| 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 ?l) which contained Takara Ex Taq buffer, 0.2 mM each deoxyribonucleoside triphosphate, 0.5 ?M 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). Station Longitude (E) Latitude (N) SFF file MID sequences 04 152.10 6.90 mid41 TAGTGTAGAT 08 151.43 7.58 mid48 ACAGTATATA A1 150.52 8.79 mid68 TCGCTGCGTA A2 147.69 9.53 mid69 TCTGACGTCA A3 145.60 9.86 mid70 TGAGTCAGTA A4 142.05 10.39 mid71 TGTAGTGTGA A5 140.05 10.70 mid72 TGTCACACGA A6 136.66 11.23 mid73 TGTCGTCGCA 09 135.00 11.50 mid52 AGTATACATA 14 135.00 14.00 mid54 AGTGCTACGA 16 135.00 15.00 mid55 CGATCGTATA 18 135.00 16.00 mid56 CGCAGTACGA 20 135.00 17.00 mid57 CGCGTATACA 22 133.33 15.00 mid58 CGTACAGTCA 24 131.67 13.00 mid59 CGTACTCAGA 27 130.00 12.00 mid60 CTACGCTCTA 31 130.00 16.00 mid61 CTATAGCGTA 34 130.00 19.00 mid62 TACGTCATCA B1 128.83 22.17 mid74 ACACATACGC B2 126.46 26.73 mid75 ACAGTCGTGC B3 126.20 28.73 mid76 ACATGACGAC 36 126.00 30.46 mid63 TAGTCGCATA 38 125.25 32.08 mid114 ACGTGCAGCG 39 127.20 32.25 mid65 TATGCTAGTA 40 127.67 33.25 mid66 TCACGCGAGA 41 128.45 34.17 mid67 TCGATAGTGA |
