16S rRNA gene amplicon sequencing of negative control: DNA extraction controls
DNA was extracted from NO sample using the blood and tissue DNA extraction kit and the standard protocol designed for purification of total DNA for gram-positive bacteria(QIAGEN) (i.e., DNA extraction negative controls). PCR amplifications were performed in 20 l mixtures containing 2 l of diluted DNA extract, 1  AmpliTaq Gold 360 master mix (Applied Biosystems), 0.5 m of each primer and 3.2 g of bovine serum albumin (Roche Diagnostics). PCR conditions consisted of an initial denaturation at 95C for 10 min, followed by 30 cycles of denaturation (at 95C for 30 s), annealing (at 57C for 30 s) and elongation (at 72C for 90 s). A final elongation step was run at 72C for 7 min. We used universal primers that specifically amplified the V5V6 region (~295 bp in length) of the bacterial 16S rRNA gene (BACTB-F: GGATTAGATACCCTGGTAGT and BACTB-R: CACGACACGAGCTGACG; Fliegerova et al., 2014). To discriminate between samples after sequencing, the 5 end of both forward and reverse primers included a combination of two different 8-bp tags. The PCR products were pooled (4 l of each PCR product in the pool) and the pool was purified using the MiniElute PCR purification kit (Qiagen). Amplicons were sequenced with an Illumina MiSeq platform, using the 2  300-bp protocol (Fasteris SA). Paired-end reads were aligned and assigned to the corresponding sample (number of errors allowed for matching primers = 2) using the OBITools package.