BINF 634 Bioinformatics
Programming



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Instructor: Jeff Solka Ph.D.
Phone: 540-809-9799
Email: jlsolka@gmail.com
Office Hours: By appointment
Required texts:





Beginning Perl for Bioinformatics by Tisdall and Waliszewski
Programming Perl (3rd Edition) by Wall, Christiansen and Orwant
Course Meeting Place: Ocaquan Prince William Rm. 304B
Course Meeting Times: M: 4:30 pm – 7:10 pm
Course webpage

http://binf.gmu.edu/jsolka/fall15/binf634/Fall_2015BINF_634_Sylla
bus_rev1.html
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Acknowledgements

Some of the material used in this course was
previously developed by
John Grefenstette
 John Kopecky

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2
Experimental Biology Computational Biology
and Bioinformatics
Computational Biology
Problem Statement
Results
Simulation
SIMS
Analysis Tools
Database
LIMS
Problem Statement
Experiment
Results
Rick Stevens
Experimental Biology
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Bioinformatics Programming
Tasks



Manage large experimental data sets

Sequence data

Microarray data (gene expression)

Mass spec data (proteomics)

Genotype project data (HapMap)

Clinical data
Build tools for Knowledge Discovery

Find motifs in sequence data

Data clustering

Visualization
Build analysis pipelines

Glue several analysis steps together into a single automated process

"Munge" data: Take data from one application or database and format it for
input to another application of database
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Objectives

Programming skills


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Problem solving and Debugging
Reading and Writing Documentation
Data Munging: Data filtering and transformation
Pattern matching and data mining
Visualization and web presentation
Object-oriented programming
Bioinformatics skills



Biological sequence analysis
Interacting with biological databases
Using Bioperl
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Background and Prerequisites

Molecular Biology
 BIOL 482 or similar course
 Recombinant DNA - Watson, Gilman, Witlowski, Zoller


Online Tutorials


http://www.amazon.com/Recombinant-DNA-Genes-GenomesCourse/dp/0716728664/ref=dp_ob_title_bk
http://www.biology-online.org/1/5_DNA.htm
Computer Science
 IT 108, CS 112 or similar
 Previous programming experience
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Course Policies

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Programming assignments (50%)

3-4 graded programming assignments
Exams: Midterm (20%) and Final (20%)

May include both closed-book section and open-book programming
problems
In-class Quizzes (10%)
Weekly homework assignments

All HW assignments must be submitted to me via Blackboard by the
beginning of the next class. HW assignments will not be graded individually,
but you may be called upon to discuss your work during the next class.
Therefore, late assignments will not be accepted.
Grading will be on the following scale. 93-100 (A), 90-92 (A-), 87-89 (B+), 8386 (B), 80-82(B-), below 80 (C). Student averages will be rounded to the closest
integer to determine final letter grades.
Keep an eye on the webpage

http://binf.gmu.edu/jsolka/fall15/binf634/Fall_2015BINF_634_Syllabus_rev1
.html
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Honor Code Policies



I take honor code violations very seriously.
Programming assignments must be your work. Each assignment
will specify whether you may use code from other sources. Any
material you take from another source must be acknowledged
within the program documentation. You must read and
understand the honor code handout. Violations of the honor
code WILL be referred to the Honor Council.
All students must adhere to the GMU Honor Code:

See: http://www.gmu.edu/academics/catalog/9798/honorcod.html
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Pragmatics
Assignments and Announcement


Will be posted on course wepage; check daily
Class email will be sent to your email address from Patriot Web
Accounts


You should have an account on the server binf.gmu.edu
Systems administrator: Chris Ryan, cryan1@gmu.edu
Accessing perl:


Method 1a:Login from Rooms 304B or 320
Method 1b:Login from off-campus using ssh
 Go to ftp://ftp.ssh.com/pub/ssh/ for academic Windows client
 Alternatively go to
http://www.chiark.greenend.org.uk/~sgtatham/putty/

Method 2:Install perl on your own computer -- see textbooks and
backup slide materials
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Pragmatics

Unix

This class will focus on using the Unix operating system


There are numerous UNIX tutorials


We will be using Mac OS X (at least in the classroom)
http://www.unixtools.com/tutorials.html
Text Editors


Perl program are stored in plain text files
I recommend emacs or vim for a Unix text editor (see links for
windows support)



If you are interested in an integrated development environment I
recommend Eclipse (see backup slides)


http://www.claremontmckenna.edu/math/ALee/emacs/emacs.html
http://www.vim.org
www.eclipse.org
There is a tutorials for each online


http://www.gnu.org/software/emacs/tour/
http://www.yolinux.com/TUTORIALS/LinuxTutorialAdvanced_vi.html
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Review: Molecular Biology


Life evolved from common
origin about 3.5 billion
years ago
All life shares similar
biochemistry



Proteins: active elements
Nucleic acids:
informational elements
Molecular Biology: the
study of structure and
function of proteins and
nucleic acids
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Proteins

Functions:

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Structural proteins
Enzymes
Transport
Antibody defense
Structure:



Chains of amino acids
Typical size ~300 residues
Range from about 100 to over
5000 residues
N.B. – A residue is one of the 20 building blocks of proteins also called an
amino acid.
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DNA

Double stranded

Four bases: adenine (A),
guanine (G), cytosine (C) and
thymine (T)

A and G are purines

C and T are pyrimidines

A always paired with T
(complementary)

C always paired with G
(complementary)
=> Watson-Crick base pairs (bp)

DNA may consist of hundreds of
millions bp

A short sequence (<100) is
called an oligonucleotide
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RNA:
 single stranded
 uses U (uracil)
instead of T
 less stable than DNA
 also used in
functional molecules
(e.g. rRNA, tRNA)
•introns are not translated
rRNA = ribosomal RNA
tRNA = transfer RNA
•exons are translated
 important regulatory
functions (siRNA)
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siRNA = small interferring
RNA
14
Translation




Translation involves mRNA and
ribosomes
Ribosomes made of protein and
ribosomal RNA (rRNA)
Transfer RNA (tRNA) make connection
between specific codons in mRNA and
amino acids

As tRNA binds to the next codon in
mRNA, its amino acid is bound to
the last amino acid in the protein
chain
When a STOP codon is encountered, the
ribosome releases the mRNA and
synthesis ends
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DNA Structure

DNA contains:

Genes

"a locatable region of genomic
sequence, corresponding to a unit of
inheritance, which is associated with
regulatory regions, transcribed
regions, and or other functional
sequence regions ".[1]

Promoters


Non-coding regions



“a promoter is a region of DNA that
facilitates the transcription of a
particular gene”
DNA which does not contain
instructions for making proteins
Reading frames
An open reading frames
(ORF): a contiguous
sequence of DNA starting at
a start codon and ending at a
STOP codon
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Shotgun DNA Sequencing
More discussions can be found here
http://en.wikipedia.org/wiki/Shotgun_sequencing
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Sequence Files -- FASTA Format
>gi|40457238|HIV-1 isolate 97KE128 from Kenya gag gene, partial cds
CTTTTGAATGCATGGGTAAAAGTAATAGAAGAAAGAGGTTTCAGTCCAGAAGTAATACCCATGTTCTCAG
CATTATCAGAAGGAGCCACCCCACAAGATTTAAATACGATGCTGAACATAGTGGGGGGACACCAGGCAGC
TATGCAAATGCTAAAGGATACCATCAATGAGGAAGCTGCAGAATGGGACAGGTTACATCCAGTACATGCA
GGGCCTATTCCGCCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCCTC
AAGAACAAGTAGGATGGATGACAAACAATCCACCTATCCCAGTGGGAGACATCTATAAAAGATGGATCAT
CCTGGGCTTAAATAAAATAGTAAGAATGTATAGCCCTGTTAGCATTTTGGACATAAAACAAGGGCCAAAA
GAACCCTTTAGAGACTATGTAGATAGGTTCTTTAAAACTCTCAGAGCCGAACAAGCTT
>gi|40457236| HIV-1 isolate 97KE127 from Kenya gag gene, partial cds
TTGAATGCATGGGTGAAAGTAATAGAAGAAAAGGCTTTCAGCCCAGAAGTAATACCCATGTTCTCAGCAT
TATCAGAAGGAGCCACCCCACAAGATTTAAATATGATGCTGAATATAGTGGGGGGACACCAGGCAGCTAT
GCAAATGTTAAAAGATACCATCAATGAGGAAGCTGCAGAATGGGACAGGTTACATCCAATACATGCAGGG
CCTATTCCACCAGGCCAAATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCCTCAAG
AGCAAATAGGATGGATGACAAGCAACCCACCTATCCCAGTGGGAGACATCTATAAAAGATGGATAATCCT
GGGATTAAATAAAATAGTAAGAATGTATAGCCCTGTTAGCATTTTGGACATAAAACAAGGGCCAAAAGAA
CCTTTCAGAGACTATGTAGATAGGTTTTTTAAAACTCTCAGAGCCGAACAAGCTT
>gi|40457234| HIV-1 isolate 97KE126 from Kenya gag gene, partial cds
CCTTTGAATGCATGGGTGAAAGTAATAGAAGAAAAGGCTTTCAGCCCAGAAGTAATACCCATGTTTTCAG
CATTATCAGAAGGAGCCACCCCACAAGATTTAAATATGATGCTGAACATAGTGGGGGGGCACCAGGCAGC
TATGCAAATGTTAAAAGATACCATCAATGAGGAAGCTGCAGAATGGGACAGGCTACATCCAGCACAGGCA
GGGCCTATTGCACCAGGCCAGATAAGAGAACCAAGGGGAAGTGATATAGCAGGAACTACTAGTACCCCTC
AAGAACAAATAGCATGGATGACAGGCAACCCGCCTATCCCAGTGGGAGACATCTATAAAAGATGGATAAT
CCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTGTTAGCATTTTGGATATAAAACAAGGGCCAAAA
GAACCATTCAGAGACTATGTAGACAGGTTCTTTAAAACTCTCAGAGCCGAACAAGCTT
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GenBank Record
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
SOURCE
ORGANISM
AK091721
2234 bp
mRNA
linear
PRI 20-JAN-2006
Homo sapiens cDNA FLJ34402 fis, clone HCHON2001505.
AK091721
AK091721.1 GI:21750158
oligo capping; fis (full insert sequence).
Homo sapiens (human)
Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Euarchontoglires; Primates; Catarrhini;
Hominidae; Homo.
TITLE
Complete sequencing and characterization of 21,243 full-length
human cDNAs
JOURNAL
Nat. Genet. 36 (1), 40-45 (2004)
FEATURES
Location/Qualifiers
source
1..2234
/organism="Homo sapiens"
/mol_type="mRNA"
CDS
529..1995
/note="unnamed protein product"
/codon_start=1
/protein_id="BAC03731.1"
/db_xref="GI:21750159"
/translation="MVAERSPARSPGSWLFPGLWLLVLSGPGGLLRAQEQPSCRRAFD
...
RLDALWALLRRQYDRVSLMRPQEGDEGRCINFSRVPSQ"
ORIGIN
1 gttttcggag tgcggaggga gttggggccg ccggaggaga agagtctcca ctcctagttt
61 gttctgccgt cgccgcgtcc cagggacccc ttgtcccgaa gcgcacggca gcggggggaa
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...
20
Why Perl?

Widely used in Bioinformatics

Bioperl

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Ease of Programming

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Few lines of code needed for many problems
One-liners
Portability

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Excellent pattern matching features
Good for gluing other programs together
Easy to learn (enough to get started)
Rapid Prototyping


http://www.bioperl.org/wiki/Main_Page
Runs on Unix, Windows, Macs
Open Source Culture

Many sources of help ( try: %perldoc perldoc)



%perldoc –f print
http://perldoc.perl.org/index-tutorials.html
Many sources of useful modules ( http://www.cpan.org/ )
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Variables
The types of Perl variables are indicated by the initial symbol:
$var stores a scalar (a single string or number)
$x = 10;
$s = "ATTGCGT";
$x = 3.1417;
@var stores an array (a list of values)
@a = (10, 20, 30);
@a = (100, $x, "Jones", $s);
print "@a\n"; # prints "100 3.1417 Jones ATTGCGT"
%var stores a hash (associative array)
%ages = { John => 30, Mary => 22, Lakshmi => 27 };
print $age{"Mary"}, "\n";
# prints 22
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Declaring Variables
use strict;



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Putting use strict; at the top of your programs will tell perl to slap
your hands with a fatal error whenever you break certain rules.
Requires us to declare all variables
Avoids creating variable by typos
variables may be declaring using my, our or local
for now, we only need to use my:
my $a;
my ($a, $b, $c);
my @array;

# value of $a is undef
# $a, $b, $c are all undef
# value of @array is ()
Can combine declaration and initialization:
my @array = qw/A list of words/;
my $a = "A string";
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How Things Can Go Wrong
We will come back and
examine this after we have
discussed references.
http://www.perlmonks.org/?node_id=269642
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Scalar and List Context

All operations in Perl are evaluated in either scalar or list
context, and may behave differently depending on context
@array = ('one', 'two', 'three');
$a = @array;
# scalar context for assignment, return size
print $a;
# prints 3
($a) = @array;
print $a;
# list context for assignment
# prints 'one'
($a, $b) = @array;
print "$a, $b";
($a, $b, $c, $d) = @array;
# prints 'one, two'
# $d is undefined
In computer science a list is an ordered collection of values
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String Operations

Ways to concatenate strings
$DNA1
$DNA2
$DNA3
$DNA3
print
= "ATG";
= "CCC";
= $DNA1 . $DNA2;
= "$DNA1$DNA2";
"$DNA3";
$DNA3 = '$DNA1$DNA2';
print "$DNA3";
# concatenation operator
# string interpolation
# prints ATGCCC
# no string interpolation
# prints $DNA1$DNA2
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Arrays
An array stores an ordered list of scalars:
@gene_array = (‘EGF1’, ‘TFEC’, ‘CFTR’, ‘LOC1691’);
print “@gene_array\n”;
Output:
EGF1 TFEC CFTR LOC1691
# there’s more than one way to do it (see previous slide
on declaring variables)
@gene_array = qw/EGF1 TFEC CFTR LOC1691/;
http://www.perlmeme.org/howtos/perlfunc/qw_function.html
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Arrays
An array stores an ordered list of scalars:
@a = (‘one’, ‘two’, ‘three’, ‘four’);
The array is indexed by integers starting with 0:
print “$a[1] $a[0] $a[3]\n”;
prints:
two one four
Notice: $a[i] is a scalar since we used the $ method of
addressing the variable
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Unix Commands I


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cat --- for creating and displaying short files
chmod --- change permissions
cd --- change directory
cp --- for copying files
date --- display date
echo --- echo argument
ftp --- connect to a remote machine to download or upload files
grep --- search file
head --- display first part of file
ls --- see what files you have
lpr --- standard print command
more --- use to read files
mkdir --- create directory
mv --- for moving and renaming files
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Unix Commands II


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
pwd --- find out what directory you are in
rm --- remove a file
rmdir --- remove directory
setenv --- set an environment variable
sort --- sort file
tail --- display last part of file
tar --- create an archive, add or extract files
ssh --- log in to another machine
wc --- count characters, words, lines
This site has a nice reference card

http://www.digilife.be/quickreferences/QRC/UNIX%20commands%
20reference%20card.pdf
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chmod and tar

chmod

There is a nice tutorial here


http://www.perlfect.com/articles/chmod.shtml
tar

There is a nice tutorial here

http://www.thegeekstuff.com/2010/04/unix-tar-command-examples/
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Running perl on binf.gmu.edu
% ssh binf.gmu.edu
Password: ******
-- Create binf634 directory (don't type stuff in red)
% mkdir binf634
% cd binf634
% ls
-- Copy a file to current directory
-- (the "." means :current directory")
% cp ~jsolka/public_html/fall15/binf634/bookcode/examples/example4-1.pl .
% ls
-- This is a one not a l
% ls -l
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Running perl on binf.gmu.edu
% cat example4-1.pl
#!/usr/bin/perl -w
# Example 4-1
Storing DNA in a variable, and printing it out
# First we store the DNA in a variable called $DNA
$DNA = 'ACGGGAGGACGGGAAAATTACTACGGCATTAGC';
# Next, we print the DNA onto the screen
print $DNA;
# Finally, we'll specifically tell the program to exit.
exit;
-- Changing permissions
% chmod 755 example4-1.pl
-- Running a perl script
% example4-1.pl
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Editing a Perl Script
-- Read the Emacs or vi tutorial.
-- Make a copy and edit the copy
% cp example4-1.pl first.pl
% l
% vim first.pl
-- 1. Change 'print $DNA;' to 'print $DNA, "\n";'
-- 2. Now add a comment:
# Author: your name
% cat first.pl
#!/usr/bin/perl -w
# Author: Jeff Solka
# Example 4-1
Storing DNA in a variable, and printing it out
# First we store the DNA in a variable called $DNA
$DNA = 'ACGGGAGGACGGGAAAATTACTACGGCATTAGC';
# Next, we print the DNA onto the screen
print $DNA, "\n";
# Finally, we'll specifically tell the program to exit.
exit;
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For Next Week

Read Tisdall chapters 1-5.



HW 1: Write programs as described in the following exercises
from "Beginning Perl for Bioinformatics" by Tisdall:




Be ready to ask questions
Be ready to answer questions
4.3, 4.4, 4.5, 5.2, 5.4 and 5.6
For each exercise, create a perl script called exX.Y.pl, for example,
ex4.3.pl for the first exercise.
Submit these via Blackboard
No class next week because of labor day
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Some of the Details
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Alternative Development
Environments
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What is Eclipse?




Eclipse is a multi-language software development platform comprising
an IDE and a plug-in system to extend it. It is written primarily in Java
and is used to develop applications in this language and, by means of
the various plug-ins, in other languages as well—C/C++, Cobol,
Python, Perl, PHP and more.
The initial codebase originated from VisualAge.[1] In its default form it
is meant for Java developers, consisting of the Java Development Tools
(JDT). Users can extend its capabilities by installing plug-ins written for
the Eclipse software framework, such as development toolkits for other
programming languages, and can write and contribute their own plugin modules. Language packs provide translations into over a dozen
natural languages.[2]
Released under the terms of the Eclipse Public License, Eclipse is free
and open source software.
http://en.wikipedia.org/wiki/Eclipse_(software)
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What Operating Systems Does
Eclipse Run Under?

LINUX

MAC OSX

WINDOWS



7
8
10 ?
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Languages Supported by the
Eclipse IDE

JAVA


Out of the box
PERL

Via EPIC library


Note one must also have a PERL compiler
PYTHON

Via PyDev library

Note one must also have a PYTHON compiler installed
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Advantages and Disadvantages of the
Eclipse Development Environment

Advantages


Support for a plethora of languages
Industrial strength



Used by many professional software developer
Has support for configuration management
Disadvantages

Can be slow when developing in languages other than JAVA (may
be mere anecdotal evidence)
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Installing Eclipse Under Windows
XP - I

First make sure that you have a Java Runtime Environment installed
Microsoft Windows XP [Version 5.1.2600]
(C) Copyright 1985-2001 Microsoft Corp.
C:\Users\jlsol_000>C:"\Program
Files\Java\jdk1.7.0_12\bin"\java –version

If you don’t have a JRE installed go to

http://www.oracle.com/technetwork/java/javase/downloads/jre8downloads-2133155.html
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Installing Eclipse Under Windows
XP - II

Obtain the Eclipse zipped file from the Eclipse downloads link at
http://www.eclipse.org/downloads/

I believe that I chose this one




Eclipse IDE for Java Developers (163MB)
I think that the standard version works also
I think that the current version of Eclipse is Mars
Unzip it into an eclipse folder under your windows Program Files
directory

In my case here


C:\Program Files\eclipse
Note that Eclipse does not modify your system’s registry
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Installing Eclipse Under Windows
XP - III

Once installed (unzipped)



Double click on the eclipse.exe icon
There is a “hello world” java tutorial
There are a number of other tutorials


Eclipse3-1.pdf (http://www.cs.umanitoba.ca/~eclipse/Eclipse31.pdf)
http://wiki.eclipse.org/Eclipse_Articles,_Tutorials,_Demos,_Books,_
and_More
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Downloading ActiveStates
ActivePerl

Go here and click on the Windows download link




http://www.activestate.com/activeperl/
I previously set this up using version 5.10
Current version is 5.20.2
Use this self extracting binary to install the program

This takes a long time (30 minutes or more, go enjoy your favorite
beverage)
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Installing the Eclipse EPIC Library

This is my synopsis of this EPIC webpage tutorial


This is also a helpful site




http://www.epic-ide.org/faq.php
Under Eclipse user the Help->Software Updates Tab
Switch to the Available Software tab
Choose Add Site and choose


http://www.epic-ide.org/download.php
http://e-p-i-c.sf.net/updates
Tick the newly created site and click the install button
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Creating Your First PERL Program
Under the Eclipse IDE - I

Under Eclipse go to Window -> Open Perspective -> Other


Choose PERL
Under Eclipse go to Window -> Preferences

Click on the PERL + and enter in the full path to the ActiveStates
PERL executable


In my case it is
"C:\Perl\bin\perl5.10.0.exe"
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Creating Your First PERL Program
Under the Eclipse IDE - II

Click on File -> New PERL Project


Click on File -> New PERL File



Call it something like HelloWorld
Call it something like HelloWorldPerl
Left click on this file symbol and make sure its extension is .pl (Now it
should have a camel symbol)
Enter in your code
print "Hello from ActivePerl!\n";


Now you should be able to choose Run from the top menu or left click
on the program symbol and choose Run As Perl Local
If all goes well a console window with the output
Hello from ActivePerl!

should show up
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Debugging With Eclipse and PERL


The Perl PPM package PadWalker has to be installed before one
can debug your PERL programs under Eclipse
Follow the steps on the next two slides to install PadWalker
within ActiveStates PERL
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First Find the Package
(PadWalker)






Find a package.
To find a package in the repository:
Click the All packages button,
Enter text from the package's name or abstract in the Filter
field
As text is entered in the Filter field, the list of packages is
automatically updated as the substring match becomes more
precise. Click the magnifying glass icon to filter on different
meta-data (e.g. Author).
Alternatively, just start typing the name of the package. The
Package List will highlight the first package that matches the
string you have typed.
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Next Install the Package
(PadWalker)




Install a package.
To install a package from the repository:
Click on the desired package in the Package List to select it.
Mark the package by:






Clicking the Mark for install button or,
Hitting the "+" key or,
Selecting Install <package-name> from the Action menu or,
Right-clicking the selection and choosing Install <package-name>
from the context menu.
Click the Run marked actions button or select Run Marked
Actions (Ctrl-Enter) from the File menu.
In my case I installed PadWalker 1.7
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Installing PadWalker Via ppm

There are other interesting discussions here but they seem to
have been somewhat relegated by the gui-based ActiveStates
PERL ppm interface

http://trouchelle.com/perl/ppmrepview.pl
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Editors
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http://www.viemu.com/vi-vimcheat-sheet.gif
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https://www.gnu.org/software/e
macs/refcards/pdf/refcard.pdf
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winscp

A graphical user interface to scp

Available here

https://winscp.net/eng/download.php
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Notepad ++

A nice editor for windows

Can be configured to execute Perl via the Nppexec plugin

Available here

https://notepad-plus-plus.org
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Suggested Paths



Do editing on own system using notepad ++ copy to binf using
scp and run there
 This is best
Can do development on your own system using Perl and
notepad ++
 Always test programs on binf
If you are adventurous use Eclipse but it can be confusing to
the computer science novice
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