rpoC1 gene of Synechococcus
The first round of PCR used the primer rpoC1-N5 and the C-terminal primer rpoC1-C, and the PCR products were used as templates for the second round PCR with modified primer rpoC1-39F (5-adaptor+barcode+GGNATNGTNTGYGAGCGYTG) and rpoC1-462R (5-adaptor+CGYAGRCFCTTGRTCAGCTT). PCR products were gel-purified using the Qiaquick gel purification kit (Qiagen, Hilgen, Germany) as described by the manufacturer. Library quantification was done by fluorometry using the Quant-iT picoGreen dsDNA Assay Kit (Invitrogen, USA). Amplicon was sequenced in a two-region 454 run on a GS PicoTiterPlate using a GS Junior pyrosequencing system according to manufacturer instructions (Roche, 454 Life Sciences, Branford, CT, USA).