After establishing our ability to detect all 28 targets using synthetic DNA at relative abundances of 0.03% or more (S2 Doc, S4 Table), we tested 40 reference isolates from Luminex’s xTAG Gastrointestinal Pathogen Panel to establish the clinical relevance of our pipeline. These verification samples comprise real or synthetic stool samples with live or recombinant material of known composition. Two of the samples were excluded due to poor sequencing depth. The remaining samples were positive for 1 of 8 different bacterial strains corresponding to 5 of our clinical targets: V. cholerae (5), S. enterica (5), Escherichia-Shigella (13), Campylobacter (5) and C. difficile (5). All of these verification samples were correctly identified as having a relative abundance of the clinical target well above our defined healthy reference range (Fig 4). Five samples containing Yersinia were tested as a negative control. Although Yersinia was included in our preliminary target list, it did not pass our stringent bioinformatics QC thresholds for accurate identification. As expected, the relative abundance of the 28 clinical targets was in the healthy range for the Yersinia positive samples, as shown for Escherichia-Shigella (Fig 4).