title: Pre-treatment pulmonary tuberculosis metagenome samples
design: Amplicon library preparation was performed  using an automated platform (Biomek 4000) using a custom liquid handling  method. For each sample, the V4 region of the bacterial 16S rRNA gene was  amplified in duplicate reactions, using primer set 515F/806R, which nearly  universally amplifies bacterial and archaeal 16S rRNA genes.1,2 Each unique  barcoded amplicon was generated in pairs of 25_l reactions with the following  reaction conditions: 11_l PCR-grade H2O, 10_l Hot MasterMix (5 Prime Cat#  2200410), 2_l of forward and reversed barcoded primer (5_M) and 2_l template  DNA. Reactions were run on a C1000 Touch Thermal Cycler (Bio-Rad) with the  following cycling conditions: initial denaturing at 94C for 3 min followed  by 35 cycles of denaturation at 94C for 45 seconds, annealing at 58C for 1  minute, and extension at 72 C for 90 seconds, with a final extension of 10  min at 72C. Amplicons were quantified using Agilent 2200 TapeStation system  and pooled. Purification was then performed using Ampure XT (Beckman Coulter  Cat# A63882) as per the manufacturer instructions