| Construction Protocol | Primers 530F (xxxxGTGCCAGCMGCNGCGG)and 803R (xxxxCTACCNGGGTATCTAAT) were used to amplify a 16S rRNA gene fragment of the appropriate size and sequence variability for 454 pyrosequencing, and xxxx was designed for the sample identification barcoding key recommended by the manufacturer (Beckman Coulter Genomics). For each treatment and sampling date analyzed, DNA extracted from the three replicates was pooled to perform the PCR. 2 ng aliquots of DNA from each pool were used for a 50 ֬ PCR reaction under the following conditions: 95у for 3 min, 30 cycles of 1 min at 95у (denaturation), 55у for 30 s (annealing) and 72у for 30 s (extension), followed by 5 min at 72у. For each sample, amplicons of five replicated PCRs were purified using a MinElute gel extraction kit (Qiagen, Courtaboeuf, France) following the manufacturer's protocol. On 300 ng per sample, adapter sequences were added by the manufacturer and pyrosequencing was carried out on a Genome Sequencer Flx 454 (Beckman Coulter Genomics). The 16S rRNA fragments provided were mainly between 250 and 300 bp. |
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