Stool DNA (3 ng) was amplified for the V2, V4, and V8 hypervariable regions of the 16S rRNA gene using the Ion 16S Metagenomics Kit. Amplicons were purified and 100 ng of PCR product per sample was used to construct single-end libraries with the Ion Plus Fragment Library Kit and Ion Xpress barcodes. Libraries were quantified, diluted to 20 pM, and pooled in equimolar amounts. Template preparation, emulsion PCR, and chip loading were performed on the Ion Chef system using the Ion 510/520/530 kit, and sequencing was carried out on the Ion GeneStudio S5 Plus with a planned run configuration of 400 bp read length and 850 flows.