This book provides a practical and self-contained overview of the Gene Ontology (GO), the leading project to organize biological knowledge on genes and their products across genomic resources. Written for biologists and bioinformaticians, it covers the state-of-the-art of how GO annotations are made, how they are evaluated, and what sort of analyses can and cannot be done with the GO. In the spirit of the Methods in Molecular Biology book series, there is an emphasis throughout the chapters on providing practical guidance and troubleshooting advice. Authoritative and accessible, The Gene Ontology Handbook serves non-experts as well as seasoned GO users as a thorough guide to this powerful knowledge system. This work was published by Saint Philip Street Press pursuant to a Creative Commons license permitting commercial use. All rights not granted by the work's license are retained by the author or authors.