title: 16S RNA amplicon sequencing from anaerobic batch cultures inoculated with human faecal samples
design: Aliquots of extracted DNA were amplified with universal primers for the V4 and V5 regions of the 16S rRNA gene. Primers U515F and U927R  (Ellis et al., 2013) were designed to permit amplification of both bacterial and archaeal rRNA gene regions. Amplification was performed with FastStart HiFi Polymerase (Roche Diagnostics Ltd, UK) using the following cycling conditions: 95 C for 3 min; 25 cycles of 95 C for 30 s, 55 C for 35 s, 72 C for 1 min; followed by 72 C for 8 min. Amplicons were purified using 0.8 volumes of Ampure XP magnetic beads (Beckman Coulter). Each sample was then tagged with a unique pair of indices and the sequencing primer, using Nextera XT v2 Index kits, and 2x KAPA HiFi HotStart ReadyMix using the following cycling conditions: 95 C for 3 min; 10 cycles of 95 C for 30 s, 55 C for 30 s, 72 C for 30 s; followed by 72 C for 5 min. Index-tagged amplicons were purified using 0.8 volumes of Ampure XP magnetic beads (Beckman Coulter). Concentrations were normalised before pooling all samples, which were subsequently identified by their unique index combinations