An introduction to Linux for bioinformatics

Paul Stothard

April 6, 2010

Contents
1 Introduction 2

2 Getting started 3
2.1 Obtaining a Linux user account . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 How to access your account from Mac OS X . . . . . . . . . . . . . . . . 3
2.3 How to access your account from Windows . . . . . . . . . . . . . . . . 4
2.3.1 Using PuTTY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3.2 Using Cygwin . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.4 Your home directory . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.5 Some basic commands . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.6 More commands and command-line options . . . . . . . . . . . . . . . . 8

3 Transferring files to and from your Linux account 10

3.1 Transferring files between Mac OS X and Linux . . . . . . . . . . . . . . 10
3.1.1 Using the Terminal application . . . . . . . . . . . . . . . . . . . 10
3.1.2 Using Fugu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.2 Transferring files between Windows and Linux . . . . . . . . . . . . . . 12
3.3 File transfer exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1
1 INTRODUCTION 2

4 Understanding Linux 14
4.1 Paths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2 Typing shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.3 File permissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.4 Redirecting output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.5 Piping output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.6 Using locate and find . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.7 Working with tar and zip files . . . . . . . . . . . . . . . . . . . . . . . . 19
4.8 Wildcard characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.9 The grep command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.10 The root user . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.11 The Linux file system . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.12 Editing a text file using vi . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5 Bioinformatics tools 25
5.1 EMBOSS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.2 Using ClustalW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5.3 Performing a BLAST search . . . . . . . . . . . . . . . . . . . . . . . . 27
5.4 Performing a BLAT search . . . . . . . . . . . . . . . . . . . . . . . . . 29

6 Streamlining data analysis 31

6.1 The .bashrc file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6.2 Modifying $PATH and other environment variables . . . . . . . . . . . . 33
6.3 Writing a simple BASH script . . . . . . . . . . . . . . . . . . . . . . . 34

7 Summary 35

1 Introduction
Linux is a free operating system for computers that is similar in many ways to propri-
etary Unix operating systems. The field of bioinformatics relies heavily on Linux-based
computers and software. Although most bioinformatics programs can be compiled to run
2 GETTING STARTED 3

on Mac OS X and Windows systems, it is often more convenient to install and use the
software on a Linux system, as pre-compiled binaries are usually available, and much of
the program documentation is often targeted to the Linux user. For most users, the sim-
plest way to access a Linux system is by connecting from their primary Mac or Windows
machine. This type of arrangement allows several users to run software on a single Linux
system, which can be maintained by an experienced systems administrator. Although
there are other ways for inexperienced users to become familiar with Linux (installing
Linux on a PC, using a Live CD to run Linux, running a Linux virtual machine), this doc-
ument focuses on accessing a remote Linux machine using a text-based terminal. Many
powerful statistics and bioinformatics programs can be run in this manner.

2 Getting started
2.1 Obtaining a Linux user account
To gain access to a Linux-based machine you first need to speak a system administrator
(sysadmin) to obtain a user name, hostname (or IP address), and password. Once you
have this information you can access your account. Alternatively, you can run a Linux
virtual machine on top of your present operating system. If you are using a Linux virtual
machine you can skip ahead to the section called “Your home directory” after launching
the machine and opening a BASH terminal).

2.2 How to access your account from Mac OS X

Mac OS X includes a Terminal application (located in the Applications >> Utilities
folder), which can be used to connect to other systems (Figure 2.2). Launch Terminal
and at the command prompt, enter ssh user@hostname, replacing “user” and “hostname”
with the user name and machine name you have been assigned. Press enter and you should
be prompted for a password. The first time you try to connect to your account, a warning
message may appear. Ignore the message and allow the connection to be established.
2 GETTING STARTED 4

Figure 1: The Mac OS X Terminal application.

2.3 How to access your account from Windows

2.3.1 Using PuTTY

On Windows systems you can use a variety of programs to connect to a Linux system.
PuTTY1 is a free program already available on many Windows machines, including most
of the student-accessible computers at the University of Alberta. If PuTTY is not installed
you can download an executable from the PuTTY website. Launching PuTTY will open
a configuration window resembling the one shown in Figure 2.3.1. Click Session in the
left pane and then in the Host Name (or IP address) text box enter user@hostname,
replacing “user” and “hostname” with the user name and machine name you have been
assigned. Click Open to establish a connection with the remote system. The first time
you try to connect to your account, a warning message may appear. Ignore the message
and allow the connection to be established. A new terminal window will open, and you
will be prompted for a password.
1 http://www.chiark.greenend.org.uk/~sgtatham/putty/
2 GETTING STARTED 5

Figure 2: The PuTTY Telnet/SSH client running on Windows.

2.3.2 Using Cygwin

Cygwin2 is a program that can be installed on Windows to provide a Linux-like environ-

ment. An advantage of using Cygwin is that you gain access to a lot of the standard Linux
utilities, without having to connect to another computer. For example, after launching
Cygwin, you can use many of the Linux commands described elsewhere in this guide,
such as pwd and mkdir. You can also edit and run BASH scripts and Perl programs, and
you can manage software repositories using subversion (these topics, apart from BASH
scripts, are not covered in the tutorial). If you choose to install Cygwin, you can use it
to access your remote Linux account by entering ssh user@hostname (replace “user”
and “hostname” with the user name and machine name you have been assigned) on the
command line (Figure 2.3.2).
2 http://www.cygwin.com/
2 GETTING STARTED 6

Figure 3: Cygwin, a Linux-like environment for Windows.

2.4 Your home directory

Once you have successfully signed in to your account, you can start exploring the di-
rectory structure of the remote computer. In your terminal you should see a command
prompt, which usually consists of your user name and the name of the computer on which
your account resides. For example, on my system the command prompt I see is the fol-
lowing:

paul@agfor $

The examples in this guide will include $ as the command prompt to illustrate that the
commands should be entered into the terminal, and to differentiate the entered commands
from the output they return.
When you sign in you will be located in your home directory. To see where this
directory is located in the file system, use the pwd command:

$ pwd
/home/paul
2 GETTING STARTED 7

In this case the output indicates that the current working directory is paul and that the
paul directory is located inside of the home directory. When you enter pwd after signing
in it should show that you are in a directory with the same name as your user name. The
home directory is located inside the / directory, which is also called the “root” directory.
If at any time you want to return to your home directory, use the cd ˜ command. As
you will see, your home directory is where you can create and delete your own files and
directories.

2.5 Some basic commands

As in the previous example, you can see which directory is the current directory by using
the pwd command:

$ pwd
/home/paul

To change to a different directory, use the cd command (cd means change directory):

$ cd /home
$ pwd
/home

Now you should be in the home directory. To see what is inside of this directory, use
the ls command (ls stands for list):

$ ls
craig joseph lost+found nmrq paul svn

The folders you see will likely differ from these. Now switch back to your home
directory:

$ cd ˜

In addition to real directory names, you can supply certain alias terms to the cd com-
mand. One of these is the ˜ character, which represents your home directory. Another is
.., which represents the directory above the current directory. Try the following:
2 GETTING STARTED 8

$ cd ˜
$ cd ..
$ ls
craig joseph lost+found nmrq paul svn
$ pwd
/home
$ cd ˜
$ pwd
/home/paul

As you can see, cd, ls, and pwd can be used to explore the Linux file system. Don’t
forget that you can always use cd ˜ to move back to your home directory.

2.6 More commands and command-line options

The pwd, ls, and cd commands point to programs on the Linux system that perform
specific tasks and return output. When you enter the commands, the system runs the
corresponding program for you. There are many other useful commands available.
To see which user you are signed in as, use the whoami command:

$ whoami
paul

To see who else is signed in to the same system, use the who command:

$ who
paul :0 2006-09-15 16:54

To see the current time and date, using the date command:

$ date
Tue Sep 19 16:14:23 MDT 2006

To create your own directories use the mkdir (make directory) command:
2 GETTING STARTED 9

$ cd ˜
$ mkdir sequences
$ cd sequences
$ mkdir proteins
$ cd proteins
$ pwd
/home/paul/sequences/proteins

To create a new file, use the touch command:

$ cd ˜ /sequences/proteins/
$ touch my_sequence.txt
$ ls -l
-rw-r--r-- 1 paul users 0 Sep 19 15:56 my_sequence.txt

As you will see later, there are other ways to create new files (output redirection for
example). In the last command above, the -l (a lowercase “L”, not a “1”) option was used
with the ls command. The -l indicates that you want the directory contents shown in the
“long listing” format. Most commands accept a variety of options. To see which options
are available for a certain command, you can try typing man followed by the command
name (man ls for example to see what options are available for the ls command), or the
command name followed by --help (ls --help for example). One of these two methods
usually provides information. To see what options can be used with ls, enter man ls.
To get through the list of options that appears, keep pressing Space until the page stops
scrolling, then enter “q” to return to the command prompt:

$ man ls

To delete a file, use the rm (remove) command:

$ cd ˜ /sequences/proteins/
$ ls
my_sequence.txt
$ rm my_sequence.txt
$ ls
$

To remove a directory, use the rmdir (remove directory) command:

3 TRANSFERRING FILES TO AND FROM YOUR LINUX ACCOUNT 10

$ cd ˜ /sequences/
$ ls
proteins
$ rmdir proteins
$ ls
$

To copy a file, use the cp (copy) command:

$ cd ˜ /sequences
$ touch testfile1
$ ls
testfile1
$ cp testfile1 testfile2
$ ls
testfile1 testfile2

To rename a file, or to move it to another directory, use the mv (move) command:

$ cd ˜
$ touch testfile3
$ mv testfile3 junk
$ mkdir testdir
$ mv junk testdir
$ cd testdir
$ ls
junk

The commands covered so far represent a small but useful subset of the many com-
mands available on a typical Linux system [1].

3 Transferring files to and from your Linux account

3.1 Transferring files between Mac OS X and Linux
3.1.1 Using the Terminal application

Recall that Mac OS X includes a Terminal application (located in the Applications >>
Utilities folder), which can be used to connect to other systems. This terminal can also
3 TRANSFERRING FILES TO AND FROM YOUR LINUX ACCOUNT 11

be used to transfer files, thanks to the scp command.

Try transferring a file from your Mac to your Linux account using the Terminal ap-
plication:

1. Launch the Terminal program.

2. Switch to your home directory on the Mac using the command cd ˜ .

3. Create a text file containing your home directory listing using ls -l > myfiles.txt
(you will learn more about the meaning of > later).

4. Now use the scp command on your Mac to transfer the file you created to your
Linux account. This command requires two values: the file you want to transfer
and the destination. Be sure to replace “user” with your user name, and replace
hostname with the real hostname or IP address of the Linux system you want to
connect to:

$ scp myfiles.txt user@hostname: /

You should be prompted for your user account password. Remember, in the above
example you are running the scp command on your Mac, not from your Linux account.
Now, delete the myfiles.txt file on your Mac, and see if you can use scp to retrieve
the file from your Linux account:

1. In the terminal on your Mac, switch to your home directory using cd ˜ .

2. Delete the myfiles.txt file using rm myfiles.txt.

3. Use the scp command to copy myfiles.txt from your Linux account back to your
Mac. Remember to replace “hostname” and “user” with the appropriate values
when you enter the command:

$ scp user@hostname: /myfiles.txt ./

The above command will prompt you for your Linux account password. Remember
that ./ means “current directory”. This informs the scp program that you would like the
file myfiles.txt in your home directory on the remote system to be copied to the current
directory on your computer.
3 TRANSFERRING FILES TO AND FROM YOUR LINUX ACCOUNT 12

3.1.2 Using Fugu

For users who prefer to use a graphical interface when transferring files between Mac
and Linux, there is the freely available Fugu program.3 To use Fugu, launch the program
and enter the hostname of the computer you wish to connect to in the Connect to text
area, and enter your Linux account name in the Username text area (Figure 3.1.2). Click
Connect to connect to the remote system. You will be prompted for your Linux account
password. Once you are connected to your Linux account you should be able to copy files
between systems by dragging files and folders.

Figure 4: Fugu, a graphical SSH and SCP tool for Mac OS X.

3.2 Transferring files between Windows and Linux

The simplest way to transfer files between Linux and Windows is to use the freely avail-
able WinSCP program.4 WinSCP is already installed on many Windows systems, includ-
3 http://rsug.itd.umich.edu/software/fugu/
4 http://sourceforge.net/projects/winscp/
3 TRANSFERRING FILES TO AND FROM YOUR LINUX ACCOUNT 13

ing those provided for student use at the University of Alberta. To use WinSCP, launch
the program and enter the appropriate information into the Host name, User name, and
Password text areas (Figure 3.2). Click Login to connect to the remote system. Once you
are connected you should be able to transfer files and directories between systems using
the simple graphical interface.

Figure 5: WinSCP.

3.3 File transfer exercise

To test your ability to transfer files to your Linux account, download the following file to
your Mac or Windows system using a web browser:
http://www.ualberta.ca/~stothard/downloads/sample_sequences.zip
Once the file has been downloaded to your system, use the file transfer methods out-
lined above to transfer the file to your Linux account. Be sure to perform this exercise, as
the sample_sequences.zip file will be used later on in this tutorial.
4 UNDERSTANDING LINUX 14

4 Understanding Linux
4.1 Paths
Many Linux commands require that you supply a directory or file name. For example,
if you enter the command touch without specifying a file name, an error message is
returned:

$ touch
touch: missing file operand
Try ‘touch --help’ for more information.

Directory and file names like “testdir” and “my_sequences.txt” are called relative
paths, since they specify the location of the file or directory in relation to the current
working directory. For example, if you are located in your home directory, the command
mkdir some_dir will create a directory called “some_dir” in your home directory.
In the following example two directories are created, and cd is used to switch to
“dir2” so that a new file can be created there using touch:

$ cd ˜
$ mkdir dir1
$ cd dir1
$ mkdir dir2
$ cd dir2
$ touch somefile

Alternatively, by specifying the names of the directories in the paths, the same direc-
tory structure and file can be created without using cd to switch to the new directories (in
this example the rm -rf dir1 is used to remove the existing “dir1” and all of its contents):

$ cd ˜
$ rm -rf dir1
$ mkdir dir1
$ mkdir dir1/dir2
$ touch dir1/dir2/somefile
4 UNDERSTANDING LINUX 15

Relative paths can use .. to refer to the parent directory (i.e. the directory above the
current directory). In the following example, .. is used twice in the path passed to touch,
to create a file called “somefile2” in the directory two levels up from the current directory
(which in this case is your home directory):

$ cd ˜
$ cd dir1/dir2
$ touch ../../somefile2

In contrast to relative paths, absolute paths specify the name of a file or directory in
relation to the root (top) directory. Absolute paths always begin with a forward slash (the
forward slash at the beginning of a path represents the root directory). In the following
example, an absolute path is used to instruct ls to list the contents of the “etc” directory,
which is used by Linux to store configuration files (i.e. the “etc” directory located in the
root directory):

$ ls /etc

Absolute paths are useful because their interpretation doesn’t depend on which direc-
tory is the current working directory.

4.2 Typing shortcuts

There are some useful tricks to save typing on the command line. One is to use Tab
to complete a command name or a file name. When you press Tab, the system will try
to complete the text you have partially entered, based on which characters are found in
known command names and file names. If there are multiple possible matches, the system
will not guess the matching text, however, if you press Tab twice a list of the possible
matches will be given. This method of using Tab on the command line is called “Tab
completion”.
Try the following:

$ cd ˜
$ mkdir a_new_directory

Now begin by typing “cd a” and press Tab instead of entering the full directory name.
The full name should appear automatically.
4 UNDERSTANDING LINUX 16

To see what happens when there are multiple possibilities for a command or filename,
begin by typing “mk” and press Tab twice. You should see a list of commands starting
with the letters “mk”.
Another useful command-line shortcut is to use the up and down arrow keys to scroll
through commands you have recently used (ls is sometimes not stored in this list since it
is easy to type). If you scroll to a command you want to use again, press Enter to execute
the command.

4.3 File permissions

Linux uses file permissions to prevent accidental file deletion and to protect data from
being manipulated by others. Each file and directory is associated with three types of
file permissions: “user”, “group”, and “other” permissions. The meanings of these terms
are discussed below. To see the permission information for a directory or file, use the ls
command with the -l option. Try the following set of commands:

$ cd ˜
$ mkdir somedir
$ cd somedir
$ touch somefile
$ mkdir anotherdir
$ ls -l
$ mkdir anotherdir
drwxr-xr-x 2 paul users 4096 Sep 19 16:20 anotherdir
-rw-r--r-- 1 paul users 0 Sep 19 16:18 somefile

The permission information for the directory anotherdir and the file somefile is given
in the file listing. The first column is the file type and the file permissions (drwxr-xr-x for
example). The third column is the owner of the file or directory (paul in this example), and
the column after that is the group that owns the file (users). A group is simply a collection
of users (groups are created by the sysadmin). A group can be used, for example, to allow
different users to collaborate on a particular set of files, while protecting the files from
editing by users not in the designated group.
As mentioned above, the first column contains the file type and permission informa-
tion. The anotherdir entry begins with d, indicating it is a directory. The somefile entry
begins with a -, which indicates that it is a file. The first three letters after the file type
letter are the permissions for the user who owns the file or the directory. The next three
4 UNDERSTANDING LINUX 17

letters are the permissions for the group that owns the file or directory. The final three
letters define the access permissions for other users. The meanings of the letters are the
following:

r (read permission) indicates that the file can be read. In the case of a directory this
means that the contents of the directory can be listed.
w (write permission) indicates that the file can be modified. In the case of a directory
this means that the contents of the directory can be changed (i.e. create new files,
delete existing files, or rename files).
x (execute permission) indicates that the file can be executed as a program. In the case
of a directory, the execute attribute means you have permission to enter a directory
(i.e. make it the current working directory).

Returning to the example above, the permissions for somefile are rw-r--r--. This
series of characters means that the owner of the file has the read and write permissions
(rw-). Other users in the group users can read the file but not write or execute it (r--).
Similarly, all other users can only read the file (r--).
To change file permissions, use the chmod command. For example, the following
changes the permissions associated with somefile so that only the owner of the file (paul
in this case) can read it (along with the root user, who will be discussed later):

$ chmod go-r somefile

$ ls -l
drwxr-xr-x 2 paul users 4096 Sep 19 16:20 anotherdir
-rw------- 1 paul users 0 Sep 19 16:18 somefile

The go-r portion of the above chmod command means “from the group (g) and other
(o) permission sets take away the read permission (r).”
To allow everyone to read the file somefile you could modify the permissions using
the following:

$ chmod a+r somefile

The a in the above command means “all users”. To refer to different types of users
separately, use u (user who owns the file), g (group that owns the file), and o (other users).
To see how permissions protect files and directories, try to delete the /etc directory,
which contains important system files:
4 UNDERSTANDING LINUX 18

$ rmdir /etc
rmdir: /etc: Permission denied

Although the full rationale behind permissions may not be apparent to you at this
time, it is important to remember that they do exist and that they control who can do
what to specific files and directories. These permissions also automatically apply to any
program you run on a Linux system. For example, if you run a program that attempts to
copy a file for which you do not have the read permission, the program will be denied
access to the file and it will not be able to make the copy.

4.4 Redirecting output

Many commands return textual output (i.e. the ls command) that is written to the terminal
window. You can redirect the output to a file instead, by providing a filename preceded
by the ‘>’ character. Use the following to create a file called my_listing.txt containing
the output of the ls -l command:
$ cd ˜
$ ls -l > my_listing.txt

To examine the contents of the my_listing.txt file, use the more command:
$ more my_listing.txt

Note that more is useful for viewing the contents of a file, one page at a time. To
advance a page press Space. To return to the command prompt, enter “q”.
Output redirection is useful for commands that return a lot of output. It is often used
so that the output can be used or processed at a later time.

4.5 Piping output

With pipes, which are represented by the | character, it is possible to send the output of
one program to another program as input. Consider the following command:
$ cat /etc/services | sort | tail -n 10

The above example uses the cat command to extract the text from the file /etc/ser-
vices. The text is then piped to the sort program, which sorts the lines alphanumerically.
Finally, the sorted text is piped to the tail program, which displays the last 10 lines of the
text. Piping provides a convenient way to perform a series of data manipulations.
4 UNDERSTANDING LINUX 19

4.6 Using locate and find

Occasionally you may want to search a Linux system for a particular file. A simple way
to do this is to use the locate command:

$ locate blastall
/usr/local/wublast/wu-blastall
/usr/local/blast/bin/blastall
/usr/local/blast/doc/blastall.html

In this example the locate program was used to search for files or directories matching
the name “blastall” (blastall is a program that can be used to search DNA and protein
sequence databases). The locate program does not actually search the Linux file system.
Instead it uses a database that is usually updated daily. By using an optimized database,
locate is able to find items quickly, however you may not obtain results for files recently
added to the system.
Another tool for searching for files of interest is find. This command accepts several
options for specifying the types of files you want. For example, you can search for files
based on name, size, owner, modification date, and permissions.
To find files in the /etc directory that end with “.conf” and that are more than 10
kilobytes in size you could use this command:

$ find /etc -name “*.conf” -size +10k

4.7 Working with tar and zip files

Sometimes you may want to compress a file or a group of files into a zip file or a tar file.
Alternatively, you may have a tar or zip file you wish to extract. Note that tar files, which
are similar to zip files, are frequently used on Linux-based systems.
To explore the zip and tar commands, first, create a text file using the following:

$ cd ˜
$ wget www.google.ca -O google.html

The wget command can be used to download web-based files. In this example it is
used to write the google homepage to a file called google.html.
To create a zip file of google.html use the zip command:
4 UNDERSTANDING LINUX 20

$ zip -r google.zip google.html

The -r (for recursive) is not necessary in the above example. However, it is needed if
you want to if you want to zip the contents.
To extract this zip file use the unzip command:

$ unzip google.zip

To create a tar file use the tar command:

$ tar -cvf google.tar google.html

The -c option tells tar that you would like to create an archive (as opposed to extract
one), and -v indicates that you want the tar program to be verbose (i.e. print warnings
and progress messages). -f is used to specify the name of the archive you would like to
create (google.tar).
To extract this tar file use the tar command again, this time with the -x (extract)
option:

$ tar -xvf google.tar

To create a tar file that is also compressed (like a zip file), use the -z option:

$ tar -cvzf google.tar.zip google.html

To extract tar.gz or tar.zip files use tar with the -z option:

$ tar -xvzf google.tar.zip

Note that when you extract a tar or zip file, the tar or zip file is not deleted.
4 UNDERSTANDING LINUX 21

4.8 Wildcard characters

Sign in to your Linux account and locate the sample_sequences.zip file you transferred
to your home directory (see the File transfer exercise section). Extract the file and then
use ls -l to examine the files that are produced:

$ unzip sample_sequences.zip
Archive: sample_sequences.zip
inflating: 16S_rRNA.fasta
inflating: bos_taurus_chromosome_29.fasta
inflating: bos_taurus_insulin_cDNA.fasta
inflating: bos_taurus_p53.fasta
inflating: e_coli.fasta
inflating: felis_catus_p53.fasta
inflating: macaca_mulatta_p53.fasta
inflating: mus_musculus_p53.fasta
inflating: xenopus_laevis_p53.fasta
$ ls -l
-rw-r--r-- 1 paul users 16S_rRNA.fasta
-rw-r--r-- 1 paul users bos_taurus_chromosome_29.fasta
-rw-r--r-- 1 paul users bos_taurus_insulin_cDNA.fasta
-rw-r--r-- 1 paul users bos_taurus_p53.fasta
-rw-r--r-- 1 paul users e_coli.fasta
-rw-r--r-- 1 paul users felis_catus_p53.fasta
-rw-r--r-- 1 paul users macaca_mulatta_p53.fasta
-rw-r--r-- 1 paul users mus_musculus_p53.fasta
-rw-r--r-- 1 paul users sample_sequences.zip
-rw-r--r-- 1 paul users xenopus_laevis_p53.fasta

As you can see, several “.fasta” files were extracted from the sample_sequences.zip
archive. Suppose you want to organize your home directory by placing these new se-
quence files into a single directory. You can do this easily using the * wildcard character.
Try the following:

$ cd ˜
$ mkdir sequences
$ mv *.fasta sequences
4 UNDERSTANDING LINUX 22

The * represents any text. The *.fasta instructs the mv command to move any file
that ends with “.fasta” from the current directory to the sequences directory. The * can be
used will other commands as a simple way to refer to multiple files with similar names.

4.9 The grep command

A useful command for searching the contents of files is grep. grep can be used to look for
specific text in one or more files. In this example you will use grep to examine whether
the fasta files you downloaded contain a properly formatted title line. The title line should
start with the > character, and there should not be any additional > characters in the file.
Try the following command:

$ grep -r ‘>’ sequences

The -r option stands for “recursive” and tells grep to examine all the files inside
the specified directory. The ‘>’ is the text you want to search for, and sequences is the
directory you want to search. For each match encountered, grep returns the name of the
file and the contents of the line containing the match. As you will see when you run the
above command, each file contains a single title line as expected.

4.10 The root user

You may have noticed that when you are signed in to your user account you are unable to
access many of the files and directories on the Linux system. One way to gain access to
these files is to sign in as user root. However, you are unlikely to be given the password for
the root user, since it is usually reserved for sysadmins, so that they install new programs,
create new user accounts, etc. Even sysadmins do not usually sign in as the root user,
since a small mistake when typing a command can have drastic consequences. Instead,
they switch to the root user only when they need to perform a specific task that they are
unable to perform as a regular user.

4.11 The Linux file system

So far you have worked inside your home directory, which is located in /home. You may
wonder what the other directories found on the typical Linux system are used for. Here is
a short description of what is typically stored in the directories:
4 UNDERSTANDING LINUX 23

/bin contains several useful programs that can be used by the root user and standard
users. For example, the ls program is located in /bin.
/boot contains files used during startup.
/dev contains files that represent hardware components of the system. When data is writ-
ten to these files it is redirected to the corresponding hardware device.
/etc contains system configuration files.
/home the user home directories.
/initrd information used for booting.
/lib software components used by many different programs.
/lost+found files saved during system failures are stored here.
/misc for miscellaneous purposes.
/mnt a directory that can be used to access external file systems, such as CD-ROMs and
digital cameras.
/opt usually contains third-party software.
/proc a virtual file system containing information about system resources.
/root the root user’s home directory.
/sbin essential programs used by the system and by the root user.
/tmp temporary space that can be used by the system and by users.
/usr programs, libraries, and documentation for all user-related programs.
/var contains log files and files created during processes such as printing and download-
ing.

To see which of these directories is present on the Linux system you are using, per-
form the following:

$ cd /
$ ls
bin boot data data2 dev etc home initrd lib
lost+found misc mnt opt proc root sbin tmp usr var
4 UNDERSTANDING LINUX 24

4.12 Editing a text file using vi

Sometimes you may want to make changes to a text file while signed in to your Linux
account. There are several programs available for this purpose, one of which is called vi.
vi is somewhat difficult to operate, since you have to use keyboard shortcuts for all the
commands you typically access using menus in other text editing applications. However,
by learning a few key commands you can comfortably edit text files using vi. In the
example below, you will use vi to edit a text file containing multiple DNA sequences.
Many bioinformatics programs, such as clustalw, read in multiple sequences from a
single file. Each sequence in the file usually needs to be in fasta format, as in the following
example:

>seq 1
gatattta
>seq2
attatcc
>seq3
etc

To combine the p53 sequences in your sequences directory into a single file, use the
following:

$ cd ˜ /sequences
$ cat *p53.fasta > all_p53_seqs.fasta

Now examine the all_p53_seqs.fasta file using the more command. In a fasta file
containing multiple sequences, each sequence should have a separate title (titles normally
begin with a > character). In the current all_p53_seqs.fasta file the first sequence record
is missing the >. To edit this sequence’s title, begin by opening the file in vi:

$ vi all_p53_seqs.fasta

Next, press i to enter insert mode. Use the arrows on the keyboard to move the cursor
to top left if it isn’t already there, and then type “>”. Press Esc to leave the insert mode.
To save the changes and quit, type :wq and press Enter. If you had problems editing the
file and wish to quit without saving, press Esc and then type :q! and press Enter.
Use vi to correct the sequence title in the bos_taurus_p53.fasta file too, as we will
be using this file in the future.
5 BIOINFORMATICS TOOLS 25

Note that the goal of this exercise was to introduce you to vi. Usually you will not
need to edit your sequence files in this manner. However, you may find vi useful for
making changes to your .bashrc file and for creating and modifying BASH scripts (both
of these are described below).

5 Bioinformatics tools
5.1 EMBOSS
Now that you have been exposed to several of the built-in Linux commands and the Linux
file system, you are ready to use some third party bioinformatics applications. One of
these applications is called EMBOSS (The European Molecular Biology Open Software
Suite).5 EMBOSS contains several powerful bioinformatics programs for performing
tasks such as sequence alignment, PCR primer design, and protein property prediction
[2]. To see whether EMBOSS is installed on the Linux system you are using, try the
following:

$ which showalign
/usr/local/bin/showalign

showalign is one of the programs included with the EMBOSS package. In the above
command, which is used to look for the showalign program on your PATH (the meaning
of “PATH” is explained in more detail below). If this command returns something like
“/usr/local/bin/showalign”, then EMBOSS is likely installed. If instead it returns “no
showalign in ..”, then talk to your sysadmin.
EMBOSS includes numerous applications. In the following examples you will ex-
plore just a few of them. First, switch to your sequences directory, which should contain
several sequences in fasta format.

$ cd ˜ /sequences

Now, use the EMBOSS transeq program to translate the Bos taurus p53 nucleotide
sequence into a protein sequence (note that the \ below is used to split the command
across multiple lines–when typing the command press Enter after the \ or omit the \ and
type the entire command on one line):
5 http://emboss.sourceforge.net/
5 BIOINFORMATICS TOOLS 26

$ transeq -sequence bos_taurus_p53.fasta \

-outseq bovine_p53_protein

To see the resulting protein sequence use:

$ cat bovine_p53_protein

Next, perform a global sequence alignment of two of the p53 sequences using needle.
Note that when you run this command you will be prompted for some additional informa-
tion. For this example you can press Enter each time you are prompted for information,
to indicate that you would like to use the default program settings:

$ needle macaca_mulatta_p53.fasta \
xenopus_laevis_p53.fasta -outfile alignment

To examine the alignment that is generated use:

$ more alignment

Finally, use the pepstats program to obtain protein statistics for the protein sequence
you created using transeq:

$ pepstats bovine_p53_protein -outfile stats

To examine the output use:

$ more stats

5.2 Using ClustalW

clustalw6 is a powerful sequence alignment program that can be used to generate large
multiple alignments [3]. To see whether clustalw is installed on the Linux system you are
using, use the which command again:

$ which clustalw

6 http://www.ebi.ac.uk/Tools/clustalw2/index.html
5 BIOINFORMATICS TOOLS 27

This command should return the full path to the clustalw program. If it returns “no
clustalw in ..”, talk to your sysadmin.
The clustalw program offers several command-line options for controlling the se-
quence alignment process. To see these options, enter clustalw -options. In the following
example clustalw is used to align the sequences in the all_p53_seqs.fasta file:

$ cd ˜
$ clustalw -infile=sequences/all_p53_seqs.fasta \
-outfile=alignment -align

To view the completed alignment, use more:

$ more alignment

5.3 Performing a BLAST search

BLAST7 is a powerful program for comparing a sequence of interest to large databases
of existing sequences [4]. By identifying related sequences you can gain insight into the
function and evolution of the genes and proteins you are interested. The BLAST pro-
gram can be installed on Windows, Mac, and Linux machines. However, to run BLAST
on your own computer you also need to download the sequence databases you wish to
search. These databases can be very large, and they become outdated quickly, since new
sequences are continually added. For these reasons, many users prefer to submit se-
quences using the web interfaces provided by NCBI. The main drawback of using the
web interface is that you can only submit one sequence at a time. If you have a large
collection of sequences you wish to analyze, this approach can be very time consuming.
To avoid these issues you can use the remote_blast_client.pl program.8 To download
remote_blast_client.pl to your Linux account, use the following command:

$ cd ˜
$ wget http://www.ualberta.ca/˜ stothard/\
downloads/remote_blast_client.zip \
--user-agent=IE

7 http://blast.ncbi.nlm.nih.gov/Blast.cgi
8 Note
that NCBI provides a similar tool, called netblast, which is available at ftp://ftp.ncbi.nih.
gov/blast/executables/LATEST/
5 BIOINFORMATICS TOOLS 28

Now unzip the file you downloaded (don’t forget about Tab completion–you can type
“unzip re” and then press Tab to get the full file name):

$ unzip remote_blast_client.zip

Change the permissions on the remote_blast_client.pl file so that you can execute it:

$ chmod u+x remote_blast_client/remote_blast_client.pl

Now use the remote_blast_client.pl program to perform a BLAST search for each
of the sequences in the all_p53_seqs.fasta file you created in your sequences directory:

$ cd ˜
$ ./remote_blast_client/remote_blast_client.pl \
-i sequences/all_p53_seqs.fasta \
-o blast_results.txt -b blastn -d nr

The -i option in the previous command is used to specify which file contains the
sequences you wish to submit and the -o is used to specify where you want the results
saved. The -b and -d options are used to specify which BLAST program and database
you want to use. The BLAST search may take a few minutes to complete. As the script
runs it will give you information about what it is doing. If you wish to cancel the search,
use Ctrl-C. Note that Ctrl-C can be used to return to the command prompt for many other
programs too.
Once the program has stopped running you can examine the results using more. Note
that this script returns results in a compact tabular format that does not include alignments.
To perform a BLAST search without relying on NCBI’s servers you can use the
blastall program. First you need to format a sequence database using the formatdb pro-
gram. The following command formats the genomic sequence of E. coli (which was
included in the sample_sequences.zip file) so that it can be used as a BLAST database:

$ cd ˜
$ formatdb -i sequences/e_coli.fasta -p F

To see what the -i and -p options are used to indicate, try the following:

$ formatdb --help
5 BIOINFORMATICS TOOLS 29

You are now ready to search the E. coli database using any fasta or multi-fasta se-
quence as the query. The following command compares the two 16S rRNA sequences in
16S_rRNA.fasta to the E. coli genome:

$ cd ˜
$ blastall -i sequences/16S_rRNA.fasta \
-d sequences/e_coli.fasta \
-p blastn -o local_blast_results.txt

To examine the results, use more:

$ more local_blast_results.txt

5.4 Performing a BLAT search

BLAT9 is a powerful tool for searching for sequences of interest in a completed genome
or proteome. It is faster than BLAST, and is much better at aligning cDNA sequences to
genomic sequence, because it looks for splice site consensus sequences [5]. BLAT is less
sensitive than BLAST, and is thus most useful for comparisons involving sequences from
the same species (e.g. a human cDNA vs. the human genome) or closely related species
(e.g. a human cDNA vs. the chimp genome).
The following command uses the blat program to compare a bovine insulin cDNA to
bovine chromosome 29:

$ cd ˜
$ blat sequences/bos_taurus_chromosome_29.fasta \
sequences/bos_taurus_insulin_cDNA.fasta blat_chr_29_output.txt

Note that blat interprets the first file to be the database, the second to be the query,
and the third to be the output. The output returned by blat contains the coordinates of
similar regions but not a sequence alignment. To generate a sequence alignment from the
coordinates, use the pslPretty program, which is included with BLAT:

$ pslPretty blat_chr_29_output.txt \
sequences/bos_taurus_chromosome_29.fasta \
sequences/bos_taurus_insulin_cDNA.fasta blat_chr_29_alignment.txt

9 http://www.kentinformatics.com/
5 BIOINFORMATICS TOOLS 30

To view the alignment, use more:

$ more blat_chr_29_alignment.txt

The blat program is usually used to compare sequences to a full genome rather than
a single chromosome. The following commands download a complete bovine genome
sequence:

$ cd ˜
$ mkdir bovine_genome
$ cd bovine_genome
$ wget -nd -r -A.fa.gz \
ftp://ftp.hgsc.bcm.tmc.edu/pub/data/Btaurus/fasta/\
Btau20070913-freeze/LinearScaffolds/*
$ gunzip *.fa.gz

If you plan on performing several blat searches against a genome, you may want
to convert the chromosome sequence text files to “2bit” files. The 2bit format is more
compact and can lead to faster searches (the faToTwoBit program used below is included
with blat):

$ faToTwoBit Chr1.fa Chr1.2bit

To convert all the chromosome text files to 2bit files you can use find followed by
xargs. In the command below, find is used to obtain a list of the chromosome sequence
files. The file list is passed to xargs, which builds a faToTwoBit command for each file,
replacing all instances of “{}” with the name of file:

$ cd ˜
$ find bovine_genome -name “*.fa” | xargs -I{} \
faToTwoBit {} {}.2bit

The resulting 2bit files (one per chromosome) can now be used as the target sequences
in blat searches. The following uses find, xargs, and blat to compare each bovine chro-
mosome to a single query sequence (bos_taurus_insulin_cDNA.fasta):

$ cd ˜
$ find bovine_genome -name “*.2bit” | xargs -I{} \
blat {} sequences/bos_taurus_insulin_cDNA.fasta {}.insulin.out
6 STREAMLINING DATA ANALYSIS 31

To quickly examine all the output files produced by blat you can use the following:

$ cat bovine_genome/*insulin.out | more

To perform searches for multiple queries, first create a text file containing a list of
the query sequence files, one filename per line. Pass this list file to blat in place of the
query. This approach is faster than manually running a separate search for each query, in
part because each chromosome sequence needs to be loaded into memory just once. The
following creates a file called query_list.txt containing the names of all the files in the
sequences directory that have “bos_taurus” in their title and are less than 1 MB in size.
The list file is then used as the query in blat searches against each bovine chromosome:

$ cd ˜
$ find ./sequences -name “*bos_taurus*” -size -1000k > query_list.txt
$ find bovine_genome -name “*.2bit” | xargs -I{} \
blat {} query_list.txt {}.list.out

To convert the blat results to alignments, first create a file containing a list of the
chromosome sequence files that were searched. This “targets” file can then be passed to
pslPretty along with the query_list.txt file you created earlier. These filename lists are
used by pslPretty when it obtains the sequences located between the match coordinates
given in the “list.out” files:

$ cd ˜
$ find bovine_genome -name “*.fa” > target_list.txt
$ find bovine_genome -name “*list.out” | xargs -I{} \
pslPretty {} target_list.txt query_list.txt {}.pretty

The resulting alignments can be viewed using the following:

$ cat bovine_genome/*pretty | more

6 Streamlining data analysis

6.1 The .bashrc file
When you sign in to your Linux account, a file in your home directory called .bashrc is
run by the system. This file contains commands that are used to control the behavior of
6 STREAMLINING DATA ANALYSIS 32

the bash program (bash is the program that passes the commands you type to the actual
programs that do the work). You may not have noticed this file in your home directory,
because by default the ls command does not show files that start with a “.” character. To
see all the files in your home directory, use ls with the -a option.

$ cd ˜
$ ls -a

In the following exercise you will make a few minor changes to your .bashrc file
using using vi. Start by copying your .bashrc file so that you can go back to the existing
version if the changes you make create problems:

$ cp .bashrc bashrc_backup

Now open your .bashrc file in vi:

$ vi .bashrc

Remember to press i to enter insert mode. Add the following text below the existing
contents:

alias rm=‘rm -i’

alias la=‘ls -al’

Now press Esc to leave insert mode, and then type :wq to save your changes and exit
vi. The first line you added to your .bashrc file will tell bash (the program that handles
the commands you type) to always pass the -i option to the rm program when you enter
the rm command. The -i option tells rm that you want to be warned before any files are
actually deleted, and that you want to have the option of canceling the delete process.
The second line tells bash that you want to use the command la to call the ls program
with the -a and -l options (show all files and use the long listing format). Defining the la
command in your .bashrc simply saves you the trouble of remembering and typing the
full command for listing all files.
Try the new la command:

$ la
-bash: la: command not found
6 STREAMLINING DATA ANALYSIS 33

Notice that the bash program is saying that it doesn’t know what is meant by la, even
though you defined it in the .bashrc file. Remember that the .bashrc file is only read
when you sign in. To tell bash to read your .bashrc file again, use the source command:

$ source .bashrc

The la command should now be recognized by bash.

6.2 Modifying $PATH and other environment variables

Try running the remote_blast_client.pl program from your home directory by typing the
name of the program:

$ remote_blast_client.pl
-bash: remote_blast_client.pl: command not found

The bash program, which interprets the commands you enter, doesn’t know anything
about the remote_blast_client.pl program. This is the reason you had to enter the exact
location of the remote_blast_client.pl script when you ran it in the previous example
(./remote_blast_client/remote_blast_client.pl). Remember that the ./ means the current
directory.
Whenever you type a command, bash searches for a program with the same name as
the command you enter, and for alias commands you specified in your .bashrc file. The
bash program does not however, search the entire file system for a matching program, as
this would be very time consuming. Instead, it searches a specified set of directories. The
names of these directories are stored in an environment variable called $PATH. To see
what is currently stored in your path, use the following:

$ echo $PATH

Notice that the directory containing remote_blast_client.pl is not stored in the $PATH
variable. You can temporarily add it using the following

$ export PATH=$PATH:˜ /remote_blast_client

6 STREAMLINING DATA ANALYSIS 34

To see that it was added, enter echo $PATH again. Note that this change to $PATH
only lasts while you are signed in. To make the change permanent, you could add the
above export command to the end of your .bashrc file using vi.
Now that the $PATH variable contains information about where to find remote_blast_client.pl,
try entering the following in your home directory:

$ remote_blast_client.pl

The remote_blast_client.pl program should start. Press Ctrl-C to return to the com-
mand line.
Although the benefits of editing the $PATH variable are minor in this case (it isn’t
difficult to enter the full path to the remote_blast_client.pl), understanding environment
variables and how to modify them is very important. Indeed, many programs require that
you add new environment variables to your .bashrc file.

6.3 Writing a simple BASH script

Sometimes you may find it hard to remember what command-line options a program like
remote_blast_client.pl or clustalw requires. Furthermore, you may always use the same
options, making all the typing seem quite repetitive. Suppose you want to be able to sign
in to your user account and quickly perform an alignment of whatever sequences you have
stored in a file called dna.fasta. You can do this by writing a simple BASH script that
contains the command you want to use.
First create the file that will contain your script:

$ touch align_dna.sh

Now edit the file in vi:

$ vi align_dna.sh

Using vi, add the following text (remember to press i to enter insert mode):

#!/bin/bash
clustalw -infile=dna.fasta -outfile=dna.alignment -align -type=dna
7 SUMMARY 35

Save your changes and exit vi.

Use chmod to make your script executable:

$ chmod u+x align_dna.sh

To test your script, first create a file called dna.fasta in your home directory by copy-
ing the fasta file you created previously in your sequences directory:

$ cd ˜
$ cp sequences/all_p53_seqs.fasta ./dna.fasta

Now execute your script:

$ ./align_dna.sh

You should see output from clustalw appear, and a file called dna.alignment should
be created. BASH scripts are useful because they help to automate analysis steps, since
you do not need to enter a lot of text, and you can be sure the same parameters are used
each time. It is possible to build complex scripts consisting of many commands.

7 Summary
This tutorial has provided a brief introduction to the Linux operating system, and in par-
ticular the use of the command line. There are numerous web sites and books that go into
much more detail about how to set up and use a Linux system [6]. Although the transition
to Linux can seem difficult at first, it is well worth the effort if you plan on working with
large data sets, such as those arising from sequencing projects or microarray experiments.
If you would like to become even more proficient at analyzing sequences and other bioin-
formatics data you may want to learn a programming language. Linux is well-suited to
such an endeavor, because of the wealth of programming tools and guides available. Once
you can program you can perform almost any analysis you can imagine.
REFERENCES 36

References
[1] http://www.oreillynet.com/linux/cmd/

[2] Rice P, Longden I, Bleasby A (2000) EMBOSS: The European Molecular Biology
Open Software Suite (2000) Trends Genet 16:276-277.

[3] Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD
(2003) Multiple sequence alignment with the Clustal series of programs. Nucleic
Acids Res 31:3497-500.

[4] Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ
(1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25:3389-3402.

[5] Kent WJ (2002) BLAT–the BLAST-like alignment tool. Genome Res 12:656-664.

[6] http://oreilly.com/pub/topic/linux