This site contains lessons for analyzing 16S rRNA amplicon sequencing data in R based on data collected at the University of Wisconsin - Madison through the Micro 551 Senior Capstone class. Introduction to R materials were modified from the Data Carpentry course materials (Copyright (c) Data Carpentry) on Data Analysis and Visualization in R for Ecologists. These lessons provide a brief overview of the experimental design, how amplicons are preprocessed, and how to analyze the resulting OTU (operational taxonomical unit) table of abundances for each taxa in R.

All Data Carpentry instructional material is freely available under the Creative Commons Attribution license, and therefore this material is freely available under the same license.

Datasets

You will need to download the data files for the Introduction to R lesson and OTU tables for the Amplicon Analysis lesson. Downloading the raw sequencing files to perform preprocessing on your own is optional, and can be accessed through your UW Box Account.

Installation Instructions

R and RStudio are separate downloads and installations. R is the underlying statistical computing environment, but using R alone is no fun. RStudio is a graphical integrated development environment (IDE) that makes using R much easier and more interactive. You need to install R before you install RStudio.

Windows

If you already have R and RStudio installed

  • Open RStudio, and click on “Help” > “Check for updates”. If a new version is available, quit RStudio, and download the latest version for RStudio.
  • To check which version of R you are using, start RStudio and the first thing that appears in the console indicates the version of R you are running. Alternatively, you can type sessionInfo(), which will also display which version of R you are running. Go on the CRAN website and check whether a more recent version is available. If so, please download and install it. You can check here for more information on how to remove old versions from your system if you wish to do so.

If you don’t have R and RStudio installed

  • Download R from the CRAN website.
  • Run the .exe file that was just downloaded
  • Go to the RStudio download page
  • Under Installers select RStudio x.yy.zzz - Windows Vista/7/8/10 (where x, y, and z represent version numbers)
  • Double click the file to install it
  • Once it’s installed, open RStudio to make sure it works and you don’t get any error messages.

macOS

If you already have R and RStudio installed

  • Open RStudio, and click on “Help” > “Check for updates”. If a new version is available, quit RStudio, and download the latest version for RStudio.
  • To check the version of R you are using, start RStudio and the first thing that appears on the terminal indicates the version of R you are running. Alternatively, you can type sessionInfo(), which will also display which version of R you are running. Go on the CRAN website and check whether a more recent version is available. If so, please download and install it.

If you don’t have R and RStudio installed

  • Download R from the CRAN website.
  • Select the .pkg file for the latest R version
  • Double click on the downloaded file to install R
  • It is also a good idea to install XQuartz (needed by some packages)
  • Go to the RStudio download page
  • Under Installers select RStudio x.yy.zzz - Mac OS X 10.6+ (64-bit) (where x, y, and z represent version numbers)
  • Double click the file to install RStudio
  • Once it’s installed, open RStudio to make sure it works and you don’t get any error messages.

Linux

  • Follow the instructions for your distribution from CRAN, they provide information to get the most recent version of R for common distributions. For most distributions, you could use your package manager (e.g., for Debian/Ubuntu run sudo apt-get install r-base, and for Fedora sudo yum install R), but we don’t recommend this approach as the versions provided by this are usually out of date. In any case, make sure you have at least R 3.3.1.
  • Go to the RStudio download page
  • Under Installers select the version that matches your distribution, and install it with your preferred method (e.g., with Debian/Ubuntu sudo dpkg -i rstudio-x.yy.zzz-amd64.deb at the terminal).
  • Once it’s installed, open RStudio to make sure it works and you don’t get any error messages.

Installing Packages

After installing R and RStudio, you need to install the following packages.

  • After starting RStudio, at the console type these commands in order (you can copy and paste the following lines):
install.packages(c("tidyverse", "remotes", "devtools"))
remotes::install_github("MadsAlbertsen/ampvis2")
if (!requireNamespace("BiocManager", quietly = TRUE)) install.packages("BiocManager")
BiocManager::install("phyloseq")

If you are asked to update packages, type a for all. If you are asked to install packages from the source compilation, type Yes. You will know if the package installed successfully if you type library(tidyverse) and so on for each package and you do not get error messages. Note, when the tidyverse package is loaded, it will show some messages describing the package - this is not an error.

  • You can also do this by going to Tools -> Install Packages and typing the names of the packages separated by a comma.

If you cannot install the entire tidyverse because of lots of errors, we only need these packages from the larger set:

install.packages(c("dplyr", "tidyr", "ggplot2"))

Resources

These materials were created by referencing several other resources, and this course website is by no means a completely exhaustive resource or tutorial on 16S rRNA amplicon analysis. I used DADA2 within R to preprocess the samples, and various other resources to help create these tutorials.

Elizabeth McDaniel, 2020. License.

Github Repository.