(2026) CBW/CS-DCC Microbiome Analysis: Guelph, ON
I Introduction
Workshop Info
Pre-work
Class Photo
Schedule: Microbiome Analysis
Meet Your Faculty
Data and Compute Setup
II Pre-Session
Meet Your Pre-Session Faculty
AWS/UNIX
1. Logging into AWS
Logging into the Amazon Cloud During the Workshop
Logging in with ssh (Mac/Linux)
Logging in with OpenSSH client (Windows)
Alternative: logging in to the terminal with the browser (all operating systems)
File System Layout
Workspace
RStudio
Jupyter Notebook
2. Introduction to the command line
Exercise: Exploring the filesystem
3. File manipulation
Exercise: Reading text files
Exercise: Editing text files
Reference material
4. Searching and sorting through files
Setup
Data Exploration
Sorting
What Does >> Mean?
Running Commands Together
Piping Commands Together
Pipe Reading Comprehension
Pipe Construction
Which Pipe?
Checking Files
Wildcard Expressions
Key Points
5. Putting it all together
Writing your first script
Awk
Key Points
Conda
Getting Started with Conda
Packages and Environment
Modules, packages, libraries
Dependencies
Environments
Environment management systems for Python
Package Management
Conda
Why use Conda?
Key Points
Working with Environments
Workspace for Conda environments
Creating a conda environment
Activating a Conda environment
Installing a package in a Conda environment
Using software from within a Conda environment
Activity
How to find environments that exist in my machine
How to find out packages installed in an environment
How to delete an environment
Sharing environments
How do I share my enviroment with others?
R
Introduction to R/RStudio
Loging in to RStudio via the AWS instance
RStudio default interface
Using R as a calculator
Mathematical functions
Variables and assignment
R packages
Seeking Help with R
Reading Help Files
Data Objects
Special Operators
Getting Help with Packages
When You Remember Part of the Function Name
When You Have No Idea Where to Begin
Other Ways to Get Help
Resources
Data Structures, Data Types and Data frames
Data Types
Manipulating data frames
Reading and Writing Data Frames
Creating Plots with ggplot2
Building a ggplot
Changing point aethetics
Mapping aesthetics to variables
Adding labels and themes
Saving a ggplot
III Modules
1 | Marker Gene Profiling
Lecture
Lab
Introduction
Reminder on logging into the server
1. 16S
1.1. 16S First steps
1.1.6. Trim primers with cutadapt
1.2. 16S Denoising the reads into amplicon sequence variants
1.3. 16S Assign taxonomy to ASVs
1.3.2 Assess subset of taxonomic assignments with BLAST
1.4. 16S Filtering resultant table
1.5. Build tree with SEPP QIIME 2 plugin
1.6. Exporting data from QIIME 2 for use in other software
16S Answers
2. 18S
2.1. 18S First steps
2.1.6. Trim primers with cutadapt
2.2. 18S Denoising the reads into amplicon sequence variants
2.3. 18S Assign taxonomy to ASVs
2.3.2 Assess subset of taxonomic assignments with BLAST
2.4. 18S Filtering resultant table
2.5. Build a tree
2.6. Exporting data from QIIME 2 for use in other software
18S Answers
3. ITS
2.1. ITS First steps
2.1.6. Trim primers with cutadapt
2.2. ITS Denoising the reads into amplicon sequence variants
2.3. ITS Assign taxonomy to ASVs
2.3.2 Assess subset of taxonomic assignments with BLAST
2.4. ITS Filtering resultant table
2.5. Build a tree
2.6. Exporting data from QIIME 2 for use in other software
ITS Answers
2 | Microbiome ecology, statistics, and visualization
Lecture
Lab
Setup
Loading the Data
Alpha Diversity
Rarefaction
Testing for differences in alpha diversity
Beta Diversity
Calculating Distance and Dissimilarity Metrics
Jaccard Distance
Principal Coordinates Analysis
PERMANOVA
Bray–Curtis Dissimilarity
Weighted UniFrac Distance
Differential Abundance Testing with MaAsLin3
Explanation of MaAsLin3 Outputs
If There Is Time
Collapsing Tables by Taxa
3 | Intro to metagenomics and read-based profiling
Lecture
Lab
Overview
About the samples
3.1. Initial setup for this module
tmux - keeping your commands running when you’re not logged into the server
A Crash Course in GNU Parallel
Get the files
3.2. Filtering with KneadData
3.3. Generating taxonomic profiles with Kraken 2
3.4. Confirmation of taxonomic annotations with GeCoCheck
3.5. Annotation with MetaPhlAn
3.6. Visualisation of Kraken results in R
3.7. Visualisation of MetaPhlAn results in R
4.1. Preparing for module 4
Answers
4 | Metagenomic Assembly and Binning
Lecture
Lab
Overview
Anvi’o
4.1. Initial setup
4.2. Assembly of raw reads with MEGAHIT
4.3. Make an Anvi’o contigs databases
4.4. Run HMMs to identify single copy genes
4.5. Identify taxonomy of single-copy genes
4.6. Map samples onto contigs with Bowtie2
Add coverage and detection statistics to the Anvi’o profile
Merge sample profiles
4.7. Cluster contigs into bins
4.8. Interactive viewing of bins
If you are using Putty
In the browser Anvi’o page
4.9. Running the other binning algorithms
4.10. Combining the clustering results with DAS Tool
4.11. Run GTDB-tk
4.12. Visualise our MAGs
4.13. Run CheckM
Extras
Answers
5 | Assigning Functions
Lecture
Lab
Overview
5.1. Functional annotation of MAGs using Anvi’o NCBI COGs
5.2. AMR annotation of MAGs with CARD RGI
5.3. Visualisation of MAG functional annotations
5.4. MMSeqs initial setup
MMSeqs
HUMAnN
MMSeqs setup
5.5. Run MMSeqs
5.6. Get MMSeqs top hits
5.7. Combine Kraken taxonomy and MMSeqs functions
5.8. Visualise MMSeqs and Kraken in JarrVis
5.9. General functional annotation of MAG fasta files using Bakta
Extras
Answers
6 | Visualization and finding functional significance
Lecture
Lab
Data loading
Libraries we will use:
Metatranscriptomic data (MTX)
Metagenomic data (MGX)
Metadata
Advanced Modeling with MaAsLin 3
Using formulas in R
Running MaAsLin 3 with both fixed effects and random effects
Metatranscriptomic analysis with MaAsLin 3
Unadjusted MTX model
DNA adjusted MTX model
Supervised Learning with Random Forests and
caret
Data splitting
Training the model
Predicting on the test set
Training a model with K-fold cross validation using
caret
Sponsors
(2026) CBW/CS-DCC Microbiome Analysis: Guelph, ON 2026
Data and Compute Setup
Course data downloads
Coming soon!
Compute setup
Coming soon!