Setting up your computing environment
Next-generation sequencing analysis is a compute intensive
process. Your average laptop is probably not up to the
challenge. Most software is geared toward UNIX style
operating systems, with large servers in mind.
Typically, analysis algorithms will be distributed by
researchers in one of three ways:
- Stand alone program to run in a UNIX environment (most
- Webserver where you upload data to be analyzed (i.e.
- R software package/library for the R computing
Some tools are available on multiple platforms, but a vast
majority of tools are available as stand alone Linux/UNIX
Types of Software Environments
Linux/UNIX [this tutorial]
Linux, BSD, Solaris, Mac OS X, or really any
UNIX-based operating system will work well. Linux
systems tend to be the most compatible with academic
software, and I find it is easier to install analysis
software on Linux than any other operating system.
Mac is an attractive choice for many users given it's a
good blend between usability and a native UNIX-style
operating system. Windows PCs are not ideal given
much of the analysis software is not configured to run
on them. However, Cygwin
is available as a Linux emulator that runs on Windows
computers. You can also always consider dual
booting your windows system, or using some sort of
virtualization software and run Linux at the same time.
Another option is to run your analysis on a
web server. These days we call this "analyzing
your data in the cloud". By far the most popular
"cloud" based analysis tool is Galaxy, which is a
compendium of programs (and user/data management tools)
for bioinformatics analysis.
This tutorial assumes you will analyze data on your own
computer/server. Personally, I feel researchers
get more out of it if they learn how to do things on
their own, including installing software. Learning
things this way also makes you self-reliant. Lets
say a new method or new piece of software becomes
available to help address a key question with your
data. That software is unlikely to be available on
Galaxy, and if it does end up being incorporated into
Galaxy, it may not end up there for another year.
So... you're stuck running it yourself.
Another popular option for next-gen analysis
is the statistical computing environment R/Bioconductor(similar
to Matlab). R/Bioconductor has a strong community, and R
has many built in statistical routines and graphics
functions that make developing algorithms in R more
attractive than writing your own code in python or
C++. Many R programs also run well under Windows,
making it a decent option for Windows users
This tutorial will cover the use of a UNIX-style operating
system on a local server or individual computer. I
feel understanding how to perform analysis from the
command line provides researchers with the greatest
flexibility, which is key in the fast moving world of
next-gen sequencing. For tutorials on how to use Galaxy or R/Bioconductor,
visit their respective websites (they are both excellent
sources of knowledge).
In theory, any computer with enough RAM, hard
drive space, and CPU power can be used for analysis - and
this includes many laptops. In general, you will
4-10 Gb of RAM Minimum (better to have 96+ Gb)
For example, if you have a brand new Mac Book Pro laptop
with lots of RAM, you can probably do sequencing analysis
on it. You may want to sit the computer on a table,
because it will get HOT while trying to map 100 million
reads to the genome, and might burn your lap if you're
sitting with it at home on the couch.
500 Gb of disk space (better to have 10+ Tb of space)
Fast CPU (better to have at least 8 cores, more the
Starter Computer: Mac Pro
Optimally, you'll have a dedicated
computer/lab server do store data on and perform
analysis. This way, you can log on to the server
from your personal computer and run analysis
remotely. For labs starting out, a popular option
is to get a Mac
Pro. Not only is it a fast, capable
computer, it can double as a shared lab computer that
can shared software, like Illustrator.
Ideal if you are a lab that does a lot of
Often you university or institution provides
access to a computing cluster for pennies on the dollar
(5-10 cents per CPU hour). These can be great
resources for doing genomic mapping if you don't want to
invest in more computing power. Usually accessed
via UNIX terminal...
Another option (similar to supercomputer
clusters) for accessing computing power
Basic Setup of OS/Environment
Linux/UNIX - Not much to do - a basic
Linux/UNIX install includes almost all of the basic tools
Mac OS - You need to install Xcode,
which includes GNU compilers and many other standard UNIX
development tools. What sucks is that Xcode is now
distributed through the App store, which I hate with a
passion. Even though Xcode is free, they'll still
basically force you to give them your credit card
number... Remember to install the "Command Line tools"
package while installing Xcode (you can also install this
separately from the Xcode application after
installation). I also recommend downloading and
since it's such a useful program after getting Xcode.
Windows - You'll need to install Cygwin. During
the Cygwin install, you'll want to install a bunch of the
Run the install program, and when prompted to
choose which packages to install, make sure to install
- GCC compiler, gcc-core, gcc-g++
- make (in Devel)
- mingw64 (in Devel, for pthread
- perl (in perl)
- zip/unzip (in Archive)
- wget (in Web) – very useful for
- ghostscript (in Graphics)
Cygwin configuration screen in the setup
program (if you already installed cygwin and need to
modify it, just rerun the setup program):
Using the Linux/UNIX terminal
Secure Shell (ssh) - accessing remote computers
One of the best features of the UNIX terminal
is that it can be used to access remote computer
systems. If you have an account on a remote
computer and it is running sshd (Secure shell daemon),
then you can log onto that computer using ssh client
To access a remote computer, you can either use the ssh
command from a local terminal (use this if you have Mac
OS, Linux, or use Cyginw), or on windows, you may want
to use a FREE ssh client program such as PuTTY or select one
To access a remote computer, you must know the hostname
or IP address of the computer, and your
username/password. To run ssh from the terminal,
run the command:
It will then prompt me for my password, and give me a
fresh terminal session on the remote computer.
i.e. ssh firstname.lastname@example.org
If you are accessing a server that is always "on", I
would highly recommend managing your sessions using screen,
which is a persistent terminal session manager that will
allow your commands to run even if you log off.
In UNIX, whenever you type something into the
terminal, the first word is considered the
"command". Every command works like this:
Here are some common commands:
cd <directory name> : change your
current directory to the directory specified
ls : list the files and directories in your
current directory. Add options "-al" to list all
details about the files.
pwd : return the full path of your current
more <filename> : display the contents of a
gzip <filename> or gunzip
<filename> : GNU zip or unzip a file
tar xvf <filename.tar> : expand a tar
tar zxvf <filename.tar.gz> : expand a GNU
zipped tar archive. Sometimes these have the file
top : Useful utility to monitor programs that are
Getting info about how to run various commands:
<command> --help : will usually
return information about how to run the command
man <command> : will display the
manual for the command (may not work for every command)
"ctrl+c" : If a command it taking
too long, or seems out of control, hit control+C at
the same time to quit execution of the current program
<tab>/Autocomplete : Most UNIX terminal
programs support auto-complete for file names and
program names. While typing a file name in the
terminal, if you hit "tab", and there is only one
logical choice, the file name will be automatically
Capturing program output: stdout and stderr
Many UNIX programs will send their output to a
special I/O stream called stdout. Normally, stdout
is displayed on the screen. However, if you wish
to capture this output into a file, you need to add "
> filename" to the end of the command.
This will capture the output content and save it into
the text file "filename".
Understanding the file system
You can think of the UNIX file system as a hierarchical
tree. UNIX file systems use a "/" between
directory names when specifying a file (windows uses
"\"). There are two concepts you must get a handle
on when thinking about UNIX filesystems:
To specify the current directory (in a relative sense),
use the ".". If you want to specify the
parent directory relative to your current position, use
"..". This link
does an decent job describing how to use file names.
- Absolute File Names (also called the absolute
path): Each file has an absolute file name, which
starts with a "/". For example,
"/home/cbenner/beerRecipe.txt". The absolute
file name provides the exact location of the file in
the system tree relative to the root (i.e. "/").
- Relative File Names: You have a current location
(or present working directory). You can learn
what it is using the command "pwd". Any filename
that doesn't start with a "/" is a relative file
name, and will be describe relative to your current
For example, lets say your present working directory
is "/home/cbenner". If you specify a file by
calling it "data/reads.txt", the system will think
you are referring to an absolute file name that is
Home Directory: This is the directory
where your personal configuration files are stored,
and is also usually the first directory that you will
be "in" once you log onto the server. There is a
special symbol for your home directory: "~/".
For example, if your home directory is
"/home/chucknorris/", then the file "~/data.txt" is
equivalent to "/home/chucknorris/data.txt".
Each file has permissions that allow you to
read (e.g. access), write (e.g. modify), and execute
(e.g. run the file as a program). These three
types of permissions allow you to restrict access to
the file. These 3 permissions also apply to
yourself, your group, and everyone else on the
system. For example if you type "ls -al",
you might see something like this:
cbenner$ ls -al
drwxr-xr-x 40 cbenner
staff 1360 Sep 27
drwxr-xr-x 7 cbenner
staff 238 Sep
22 17:28 ..
-rw-r--r-- 1 cbenner staff
84930997 Sep 5 12:44 chimp.L1.txt
-rw-r--r-- 1 cbenner
staff 342187 Sep 5 12:44
-rwxr-xr-x 1 cbenner
Sep 5 21:43 combine.pl
The first several symbols (i.e. drwxr-xr-x) give the
permissions on each file. Normally, you don't
have to worry much about the permissions until you
become comfortable with UNIX, but if you do need to
make a program executable, you should run the
chmod 755 program.pl
This will allow you to run the file as a
program (i.e. "$ ./program.pl").
Editing text files
In general, there are really only two types of
files. ASCII text files, and binary files.
ASCII text files are formatted such that each "byte" is
represents a character. A byte is composed of a
value between 0 and 255, and the meaning of each value
is encoded in the ASCII
Table. Text files like this are very powerful
because virtually any program can interpret them.
Most specialized files you may know of are ASCII text
files, such as XML, HTML, CSV, and of course TXT.
Binary files, on the other hand, can adhere to any
format, and are usually meant to be read and written by
a single program. An example would be a word doc
file, which has a special encoding that is only meant to
be accessed my Microsoft Word. For sequencing, a
classic example of a text files is a FASTQ
file, while a common binary file is a BAM file.
Text files are heavily used in the UNIX world due to
their general accessibility. Not only are most
data files encoded as text files (like FASTQ), most UNIX
configuration files are also text files.
There are several common utilities included in UNIX to
manipulate text files, such as more, cut,
wc, cat, etc.
Knowing how to use UNIX Text Editors is
important when achieving comfort with UNIX.
Below are three common editors, with links to pages
the give some background information about how to use
them. Each will have countless tutorials
covering their use (just google "<editor>
: the most common UNIX text editor, found on any
system. Very powerful, but unfortunately a
little difficult to use for beginners. Worth
learning, but takes some effort.
: one of the easiest UNIX text editors to use, good
for beginners, not as popular as vi.
: one of the most extensible UNIX editors, common for
There are two general approaches to installing
software on a Linux/UNIX system. The first is to
use a package manager (like yum or apt-get). The
second is to directly download the software and install
it from source (or simply download the executable
Installing software with package managers is
great because it will make sure all "prerequisites"
are also installed to make sure your software runs as
planned. This is typically only an option for
well known programs (most academic software must be
installed directly from source).
Installing software from source:
If running a debian flavor of Linux (i.e. Ubuntu),
then you will use something like this:
apt-get install <package name>
If running a redhat flavor of Linux (i.e. CentOS),
then you will use a different package manager, such
yum install <package name>
In many cases, you must be the root (or superuser) to
install system software, so you may have to add "sudo"
to the beginning of these commands.
Most academic software must be directly
downloaded from the author's website. Software
like this can come in two flavors, it can be source
files or it can be pre-compiled/binary files (programs
ready to run). If you are downloading programs
that are pre-compiled, make sure you get the files
specific to your system (i.e. Mac OSX, Linux x86_64,
It might sound daunting to install software from
source, but usually it is not bad at all. In 90%
of cases, you'll simply run the next couple commands:
- Download the software to your current directory
- type: "tar zxvf program.tar.gz"
- This will likely make a new directory, use "cd
programDirectory" to move into the new
- Read the "README.txt" and "INSTALL.txt" files,
if they exist. These files should include
directions about how to install the software.
- Usually, this means running the next three
sudo make install
Changing your executable PATH variable
Every program in UNIX is actually a file. Most
program files for system commands, like "cd" or
"ls" are found in special directories such as "/usr/bin"
or "/usr/local/bin". Whenever you type
something into the terminal, the first word you type
is the command. When you press enter, the
operating system attempt to find the program file
corresponding to the command. For example, if
you type "ls", it needs to find the file with
the execution instructions that will list your files.
The location of program files is managed in UNIX
systems using the PATH variable. This is
a variable maintained by the shell (e.g. terminal
program) that stores the directories that contain
program files. If you run the command "echo
$PATH", the contents of the PATH variable will be
What is shown is a list of directories separated by
colons ":". When you type a command, the
operating system will go to each of these directories
(in order) and look for an executable program file
matching the name of the one you typed. If you
want to figure out where the command is, you can use
the "which <command>" command to return
the location of the command you ran. For
This shows that the ls command is actually a
program file located in the /bin/ directory.
Why is this important? Often, you may download
a program and put it in a directory - lets say I
download a program called "roundhouse" and
place it in the directory "/home/chucknorris/software/".
I then make sure it is executable by changing it's
permissions: "chmod 755 roundhouse". To
run the program from anywhere, I must specify the
program file location explicitly: "/home/chucknorris/software/roundhouse".
However, if I want to run it like most commands (i.e.
just type "roundhouse"), I need to place it in
a directory that the operating system will search for
(e.g. a directory that is included with the PATH
For that, you have two options. First, you could
copy the roundhouse program to a directory
that is in the PATH, such as copying it to the
/bin/ directory. However, this is usually
not a good idea since the bin directory is normally
reserved for system applications. Another
approach is to modify your PATH variable to
include the /home/chucknorris/software/
directory so that the operating system will search it
when looking for program files.
The best way to do this (assuming you are using the
bash shell, which is the most common), is to edit the
"~/.bashrc" resource file. Using vi
or pico, edit the file and include a line such
as this one:
The "~/.bashrc" file is a special resource
file that is executed whenever you login. This
way, whenever you log into your computer, your PATH
variable will be updated to include the special