Pyrosequencing TB microbiome
PCR was performed using composite primers flanking the hypervariable V1-V2 region of the 16S rRNA gene: A-8F and B-357R. Sequencing adaptors and sample-specific MIDs were also added to the 5’ ends of the primers according to the manufacturer’s protocol. PCR reactions were carried out using the Platinum* PCR SuperMix High Fidelity Kit following the manufacturer's instructions. PCR products were purified using the MinElute PCR Purification Kit and the quality of the purified products was assessed using Agilent Bioanalyzer 2100. Pyrosequencing of the purified PCR products was performed uni-directionally from the A-8F primer end and on a GS FLX-Titanium platform in a single full-plate run.