Introduction to

Bioinformatics
Esa Pitkänen
[email protected]
Autumn 2008, I period
www.cs.helsinki.fi/mbi/courses/08-09/itb

582606 Introduction to Bioinformatics, Autumn 2008

 Introduction to
Bioinformatics
Lecture 1:
Administrative issues
MBI Programme, Bioinformatics courses
What is bioinformatics?
Molecular biology primer
How to enrol for the course?
p Use the registration system of the Computer
Science department: https://ilmo.cs.helsinki.fi
n You need your user account at the IT department (“cc
account”)
p If you cannot register yet, don’t worry: attend the
lectures and exercises; just register when you are
able to do so

3
 Teachers
p Esa Pitkänen, Department of Computer Science,
University of Helsinki
p Elja Arjas, Department of Mathematics and
Statistics, University of Helsinki
p Sami Kaski, Department of Information and
Computer Science, Helsinki University of
Technology
p Lauri Eronen, Department of Computer Science,
University of Helsinki (exercises)

4
 Lectures and exercises
p Lectures: Tuesday and Friday 14.15-16.00
Exactum C221

p Exercises: Tuesday 16.15-18.00 Exactum

C221
n First exercise session on Tue 9 September

5
 Status & Prerequisites
p Advanced level course at the Department
of Computer Science, U. Helsinki
p 4 credits
p Prerequisites:
n Basic mathematics skills (probability calculus,
basic statistics)
n Familiarity with computers
n Basic programming skills recommended
n No biology background required

6
 Course contents
p What is bioinformatics?
p Molecular biology primer
p Biological words
p Sequence assembly
p Sequence alignment
p Fast sequence alignment using FASTA and BLAST
p Genome rearrangements
p Motif finding (tentative)
p Phylogenetic trees
p Gene expression analysis

7
 How to pass the course?
p Recommended method:
n Attend the lectures (not obligatory though)
n Do the exercises
n Take the course exam
p Or:
n Take a separate exam

8
 How to pass the course?
p Exercises give you max. 12 points
n 0% completed assignments gives you 0 points,
80% gives 12 points, the rest by linear
interpolation
n “A completed assignment” means that
p You are willing to present your solution in the
exercise session and
p You return notes by e-mail to Lauri Eronen (see
course web page for contact info) describing the main
phases you took to solve the assignment
n Return notes at latest on Tuesdays 16.15

p Course exam gives you max. 48 points

9
 How to pass the course?
p Grading: on the scale 0-5
n To get the lowest passing grade 1, you need to get at
least 30 points out of 60 maximum
p Course exam: Wed 15 October 16.00-19.00
Exactum A111
p See course web page for separate exams
p Note: if you take the first separate exam, the
best of the following options will be considered:
n Exam gives you 48 points, exercises 12 points
n Exam gives you 60 points
p In second and subsequent separate exams, only
the 60 point option is in use

10
 Literature
p Deonier, Tavaré,
Waterman: Computational
Genome Analysis, an
Introduction. Springer,
2005
p Jones, Pevzner: An
Introduction to
Bioinformatics Algorithms.
MIT Press, 2004
p Slides for some lectures
will be available on the
course web page

11
 Additional literature
p Gusfield: Algorithms on
strings, trees and
sequences
p Griffiths et al: Introduction
to genetic analysis
p Alberts et al.: Molecular
biology of the cell
p Lodish et al.: Molecular cell
biology

p Check the course web site

12
 Questions about administrative &
practical stuff?

13
Master's Degree Programme in
Bioinformatics (MBI)
p Two-year MSc programme
p Admission for 2009-2010 in January 2009
n You need to have your Bachelor’s degree ready by
August 2009

www.cs.helsinki.fi/mbi

14
MBI programme organizers

Department of Computer Science, Laboratory of Computer and

Department of Mathematics and Statistics Information Science, Laboratory of
Faculty of Science, Kumpula Campus, HY CS and Engineering,TKK

Faculty of Biosciences
Faculty of Agriculture and Forestry
Viikki Campus, HY

Faculty of Medicine, Meilahti Campus, HY

15
 Four MBI campuses
HY, Viikki

HY, Meilahti
HY, Kumpula

TKK, Otaniemi

16
 MBI highlights
p You can take courses from both HY and
TKK
p Two biology courses tailored specifically
for MBI
p Bioinformatics is a new exciting field, with
a high demand for experts in job market

p Go to www.cs.helsinki.fi/mbi/careers to
find out what a bioinformatician could do
for living

17
 Admission
p Admission requirements
n Bachelor’s degree in a suitable field (e.g., computer
science, mathematics, statistics, biology or medicine)
n At least 60 ECTS credits in total in computer science,
mathematics and statistics
n Proficiency in English (standardized language test:
TOEFL, IELTS)
p Admission period opens in late Autumn 2009 and
closes in 2 February 2009
p Details on admission will be posted in
www.cs.helsinki.fi/mbi during this autumn

18
 Bioinformatics courses in Helsinki
region: 1st period
p Computational genomics (4-7 credits, TKK)
p Seminar: Neuroinformatics (3 credits, Kumpula)
p Seminar: Machine Learning in Bioinformatics (3
credits, Kumpula)
p Signal processing in neuroinformatics (5 credits,
TKK)

19
 A good biology course for computer
scientists and mathematicians?
p Biology for methodological scientists (8 credits, Meilahti)
n Course organized by the Faculties of Bioscience and Medicine
for the MBI programme
n Introduction to basic concepts of microarrays, medical genetics
and developmental biology
n Study group + book exam in I period (2 cr)
n Three lectured modules, 2 cr each
n Each module has an individual registration so you can
participate even if you missed the first module
n www.cs.helsinki.fi/mbi/courses/08-09/bfms/

20
 Bioinformatics courses in Helsinki
region: 2nd period
p Bayesian paradigm in genetic bioinformatics (6
credits, Kumpula)
p Biological Sequence Analysis (6 credits, Kumpula)
p Modeling of biological networks (5-7 credits, TKK)
p Statistical methods in genetics (6-8 credits,
Kumpula)

21
 Bioinformatics courses in Helsinki
region: 3rd period
p Evolution and the theory of games (5 credits, Kumpula)
p Genome-wide association mapping (6-8 credits, Kumpula)
p High-Throughput Bioinformatics (5-7 credits, TKK)
p Image Analysis in Neuroinformatics (5 credits, TKK)
p Practical Course in Biodatabases (4-5 credits, Kumpula)
p Seminar: Computational systems biology (3 credits,
Kumpula)
p Spatial models in ecology and evolution (8 credits,
Kumpula)
p Special course in bioinformatics I (3-7 credits, TKK)

22
 Bioinformatics courses in Helsinki region:
4th period
p Metabolic Modeling (4 credits, Kumpula)
p Phylogenetic data analyses (6-8 credits,
Kumpula)

23
 1. What is bioinformatics?

24
What is bioinformatics?
p Bioinformatics, n. The science of information
and information flow in biological systems,
esp. of the use of computational methods in
genetics and genomics. (Oxford English
Dictionary)

p "The mathematical, statistical and computing

methods that aim to solve biological problems
using DNA and amino acid sequences and
related information." -- Fredj Tekaia

25
 What is bioinformatics?
p "I do not think all biological computing is
bioinformatics, e.g. mathematical modelling is
not bioinformatics, even when connected with
biology-related problems. In my opinion,
bioinformatics has to do with management and
the subsequent use of biological information,
particular genetic information."
-- Richard Durbin

26
What is not bioinformatics?
p Biologically-inspired computation, e.g., genetic algorithms
and neural networks
p However, application of neural networks to solve some
biological problem, could be called bioinformatics
p What about DNA computing?

27 http://www.wisdom.weizmann.ac.il/~lbn/new_pages/Visual_Presentation.html
Computational biology
p Application of computing to biology (broad
definition)
p Often used interchangeably with bioinformatics
p Or: Biology that is done with computational
means

28
 Biometry & biophysics
p Biometry: the statistical analysis of biological
data
n Sometimes also the field of identification of individuals
using biological traits (a more recent definition)
p Biophysics: "an interdisciplinary field which
applies techniques from the physical sciences
to understanding biological structure and
function" -- British Biophysical Society

29
 Mathematical biology
p Mathematical biology
“tackles biological
problems, but the methods
it uses to tackle them need
not be numerical and need
not be implemented in
software or hardware.”
-- Damian Counsell

Alan Turing

30
 Turing on biological complexity
p “It must be admitted that the biological examples which
it has been possible to give in the present paper are very
limited.

This can be ascribed quite simply to the fact that

biological phenomena are usually very complicated.
Taking this in combination with the relatively elementary
mathematics used in this paper one could hardly expect to
find that many observed biological phenomena would be
covered.

It is thought, however, that the imaginary biological

systems which have been treated, and the principles which
have been discussed, should be of some help in
interpreting real biological forms.”

– Alan Turing, The Chemical Basis of Morphogenesis, 1952

31
 Related concepts
p Systems biology
n “Biology of networks”
n Integrating different levels
of information to
understand how biological
systems work
p Computational systems biology

Overview of metabolic pathways in

KEGG database, www.genome.jp/kegg/

32
 Why is bioinformatics important?
p New measurement techniques produce
huge quantities of biological data
n Advanced data analysis methods are needed to
make sense of the data
n Typical data sources produce noisy data with a
lot of missing values
p Paradigm shift in biology to utilise
bioinformatics in research

33
 Bioinformatician’s skill set
p Statistics, data analysis methods
n Lots of data
n High noise levels, missing values
n #attributes >> #data points
p Programming languages
n Scripting languages: Python, Perl, Ruby, …
n Extensive use of text file formats: need
parsers
n Integration of both data and tools
p Data structures, databases

34
 Bioinformatician’s skill set
p Modelling
n Discrete vs continuous domains
n -> Systems biology
p Scientific computation packages
n R, Matlab/Octave, …
p Communication skills!

35
 Communication skills: case 1
? Biologist presents a problem
to computer scientists /
mathematicians

”I am interested in finding what affects the

regulation gene x during condition y and how
that relates to the organism’s phenotype.”

”Define input and output of the problem.”

36
 Communication skills: case 2
Bioinformatician is a part
of a group that consists
mostly of biologists.

37
 Communication skills: case 2

...biologist/bioinformatician ratio is important!

38
 Communication skills: case 3
A group of
bioinformaticians
offers their services to
more than one group

39
 Bioinformatician’s skill set
p How much biology you should know?

40
 Bioinformatician’s skill set
Bioinformatics
Biology & Medicine • Biological sequence analysis
• Basics in molecular and • Biological databases
cell biology • Analysis of gene expression
• Measurement techniques • Modeling protein structure and
function
• Gene, protein and metabolic
networks
Computer Science •…
• Programming Mathematics and
• Databases statistics
• Algorithmics • Calculus
• Probability calculus
• Linear algebra

Where would you be in this triangle?

41 Prof. Juho Rousu, 2006

 A problem involving bioinformatics?

- ”I found a fruit fly that is immune to all diseases!”

- ”It was one of these”

42 Pertti Jarla, http://www.hs.fi/fingerpori/

 Molecular biology primer

Molecular Biology Primer by Angela Brooks, Raymond Brown,

Calvin Chen, Mike Daly, Hoa Dinh, Erinn Hama, Robert Hinman,
Julio Ng, Michael Sneddon, Hoa Troung, Jerry Wang, Che Fung
Yung
Edited for Introduction to Bioinformatics (Autumn 2007, Summer
43 2008, Autumn 2008) by Esa Pitkänen
 Molecular biology primer
p Part 1: What is life made of?
p Part 2: Where does the variation in
genomes come from?

44
 Life begins with Cell

p A cell is a smallest structural unit of an

organism that is capable of independent
functioning
p All cells have some common features
45
 Cells
p Fundamental working units of every living system.
p Every organism is composed of one of two radically different types of
cells:
n prokaryotic cells or
n eukaryotic cells.
p Prokaryotes and Eukaryotes are descended from the same
primitive cell.
n All prokaryotic and eukaryotic cells are the result of a total of 3.5
billion years of evolution.

46
Two types of cells: Prokaryotes and
Eukaryotes

47
 Prokaryotes and Eukaryotes
p According to the most
recent evidence, there
are three main
branches to the tree of
life
p Prokaryotes include
Archaea (“ancient
ones”) and bacteria
p Eukaryotes are
kingdom Eukarya and
includes plants,
animals, fungi and
certain algae Lecture: Phylogenetic trees

48
 All Cells have common Cycles

p Born, eat, replicate, and die

49
 Common features of organisms
p Chemical energy is stored in ATP
p Genetic information is encoded by DNA
p Information is transcribed into RNA
p There is a common triplet genetic code
p Translation into proteins involves ribosomes
p Shared metabolic pathways
p Similar proteins among diverse groups of
organisms

50
 All Life depends on 3 critical molecules
p DNAs (Deoxyribonucleic acid)
n Hold information on how cell works
p RNAs (Ribonucleic acid)
n Act to transfer short pieces of information to different
parts of cell
n Provide templates to synthesize into protein
p Proteins
n Form enzymes that send signals to other cells and
regulate gene activity
n Form body’s major components (e.g. hair, skin, etc.)
n “Workhorses” of the cell

51
 DNA: The Code of Life

p The structure and the four genomic letters code for all living
organisms
p Adenine, Guanine, Thymine, and Cytosine which pair A-T and C-G
on complimentary strands.
Lecture: Genome sequencing
52 and assembly
 Discovery of the structure of DNA
p 1952-1953 James D. Watson and Francis H. C. Crick
deduced the double helical structure of DNA from X-ray
diffraction images by Rosalind Franklin and data on amounts
of nucleotides in DNA

”Photo 51” Rosalind

Franklin

James Watson and

Francis Crick
53
 DNA, continued
p DNA has a double helix
structure which is
composed of
n sugar molecule
n phosphate group
n and a base (A,C,G,T)

p By convention, we read
DNA strings in direction of
transcription: from 5’ end
to 3’ end
5’ ATTTAGGCC 3’
3’ TAAATCCGG 5’

54
 DNA is contained in chromosomes

p In eukaryotes, DNA is packed into chromatids

n In metaphase, the “X” structure consists of two identical
chromatids
p In prokaryotes, DNA is usually contained in a single,
circular chromosome

55
http://en.wikipedia.org/wiki/Image:Chromatin_Structures.png
 Human chromosomes
p Somatic cells in humans
have 2 pairs of 22
chromosomes + XX
(female) or XY (male) =
total of 46 chromosomes
p Germline cells have 22
chromosomes + either X or
Y = total of 23
chromosomes

Karyogram of human male using Giemsa staining

(http://en.wikipedia.org/wiki/Karyotype)

56
 Length of DNA and number of chromosomes
Organism #base pairs #chromosomes (germline)

Prokayotic
Escherichia coli (bacterium) 4x106 1

Eukaryotic
Saccharomyces cerevisia (yeast) 1.35x107 17
Drosophila melanogaster (insect) 1.65x108 4
Homo sapiens (human) 2.9x109 23
Zea mays (corn / maize) 5.0x109 10

57
 Hepatitis delta virus, complete genome
1 atgagccaag ttccgaacaa ggattcgcgg ggaggataga tcagcgcccg agaggggtga
61 gtcggtaaag agcattggaa cgtcggagat acaactccca agaaggaaaa aagagaaagc
121 aagaagcgga tgaatttccc cataacgcca gtgaaactct aggaagggga aagagggaag
181 gtggaagaga aggaggcggg cctcccgatc cgaggggccc ggcggccaag tttggaggac
241 actccggccc gaagggttga gagtacccca gagggaggaa gccacacgga gtagaacaga
301 gaaatcacct ccagaggacc ccttcagcga acagagagcg catcgcgaga gggagtagac
361 catagcgata ggaggggatg ctaggagttg ggggagaccg aagcgaggag gaaagcaaag
421 agagcagcgg ggctagcagg tgggtgttcc gccccccgag aggggacgag tgaggcttat
481 cccggggaac tcgacttatc gtccccacat agcagactcc cggaccccct ttcaaagtga
541 ccgagggggg tgactttgaa cattggggac cagtggagcc atgggatgct cctcccgatt
601 ccgcccaagc tccttccccc caagggtcgc ccaggaatgg cgggacccca ctctgcaggg
661 tccgcgttcc atcctttctt acctgatggc cggcatggtc ccagcctcct cgctggcgcc
721 ggctgggcaa cattccgagg ggaccgtccc ctcggtaatg gcgaatggga cccacaaatc
781 tctctagctt cccagagaga agcgagagaa aagtggctct cccttagcca tccgagtgga
841 cgtgcgtcct ccttcggatg cccaggtcgg accgcgagga ggtggagatg ccatgccgac
901 ccgaagagga aagaaggacg cgagacgcaa acctgcgagt ggaaacccgc tttattcact
961 ggggtcgaca actctgggga gaggagggag ggtcggctgg gaagagtata tcctatggga
1021 atccctggct tccccttatg tccagtccct ccccggtccg agtaaagggg gactccggga
1081 ctccttgcat gctggggacg aagccgcccc cgggcgctcc cctcgttcca ccttcgaggg
1141 ggttcacacc cccaacctgc gggccggcta ttcttctttc ccttctctcg tcttcctcgg
1201 tcaacctcct aagttcctct tcctcctcct tgctgaggtt ctttcccccc gccgatagct
1261 gctttctctt gttctcgagg gccttccttc gtcggtgatc ctgcctctcc ttgtcggtga
1321 atcctcccct ggaaggcctc ttcctaggtc cggagtctac ttccatctgg tccgttcggg
1381 ccctcttcgc cgggggagcc ccctctccat ccttatcttt ctttccgaga attcctttga
1441 tgtttcccag ccagggatgt tcatcctcaa gtttcttgat tttcttctta accttccgga
1501 ggtctctctc gagttcctct aacttctttc ttccgctcac ccactgctcg agaacctctt
1561 ctctcccccc gcggtttttc cttccttcgg gccggctcat cttcgactag aggcgacggt
1621 cctcagtact cttactcttt tctgtaaaga ggagactgct ggccctgtcg cccaagttcg
58
1681 ag
 RNA
p RNA is similar to DNA chemically. It is usually only a
single strand. T(hyamine) is replaced by U(racil)
p Several types of RNA exist for different functions in
the cell.

tRNA linear and 3D view: http://www.cgl.ucsf.edu/home/glasfeld/tutorial/trna/trna.gif

59
 DNA, RNA, and the Flow of
Information
Replication
”The central dogma”

Transcription Translation

Is this true?

Denis Noble: The principles of Systems Biology illustrated using the virtual heart
60 http://velblod.videolectures.net/2007/pascal/eccs07_dresden/noble_denis/eccs07_noble_psb_01.ppt
 Proteins
p Proteins are polypeptides
(strings of amino acid
residues)
p Represented using strings
of letters from an alphabet
of 20: AEGLV…WKKLAG
p Typical length 50…1000
residues

Urease enzyme from Helicobacter pylori

61
 http://upload.wikimedia.org/wikipedia/commons/c/c5/Amino_acids_2.png

Amino acids

62
 How DNA/RNA codes for protein?
p DNA alphabet contains four
letters but must specify
protein, or polypeptide
sequence of 20 letters.
p Dinucleotides are not
enough: 42 = 16 possible
dinucleotides
p Trinucleotides (triplets)
allow 43 = 64 possible
trinucleotides
p Triplets are also called
codons

63
 How DNA/RNA codes for protein?
p Three of the possible
triplets specify ”stop
translation”
p Translation usually starts
at triplet AUG (this codes
for methionine)
p Most amino acids may be
specified by more than
triplet
p How to find a gene? Look
for start and stop codons
(not that easy though)

64
 Proteins: Workhorses of the Cell
p 20 different amino acids
n different chemical properties cause the protein chains to fold
up into specific three-dimensional structures that define their
particular functions in the cell.
p Proteins do all essential work for the cell
n build cellular structures
n digest nutrients
n execute metabolic functions
n mediate information flow within a cell and among cellular
communities.
p Proteins work together with other proteins or nucleic acids
as "molecular machines"
n structures that fit together and function in highly specific, lock-
and-key ways.
Lecture 8: Proteomics
65
 Genes
p “A gene is a union of genomic sequences encoding a
coherent set of potentially overlapping functional products”
--Gerstein et al.
p A DNA segment whose information is expressed either as
an RNA molecule or protein

(translation) (folding)
MSG …
a ...
ugg
g
uga
a (transcription)

5’ … a t g a g t g g a … 3’

3’ … t a c t c a c c t … 5’

66 http://fold.it
 FoldIt: Protein folding game

http://fold.it
67
 Genes & alleles
p A gene can have different variants
p The variants of the same gene are called
alleles

... ...
a a
ugg uc g
g MSG … g
ga g a MSR …
au au

5’ … a t g a g t g g a … 5’ … a t g a g t c g a …

3’ … t a c t c a c c t … 3’ … t a c t c a g c t …

68
 Genes can be found on both strands

5’ 3’

3’ 5’

69
 Exons and introns & splicing
Exons

5’ 3’

3’ 5’

Introns are removed from RNA after transcription

Exons are joined:

This process is called splicing

70
 Alternative splicing
Different splice variants may be generated

5’ A B C 3’

3’ 5’

A B C

B C

A C

…
71
 Where does the variation in genomes come
from?
p Prokaryotes are typically
haploid: they have a single
(circular) chromosome
p DNA is usually inherited
vertically (parent to
daughter)
p Inheritance is clonal
n Descendants are faithful
copies of an ancestral DNA
n Variation is introduced via
mutations, transposable
elements, and horizontal
transfer of DNA
Chromosome map of S. dysenteriae, the nine rings
describe different properties of the genome
http://www.mgc.ac.cn/ShiBASE/circular_Sd197.htm

72
 Causes of variation
p Mistakes in DNA replication
p Environmental agents (radiation, chemical
agents)
p Transposable elements (transposons)
n A part of DNA is moved or copied to another location in
genome
p Horizontal transfer of DNA
n Organism obtains genetic material from another
organism that is not its parent
n Utilized in genetic engineering

73
 Biological string manipulation
p Point mutation: substitution of a base
n …ACGGCT… => …ACGCCT…
p Deletion: removal of one or more contiguous
bases (substring)
n …TTGATCA… => …TTTCA…
p Insertion: insertion of a substring
n …GGCTAG… => …GGTCAACTAG…

Lecture: Sequence alignment

Lecture: Genome rearrangements

74
 Meiosis
p Sexual organisms are usually
diploid
n Germline cells (gametes)
contain N chromosomes
n Somatic (body) cells have 2N
chromosomes
p Meiosis: reduction of
chromosome number from
2N to N during reproductive
cycle Major events in meiosis
n One chromosome doubling is http://en.wikipedia.org/wiki/Meiosis
followed by two cell divisions
http://www.ncbi.nlm.nih.gov/About/Primer

75
 Recombination and variation
p Recap: Allele is a viable DNA
coding occupying a given locus
(position in the genome)
p In recombination, alleles from
parents become suffled in
offspring individuals via
chromosomal crossover over
p Allele combinations in
offspring are usually different
from combinations found in
parents
p Recombination errors lead into
additional variations
Chromosomal crossover as described by
T. H. Morgan in 1916
76
 Mitosis

p Mitosis: growth and development of the organism

n One chromosome doubling is followed by one cell
division

77
http://en.wikipedia.org/wiki/Image:Major_events_in_mitosis.svg
 Recombination frequency and linked genes
p Genetic marker: some DNA sequence of interest
(e.g., gene or a part of a gene)

p Recombination is more likely to separate two

distant markers than two close ones

p Linked markers: ”tend” to be inherited together

p Marker distances measured in centimorgans: 1

centimorgan corresponds to 1% chance that two
markers are separated in recombination

78
 Biological databases
p Exponential growth of
biological data
n New measurement
techniques
n Before we are able to use
the data, we need to store
it efficiently -> biological
databases
n Published data is
submitted to databases
p General vs specialised
databases
p This topic is discussed
extensively in Practical
course in biodatabases (III
period)

79
 10 most important biodatabases… according
to ”Bioinformatics for dummies”
p GenBank/DDJB/EMBL www.ncbi.nlm.nih.gov Nucleotide sequences
p Ensembl www.ensembl.org Human/mouse genome
p PubMed www.ncbi.nlm.nih.gov Literature references
p NR www.ncbi.nlm.nih.gov Protein sequences
p UniProt www.expasy.org Protein sequences
p InterPro www.ebi.ac.uk Protein domains
p OMIM www.ncbi.nlm.nih.gov Genetic diseases
p Enzymes www.expasy.org Enzymes
p PDB www.rcsb.org/pdb/ Protein structures
p KEGG www.genome.ad.jp Metabolic pathways

80 Sophia Kossida, Introduction to Bioinformatics, Summer 2008

 FASTA format
p A simple format for DNA and protein sequence
data is FASTA
Header line,
begins with >
>Hepatitis delta virus, complete genome
atgagccaagttccgaacaaggattcgcggggaggatagatcagcgcccgagaggggtga
gtcggtaaagagcattggaacgtcggagatacaactcccaagaaggaaaaaagagaaagc
aagaagcggatgaatttccccataacgccagtgaaactctaggaaggggaaagagggaag
gtggaagagaaggaggcgggcctcccgatccgaggggcccggcggccaagtttggaggac
actccggcccgaagggttgagagtaccccagagggaggaagccacacggagtagaacaga
gaaatcacctccagaggaccccttcagcgaacagagagcgcatcgcgagagggagtagac
catagcgataggaggggatgctaggagttgggggagaccgaagcgaggaggaaagcaaag
agagcagcggggctagcaggtgggtgttccgccccccgagaggggacgagtgaggcttat
cccggggaactcgacttatcgtccccacatagcagactcccggaccccctttcaaagtga
…

81
Introduction to
Bioinformatics
Biological words
 Recap
p DNA codes information with alphabet of 4
letters: A, C, G, T
p In proteins, the alphabet size is 20
p DNA -> RNA -> Protein (genetic code)
n Three DNA bases (triplet, codon) code for one
amino acid
n Redundancy, start and stop codons

83
Given a DNA sequence, we might ask a number of questions
1 atgagccaag ttccgaacaa ggattcgcgg ggaggataga tcagcgcccg agaggggtga
61 gtcggtaaag agcattggaa cgtcggagat acaactccca agaaggaaaa aagagaaagc
121 aagaagcgga tgaatttccc cataacgcca gtgaaactct aggaagggga aagagggaag
What sort of statistics
gaagggttga should begagggaggaa
used to gccacacgga
describe the sequence?
181 gtggaagaga aggaggcggg cctcccgatc cgaggggccc ggcggccaag tttggaggac
241 actccggccc gagtacccca gtagaacaga
301 gaaatcacct ccagaggacc ccttcagcga acagagagcg catcgcgaga gggagtagac
361 catagcgata ggaggggatg ctaggagttg ggggagaccg aagcgaggag gaaagcaaag
421 agagcagcgg ggctagcagg tgggtgttcc gccccccgag aggggacgag tgaggcttat
481 cccggggaac tcgacttatc gtccccacat agcagactcc cggaccccct ttcaaagtga
541 ccgagggggg tgactttgaa cattggggac cagtggagcc atgggatgct cctcccgatt
What sort of organism
601 ccgcccaagc tccttcccccdid this sequence
caagggtcgc ccaggaatggcome from?
cgggacccca ctctgcaggg
661 tccgcgttcc atcctttctt acctgatggc cggcatggtc ccagcctcct cgctggcgcc
721 ggctgggcaa cattccgagg ggaccgtccc ctcggtaatg gcgaatggga cccacaaatc
781 tctctagctt cccagagaga agcgagagaa aagtggctct cccttagcca tccgagtgga
841 cgtgcgtcct ccttcggatg cccaggtcgg accgcgagga ggtggagatg ccatgccgac
901 ccgaagagga aagaaggacg cgagacgcaa acctgcgagt ggaaacccgc tttattcact
Does the
961 ggggtcgaca actctgggga description
gaggagggag of this
ggtcggctgg sequence
gaagagtata differ from
tcctatggga
the description of other DNA in the organism?
1021 atccctggct tccccttatg tccagtccct ccccggtccg agtaaagggg gactccggga
1081 ctccttgcat gctggggacg aagccgcccc cgggcgctcc cctcgttcca ccttcgaggg
1141 ggttcacacc cccaacctgc gggccggcta ttcttctttc ccttctctcg tcttcctcgg
1201 tcaacctcct aagttcctct tcctcctcct tgctgaggtt ctttcccccc gccgatagct
1261 gctttctctt gttctcgagg gccttccttc gtcggtgatc ctgcctctcc ttgtcggtga
1321 atcctcccct ggaaggcctc ttcctaggtc cggagtctac ttccatctgg tccgttcggg
What sort of sequence
1381 ccctcttcgc is this? What
cgggggagcc ccctctccat doesctttccgaga
ccttatcttt it do? attcctttga
1441 tgtttcccag ccagggatgt tcatcctcaa gtttcttgat tttcttctta accttccgga
1501 ggtctctctc gagttcctct aacttctttc ttccgctcac ccactgctcg agaacctctt
1561 ctctcccccc gcggtttttc cttccttcgg gccggctcat cttcgactag aggcgacggt
1621 cctcagtact cttactcttt tctgtaaaga ggagactgct ggccctgtcg cccaagttcg
84 1681 ag
 Biological words
p We can try to answer questions like these
by considering the words in a sequence
p A k-word (or a k-tuple) is a string of length
k drawn from some alphabet
p A DNA k-word is a string of length k that
consists of letters A, C, G, T
n 1-words: individual nucleotides (bases)
n 2-words: dinucleotides (AA, AC, AG, AT, CA, ...)
n 3-words: codons (AAA, AAC, …)
n 4-words and beyond

85
 1-words: base composition
p Typically DNA exists as duplex molecule
(two complementary strands)

5’-GGATCGAAGCTAAGGGCT-3’
3’-CCTAGCTTCGATTCCCGA-5’

Top strand: 7 G, 3 C, 5 A, 3 T
These are something
Bottom strand: 3 G, 7 C, 3 A, 5 T
we can determine
Duplex molecule: 10 G, 10 C, 8 A, 8 T
experimentally.
Base frequencies: 10/36 10/36 8/36 8/36

fr(G + C) = 20/36, fr(A + T) = 1 – fr(G + C) = 16/36

86
 G+C content
p fr(G + C), or G+C content is a simple
statistics for describing genomes
p Notice that one value is enough
characterise fr(A), fr(C), fr(G) and fr(T)
for duplex DNA
p Is G+C content (= base composition) able
to tell the difference between genomes of
different organisms?
n Simple computational experiment, if we have
the genome sequences under study
(-> exercises)

87
 G+C content and genome sizes (in
megabasepairs, Mb) for various organisms
p Mycoplasma genitalium 31.6% 0.585
p Escherichia coli K-12 50.7% 4.693
p Pseudomonas aeruginosa PAO1 66.4% 6.264
p Pyrococcus abyssi 44.6% 1.765
p Thermoplasma volcanium 39.9% 1.585
p Caenorhabditis elegans 36% 97
p Arabidopsis thaliana 35% 125
p Homo sapiens 41% 3080

88
 Base frequencies in duplex molecules
p Consider a DNA sequence generated randomly,
with probability of each letter being independent
of position in sequence
p You could expect to find a uniform distribution of
bases in genomes…

5’-...GGATCGAAGCTAAGGGCT...-3’
3’-...CCTAGCTTCGATTCCCGA...-5’
p This is not, however, the case in genomes,
especially in prokaryotes
n This phenomena is called GC skew

89
 DNA replication fork
p When DNA is replicated, Replication fork movement
the molecule takes the
replication fork form Leading strand
p New complementary
DNA is synthesised at
both strands of the
”fork”
p New strand in 5’-3’
direction corresponding
to replication fork
movement is called
leading strand and the
other lagging strand Lagging strand

Replication fork
90
 DNA replication fork
p This process has Replication fork movement
specific starting
points in genome Leading strand
(origins of
replication)
p Observation:
Leading strands
have an excess of G
over C
p This can be
described by GC
skew statistics Lagging strand

Replication fork
91
 GC skew
p GC skew is defined as (#G - #C) / (#G +
#C)
p It is calculated at successive positions in
intervals (windows) of specific width

5’-...GGATCGAAGCTAAGGGCT...-3’
3’-...CCTAGCTTCGATTCCCGA...-5’
(4 – 2) / (4 + 2) = 1/3

(3 – 2) / (3 + 2) = 1/5

92
 G-C content & GC skew
p G-C content &
GC skew
statistics can be
displayed with a
circular genome
map

G+C content

GC skew

(10kb window size) Chromosome map of S. dysenteriae, the nine rings

describe different properties of the genome
93
http://www.mgc.ac.cn/ShiBASE/circular_Sd197.htm
 GC skew
p GC skew
often
changes sign
at origins
and termini
of replication

G+C content

GC skew

(10kb window size)

94
Nie et al., BMC Genomics, 2006
 2-words: dinucleotides
p Let’s consider a sequence L1,L2,...,Ln
where each letter Li is drawn from the
DNA alphabet {A, C, G, T}
p We have 16 possible dinucleotides lili+1:
AA, AC, AG, ..., TG, TT.

95
 i.i.d. model for nucleotides
p Assume that bases
n occur independently of each other
n bases at each position are identically
distributed
p Probability of the base A, C, G, T occuring
is pA, pC, pG, pT, respectively
n For example, we could use pA=pC=pG=pT=0.25
or estimate the values from known genome
data
p Probability of lili+1 is then PliPli+1
n For example, P(TG) = pT pG

96
 2-words: is what we see surprising?
p We can test whether a sequence is ”unexpected”,
for example, with a 2 test

p Test statistic for a particular dinucleotide r1r2 is

2 = (O – E)2 / E where

n O is the observed number of dinucleotide r1r2

n E is the expected number of dinucleotide r1r2
n E = (n – 1)pr1pr2 under i.i.d. model

p Basic idea: high values of 2 indicate deviation

from the model
n Actual procedure is more detailed -> basic statistics
courses

97
 Refining the i.i.d. model
p i.i.d. model describes some organisms well
(see Deonier’s book) but fails to
characterise many others

p We can refine the model by having the

DNA letter at some position depend on
letters at preceding positions

…TCGTGACGC C G ?
Sequence context to
consider
98
 First-order Markov chains
Xt

…TCGTGACGC C G ?
Xt-1

p Lets assume that in sequence X the letter at

position t, Xt, depends only on the previous letter
Xt-1 (first-order markov chain)
p Probability of letter j occuring at position t given
Xt-1 = i: pij = P(Xt = j | Xt-1 = i)
p We consider homogeneous markov chains:
probability pij is independent of position t
99
 Estimating pij
p We can estimate probabilities pij (”the probability
that j follows i”) from observed dinucleotide
frequencies
Frequency
of dinucleotide AT
A C G T in sequence
A pAA pAC pAG pAT
C pCA + pCC + pCG + pCT Base frequency
fr(C)
G pGA pGC pGG pGT
T pTA pTC pTG pTT

…the values pAA, pAC, ..., pTG, pTT sum to 1

100
 Estimating pij Dinucleotide frequency

p pij = P(Xt = j | Xt-1 = i) = P(Xt = j, Xt-1 = i)

P(Xt-1 = i)
Probability of transition i -> j Base frequency of nucleotide i,
fr(i)
0.052 / 0.345 0.151

A C G T A C G T
A 0.146 0.052 0.058 0.089 A 0.423 0.151 0.168 0.258
C 0.063 0.029 0.010 0.056 C 0.399 0.184 0.063 0.354
G 0.050 0.030 0.028 0.051 G 0.314 0.189 0.176 0.321
T 0.086 0.047 0.063 0.140 T 0.258 0.138 0.187 0.415

101 P(Xt = j, Xt-1 = i) P(Xt = j | Xt-1 = i)

 Simulating a DNA sequence
p From a transition matrix, it is easy to generate a
DNA sequence of length n:
n First, choose the starting base randomly according to
the base frequency distribution
n Then, choose next base according to the distribution
P(xt | xt-1) until n bases have been chosen

T T C T T CA A A C G T
A 0.423 0.151 0.168 0.258
C 0.399 0.184 0.063 0.354
Look for R code in Deonier’s G 0.314 0.189 0.176 0.321
book
T 0.258 0.138 0.187 0.415

102 P(Xt = j | Xt-1 = i)

 Simulating a DNA sequence
p Now we can quickly generate sequences of
arbitrary length...

ttcttcaaaataaggatagtgattcttattggcttaagggataacaatttagatcttttttcatgaatcatgtatgtcaacgttaaaagttgaactgcaataagttc
ttacacacgattgtttatctgcgtgcgaagcatttcactacatttgccgatgcagccaaaagtatttaacatttggtaaacaaattgacttaaatcgcgcacttaga
gtttgacgtttcatagttgatgcgtgtctaacaattacttttagttttttaaatgcgtttgtctacaatcattaatcagctctggaaaaacattaatgcatttaaac
cacaatggataattagttacttattttaaaattcacaaagtaattattcgaatagtgccctaagagagtactggggttaatggcaaagaaaattactgtagtgaaga
ttaagcctgttattatcacctgggtactctggtgaatgcacataagcaaatgctacttcagtgtcaaagcaaaaaaatttactgataggactaaaaaccctttattt
ttagaatttgtaaaaatgtgacctcttgcttataacatcatatttattgggtcgttctaggacactgtgattgccttctaactcttatttagcaaaaaattgtcata
gctttgaggtcagacaaacaagtgaatggaagacagaaaaagctcagcctagaattagcatgttttgagtggggaattacttggttaactaaagtgttcatgactgt
tcagcatatgattgttggtgagcactacaaagatagaagagttaaactaggtagtggtgatttcgctaacacagttttcatacaagttctattttctcaatggtttt
ggataagaaaacagcaaacaaatttagtattattttcctagtaaaaagcaaacatcaaggagaaattggaagctgcttgttcagtttgcattaaattaaaaatttat
ttgaagtattcgagcaatgttgacagtctgcgttcttcaaataagcagcaaatcccctcaaaattgggcaaaaacctaccctggcttctttttaaaaaaccaagaaa
agtcctatataagcaacaaatttcaaaccttttgttaaaaattctgctgctgaataaataggcattacagcaatgcaattaggtgcaaaaaaggccatcctctttct
ttttttgtacaattgttcaagcaactttgaatttgcagattttaacccactgtctatatgggacttcgaattaaattgactggtctgcatcacaaatttcaactgcc
caatgtaatcatattctagagtattaaaaatacaaaaagtacaattagttatgcccattggcctggcaatttatttactccactttccacgttttggggatatttta
acttgaatagttcacaatcaaaacataggaaggatctactgctaaaagcaaaagcgtattggaatgataaaaaactttgatgtttaaaaaactacaaccttaatgaa
ttaaagttgaaaaaatattcaaaaaaagaaattcagttcttggcgagtaatatttttgatgtttgagatcagggttacaaaataagtgcatgagattaactcttcaa
atataaactgatttaagtgtatttgctaataacattttcgaaaaggaatattatggtaagaattcataaaaatgtttaatactgatacaactttcttttatatcctc
catttggccagaatactgttgcacacaactaattggaaaaaaaatagaacgggtcaatctcagtgggaggagaagaaaaaagttggtgcaggaaatagtttctacta
acctggtataaaaacatcaagtaacattcaaattgcaaatgaaaactaaccgatctaagcattgattgatttttctcatgcctttcgcctagttttaataaacgcgc
cccaactctcatcttcggttcaaatgatctattgtatttatgcactaacgtgcttttatgttagcatttttcaccctgaagttccgagtcattggcgtcactcacaa
atgacattacaatttttctatgttttgttctgttgagtcaaagtgcatgcctacaattctttcttatatagaactagacaaaatagaaaaaggcacttttggagtct
gaatgtcccttagtttcaaaaaggaaattgttgaattttttgtggttagttaaattttgaacaaactagtatagtggtgacaaacgatcaccttgagtcggtgacta
taaaagaaaaaggagattaaaaatacctgcggtgccacattttttgttacgggcatttaaggtttgcatgtgttgagcaattgaaacctacaactcaataagtcatg
ttaagtcacttctttgaaaaaaaaaaagaccctttaagcaagctc
103
 Simulating a DNA sequence
Dinucleotide frequencies
Simulated Observed
aa 0.145 0.146
ac 0.050 0.052
ag 0.055 0.058
at 0.092 0.089
ca 0.065 0.063
cc 0.028 0.029
cg 0.011 0.010
ct 0.058 0.056 n = 10000
ga 0.048 0.050
gc 0.032 0.030
gg 0.029 0.028
gt 0.050 0.051
ta 0.084 0.086
tc 0.052 0.047
tg 0.064 0.063
tt 0.138 0.0140
104
 Simulating a DNA sequence
p The model is able to generate correct proportions
of 1- and 2-words in genomes...
p ...but fails with k=3 and beyond.
ttcttcaaaataaggatagtgattcttattggcttaagggataacaatttagatcttttttcatgaatcatgtatgtcaacgttaaaagttgaactgcaataagttc
ttacacacgattgtttatctgcgtgcgaagcatttcactacatttgccgatgcagccaaaagtatttaacatttggtaaacaaattgacttaaatcgcgcacttaga
gtttgacgtttcatagttgatgcgtgtctaacaattacttttagttttttaaatgcgtttgtctacaatcattaatcagctctggaaaaacattaatgcatttaaac
cacaatggataattagttacttattttaaaattcacaaagtaattattcgaatagtgccctaagagagtactggggttaatggcaaagaaaattactgtagtgaaga
ttaagcctgttattatcacctgggtactctggtgaatgcacataagcaaatgctacttcagtgtcaaagcaaaaaaatttactgataggactaaaaaccctttattt
ttagaatttgtaaaaatgtgacctcttgcttataacatcatatttattgggtcgttctaggacactgtgattgccttctaactcttatttagcaaaaaattgtcata
gctttgaggtcagacaaacaagtgaatggaagacagaaaaagctcagcctagaattagcatgttttgagtggggaattacttggttaactaaagtgttcatgactgt
tcagcatatgattgttggtgagcactacaaagatagaagagttaaactaggtagtggtgatttcgctaacacagttttcatacaagttctattttctcaatggtttt
ggataagaaaacagcaaacaaatttagtattattttcctagtaaaaagcaaacatcaaggagaaattggaagctgcttgttcagtttgcattaaattaaaaatttat
ttgaagtattcgagcaatgttgacagtctgcgttcttcaaataagcagcaaatcccctcaaaattgggcaaaaacctaccctggcttctttttaaaaaaccaagaaa
agtcctatataagcaacaaatttcaaaccttttgttaaaaattctgctgctgaataaataggcattacagcaatgcaattaggtgcaaaaaaggccatcctctttct
ttttttgtacaattgttcaagcaactttgaatttgcagattttaacccactgtctatatgggacttcgaattaaattgactggtctgcatcacaaatttcaactgcc
caatgtaatcatattctagagtattaaaaatacaaaaagtacaattagttatgcccattggcctggcaatttatttactccactttccacgttttggggatatttta
acttgaatagttcacaatcaaaacataggaaggatctactgctaaaagcaaaagcgtattggaatgataaaaaactttgatgtttaaaaaactacaaccttaatgaa
ttaaagttgaaaaaatattcaaaaaaagaaattcagttcttggcgagtaatatttttgatgtttgagatcagggttacaaaataagtgcatgagattaactcttcaa
atataaactgatttaagtgtatttgctaataacattttcgaaaaggaatattatggtaagaattcataaaaatgtttaatactgatacaactttcttttatatcctc
catttggccagaatactgttgcacacaactaattggaaaaaaaatagaacgggtcaatctcagtgggaggagaagaaaaaagttggtgcaggaaatagtttctacta
acctggtataaaaacatcaagtaacattcaaattgcaaatgaaaactaaccgatctaagcattgattgatttttctcatgcctttcgcctagttttaataaacgcgc
cccaactctcatcttcggttcaaatgatctattgtatttatgcactaacgtgcttttatgttagcatttttcaccctgaagttccgagtcattggcgtcactcacaa
atgacattacaatttttctatgttttgttctgttgagtcaaagtgcatgcctacaattctttcttatatagaactagacaaaatagaaaaaggcacttttggagtct
gaatgtcccttagtttcaaaaaggaaattgttgaattttttgtggttagttaaattttgaacaaactagtatagtggtgacaaacgatcaccttgagtcggtgacta
taaaagaaaaaggagattaaaaatacctgcggtgccacattttttgttacgggcatttaaggtttgcatgtgttgagcaattgaaacctacaactcaataagtcatg
ttaagtcacttctttgaaaaaaaaaaagaccctttaagcaagctc
105
 3-words: codons
p We can extend the previous method to 3-
words
p k=3 is an important case in study of DNA
sequences because of genetic code

MSG…

a u g a g u g g a ...

5’ … a t g a g t g g a … 3’

3’ … t a c t c a c c t … 5’

106
 3-word probabilities
p Let’s again assume a sequence L of
independent bases
p Probability of 3-word r1r2r3 at position
i,i+1,i+2 in sequence L is
P(Li = r1, Li+1 = r2, Li+2 = r3) =
P(Li = r1)P(Li+1 = r2)P(Li+2 = r3)

107
 3-words in Escherichia coli genome
Word Count Observed Expected Word Count Observed Expected
AAA 108924 0.02348 0.01492 CAA 76614 0.01651 0.01541
AAC 82582 0.01780 0.01541 CAC 66751 0.01439 0.01591
AAG 63369 0.01366 0.01537 CAG 104799 0.02259 0.01588
AAT 82995 0.01789 0.01490 CAT 76985 0.01659 0.01539
ACA 58637 0.01264 0.01541 CCA 86436 0.01863 0.01591
ACC 74897 0.01614 0.01591 CCC 47775 0.01030 0.01643
ACG 73263 0.01579 0.01588 CCG 87036 0.01876 0.01640
ACT 49865 0.01075 0.01539 CCT 50426 0.01087 0.01589
AGA 56621 0.01220 0.01537 CGA 70938 0.01529 0.01588
AGC 80860 0.01743 0.01588 CGC 115695 0.02494 0.01640
AGG 50624 0.01091 0.01584 CGG 86877 0.01872 0.01636
AGT 49772 0.01073 0.01536 CGT 73160 0.01577 0.01586
ATA 63697 0.01373 0.01490 CTA 26764 0.00577 0.01539
ATC 86486 0.01864 0.01539 CTC 42733 0.00921 0.01589
ATG 76238 0.01643 0.01536 CTG 102909 0.02218 0.01586
ATT 83398 0.01797 0.01489 CTT 63655 0.01372 0.01537

108
 3-words in Escherichia coli genome
Word Count Observed Expected Word Count Observed Expected
GAA 83494 0.01800 0.01537 TAA 68838 0.01484 0.01490
GAC 54737 0.01180 0.01588 TAC 52592 0.01134 0.01539
GAG 42465 0.00915 0.01584 TAG 27243 0.00587 0.01536
GAT 86551 0.01865 0.01536 TAT 63288 0.01364 0.01489
GCA 96028 0.02070 0.01588 TCA 84048 0.01812 0.01539
GCC 92973 0.02004 0.01640 TCC 56028 0.01208 0.01589
GCG 114632 0.02471 0.01636 TCG 71739 0.01546 0.01586
GCT 80298 0.01731 0.01586 TCT 55472 0.01196 0.01537
GGA 56197 0.01211 0.01584 TGA 83491 0.01800 0.01536
GGC 92144 0.01986 0.01636 TGC 95232 0.02053 0.01586
GGG 47495 0.01024 0.01632 TGG 85141 0.01835 0.01582
GGT 74301 0.01601 0.01582 TGT 58375 0.01258 0.01534
GTA 52672 0.01135 0.01536 TTA 68828 0.01483 0.01489
GTC 54221 0.01169 0.01586 TTC 83848 0.01807 0.01537
GTG 66117 0.01425 0.01582 TTG 76975 0.01659 0.01534
GTT 82598 0.01780 0.01534 TTT 109831 0.02367 0.01487

109
 2nd order Markov Chains
p Markov chains readily generalise to higher orders
p In 2nd order markov chain, position t depends on
positions t-1 and t-2 A C G T
p Transition matrix: AA
AC
AG
AT
CA
...

p Probabilistic models for DNA and amino acid

sequences will be discussed in Biological
sequence analysis course (II period)

110
 Codon Adaptation Index (CAI)
p Observation: cells prefer certain codons
over others in highly expressed genes
n Gene expression: DNA is transcribed into RNA
(and possibly translated into protein)
Moderately
expressed
Amino
acid Codon Predicted Gene class I Gene class II
Phe TTT 0.493 0.551 0.291
TTC 0.507 0.449 0.709
Highly
Ala GCT 0.246 0.145 0.275
expressed
GCC 0.254 0.276 0.164
GCA 0.246 0.196 0.240
GCG 0.254 0.382 0.323
Asn AAT 0.493 0.409 0.172
AAC 0.507 0.591 0.828
111 Codon frequencies for some genes in E. coli
 Codon Adaptation Index (CAI)
p CAI is a statistic used to compare the
distribution of codons observed with the
preferred codons for highly expressed
genes

112
 Codon Adaptation Index (CAI)
p Consider an amino acid sequence X = x1x2...xn
p Let pk be the probability that codon k is used in
highly expressed genes
p Let qk be the highest probability that a codon
coding for the same amino acid as codon k has
n For example, if codon k is ”GCC”, the
corresponding amino acid is Alanine (see genetic
code table; also GCT, GCA, GCG code for Alanine)
n Assume that pGCC = 0.164, pGCT = 0.275, pGCA =
0.240, pGCG = 0.323
n Now qGCC = qGCT = qGCA = qGCG = 0.323

113
 Codon Adaptation Index (CAI)
p CAI is defined as
n 1/n
CAI = ( pk / qk )
k=1

p CAI can be given also in log-odds form:

n

log(CAI) = (1/n) log(pk / qk)

k=1

114
 CAI: example with an E. coli gene qk
pk

M A L T K A E SM E Y L …
ATG GCG CTT ACA AAA GCT GAA ATG
TCA GAA TATCTG
1.00 0.47 0.02 0.45 0.80 0.47 0.79 1.00
0.43 0.79 0.19
0.02
0.06 0.02 0.47 0.20 0.06 0.210.32 0.21 0.81
0.02
0.28 0.04 0.04 0.28 0.03 0.04
0.20 0.03 0.05 0.20 0.01 0.03
0.01 0.04 0.01
0.89 0.18 0.89
ATG GCT TTA ACT AAA GCT GAA ATG TCT GAA TAT TTA
GCC TTG ACC AAG GCC GAG TCC GAG TAC TTG
GCA CTT ACA GCA TCA CTT
GCG CTC ACG GCG TCG CTC
CTA AGT CTA
CTG AGC CTG 1/n
1.00 0.20 0.04 0.04 0.80 0.47 0.79 1.00 0.03 0.79 0.19 0.89…
1.00 0.47 0.89 0.47 0.80 0.47 0.79 1.00 0.43 0.79 0.81 0.89

115
 CAI: properties
p CAI = 1.0 : each codon was the most frequently
used codon in highly expressed genes
p Log-odds used to avoid numerical problems
n What happens if you multiply many values <1.0
together?
p In a sample of E.coli genes, CAI ranged from 0.2
to 0.85
p CAI correlates with mRNA levels: can be used to
predict high expression levels

116
 Biological words: summary
p Simple 1-, 2- and 3-word models can
describe interesting properties of DNA
sequences
n GC skew can identify DNA replication origins
n It can also reveal genome rearrangement
events and lateral transfer of DNA
n GC content can be used to locate genes:
human genes are comparably GC-rich
n CAI predicts high gene expression levels

117
 Biological words: summary
p k=3 models can help to identify correct
reading frames
n Reading frame starts from a start codon and
stops in a stop codon
n Consider what happens to translation when a
single extra base is introduced in a reading
frame
p Also word models for k > 3 have their
uses

118
 Next lecture
p Genome sequencing & assembly – where
do we get sequence data?

119
 Note on programming languages
p Working with probability distributions is
straightforward with R, for example
n Deonier’s book contains many computational
examples
n You can use R in CS Linux systems
p Python works too!

120
 Example Python code for generating
DNA sequences with first-order
#!/usr/bin/env python Markov chains.
import sys, random
Initialisation: use packages ’sys’ and ’random’,
n = int(sys.argv[1]) read sequence length from input.

tm = {'a' : {'a' : 0.423, 'c' : 0.151, 'g' : 0.168, 't' : 0.258},

'c' : {'a' : 0.399, 'c' : 0.184, 'g' : 0.063, 't' : 0.354}, Transition matrix
'g' : {'a' : 0.314, 'c' : 0.189, 'g' : 0.176, 't' : 0.321},
tm and initial
't' : {'a' : 0.258, 'c' : 0.138, 'g' : 0.187, 't' : 0.415}}
distribution pi.
pi = {'a' : 0.345, 'c' : 0.158, 'g' : 0.159, 't' : 0.337}

def choose(dist):
r = random.random()
sum = 0.0
keys = dist.keys() Function choose(), returns a key (here ’a’, ’c’, ’g’ or
for k in keys: ’t’) of the dictionary ’dist’ chosen randomly
sum += dist[k] according to probabilities in dictionary values.
if sum > r:
return k
return keys[-1]

c = choose(pi)
for i in range(n - 1):
sys.stdout.write(c)
Choose the first letter, then choose
c = choose(tm[c])
sys.stdout.write(c) next letter according to P(xt | xt-1).
121
sys.stdout.write("\n")
 Introduction to
Bioinformatics
Genome sequencing & assembly
 Genome sequencing & assembly
p DNA sequencing
n How do we obtain DNA sequence information from
organisms?
p Genome assembly
n What is needed to put together DNA sequence
information from sequencing?
p First statement of sequence assembly problem
(according to G. Myers):
n Peltola, Söderlund, Tarhio, Ukkonen: Algorithms for
some string matching problems arising in molecular
genetics. Proc. 9th IFIP World Computer Congress, 1983

123
 Recovery of shredded newspaper

124
 DNA sequencing
p DNA sequencing: resolving a nucleotide
sequence (whole-genome or less)
p Many different methods developed
n Maxam-Gilbert method (1977)
n Sanger method (1977)
n High-throughput methods

125
 Sanger sequencing: sequencing by
synthesis
p A sequencing technique developed by Fred
Sanger
p Also called dideoxy sequencing

126
 DNA polymerase
p A DNA polymerase is an
enzyme that catalyzes
DNA synthesis
p DNA polymerase needs
a primer
n Synthesis proceeds
always in 5’->3’ direction

127
http://en.wikipedia.org/wiki/DNA_polymerase
 Dideoxy sequencing
p In Sanger sequencing, chain-terminating
dideoxynucleoside triphosphates (ddXTPs)
are employed
n ddATP, ddCTP, ddGTP, ddTTP lack the 3’-OH
tail of dXTPs
p A mixture of dXTPs with small amount of
ddXTPs is given to DNA polymerase with
DNA template and primer
p ddXTPs are given fluorescent labels

128
 Dideoxy sequencing
p When DNA polymerase encounters a
ddXTP, the synthesis cannot proceed
p The process yields copied sequences of
different lengths
p Each sequence is terminated by a labeled
ddXTP

129
 Determining the sequence
p Sequences are sorted
according to length by
capillary
electrophoresis
p Fluorescent signals
corresponding to
labels are registered
p Base calling:
identifying which base
corresponds to each
position in a read Output sequences from
n Non-trivial problem! base calling are called reads

130
 Reads are short!
p Modern Sanger sequencers can produce
quality reads up to ~750 bases1
n Instruments provide you with a quality file for
bases in reads, in addition to actual sequence
data
p Compare the read length against the size
of the human genome (2.9x109 bases)

p Reads have to be assembled!

131
1 Nature Methods - 5, 16 - 18 (2008)
 Problems with sequencing
p Sanger sequencing error rate per base
varies from 1% to 3%1
p Repeats in DNA
n For example, ~300 base Alu sequence
repeated is over million times in human
genome
n Repeats occur in different scales
p What happens if repeat length is longer
than read length?
n We will get back to this problem later

132 1 Jones, Pevzner (2004)

 Shortest superstring problem
p Find the shortest string that ”explains” the
reads
p Given a set of strings (reads), find a
shortest string that contains all of them

133
 Example: Shortest superstring
Set of strings: {000, 001, 010, 011, 100, 101, 110, 111}

Concetenation of strings: 000001010011100101110111

010
110
011
000
Shortest superstring: 0001110100
001
111
101
100

134
 Shortest superstrings: issues
p NP-complete problem: unlike to have an
efficient (exact) algorithm
p Reads may be from either strand of DNA
p Is the shortest string necessarily the
correct assembly?
p What about errors in reads?
p Low coverage -> gaps in assembly
n Coverage: average number of times each base
occurs in the set of reads (e.g., 5x coverage)

135
 Sequence assembly and combination
locks
p What is common with sequence assembly
and opening keypad locks?

136
 Whole-genome shotgun sequence
p Whole-genome shotgun sequence
assembly starts with a large sample of
genomic DNA
1. Sample is randomly partitioned into inserts of
length > 500 bases
2. Inserts are multiplied by cloning them into a
vector which is used to infect bacteria
3. DNA is collected from bacteria and sequenced
4. Reads are assembled

137
 Assembly of reads with Overlap-Layout-
Consensus algorithm
p Overlap
n Finding potentially overlapping reads
p Layout
n Finding the order of reads along DNA
p Consensus (Multiple alignment)
n Deriving the DNA sequence from the layout

p Next, the method is described at a very

abstract level, skipping a lot of details

Kececioglu, J.D. and E.W. Myers. 1995. Combinatorial algorithms for

138 DNA sequence assembly. Algorithmica 13: 7-51.
 Finding overlaps
p First, pairwise overlap
alignment of reads is acggagtcc
resolved agtccgcgctt
p Reads can be from
either DNA strand: r1
The reverse
complement r* of 5’ … a t g a g t g g a … 3’
each read r has to be 3’ … t a c t c a c c t … 5’
considered
r2

r1: tgagt, r1*: actca

r2: tccac, r2*: gtgga
139
 Example sequence to assemble
5’ – CAGCGCGCTGCGTGACGAGTCTGACAAAGACGGTATGCGCATCG
TGATTGAAGTGAAACGCGATGCGGTCGGTCGGTGAAGTTGTGCT - 3’

p 20 reads:
# Read Read* # Read Read*
1 CATCGTCA TCACGATG 11 GGTCGGTG CACCGACC
2 CGGTGAAG CTTCACCG 12 ATCGTGAT ATCACGAT
3 TATGCGCA TGCGCATA 13 GCGCTGCG CGCAGCGC
4 GACGAGTC GACTCGTC 14 GCATCGTG CACGATGC
5 CTGACAAA TTTGTCAG 15 AGCGCGCT AGCGCGCT
6 ATGCGCAT ATGCGCAT 16 GAAGTTGT ACAACTTC
7 ATGCGGTC GACCGCAT 17 AGTGAAAC GTTTCACT
8 CTGCGTGA TCACGCAG 18 ACGCGATG CATCGCGT
9 GCGTGACG CGTCACGC 19 GCGCATCG CGATGCGC
10 GTCGGTGA TCACCGAC 20 AAGTGAAA TTTCACTT

140
 Finding overlaps
p Overlap between two reads Overlap(1, 6) = 3
can be found with a
6 ATGCGCAT
dynamic programming
algorithm 1 CATCGTCA
n Errors can be taken into
account 12 ATCGTGAT
p Dynamic programming will
Overlap(1, 12) = 7
be discussed more on next
lecture 6 12
p Overlap scores stored into 1 3 7
the overlap matrix
n Entries (i, j) below the
diagonal denote overlap of
read ri and rj*

141
 Finding layout & consensus
p Method extends the
assembly greedily by Ambiguous bases
choosing the best
overlaps
7* GACCGCAT
p Both orientations are 6=6* ATGCGCAT
considered 14 GCATCGTG
p Sequence is extended 1 CATCGTGA
as far as possible 12 ATCGTGAT
19 GCGCATCG
13* CGCAGCGC
---------------------
Consensus sequence CGCATCGTGAT

142
 Finding layout & consensus
p We move on to next best
overlaps and extend the
sequence from there
p The method stops when
there are no more overlaps 2 CGGTGAAG
to consider 10 GTCGGTGA
p A number of contigs is 11 GGTCGGTG
produced
7 ATGCGGTC
p Contig stands for
contiguous sequence, ---------------------
resulting from merging ATGCGGTCGGTGAAG
reads

143
 Whole-genome shotgun sequencing:
summary
Original genome sequence
… …

Reads
Non-overlapping Overlapping reads
read => Contig

p Ordering of the reads is initially unknown

p Overlaps resolved by aligning the reads
p In a 3x109 bp genome with 500 bp reads and 5x
coverage, there are ~107 reads and ~107(107-1)/2
= ~5x1013 pairwise sequence comparisons

144
 Repeats in DNA and genome assembly
Two instances of the same repeat

145
Pop, Salzberg, Shumway (2002)
 Repeats in DNA cause problems in
sequence assembly
p Recap: if repeat length exceeds read
length, we might not get the correct
assembly
p This is a problem especially in eukaryotes
n ~3.1% of genome consists of repeats in
Drosophila, ~45% in human
p Possible solutions
1. Increase read length – feasible?
2. Divide genome into smaller parts, with known
order, and sequence parts individually

146
 ”Divide and conquer” sequencing
approaches: BAC-by-BAC
Whole-genome shotgun sequencing
Genome

Divide-and-conquer
Genome

BAC library

147
 BAC-by-BAC sequencing
p Each BAC (Bacterial Artificial
Chromosome) is about 150 kbp
p Covering the human genome requires
~30000 BACs
p BACs shotgun-sequenced separately
n Number of repeats in each BAC is
significantly smaller than in the whole
genome...
n ...needs much more manual work compared
to whole-genome shotgun sequencing

148
 Hybrid method
p Divide-and-conquer and whole-genome
shotgun approaches can be combined
n Obtain high coverage from whole-genome
shotgun sequencing for short contigs
n Generate of a set of BAC contigs with low
coverage
n Use BAC contigs to ”bin” short contigs to
correct places
p This approach was used to sequence the
brown Norway rat genome in 2004

149
 Paired end sequencing
p Paired end (or mate-pair) sequencing is
technique where
n both ends of an insert are sequenced
n For each insert, we get two reads
n We know the distance between reads, and that
they are in opposite orientation

Read 1 k Read 2

n Typically read length < insert length

150
 Paired end sequencing
p The key idea of paired end sequencing:
n Both reads from an insert are unlikely to be in repeat
regions
n If we know where the first read is, we know also
second’s location

Repeat region

Read 1 k Read 2

p This technique helps to WGSS higher organisms

151
 First whole-genome shotgun sequencing
project: Drosophila melanogaster
p Fruit fly is a common
model organism in
biological studies
p Whole-genome
assembly reported in
Eugene Myers, et al.,
A Whole-Genome
Assembly of
Drosophila, Science
24, 2000
p Genome size 120 Mbp

152
http://en.wikipedia.org/wiki/Drosophila_melanogaster
 Sequencing of the Human Genome
p The (draft) human
genome was published
in 2001
p Two efforts:
n Human Genome Project
(public consortium)
n Celera (private
company) HGP: Nature 15 February 2001
p HGP: BAC-by-BAC Vol 409 Number 6822
approach
p Celera: whole-genome Celera: Science 16 February 2001
Vol 291, Issue 5507
shotgun sequencing

153
 Genome assembly software
p phrap (Phil’s revised assembly program)
p AMOS (A Modular, Open-Source whole-
genome assembler)
p CAP3 / PCAP
p TIGR assembler

154
 Next generation sequencing techniques
p Sanger sequencing is the prominent first-
generation sequencing method
p Many new sequencing methods are
emerging

p See Lars Paulin’s slides (course web page)

for details

155
 Next-gen sequencing: 454
p Genome Sequencer FLX (454 Life Science
/ Roche)
n >100 Mb / 7.5 h run
n Read length 250-300 bp
n >99.5% accuracy / base in a single run
n >99.99% accuracy / base in consensus

156
 Next-gen sequencing: Illumina Solexa
p Illumina / Solexa Genome Analyzer
n Read length 35 - 50 bp
n 1-2 Gb / 3-6 day run
n > 98.5% accuracy / base in a single run
n 99.99% accuracy / consensus with 3x
coverage

157
 Next-gen sequencing: SOLiD
p SOLiD
n Read length 25-30 bp
n 1-2 Gb / 5-10 day run
n >99.94% accuracy / base
n >99.999% accuracy / consensus with 15x
coverage

158
 Next-gen sequencing: Helicos
p Helicos: Single Molecule Sequencer
n No amplification of sequences needed
n Read length up to 55 bp
p Accuracy does not decrease when read length is
increased
p Instead, throughput goes down
n 25-90 Mb / h
n >2 Gb / day

159
 Next-gen sequencing: Pacific
Biosciences
p Pacific Biosciences
n Single-Molecule Real-Time (SMRT) DNA
sequencing technology
n Read length “thousands of nucleotides”
p Should overcome most problems with repeats
n Throughput estimate: 100 Gb / hour
n First instruments in 2010?

160
 Exercise groups
p 1st group: Tuesdays 16.15-18.00 C221
p 2nd group: Wednesday 14.15-16.00 B120

p You can choose freely which group you

want to attend to
p Send exercise notes before the 1st group
starts (Tue 16.15), even if you go to the
2nd group

161
Introduction to
Bioinformatics
Lecture 3:
Sequence alignment
 Sequence alignment
p The biological problem
p Global alignment
p Local alignment
p Multiple alignment

163
 Background: comparative genomics
p Basic question in biology: what properties
are shared among organisms?
p Genome sequencing allows comparison of
organisms at DNA and protein levels
p Comparisons can be used to
n Find evolutionary relationships between
organisms
n Identify functionally conserved sequences
n Identify corresponding genes in human and
model organisms: develop models for human
diseases
164
 Homologs
p Two genes (sequences in
general) gB and gC
evolved from the same gA = agtgtccgttaagtgcgttc
ancestor gene gA are
called homologs
gB = agtgccgttaaagttgtacgtc

p Homologs usually exhibit

gC = ctgactgtttgtggttc
conserved functions

p Close evolutionary
relationship => expect a
high number of homologs

165
 Sequence similarity
p We expect homologs to be ”similar” to each other
p Intuitively, similarity of two sequences refers to
the degree of match between corresponding
positions in sequence
agtgccgttaaagttgtacgtc

ctgactgtttgtggttc
p What about sequences that differ in length?

166
 Similarity vs homology
p Sequence similarity is not sequence
homology
n If the two sequences gB and gC have accumulated
enough mutations, the similarity between them is likely
to be low
#mutations #mutations
0 agtgtccgttaagtgcgttc 64 acagtccgttcgggctattg
1 agtgtccgttatagtgcgttc 128 cagagcactaccgc
2 agtgtccgcttatagtgcgttc 256 cacgagtaagatatagct
4 agtgtccgcttaagggcgttc 512 taatcgtgata
8 agtgtccgcttcaaggggcgt 1024 acccttatctacttcctggagtt
16 gggccgttcatgggggt 2048 agcgacctgcccaa
32 gcagggcgtcactgagggct 4096 caaac

Homology is more difficult to detect over greater

evolutionary distances.
167
 Similarity vs homology (2)
p Sequence similarity can occur by chance
n Similarity does not imply homology

p Consider comparing two short sequences

against each other

168
 Orthologs and paralogs
p We distinguish between two types of homology
n Orthologs: homologs from two different species,
separated by a speciation event
n Paralogs: homologs within a species, separated by a
gene duplication event Organism A
gA
gA
Gene duplication event

gA gA’

gB gC

Organism B Organism C gB gC Paralogs

Orthologs
169
 Orthologs and paralogs (2)
p Orthologs typically retain the original function
p In paralogs, one copy is free to mutate and
acquire new function (no selective pressure)
Organism A
gA

gA

gA gA’

gB gC
gB gC
Organism B Organism C

170
 Paralogy example: hemoglobin
p Hemoglobin is a protein
complex which transports
oxygen
p In humans, hemoglobin
consists of four protein
subunits and four non-
protein heme groups

Sickle cell diseases

Hemoglobin A,
are caused by mutations
www.rcsb.org/pdb/explore.do?structureId=1GZX
in hemoglobin genes

http://en.wikipedia.org/wiki/Image:Sicklecells.jpg
171
 Paralogy example: hemoglobin
p In adults, three types are
normally present
n Hemoglobin A: 2 alpha and
2 beta subunits
n Hemoglobin A2: 2 alpha
and 2 delta subunits
n Hemoglobin F: 2 alpha and
2 gamma subunits
p Each type of subunit
(alpha, beta, gamma,
delta) is encoded by a
separate gene
Hemoglobin A,
www.rcsb.org/pdb/explore.do?structureId=1GZX

172
 Paralogy example: hemoglobin
p The subunit genes are
paralogs of each other, i.e.,
they have a common ancestor
gene
p Exercise: hemoglobin human
paralogs in NCBI sequence
databases
http://www.ncbi.nlm.nih.gov/sites/entre
z?db=Nucleotide
n Find human hemoglobin alpha, beta,
gamma and delta
n Compare sequences

Hemoglobin A,
www.rcsb.org/pdb/explore.do?structureId=1GZX

173
 Orthology example: insulin
p The genes coding for insulin in human
(Homo sapiens) and mouse (Mus
musculus) are orthologs:
n They have a common ancestor gene in the
ancestor species of human and mouse
n Exercise: find insulin orthologs from human
and mouse in NCBI sequence databases

174
 Sequence alignment
p Alignment specifies which positions in two
sequences match

acgtctag acgtctag acgtctag

|| ||||| || |||||
actctag- -actctag ac-tctag

2 matches 5 matches 7 matches

5 mismatches 2 mismatches 0 mismatches
1 not aligned 1 not aligned 1 not aligned

175
 Sequence alignment
p Maximum alignment length is the total length of
the two sequences

acgtctag------- -------acgtctag

--------actctag actctag--------

0 matches 0 matches
0 mismatches 0 mismatches
15 not aligned 15 not aligned

176
 Mutations: Insertions, deletions and
substitutions
Indel: insertion or acgtctag Mismatch: substitution
deletion of a base ||||| (point mutation) of
with respect to the a single base
-actctag
ancestor sequence

p Insertions and/or deletions are called

indels
n We can’t tell whether the ancestor sequence
had a base or not at indel position!

177
 Problems

p What sorts of alignments should be considered?

p How to score alignments?
p How to find optimal or good scoring alignments?
p How to evaluate the statistical significance of
scores?

In this course, we discuss each of these problems

briefly.

178
 Sequence Alignment (chapter 6)
p The biological problem
p Global alignment
p Local alignment
p Multiple alignment

179
 Global alignment
p Problem: find optimal scoring alignment between
two sequences (Needleman & Wunsch 1970)
p Every position in both sequences is included in
the alignment
p We give score for each position in alignment
n Identity (match) +1
n Substitution (mismatch) -µ
n Indel -
p Total score: sum of position scores

180
 Scoring: Toy example
p Consider two sequences
WHAT
with characters drawn
from the English
||
language alphabet: WH-Y
WHAT, WHY
S(WHAT/WH-Y) = 1 + 1 – –µ

WHAT

-WHY
S(WHAT/-WHY) = – –µ–µ–µ
181
 Dynamic programming
p How to find the optimal alignment?
p We use previous solutions for optimal
alignments of smaller subsequences
p This general approach is known as
dynamic programming

182
 Introduction to dynamic programming:
the money change problem
p Suppose you buy a pen for 4.23€ and pay for
with a 5€ note
p You get 77 cents in change – what coins is the
cashier going to give you if he or she tries to
minimise the number of coins?
p The usual algorithm: start with largest coin
(denominator), proceed to smaller coins until no
change is left:
n 50, 20, 5 and 2 cents
p This greedy algorithm is incorrect, in the sense
that it does not always give you the correct
answer

183
 The money change problem
p How else to compute the 77
change?
50 20 5
p We could consider all possible
ways to reduce the amount of 27 57 72
change
p Suppose we have 77 cents
change, and the following 7 22 7 37 52 22 52 67
coins: 50, 20, 5 cents
p We can compute the change …
with recursion p Many values are computed more
n C(n) = min { C(n – 50) + 1, than once!
C(n – 20) + 1, C(n – 5) + 1 } p This leads to a correct but very
p Figure shows the recursion inefficient algorithm
tree for the example

184
 The money change problem
p We can speed the computation up by
solving the change problem for all i n
n Example: solve the problem for 9 cents with
available coins being 1, 2 and 5 cents
p Solve the problem in steps, first for 1
cent, then 2 cents, and so on
p In each step, utilise the solutions from the
previous steps

185
 The money change problem

Amount of
change left 0 1 2 3 4 5 6 7 8 9

Number of 0 1 1 2 2 1 2 2 3 3
coins used

p Algorithm runs in time proportional to Md, where M is the

amount of change and d is the number of coin types
p The same technique of storing solutions of subproblems can
be utilised in aligning sequences

186
 Representing alignments and scores
Alignments can be
represented in the
following tabular form. - W H A T
Each alignment
corresponds to a path -
through the table.
W
WHAT
H
||
Y
WH-Y

187
 Representing alignments and scores
WH-AT - W H A T
||
-
WHY--
W
WHAT---
H

Y
----WHY

188
 Representing alignments and scores

WHAT - W H A T

|| - 0
WH-Y W 1

H 2 2-

Global alignment Y 2- -µ
score S3,4 = 2- -µ

189
 Filling the alignment matrix
- W H A T Consider the alignment process
at shaded square.
- Case 1. Align H against H
(match)
W Case 1
Case 2 Case 2. Align H in WHY against
– (indel) in WHAT
H
Case 3 Case 3. Align H in WHAT
Y against – (indel) in WHY

190
 Filling the alignment matrix (2)
- W H A T Scoring the alternatives.
Case 1. S2,2 = S1,1 + s(2, 2)
-
Case 2. S2,2 = S1,2
W Case 1 Case 3. S2,2 = S2,1
Case 2
s(i, j) = 1 for matching positions,
H
Case 3 s(i, j) = - µ for substitutions.
Y Choose the case (path) that
yields the maximum score.
Keep track of path choices.

191
 Global alignment: formal
development
A = a1a2a3…an, 0 1 2 3 4
B = b1b2b3…bm
- b1 b2 b3 b4
b1 b2 b3 b4 -
- a1 - a2 a3 0 -
lAny alignment can be written
as a unique path through the 1 a1
matrix
2 a2
l Score for aligning A and B up
to positions i and j:
3 a3
Si,j = S(a1a2a3…ai, b1b2b3…bj)

192
 Scoring partial alignments
p Alignment of A = a1a2a3…ai with B = b1b2b3…bj
can be end in three possible ways
n Case 1: (a1a2…ai-1) ai
(b1b2…bj-1) bj
n Case 2: (a1a2…ai-1) ai
(b1b2…bj) -
n Case 3: (a1a2…ai) –
(b1b2…bj-1) bj

193
 Scoring alignments
p Scores for each case:
+1 if ai = bj
n Case 1: (a1a2…ai-1) ai
(b1b2…bj-1) bj
s(ai, bj) = { -µ otherwise
n Case 2: (a1a2…ai-1) ai
(b1b2…bj) –

n Case 3: (a1a2…ai) – s(ai, -) = s(-, bj) = -

(b1b2…bj-1) bj

194
 Scoring alignments (2)
p First row and first column 0 1 2 3 4
correspond to initial alignment
against indels:
- b1 b2 b3 b4
S(i, 0) = -i
S(0, j) = -j
0 - 0 - -2 -3 -4

p Optimal global alignment score

S(A, B) = Sn,m 1 a1 -

2 a2 -2

3 a3 -3

195
 Algorithm for global alignment
Input sequences A, B, n = |A|, m = |B|
Set Si,0 := - i for all i
Set S0,j := - j for all j
for i := 1 to n
for j := 1 to m
Si,j := max{Si-1,j – , Si-1,j-1 + s(ai,bj), Si,j-1 – }
end
end

Algorithm takes O(nm) time

196
 Global alignment: example
- T G G T G
µ=1 - 0 -2 -4 -6 -8 -10
=2 A -2
T -4
C -6
G -8
T -10 ?

197
 Global alignment: example
- T G G T G
µ=1 - 0 -2 -4 -6 -8 -10
=2 A -2 -1 -3
T -4
C -6
G -8
T -10 ?

198
 Global alignment: example (2)
- T G G T G
µ=1 - 0 -2 -4 -6 -8 -10
=2 A -2 -1 -3 -5 -7 -9
T -4 -1 -2 -4 -4 -6
C -6 -3 -2 -3 -5 -5
ATCGT- G -8 -5 -2 -1 -3 -4
T -10 -7 -4 -3 0 -2
| ||
-TGGTG

199
 Sequence Alignment (chapter 6)
p The biological problem
p Global alignment
p Local alignment
p Multiple alignment

200
 Local alignment: rationale
p Otherwise dissimilar proteins may have local regions of
similarity
-> Proteins may share a function

Human bone
morphogenic protein
receptor type II
precursor (left) has a
300 aa region that
resembles 291 aa
region in TGF-
receptor (right).
The shared function
here is protein kinase.

201
 Local alignment: rationale
A

B
Regions of
similarity

p Global alignment would be inadequate

p Problem: find the highest scoring local alignment
between two sequences
p Previous algorithm with minor modifications solves this
problem (Smith & Waterman 1981)

202
 From global to local alignment
p Modifications to the global alignment
algorithm
n Look for the highest-scoring path in the
alignment matrix (not necessarily through the
matrix), or in other words:
n Allow preceding and trailing indels without
penalty

203
 Scoring local alignments
A = a1a2a3…an, B = b1b2b3…bm

Let I and J be intervals (substrings) of A and B, respectively:

Best local alignment score:

where S(I, J) is the alignment score for substrings I and J.

204
 Allowing preceding and trailing
indels
p First row and column 0 1 2 3 4
initialised to zero:
Mi,0 = M0,j = 0 - b1 b2 b3 b4

0 - 0 0 0 0 0

1 a1 0

b1 b2 b3 2 a2 0
- - a1
3 a3 0

205
 Recursion for local alignment
p Mi,j = max { - T G G T G
Mi-1,j-1 + s(ai, bi), - 0 0 0 0 0 0
Mi-1,j – ,
Mi,j-1 – , A 0 0 0 0 0 0
0 T 0 1 0 0 1 0
}
C 0 0 0 0 0 0

Allow alignment to G 0 0 1 1 0 1
start anywhere in
sequences
T 0 1 0 0 2 0

206
 Finding best local alignment
p Optimal score is the highest - T G G T G
value in the matrix
- 0 0 0 0 0 0

= maxi,j Mi,j A 0 0 0 0 0 0

p Best local alignment can be

T 0 1 0 0 1 0
found by backtracking from the
highest value in M C 0 0 0 0 0 0

G 0 0 1 1 0 1
p What is the best local
alignment in this example?
T 0 1 0 0 2 0

207
 Local alignment: example
0 1 2 3 4 5 6 7 8 9 10
Mi,j = max {
Mi-1,j-1 + s(ai, - G G C T C A A T C A
bi), 0 - 0 0 0 0 0 0 0 0 0 0 0
Mi-1,j ,
Mi,j-1 , 1 A 0 0
0 2 C 0
}
3 C 0
4 T 0
5 A 0
Scoring (for example) 6 A 0
Match: +2 7 G 0
Mismatch: -1
8 G 0
Indel: -2

208
 Local alignment: example
0 1 2 3 4 5 6 7 8 9 10
Mi,j = max {
Mi-1,j-1 + s(ai, - G G C T C A A T C A
bi), 0 - 0 0 0 0 0 0 0 0 0 0 0
Mi-1,j ,
Mi,j-1 , 1 A 0 0 0 0 0 0 2
0 2 C 0
}
3 C 0
4 T 0
5 A 0
Scoring (for example) 6 A 0
Match: +2 7 G 0
Mismatch: -1
8 G 0
Indel: -2

209
 Local alignment: example
Optimal local 0 1 2 3 4 5 6 7 8 9 10

alignment:
- G G C T C A A T C A
C T – A A 0 - 0 0 0 0 0 0 0 0 0 0 0
C T C A A 1 A 0 0 0 0 0 0 2 2 0 0 2
2 C 0 0 0 2 0 2 0 1 1 2 0
3 C 0 0 0 2 1 2 1 0 0 3 1
4 T 0 0 0 0 4 2 1 0 2 1 2
5 A 0 0 0 0 2 3 4 3 1 1 3
6 A 0 0 0 0 0 1 5 6 4 2 3
Scoring (for example)
Match: +2 7 G 0 2 2 0 0 0 3 4 5 3 1
Mismatch: -1 8 G 0 2 4 2 0 0 1 2 3 4 2
Indel: -2

210
 Multiple optimal alignments
Non-optimal, good-scoring alignments
0 1 2 3 4 5 6 7 8 9 10

How can you find - G G C T C A A T C A

0 - 0 0 0 0 0 0 0 0 0 0 0
1. Optimal
alignments if 1 A 0 0 0 0 0 0 2 2 0 0 2
more than one 2 C 0 0 0 2 0 2 0 1 1 2 0
exist?
3 C 0 0 0 2 1 2 1 0 0 3 1
2. Non-optimal, 4 T 0 0 0 0 4 2 1 0 2 1 2
good-scoring 5 A 0 0 0 0 2 3 4 3 1 1 3
alignments?
6 A 0 0 0 0 0 1 5 6 4 2 3
7 G 0 2 2 0 0 0 3 4 5 3 1
8 G 0 2 4 2 0 0 1 2 3 4 2

211
 Overlap alignment
p Overlap matrix used by Overlap-Layout-
Consensus algorithm can be computed with
dynamic programming
p Initialization: Oi,0 = O0,j = 0 for all i, j
p Recursion:
Oi,j = max {
Oi-1,j-1 + s(ai, bi),
Oi-1,j – ,
Oi,j-1 – ,
}
Best overlap: maximum value from rightmost
column and bottom row

212
 Non-uniform mismatch penalties
p We used uniform penalty for mismatches:
s(’A’, ’C’) = s(’A’, ’G’) = … = s(’G’, ’T’) = µ
p Transition mutations (A->G, G->A, C->T, T->C) are
approximately twice as frequent than transversions (A->T,
T->A, A->C, G->T)
n use non-uniform mismatch
penalties collected into a
substitution matrix
A C G T
A 1 -1 -0.5 -1
C -1 1 -1 -0.5
G -0.5 -1 1 -1
T -1 -0.5 -1 1
213
 Gaps in alignment
p Gap is a succession of indels in alignment
C T – - - A A
C T C G C A A

p Previous model scored a length k gap as

w(k) = -k
p Replication processes may produce longer
stretches of insertions or deletions
n In coding regions, insertions or deletions of
codons may preserve functionality

214
 Gap open and extension penalties (2)
p We can design a score that allows the
penalty opening gap to be larger than
extending the gap:
w(k) = - – (k – 1)
p Gap open cost , Gap extension cost
p Alignment algorithms can be extended to
use w(k) (not discussed on this course)

215
 Amino acid sequences
p We have discussed mainly DNA sequences
p Amino acid sequences can be aligned as
well
p However, the design of the substitution
matrix is more involved because of the
larger alphabet
p More on the topic in the course Biological
sequence analysis

216
 Demonstration of the EBI web site
p European Bioinformatics Institute (EBI)
offers many biological databases and
bioinformatics tools at
http://www.ebi.ac.uk/
n Sequence alignment: Tools -> Sequence
Analysis -> Align

217
 Sequence Alignment (chapter 6)
p The biological problem
p Global alignment
p Local alignment
p Multiple alignment

218
 Multiple alignment
p Consider a set of n sequences
on the right aggcgagctgcgagtgcta
n Orthologous sequences from cgttagattgacgctgac
different organisms ttccggctgcgac
n Paralogs from multiple gacacggcgaacgga
duplications agtgtgcccgacgagcgaggac
p How can we study gcgggctgtgagcgcta
relationships between these aagcggcctgtgtgcccta
sequences? atgctgctgccagtgta
agtcgagccccgagtgc
agtccgagtcc
actcggtgc

219
 Optimal alignment of three
sequences
p Alignment of A = a1a2…ai and B = b1b2…bj can
end either in (-, bj), (ai, bj) or (ai, -)
p 22 – 1 = 3 alternatives
p Alignment of A, B and C = c1c2…ck can end in 23 –
1 ways: (ai, -, -), (-, bj, -), (-, -, ck), (-, bj, ck),
(ai, -, ck), (ai, bj, -) or (ai, bj, ck)
p Solve the recursion using three-dimensional
dynamic programming matrix: O(n3) time and
space
p Generalizes to n sequences but impractical with
even a moderate number of sequences

220
 Multiple alignment in practice
p In practice, real-world multiple alignment
problems are usually solved with heuristics
p Progressive multiple alignment
n Choose two sequences and align them
n Choose third sequence w.r.t. two previous sequences
and align the third against them
n Repeat until all sequences have been aligned
n Different options how to choose sequences and score
alignments
n Note the similarity to Overlap-Layout-Consensus

221
 Multiple alignment in practice
p Profile-based progressive multiple
alignment: CLUSTALW
n Construct a distance matrix of all pairs of
sequences using dynamic programming
n Progressively align pairs in order of decreasing
similarity
n CLUSTALW uses various heuristics to
contribute to accuracy

222
 Additional material
p R. Durbin, S. Eddy, A. Krogh, G.
Mitchison: Biological sequence analysis
p N. C. Jones, P. A. Pevzner: An introduction
to bioinformatics algorithms
p Course Biological sequence analysis in
period II, 2008

223
 Rapid alignment methods: FASTA and
BLAST
p The biological problem
p Search strategies
p FASTA
p BLAST

224
 The biological problem
p Global and local
alignment algoritms are
slow in practice
p Consider the scenario of
aligning a query
sequence against a large
database of sequences
n New sequence with
unknown function n NCBI GenBank size in January
2007 was 65 369 091 950
bases (61 132 599 sequences)
n Feb 2008: 85 759 586 764
bases (82 853 685 sequences)

225
 Problem with large amount of sequences
p Exponential growth in both number and
total length of sequences
p Possible solution: Compare against model
organisms only
p With large amount of sequences, chances
are that matches occur by random
n Need for statistical analysis

226
 Rapid alignment methods: FASTA and
BLAST
p The biological problem
p Search strategies
p FASTA
p BLAST

227
 FASTA
p FASTA is a multistep algorithm for sequence
alignment (Wilbur and Lipman, 1983)
p The sequence file format used by the FASTA
software is widely used by other sequence
analysis software
p Main idea:
n Choose regions of the two sequences (query and
database) that look promising (have some degree of
similarity)
n Compute local alignment using dynamic programming in
these regions

228
 FASTA outline
p FASTA algorithm has five steps:
n 1. Identify common k-words between I and J
n 2. Score diagonals with k-word matches,
identify 10 best diagonals
n 3. Rescore initial regions with a substitution
score matrix
n 4. Join initial regions using gaps, penalise for
gaps
n 5. Perform dynamic programming to find final
alignments

229
 Search strategies
p How to speed up the computation?
n Find ways to limit the number of pairwise
comparisons
p Compare the sequences at word level to
find out common words
n Word means here a k-tuple (or a k-word), a
substring of length k

230
 Analyzing the word content
p Example query string I: TGATGATGAAGACATCAG
p For k = 8, the set of k-words (substring of length
k) of I is

TGATGATG
GATGATGA
ATGATGAA
TGATGAAG
…
GACATCAG

231
 Analyzing the word content
p There are n-k+1 k-words in a string of length n

p If at least one word of I is not found from

another string J, we know that I differs from J

p Need to consider statistical significance: I and J

might share words by chance only

p Let n=|I| and m=|J|

232
 Word lists and comparison by content
p The k-words of I can be arranged into a table of
word occurences Lw(I)
p Consider the k-words when k=2 and
I=GCATCGGC:
GC, CA, AT, TC, CG, GG, GC
AT: 3
CA: 2
CG: 5
GC: 1, 7 Start indecies of k-word GC in I
GG: 6
TC: 4 Building Lw(I) takes O(n) time

233
 Common k-words
p Number of common k-words in I and J can
be computed using Lw(I) and Lw(J)
p For each word w in I, there are |Lw(J)|
occurences in J
p Therefore I and J have
common words
p This can be computed in O(n + m + 4k)
time
n O(n + m) time to build the lists
n O(4k) time to calculate the sum (in DNA
234
strings)
 Common k-words
p I = GCATCGGC
p J = CCATCGCCATCG
Lw(I) Lw(J) Common words
AT: 3 AT: 3, 9 2
CA: 2 CA: 2, 8 2
CC: 1, 7 0
CG: 5 CG: 5, 11 2
GC: 1, 7 GC: 6 2
GG: 6 0
TC: 4 TC: 4, 10 2
10 in total

235
 Properties of the common word list
p Exact matches can be found using binary search
(e.g., where TCGT occurs in I?)
n O(log 4k) time
p For large k, the table size is too large to compute
the common word count in the previous fashion
p Instead, an approach based on merge sort can be
utilised (details skipped)
p The common k-word technique can be combined
with the local alignment algorithm to yield a rapid
alignment approach

236
 FASTA outline
p FASTA algorithm has five steps:
n 1. Identify common k-words between I and J
n 2. Score diagonals with k-word matches,
identify 10 best diagonals
n 3. Rescore initial regions with a substitution
score matrix
n 4. Join initial regions using gaps, penalise for
gaps
n 5. Perform dynamic programming to find final
alignments

237
 Dot matrix comparisons
p Word matches in two sequences I and J can be
represented as a dot matrix
p Dot matrix element (i, j) has ”a dot”, if the word
starting at position i in I is identical to the word
starting at position j in J
p The dot matrix can be plotted for various k

j
i
I = … ATCGGATCA …
i J = … TGGTGTCGC …
j

238
 k=1 k=4

Dot matrix (k=1,4,8,16)

for two DNA sequences
X85973.1 (1875 bp)
Y11931.1 (2013 bp)

k=8 k=16

239
 k=1 k=4

Dot matrix
(k=1,4,8,16) for two
protein sequences
CAB51201.1 (531 aa)
CAA72681.1 (588 aa)

k=8 k=16

Shading indicates
now the match score
according to a
score matrix
(Blosum62 here)

240
 Computing diagonal sums
p We would like to find high scoring diagonals of the dot
matrix
p Lets index diagonals by the offset, l = i - j

J
C C A T C G C C A T C G
G * k=2
C * *
A * *
I T * *
C * *
G
G *
Diagonal l = i – j = -6
C
241
 Computing diagonal sums
p As an example, lets compute diagonal sums for
I = GCATCGGC, J = CCATCGCCATCG, k = 2
p 1. Construct k-word list Lw(J)
p 2. Diagonal sums Sl are computed into a table,
indexed with the offset and initialised to zero

l -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
Sl 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

242
 Computing diagonal sums
p 3. Go through k-words of I, look for matches in
Lw(J) and update diagonal sums

J
For the first 2-word in I,
GC, LGC(J) = {6}.
C C A T C G C C A T C G
G * We can then update
C * * the sum of diagonal
A * * l = i – j = 1 – 6 = -5 to
I T * * S-5 := S-5 + 1 = 0 + 1 = 1
C * *
G
G *
C
243
 Computing diagonal sums
p 3. Go through k-words of I, look for matches in
Lw(J) and update diagonal sums

J
Next 2-word in I is CA,
for which LCA(J) = {2, 8}.
C C A T C G C C A T C G
G * Two diagonal sums are
C * * updated:
A * * l=i–j=2–2=0
I T * * S0 := S0 + 1 = 0 + 1 = 1
C * *
G I = i – j = 2 – 8 = -6
G * S-6 := S-6 + 1 = 0 + 1 = 1
C
244
 Computing diagonal sums
p 3. Go through k-words of I, look for matches in
Lw(J) and update diagonal sums

J
Next 2-word in I is AT,
for which LAT(J) = {3, 9}.
C C A T C G C C A T C G
G * Two diagonal sums are
C * * updated:
A * * l=i–j=3–3=0
I T * * S0 := S0 + 1 = 1 + 1 = 2
C * *
G I = i – j = 3 – 9 = -6
G * S-6 := S-6 + 1 = 1 + 1 = 2
C
245
 Computing diagonal sums
After going through the k-words of I, the result is:
l -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
Sl 0 0 0 0 4 1 0 0 0 0 4 1 0 0 0 0 0

J
C C A T C G C C A T C G
G *
C * *
A * *
I T * *
C * *
G
G *
C
246
 Algorithm for computing diagonal sum of scores

Sl := 0 for all 1 – m l n–1

Compute Lw(J) for all words w
for i := 1 to n – k – 1 do
w := IiIi+1…Ii+k-1
for j Lw(J) do
l := i – j
Sl := Sl + 1 Match score is here 1
end
end

247
 FASTA outline
p FASTA algorithm has five steps:
n 1. Identify common k-words between I and J
n 2. Score diagonals with k-word matches,
identify 10 best diagonals
n 3. Rescore initial regions with a substitution
score matrix
n 4. Join initial regions using gaps, penalise for
gaps
n 5. Perform dynamic programming to find final
alignments

248
 Rescoring initial regions
p Each high-scoring diagonal chosen in the
previous step is rescored according to a score
matrix
p This is done to find subregions with identities
shorter than k
p Non-matching ends of the diagonal are trimmed

I: C C A T C G C C A T C G
75% identity, no 4-word identities
J: C C A A C G C A A T C A

I’: C C A T C G C C A T C G
33% identity, one 4-word identity
J’: A C A T C A A A T A A A
249
 Joining diagonals
p Two offset diagonals can be joined with a gap, if
the resulting alignment has a higher score
p Separate gap open and extension are used
p Find the best-scoring combination of diagonals

High-scoring
diagonals

Two diagonals
joined by a gap

250
 FASTA outline
p FASTA algorithm has five steps:
n 1. Identify common k-words between I and J
n 2. Score diagonals with k-word matches,
identify 10 best diagonals
n 3. Rescore initial regions with a substitution
score matrix
n 4. Join initial regions using gaps, penalise for
gaps
n 5. Perform dynamic programming to find final
alignments

251
 Local alignment in the highest-scoring
region
p Last step of FASTA: perform local
alignment using dynamic
programming around the highest-
scoring
p Region to be aligned covers –w and
+w offset diagonal to the highest-
scoring diagonals w
p With long sequences, this region is
typically very small compared to the
whole n x m matrix w
Dynamic programming matrix
M filled only for the green region

252
 Properties of FASTA
p Fast compared to local alignment using dynamic
programming only
n Only a narrow region of the full matrix is aligned
p Increasing parameter k decreases the number of
hits:
n Increases specificity
n Decreases sensitivity
n Decreases running time
p FASTA can be very specific when identifying long
regions of low similarity
n Specific method does not find many incorrect results
n Sensitive method finds many of the correct results

253
 Properties of FASTA
p FASTA looks for initial exact matches to
query sequence
n Two proteins can have very different amino
acid sequences and still be biologically similar
n This may lead into a lack of sensitivity with
diverged sequences

254
 Demonstration of FASTA at EBI
p http://www.ebi.ac.uk/fasta/
p Note that parameter ktup in the software
corresponds to parameter k in lectures

255
 Note on sequences and alignment
matrices in exercises
p Example solutions to alignment problems
will have sequences arranged like this:
n Perform global alignment of the sequences
p s = AGCTGCGTACT
p t = ATGAGCGTTA ATGAGCGTTA

AGCTGCGTACT
So if you want to be able to
compare your solution easily
against the example, use this
convention.

256
 Rapid alignment methods: FASTA and
BLAST
p The biological problem
p Search strategies
p FASTA
p BLAST

257
 BLAST: Basic Local Alignment Search
Tool
p BLAST (Altschul et al., 1990) and its variants are
some of the most common sequence search tools
in use
p Roughly, the basic BLAST has three parts:
n 1. Find segment pairs between the query sequence and
a database sequence above score threshold (”seed hits”)
n 2. Extend seed hits into locally maximal segment pairs
n 3. Calculate p-values and a rank ordering of the local
alignments
p Gapped BLAST introduced in 1997 allows for gaps
in alignments

258
 Finding seed hits
p First, we generate a set of neighborhood
sequences for given k, match score matrix and
threshold T
p Neighborhood sequences of a k-word w include
all strings of length k that, when aligned against
w, have the alignment score at least T
p For instance, let I = GCATCGGC, J =
CCATCGCCATCG and k = 5, match score be 1,
mismatch score be 0 and T = 4

259
 Finding seed hits
p I = GCATCGGC, J = CCATCGCCATCG, k = 5,
match score 1, mismatch score 0, T = 4
p This allows for one mismatch in each k-word
p The neighborhood of the first k-word of I, GCATC,
is GCATC and the 15 sequences

A A C A A
CCATC,G GATC,GC GTC,GCA CC,GCAT G
T T T G T

260
 Finding seed hits
p I = GCATCGGC has 4 k-words and thus 4x16 =
64 5-word patterns to locate in J
n Occurences of patterns in J are called seed hits
p Patterns can be found using exact search in time
proportional to the sum of pattern lengths +
length of J + number of matches (Aho-Corasick
algorithm)
n Methods for pattern matching are developed on course
58093 String processing algorithms
p Compare this approach to FASTA

261
 Extending seed hits: original BLAST
p Initial seed hits are extended into
locally maximal segment pairs
or High-scoring Segment Pairs
(HSP)
p Extensions do not add gaps to the
alignment
p Sequence is extended until the
alignment score drops below the
maximum attained score minus a
Extension
threshold parameter value
p All statistically significant HSPs
AACCGTTCATTA
reported
| || || ||
Altschul, S.F., Gish, W., Miller, W., Myers, E. W. and
TAGCGATCTTTT
Lipman, D. J., J. Mol. Biol., 215, 403-410, 1990
Initial seed hit
262
 Extending seed hits: gapped BLAST
p In a later version of BLAST, two
seed hits have to be found on the
same diagonal
n Hits have to be non-overlapping
n If the hits are closer than A
(additional parameter), then they
are joined into a HSP
p Threshold value T is lowered to
achieve comparable sensitivity
p If the resulting HSP achieves a
score at least Sg, a gapped
extension is triggered

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, and
Lipman DJ, Nucleic Acids Res. 1;25(17), 3389-402, 1997

263
 Gapped extensions of HSPs
p Local alignment is performed
starting from the HSP
p Dynamic programming matrix
filled in ”forward” and
”backward” directions (see
figure)
HSP
p Skip cells where value would
be Xg below the best
alignment score found so far

Region searched with score Region potentially searched

above cutoff parameter by the alignment algorithm

264
 Estimating the significance of results
p In general, we have a score S(D, X) = s for a
sequence X found in database D
p BLAST rank-orders the sequences found by p-
values
p The p-value for this hit is P(S(D, Y) s) where Y
is a random sequence
n Measures the amount of ”surprise” of finding sequence X
p A smaller p-value indicates more significant hit
n A p-value of 0.1 means that one-tenth of random
sequences would have as large score as our result

265
 Estimating the significance of results
p In BLAST, p-values are computed roughly as
follows
p There are nm places to begin an optimal
alignment in the n x m alignment matrix
p Optimal alignment is preceded by a mismatch
and has t matching (identical) letters
n (Assume match score 1 and mismatch/indel score - )
p Let p = P(two random letters are equal)
p The probability of having a mismatch and then t
matches is (1-p)pt

266
 Estimating the significance of results
p We model this event by a Poisson distribution
(why?) with mean = nm(1-p)pt
p P(there is local alignment t or longer)
1 – P(no such event)
= 1 – e- = 1 – exp(-nm(1-p)pt)
p An equation of the same form is used in Blast:
p E-value = P(S(D, Y) s) 1 – exp(-nm t) where
> 0 and 0 < < 1
p Parameters and are estimated from data

267
 Scoring amino acid
alignments
p We need a way to compute the
score S(D, X) for aligning the
sequence X against database D
p Scoring DNA alignments was
discussed previously
p Constructing a scoring model for
amino acids is more challenging
n 20 different amino acids vs. 4
bases
p Figure shows the molecular
structures of the 20 amino acids

http://en.wikipedia.org/wiki/List_of_standard_amino_acids

268
 Scoring amino acid
alignments
p Substitutions between chemically
similar amino acids are more
frequent than between dissimilar
amino acids
p We can check our scoring model
against this

http://en.wikipedia.org/wiki/List_of_standard_amino_acids

269
 Score matrices
p Scores s = S(D, X) are obtained from score
matrices
p Let A = A1a2…an and B = b1b2…bn be sequences
of equal length (no gaps allowed to simplify
things)
p To obtain a score for alignment of A and B, where
ai is aligned against bi, we take the ratio of two
probabilities
n The probability of having A and B where the characters
match (match model M)
n The probability that A and B were chosen randomly
(random model R)

270
 Score matrices: random model
p Under the random model, the probability
of having X and Y is

where qxi is the probability of occurence of

amino acid type xi
p Position where an amino acid occurs does
not affect its type

271
 Score matrices: match model
p Let pab be the probability of having amino acids
of type a and b aligned against each other given
they have evolved from the same ancestor c
p The probability is

272
 Score matrices: log-odds ratio score
p We obtain the score S by taking the ratio
of these two probabilities

and taking a logarithm of the ratio

273
 Score matrices: log-odds ratio score

p The score S is obtained by summing over

character pair-specific scores:

p The probabilities qa and pab are extracted

from data

274
 Calculating score matrices for amino
acids
p Probabilities qa are in
principle easy to obtain:
n Count relative frequencies of
every amino acid in a sequence
database

275
Calculating score matrices for amino
acids
p To calculate pab we can use a
known pool of aligned sequences
p BLOCKS is a database of highly
conserved regions for proteins Block PR00851A
ID XRODRMPGMNTB; BLOCK
p It lists multiply aligned, ungapped AC PR00851A; distance from previous block=(52,131)
DE Xeroderma pigmentosum group B protein signature
and conserved protein segments BL adapted; width=21; seqs=8; 99.5%=985; strength=1287
XPB_HUMAN|P19447 ( 74) RPLWVAPDGHIFLEAFSPVYK 54
p Example from BLOCKS shows XPB_MOUSE|P49135 ( 74) RPLWVAPDGHIFLEAFSPVYK 54
P91579 ( 80) RPLYLAPDGHIFLESFSPVYK 67
genes related to human gene XPB_DROME|Q02870 ( 84) RPLWVAPNGHVFLESFSPVYK 79
RA25_YEAST|Q00578 ( 131) PLWISPSDGRIILESFSPLAE 100
associated with DNA-repair Q38861 ( 52) RPLWACADGRIFLETFSPLYK 71
O13768 ( 90) PLWINPIDGRIILEAFSPLAE 100
defect xeroderma pigmentosum O00835 ( 79) RPIWVCPDGHIFLETFSAIYK 86

http://blocks.fhcrc.org

276
 BLOSUM matrix
p BLOSUM is a score matrix RPLWVAPD
for amino acid sequences RPLWVAPR
derived from BLOCKS data
RPLWVAPN
p First, count pairwise
matches fx,y for every amino PLWISPSD
acid type pair (x, y) RPLWACAD
p For example, for column 3 PLWINPID
and amino acids L and W,
we find 8 pairwise matches: RPIWVCPD
fL,W = fW,L = 8

277
Creating a BLOSUM matrix
p Probability pab is obtained by RPLWVAPD
dividing fab with the total RPLWVAPR
number of pairs (note
difference with course book): RPLWVAPN
PLWISPSD
RPLWACAD
PLWINPID
RPIWVCPD
p We get probabilities qa by

278
 Creating a BLOSUM matrix
p The probabilities pab and qa can now be plugged
into

to get a 20 x 20 matrix of scores s(a, b).

p Next slide presents the BLOSUM62 matrix
n Values scaled by factor of 2 and rounded to integers
n Additional step required to take into account expected
evolutionary distance
n Described in Deonier’s book in more detail

279
 BLOSUM62
A R N D C Q E G H I L K M F P S T W Y V B Z X *
A 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0 -2 -1 0 -4
R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3 -1 0 -1 -4
N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3 3 0 -1 -4
D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 4 1 -1 -4
C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 -3 -3 -2 -4
Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2 0 3 -1 -4
E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2 1 4 -1 -4
G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 -1 -2 -1 -4
H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3 0 0 -1 -4
I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3 -3 -3 -1 -4
L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1 -4 -3 -1 -4
K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2 0 1 -1 -4
M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1 -3 -1 -1 -4
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1 -3 -3 -1 -4
P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2 -2 -1 -2 -4
S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2 0 0 0 -4
T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0 -1 -1 0 -4
W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3 -4 -3 -2 -4
Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1 -3 -2 -1 -4
V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 -3 -2 -1 -4
B -2 -1 3 4 -3 0 1 -1 0 -3 -4 0 -3 -3 -2 0 -1 -4 -3 -3 4 1 -1 -4
Z -1 0 0 1 -3 3 4 -2 0 -3 -3 1 -1 -3 -1 0 -1 -3 -2 -2 1 4 -1 -4
X 0 -1 -1 -1 -2 -1 -1 -1 -1 -1 -1 -1 -1 -1 -2 0 0 -2 -1 -1 -1 -1 -1 -4
280
* -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 -4 1
 Using BLOSUM62 matrix
MQLEANADTSV
| | |
LQEQAEAQGEM

=2+5–3–4+4+0+4+0–2+0+
1
=7

281
 Demonstration of BLAST at NCBI
p http://www.ncbi.nlm.nih.gov/BLAST/

282
Introduction to
Bioinformatics
Lecture 4:
Genome rearrangements
 Why study genome rearrangements?
p Provide insight into evolution of species
p Fun algorithmic problem!

p Structure of this lecture:

n The biological phenomenon
n How to computationally model it?
n How to compute interesting things?
n Studying the phenomenon using existing tools
(continued in exercises)

284
 Genome rearrangements as an
algorithmic problem

285
 Background
p Genome sequencing enables us to
compare genomes of two or more
different species
n -> Comparative genomics
p Basic observation:
n Closely related species (such as human and
mouse) can be almost identical in terms of
genome contents...
n ...but the order of genomic segments can be
very different between species

286
 Synteny blocks and segments
p Synteny – derived from Greek ’on the
same ribbon’ – means genomic segments
located on the same chromosome
n Genes, markers (any sequence)
p Synteny block (or syntenic block)
n A set of genes or markers that co-occur
together in two species
p Synteny segment (or syntenic segment)
n Syntenic block where the order of genes or
markers is preserved

287
 Synteny blocks and segments
Homologs
Chromosome i, species B
of the same
gene

Synteny segment

Synteny block

Chromosome j, species C

288
 Observations from sequencing
1. Large chromosome inversions and
translocations (we’ll get to these shortly)
are common
n ...Even between closely related species
2. Chromosome inversions are usually
symmetric around the origin of DNA
replication
3. Inversions are less common within
species...

289
 What causes rearrangements?
p RecA, Recombinase A,
is a protein used to
repair chromosomal
damage
p It uses a duplicate
copy of the damaged
sequence as template
p Template is usually a
homologous sequence
on a sister
chromosome

Diarmaid Hughes: Evaluating genome dynamics: the constraints on

290
rearrangements within bacterial genomes, Genome Biology 2000, 1
 Chromosomes: recap
p Linear chromosomes
centromere
n Eukaryotes (mostly)

chromatid
p Circular chromosomes
gene 2
n Prokaryotes (mostly)
gene 1
n Mitochondria

gene 3
Also double-stranded: genes can be
291
found on both strands (orientations)
 What effects does RecA have on
genome?
p Repeated sequences cause RecA to fail to
choose correct recombination start
position
p This leads to
Damaged sequence
n Tandem duplications
n Translocations
RecA
n Inversions
?

Repeat 1 Repeat 2

292
 X, Y, Z and W are repeats of
the same sequence.

a, b, c and d are sequences on genome

bounded by repeats.

In a tandem duplication example,

RecA recombines a sequence that
starts from Y instead of Z after Z.

This leads to duplication of segment Y-Z.

Diarmaid Hughes: Evaluating genome dynamics: the constraints on

293
rearrangements within bacterial genomes, Genome Biology 2000, 1
Recombination of two
repeat sequences in the
same chromosome can
lead to a fragment translocation

Here sequence d is translocated

Diarmaid Hughes: Evaluating genome dynamics: the constraints on

294
rearrangements within bacterial genomes, Genome Biology 2000, 1
Inversion happens when two
sequences of opposite orientations
are recombined.

Diarmaid Hughes: Evaluating genome dynamics: the constraints on

295
rearrangements within bacterial genomes, Genome Biology 2000, 1
 Example: human vs mouse genome
p Human and mouse genomes share
thousands of homologous genes, but they
are
n Arranged in different order
n Located in different chromosomes
p Examples
n Human chromosome 6 contains elements from
six different mouse chromosomes
n Analysis of X chromosome indicates that
rearrangements have happened primarily
within chromosome

296
297
Jones & Pevzner, 2004
298
 Representing genome rearrangments
p When comparing two genomes, we can
find homologous sequences in both using
BLAST, for example
p This gives us a map between sequences in
both genomes

299
 Representing genome rearrangments
p We assign numbers 1,...,n to Human Mouse
the found homologous 1 (gnat2) 12 (inpp1)
sequences 2 (nras) 13 (cd28)
p By convention, we number the 3 (ngfb) 14 (fn1)
sequences in the first genome 4 (gba) 15 (pax3)
by their order of appearance 5 (pklr) -9 (il10)
in chromosomes 6 (at3) -8 (pdc)

p If the homolog of i is in 7 (lamc1) -7 (lamc1)

reverse orientation, it receives 8 (pdc) -6 (at3)

number –i (signed data) 9 (il10)

p For example, consider human

vs mouse gene numbering on
the right List order corresponds to
300
physical order on chromosomes!
 Permutations
p The basic data structure in the study of
genome rearrangements is permutation
p A permutation of a sequence of n numbers
is a reordering of the sequence
p For example, 4 1 3 2 5 is a permutation of
12345

301
 Genome rearrangement problem
p Given two genomes (set of markers), how
many
n duplications,
n inversions and
n translocations
do we need to do to transform the first
genome to the second?
Minimum number of operations?
What operations? Which order?

302
 Genome rearrangement problem

#duplications?
#inversions?
#translocations?
612345 123456

303
 Genome rearrangement problem
Permutation
1 2 3 4 5 6
of 1,...,6

612345 123456

Keep in mind, that the two genomes

have been evolved from a common
ancestor genome!

304
 Genome rearrangements using reversals
(=inversions) only
p Lets consider a simpler problem where we just
study reversals with unsigned data
p A reversal p(i, j) reverses the order of the
segment i i+1 ... j-1 j (indexing starts from 1)
p For example, given permutation
6 1 2 3 4 5 and reversal p(3, 5) we get
permutation 6 1 4 3 2 5

p(3, 5)

...note that we do not care about exact positions on the genome

305
 Reversal distance problem
p Find the shortest series of reversals that, given
a permutation , transforms it to the identity
permutation (1, 2, ..., n)
p This quantity is denoted by d( )

p Reversal distance for a pair of chromosomes:

n Find synteny blocks in both
n Number blocks in the first chromosome to identity
n Set to correspond matching of second chromosome’s
blocks against the first
n Find reversal distance

306
 Reversal distance problem: discussion
p If we can find the minimal series of
reversals for some pair of genomes
n Is that what happened during evolution?
n If not, is it the correct number of reversals?

p In any case, reversal distance gives us a

measure of evolutionary distance between
the two genomes and species

307
 Solving the problem by sorting
p Our first approach to solve the reversal
distance problem:
n Examine each position i of the permutation
n At each position, if i i, do a reversal such
that i = i
p This is a greedy approach: we try to
choose the best option at each step

308
 Simple reversal sort: example
6 1 2 3 4 5 -> 1 6 2 3 4 5 -> 1 2 6 3 4 5 -> 1 2 3 4 6 5

-> 1 2 3 4 5 6

Reversal series: p(1,2), p(2,3), p(3,4), p(5,6)

Is d(6 1 2 3 4 5) then 4?

6 1 2 3 4 5 -> 5 4 3 2 1 6 -> 1 2 3 4 5 6

D(6 1 2 3 4 5) = 2

309
 Pancake flipping problem
p No pancake made by
the chef is of the
same size
p Pancakes need to be
rearranged before
delivery
p Flipping operation:
take some from the
top and flip them over
p This corresponds to 1 2 3 6 4 5 -> 6 3 2 1 4 5 ->
always reversing the 5 4 1 2 3 6 -> 3 2 1 4 5 6 ->
sequence prefix
123456

310
 How good is simple reversal sort?
p Not so good actually
p It has to do at most n-1 reversals with
permutation of length n
p The algorithm can return a distance that is
as large as (n – 1)/2 times the correct
result d( )
n For example, if n = 1001, result can be as bad
as 500 x d( )

311
 Estimating reversal distance by cycle
decomposition
p We can estimate d( ) by cycle
decomposition
p Lets represent permutation =12453
with the following graph

0 1 2 4 5 3 6

where edges correspond to adjacencies

(identity, permutation F)
312
 Estimating reversal distance by cycle
decomposition
p Cycle decomposition: a set of cycles that
n have edges with alternating colors
n do not share edges with other cycles (=cycles
are edge disjoint)

0 1 2 4 5 3 6

1 2 4 5
313
 Cycle decompositions
p Let c( ) the maximum number of alternating,
edge-disjoint cycles in the graph representation
of
p The following formula allows estimation of d( )
n d( ) n + 1 – c( ), where n is the permutation length

0 1 2 4 5 3 6
d( ) 5+1–4=2

1 2 4 5
Claim in Deonier: equality holds for ”most of the usual and
314
interesting biological systems.
 Cycle decompositions
p Cycle decomposition is NP-complete
n We cannot solve the general problem exactly
for large instances
p However, with signed data the problem
becomes easy
n Before going into signed data, lets discuss
another algorithm for the general case

315
 Computing reversals with breakpoints
p Lets investigate a better way to compute
reversal distance
p First, some concepts related to
permutation 1 2,,, n-1 n
n Breakpoint: two elements i and i+1 are a
breakpoint, if they are not consecutive
numbers
n Adjacency: if i and i+1 are consecutive, they
are called adjacency

316
 Breakpoints and adjacencies
This permutation contains
four breakpoints begin-2, 13, 58, 6-end and
five adjacencies 21, 34, 45, 87, 76

21345876

Breakpoints

317
 Breakpoints
p Each breakpoint in permutation needs to be
removed to get to the identity permutation (=our
target)
n Identity permutation does not contain any breakpoints

21345876 b( ) = 4

p First and last positions special cases

p Note that each reversal can remove at most two
breakpoints
p Denote the number of breakpoints by b( )

318
 Breakpoint reversal sort
p Idea: try to remove as many breakpoints
as possible (max 2) in every step

1. While b( ) > 0
2. Choose reversal p that removes most breakpoints
3. Perform reversal p to
4. Output
5. return

319
 Breakpoint removal: example
82765143 b( ) = 6

28765143 b( ) = 5

23415678 b( ) = 3

43215678 b( ) = 2

12345678 b( ) = 0

320
 Breakpoint removal
p The previous algorithm needs refinement
to be correct
p Consider the following permutation:

15672348

p There is no reversal that decreases the

number of breakpoints!
p See Jones & Pevzner for detailed
description on this

321
 Strip: maximal segment without breakpoints
Increasing strip
Breakpoint removal Decreasing strip

p Reversal can only decrease breakpoint

count if permutation contains decreasing
strips

15672348

15674328

12347658

322
 Improved breakpoint reversal sort
1. While b( ) > 0
2. If has a decreasing strip
3. Do reversal p that removes most BPs
4. Else
5. Reverse an increasing strip
6. Output
7. return

323
 Is Improved BP removal enough?
p The algorithm works pretty well:
n It produces a result that is at most four times
worse than the optimal result
n ...is this good?

p We considered only reversals

p What about translocations & duplications?

324
 Translocations via reversals

12345678
Translocation of 2,3,4

15678234
p(2,8)
14328765
p(2,4)

12348765
p(5,8)

12345678
325
 Genome rearrangements with reversals
p With unsigned data, the problem of finding
minimum reversal distances is NP-
complete
n Why is this so if sorting is easy?
p An algorithm has been developed that
achieves 1.375-approximation
p However, reversal distance in signed data
can be computed quickly!
n It takes linear time w.r.t. the length of
permutation (Bader, Moret, Yan, 2001)

326
 Cycle decomposition with signed data
p Consider the following two permutations
that include orientation of markers
n J: +1 +5 -2 +3 +4
n K: +1 -3 +2 +4 -5

p We modify this representation a bit to

include both endpoints of each marker:
n J’: 0 1a 1b 5a 5b 2b 2a 3a 3b 4a 4b 6
n K’: 0 1a 1b 3b 3a 2a 2b 4a 4b 5b 5a 6

327
 Graph representation of J’ and K’
p Drawn online in lecture!

328
 Multiple chromosomes
p In unichromosomal genomes, inversion
(reversal) is the most common operation
p In multichromosomal genomes,
inversions, translocations, fissions and
fusions are most common

329
 Multiple chromosomes
p Lets represent multichromosomal genome
as a set of permutations, with $ denoting
the boundary of a chromosome:
59$ Chr 1

1328$ Chr 2

764$ Chr 3

This notation is frequently used in software

used to analyse genome rearrangements.

330
 Multiple chromosomes
p Note that when dealing with multiple
chromosomes, you need to specify
numbering for elements on both genomes

331
 Reversals & translocations
p Reversal p( , i, j)
p Translocation p( , , i, j)

Translocation

332
 Fusions & fissions
p Fusion: merging of two chromosomes
p Fission: chromosome is split into two
chromosomes
p Both events can be represented with a
translocation

333
 Fusion
p Fusion by translocation p( , , n+1, 1)

i=n+1

j=1
Fusion

334
 Empty chromosome
Fission
p Fission by translocation p( , , i, 1)
i

Fission

335
 Algorithms for general genomic distance
problem
p Hannenhalli, Pevzner: Transforming Men into
Mice (polynomial algorithm for genomic distance
problem), 36th Annual IEEE Symposium on
Foundations of Computer Science, 1995

336
 Human & mouse revisited
p Human and mouse are separated by about
75-83 million years of evolutionary history
p Only a few hundred rearrangements have
happened after speciation from the
common ancestory
p Pevzner & Tesler identified in 2003 for 281
synteny blocks a rearrangement from
mouse to human with
n 149 inversions
n 93 translocations
n 9 fissions

337
 Discussion
p Genome rearrangement events are very
rare compared to, e.g., point mutations
n We can study rearrangement events further
back in the evolutionary history
p Rearrangements are easier to detect in
comparison to many other genomic events
p We cannot detect homologs 100%
correctly so the input permutation can
contain errors

338
 Discussion
p Genome rearrangement is to some degree
constrained by the number and size of
repeats in a genome
n Notice how the importance of genomic repeats
pops up once again
p Sequencing gives us (usually) signed data
so we can utilize faster algorithms
p What if there are more than one optimal
solution?

339
 Two different genome rearrangement scenarios
giving the same result.

340
 GRIMM demonstration

Glenn Tesler, GRIMM: genome rearrangements web server.

341
Bioinformatics, 2002,
 GRIMM file format
# useful comment about first genome
# another useful comment about it
>name of first genome
1 -4 2 $ # chromosome 1
-3 5 6 # chromosome 2
>name of second genome
5 -3 $
6$
2 -4 1 $

GRIMM supports analysis of

one, two or more genomes

342
http://grimm.ucsd.edu/GRIMM/grimm_instr.html
Introduction to
Bioinformatics
Phylogenetic trees
 Inferring the Past: Phylogenetic
Trees
p The biological problem
p Parsimony and distance methods
p Models for mutations and estimation of
distances
p Maximum likelihood methods

344
 Phylogeny
p We want to study ancestor-
descendant relationships, or
phylogeny, among groups of
organisms
p Groups are called taxa (singular:
taxon)
p Organisms are usually called
operational taxonomic units or
OTUs in the context of
phylogeny

345
 Phylogenetic trees
p Leaves (external nodes)
~ species, observed 3
(OTUs)
p Internal nodes ~ 2 4
ancestral 7
species/divergence 6 8
events, not observed 1 5
p Unrooted tree does not
specify ancestor- Unrooted tree with 5
descendant relationships
leaves and 3 internal
beyond the observation
nodes.
”leaves are not
ancestors” Is node 7 ancestor of node
6?

346
 Phylogenetic trees 3
R1 R2
p Rooting a tree specifies all
ancestor-descendant 2 7 4
relationships in the tree 6 8
p Root is the ancestor to the
other species 1 5

roo
1)
There are n-1 ways to root

t(R 2
(R
p

ot
a tree with n nodes

ro

)
R 1
R2
8
7 7
6 8 6

1 2 3 4 5 1 2 3 5 4

347
 Questions
p Can we enumerate all possible
phylogenetic trees for n species (or
sequences?)
p How to score a phylogenetic tree with
respect to data?
p How to find the best phylogenetic tree
given data?

348
 Finding the best phylogenetic tree:
naive method
p How can we find the phylogenetic tree
that best represents the data?
p Naive method: enumerate all possible
trees
p How many different trees are there of n
species?
p Denote this number by bn

349
 Enumerating unordered trees
p Start with the only 1 2 1 2 1 2
unordered tree with 3
leaves (b3 = 1) 4 4 4
3 3 3
1 2
p Fourth node can be added
to 3 different branches
3 (edges), creating 1 new
internal branch
p Consider all ways to add p Total number of branches
a leaf node to this tree is n external and n – 3
internal branches
p Unrooted tree with n
leaves has 2n – 3 branches

350
 Enumerating unordered trees
p Thus, we get the number of unrooted trees
bn = (2(n – 1) – 3)bn-1 = (2n – 5)bn-1
= (2n – 5) * (2n – 7) * …* 3 * 1
= (2n – 5)! / ((n-3)!2n-3), n > 2
p Number of rooted trees b’n is
b’n = (2n – 3)bn = (2n – 3)! / ((n-2)!2n-2),
n>2
that is, the number of unrooted trees times the
number of branches in the trees

351
 Number of possible rooted and
unrooted trees
n Bn b’n
3 1 3
4 3 15
5 15 105
6 105 945
7 954 10395
8 10395 135135
9 135135 2027025
10 2027025 34459425
20 2.22E+020 8.20E+021
30 8.69E+036 4.95E+038

352
 Too many trees?
p We can’t construct and evaluate every
phylogenetic tree even for a smallish
number of species
p Better alternative is to
n Devise a way to evaluate an individual tree
against the data
n Guide the search using the evaluation criteria
to reduce the search space

353
 Inferring the Past: Phylogenetic
Trees (chapter 12)
p The biological problem
p Parsimony and distance methods
p Models for mutations and estimation of
distances
p Maximum likelihood methods

354
 Parsimony method
p The parsimony method finds the tree that
explains the observed sequences with a
minimal number of substitutions
p Method has two steps
n Compute smallest number of substitutions for
a given tree with a parsimony algorithm
n Search for the tree with the minimal number of
substitutions

355
 Parsimony: an example
p Consider the following short sequences
1 ACTTT
2 ACATT
3 AACGT
4 AATGT
5 AATTT
p There are 105 possible rooted trees for 5
sequences
p Example: which of the following trees
explains the sequences with least number
356
of substitutions?
 9 AATTT
A->C
T->G 7 AATTT

T->C 6 AATGT T->A 8 ACTTT

3 4 5 2 1
AACGT AATGT AATTT ACATT ACTTT

This tree explains the sequences

with 4 substitutions

357
4 9 AATTT First tree is
substitutions… A->C more
T->G 7 AATTT
parsimonious!
T->C 6 AATGT T->A 8 ACTTT

3 4 5 2 1
AACGT AATGT AATTT ACATT ACTTT

T->G 9 AATTT
6 T->C 8 AATGT
A->C
substitutions… G->T 7 AACGT

C->T 6 ACCTT C->A

1 2 3 4 5
ACTTT ACATT AACGT AATGT AATTT

358
 Computing parsimony
p Parsimony treats each site (position in a
sequence) independently
p Total parsimony cost is the sum of parsimony
costs (=required substitutions) of each site
p We can compute the minimal parsimony cost for
a given tree by
n First finding out possible assignments at each node,
starting from leaves and proceeding towards the root
n Then, starting from the root, assign a letter at each
node, proceeding towards leaves

359
 Labelling tree nodes
p An unrooted tree with n leaves contains 2n-1
nodes altogether
p Assign the following labels to nodes in a rooted
tree
n leaf nodes: 1, 2, …, n
n internal nodes: n+1, n+2, …, 2n-1 9
n root node: 2n-1
p The label of a child node is always
smaller than the label of the 8

parent node 6 7

1 2 3 4 5

360
 Parsimony algorithm: first phase
p Find out possible assignments at every node for each
site u independently. Denote site u in sequence i by
si,u.
For i := 1, …, n do
Fi := {si,u} % possible assignments at node i
Li := 0 % number of substitutions up to node i

For i := n+1, …, 2n-1 do

Let j and k be the children of node i
If Fj Fk =
then Li := Lj + Lk + 1, Fi := Fj Fk
else Li := Lj + Lk, Fi := Fj Fk
361
 Parsimony algorithm: first phase
Choose u = 3 (for example, in general we do this for all sites)
F1 := {T}
L1 := 0
9
F2 := {A}
L2 := 0 7
F3 := {C}, L3 := 0 6 8

F4 := {T}, L4 := 0 3 4 5 2 1
F5 := {T}, L5 := 0 AACGT AATGT AATTT ACATT ACTTT

362
 Parsimony algorithm: first phase
F6 := F3 F4 = {C, T}
L6 := L3 + L4 + 1 = 1
9T
F7 := F5 F6 = {T}
L7 := L5 + L6 = 1 7T

6 {C,T} 8 {A, T}
F8 := F1 F2 = {A, T}
L8 := L1 + L2 + 1 = 1 3 4 5 2 1
AACGT AATGT AATTT ACATT ACTTT
F9 := F7 F8 = {T}
L9 := L7 + L8 = 2
Parsimony cost for site 3 is 2

363
 Parsimony algorithm: second phase
p Backtrack from the root and assign x Fi
at each node
p If we assigned y at parent of node i and y
Fi, then assign y
p Else assign x Fi by random

364
 Parsimony algorithm: second phase
At node 6, the
algorithm assigns T 9T
because T was
assigned to parent 7T
node 7 and T F6. 6 {C,T} 8 {A,T}
T is assigned to node 8
3 4 5 2 1
for the same reason.
AACGT AATGT AATTT ACATT ACTTT

The other nodes have

only one possible letter
to assign

365
 Parsimony algorithm
First and second phase are
repeated for each site in 9T
the sequences,
summing the parsimony 7T
costs at each site
6T 8T

3 4 5 2 1
AACGT AATGT AATTT ACATT ACTTT

366
 Properties of parsimony algorithm
p Parsimony algorithm requires that the sequences
are of same length
n First align the sequences against each other and,
optionally, remove indels
n Then compute parsimony for the resulting sequences
n Indels (if present) considered as characters
p Is the most parsimonious tree the correct tree?
n Not necessarily but it explains the sequences with least
number of substitutions
n We can assume that the probability of having fewer
mutations is higher than having many mutations

367
 Finding the most parsimonious tree
p Parsimony algorithm calculates the
parsimony cost for a given tree…
p …but we still have the problem of finding
the tree with the lowest cost
p Exhaustive search (enumerating all trees)
is in general impossible
p More efficient methods exist, for example
n Probabilistic search
n Branch and bound

368
 Branch and bound in parsimony
p We can exploit the fact that adding edges
to a tree can only increase the parsimony
cost {C, T}

{T}
{T}
1 2 3 1 2
AATGT AATTT AACGT AATGT AATTT

cost 0 cost 1

369
 Branch and bound in parsimony
Branch and bound is
In parsimony…
a general search
p Start from a tree with 1
strategy where
sequence
p Each solution is p Add a sequence to the tree
potentially generated and calculate parsimony
p Track is kept of the cost
best solution found p If the tree is complete,
check if found the best tree
p If a partial solution so far
cannot achieve better p If tree is not complete and
score, we abandon cost exceeds best tree
the current search cost, do not continue
path adding edges to this tree

370
 Complete tree:

Branch and bound example compute parsimony

cost

Example with 4 sequences

1 1 2 1 2 3 1 2 3 4

1 3 1 2 3 4
1 3 2
1 3 2 4

Partial tree:
… Compute parsimony cost
and compare against best
so far;
1 3 2 4 Do not continue expansion
if above cost of the best tree
371
 Distance methods
p The parsimony method works on sequence
(character string) data
p We can also build phylogenetic trees in a
more general setting
p Distance methods work on a set of
pairwise distances dij for the data
p Distances can be obtained from
phenotypes as well as from genotypes
(sequences)

372
 Distances in a phylogenetic tree
p Distance matrix D = (dij)
gives pairwise distances
for leaves of the
phylogenetic tree 7

p In addition, the 6 8

phylogenetic tree will

1 2 3 4 5
now specify distances
between leaves and Distance dij states how
internal nodes far apart species i and j
n Denote these with dij as are evolutionary (e.g.,
well number of mismatches in
aligned sequences)

373
 Distances in evolutionary context
p Distances dij in evolutionary context
satisfy the following conditions
n Symmetry: dij = dji for each i, j
n Distinguishability: dij 0 if and only if i j
n Triangle inequality: dij dik + dkj for each i, j, k
p Distances satisfying these conditions are called
metric
p In addition, evolutionary mechanisms may
impose additional constraints on the distances
additive and ultrametric distances
374
 Additive trees
p A tree is called additive, if the distance
between any pair of leaves (i, j) is the
sum of the distances between the leaves
and a node k on the shortest path from i
to j in the tree
dij = dik + djk

p ”Follow the path from the leaf i to the leaf

j to find the exact distance dij between the
leaves.”
375
 Additive trees: example

A B C D
A C
A 0 2 4 4
1 1
B 2 0 4 4 2

C 4 4 0 2 1 1

D 4 4 2 0 B D

376
 Ultrametric trees
p A rooted additive tree is called an ultrametric
tree, if the distances between any two leaves i
and j, and their common ancestor k are equal
dik = djk

p Edge length dij corresponds to the time elapsed

since divergence of i and j from the common
parent
p In other words, edge lengths are measured by a
molecular clock with a constant rate

377
 Identifying ultrametric data
p We can identify distances to be ultrametric
by the three-point condition:
D corresponds to an ultrametric tree if
and only if for any three species i, j and
k, the distances satisfy dij max(dik, dkj)

p If we find out that the data is ultrametric, we can

utilise a simple algorithm to find the
corresponding tree

378
 Ultrametric trees
9

8
Time

Observation time
5 4 3 2 1

379
 Ultrametric trees
9
d8,9
Only vertical segments of the
tree have correspondence to
8 some distance dij:
Time

7 Horizontal segments act as

connectors.

Observation time
5 4 3 2 1

380
 Ultrametric trees
9
dik = djk for any two leaves
i, j and any ancestor k of
i and j
8
Time

Observation time
5 4 3 2 1

381
 Ultrametric trees
9

Three-point condition: there are

8 no leafs i, j for which dij > max(dik, djk)
Time

for some leaf k.

7

Observation time
5 4 3 2 1

382
 UPGMA algorithm
p UPGMA (unweighted pair group method
using arithmetic averages) constructs a
phylogenetic tree via clustering
p The algorithm works by at the same time
n Merging two clusters
n Creating a new node on the tree
p The tree is built from leaves towards the
root
p UPGMA produces a ultrametric tree

383
 Cluster distances
p Let distance dij between clusters Ci and Cj
be

that is, the average distance between

points (species) in the cluster.

384
 UPGMA algorithm
p Initialisation
n Assign each point i to its own cluster Ci
n Define one leaf for each sequence, and place it at height
zero
p Iteration
n Find clusters i and j for which dij is minimal
n Define new cluster k by Ck = Ci Cj, and define dkl for all l
n Define a node k with children i and j. Place k at height dij/2
n Remove clusters i and j
p Termination:
n When only two clusters i and j remain, place root at height dij/2

385
 1 2

386
 1 2

4 6
1 2
5

387
 1 2

6 7
4

1 2 4 5
5

388
 1 2

3
8

6 7
4

1 2 4 5 3
5

389
 1 2

9
3
8

6 7
4

1 2 4 5 3
5

390
 UPGMA implementation
p In naive implementation, each iteration
takes O(n2) time with n sequences =>
algorithm takes O(n3) time

p The algorithm can be implemented to take

only O(n2) time (see Gronau & Moran,
2006, for a survey)

391
 Problem solved?
p We now have a simple algorithm which finds a
ultrametric tree
n If the data is ultrametric, then there is exactly one
ultrametric tree corresponding to the data (we skip the
proof)
n The tree found is then the ”correct” solution to the
phylogeny problem, if the assumptions hold
p Unfortunately, the data is not ultrametric in
practice
n Measurement errors distort distances
n Basic assumption of a molecular clock does not hold
usually very well

392
 Incorrect reconstruction of non-
ultrametric data by UPGMA

2 3

4
1 1 4 2 3

Tree which corresponds Incorrect ultrametric reconstruction

to non-ultrametric by UPGMA algorithm
distances
393
 Checking for additivity
p How can we check if our data is additive?
p Let i, j, k and l be four distinct species

p Compute 3 sums: dij + dkl, dik + djl, dil

+ djk

394
 Four-point condition
i dik k i dil k i k
dij dkl
j djl l j djk l j l

p The sums are represented by the three figures

n Left and middle sum cover all edges, right sum does not
p Four-point condition: i, j, k and l satisfy the four-
point condition if two of the sums dij + dkl, dik +
djl, dil + djk are the same, and the third one is
smaller than these two

395
 Checking for additivity
p An n x n matrix D is additive if and only if
the four point condition holds for every 4
distinct elements 1 i, j, k, l n

396
 Finding an additive phylogenetic tree
p Additive trees can be found with, for example,
the neighbor joining method (Saitou & Nei, 1987)
p The neighbor joining method produces unrooted
trees, which have to be rooted by other means
n A common way to root the tree is to use an outgroup
n Outgroup is a species that is known to be more distantly
related to every other species than they are to each
other
n Root node candidate: position where the outgroup would
join the phylogenetic tree
p However, in real-world data, even additivity
usually does not hold very well

397
 Neighbor joining algorithm
p Neighbor joining works in a similar fashion
to UPGMA
n Find clusters C1 and C2 that minimise a
function f(C1, C2)
n Join the two clusters C1 and C2 into a new
cluster C
n Add a node to the tree corresponding to C
n Assign distances to the new branches
p Differences in
n The choice of function f(C1, C2)
n How to assign the distances
398
 Neighbor joining algorithm
p Recall that the distance dij for clusters Ci and Cj
was

p Let u(Ci) be the separation of cluster Ci from

other clusters defined by

where n is the number of clusters.

399
 Neighbor joining algorithm
p Instead of trying to choose the clusters Ci
and Cj closest to each other, neighbor
joining at the same time
n Minimises the distance between clusters Ci and
Cj and
n Maximises the separation of both Ci and Cj
from other clusters

400
 Neighbor joining algorithm
p Initialisation as in UPGMA
p Iteration
n Find clusters i and j for which dij – u(Ci) – u(Cj) is minimal
n Define new cluster k by Ck = Ci Cj, and define dkl for all l
n Define a node k with edges to i and j. Remove clusters i and j
n Assign length ½ dij + ½ (u(Ci) – u(Cj)) to the edge i -> k
n Assign length ½ dij + ½ (u(Cj) – u(Ci)) to the edge j -> k
p Termination:
n When only one cluster remains

401
 Neighbor joining algorithm: example
a b c d i u(i)
a 0 6 7 5 a (6+7+5)/2 = 9
b 0 11 9 b (6+11+9)/2 = 13
c 0 6 c (7+11+6)/2 = 12
d 0 d (5+9+6)/2 = 10

i,j dij – u(Ci) – u(Cj)

a,b 6 - 9 - 13 = -16
a,c 7 - 9 - 12 = -14
a,d 5 - 9 - 10 = -14
Choose either pair
b,c 11 - 13 - 12 = -14
to join
b,d 9 - 13 - 10 = -14
c,d 6 - 12 - 10 = -16

402
 Neighbor joining algorithm: example
a b c d i u(i)
a 0 6 7 5 a (6+7+5)/2 = 9
b 0 11 9 b (6+11+9)/2 = 13
c 0 6 c (7+11+6)/2 = 12
d 0 d (5+9+6)/2 = 10

i,j dij – u(Ci) – u(Cj) e

a,b 6 - 9 - 13 = -16 dae dbe
a,c 7 - 9 - 12 = -14
a b c d
a,d 5 - 9 - 10 = -14
b,c 11 - 13 - 12 = -14 dae = ½ 6 + ½ (9 – 13) = 1
b,d 9 - 13 - 10 = -14 dbe = ½ 6 + ½ (13 – 9) = 5
c,d 6 - 12 - 10 = -16
This is the first step only…
403
 Inferring the Past: Phylogenetic
Trees (chapter 12)
p The biological problem
p Parsimony and distance methods
p Models for mutations and estimation of
distances
p Maximum likelihood methods
n These parts of the book is skipped on this
course (see slides of 2007 course for material
on these topics)
n No questions in exams on these topics!

404
 Problems with tree-building
p Assumptions
n Sites evolve independently of one other
n (Sites evolve according to the same stochastic
model; not really covered this year)
n The tree is rooted
n The sequences are aligned
n Vertical inheritance

405
 Additional material on phylogenetic
trees
p Durbin, Eddy, Krogh, Mitchison: Biological
sequence analysis
p Jones, Pevzner: An introduction to
bioinformatics algorithms
p Gusfield: Algorithms on strings, trees, and
sequences

p Course on phylogenetic analyses in Spring

2009

406
Introduction to
Bioinformatics
Wrap-up
 201
0
i n 1 : Hum
5m a
inut n geno
es! me
Topics Reads
Sequence
assembler
atgagccaag ttccgaacaa ggattcgcgg
gtcggtaaag agcattggaa cgtcggagat
aagaagcgga tgaatttccc cataacgcca
gtggaagaga aggaggcggg cctcccgatc
actccggccc gaagggttga gagtacccca
Sequencing gaaatcacct ccagaggacc ccttcagcga
-Sanger catagcgata ggaggggatg ctaggagttg

-High-throughput

Contigs Comparative genomics

=> Genomes - Phylogenetics
- Genome rearrangements
-…
Gene expression BLAST query
analysis gtggaagagaaggaggcgg…

gaagggttgagagtacccca...
DNA chips
ccagaggaccccttcagcga…
Biodatabases ggaggggatgctaggagttg…
Similar
sequences = homologs?

Systems
Proteomics Biology
Protein gels
MS/MS techniques atgagccaag
408 aagaagcgga
cagcggaaga
 Exams
p Course exam Wednesday 15 October
16.00-19.00 Exactum A111
p Separate exams
n Tue 18 November 16.00-20.00 Exactum A111
n Fri 16 January 16.00-20.00 Exactum A111
n Tue 31 March 16.00-20.00 Exactum A111
p Check exam date and place before taking
the exam! (previous week or so)

409
 Exam regulations
p If you are late more than 30 min, you
cannot take the exam
p You are not allowed to bring material such
as books or lecture notes to the exam
p Allowed stuff: blank paper (distributed in
the exam), pencils, pens, erasers,
calculators, snacks
p Bring your student card or other id!

410
 Grading
p Grading: on the scale 0-5
n To get the lowest passing grade 1, you need to get at
least 30 points out of 60 maximum
p Course exam gives you maximum of 48 points
p Note: if you take the first separate exam, the
best of the following options will be considered:
n Exam gives you max 48 points, exercises max 12 points
n Exam gives you max 60 points
p In second and subsequent separate exams, only
the 60 point option is in use

411
 Exercise points
p Max. marks: 31
p 80% of 31 ~= 24 marks -> 12 points
p 2 marks = 1 point

412
 Topics covered by exams
p Exams cover everything presented in lectures
(exception: biological background not covered)

p Word distributions and occurrences (course book

chapters 2-3)
p Genome rearrangements (chapter 5)
p Sequence alignment (chapter 6)
p Rapid alignment methods: FASTA and BLAST
(chapter 7)
p Sequencing and sequence assembly (chapter 8)

413
 Topics covered by exams
p Similarity, distance and clustering
(chapter 10)
p Expression data analysis (chapter 11)
p Phylogenetic trees (chapter 12)
p Systems biology: modelling biological
networks (no chapter in course book)

414
 Bioinformatics courses in 2008
p Biological sequence analysis (II period,
Kumpula)
n Focus on probabilistic methods: Hidden Markov
Models, Profile HMMs, finding regulatory
elements, …
p Modeling of biological networks (20-
24.10., TKK)
n Biochemical network modelling and parameter
estimation in biochemical networks using
mechanistic differential equation models.

415
 Bioinformatics courses in autumn 2008
p Bayesian paradigm in genetic
bioinformatics (II period, Kumpula)
n Applications of Bayesian approach in computer
programs and data analysis of
p genetic past,
p phylogenetics,
p coalescence,
p relatedness,
p haplotype structure,
p disease gene associations.

416
 Bioinformatics courses in autumn 2008
p Statistical methods in genetics (II period,
Kumpula)
n Introduction to statistical methods in gene
mapping and genetic epidemiology.
n Basic concepts of linkage and association
analysis as well as some concepts of
population genetics will be covered.

417
 Bioinformatics courses in Spring 2009
p Practical Course in Biodatabases (III
period, Kumpula)
p High-throughput bioinformatics (III-IV
periods, TKK)
p Phylogenetic data analyses (IV period,
Kumpula)
n Maximum likelihood methods, Bayesian
methods, program packages
p Metabolic modelling (IV period, Kumpula)

418
 Genomes sequenced – all done?
p Sequencing is just the beginning
n What do genes and proteins do?
p Functional genomics
n How do they interact with other genes
and proteins?
p Systems biology

Two sides of the same question!

419
 Bioinformatics (at least mathematical
biology) can exist outside molecular biology

Melitaea cinxia, Glanville Fritillary butterfly

The metapopulation capacity of a fragmented landscape

Ilkka Hanski and Otso Ovaskainen
420
Nature 404, 755-758(13 April 2000)
 Metagenomics
p Metagenomics or environmental genomics
n ”At the last count 1.8 million species were known to
science. That sounds like a lot, but in truth it's no big
deal. We may have done a reasonable job of describing
the larger stuff, but the fact remains that an average
teaspoon of water, soil or ice contains millions of micro-
organisms that have never been counted or named. ”
-- Henry Nicholls

421
 Omics
p Genome p Phenome
p Transcriptome p Exposome
p Metabolome p Textome
p Metallome p Receptorome
p Lipidome p Kinome
p Glycome p Neurome
p Interactome p Cytome
p Spliceome p Predictome
p ORFeome p Omeome
p Speechome p Reactome
p Mechanome p Connectome

422
http://en.wikipedia.org/wiki/-omics
 Take-home messages
p Don’t trust biodatabases blindly!
n Annotation errors tend to accumulate
p Consider
n Statistical significance
n Sensitivity
of your results
p Think about the whole ”bioinformatics workflow”:
n Biological phenomenon -> Modelling -> Computation ->
Validation of results
p Results from bioinformatics tools and methods
must be validated!
p Actively seek cooperation with experts

423
 Bioinformatics journals
p Bioinformatics, http://bioinformatics.oupjournals.org/
p BMC Bioinformatics,
http://www.biomedcentral.com/bmcbioinformatics
p Journal of Bioinformatics and Computational Biology
(JBCB), http://www.worldscinet.com/jbcb/jbcb.shtml
p Journal of Computational Biology,
http://www.liebertpub.com/CMB/
p IEEE/ACM Transactions on Computational Biology and
Bioinformatics , http://www.computer.org/tcbb/
p PLoS Computational Biology, www.ploscompbiol.org
p In Silico Biology, http://www.bioinfo.de/isb/
p Nature, Science (bedtime reading)

424
 Bioinformatics conferences
p ISMB, Intelligent Systems for
Molecular Biology (Toronto, July 2008)
p ICSB, International Conference on
Systems Biology (Göteborg, Sweden;
22-28 August)
p RECOMB, Research in Computational
Molecular Biology
p ECCB, European Conference on
Computational Biology
p WABI, Workshop on Algorithms in
Bioinformatics
p PSB, Pacific Symposium on
Biocomputing
January 5-9, 2009
The Big Island of Hawaii
425
 Master’s degree in bioinformatics?
p You can apply to MBI during the
application period November ’08 – 2
February ’09
n Bachelor’s degree in suitable field
n At least 60 ECTS credits in CS or mathstat
n English language certificate
p Passing this course gives you the first 4
credits for Bioinformatics MSc!

426
 Information session on MBI
p Wednesday 19.11. 13.00-15.00 Exactum
D122
p www.cs.helsinki.fi/mbi/events/info08
p Talks in Finnish

427
 Mailing list for bioinformatics courses
and events
p MBI maintains a mailing list for
announcement on bioinformatics courses
and events
p Send email to bioinfo a t cs.helsinki.fi if
you want to subscribe to the list (you can
unsubscribe in the same way)
p List is moderated

428
 The aim of this course
Biology & Medicine

Bioinformatics
Mathematics
Computer Science
and Statistics

Where would you be in this triangle?

Has your position shifted during the course?
429
 Feedback
p Please give feedback on the course!
n https://ilmo.cs.helsinki.fi/kurssit/servlet/Valint
a?kieli=en
p Don’t worry about your grade – you can
give feedback anonymously

430
 Thank you!
p I hope you enjoyed the course!

Halichoerus grypus, Gray seal or harmaahylje in Finnish

431
taivasalla.net