QIIME is a software package for analyzing microbial communities, designed to process and interpret nucleic acid sequence data from fungal, viral, bacterial, and archaeal communities. This unit describes how to install QIIME on a single computer and use it to analyze microbial 16S rRNA gene sequences from nine distinct microbial communities. The protocols include installing QIIME via VirtualBox, acquiring and demultiplexing DNA sequences, picking OTUs, assigning taxonomy, inferring phylogeny, creating an OTU table, calculating alpha diversity, and generating beta diversity plots. The example uses data from a study on the response of mouse gut microbial communities to fasting. The process involves installing QIIME, processing sequence data, and analyzing the results to compare community structures and diversity metrics. The unit also includes troubleshooting information and references to additional resources. QIIME provides a comprehensive workflow for microbial community analysis, with customizable parameters and support for various sequencing technologies. The software is designed to be user-friendly, with default settings that allow for quick analysis, while also offering flexibility for researchers with specific needs. The unit emphasizes the importance of proper data formatting and the use of established databases for taxonomic assignments. The workflow includes steps for quality filtering, sequence alignment, phylogenetic tree construction, and visualization of results. The final output includes diversity metrics, phylogenetic trees, and visualizations such as heatmaps and PCoA plots, which help in understanding microbial community composition and diversity.QIIME is a software package for analyzing microbial communities, designed to process and interpret nucleic acid sequence data from fungal, viral, bacterial, and archaeal communities. This unit describes how to install QIIME on a single computer and use it to analyze microbial 16S rRNA gene sequences from nine distinct microbial communities. The protocols include installing QIIME via VirtualBox, acquiring and demultiplexing DNA sequences, picking OTUs, assigning taxonomy, inferring phylogeny, creating an OTU table, calculating alpha diversity, and generating beta diversity plots. The example uses data from a study on the response of mouse gut microbial communities to fasting. The process involves installing QIIME, processing sequence data, and analyzing the results to compare community structures and diversity metrics. The unit also includes troubleshooting information and references to additional resources. QIIME provides a comprehensive workflow for microbial community analysis, with customizable parameters and support for various sequencing technologies. The software is designed to be user-friendly, with default settings that allow for quick analysis, while also offering flexibility for researchers with specific needs. The unit emphasizes the importance of proper data formatting and the use of established databases for taxonomic assignments. The workflow includes steps for quality filtering, sequence alignment, phylogenetic tree construction, and visualization of results. The final output includes diversity metrics, phylogenetic trees, and visualizations such as heatmaps and PCoA plots, which help in understanding microbial community composition and diversity.