16S rRNA gene Bacteria V3V4 amplification of JBL_J07_BT
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’) (note that 454 adaptor and seven-base long bar-code sequences are not shown here). Primer 341F matched perfectly to 891 287 of 933 229 bacterial sequences spanning Escherichia coli positions 300–400, while primer 805R matched perfectly to 727 896 of 815 023 bacterial sequences spanning positions 750–850 (Ribosomal Database Project [RDP], v10.20, http://rdp.cme.msu.edu 59). PCR reactions were performed in a 20-µL reaction volume comprising 0.4 U Phusion™ high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 1X Phusion™ HF reaction buffer (Finnzymes), 200 µM of each dNTP (Invitrogen), 500 nM of each primer (Eurofins MWG, Ebersberg, Germany), 8 µg BSA (New England Biolabs, Ipswich, UK) and finally 5-10 ng of extracted nucleic acid. Thermocycling (DNA Engine [PTC-200] Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) was conducted with an initial denaturation step at 95 °C for 5 min, followed by 25 cycles of denaturation at 95 °C for 40 sec, annealing at 53 °C for 40 sec and extension at 72 °C for 1 min, and finalised with a 7-min extension step at 72 °C. Four technical replicates were run per sample, pooled after PCR amplification and purified using the Agencourt AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Nucleic acid yields were checked on a fluorescence microplate reader (Ultra 384; Tecan Group Ltd., Männedorf, Switzerland) employing the Quant-iT PicoGreen dsDNA quantification kit (Invitrogen). Finally, PCR amplicons were pooled in equal proportions to obtain a similar number of 454 pyrosequencing reads per sample.