title: Spatiotemporal dynamics of bacterial diversity and community composition in large shallow eutrophic Lake Taihu using pyrosequencing: high overlap between free-living and particle-attached assemblages
design: We explored the diversity and phylogenetic composition of free-living and particle-attached bacterial assemblages from Lake Taihu at four seasons and four stations by high-throughput 454-tag pyrosequencing V1-V3 regions of 16S rRNA genes.       For 454-tag pyrosequencing, bacterial 16S rRNA genes were amplified using the 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) primer with the Roche 454 ‘A’ sequencing adapter, and the 533R (5’- TTACCGCGGCTGCTGGCAC -3’) primer with a 10 bp barcode sequence and the Roche 454 ‘B’ sequencing adapter. PCR amplifications were performed in triplicate for each sample using 20-μl reaction mixtures containing 1 × PCR buffer, 1.5 mM MgCl2, 2.5 mM dNTPs, 4 μM of each primer, 1.0 U of TransStart Fastpfu DNA Polymerase (TransGen Biotech, China), and 10 ng diluted DNA extract.   Amplification was carried out in GeneAmp® PCR System 9700 (Applied Biosystems, USA) under the following conditions: denaturation at 95°C for 2 min, 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. PCR products were pooled, run on a 2% agarose gel, purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA), and then quantified with QuantiFluor® ONE dsDNA System (Promega, USA). An equimolar amount of barcoded amplicons for each sample were used for subsequent emulsion-based clonal amplification and sequencing on a Roche 454 FLX pyrosequencer. Reads were produced from the backward direction (533R to 27F) at Majorbio Bio-pharm Technology Co. (Shanghai, China).