Microbiome marker gene analysis with QIIME 2: the "gut-to-soil microbiome axis" tutorial

QIIME 2 is a widely used platform for microbiome marker gene (i.e., amplicon) data analysis. Here we present the "QIIME 2 gut-to-soil microbiome axis" tutorial, a hands-on tutorial that takes readers from raw sequencing data through summary visualizations and statistics for a real-world microbiome dataset. The tutorial is designed such that readers can follow along on their own computer. Exercises are presented throughout, guiding readers through exploring the tutorial outputs, and solutions to the exercises are presented in an Appendix. The tutorial uses QIIME 2 2026.7 (July 2026), which readers can access through a Docker container. Readers interested in using more recent versions of QIIME 2 can refer to the online "living version" of this tutorial, which is kept up-to-date with QIIME 2 (see https://amplicon-docs.qiime2.org/en/stable/tutorials/gut-to-soil/). In addition to the tutorial, the figures presented here follow a simplified, constructed dataset through the analysis steps. This constructed data allows readers to visualize what the data looks like at each analysis step: something that is often difficult to assess because the data we're working with is too large to view directly. The figures are inspired by years of workshops taught by the QIIME 2 development team, and they are licensed such that they can be reused in class materials or other educational contexts. After working through this tutorial, readers should be comfortable applying QIIME 2 to their own microbiome data. About this record. This is the preprint of a manuscript submitted for peer review (version submission-1). It contains the manuscript, its figures and its appendices as one PDF; each figure as an individual SVG or PNG file, provided so that the online tutorial can embed them; and a zip archive of every figure in PNG, SVG and PDF, with and without its caption, for reuse. Copyright © 2026–present J. Gregory Caporaso. The content is licensed under CC BY-NC-SA 4.0: it may be shared and adapted with attribution for non-commercial purposes, and adaptations must carry the same licence. The glossary in Appendix 1 is reproduced from the rachis project documentation under the same licence.