title: 16S rRNA gene sequencing of souring  enrichement +GA750_replicate 2+T06
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.