16S rRNA gene, V3-V4 of DispExp
The bacterial hypervariable regions V3 and V4 of the 16S rRNA gene were PCR amplified using forward primer 341 5’- CCTACGGGNGGCWGCAG-3’ and individually bar-coded reverse primers 805 5’- GACTACHVGGGTATCTAATCC-3’ (Herlemann et al., 2011). Note that 454 adaptor [454 Life Sciences, Branford, CT, USA] and seven-base long bar-code sequences are not shown here. Each 20 µL PCR reaction contained 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen, Carlsbad, CA, USA), either 500 (Water07) or 250 (Water10, Sediments) nM of each primer (Eurofins MWG, Ebersberg, Germany), 0.4 mg mL-1 BSA (New England Biolabs, Ipswich, UK) and finally 5–10 ng of extracted nucleic acid. Thermocycling was conducted with an initial denaturation step at 95 °C for 5 min (98 °C for 30 sec, Water10 and Sediments), followed by 25 cycles of denaturation at 95 °C for 40 sec (98 °C for 10 sec, Water10 and Sediments), annealing at 53 °C for 40 sec (50 °C for 30 sec, Water10 and Sediments) and extension at 72 °C for 1 min (30 sec, Water10 and Sediments), and finalised with a 7-min extension step at 72 °C. Three to four technical replicates were run per sample, pooled after PCR amplification and quality-checked on a 1% agarose gel. Purification was carried out using the AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were then checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) applying the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were combined equimolarly, i.e. in equal proportions, to obtain a similar number of 454 pyrosequencing reads per sample.