(1) Login to your AWS-hosted RStudio Instance (URL/username in the Google Sheet)

You are provided an instance of the R editor RStudio on AWS already waiting for you to use. Login to your AWS-hosted RStudio instance using the URL, username, and password assigned to you in the Google Sheet.

Please make sure the RStudio user interface dashboard looks as follows: Console Pane (Lower Left Quadrant), Scripting Pane (Upper Left Quadrant), File Manager/Plots Display/Help-Tab Pane (Lower Right Quadrant), Environment/History-Tab Pane (Upper Right Quadrant).

(2) Generate barplot(s) using subset of kraken samples

In this section, you will learn how to use R to generate two plots that help visualize the similarities and differences between a subset of the Metasub samples and compare it to metagenomics samples from the Human Microbiome project.

We have provided a subset of of metagnomics data (taxa_table.csv) and one with, for your convenience. This file contains taxonomically characterized and quantified metagenomics data from nine microbiome samples: three from the human microbiome project, three from the Metasub project, and three from a mystery source.

First we will plot our samples as a stacked-barplot at the phylum level. A stacked-barplot shows a set of numbers as a series of columns one on top of each other colored by a label. In our case, a taxonomic profile is a set of numerical abundances labeled by the microbial species it belongs to.

Here’s how we make this plot in R:

library(ggplot2)   # These lines load additional libraries into our environment

taxa = read.csv('taxa_table.csv', header=TRUE, sep=',')  # Read our taxonomic table into a computational object from a file

taxa = taxa[taxa$rank == 'phylum',]  # This filters our taxonomic table to a specific taxonomic rank. One of Kingdom, Phylum, Class, Order, Family, Genus, Species. Play around with a few other ranks.

ggplot(taxa, aes(x=sample, y=percent_abundance, fill=taxon)) + # this creates a ggplot object. Can you figure out what the aes(...) section is doing?
  geom_bar(stat="identity") +  # this tells ggplot how we want our data to be displayed
  xlab('Sample') +  # These lines tell ggplot what our axis labels should be
  ylab('Abundance') +
  labs(fill='Phylum') +
  theme(axis.text.x = element_text(angle = 90)) # this rotates the x-axis text 90 degrees

Here is a video of this step being performed:

(3) Clear your Console, Environment Tab, and Plots tab using the brush button and re-run the script chunk-by-chunk (noting what each chunk appears to be doing):

Chunk 1:

library(ggplot2)   # These lines load additional libraries into our environment

Chunk 1+2:

library(ggplot2)   # These lines load additional libraries into our environment

taxa = read.csv('taxa_table.csv', header=TRUE, sep=',')  # Read our taxonomic table into a computational object from a file

Chunk 1+2+3:

library(ggplot2)   # These lines load additional libraries into our environment

taxa = read.csv('taxa_table.csv', header=TRUE, sep=',')  # Read our taxonomic table into a computational object from a file

taxa = taxa[taxa$rank == 'phylum',]  # This filters our taxonomic table to a specific taxonomic rank. One of Kingdom, Phylum, Class, Order, Family, Genus, Species. Play around with a few other ranks.

Chunk 1+2+3+4:

library(ggplot2)   # These lines load additional libraries into our environment

taxa = read.csv('taxa_table.csv', header=TRUE, sep=',')  # Read our taxonomic table into a computational object from a file

taxa = taxa[taxa$rank == 'phylum',]  # This filters our taxonomic table to a specific taxonomic rank. One of Kingdom, Phylum, Class, Order, Family, Genus, Species. Play around with a few other ranks.

ggplot(taxa, aes(x=sample, y=percent_abundance, fill=taxon)) + # this creates a ggplot object. Can you figure out what the aes(...) section is doing?
  geom_bar(stat="identity") +  # this tells ggplot how we want our data to be displayed
  xlab('Sample') +  # These lines tell ggplot what our axis labels should be
  ylab('Abundance') +
  labs(fill='Phylum') +
  theme(axis.text.x = element_text(angle = 90)) # this rotates the x-axis text 90 degrees

(4) Generate barplot at the family level

2. Modify code: edit your script to generate a barplot at the “family” level, by modifying this line of code:

taxa = taxa[taxa$rank == 'phylum',] 

CP Template:

Checkpoint Example:

Checkpoint (CP) 1 In a single message in your assigned Slack channel, please:

1. Upload barplot at the phylum level
2. Upload barplot at the family level
3. Answer to these questions:

• Which line of code ingests data from the taxa_table.csv file into the taxa object (copy paste the exact line of code)?

• Which line of code allows you to filter down to a specific taxonomic level?

*What is the $ operator doing in this line of code: taxa = taxa[taxa$rank == 'phylum',] ?

• Why wouldn’t you want to plot a barplot at the species level? (If you’re not sure, try it!)

• What can the barplots tell you about the similarities and dissimilarities of microbiome samples?

• What questions do barlots leave unanswered about microbiome samples?

(5.) Access your Linux terminal via your AWS-hosted RStudio instance

Access your Linux terminal by logging into RStudio via the web browser (can be found in the the Google Sheet.

Setup to your Linux environment:

It should look like this once you’re finished setting up your Linux environment:

(6.) Navigating your Linux terminal.

Linux is a command-line based operating system. For our purposes Linux = Unix.

Let’s do a brief review of Linux commands on our AWS-hosted Linux instance in RStudio. This the language and structure of this tutorial is heavily borrowed from http://www.ee.surrey.ac.uk/Teaching/Unix/. Go to this website and read the “Introduction to the UNIX Operating System”. For our purposes, you can treat Unix and Linux as interchangeable.

ls (list) When you first login, your current working directory is your home directory by default. Your home directory in every Linux system is denoted by the shorthand ~.

To find out what is in your home directory, type:

$ ls

The ls command ( lowercase L and lowercase S ) lists the contents of your current working directory.

You should see these Directories and files: R databases miniconda3 pass.txt reads rstudio taxa_table.csv

Note that the directories and files are colored differently.

ls does not cause all the files in your home directory to be listed—only those ones whose name does not begin with a dot (.). Files beginning with a dot (.) are known as hidden files and usually contain important program configuration information. They are hidden because you should not change them unless you are very familiar with Linux!!!

To list all files in your home directory including those whose names begin with a dot, type:

$ ls -a

As you likely guessed, the a in -a stands for all.

As you should now see, ls -a lists files that are normally hidden.

ls is an example of a command which can take options: -a is an example of an option. The options change the behaviour of the command. There are many online resources that tell you which options a particular command can take, and how each option modifies the behaviour of the command. We’ve posted a Linux command “Cheat Sheet” in the channel with Cody.

Making Directories

mkdir (make directory)

We will now make a subdirectory in your home directory to hold the files you will be creating and using in the course of this tutorial. To make a subdirectory calledunixstuff in your current working directory type:

$ mkdir unixstuff

To see the directory you have just created, type:

$ ls

Changing to a different directory

cd (change directory)

The command cd directory means change the current working directory to ‘directory’. The current working directory may be thought of as the directory you are in, i.e. your current position in the file-system tree.

To change to the directory you have just made, type:

$ cd unixstuff

Type ls to see the contents (which should be empty)

Make another directory inside the unixstuff directory called backups

The directories . and ..

Still in the unixstuff directory, type:

$ ls -a

As you can see, in the unixstuff directory (and in all other directories), there are two special directories called (.) and (..)

The current directory (.) In UNIX, (.) means the current directory, so typing

$ cd .

NOTE: there is a space between cd and the dot

means stay where you are (the unixstuff directory).

This may not seem very useful at first, but using (.) as the name of the current directory will save a lot of typing, as we shall see later in the tutorial.

The parent directory (..) (..) means the parent of the current directory, so typing

$ cd ..

will take you one directory up the hierarchy (back to your home directory). Try it now.

Note: typing cd with no argument always returns you to your home directory. This is very useful if you are lost in the file system.

Pathnames pwd (print working directory) Pathnames (also called filepaths) enable you to work out where you are in relation to the whole file-system. For example, to find out the absolute pathname of your home-directory, type cd to get back to your home-directory and then type:

$ pwd

The full pathname will look something like this:

/home/user#

which means that user# (your home directory) is in the sub-directory home (the group directory), which is in the top-level root directory called " / " .

Unix File structure

Use the commands cd, ls and pwd to explore the file system.

(Remember, if you get lost, type cd by itself to return to your home-directory)

More about home directories and pathnames Understanding pathnames First type cd to get back to your home-directory, then type:

$ ls unixstuff

to list the conents of your unixstuff directory.

Now type:

$ ls backups

You will get a message like this: backups: No such file or directory

The reason is, backups is not in your current working directory. To use a command on a file (or directory) not in the current working directory (the directory you are currently in), you must either cd to the correct directory, or specify its full pathname. To list the contents of your backups directory, you must type:

$ ls unixstuff/backups

~ (your home directory)

Home directories can also be referred to by the tilde ~ character. It can be used to specify paths starting at your home directory. So typing

$ ls ~/unixstuff

will list the contents of your unixstuff directory, no matter where you currently are in the file system.

What do you think ls ~ list?

At this point, you have completed through Introduction and Tutorial One on http://www.ee.surrey.ac.uk/Teaching/Unix/. Continue with Tutorial Two (Copying Files, Moving Files, Removing Files and directories, Displaying the contents of a file on the screen, Searching the contents of a file). After those are complete, you can optionally complete Tutorials 3-8, but they are outside of the scope of the Linux skills you’ll need to complete the VTP.

Once you have finished all of the above exercises, please execute:

$ history > yourName_linux_work.txt

yourName_linux_work.txt is a file that should contain all of the commands you’ve executed in your Linux terminal.

Checkpoint (CP) 2: Please take a few screenshots of your work up to this point and post them in a single post on the group Slack channel. In the same post, please upload the yourName_linux_work.txt file so we can review it.