title: 16S rRNA gene  sequencing of souring enrichement +MBD_inoculum_autoclaved control
design: DNA  extracted with CTAB ((R. I. Griffiths et al., 2000)), 16S rRNA gene  sequencing of the variable region 4 (V4) was carried out by NU-OMICS  (Northumbria University) based on the Schloss wet-lab MiSeq SOP (Kozich et  al., 2013). The primers F515 and R806 (Caporaso et al., 2011) were used in  the sequencing. PCR was carried out using 1x Accuprime Pfx Supermix, 0.5 M  each primer and 1 l of template DNA under the following conditions: 95 C  for 2 minutes, 30 cycles of 95 C for 20 s, 55 C for 15 s, 72 C for 5  minutes with a final extension at 72 C for 10 minutes. One positive and one  negative control sample were included in each 96 well plate and carried  through to sequencing. PCR products were normalised using SequalPrep  Normalization kit (Invitrogen) as described in the manufacturers  instructions and combined into four pools. Each pool was quantified using  fragment size determined by BioAnalyzer (Agilent Technologies) and  concentration by Qubit (ThermoScientific). Pools were combined in equimolar amounts  to create a single library then denatured using 0.2 M NaOH for 5 minutes  followed by a 2 minute incubation at 96 C. The library was diluted to a  final concentration of 5 pM, supplemented with 15% PhiX and loaded onto a  MiSeq V2 500 cycle cartridge.