16S rRNA controls
Two steps of amplification were performed to prepare the sequencing library using the AccuPrime Taq high fidelity DNA polymerase system (Invitrogen, Carlsbad, CA). Negative controls (DNA extraction and environment) and a positive control (Microbial mock community HM-276D, BEI Resources, Manassas, VA) were also processed during amplicon library preparation. The hypervariable V4 region was amplified using 20ng genomic DNA and gene-specific primers with Illumina adaptors. The PCR conditions were as follows: 95C for 3 min, 25 cycles of 95C for 30 s, 55C for 30 s and 72C for 30 s, 72C for 5 min and holding at 4C. PCR products were purified using AMPure XP beads with beads added as 0.85x the PCR volume. Four microliters of purified amplicon DNA from the previous step was amplified for 8 cycles with the same PCR conditions using primers with different molecular barcodes. The indexed amplicon libraries were purified using AMPure XP beads and quantified using Quant-iT PicoGreen (Invitrogen), then normalized and pooled. The pooled library was quantified using the KAPA Library Quantification Kit (Kapa Biosystems, Cape Town, South Africa), diluted and denatured as per Illumina guidelines. Ten per cent Illumina PhiX was added to the sequencing library as an internal control and to increase diversity of the 16s rRNA amplicon library.