Overview:

This portion of the class is devoted to making sure we are all starting from the same starting point on stampede. This tutorial was developed as a combined version of multiple other tutorials which were previously given credit here. Anyone wishing to use this tutorial is welcome.

This is probably the longest tutorial in the entire class. It is designed to take between 1/2 and 3/4 of the first class. Do not stress if you feel people are moving through it faster than you are, or if you do not get it done before the next presentation. There will be links back to this tutorial from other tutorials as needed, and by the 2nd half of Wednesday's class when we start with the specialized tutorials, you can circle back to this tutorial as well.

Objectives:

Familiarize yourself with the way course material will be presented.
Log into stampede2.
Change your stampede2 profile to the course specific format.
Refresh understanding of basic linux commands with some course organization.
Review use of the nano text editor program, and become familiar with several other text editor programs.

Example things you will encounter in the course:

As this is the first real tutorial you are encountering in this course, some housekeeping matters to familiarize you with how information will be presented.

Code blocks

There will be 4 types of code blocks used throughout this class. Text inside of code blocks represent at least 1 possible correct answer, and should either be typed EXACTLY into the terminal window as they are, or copy pasted. There is a notable exception that text between <> symbols represent something that you need to replace before sending it to the terminal. Yes, the <> marks themselves also need to be replaced. We try to put informative text within the brackets so you know what to replace it with. If you are ever unsure of what to replace the <> text with, just ask.

Visible
1. These are code blocks that you would have no idea what to type without help. (like when a new command is being introduced)
2. These will typically be associated with longer/more detailed text above the text box explaining things.
3. An example code block showing you the command you need to type into the prompt to list what directory you are currently in:
```
pwd
```
Hinted
1. These are code blocks that you can probably figure out what to type with a hint that goes beyond what the tutorial is requesting. Access the hint by clicking the triangle or hint hyperlink text.
2. These exist to force you to think about what command you need, and hopefully make some connections to help you remember what you will need to type in the future.
3. These should all come with additional explanation as to what is going on.
4. Rather than just expanding these by reflex, I strongly suggest seeing if you can figure out what the command will be, and checking your work
5. Example:
  what command would you use to Print your current Working Directory
  In this example the letters P W and D are all capitalized to try to help you focus on the command itself
  pwd
Hidden:
1. These code blocks represent things that you should have seen several times already, or things that can be succinctly explained.
2. Example:
  use the pwd command to print your current working directory Expand source
```
pwd
```
Speed bump:
1. This combines the previous 2 types to deliberately slow you down and be cumbersome.
2. If you find yourself consistently wrong about what eventually shows up in the text box, slow down, step back, think about whats going on, and consider asking a question.
3. These should only come after you have seen the same (or very similar) commands in the other formats previously
4. Example:
  print your current working directory
  Remember, the command you need is "pwd".
  This command needs no options Expand source
  
  pwd

Warnings

Why the tutorials have warnings?

Warnings exist for 2 reasons:

Something you are about to do can have negative impact on you
1. You saw an example of this talking about paying attention to warnings when using ssh to access new remote computers
Something you are about to do can have negative impacts on others
1. this will be related mostly to the use of "idev" sessions beginning tomorrow.

Info boxes

These are used to give more general background about things

These were introduced last year, and not much feedback was provided. If you find them useful (or have ideas of how they might be more useful) please remember to mention them in the post class survey.

Tip boxes

Things I wish I knew sooner

As an example: On the command line, you can use the tab key to try to autofill the "rest" of whatever you are typing, whether it is the name of the directory, a long file, or even a command. Hitting tab twice will list all possible matches to whatever you have already typed when there are multiple different possibilities

Tutorial:

Logging into stampede2

Hopefully you were able to log into stampede2 last week as part of the pre-class assignment. If not make sure the instructor is aware as there are additional elements that still need to be addressed (potentially adding you to the project allocation and definitely being added to the reservation that we will use starting tomorrow).

log into lonestar 5 (aka ls5) with the ssh command

ssh <username>@stampede2.tacc.utexas.edu

When prompted enter your password, and digital security code from the app, and answer "yes" to the security question if you see one.

Logging into remote computers

You are blindly told to enter yes here, only because you are given a command above to copy which will take you to a remote computer system that I know to be safe, and as this is an introductory class, it is likely you have not logged into it before. If you have previously logged into this remote computer from the local computer you are sitting at, you will not be issued a security warning prompt.

The same will be true the first time you log into any of the other TACC resource, or other remote computer. If you ever see a security warning logging into somewhere that you use commonly you should answer no and try to figure out why you were warned. Otherwise type "yes" to bypass the security check.

As a reminder, the ssh command, and launching programs to give you the prompt to type them was provided as part of the pre-class assignment. Convenient links incase you need them or want to refresh your memory:

Windows users

Mac users

Setting up your stampede2 profile

There are many flavors of Linux/Unix shells. The default for TACC's Linux (and most other Linuxes) is bash (bourne again shell), which we will use throughout.

Whenever you login via an interactive shell as you did above, a well-known script is executed by the shell to establish your favorite environment settings. I've set up a common profile for you to start with that will help you know where you are in the file system and make it easier to access some of our shared resources. If you already have a profile set up on stampede2 that you like, we want to make sure that we don't destroy it but it is critical to make sure that we change it temporarily so everyone is working from the same place through the class. Use the ls command to check if you have a profile already set up in your home directory.

Use ls to check if particular file exists

cdh
ls .profile
ls .bashrc

If you already have a .profile or .bashrc file, use the mv command to change the name to something descriptive (for example ".profile_pre_GVA_backup"). Otherwise continue to creating a new files.

Use mv to change your .profile file to a backup copy

mv .profile profile_pre_GVA_backup
mv .bashrc bashrc_pre_GVA_backup

A warning about deleting files

Most of us are used to having an 'undo' button, trash/recycling collection of deleted files, or warnings when we tell a computer to do something that can't be undone. The command line offers none of these options. In extreme situations on TACC, you can use the help desk ticket system to recover a deleted file, but there is no guarantee files can be recovered under normal circumstances (we will cover exceptions to this later).

The specific warning right now is that if you have an existing profile, and have not done the above commands correctly, you will not be able to recover your existing profile. Thus this is a great opportunity to interact with your instructor and make 100% the above steps have been correctly performed. Type ls -al onto the command line and then share your screen on zoom if you are not sure

Now that we have backed up your profiles so you won't lose any previous settings, you can copy our predefined GVA2021.bashrc file from the /corral-repl/utexas/BioITeam/scripts/ folder to your $HOME folder as .bashrc and the predefined GVA2021.profile as .profile from the same location before using the chmod command to change the permissions to read and write for the user only.

Copy the course provided .profile file and change its name and permissions

cp /corral-repl/utexas/BioITeam/gva_course/GVA2021.bashrc .bashrc
cp /corral-repl/utexas/BioITeam/gva_course/GVA2021.profile .profile
chmod 700 .bashrc
chmod 700 .profile

The chmod 700 <FILE> command marks the file as readable/writable/executable only by you. The .bashrc script file will not be executed unless it has these permissions settings.

Understanding why some files start with a "."

In the above code box, you see that the names start with a . when a filename starts with a . it conveys a special meaning to the operating system/command line. Specifically, it prevents that file from being displayed when you use the ls command unless you specifically as for hidden files to be displayed using the -a option. Such files are termed "dot-files" if you are interested in researching them further.

Let's look at a few different ways we will use the ls command throughout the course. Compare the output of the following 4 commands:

Standard output

ls              #ignore everything that comes after the # mark. There is a problem on this wiki page but things after a # wont effect commands

Standard output plus hidden files

ls -a

Standard output plus hidden files in a single column

ls -a -1

Standard output plus hidden files in a single column with additional information

ls -a -l

Throughout the course you will notice that many options are supplied to commands via a single dash immediately followed by a single letter. Usually when you have multiple commands supplied in this manner you can combine all the letters after a single dash to make things easier/faster to type. Experiment a little to prove to yourself that the following 2 commands give the same output.

Standard output plus hidden files in a single column

ls -a -1

ls -al

While knowing that you can combine options in this way helps you analyze data faster/better, the real value comes from being able to decipher commands you come across on help forums, or in publications.

For ls specifically the following association table is worth making note of, but if you want the 'official' names consider using the man command to bring up the ls manual.

flag	association
-a	"all" files
-l	"long" listing of file information
-1	1 column

Getting back to your profile... Since .bashrc is executed when you login, to ensure it is set up properly you should first logout:

How to leave Lonestar by logout or exit from a remote connection

logout
# or
exit

then log back in:

Go log back in to Lonestar

ssh <username>@stampede2.tacc.utexas.edu

If everything is working correctly you should now see this as your prompt:

tacc:~$

It is also likely or expected that upon logging in you see the following:

The following have been reloaded with a version change:
  1) impi/18.0.2 => impi/17.0.3     2) intel/18.0.2 => intel/17.0.4     3) python2/2.7.15 => python2/2.7.14

These messages have to do with some of the core compilers and associated tools on TACC. You could use the module spider commands detailed below to find out more information of any of these modules and track down why such changes might be being made, but they are not concerning.

If you see anything besides "tacc:~$" as your prompt, get my attention and be ready to share your screen rather than continuing forward.

Setting up other shortcuts:

In order to make navigating to the different file systems on stampede2 a little easier ($SCRATCH and $WORK), you can set up some shortcuts with these commands that create folders that "link" to those locations. Run these commands when logged into stampede2 with a terminal, from your home directory.

Creating a shortcut to the main Lonestar working directories

cdh
ln -s $SCRATCH scratch
ln -s $WORK work
ln -s $BI BioITeam

Several people report seeing an error message stating "ln: failed to create symbolic link 'BioITeam/BioITeam': Permission denied." This is being investigated, but is not expected to impact today's tutorial.

Understanding what your .bashrc file actually does.

While interesting and useful information to have, understanding it is not critical to variant analysis. I suggest you to look through this information after you complete the rest of the tutorial, in your free time, or when you need to modify your profile or bashrc files in the future.

Let's look at what your .bashrc profile actually does. Use the cat command to print contents of the .bashrc file to the screen.

Print the contents of the .profile file to the screen

cat .bashrc

This will print several lines of text to the terminal window. Let's look at what some of these lines do with a little more information:

lines that start with #
- Any line begins with a # symbol, is "commented out". Anything after a # symbol will not be executed by any program. Programers commonly make use of behavior to leave notes for others, or even themselves at a later date as to what particular lines of a script are actually doing.
Section 1 has multiple lines involving "module load <NAME>"
- This loads different modules by default. We have included basic ones that will help with basic TACC things. After we review the use of the nano text editor we'll go into more depth with TACC modules. But for now trust us when we say that not having to load a bunch of modules every time you log into TACC is a good thing.
- In previous years the module system was used more extensively. Here we will attempt to rely more on miniconda installations for increased portability.
Section 2 has multiple lines starting with "export"
- The export lines define shell variables for example BI and PATH. You've already seen how using $BI can come in handy accessing our shared course directory. As for PATH, that is a well-known environment variable that defines a set of directories where the shell will look when you type in a program's name. Our shared profile adds the common course directories that we copied at the start of this tutorial and your local ~/local/bin directory (which does not exist yet) to the location list. You can see the entire list of locations by doing this:
  How to see where the bash shell looks for programs
```
echo $PATH
```
  As you can see, there are a lot of locations on the path. That's because when you load modules at TACC (see above), that mechanism makes the programs available to you by putting their installation directories on your $PATH.
umask 002
- The umask command is used to set the default permissions of newly created files and directories limiting the need to use the chmod command. umask functions as the inverse of chmod meaning that it subtracts the values from the default permissions. In this case the command umask 002 is the equivalent of the command chmod 775 for directories, and chmod 664 for files. in summary, having this command in your .profile gives all new files you create read and write access to both you and your group while giving read only access to everyone else.
PS1='tacc:\w$ '
- The PS1='tacc:\w$ ' line is a special setting that tells the shell to display the current directory as part of its prompt. It saves you typing pwd all the time to see where you are in the directory hierarchy. Try using the mkdir command to make a new directory called tmp and change into that directory to see what it does to your prompt.
  See how your prompt reflects your current directory Expand source
```
mkdir tmp
cd tmp
```
- Your prompt should have changed from: "tacc:~$"to now be "tacc:~/tmp$". Your prompt now tells you you are in the tmp subdirectory of your home directory (~). See if you can figure out how to return to your home directory without expanding the code block. Expand the following code block to see the different ways of returning to your home directory.
  How to return to your home directory Expand source
```
cd
cdh
cd $HOME
cd ~
cd -
```
  The last example in the above code block will return you to your previous directory. In this case, that means the home directory, but it can be very useful in other situations when you change directories to do something in 1 place then need to hop back to where you were, or if you mistakenly leave a directory.

Editing files

There are a number of options for editing files at TACC. These fall into three categories:

Linux text editors installed at TACC (nano, vi, emacs). These run in your terminal window. vi and emacs are extremely powerful but also quite complex, so nano is the best choice as a first local text editor. If you are already familiar with one of the other programs you are welcome to continue using it.
Text editors or IDEs that run on your local computer but have an SFTP (secure FTP) interface that lets you connect to a remote computer (Notepad++ or Komodo Edit). Once you connect to the remote host, you can navigate its directory structure and edit files. When you open a file, its contents are brought over the network into the text editor's edit window, then saved back when you save the file.
Software that will allow you to mount your home directory on TACC as if it were a normal disk e.g. MacFuse/MacFusion for Mac, or ExpanDrive for Windows or Mac ($$, but free trial). Then, you can use any text editor to open files and copy them to your computer with the usual drag-drop.

We'll go over nano together in class, but you may find these other options more useful for your day-to-day work so feel free to go over these sections in your free time to familiarize yourself with their workings to see if one is better for you.

Komodo Edit for Mac and Windows

Komodo Edit is another free, full-featured text editor with syntax coloring for many programming languages and a remote file editing interface. It has versions for both Macintosh and Windows. Download the appropriate install image here.

Once installed, start Komodo Edit and follow these steps to configure it:

Configure the default line separator for Unix
- On the Edit menu select Preferences
- Select the New Files Category
- For Specify the end-of-line (EOL) indicator for newly created files select UNIX (\n)
- Select OK
Configure a connection to TACC
- On the Edit menu select Preferences
- Select the Servers Category
- For Server type select SFTP
- Give this profile the Name of stampede2
- For Hostname enter stampede2.tacc.utexas.edu
- Enter your TACC user ID for Username
- Leave Port and Default path blank
- Select OK

When you want to open an existing file at stampede2, do the following:

Select the File menu -> Open -> Remote File
- Select your stampede2 profile from the top Server drop-down menu
- Once you log in, it should show you all the files and directories in your stampede2 $HOME directory
Navigate to the file you want and open it
- Often you will use the work or scratch directory links to help you here

To create and save a new file, do the following:

From the Komodo Edit Start Page, select New File
- Select the file type (Text is good for commands files)
Edit the contents
Select the File menu -> Save As Other -> Remote File
- Select your Stampede2 profile from the Server drop-down menu
- Once you log in, it should show you all the files and directories in your stampede $HOME directory
Navigate to where you want the put the file and save it
- Often you will use the work or scratch directory links to help you here

Notepad++ for Windows

Notepad++ is an open source, full-featured text editor for Windows PCs (not Macs). It has syntax coloring for many programming languages (Python, Perl, shell), and a remote file editing interface.

If you're on a Windows PC download the installer here.

Once it has been installed, start Notepad++ and follow these steps to configure it:

Configure the default line separator for Unix
- In the Settings menu, select Preferences
- In the Preferences dialog, select the New Document/Default Directory tab.
- Select Unix in the Format section
- Close
Configure a connection to TACC
- In the Plugins menu, select NppFTP, then select Focus NppFTP Window. The top bar of the NppFTP panel should become blue.
- Click the Settings icon (looks like a gear), then select Profile Settings
- In the Profile settings dialog click Add new
- Call the new profile stampede
- Fill in Hostname (stampede2.tacc.utexas.edu) and your TACC user ID
- Connection type must be SFTP
- Close

To open the connection, click the blue (Dis)connect icon then select stampede connection. It should prompt for your password. Once you've authenticated, a directory tree ending in your home directory will be visible in the NppFTP window. You can click the the (Dis)connect icon again to Disconnect when you're done.

Since much of the editing we'll do will be in your SCRATCH area at TACC, rather than having to navigate around TACC's complex file system tree, it helps to create symbolic links to your WORK and SCRATCH directory in your home directory. Then you'll be able to get there just by clicking on the scratch or work folder in the Notepad++ Remote directory tree. See below for how to do this.

MacFuse/MacFusion/TextWrangler for Mac

Want your stampede2 files to appear like any other place on your hard drive? You can do this using MacFuse/MacFusion on a Mac.

Want to edit files on TACC without having to use nano? You might want to use BBedit, a text editor that can edit files over ssh.

Editing Text Files on TACC: BBedit

BBedit is a recommended FreeWare text editor for MacOS X. You can use it to directly edit text files on stampede2 with OSXFuse/MacFusion using a nice GUI. It is a much more powerful text editor than TextEdit, and won't trip you up by wrapping lines etc., if you learn to use it.

Even if you cannot install OSXFuse/MacFusion, BBedit allows you to edit a remote file via SSH. To do this:

Select *File > Open from FTP/SFTP Server...
Type stampede2.tacc.utexas.edu, your username, and your password into the appropriate boxes.
Check the You need to check the SFTP box.
Click connect.
You will now have a file browser window. You can create new files and edit existing files on stampede, but won't be able to drag-and-drop copy files.

Tip: Files beginning in a dot (.) like (.bashrc) are "hidden" and won't show up when you are navigating in Finder (if using OSXFuse/MacFusion). There is a way to turn on showing these files in finder, but it can get annoying because they will show up everywhere. If you use the TextWrangler "open" command to open a file, there is a box that you can check to show these files.

Connecting to TACC Like a Hard Drive: MacFuse/MacFusion

Here are the steps for an installation:

Download and install FUSE for OS X.
- Check the option to install the "compatibility layer"
Download MacFusion.
- Move the app that gets downloaded to your Applications folder
Restart your computer.
Open the MacFusion application.
Click the + menu in the window and select SSHFS. Enter your login information for stampede2. Choose connect. The remote file system will appear in Finder (depending on your settings it may be on the desktop or inside the computer shortcut in the side of a Finder window). You can also click on the mounted volume within MacFusion and choose "Reveal" from the gear menu.

Copying Files To and From TACC: SFTP Clients

If you can't get OSXFuse/MacFusion to work, you can still copy files back and forth between your computer and TACC using a secure FTP (SFTP) client. Some examples of free programs for Mac are:

As we will be using nano throughout the class, it is a good idea to review some of the basics. nano is a very simple editor available on most Linux systems. If you are able to use ssh, you can use nano. To invoke it, just type:

How to start the nano text editor

nano

You'll see a short menu of operations at the bottom of the terminal window. The most important are:

ctl-o - write out the file
ctl-x - exit nano
You can just type in text, and navigate around using arrow keys. A couple of other navigation shortcuts:
ctl-a - go to start of line
ctl-e - go to end of line

Be careful with long lines – sometimes nano will split long lines into more than one line, which can cause problems in our commands files, and if you copy paste code into a nano editor.

What can you do to see contents of a file without opening it for editing?

Command	useful for	bad if
head	seeing the first lines of a file (10 by default)	file is binary
tail	seeing the last lines of a file (10 by default)	file is binary
cat	print all lines of a file to the screen	the file is big and/or binary
less	opens the entire file in a separate program but does not allow editing	if you are going to type a new command based on the content, or forget the q key exits the view, or file is binary
more	prints 1 page worth of a file to the screen, can hold enter key down to see next line repeatedly. Contents will remain when you scroll back up.	you forget that you hit the q key to stop stop looking at the file, or file is binary

Note that all of the above state that it is bad to view binary files. Binary files exist for computers to read, not humans, and are thus best ignored. We'll go over this in more detail as well as some conversion steps when we deal with .sam and .bam files later in the course.

Many expect to see something that looks like something out of The Matrix, but unfortunately, you actually just see a bunch of gibberish and it can mess with your terminal. Typically if you accidentally or unknowingly try to view such a file, its best to just close your terminal window and start a new session.

How should we name files and folders?

In general you will want to adopt a consistent pattern of naming, and it should be your own and something that makes sense to you. After that there are some tips:

The most important thing to get used to is the convention of using . _ or capitalizing the first letter in each word in names rather than spaces in names, and limiting your use of any other punctuation. Spaces are great for mac and windows folder names when you are using visual interfaces, but on the command line, a space is a signal to start doing something different. Imagine instead of a BioITeam folder you wanted to make it a little easier to read and wanted to call it "Bio I Team" certainly everyone would agree its easier to read that way, but because of the spaces, bash will think you want to create 3 folders, 1 named Bio another named I and a third named Team. Now this is certainly behavior you can use when appropriate to your advantage, but generally speaking spaces will not be your friend. Early on in my computational learning I was told "A computer will always do exactly what you told it to do. The trick is telling it to do what you want it to do".
Name things something that makes it obvious to you what the contents are not just today but next week, next month, and next year even if you don't touch the it for weeks-months-years.
Prefixing file/folder names with international date format (YYYY-MM-DD) will ensure that listing the contents will print in an order in which they were created. This can be useful when doing the same or similar analysis on new samples as new data is generated.

People always ask but can you name new directories or files to have spaces in them?

To answer the question, Yes, files/folders can have spaces. This is hidden away to keep you from accidentally thinking that this is a good idea. LET ME STRESS AGAIN this is a horrible habit to get into and will lead to unforced errors.

Instead let's think about this from the prospective of encountering files or directories that you are working with but didn't create that have spaces in them. Assumably because a colleague who didn't take this course sent you some data, and not because you thought it was a good idea personally. Spaces can be "escaped" like many other special characters. Imagine someone sent you directory name "This is really annoying to use but I don't know it yet" to change into that directory you would have to type:

cd this\ is\ really\ annoying\ to\ use\ but\ I\ don\'t\ know\ it\ yet

Notice that the apostrophe also had to be escaped, which should help show you not to use other punctuation.

The tab key would automatically add the escape character for you.

Understanding TACC

Now that we've been using stampede2 for a little bit, and have it behaving in a way that is a little more useful to us, let's get more of a functional understanding of what exactly it is and how it works.

Diagram of Stampede2 directories: What connects to what, how fast, and for how long.

Stampede2 is a computer cluster connected to three file servers (each with unique characteristics), and other computer infrastructure. For the purpose of this class, and your own work, you only need to understand the basics of the 3 file servers to know how to use them effectively. The 3 servers are named, "HOME", "WORK2", and "SCRATCH", and we will work with them all over the next 5 days

	$HOME	$WORK2	$SCRATCH
Purged?	No	No	Files can be purged if not accessed for 10 days.
Backed Up?	Yes	No	No
Capacity	10GB	1TB	Basically infinite.
Commands to Access	cdh cd $HOME/	cdw cd $WORK/	cds cd $SCRATCH/
Purpose	Store Executables	Store Files and Programs	Run Jobs
Time spent	When modifying basic settings	When installing new programs; Storing raw or final data	When analyzing data

Executables that aren't available on TACC through the "module" command should be stored in $HOME.

If you plan to be using a set of files frequently or would like to save the results of a job, they should be stored in $WORK. While 1TB may seem like a lot of space you can easily fill it up with just a few sequencing projects, particularly if you store files in a non-compressed manner, or wish to store analyzed intermediates. Best practice would be to store the most important files (raw > scripts > final > analysis files) on a system such as corral or backed up to something else such as UTbox.

If you're going to run a job, it's a good idea to keep your input files in a directory in $WORK and copy them to a directory in $SCRATCH where you plan to run your job.

Example command for copying data from a $WORK directory to $SCRATCH . This command is only an example of something you may use in the future. As you do not have any fastq files on $WORK, or at least likely do not have them in a folder titled 'my_fastq_data' if you tried this command you would be expected to get a message stating no such file or directory found.

 cp $WORK2/my_fastq_data/*fastq $SCRATCH/my_project/

Understanding "jobs" and compute nodes.

When you log into stampede2 using ssh you are connected to what is known as the login node or "the head node". There are several different head nodes, but they are shared by everyone that is logged into stampede2 (not just in this class, or from campus, or even from Texas, but everywhere in the world). Anything you type onto the command line has to be executed by the head node. The longer something takes to complete, or the more commands you send at once the slower the head node will work for you and everybody else. Get enough people running large jobs on the head node all at once (say a zoom class of summer school students) and stampede2 can actually crash leaving nobody able to execute commands or even log in for minutes -> hours -> perhaps even days if something goes really wrong. To try to avoid crashes, TACC tries to monitor things and proactively stop things before they get too out of hand. If you guess wrong on if something is safe to run on the head node, you may eventually see a message like the one pasted below. If you do, it's not the end of the world, but repeated messages will lead to revoked TACC access and emails where you have to explain what you are doing to TACC and your PI and how you are going to fix it and avoid it in the future.

Example of how you learn you shouldn't have been on the head node

Message from root@login1.ls4.tacc.utexas.edu on pts/127 at 09:16 ...
Please do not run scripts or programs that require more than a few minutes of
CPU time on the login nodes.  Your current running process below has been
killed and must be submitted to the queues, for usage policy see
http://www.tacc.utexas.edu/user-services/usage-policies/
If you have any questions regarding this, please submit a consulting ticket.

So you may be asking yourself what the point of using stampede2 is at all if it is wrought with so many issues. The answer comes in the form of compute nodes. There are nearly 6,000 compute nodes with different configurations that can only be accessed by a single person for a specified amount of time. For the duration of the class, each student will interact with a single compute node using an interactive DEVelopment (iDEV) session so that you get immediate feedback of seeing commands being run and know when to use the next command. This is not the typical way you will analyze your own data. Friday's tutorial will deal with the queue system.

While stampede2 is tremendously powerful and will greatly speed up your analysis, it doesn't have much in the way of a GUI (graphical user interface). The lack of a GUI means it can't visualize graphs or other meaningful representations of our data that we are used to seeing. In order to do these types of things, we have to get our data off of stampede2 and onto our own computers. This course uses the scp ("secure copy command") exclusively to move files back to your local computer, as mentioned there are other programs that can be configured to more easily transfer files back and forth as you progress in your analysis.

Transferring files to and from stampede2 with scp

When this class was taught in person, it was helpful to have a small set of steps on transferring files between stampede2 and your local computer which tended to give people problems. The idea being that some problems on the first day would eventually work themselves out through the week as the SCP command was repeatedly used. Given how zoom makes it more difficult for problems to be identified, this tutorial has been moved to its own tutorial page so that it can be more referenced more easily when files are to be transferred in future tutorials. for now, focus on transferring the README file from the BioITeam. Once done with the transfer tutorial come back to this page to install a few extra programs and learn about the module system.

scp tutorial page.

Moving beyond the preinstalled commands on TACC

If (or when) you looked at what our edits to the .bashrc file did, you would have seen that section 1 has a series of "module load XXXX" commands, and a promise to talk more about them later. I'm sure you will be thrilled to learn that now is that time... As a "classically trained wet-lab biologist" one of the most difficult things I have experienced in computational analysis has been in installing new programs to improve my analysis. Programs and their installation instructions tend (or appear) to be written by computational biologists in what at times feels like a foreign language, particularly when things start going wrong. Here we will discuss 3 ways of accessing new commands/programs/scripts and explain their benefit. This is an incomplete list of ways to install new programs to use, but is meant to be a good working example that you can adapt to install other programs in your future work.

1. TACC modules

Modules are programs or sets of programs that have been set up to run on TACC. They make managing your computational environment very easy. All you have to do is load the modules that you need and a lot of the advanced wizardry needed to set up the linux environment has already been done for you. New commands just appear.

To see all modules available in the current context, type:

list all modules

 module avail

Remember you can hit the "q" key to exit out of the "more" system, or just keep hitting return to see all of the modules available. The "module avail" command is not the most useful of commands if you have some idea of what you are looking for. For example imagine you want to align a few million next generation sequencing reads to a genome, but you don't know what your options are. You can use the following command to get a list of programs that may be useful:

List all modules containing a particular term

module keyword alignment

Note that this may not be an inclusive list as it requires the name of the program, or its description to contain the word "alignment". Looking through the results you may notice some of the programs you already know and use for aligning 2 sequences to each other such as blast. Try broadening your results a little by searching for "align" rather than "alignment" to see how important word choice is. When you compare the two sets of results you will see that one of the new results is:

bowtie: bowtie/2.3.2
	Memory-efficient short read (NGS) aligner

This may sound much better, but you still only have limited information about it. To learn more about a particular program, try the following 2 commands:

Get more information on particular module

module spider bowtie

module spider bowtie/2.3.2

In the first case, we see information about what versions of bowtie stampede2 has available for us, but really that is just the same information as we had from our previous search. This can be particularly useful when you know what program you want to use but don't know what versions are available. In the second case we now have more detailed information about the particular version of interest including websites we can go to to learn more about the program itself.

Once you have identified the module that you want to use, you install it using the following command:

 module load bowtie/2.3.2

Using the version numbers for module commands

While not always strictly necessary, using the version number (in this case "/2.3.2") is a very good habit to get into as it controls what version is to be loaded. In this case the because there are 2 very different versions available (2.3.2 and 1.2.1.1) module load bowtie will actually throw an error which tells you to use the module spider command to figure out how to correctly load the module.

While it is tempting to only use "module load name" without the version numbers, using the version numbers can help keep track of what versions were used for referencing in your future publications, and make it easier to identify what went wrong when scripts that have been working for months or years suddenly stop working (ie TACC changed the default version of a program you are using).

Since the module load command doesn't give any output, it is often useful to check what modules you have installed with either of the following commands:

module list
module list bowtie

The first example will list all currently installed modules while the second will only list modules containing bowtie in the name. If you see that you have installed the wrong version of something, a module is conflicting with another, or just don't feel like having it turned on anymore, use the following command:

 module unload bowtie

You will notice when you type module list you have several different modules installed already. These come from both TACC defaults (TACC, linux, etc), and several that are used so commonly both in this class and by biologists that it becomes cumbersome to type "module load python3" all the time and therefore we just have them turned on by default by putting them in our profile to load on startup. As you advance in your own data analysis you may start to find yourself constantly loading modules as well. When you become tiered of doing this (or see jobs fail to run because the modules that load on the compute nodes are based on your .bashrc file plus commands given to each node), you may want to add additional modules to your .bashrc file. This can be done using the "nano .bashrc" command from your home directory.

2. Downloading from the web directly to TACC

When files are hosted online as direct downloads, you can use the wget (Web get) command to skip your local computer and download the file directly to TACC. Typically this makes use of the "Copy Link Address" option when you right click on a link in a web browser that would otherwise start a download to your computer.

Here we will download the installation file for miniconda (which we will use in the next section and throughout the course) using both scp and wget to compare and contrast their functionality.

Using wget.

In a new browser or tab navigate to https://docs.conda.io/en/latest/miniconda.html and right click on the "Miniconda3 Linux 64-bit" in the linux installers section and choose copy link address.

Using the mkdir command to create a folder named 'src' inside of your $WORK2 directory

cd $WORK2
mkdir src
cd src

Use the wget command to download the linux installer directly to your current directory

wget https://repo.anaconda.com/miniconda/Miniconda3-py39_4.9.2-Linux-x86_64.sh

You should see a download bar showing you the file has begun downloading, when complete the ls command will show you a new compressed file named 'Miniconda3-py39_4.9.2-Linux-x86_64.sh'

Using scp.

This is not necessary if you followed the wget commands above. Again In a new browser or tab you would navigate to https://docs.conda.io/en/latest/miniconda.html but instead of right clicking on the "Miniconda3 Linux 64-bit" in the linux installers section and choosing copy link address you would simply left click and allow the file to download directly to your browser's Downloads folder. Using information from the SCP tutorial you would then transfer the local 'Miniconda3-py39_4.9.2-Linux-x86_64.sh' file to the stampede2 remote location '$WORK2/src'.

Given that the wget command doesn't involve having to use MFA, or the somewhat cumbersome use of 2 differnt windows, and is subject to many fewer typos, hopefully you see how wget is preferable provided left clicking on a link directly downloads a file.

Finishing conda installation, and

Regardless of what method you chose to use, the following set of commands will work to install conda. For later reference, if you are planning to install miniconda on other systems or your local laptop, the 'regular installation' links on this link may be useful.

The following command is then used to install miniconda

bash Miniconda3-py39_4.9.2-Linux-x86_64.sh
logout
#log back in using the ssh command. 
conda config --set auto_activate_base false

Following the installation prompts you will need to:

hit enter to page through the license agreement
enter 'yes' to agree to said license agreement
enter to confirm the default installation location
enter 'yes' to initialize Miniconda3 by running conda init?

as per the directions at the end of the installation process, logout, log back in, and disable conda base environment being activated

logout
#log back in using the ssh command. 
conda config --set auto_activate_base false

For help with the ssh command please refer back to Windows10 or MacOS tutorials. If you log out and back in 1 more time, what do you notice is different?

The first time you logged back in, your prompt should have looked like this:

(base) tacc:~$

The second time you logged back in, your prompt should go back to looking like it did before you installed conda:

tacc:~$

If your prompt is different, please get the instructor's attention.

Setting up your first environment

Now that you have installed conda, we want to get started with our first environment. More information about environments and their purpose can be found here, but for now we will just think about them as different sets of programs and relevant dependencies being installed together.

using the conda create command, make a new environment named "GVA2021", and activate it

conda create --name GVA2021
# enter 'y' to proceed
conda activate GVA2021

This will once again change your prompt. This time the expected prompt is:

Again if you see something different, you need to get the instructors attention. For the rest of the course, it is assumed that your prompt will start with (GVA2021) if not, remember that you need to use the conda activate GVA2021 command to enter the environment.

3. Using miniconda on TACC

The anaconda or miniconda interfaces to the conda system is becoming increasingly popular for controlling one's environment, streamlining new program installation, and tracking what versions of programs are being used. A comparison of the two different interfaces can be found here. The deciding factor on which interface we will use is hinted at, but not explicitly stated in the referenced comparison: TACC does not have a GUI and therefore anacondaa will not work, which is why we installed miniconda above.

Similar to the module system that TACC uses, the "conda" system allows for simple commands to download required programs/packages, and modify environmental variables (like $PATH discussed above). Two huge advantages of conda over the module system, are: #1 instead of relying on the employees at TACC to take a program and package it for use in the module system, anyone (including the same authors publishing a new tool they want the community to use) can create a conda package for a program; #2 rather than being restricted to use on the TACC clusters, conda works on all platforms (including windows and macOS), and deal with all the required dependency programs in the background for you.

Conda environments in the instructors work

In my own work, I recently remarked to my PI that "I wish I had started using this 5 years ago", and was reminded that "it didn't exist 5 years ago, at least in its current super usable and popular format". It is entirely possible that future classes will be taught with only minimal references to the TACC module system, and this years course will feature far fewer than any previous year.

While you may be thinking that since the conda system can work on your personal computer, you may want to just work on your personal computer for the duration of this class and ignore all the ssh commands and working remotely. This is strongly not advised. While you would be able to use the same programs in both instances (in most cases), the tutorials are developed with the speed of the stampede2 system in mind and attempt to minimize "waiting for something to finish" to how long it takes someone to read through the next block of text on the tutorial with some exceptions. If you were to do these tutorials on your personal computer, the timing would significantly increase and it would be difficult to keep up with the rest of the class.

In Friday's lecture I will explain why installing and using conda on your local computer is still a good idea and how I am currently using it in conjuncture with TACC.

In the next tutorial we will start accessing the quality of some NGS reads using the fastqc program. Before we can use it, we must install it. Similar to the module system described above, to install a program via conda, we need 3 things:

Tell bash we want to use the conda program.
Tell conda we want to install a new program.
Name the program we want to install.

attempt to install the fastqc program using conda

conda activate GVA2021 
conda install fastqc

If you have already activated your GVA2021 environment, the first line will not do anything, but if you have not, you will see your promt has changed to now say (GVA2021) on the far left of the line. As to the second command, like we saw with the module system above, things aren't quite this simple. In this particular case, we get a very helpful error message that can guide our next steps:

PackagesNotFoundError: The following packages are not available from current channels:

  - fastqc

Current channels:

  - https://repo.anaconda.com/pkgs/main/linux-64
  - https://repo.anaconda.com/pkgs/main/noarch
  - https://repo.anaconda.com/pkgs/r/linux-64
  - https://repo.anaconda.com/pkgs/r/noarch

To search for alternate channels that may provide the conda package you're
looking for, navigate to

    https://anaconda.org

and use the search bar at the top of the page.

More information about "channels" can be found here. By the end of this course you may find that the 'bioconda' channel is full of lots of programs you want to use, and may choose to permanently add it to your list of channels so the above command conda install fastqc and others used in this course would work without having to go through the intermediate of searching for the specific installation commands, or finding what channel the program you want is in. Information about how to do this, as well as more detailed information of why it is bad practice to go around adding large numbers of channels can be found here.

For now, use the error message you saw above to try to install the fastqc program yourself.

If you are having trouble finding the fastqc page on anaconda, the answer is here, as well as a description of the most likely problem you encountered.

https://anaconda.org/bioconda/fastqc

If you were unable to find this page, the most likely error you entered fastqc into the search box, and you recognized that 360,000+ downloads was likely the program you wanted, you clicked the first bit of hyperlink you found which took you to the bioconda page instead of to the fastqc program. Personally, I think the entire box should be clickable to send you the program page, but nobody has asked me.

Click here if you are unsure what command to use to install fastqc, or want to check your understanding

conda install -c bioconda fastqc

While there are two other possible commands listed, I tend to always start with the simplest command and work my way from there. The other two commands deal with accessing specific labels/versions of the program.

If all goes well, the installation command should give you the following output with you answering "y" when prompted if you actually want to install the packages:

The following packages will be downloaded:

    package                    |            build
    ---------------------------|-----------------
    fastqc-0.11.9              |       hdfd78af_1         9.7 MB  bioconda
    font-ttf-dejavu-sans-mono-2.37|       h6964260_0         335 KB
    ------------------------------------------------------------
                                           Total:        10.0 MB

The following NEW packages will be INSTALLED:

  fastqc             bioconda/noarch::fastqc-0.11.9-hdfd78af_1
  font-ttf-dejavu-s~ pkgs/main/noarch::font-ttf-dejavu-sans-mono-2.37-h6964260_0
  openjdk            pkgs/main/linux-64::openjdk-8.0.152-h7b6447c_3


Proceed ([y]/n)? y


Downloading and Extracting Packages
fastqc-0.11.9        | 9.7 MB    | ####################################################################################################################################################################################### | 100% 
font-ttf-dejavu-sans | 335 KB    | ####################################################################################################################################################################################### | 100% 
Preparing transaction: done
Verifying transaction: done
Executing transaction: done

There are three commonly used methods to verify you have a given program installed. You should try all three in order for the fastqc program:

The 'which' command can be used to search your $PATH variable for a command with a specific name, and return the location the command is stored in
```
which fastqc
```
Many commands accept an option of '--version' to simply access the program and return what version of the program is installed
```
fastqc --version
```
Nearly all commands/programs accept "h" or "-help" options to give you basic information about how the command or program works
```
fastqc --help
```

Throughout the course, you will routinely use the above 3 commands to make sure that you have access to a given program, that it is the correct version, and to get an idea of how to construct commands to perform a given analysis step. For now, be satisfied that if you get output that is not the following that you have correctly installed fastqc. In the next tutorial we will actually use fastqc. Examples of output you do not want to see to the above commands:

/usr/bin/which: no fastqc in (<large list of directories specific to your TACC account>)
-bash: fastqc: command not found
-bash: fastqc: command not found

Github – an additional common method of getting files onto TACC

This is about using the git clone command. Git is a command often used for collaborative program development or sharing of files. Some groups also put the programs or scripts associated with a particular paper on a github project and publish the link in their paper or on their lab website. Github repositories are a great thing to add to a single location in your $WORK2 directory.

Here we will clone the github repository for the E. coli Long-Term Evolution Experiment (LTEE) originally started by Dr. Richard Lenski. These files will be used in some of the later tutorials, and are a good source of data for identifying variants in NGS data as the variants are well documented, and emerge in a controlled manner over the course of the evolution experiment. Initially cloning a github repository as exceptionally similar to using the wget command to download the repository, it involves typing 'git clone' followed by a web address where the repository is stored. As we did for installing miniconda, with wget we'll clone the repository into a 'src' directory inside of $WORK2.

Using the mkdir command to create a folder named 'src' inside of your $WORK2 directory

cd $WORK2
mkdir src
cd src

If you already have a src directory, you'll get a very benign error message stating that the folder already exists and thus can not be created.

In a web browser navigate to github and search for 'LTEE-Ecoli' in the top right corner of the page. The only result will be for barricklab/LTEE-Ecoli; click the green box for 'clone or download' and either control/command + C on the address listed, or click the clipboard icon to copy the repository address. This image may be helpful if you are are having trouble locating the green box.

Once you have copied the address and are in the $WORK2/src directory clone the repository with 'git clone'

git clone https://github.com/barricklab/LTEE-Ecoli.git

You will see several download indicators increase to 100%, and when you get your command prompt back the ls command will show a new folder named 'LTEE-Ecoli' containing a set of files. If you don't see said directory, or can't cd into that directory let the instructor know.

pip

In previous years, the pip installation program was used to install a few programs. While those programs will be installed through conda this year, the link here is provided to give a detailed walk through of how to use pip on TACC resources. This is particularly helpful for making use of the '--user' flag during the installation process as you do not have the expected permissions to install things in the default directories.

This concludes the the linux and stampede2 refresher/introduction tutorial.

Genome Variant Analysis Course 2021 home.

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