Dataset: 16S rRNA clone library
Deployment: KN192-05

Community genomic DNA was extracted from 200 ml of cell lysate using a DNeasy Blood and Tissue kit (QIAGEN, Germantown, MD, USA). DNA was extracted according to the manufacturer’s instructions. Ribosomal RNA genes were amplified from community genomic DNA for cloning by PCR with Taq polymerase (Fermentas, Hanover, MD, USA) and variations of commonly used bacterial primers, 8F and 519R. Briefly, amplifications were performed in a C1000 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using the following conditions: 35 cycles, annealing at 55 1C for 1 min, elongation at 72 1C for 2 min and denaturation at 94 1C for 30 s. A single band of the predicted length was observed by agarose gel electrophoresis, excised and purified using a DNeasy minelute kit (QIAGEN) according to the manufacturer’s instructions. Clone libraries were constructed using the resulting mixed-template amplicons and the pGEM-T-Easy vector (Promega) following the manufacturer’s instructions. Clone sequences were obtained from transformations by plating, rolling-circle amplification and cycle sequencing at the High-Throughput Genomics Unit (University of Washington, Seattle, WA, USA). Clones from each station were assigned library and station prefixes and numbered sequentially from 1 to 96 (GenBank Accession: GU460426-GU461274). Cloned 16S rRNA gene sequences were aligned in the ARB software package (Ludwig et al., 2004) using a custom database that contained 151 952 sequences from cultured organisms and environmental gene clone libraries. Unambiguously aligned nucleotide sequences were added to a custom tree using the parsimony insertion tool for phylogenetic identification available in ARB. Taxonomic assignments were determined by phylogenetic inference.