In this study, we present an NGS-based clinical gut microbiome sequencing assay to assess the relative abundance of health condition-associated microorganisms (Fig 1). The assay utilizes 16S rRNA gene sequencing to identify 28 clinically relevant microbial targets (14 species and 14 genera), including 5 intestinal pathogens, 3 beneficial bacteria, and 20 commonly present inhabitants of the human gastrointestinal tract, with high precision and sensitivity. In addition, we define the relative abundance ranges of these taxa in stool samples from a large healthy human cohort.