Algorithms for

Sequence Alignments
A. Brngger, Labhead Bioinformatics
Novartis Pharma AG
adrian.bruengger@pharma.novartis.com

Algorithms for Sequence Alignments

Definition of Sequence Alignment and Origins of Sequence
Similarity

Global Alignment
Local Alignment
Alignment of Pairs of Sequences
Alignment of Multiple Sequences

Methods for Pairwise Sequence Alignments

Dot matrix
Scoring Matrices
Dynamic Programming: Smith-Waterman, Needleman-Wunsch

Methods for Multiple Sequence Alignment

Dynamic Programming
Progressive multiple alignments: CLUSTALW, PILEUP

Database Searching for Similar Sequences

FASTA, BLAST
Patscan
Hidden Markov Models

Outline follows: David W. Mount,

"Bioionformatics - Sequence and
Genome Analysis Cold Spring
Harbour Laboratory Press, 2001.
Online:
http://www.bioinformaticsonline.or
g

Rational for Sequence Analysis, Origins of Sequence

Similarity
Function
Structure

Similar sequence leads to similar function

Protein

Sequence Analysis as the basic tool to

discover
functional, structural,evolutionary
information in biological sequences

DNA

Sequence A

y Steps

Sequence B

x Steps

common ancestor
sequence

Evolutionary relationship between two

similar sequences and a possible common
ancestor. The number of steps to convert
one sequence into the other is the
"evolutionary" distance between the
sequences (x + y). Usually, the ancestor
sequence is not available, only (x + y) can
be computed.

Origins of Homology Significance of Sequence Alignments

Possible Origins of Sequence Homology:

orthologs (panel A and B) a1 in species I and a1 in species II (same ancestor!)

paralogs (panel A and B) a1 and a2 (arose from gene duplication event)
analogs (panel C): different genes converge to same function by different
evolutionary paths
transfer of genetic material (panel D) between different species

Homology vs. Similarity

Similarity can be computed (by sequence alignments)

Homology is deduced (e.g. from similarity, but also from other evidence!)

Definition of Sequence Alignment

Computational procedure (algorithm) for comparing two/many
sequences
identify series of identical residues or patterns of identical residues
that appear in the same order in the sequences
visualized by writing sequences as follows:

MLGPSSKQTGKGS-SRIWDN*
||
| |||
| |
MLN-ITKSAGKGAIMRLGDA*

Pairwise Global Alignment

(over whole length of sequences)

GKG
|||
GKG

Pairwise Local Alignment

(similar parts of sequences)

sequence alignment is an optimiztion problem

bringing as many identical residues as possible into corresponding
positions

Pairwise Sequence Alignment

Alignment Score: Quantify the Quality of an alignment
simple scoring system (eg. for DNA sequences)
when two identical residues are aligned:
+1
when two non-identical residues are aligned: -1
when a gap is introduced
-1

total score of alignment is the sum of the scores in each position:

agaag-tagattcta
|| || ||| || ||
aggaggtag-tt-ta

Compute Score:

11 matches
1 mismatch
3 gaps
Score = 11 - 1 -3 = 7

Alignment is 'optimal' when assigned score is maximal

Note: Scoring scheme defines 'optimal' alignment

Algorithms for Pairwise Sequence Alignments: Dot Plot

Algorithm introduced by Gibbs and McIntyre (1970):
write one sequence on top from left to right
write other sequence on the left from top to bottom
place a dot whenever two residues are identical; variants use
colors
visual inspection (variants: identify diagonals with many dots

a g a c c t a g
a *
*
*
g
*
*
a *
*
*
c
* *
c
* *
t
*

Algorithms for Pairwise Sequence Alignments: Dot Plot

Example
Dot Plots of DNA and Protein of Phage l cI (horizontal) and P22 c3
(vertical)

DNA

Protein

Removing noise:
Plot a dot only if 7 ("stringency") out of the next 11 ("window size") residues are identical

Algorithms for Pairwise Sequence Alignments: Dot Plot

Example: Human LDL receptor against itself.

Window size 1, stringency 1.

Window size 23, stringency 7.

"Repeats" in first part of sequence.

Algorithms for Pairwise Sequence Alignments: Dot Plot

Strengths:

visualization of sequence similarity

finding direct and inverted repeats in sequences
finding self-complementary regions in RNA (secondary structures)
simple to compute, simple to visualize

Implementations
DNA Strider (Macintosh)
DOTTER (UNIX X-Windows)
GCG "COMPARE", "DOTPLOT"
online SIB: http://www.isrec.isb-sib.ch/java/dotlet/Dotlet.html

Computational Complexity:
two sequences of length n, m: O(nm)

compute time

sequence length

Scoring Matrices for Proteins

Observation: Protein (Function, Structure) is preserved when
substitutions in certain AAs happen
scoring system less simple in case of proteins
Example: The same protein in three different species

A W T V A S A V R T S I
A Y T V A S A V R T S I
A W T V A A A V L T S I
A

B
W to Y
L to R
A to S

Organism A
Organism B
Organism C

C
Therefore:
Assigning L to R scores higher
than assigning it to another AA

Scoring Matrices for Proteins

Generalization: Dayhoff PAM Weight Matrices (percent accepted
mutation)
observed mutations during a unit of evolutionary time
(1 PAM ~ time that is required that an AA is changed with probability
1%)
initial PAM matrix ("PAM1"): derived from 1572 changes in 71 groups
of protein sequences that are at least 85% similar
observed in proteins that have the same function => "accepted"
mutations

C
S
.
.

C
S
T
P ..
fcc fcs fct fcp
fsc fss fst fsp

Scoring Matrices for Proteins

Extrapolation to longer evolutionary distances possible!

PAM1: p(HM) = 0
PAM2: p(HM) = p(HK)*p(KM)
+p(HR)*p(RM)

Scoring Matrices for Proteins

For each pair of residues, a score is given by a "scoring matrix"
Scoring of two pairs of residues depends on:
frequency of the two residues
exchange that tends to preserves function should have high score

Scoring matrices are constructed by empirically evaluating

(manually constructed) alignments of closely related proteins
PAM
introduced by Margarete Dayhoff, 1978
inspecting 1572 changes in 71 groups of protein sequences
different matrices for varying degree of similarity (or, evolutionary
distance)

BLOSUM
inspection of about 2'000 conserved AA-stretches, "BLOCKS"
Sequence 1:
Sequence 2:
score

V
V
4

D
E
2

S S L
4 -2

C
C
4

Y
Y
4

Total 16

Dynamic Programming Approach to Sequence Alignments

Description by Example:
input:

two AA sequences VDSCY and VESLCY

scoring matrix: match +4, mismatch +2, gap -2
Some possible alignments and their score:
VDSCY
VD-SCY
VDS-CY
| |
| |
| | ||
VESLCY
V-ESLCY
VESLCY
14
8
16
Observation: Longer alignments can be derived from shorter
new score
VDS-CY
VESLCY
16
VDS-C
VESLC
12

= old score

VDS-C
VESLC
12

VDSVESL
8

+ score of new pair

Y
Y
4

C
C
4

Dynamic Programming Approach to Sequence Alignments

Alignment: Path in matrix from "top left" to "bottom right"
V D S C Y
Seq 1:
Seq 2:

V D S - C Y
V E S L C Y

V
E
S
L
C
Y
possible extensions of "current"
alignment ( )

Three situations for extending previous alignment:

introduce gap in
sequence 1

introduce gap in
sequence 2

Dynamic Programming Approach to Sequence Alignments

In each matrix element, we can write the score of the best alignment
up to this element, and a pointer to the shorter alignment it is derive
from
V D S C Y

Seq 1:
Seq 2:

V
V
4

D
E
2

S S L
4 -2

C
C
4

Y
Y
4

match +4, mismatch +2, gap -2

V D S C Y
V
E
S
L
C
Y

4 2 2 2 2
2
2
2
2
2

V
E
S
L
C
Y

Goal is to compute this path!

6
10
12
16

V D S C Y

two possibilities:
4 + (E->S) + gap = 4
2 + (E->S)
= 4
choose one (first)

three possibilities:
4 + (L->D) + 2 gaps = 2
2 + (L->D) + 1 gap = 2
2 + (L->D)
= 4
choose third (score max)

fill first
row/column

V
E
S
L
C
Y

4 2 2 2 2
2 6 4 4 4
2 4
2 4
2 4
2 4

Dynamic Programming Approach to Sequence Alignments

Formally:

Si-1, j-1 + s(ai -> bi)

Sij =
max

max [x1] (Si-x,j - wx)

max [y1] (Si,j-y - wy)

ai, bj : Residue of Sequence A at position i and Residue of Sequence B at position j

wx : Penalty for introducing gap of length x
Sij : Score of alignment at position (i, j)
s(a->b) : Score for aligning a and b (known from scoring matrix)

Iterate process until whole matrix is filled with scores and backpointers
Choose maximum score in last column or row
Follow pointers to construct alignment

Dynamic Programming Approach to Sequence Alignments

Global Alignment
Needleman and Wunsch
(1970)
Local Alignment:
Smith-Waterman
(minor modification)

Dynamic Programming Approach to Sequence Alignments

Implementations available on the web

Dynamic Programming Approach to Sequence Alignments

Strengths:

finds optimal (mathematically best) alignment

suited for both, local and global alignments
global alignment: Needleman-Wunsch
local alignment: Smith-Waterman
statistical significance can be attached
(recompute alignment when one or both sequences are randomly
changed)

Implementations
LALIGN
GCG "GAP" (global) and BESTFIT (local)
...

Complexity:
two sequences of length n, m
time: O(nm), space: O(nm)
space is crucial
example: matrix element 4 bytes, n=m=10000, space requirement: 400MB
can be improved: trade time for space

Multiple Sequence Alignment

When aligning k (k>2) sequences, the dynamic programming
idea can theoretically be used:
instead of a two-dimensional scoring schema, a k-dimensional one is
used
instead of two-dimensional residue substitution matrix, a kdimensional one is used

However, this is not feasible in practical terms

size of these matrices increase too rapidly!
example: 5 sequences, 100 residues each, matrix size = 100^5 =
10^10
in terms of computer memory: 10 GB

Consequence:
Optimal alignment for multiple sequences is not
computable!
Approximative methods used (heuristics)

Multiple Sequence Alignment: Progressive methodes

Idea for progressive methodes:

step
step
step
step

1
2
3
4

:
:
:
:

Align two of the sequences

Compute "consensus" sequence
Align a third sequence to the consensus
Repeat steps 2 and 3

Multiple Sequence Alignment: Progressive methodes,

CLUSTALW
Basic steps

step 1 : Perform pairwise sequence alignments of all possible pairs

step 2 : Use scores to produce a phylogenetic tree
step 3 : align the sequences sequentially, guided by the tree
the most closely related sequences are aligned first
other sequences or groups of sequences are added

Example: Phylogenetic tree for 4 sequences

A
B
C
D
A 100 90 85 90
B
100 95 80
C
100 97
D
100
Percentage Identities in
pairwise alignment

90

90
85

80
95

97

Join sequences with highest similarity

Multiple Sequence Alignment: Progressive methodes,

CLUSTALW
Computation of Phylogenetic tree
Join sequences with
highest similarity

A
B
C
D
A 100 95 80 90
B
100 95 85
C
100 97
D
100
Percentage Identities in
pairwise alignment

95

95

90
80

85

(80+90)/2
= 85

95
C

97

(85+95)/2
= 90

D
C, D

Join sequences with

highest similarity

A, B

A
B
C
D

(85+90)/2
= 87.5

C, D

Multiple Sequence Alignment: Progressive methodes,

CLUSTALW
Example: Seven
Globins from
SWISSPROT

Multiple Sequence Alignment: Progressive methodes,

CLUSTALW
Available Implementations

Riddle: Probability that a short string occurs in a longer text

Given:

an alphabet A = {a, c, g, t}
a random string s of length k over A
a random text t of length n (n>k) over A

Find probability p, by which the string s occurs in the text t.

Find expected number of occurrences e of s in t.

Example: s = tggtacaaatgttct (glucocorticoid response element GR

t = 10000 bp (promoter, upstream DNA to start codon

Basics of Sequence DB Searches: Definition

Given:

one short sequence q (query)

sequence DB, conceptually one long sequence s

(subject)
Find occurrences of similar sequences to q in s.
Attach to each similar occurrence a statistical significance.
Example: s = human genomic DNA, 3109 b
q = tggtacaaatgttct (glucocorticoid response
element GRE)
Dynamic programming approach not feasible!

Basics of Sequence DB Searches: Detection of identical kmers

Idea:

identify identical k-mers in s and q (seed)

expand alignment from seed in both directions

Example:
q

MAAARLCLSLLLLSTCVALLLQPLLGAQGAPLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRYGKRHKEDTLAFSEWGS
|| | |||| | || |||||||| | |||||| |||| |||||||||
|||||| ||||||||| |
... MAVAYCCLSLFLVSTWVALLLQPLQGTWGAPLEPMYPGDYATPEQMAQYETQLRRYINTLTRPRYGKRAEEENTGGLP...

1
2 Gonnet120 score: 412, 76 % identities

3
no more increase
in score

BLAST
HSP, high scoring pair
gapped alignment
starting extension also from similar (and not only identical) seeds

Basics of Sequence DB Dearches: Detection of identical kmers

Precompute position of all k-mers in DB sequence
Indexing all Peptides of length k in "database
0
1
2
3
Example:
1234567890123456789012345678901234
MAAARLCLSLLLLSTCVALLLQPLLGAQGAPLEP
MAAAR
AAARL
AARLC
....
APLEP
Sorted:
AAARL 2
AARLC 3
APLEP 30
...
MAAAR 1
...
VALLL 17

For each peptide of length k in "query", the identical peptides in

"database" are identified efficiently (binary search in sorted list)
For identical pairs, extension step in both directions is performed

Basics of Sequence DB Dearches: Detection of identical kmers

Indexing all Peptides of length k in "database: some
refinements
0
1
2
3
1234567890123456789012345678901234
MAAARLCVALLLLSTCVALLLQPLLGAQGAPLEP
8

Pointers to previous occurrences

List of all words of length k and

last occurrence in query:
Simple, yet efficient data-structure:
AAAAA
- array of integers (size=length of db)
AAAAC
AAAAD
- array of integers (size=number of words with length k
....
AARLC
3
Book-keeping:
....
- more than one db/query sequence
APLEP 30
- build database chunks that fit into main memory
...
(speeds up computation 1000x)
...
VALLL 17
...
Extension step: optimizations

For each peptide of length k in "query", the position in the wordlist can be
easily computed (no binary search!)

Significance of matches: DNA case

issue: searching with short query vs. large database found mat
could have occurred by pure chance
assume equal distribution of c,g,a,t
what is ...

the probability q, that sequence B (len=m) is contained in sequence A

(len=n)?
the expected length of a common subsequence of two sequences?
the expected score when locally aligning two sequences of length n, m
Solution for first:
a. There are (n-m) 'words' of length m in sequence A
b. In total, there are 4^m sequences of length m
c. p = (n-m) / 4^m

This is wrong. Why?

Not independent!

Significance of matches: Statistics

statistics
the statistical distribution of alignment scores found in a DB search
follows the extreme value distribution (not normal distribution)
extreme value distribution changes with length of sequences and their
residual composition
scores of actual database search results are plotted vs. expected
scores
(FASTA)
BLAST computes E-Value (number of expected hits with this score,
when comparing the query with unrelated database sequences)

Putting it together: BLAST2

A W T V A S A V R T S I

(optional) filtering for low complexity region in query

all words of length 3 are listed:
AWT VAS AVR TSI | WTV ASA VRT | TVA SAV RTS

to each word, ~50 'high scoring' additional words are added:

AWT
AWA
TWA
ART
...

VAS
IAS
LAS
ITS
...

AVR
TVR
AIR
AVS
...

TSI | WTV ASA VRT | TVA SAV RTS

...
...
...

matching words are found in DB (with datastructure as described

before)
ungapped alignment constructed from word matches, 'HSP'
statistics determines, whether HSP is significant
SW-alignment for significant HSPs

An example: comparing mus chr X vs. hum chr X, syntenic

regions
mouse chromosome X (147Mbp)

human chromosome X (149Mbp)

Each dot:
conserved stretch of AA
HSP, high scoring pair

Conclusions

types of sequence alignments: pairwise, multiple, query vs. database

local and global alignment
scoring matrices: attach score to each pair of residues
(allow similar residues to be aligned)
sequence alignment problem is mathematical optimization problem:
find optimal alignment: maximise score
optimal alignment in practical terms only feasible for pairwise alignment
Heuristics for multiple sequence alignments:
progressive alignment guided by all pairwise alignments
Sequence database searches:
efficiently find occurrence of query k-mers in db sequences (seeds)
expand (ungapped) HSP from seeds
attach statistical significance