Welcome to the UCLA – IGERT
2005 Summer Bioinformatics
Workshop
IGERT – Sponsored Bioinformatics Workshop Series
Michael Janis and Max Kopelevich, Ph.D.
Dept. of Chemistry & Biochemistry, UCLA
Who this workshop is for
• Biologists:
–Integrating bioinformatics methods into their existing projects
• Bioinformatics students:
–Preparing for and augmenting existing
bioinformatics coursework
• Computational researchers:
–Identifying and understanding common
bioinformatics language classes and data structures
Why this workshop?
• Current graduate curriculum:
– Assumes substantial knowledge base upon entry into
bioinformatics program
• Researchers:
– No general overview of computational research
environments
• some aimed at tools (suites), others deep in theory base
Why learn bioinformatics?
• The 1 gene, 1 protein research model ignores the deluge of
inferential biological data at our disposal
• This information must be combined for any thorough
study of biological phenomena
• Assembly of qualitative (sequence, etc.) and quantitative
(measured, inferential) data is beyond the capacity of a
human to interpret in a systematic fashion
• Computational methods must be addressed both to
interpret and to share research data in a meaningful way
– Yet Another Tool in a biologist’s arsenal
What is Bioinformatics?
• A tool in a molecular biologist’s arsenal such as
PCR, gels, or microarrays; or:
• A support machine for handling and storing large
data sets; or:
• A discipline in its’ own right, enabled by a critical
mass of biological data and mirroring the
evolution of a number of scientific sub-disciplines
An argument for learning: as a
tool for molecular biologists?
• 1 gene, 1 protein becomes an interactive series of
pathways and protein interactions
• A pathway allows novel interaction discovery and
ontological groupings of data
• Moving from identification of a gene that has
biological function to elucidation of a pathway
from a given biological function (just for
example)
An argument for learning: as
support for data management?
• Completion of the human genome working draft in June
2000 resulted in an absolute requirement for a logical,
hierarchical way to store, modify, and retrieve data
• Cross-referencing information is necessary to incorporate
disparate and sometimes conflicting information
• Combination of data and necessary algorithm
development to find signal among the noise of the genome
An argument for learning: as
an entirely new discipline?
• Evolutionary biology through homology
– (COG for example)
• Gene expression and structure prediction
– (alternative splicing for example)
• Protein and ncRNA modeling
• Genome mapping
– (statistical genetics and disease correlations)
• Pathway prediction
• Towards an end: Systems Biology
What has bioinformatics
allowed us to do?
• Evolutionary Biology
– Clusters of Orthologous Genes (COG database)
http://www.ncbi.nlm.nih.gov/COG/
•
Structure and alignments
– PFAM/RFAM http://www.sanger.ac.uk/Software/Rfam/
• Expression inferences
– clustering to find interactions (for example through SMD:
http://genome-www5.stanford.edu/
• Genome mapping
– (for example the UCSC Genome Browser:
http://genome.ucsc.edu/cgi-bin/hgGateway
What do we need to create our
own genomic inferences?
• That’s the problem… The data is encoded…
• AGCTAGCGACTAGCGATTATA…
– Now predict what this sequence codes for, how it is
transcriptionally regulated, in what pathway does it function …
• The human genome is 3 billion bases.
– Clearly we need a new set of tools
• Problem: no human being could ever look at all
this data, so the patterns must be discovered
computationally
The magic wand analogy
• The sum of all human knowledge can be encrypted and
stored by marking a single scratch on a metal rod. It all
depends upon RESOLUTION and LANGUAGE.
• We can apply this analogy to our analytical process:
– High RESOLUTION is required to decode the genome
– A robust LANGUAGE is required to achieve that RESOLUTION
– Computational biology seeks to describe that LANGUAGE
How do discover meaningful
biological patterns?
•
•
•
•
•
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Need to be able to pick “needles from haystacks”
Need to be able to assemble needles
Need to orient and classify needles
And so on…
This requires a strong, flexible environment
The Shell, PERL, R, PostGreSQL/MySQL – integration,
data exploration
• Statistical development environments – making inferences
from the data
• Databases and resources – combining the data
How our workshop will help
you (and how it won’t?)
• We cover common, portable, powerful resources – but
require suitable activation energy!
• Integration for a project model is key
• Thus, easily understood but limited GUI based suites are
not covered
• Likewise theory of algorithm development is covered in
other courses and will be ignored (for the time being!)
How this workshop is structured
• Two lectures a week for six weeks (fast!)
• Lots of reading material (need to keep pace)
• Hands – on practical experience with biological data
and programming tasks
• Peer interaction
What you’ll need for this workshop
• Access to either the online version of the text through
O’Reilly Media (linked on the website, $20 for two
months) or through purchase of the print bound version
(approx $130, we’ll put in an order during the second
week)
• Access to a computer. Any computer will do.
• Motivation to learn, and to ask questions
What is the text for this workshop?
• O’Reilly Media SafariU agglomerative text of 10
textbooks from O’Reilly, New Riders, and AddisonWesley publishers
• Chosen for topics applicable to modern computational
biology
• Since many texts overlap, no need to own all – cost
reduction ($130 vs. over $500)
Class information
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[email protected]
Office: 4054 Young Hall
http://www.bol.ucla.edu/~mjanis/biohackers2005.html
http://lists.ucla.edu/cgibin/mailman/listinfo/bioinformatics
IRC: #uclaBioinformatics, freenode server (mako)
T / R 2-4 pm 1054 Young Hall, Aug 11- Sept. 16 2005
Class information
BRIEF SUBJECT MATTER OUTLINE (SUBJECT TO CHANGE!)
08/11 Unix: Introduction to Unix; Bioknoppix
08/16 Unix basics, editors, remote logins
08/18 Shell programming
08/23 Regular expressions; biological data formats
08/25 Perl: data and control structures
08/30 Modular programming
09/01 Object oriented perl
09/06 Biological classes; Bioperl
09/08 NCBI tools; BLAST
09/13 Database design; perl DBI
09/15 Net programming; XML; CGI
09/16 R; Bioconductor
We used to call it “biohackers”…
• What is a hacker?
• One with technical adpetness who delights in solving
problems and overcoming limits…
• Hackers can be found in all walks of life – music, art,
science
• Hackers create things – the internet, Unix, NCBI, the
Human Genome project
• Not what the media would call a “hacker”…
The hacker attitude*
• Hackers believe the world is full of fascinating
problems waiting to be solved.
• Nobody should ever have to solve the same problem
twice.
• Boredom and drudgery are evil.
• Freedom is good
• Attitude is no substitute for competence
* From “The Cathedral and the Bazaar”
Why would I want to become a
“hacker” or a “biohacker” or
whatever?
• Spend less time brutally munging your data. Spend
more time gaining biological insight. Publish faster.
• Begin to assemble complex and disparate data in ways
not achievable by humans. Think systematically about
your data. Think systemically about your research.
• Smugly travel easily through electronic medium.
Automate your life
• Be cool. Get chicks! (or whomever you want)…
It worked for “neo”…
In reality, it worked for someone
else too …
How does one become a hacker?
• Learn to program. The more languages, the better.
(Don’t think in terms of languages… Think in terms of
PROGRAMMING)
• Get an extensible, modifiable OS and learn to use it
• Learn how to access and use information
• Practice, practice, practice. This workshop can only
show you the door. You have to walk through it.
So what is biohacking?
• Inferential analysis of disparate biological data
• Discovering and creating new ways of looking at
biological phenomenon
• Creation of novel tools
• Decoding the nature of life (BHAG)
So show me the tool to find the
gene… I’ll be in the lab…
• What if your research looks at biology from a novel
angle? What if there are no tools? Where to start?
• We need a modular framework. Something that will
scale nicely so that we can incorporate more data later.
Some set of tools that integrate well, each doing one job
and doing it well. We need COMBINATORIC
TOOLSETS!
A tale of two philosophies
• Windows works extremely well at solving one problem
at a time, if it falls within defined parameters… It does
not scale well.
• This monolithic approach is good for small scale
biology but not for complex biological analysis
We need a construct where we can
call upon modular tools to
assemble our own analysis…
I need bioinformatics tools…
Lots of bioinformatics tools…
The construct – UNIX (the other
philosophy)
• UNIX is an operating system that provides powerful
modular utilities that function together to create
computer environments that can solve a wide variety of
tasks.
• Unix Operating System
–created 1960s
–provides a multi-user, multitasking system
•It’s a server by design
•Networks extremely well due to ports – X11 is an example
–command line based
•Bioinformatics relies heavily upon UNIX – and a fundamentally different
philosophy:
•modular by its’ very nature, UNIX is scaleable, robust, and … FREE!
• But more than anything else, Unix has THE SHELL
The Unix Shell
•
•
•
•
The shell is the unix user environment
The shell is started automatically when you log in
Everything is controlled from a shell
The shell interprets commands
– It’s also a programming language itself!
• http://www.looking-glass.org/shell.html
– http://www.strath.ac.uk/CC/Courses/IntroToUnix/node46.html
• More than one shell can be open at a time
Commands to the system
• Specific commands used to perform functions in
the shell
– Ex: changing directories, copying files, mv files,
sorting, cutting, pasting, etc…
– Many of these can be done in XL, but…
• What if you need a subsequent command? ChiSquare
analysis for example?
• What if you need to do this to 10 files? 100? 10000?
• Each command is itself a program and take
command line arguments
– The Shell is the “glue” that allows us to
combinatorically daisy-chain our tools together to do a
job
• The Shell allows us to batch these commands into
a pipeline or framework for analysis
The Shell controls input / output
STDIN
Command
Or
Program
STDERR
STDOUT
Input / Output II
STDIN
-Keyboard
-File
-Previous STDOUT
Command
Or
Program
STDERR
STDOUT
-Terminal/ monitor
-File
-Next STDIN
Input / Output III
Command 1
Pipe
Command 2
Large Pipe
Command 1
Pipe
Command 2
Pipe
Command 3
Pipe
Command 4
Can daisychain commands from every shell program
UNIX then… Linux? BSD? OS
X? (over 360 distributions
according to linux.org)
How can I get *nix to use?
• Linux is open source
– Available for a variety of platforms
• BSD is likewise open source
– Forms the basis of Mac OS X, and many MSWin utilities
• There are live distributions as well
– Example: Bioknoppix - http://bioknoppix.hpcf.upr.edu/
• Bioknoppix is a customized distribution of Knoppix Linux Live CD.
With this distribution you just boot from the CD and you have a
fully functional Linux OS distribution with open source
bioinformatics (sic) applications.
• Beside using some RAM, Bioknoppix doesn't touch the host
computer.
What about administrating the
system? I heard it’s hard!
Package managers
• Most modern distributions have a package manager
–
–
–
–
Handles installation
Handles dependencies
Handles conflicts
Packages are written in such a way as to ensure that they do not
interfere with other parts of the OS install
• With BioKnoppix, you won’t really need one
• With Debian, there is aptitude, and synaptic
• There are a wealth of bioinformatics tools packaged for
use with package managers
• We’ll be exploring these as we go along…
For our Apple brethren…
There is fink
• Mac OS X forms a natural basis for a end user
bioinformatics analysis station
• It’s UNIX underneath!
• The GUI is advanced
• It’s easy to administrate
• http://fink.sourceforge.net/
• Finks installs into its own protected directory /sw
• Easy to remove if things go south…
What is fink?
• Fink is a distribution of Unix Open Source
software for Mac OS X and Darwin.
– It brings a wide range of free command-line and
graphical software developed for Linux and similar
operating systems to your Mac.
• Installation is accomplished via a tarball.
– After that, it’s simple command line- interaction
(based upon the debian model, which I’ll show in class)
• http://fink.sourceforge.net/doc/usersguide/install.php?phpLang=en
For our MS brethren…
Welcome to Earth
A subsidiary of Microsoft
There is cygwin
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Shell utilities written as windows executables
Creates its own protected / filesystem
Problems with networking
No real package manager beyond the shell utils
Can be problematic to install packages with deep
*nix library dependencies
• Created / maintained by RedHat developers
• http://www.cygwin.com/
But mainly have remote access to a
real *nix machine
Configure Putty
SSH Option
We’ll be using…
• BioKnoppix Beta 0.2.1 (but the Knoppix part is 3.3…)
• This distribution is included in your folder
• Debian stable 3.1 (“Sarge”) install – which I’ll do in class
– will be our main server. We’ll call it “subi”
(subi.chem.ucla.edu) for SUmmer BIonformatics.
• The text has details about the use of knoppix
• Let’s take a look …
The shell, files, directories,
commands, and editors
File System
visualize the Unix file system as an upside down tree.
At the very top of the tree is the root directory, named "/".
Common filesystem locations
/usr/
The majority of user utilities and
applications.
/
Root directory of the file system.
/usr/bin/
Common utilities, programming tools, and
applications.
/bin/
User utilities fundamental to both singleuser and multi-user environments.
/usr/include/
Standard C include files.
/boot/
Programs and configuration files used
during operating system bootstrap.
/usr/lib/
Archive libraries.
/usr/libdata/
Miscellaneous utility data files.
/boot/defaults/
Default bootstrapping configuration files;
see loader.conf(5).
/usr/libexec/
System daemons & system utilities
(executed by other programs).
/dev/
Device nodes; see intro(4).
/etc/
System configuration files and scripts.
/etc/defaults/
Default system configuration files; see
rc(8).
/usr/local/
/etc/mail/
Configuration files for mail transport agents
such as sendmail(8).
Local executables, libraries, etc. Also used
as the default destination for the FreeBSD
ports framework. Within /usr/local, the
general layout sketched out by hier(7) for
/usr should be used. Exceptions are the man
directory, which is directly under /usr/local
rather than under /usr/local/share, and the
ports documentation is in share/doc/port.
/etc/namedb/
named configuration files; see named(8).
/usr/obj/
Architecture-specific target tree produced
by building the /usr/src tree.
/etc/periodic/
Scripts that are run daily, weekly, and
monthly, via cron(8); see
periodic(8).
/usr/ports
The FreeBSD Ports Collection (optional).
/etc/ppp/
ppp configuration files; see ppp(8).
/usr/sbin/
System daemons & system utilities
(executed by users).
/mnt/
Empty directory commonly used by system
administrators as a temporary
mount point.
/usr/share/
Architecture-independent files.
/usr/src/
BSD and/or local source files.
/proc/
Process file system; see procfs(5),
mount_procfs(8).
/usr/X11R6/
X11R6 distribution executables, libraries,
etc (optional).
/rescue/
Statically linked programs for emergency
recovery; see rescue(8).
/var/
Multi-purpose log, temporary, transient,
and spool files.
/root/
Home directory for the root account.
/var/log/
Miscellaneous system log files.
/sbin/
System programs and administration
utilities fundamental to both singleuser and multi-user environments.
/var/mail/
User mailbox files.
/var/spool/
Miscellaneous printer and mail system
spooling directories.
/var/tmp/
Temporary files that are kept between
system reboots.
/var/yp
NIS maps.
/stand/
Programs used in a standalone environment.
/tmp/
Temporary files, usually a mfs(8) memorybased file system (the contents of
/tmp are usually NOT preserved
Types Files
• ordinary files
–
–
–
–
text files
data files
command text files (shell scripts),
executable files (binaries);
• directories;
• links special device files (physical hardware)
• In unix, everything is a file… and every file is a
device… including the display – which is a port
…
Directories
All directories are subdirectories of the root
directory
Every directory can contain files or subdirectories
Every directory can contain a device. Which is a
file. Which really means that from anyplace in
the filestructure hierarchy, you can pipe IO to
any other place (device… err, file…)
Relevant Terminology
•
•
•
•
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Path
File permission
Home directory
Current working directory
Account
Login Name / Username
Password
Root
Logging Into Machine
Red Hat Linux release 9
Kernel 2.4.18-14 on an i686
localhost login:
The Unix Shell
• http://www.looking-glass.org/shell.html
•
http://www.strath.ac.uk/CC/Courses/IntroToUnix/node46.html
• When you type commands you are
communicating with the shell
• The shell is started automatically when you log in
• Everything is controlled from a shell
• More than one shell can be open at a time
Commands
• Specific commands used to perform functions in
the shell
• Ex: changing directories, copying files, mv
files,etc…
• Each command is itself a program and take
command line arguments
• For help on a specific command type:
man command
ls
•
•
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•
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•
•
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pwd “returns current working dir path”
ls “view contents of directory”
ls “lists all files (except hidden files)”
ls –A “all files INCLUDING hidden files”
ls -lt “lists all files in descending time
ls –1 “what does this do?”
man ls
ls *.CEL wildcards can be used
cd
• cd “change directory”
• cd “changes to user’s home directory”
– ‘home directory’ –top of users dir tree
•
•
•
•
•
cd some_dir “moves you to some_dir”
cd ~ “moves you to your home directory”
cd ../.. “moves you two directories up”
Cd – “returns you to previous working dir”
mkdir – creates a new directory with default permissions.
more / less
• More “view contents of a file”
– Use return or space to move through file
– Use b to go backwards
– ASCII only -> dos2unix command
• Less “view contents of a file”
– Use page up and page down to scrolol
– Does not buffer whole file
man more
man less
“less is more!!”
grep
• Search contents of a file or directory of files
– “Get Regex” – uses regular expressions we learned
– File wildcards can be used like with ls
• grep 1sq ~/DATA/*.CEL -> array type used
rm
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•
•
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rm “remove a file or directory”
rm –R “remove all subdirectories”
rm –f “dangerous, forces, no prompt”
man rm
• HOW DO YOU UNDELETE???
UNDELETE
• THERE IS NO UNDELETE
• (no trash, no recycle bin to open / browse)
• Be careful what you do
– But, that’s why there are permissions!!!
– Also a reason for *nix stability
An Example ls –l output: Unix
Permissions
File permissions prevent users from
deleting or tampering others files
Permissions listings
chmod
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•
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chmod “change file permissions”
chmod 777 “change permissions to 777”
chmod –R 775 “change recursively”
man chmod
Permissions Explained
• ------• you group world
• --- READ WRITE EXECUTE
• rwx rwx rwx
• Binary
421 421 421 = 777
• rw- r– r-- 6 44
cp
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•
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cp “copy from x to y”
cp /home/igert/bin/a /home/igert
cp –R directory /folder/new_dir
man cp
• cd /home/igert/temp
• cp a ..
• cp /home/igert/temp/a ~
Process Control, I/O
Redirection & Pipes
Overview
• Unix provides powerful features for:
–
–
–
–
Managing your jobs (process control)
Redirecting standard input and standard output
Reading and writing files
This is the ‘power of the shell’
Input / Output
STDIN
Command
Or
Program
STDERR
STDOUT
Input / Output II
STDIN
-Keyboard
-File
-Previous STDOUT
Command
Or
Program
STDERR
STDOUT
-Terminal/ monitor
-File
-Next STDIN
Input / Output III
Command 1
Pipe
Command 2
Large Pipe
Command 1
Pipe
Command 2
Pipe
Command 3
Pipe
Command 4
Can daisychain commands from every shell program
Example
STDIN
Command
Or
Program
echo “Hello Genome Hacker!”
STDOUT
Example (details)
“Hello Genome Hacker!”
ECHO
Hello Genome Hacker!
echo “Hello Genome Hacker!”
What can we pipe to?
• Many useful shell commands exist – use apropos
to find
• awk, sed, perl –e, wc, diff, cut, paste, sort, uniq…
just to name a few
• What do these do? Try man ‘command’ (as: man
paste)
AWK is your pre-perl friend
•
•
•
•
Use to print a subset of fields
Default field delimiter is “ “ (white space)
Useful for grabbing a subset of fields
Useful for rearranging fields
field1 filed2 field3 field4 . . .
$1 $2
$3
$4 . . . .
pipe
Using AWK
| awk –F” “ ‘{print $1}’
| awk –F” “ ‘{print $1”
“$2}’
| awk –F” “ ‘{print $1”\t”$2}’
\t = TAB
\n = newline
Example of a pipe
“Hello Genome Hacker!”
Command 1
(ECHO)
Pipe
Command 2
(AWK}
Hacker! Genome Hello
echo “Hello Genome Hacker!” | awk ‘{print $3”\t”$2”\t”$1}’
Example: redirecting STDOUT
STDIN
Command
Or
Program
STDOUT
OUTPUT_FILE
echo “Hello Genome Hacker!” > output_file
more output_file
“redirection operator”
Overwrite versus Append
• > OVERWRITE – delete and replace
• >> APPEND – add to end of existing file
Example: microarray data
tracking
• grep 1sq ~/DATA/*.CEL (gives array info)
• grep 1sq ~/DATA/*.CEL | awk ‘{print $12}’ gives
array type only
• grep 1sq ~/DATA/*.CEL | awk ‘{print $12}’ >
arrayTypes.txt (store results in file)
• ls ~/DATA/*.DAT | wc (gives a count)
Process Control
• Each specific job / command is called a process
• Each process runs in a shell
– BEFORE: prompt available
– DURING: prompt NOT available
– AFTER: prompt available
Two Ways to monitor Processes
• “top”
– Lists all jobs
– Uses a table format
– Dynamically changes
• “ps”
– man ps
– static content
– Command options
Top output
Background / Foreground
• Commands running in foreground prevent
prompt from being used until command
completes
• Commands can also run in BACKGROUND
• “Backgrounded” commands DO NOT AFFECT
the prompt
Two Ways to Background jobs
• “&”
– Running a command with
“&” automacically sends it
to the background
– Backgrounded commands
return the prompt
• “bg”
– Once a command is run
from the prompt
– Stop the command
– Then background it
• Starts the command again
• Returns the prompt for
use
Creating Our Own Commands
• Use programming language to create the new command
• We will use perl
• We’ll start USING OUR ENVIRONMENT (*NIX) NEXT
WEEK!
Homework Set 1
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What is the purpose of a pipe?
What does the pipe do?
What symbol is used for pipe?
How are commands put in background?
Why would you want to background?
What is the top command?
Why is it useful?
Submit one question about the *NIX environment to the
bioinformatics mailing list
• ANSWER one question from the bioinformatics mailing
list!
(estimated time: 10 – 15 minutes)
Homework Problem 2
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Create a two command pipe
What does it do
What are the inputs and outputs for each command?
Can you extend it to three commands?
How about four?
(command hints: echo, grep, awk, wc)
Redirect output to a file
Run it a second time, and redirect (append) this output to
previous output
(estimated time: 15 to 30 minutes)
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Discovering conserved DNA