| Design Description | To elucidate the diversity of picocyanobacterial ITS sequences, bar-coded amplicon sequencing with the GS FLX Titanium was conducted. For amplification of picocyanobacterial 16S-23S ITS sequences, picocyanobacteria-specific ITS-af (5’-GGA TCA CCT CCT AAC AGG GAG-3’) forward and ITS-ar (5’-GGA CCT CAC CCT TAT CAG GG-3’) reverse primer sequences were used (Lavin et al., 2008). For bar-coding each sample, each MID sequence presented from Roche were inserted between Key and specific forward sequences. These primer sets produced amplicons with length of around 230 to 300 bps. One to 10 ng of template DNA was added to PCR reaction (total of 50 ul) which contained Takara Ex Taq buffer, 0.2 mM each deoxyribonucleoside triphosphate, 0.5 uM of each primer, and 2 units of Takara Ex Taq. PCR-amplification was conducted according to the following cycle parameters: an initial denaturation step (5 min, 94oC) was followed by 35 cycles consisting of a denaturation step (45s, 94oC), annealing (45s, 50oC) and extension step (1.5 min, 72oC) and a final 10-min extension step at 72oC at the end. The size of the PCR products was confirmed by agarose gel electrophoresis. Quantification of each PCR product was performed on agarose gel using DNA QuantLadders (Lonza Rockland Inc.). After pooling the same quantities of each PCR product, the pooled PCR product was purified using AccuPrep PCR purification kit (Bioneer). After the purified DNA was resolved on a 2% agarose gel, DNA with size of between 200 and 400 bps were excised and then extracted using the Qiagen gel extraction kit (Qiagen). Pyrosequencing of PCR products was performed on a GS-FLX Titanium system (454 Life Sciences, CT) at Macrogen Co. (Seoul, Korea). |
