The Metcalf Lab Presents:

QIIME2 Microbiome Workshop

Welcome to the CSU QIIME2 Microbiome Analysis Workshop, a hands-on, multi-day workshop covering the complete microbiome bioinformatics pipeline from raw amplicon sequences to publication-quality figures.

Day 1

Sequence Processing & Taxonomy

Import paired-end Illumina reads, denoise into Amplicon Sequence Variants with DADA2, and assign taxonomy against a reference database.

Start Day 1

Day 2

Community & Advanced Analyses

Run quality control, rarefaction, phylogenetic tree, α/β-diversity, differential abundance, machine learning, longitudinal modeling, and import into R.

Start Day 2

Day 3

Full Cow Dataset Pipeline

Run the complete QIIME2 pipeline end-to-end on the cow dataset, from import through diversity and differential abundance, applying everything learned in Days 1–2.

Start Day 3

Workshop Schedule: June 9–11, 2026 · CSU Anatomy-Zoology Building H210

Day 1 · Tues Day 2 · Wed Day 3 · Thurs (Cow Day)

8:45Coffee, Introductions & Agenda Overview

9:00Alpine Overview, Connecting to the Workshop Server & Linux RefresherKhalid

9:45Introduction to Microbial Ecology, Microbiome Analysis & QIIME2 Core ConceptsJessica M.

10:30Sample Preparation, Sequencing Technology & NECsJessica H.

11:15Buffer / Q&A

11:30Keeping Track of Code & Using VS CodeVictoria T.

11:45Metadata & the Decomposition MicrobiomeJessica M. & Khalid

12:00Lunch 60 min · catered

13:00Activating QIIME2 & Importing MetadataVictoria T.

13:30Importing Sequencing Data, Demultiplexing & Sequence QualityVictoria T.

14:30ASVs, Denoising with DADA2, Merging & Visualizing OutputsValerie S.

15:00Taxonomy Assignment, Classifier Training, Reference Databases & FilteringValerie S.

16:30Open Q&A & Individual Discussion with Instructors Q&A

8:45Coffee & Recap of Day 1Valerie S.

9:30Quality Control, Sample, Negative & Positive ControlsValerie S.

10:00Rarefaction & Even SamplingJessica M.

10:30Phylogenetics & Tree BuildingJessica M.

11:00α-Diversity Metrics, Statistics & VisualizationsKhalid

11:30β-Diversity Metrics, Statistics & VisualizationsVictoria T.

13:00Lunch catered

13:30Differential Abundance, ANCOM-BC2Anthony

14:00Machine Learning + Network PrimerAnthony

14:45Longitudinal AnalysesVictoria T.

15:30Getting QIIME2 Data Into R, Short Demo & Linked Self-StudyVictoria T.

16:30Open Q&A & Individual Discussion with Instructors Q&A

8:45Coffee & Recap of Days 1–2 PipelineVictoria N.

9:00Intro to Cow Dataset, Study Design & Metadata WalkthroughKhalid

9:30Cow: Import, Demultiplex & Sequence Quality

10:00Check-in #1, Outputs & Choosing Quality ThresholdsValerie S.

10:15Cow: DADA2 Denoising, Quality Checks & Taxonomy Assignment

11:15Check-in #2, Quality Checking DADA2 Outputs & TaxonomyVictoria T.

11:30Cow: Rarefaction & Phylogenetic Tree

12:00Lunch catered

13:30Cow: Core Metrics & α-Diversity

14:00Cow: β-Diversity

14:30Cow: Alternative Analyses (ANCOM-BC2)

15:00Cow: Results Review, Troubleshooting & Group DiscussionValerie S. & Victoria T.

15:30Open Q&A & Wrap-up Q&A

What You Will Learn

Over three days, participants will work through a full 16S rRNA amplicon sequencing analysis using QIIME2. By the end of the workshop you will be able to:

• Import and quality-filter paired-end Illumina sequences
• Denoise reads into Amplicon Sequence Variants (ASVs) using DADA2
• Assign taxonomy with the GreenGenes2 reference database
• Perform quality control and evaluate sequencing controls
• Calculate alpha and beta diversity metrics
• Test for differences in community composition using PERMANOVA and ADONIS
• Identify differentially abundant taxa using ANCOM-BC
• Build and interpret Random Forest classifiers and regressors
• Run longitudinal volatility and linear mixed effects models
• Export results and generate publication-quality figures in R

Dataset Overview

This workshop uses a soil microbiome dataset collected across multiple facilities and time points. Samples were sequenced across two Illumina runs (run 2 and run 3), which are merged during the DADA2 step. The study design includes:

• Multiple sample types (e.g., soil, environmental controls)
• Multiple facilities
• A time series component captured as accumulated degree days (ADD)

Before You Begin

Complete the Getting Started section before Day 1, this covers requesting an Alpine account and installing QIIME2 in your projects directory.

QIIME2 View

All .qzv visualization files produced during the workshop can be viewed interactively at view.qiime2.org, no installation required.