were also subjected to DNA extraction using same procedure as described above except that 20 g of sediment soil was used for ISOPLANT extraction without homogenization.For library construction of pyrosequence, almost entire length of bacterial 16S rRNA gene was amplified at first with a universal primer set, Eub11f3mx (5???-TGR GTT TGA TCM TGG CTY AG) and Eub1511r1mx (5???-TGG HTA CCT TGT TAC GAC TT) [29. Shinzato N et al. Biosci Biotechnol Biochem 69: 1145-1155, 2005.], which was performed by the following temperature regime: 3 min at 95 deg C, 10 cycles of 30 sec at 95 deg C, 30 sec at 53 deg C and 1.5 min at 72 deg C followed by a final extension step at 72 deg C for 10 min. The second PCR was performed using the product of fast PCR as template and newly designed nested primers PrimB-B16S-11F and PrimA-B16S-532R, which were complemented with sequencing adaptors B and A, respectively, as recommended by the manufacture (Roche diagnostics), and the reverse primer contained one of 4-base multiplex identifier (MID) barcodes. The second PCR conditions were as follows: initial denaturing step at 95 deg C for 3 min, 25 cycles of denaturing at 95 deg C for 30 sec, primer annealing at 54 deg C for 30 sec, and extension at 72 deg C for 1 min, followed by final extension step at 72 deg C for 10 min. The resulting PCR product was checked by agarose gel electrophoresis and their quantity was assessed by a Qubit assay (Life technologies) or quantitative PCR procedure using a KAPA library quantification kit (Kapa Biosystems, Wilmington, MA). Finally, equal amounts of PCR products were pooled and then subjected to pyrosequencing with a Roche 454 GS Junior system (Roche diagnostics) according to manufacturer???s instructions for amplicon library sequence. |
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