Molecular analysis of freshwater bacterial biofilm communities under experimentally manipulated dissolved organic carbon regimes at Llyn Brianne (2014)

Dissolved organic carbon (DOC) additions were made to replicate recirculating flumes at three upland streams in mid-Wales. Water and biofilm samples were taken at regular intervals over the following 24-hour period to monitor the effects of carbon addition. Temperature and water oxygen content were also monitored over the course of the experiment. The water samples were analysed for total carbon, inorganic carbon and organic carbon content. Epilithon were taken from unglazed ceramic tiles that had been colonised by epilithon in the river using the tooth-brush method. Samples were frozen immediately after collection. On return to the laboratory, samples were stored at -70 degrees Celsius prior to DNA extractions. Total genomic DNA was extracted from epilithon using the FastDNA (registered trademark) SPIN Kit for soil, incorporating a bead beating step. An extraction kit negative control was included in the sequencing run.

To test for the presence of DNA, amplifications were carried out in a total volume of 50 microlitre containing 10 microlitre of Polymerase chain reaction P(CR) Mix (Buffer), 0.2 micromolar of each of the forward and reverse primers, 1.5 Uracil (U) of Taq DNA polymerase and 1 microlitre of DNA (approximately 50 nanogram/microliter). Primers 357F (Turner et al. 1999) and 518R (Muyzer et al. 1993) were used for amplification. In addition (these amplifications were carried out by a commercial company - GeneTiCA), the bacterial 16S region was amplified with primers S-D-Bact-0341-b-S-17: CCTACGGGNGGCWGCAG, S-D-Bact-0785-a-A-21: GACTACHVGGGTATCTAATCC (Klindworth et al. 2013). Triplicate Polymerase chain reactions (PCRs) for each sample were cleaned with the AMPure bead method, pooled and sequenced on an Illumina MiSeq v3 (2 x 300 base pairs) (Illumina, Inc., San Diego, USA) with Nextera XT assay chemistry. Polymerase chain reaction (PCR) and sequencing were done by GeneTiCA (Prague, Czech Republic). Samples were double-barcoded to allow 192 samples to be run together; the sequencing run was shared with samples from another experiment.

Sequences were quality filtered and grouped into operational taxonomic units (OTUs) using USEARCH and QIIME. Taxonomy was assigned from the Greengenes database. Each row in the OTU table corresponds to an OTU. Column 1 contains the OTU identifiers, columns 2-98 are headed as per the sample IDs in the mapping file; columns 99-105 contain the taxonomic information.

This dataset is included in the following collections

The DURESS project - Diversity of Upland Rivers for Ecosystem Service Sustainability