Microbial Profiling via 16S rRNA Sequencing

DNA was extracted from the samples using the Qiagen MagAttract PowerSoil DNA KF Kit (Qiagen, Hilden, Germany) optimized for the ThermoFisher KingFisher robot (Thermo Fisher Scientific, Waltham, MA). Both negative controls and positive controls were used. The positive controlled consisted of cloned Thioglobaceae SUP05, a Gamma-Proteobacteria; while negative control was extraction control negative, template-free DNA. The kit used magnetic beads to capture DNA while excluding organic inhibitors specifically. Qubit (Thermo Fisher Scientific) was used for DNA quantification and quality check. The microbial composition of samples was characterized by sequencing the V4 hypervariable region of the 16S rRNA gene using the Illumina MiSeq sequencer (Illumina, San Diego, CA) (Meisel et al., 2016 (link)). Samples that failed PCR or had spurious bands were re-amplified by optimizing PCR conditions. We used a high-throughput SequalPrep96-well plate kit (Thermo Fisher Scientific) to clean up and normalize PCR reactions.