Assessment of microbial communities in Great Boiling Spring (GBS) and Sandy’s Spring West (SSW) in coordination with nitrification and denitrification rate measurements.
DNA extracted from GBS water sampled on Oct., 2008 (0810), was added as template in an amount containing 100,000-1,000,000 16S rRNA gene copies. Primers 515F and 1391R, modified with Titanium pyrosequencing adaptors A (515F) and B (1391R) and a 10 nucleotide bar code (515F; TATAGACATC for GBS_0810_water), were used to amplify 16S rRNA genes from bacteria and archaea. The reactions were done in 50 µL total volume using Platinum HiFi Master Mix (Invitrogen, Carlsbad, CA) on an MJ PTC-100 thermocycler (MJ research/BioRad Laboratories, Inc., Hercules, CA) with the following cycling conditions: 94°C for 5 min; 24 cycles of (92°C for 20 s; 50°C for 30 s; 65°C for 60 s; 75°C for 60 s; 70°C for 60 s); and a final 10 min extension step at 70°C. Products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA), followed by purification by Solid Phase Reversible Immobilization and size-selection step on calibrated Agentcourt AMPure magnetic beads (Beckman Coulter Genomics, Danvers, MA) according to the Roche/454 method. Purified libraries were quantified using previously a digital PCR method, and sequencing of the 16S rRNA gene amplicons was performed on a Roche 454 Genome Sequencer FLX System using Titanium chemistry according to the manufacturers protocols (454 Life Sciences, Branford, CT). Emulsion PCR was carried out with DNA:bead ratios between 0.08:1 and 0.3:1.