Coherent dynamics and association networks among lake bacterioplankton taxa
DNA was extracted by bead-beating and solid phase extraction using MoBio Ultra clean Soil DNA kit as recommended by the manufacturer (MoBio, Laboratories, Solana Beach, CA). The quality and amount of extracted DNA was determined by agarose-gel electrophoresis (1% agarose, 0.5 × TBE) followed by detection with ethidium bromide staining, UV-transillumination and comparison to a 1 kb DNA extension ladder (Invitrogen). The extracts contained 1 to 20 ng DNA µl –1.  Amplification of bacterial 16S rRNA genes was conducted using general bacteria primers  341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC). This primer pair matches perfectly to 891,287 of 933,229 bacterial sequences in the Ribosomal Database Project release 20.20. Primer 341F carried 454FLX adaptor B at the 5´end and primer 805R carried a 5 bp molecular barcode specific for each sample followed by 454FLX adaptor A at the 5´end. Each DNA sample was individually PCR amplified in triplicate 20 ml reactions by initial denaturation at 95 °C for 5 minutes followed by 25 cycles of 40 sec at 95 °C, 40 sec at 53 °C , 60 sec at 72°C and a final 7 min elongation step. Each reaction contained between 1-10 ng of target DNA, 1nM Phusion HF buffer (Finnzymes), 0.02 nM Phusion DNA polymerase (Finnzymes), 200 µM dNTPs and 500 nM of each primer. Replicate PCR reactions were pooled and the amplicon concentration for each sample was assessed by electrophoretic separation on a 1% agarose gel, ethidium bromide staining and UV transillumination using a cooled CCD camera and comparison to as  Low DNA Mass Ladder (Invitrogen). Equal amounts of specifically barcoded amplicons from each of the samples were combined and gel purified using the Qiaquick gel purification kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. The purified amplicons were again sized and quantified (see above) followed by high-throughput pyrosequencing from adaptor A using the 454 GS-FLX system (454 Life Sciences, Branford, CT) where the resulting reads carried the sample-specific molecular barcode and covered the V4 region of the 16S rRNA gene. All sequencing was done at the Centre for Metagenomic Sequence Analysis hosted by the Royal Institute of Technology, Stockholm, Sweden.