Lung Cancer Metagenome
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 94çC for 3 min followed by 35 cycles of denaturation at 94çC for 45 seconds, annealing at 58çC for 1 minute, and extension at 72 C for 90 seconds, with a final extension of 10 min at 72çC. 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
SUB3101150
55.RM.64