I519 Introduction to Bioinformatics, Fall 2012

Pairwise alignment of DNA/protein

sequences
 We compare biological molecules,
not any two strings!
 Sequence alignment reveals function, structure,
and evolutionary information!
 From edit distance to distance between two
biological molecules with biological meaning—
the scoring matrix
 Local alignment versus global alignment
 But still the dynamic programming algorithm is
the algorithm behind pairwise alignment of
biological sequences
Aligning DNA Sequences: scoring
matrix

V = ATCTGATG n=8 4 matches

m=7 1 mismatches
W = TGCATAC
match
2 insertions
mismatch 2 deletions

V A T C T G A T G
W T G C A T A C
indels deletion
insertion
 Simple scoring
 When mismatches are penalized by –μ, indels
are penalized by –σ,
and matches are rewarded with +1,
the resulting score is:

#matches – μ(#mismatches) – σ (#indels)

 Scoring matrices
To generalize scoring, consider a (4+1) x(4+1)
scoring matrix δ.
In the case of an amino acid sequence alignment,
the scoring matrix would be a (20+1)x(20+1)
size. The addition of 1 is to include the score
for comparison of a gap character “-”.
This will simplify the algorithm as follows:
si-1,j-1 + δ (vi, wj)
si,j = max s i-1,j + δ (vi, -)
s i,j-1 + δ (-, wj)
 Scoring matrices for DNA sequence
alignment
 A simple positive score for matches and a
negative for mismatches and gaps are most
often used.
 Transversions penalized more than transitions
– transitions: replacement of a purine base with
another purine or replacement of a pyrimidine
with another pyrimidine (A <-> G, C <-> T)
– transversions: replacement of a purine with a
pyrimidine or vice versa.
– Transition mutations are more common than
transversions
 Making a scoring matrix for
protein sequence alignment
 Scoring matrices are created based on biological
evidence.
 Alignments can be thought of as two sequences
that differ due to mutations.
 Some of these mutations have little effect on the
protein’s function, therefore some penalties,
δ(vi , wj), will be less harsh than others.
 We need to know how often one amino acid is
substituted for another in related proteins
 Scoring matrix: example
A R N K
• Notice that although
A 5 -2 -1 -1
R and K are different
R - 7 -1 3
amino acids, they
N - - 7 0 have a positive score.
K - - - 6
• Why? They are both
positively charged
amino acids will not
greatly change
function of protein.
 Conservation
 Amino acid changes that tend to preserve the
physico-chemical properties of the original
residue
– Polar to polar
• aspartate  glutamate
– Nonpolar to nonpolar
• alanine  valine
– Similarly behaving residues
• leucine to isoleucine
 Common scoring matrices for
protein sequence alignment
 Amino acid substitution matrices
– PAM
– BLOSUM

 Try to compare protein coding regions at amino

acid level
– DNA is less conserved than protein sequences
(codon degeneracy; synonymous mutations)
– Less effective to compare coding regions at
nucleotide level
Reading: Chapter 4, 4.3
 PAM
 Point Accepted Mutation (Dayhoff et al.)
 1 PAM = PAM1 = 1% average change of all
amino acid positions
– After 100 PAMs of evolution, not every residue
will have changed
• some residues may have mutated several times
• some residues may have returned to their original state
• some residues may not changed at all
 PAM1 & PAM250
Substitution PAM1 PAM250
Phe to Ala 0.0002 0.04
Phe to Arg 0.0001 0.01
Phe to Asp
..
Phe to Phe 0.9946 0.32
…

Sum 1 1

Normalized probability scores for changing Phe to other amino

acids at PAM1 and PAM250 evolutionary distances

Chapter 3, table 3.2

 Log-odds substitution matrices
 Using amino acid changes that were observed in
closely related proteins; they represented amino
acid substitutions that don’t significantly change
the structure and function of the protein.
– “accepted mutations” or “accepted” by natural
selection.
 Log-odds of the probability of matching a pair of
amino acids in this database relative to a
random one
– Ref: Amino acid substitution matrices from protein
blocks (PNAS. 1992, 89(22): 10915–10919)
 Log odd scores
Define fij as the frequency of observing amino acid pair i, j. Then the
observed probability of occurrence for each i, j pair is
 The amino acid substitution is
considered as a Markov model
 A Markov model is characterized by a series of
changes of state in a system such that a change
from one state to another does not depend on
the previous history of the state
 Use of the Markov model makes it possible to
extrapolate amino acid substitutions observed
over a relatively short period of evolutionary time
to longer periods of evolutionary time
– PAMx = PAM1x
– The multiplication of two PAM1 matrices -> PAM2
 PAM250
 PAM250 is a widely used scoring matrix:
 PAM250 = PAM1250

Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys ...
A R N D C Q E G H I L K ...
Ala A 13 6 9 9 5 8 9 12 6 8 6 7 ...
Arg R 3 17 4 3 2 5 3 2 6 3 2 9
Asn N 4 4 6 7 2 5 6 4 6 3 2 5
Asp D 5 4 8 11 1 7 10 5 6 3 2 5
Cys C 2 1 1 1 52 1 1 2 2 2 1 1
Gln Q 3 5 5 6 1 10 7 3 7 2 3 5
...
Trp W 0 2 0 0 0 0 0 0 1 0 1 0
Tyr Y 1 1 2 1 3 1 1 1 3 2 2 1
Val V 7 4 4 4 4 4 4 4 5 4 15 10
 BLOSUM
 Blocks Substitution Matrix
 Scores derived from observations of the
frequencies of substitutions in blocks of local
alignments in related proteins
 Matrix name indicates evolutionary distance
– BLOSUM62 was created using sequences
sharing no more than 62% identity
 BLOSUM versus PAM
 The PAM family
– PAM matrices are based on global alignments of closely related proteins.
– The PAM1 is the matrix calculated from comparisons of sequences with no
more than 1% divergence; Other PAM matrices are extrapolated from
PAM1.
 The BLOSUM family
– BLOSUM matrices are based on local alignments (blocks)
– All BLOSUM matrices are based on observed alignments (BLOSUM 62 is a
matrix calculated from comparisons of sequences with no less than 62%
divergence)
 Higher numbers in the PAM matrix naming scheme
denote larger evolutionary distance; BLOSUM is the
opposite.
– For alignment of distant proteins, you use PAM150 instead of PAM100, or
BLOSUM50 instead of BLOSUM62.
 Local vs. global alignment

• Global Alignment
--T—-CC-C-AGT—-TATGT-CAGGGGACACG—A-GCATGCAGA-GAC
| || | || | | | ||| || | | | | |||| |
AATTGCCGCC-GTCGT-T-TTCAG----CA-GTTATG—T-CAGAT--C

• Local Alignment—better alignment to find

conserved segment
tccCAGTTATGTCAGgggacacgagcatgcagagac
||||||||||||
aattgccgccgtcgttttcagCAGTTATGTCAGatc
 Local vs. global alignment
 The Global Alignment Problem tries to find the
longest path between vertices (0,0) and (n,m) in
the edit/alignment graph.
– The Needleman–Wunsch algorithm

 The Local Alignment Problem tries to find the

longest path among paths between arbitrary
vertices (i,j) and (i’, j’) in the edit graph.
– The Smith–Waterman algorithm
 Local alignments: why?
 Two genes in different species may be similar
over short conserved regions and dissimilar over
remaining regions.
 Example:
– Homeobox genes have a short region called the
homeodomain that is highly conserved between
species.
– A global alignment would not find the
homeodomain because it would try to align the
ENTIRE sequence
 The local alignment problem
 Goal: Find the best local alignment between two
strings
 Input : Strings v, w and scoring matrix δ
 Output : Alignment of substrings of v and w
whose alignment score is maximum among all
possible alignment of all possible substrings
 Local alignment: example

Compute a “mini”
Global Alignment to
Local alignment
get Local

Global alignment
Local alignment: running time
• Long run time O(n6):

- In the grid of size n x n

there are ~n2 vertices
(i,j) that may serve as a
source and ~n2 vertices
(i’,j’) that may serve as a
sink.
- For each such vertices
computing alignments
from (i,j) to (i’,j’) takes
O(n2) time.
We do NOT go with this algorithm!
 The local alignment recurrence

• The largest value of si,j over the whole edit

graph is the score of the best local
alignment.

• The recurrence:
Notice there is only
0
this change from the
si,j = max si-1,j-1 + δ (vi, wj) original recurrence of
s i-1,j + δ (vi, -) a Global Alignment
s i,j-1 + δ (-, wj)
 The local alignment recurrence
• The largest value of si,j over the whole edit graph
is the score of the best local alignment.
• The recurrence:
Power of ZERO: there is
0 only this change from the
si,j = max si-1,j-1 + δ (vi, wj) original recurrence of a
s i-1,j + δ (vi, -) Global Alignment - since
there is only one “free ride”
s i,j-1 + δ (-, wj) edge entering into every
vertex
• Complexity: O(N2), or O(MN)
• Initialization will be different
 Scoring indels: naive approach
 A fixed penalty σ is given to every indel:
– -σ for 1 indel,
– -2σ for 2 consecutive indels
– -3σ for 3 consecutive indels, etc.

 Can be too severe penalty for a series of 100

consecutive indels
Arbitrary gap penalty?

There are many such edges!

Adding them to the graph

increases the running time
of the alignment algorithm
by a factor of n (where n
is the number of vertices)

So the complexity increases

from O(n2) to O(n3)
 Affine gap penalties
 In nature, a series of k indels often come as a
single event rather than a series of k single
nucleotide events:

Normal scoring would

This is more give the same score This is less
likely. for both alignments likely.
 Affine gap penalties
 Score for a gap of length x is:
-(ρ + σx)
where ρ >0 is the penalty for introducing a gap:
gap opening penalty
ρ will be large relative to σ:
gap extension penalty
because you do not want to add too much of a
penalty for extending the gap.

 Reduced penalties (as compared to naïve

scoring) are given to runs of horizontal and
vertical edges
Affine gap penalty recurrences

Continue gap in y (deletion)

Start gap in y (deletion)

Continue gap in x (insertion)

Start gap in x (insertion)

Match or mismatch
End with deletion
End with insertion
 Readings
 Chapter 4, 4.1
– “Alignment can reveal homology between
sequences” (similarity vs homology)
– “It is easier to detect homology when comparing
protein sequences than when comparing nucleic
acid sequences”
 Primer article: What is dynamic programming by
Eddy
 We will continue on
 Significance of an alignment (score)
– Homologous or not?

 Faster alignment tools for database search