Microbiome analysis involves determining the composition and function of a community of microorganisms in a particular location. For the gastroenterologist, this technology opens up a rapidly evolving set of challenges and opportunities for generating novel insights into the health of patients on the basis of microbiota characterizations from intestinal, hepatic or extraintestinal samples. Alterations in gut microbiota composition correlate with intestinal and extraintestinal disease and, although only a few mechanisms are known, the microbiota are still an attractive target for developing biomarkers for disease detection and management as well as potential therapeutic applications. In this Review, we summarize the major decision points confronting new entrants to the field or for those designing new projects in microbiome research. We provide recommendations based on current technology options and our experience of sequencing platform choices. We also offer perspectives on future applications of microbiome research, which we hope convey the promise of this technology for clinical applications.