16S rRNA amplicon sequencing of soil microbiome: day 5 soil
16S rRNA gene V4 variable region PCR primers 515  806 (Caporaso et al. 2011) were used with HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C (3 mins), followed by 28 cycles (5 cycle used on PCR products) of 94C (30s), 53C for (40 s) and 72C (1 min), after which a final elongation step at 72C (5 mins) was performed. Sequencing was performed at MR DNA, Shallowater, TX, USA on an Ion Torrent PGM system. Sequence data were processed using a proprietary analysis pipeline (MR DNA, Shallowater, TX, USA). In summary, sequences were depleted of barcodes and primers, then sequences < 150bp removed, sequences with ambiguous base calls and with homopolymer runs exceeding 6bp were also removed.