title: Effects of flow intermittency on bacterial biofilm communities in a Mediterranean stream
design: 51 biofilm samples from three different compartments from the streambed, namely Epilithic (EL, 17 samples), Epipsammic (ES, 17 samples) and Hyporheic (H, 17 samples) were subjected to DNA extraction using UltraClean  Soil DNA Extraction Kit (MoBio Laboratories Inc.) according to manufacturer instructions. The 51 DNA extracts were quantified by QUBIT and used as template in PCR reactions using primers 28F/519R targeting the V1/3 region of the bacterial 16S rRNA gene complemented with 454-adapters and sample-specific barcodes using optimized protocols by the sequencing company (research and Testing Laboratories, Lubbock, Texas) according to Dowd and colleagues (Dowd et al., 2008, BMC Microbiology 2008, 8:43, doi:10.1186/1471-2180-8-43). Pyrosequencing reactions were carried out in a Roche Genome Sequencer 454 GS FLX Titanium at a sequencing depth of 5,000 seqs per sample.    Raw sequence dataset was pre-processed in Research and Testing Laboratory facilities in order to reduce noise and sequencing artefacts as previously described (Dowd et al., 2008, BMC Microbiology 2008, 8:43, doi:10.1186/1471-2180-8-43). Demultiplexing according to sample barcodes, sequence quality checking, chimera detection and downstream phylogenetic analyses were conducted in QIIME (Caporaso et al., 2010, Nat Meth 7: 335–336). Clustering of sequences into OTUs was done in QIIME using USEARCH (Edgar, 2010, Bioinformatics 26: 2460–2461). OTUs with less than 4 members were removed from downstream analysis. Representative sequences from each OTU were aligned to the Greengenes imputed core reference alignment (DeSantis et al., 2006, Appl Environ Microbiol 72: 5069–5072) using PyNAST (Caporaso et al., 2010, Bioinformatics 26: 266–267). Taxonomical assignments for each OTU were carried out using the RDP classifier (Wang et al., 2007, Appl Environ Microbiol 73: 5261–5267) retrained with the October 2012 Greengenes taxonomy database (12_10, http://greengenes.secondgenome.com).