16S rRNA amplicon sequencing of soil microbiome: day 21 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.