PROJECT SUMMARY There are no tests to aid in the diagnosis of irritable bowel syndrome (IBS), which is the most common chronic gastrointestinal condition in the U.S. and is thought to arise in part from gut host-microbe interactions. Here, we propose to identify novel stool-based IBS biomarkers by sequencing exfoliated host gut cells and microbiota to non-invasively assess gene expression and microbiome changes in the gut. Our proposed research comprises two main aims. Firstly, we will establish a stool-based RNA sequencing method optimized for IBS and combine it with gut microbiome measurements. Secondly, we will apply our method to patient cohorts to identify stool RNA and gut microbiome features that could potentially diagnose and subtype IBS-D. This project will focus on diarrhea-predominant IBS (IBS-D), using test and validation patient cohorts to determine the accuracy of our method in distinguishing patients with IBS-D from healthy controls. We anticipate that this exploratory work will deliver a novel method to generate stool-based biomarkers for IBS-D, thereby improving diagnosis, phenotyping, and subsequent management of the disorder.