title: Cerrado soil microbiome
design: Genomic DNA was extracted from 250 mg of soil sample using Soil DNA Isolation Kit (Norgen, Canada) as described by the manufacturer. The 16S rRNA gene fragments were sequenced using 454 GS FLX Titanium (Lib-L) chemistry for unidirectional sequencing of the amplicon libraries. Barcoded primers were used to multiplex the amplicon pools so they could be sequenced together and computationally separated afterward. To do this, 8-base barcodes were added to the 5’ -end of the reverse primers using the self-correcting barcode method of Hamady et al., (2008). The primers were attached to the GS FLX Titanium Adaptor A-Key (5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG -3’) and Adaptor B-Key (5’-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-3’) sequences, modified for use with GS FLX Titanium emPCR Kits (Lib-L) and a two-base linker sequence was inserted between the 454 adapter and the 16S rRNA primers to reduce any effect the composite primer might have on PCR efficiency. Twelve independent PCR reactions were performed for each soil sample with the primers 338R and 27F for the amplification of the V1 – V2 hypervariable region of the 16S rRNA gene. The PCR conditions were 94°C for 2 minutes, 30 cycles of 94°C for 45s; 55°C for 45s; and 72°C for 1 min extension; followed by 72°C for 6 minutes. The PCR products for each of the 12 samples were purified and combined in equimolar ratios with the quantitative DNA binding method (SequalPrep Kit, Invitrogen, Carlsbad, CA, USA) to create a DNA pool that was used for pyrosequencing from the A-Key adaptor.