High-Throughput Gut Microbiome Profiling

DNA was extracted from −80°C-stored stool samples using the DNeasy PowerSoil HTP 96 kit (Qiagen) and an EpMotion 5075 automated pipetting system (Eppendorf). The V4 region was amplified for 16S rRNA with the AccuPrime Pfx DNA polymerase (Thermo Fisher Scientific) using custom barcoded primers, as previously described (68 (link)). The ZymoBIOMICS microbial community DNA standards were used as a mock community control (69 (link)), and water was used as a negative control per 96-well extraction plate. The PCR amplicons were cleaned up and normalized with the SequalPrep normalization plate kit (Thermo Fisher Scientific). Amplicons were pooled and quantified with the Kapa library quantification kit (Kapa Biosystems), prior to sequencing using the MiSeq system (Illumina).