Microbiome Bioinformatics Analysis Workshop

General Information

Led by: ArrietaLab

Location: Oswaldo Cruz Foundation - Rio de Janeiro, Brazil

Dates: 3-5 April 2023

Objective: Analyzing microbial DNA sequences

Requirements:

• Laptop with a Mac, Linux, or Windows operating system (not a tablet, Chromebook, etc.) with administrative privileges
• Access to Wifi
• R and Rstudio installed (instructions below)
• Excel or any text editor installed (e.g. TextWrangler, Notepad, BBEdit, etc.)

Contact: mona.parizadeh@ucalgary.ca

Schedule

This workshop covers the following material:

Day 1

(9 a.m. to 12 p.m. & 1 p.m. to 4 p.m.)

• Introduction to metabarcoding and amplicon sequencing
• Introduction to R
• DADA2 Tutorial

Day 2

(9 a.m. to 12 p.m. & 1 p.m. to 4 p.m.)

• Introduction to phyloseq package in R
• Data exploration
• Statistical analyses of 16S rRNA gene sequences:
  • Taxonomic composition
  • Alpha diversity
  • Beta diversity (ordination)
  • PERmutational Multivariate ANalysis Of VAriance (PERMANOVA)
  • Differential analysis
• Discussion
  • Wrap up of the results of 16S rRNA gene sequence analysis
  • Differences between DADA2 pipeline workflows in analyzing 16S vs ITS sequences

Setup

To participate in this Workshop, please install the following software and packages, complete the R courses for beginners, and let us know if you need any help before attending.

Install R and RStudio

R is a free and open-source programming language that is particularly powerful for data exploration, visualization, and statistical analysis. We use RStudio to interact with R.

Windows

Please download R for Windows from CRAN to install R, and also install the RStudio IDE. If you have separate user and admin accounts, please run the installers as an administrator by right-clicking on the .exe file and selecting "Run as administrator" instead of double-clicking. Otherwise, problems may arise later when installing R packages.

Video Tutorial

Mac OS X

Please download R for macOS from CRAN to install R, and also install the RStudio IDE.

Video Tutorial

Linux (Debian, Fedora/Redhat, Ubuntu)

Please download the binary files for your distribution from CRAN to install R, or use a package manager (e.g. run sudo apt-get install r-base for Debian/Ubuntu and run sudo yum install R for Fedora/Redhat). Additionally, please install the RStudio IDE.

R for beginners

To follow the workshop, you must have a basic understanding of R. Before attending the workshop, please go through the following courses:

• Introduction to R (Chapter 1)
• Programming with R

Install R packages

Please install the following R packages in RStudio:

• dada2
• phyloseq
• vegan
• ggplot2
• tidyverse (or tidyr): Tutorial by Data Carpentry
• dplyr
• BiocGenerics
• SummarizedExperiment
• rstatix
• DESeq2
• ggpubr
• RColorBrewer
• gridExtra

Download database for DADA2

Please download the silva_nr99_v138.1_wSpecies_train_set.fa.gz file for taxonomic assignment with DADA2.

BLAST

Sequence Example