redoxcline sample from winter 2013
Pyrosequencing of total extracted genomic DNA from selected depths and seasons was performed at Research and Testing Laboratory facilities (RTL) (Texas, USA) using Roche 454 FLX-Titanium technology (Dowd et al., 2008). Amplification reactions were performed using primers 28F/519R targeting the V1/V3 region of the bacterial 16S rRNA gene complemented with 454-adapters and sample-specific barcodes. Sequence dataset was pre-processed in RTL facilities to reduce pyrosequencing noise. After this initial denoising, sequences were demultiplexed according to sample barcodes, quality-filtered, chimera checked and clustered into OTUS (97% cutoff) using MOTHUR (P D Schloss et al., 2009). MOTHUR was also used for the alignment and taxonomic classification of the representative sequences of each OTU using the SILVA reference alignment and taxonomy database, respectively, available at MOTHUR website (http://www.mothur.org). α-diversity indicators of richness (Observed richness and Chao1) and diversity (Shannon) were calculated in MOTHUR after normalization of the number of sequences in each sample by randomly selecting a subset of 1,600 sequences from each sample to minimize bias due to different sampling efforts.