title: paddy soil Prokaryotes Community Analysis
design: For 16S amplicon libraries the Prokaryotic primers pairs 515F-806R (Bergmann et al., 2011), which targets the V4-V5 hypervariable region, were used as detailed in Fierer et al. (2008) and Bates et al. (2010). These primers have been previously shown to yield accurate phylogenetic information and to have only few biases against any bacterial taxa (Liu et al., 2007; Bates et al., 2011; Bergmann et al., 2011). Each primer pairs contained a 5 6 bp-long barcode sequence used to identify each sample in multiplexed pyrosequencing runs. Four independent PCRs were performed for each DNA sample as detailed in Fierer et al. (2008) and Bates et al. (2010) and using 2 U of High Fidelity Taq (Roche) to obtain a final volume of 25 l. The DNA was amplified using a T3000 thermal cycler (Biometra, Gttingen, DE) and amplification success was checked on a agarose gel. Each of the technical replicate was amplified in duplicate, pooled, checked on 1.5% agarose gel and concentration estimated spectrophotometrically as previously described. Libraries were sequenced using a Genome Sequencer (GS) FLX Titanium 454 System (Roche) at Beckman Coulter Genomics (Beverly, MA, USA). The two 454 pyrosequencing forward primer (Primer A-Key) and reverse primer (Primer B-Key) were added to the amplicons by random ligation, so that each amplicon library will generate reads from both the 5 or 3 ends.