The biological meaning of pairwise
alignments
Arthur Gruber
Instituto de Ciências Biomédicas
Universidade de São Paulo
AG-ICB-USP
What is a pairwise alignment?
• Comparison of 2 sequences – nucleotide or protein
sequences
• We can compare a sequence to an entire database of
sequences – one pairwise alignment at a time
• Different types of alignments – global and local alignment
• Different algorithms –
Waterman, FastA, BLAST
Needleman-Wunsch,
Smith-
AG-ICB-USP
Pairwise alignment
• Output: alignment of similar blocks or whole sequences
gi|3323386|gb|U85705.1|IFU85705 Isospora felis 28S large subunit ribosomal RNA
gene, complete sequence Length = 3227 Score = 218 bits (110), Expect = 2e-54
Identities = 146/158 (92%) Strand = Plus / Minus
Query: 3 cacttttaactctctttccaaagtccttttcatctttccttcacagtacttgttcactat 62
||||||||||||||||||||||| |||||||||||||| |||| ||||||||| ||||
Sbjct: 386 cacttttaactctctttccaaagaacttttcatctttccctcacggtacttgtttgctat 327
Query: 63 cggtctcacgccaatatttagctttacgtgaaacttatcacacattttgcgctcaaatcc 122
||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||
Sbjct: 326 cggtctcgcgccaatatttagctttatgtgaaacttatcacacattttgcgctcaaatcc 267
Query: 123 caatgaacgcgactcaataaaagcgcaccgtacgtgga 160
| ||||||||||||| ||||| ||| ||||||||||||
Sbjct: 266 cgatgaacgcgactctataaaggcgtaccgtacgtgga 229
AG-ICB-USP
Some applications of pairwise
alignments
• Annotation – description of the characteristics of a sequence
• Function ascribing – similar sequences MAY share similar
functions
• Identification of structural domains – similar sequences MAY
share similar structures
• Identification
architecture
of
protein
domains
–
defines
protein
• Phylogenetic inference – identification of similar sequences
that MAY have a common ancestry
AG-ICB-USP
Some applications of pairwise
alignments
• Identification of contaminant sequences in a sequencing
project – query sequence x databases (bacterial, ribosomal,
mitochondrial, etc.)
• Identification of vector sequences in sequencing reads –
alignment and masking
AG-ICB-USP
Identity, similarity, homology
• Identity – refers to nucleotide or amino acid residues that
are identical
• Similarity - measurable quantity: percentage of identities
between two sequences, percentage of similar amino acid
residues (conserved along the evolution).
• Homology – based on a evolutionary conclusion that implies
that two sequences has a common ancestral sequence.
They are said to share the same evolutionary history.
Homology is not quantitative. Two sequences can be or not
to be homologous.
AG-ICB-USP
Identity, similarity, homology
• A high degree of similarity between two sequences MAY
suggest that they share a common evolutionary history.
Other analyses and experimental work should be done to
validate such hypothesis
AG-ICB-USP
Contaminant removal
Libraries can be contaminated by different sources
Genomic libraries:
• Other organisms and/or cells – co-purification
• Bacterial DNA - E. coli used as the host cell
• Human – contamination during manipulation
• Other genomes being manipulated in the lab – crosscontamination
AG-ICB-USP
Contaminant removal
Libraries can be contaminated by different sources
EST libraries:
• All sources already mentioned
• Ribosomal RNA – co-purification with the polyA
fraction
• Organelle transcripts – mitochondrion, plastid
AG-ICB-USP
Vector masking
A typical read contains sequence stretches that
are not originally part of the insert
insert
Sequencing reaction
Vector
sequence
Vector
sequence
AG-ICB-USP
Vector masking
Masking consists in a substitution of bases that
are not part of the insert by Xs
insert
Vector
sequence
Vector
sequence
xxxxxxxxx
Vector
sequence
insert
xxxxxxxxxxxxxxxx
Vector
sequence
• “ X ” bases will not be taken into account by
assembly/clustering programs
AG-ICB-USP
Aligning Two Sequences
Human Hemoglobin (HH):
VLSPADKTNVKAAWGKVGAHAGYEG
Sperm Whale Myoglobin (SWM):
VLSEGEWQLVLHVWAKVEADVAGHG
AG-ICB-USP
Aligning Two Sequences
(HH)
VLSPADKTNVKAAWGKVGAHAGYEG
|||
|
| || |
|
(SWM) VLSEGEWQLVLHVWAKVEADVAGHG
•
Gap Weight:
12
•
Length Weight:
•
Gaps:
•
Percent Similarity: 40.000
•
Percent Identity:
•
Matrix: blosum62
4
0
36.000
AG-ICB-USP
Gap Insertion/Deletion
(HH)
VLSPADKTNVKAAWGKVGAH-AGYEG


    
(SWM) VLSEGEWQLVLHVWAKVEADVAGH-G
- gap insertion/deletion
•
Gap Weight:
4
•
Length Weight:
•
Gaps:
•
Percent Similarity: 54.167
•
Percent Identity:
•
BLOSUM62
1
2
45.833
AG-ICB-USP
Scoring
(HH)
VLSPADKTNVKAAWGKVGAH-AGYEG
|||
|
| ||
|| |
(SWM) VLSEGEWQLVLHVWAKVEADVAGH-G
The score of the alignment is:
Matrix value at (V,V) + (L,L) + (S,S) + (P,E) + …
(penalty for gap insertion/deletion)*gaps
(penalty for gap extension)*(total length of all gaps)
AG-ICB-USP
Scoring System
• Identity: An objective and quite well defined measure
Count the number of identical matches, divide by length
of aligned region
• Similarity: A less well defined measure
Category
Amino acid
Acids and Amides
Asp (D) Glu(E) Asn (N) Gln (Q)
Basic
His (H) Lys (K) Arg (R)
Aromatic
Phe (F) Tyr (Y) Trp (W)
Hydrophilic
Ala (A) Cys (C) Gly (G) Pro (P) Ser (S) Thr (T)
Hydrophobic
Ile (I) Leu (L) Met (M) Val (V)
AG-ICB-USP
Scoring system
 Rates of amino acid substitution are not uniform
 Some amino acids are more conserved than others (e.g.
C, H, W compared to A, L, I)
 Some substitutions are more common than others
 (e.g. A
I, A
L compared to D
L)
 Conclusion: there are evolutionary pressures that
probably reflect structural and functional constraints
 Scoring matrices – matrices that are used for scoring
amino acid substitutions in pairwise alignments
 They reflect substitution rates that are originated by
evolutionary events
AG-ICB-USP
Amino acids - chemical relationships
Tiny
Aliphatic
P
Hydrophobic
I
A
L
S
V
M
F
H
Aromatic
Positive
OH
C
Polar
T
Y
W
G
Hydrophilic
K
D
N
R
E
K
Negative
NH2
Charged
AG-ICB-USP
PAM
 Stands for Point Accepted Mutation
 Dayhoff Matrix, 1978
 A series of matrices describing the extent to which two
amino acids have been interchanged in evolution
 Very similar sequences were aligned, phylogenetic trees
were built, and ancestral sequences were reconstructed
 Out of these alignments, the frequency of substitution
between each pair of amino acids was calculated. Using
this information, PAM matrices were built (PAM1 i.e. one
accepted point mutation per 100 amino acids).
AG-ICB-USP
PAM250 - amino acid substitution matrix
GAP_CREATE 12
GAP_EXTEND 4
A
B
C
D
E
A
2
0
-2
0
0
B
0
2
-4
3
2
C
-2
-4
12
-5
-5
D
0
3
-5
4
3
E
0
2
-5
3
4
F
-4
-5
-4
-6
-5
G
1
0
-3
1
0
H
-1
1
-3
1
1
I
-1
-2
-2
-2
-2
K
-1
1
-5
0
0
L
-2
-3
-6
-4
-3
M
-1
-2
-5
-3
-2
N
0
2
-4
2
1
P
1
-1
-3
-1
-1
Q
0
1
-5
2
2
R
-2
-1
-4
-1
-1
S
1
0
0
0
0
T
1
0
-2
0
0
V
0
-2
-2
-2
-2
W
-6
-5
-8
-7
-7
F
G
H
I
K
-4
1
-1
-1
-1
-5
0
1
-2
1
-4
-3
-3
-2
-5
-6
1
1
-2
0
-5
0
1
-2
0
9
-5
-2
1
-5
-5
5
-2
-3
-2
-2
-2
6
-2
0
1
-3
-2
5
-2
-5
-2
0
-2
5
2
-4
-2
2
-3
0
-3
-2
2
0
-4
0
2
-2
1
-5
-1
0
-2
-1
-5
-1
3
-2
1
-4
-3
2
-2
3
-3
1
-1
-1
0
-3
0
-1
0
0
-1
-1
-2
4
-2
0
-7
-3
-5
-3
L
M
N
P
Q
-2
-1
0
1
0
-3
-2
2
-1
1
-6
-5
-4
-3
-5
-4
-3
2
-1
2
-3
-2
1
-1
2
2
0
-4
-5
-5
-4
-3
0
-1
-1
-2
-2
2
0
3
2
2
-2
-2
-2
-3
0
1
-1
1
6
4
-3
-3
-2
4
6
-2
-2
-1
-3
-2
2
-1
1
-3
-2
-1
6
0
-2
-1
1
0
4
-3
0
0
0
1
-3
-2
1
1
-1
-2
-1
0
0
-1
2
2
-2
-1
-2
-2
-4
-4
-6
-5
R
S
T
V
W
-2
1
1
0
-6
-1
0
0
-2
-5
-4
0
-2
-2
-8
-1
0
0
-2
-7
-1
0
0
-2
-7
-4
-3
-3
-1
0
-3
1
0
-1
-7
2
-1
-1
-2
-3
-2
-1
0
4
-5
3
0
0
-2
-3
-3
-3
-2
2
-2
0
-2
-1
2
-4
0
1
0
-2
-4
0
1
0
-1
-6
1
-1
-1
-2
-5
6
0
-1
-2
2
0
2
1
-1
-2
-1
1
3
0
-5
-2
-1
0
4
-6
2
-2
-5
-6
17
AG-ICB-USP
BLOSUM
• Stands for Blocks Substitution Matrices
• Henikoff and Henikoff, 1992
• A series of matrices describing the extent to which two
amino acids are interchangeable in conserved structures
• Built by extracting replacement information from the
alignments in the BLOCKS database.
AG-ICB-USP
BLOSUM
• The number in the series (BLOSUM62) represents
the threshold percent similarity between
sequences, for considering them in the calculation.
• For example, BLOSUM62 is derived from an
alignment of sequences that share 62% similarity,
BLOSUM45 is based on 45% sequence similarity in
aligned sequences
AG-ICB-USP
BLOSUM62 - amino acid substitution matrix
Reference: Henikoff, S. and Henikoff, J. G. (1992). Amino acid substitution
matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89: 10915-10919.
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
*
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
1
AG-ICB-USP
Amino acid structure
Guidelines
• Lower PAMs and higher Blosums find short local
alignment of highly similar sequences
• Higher PAMs and lower Blosums find longer weaker
local alignment
• No single matrix answers all questions
AG-ICB-USP
BLAST – Basic Local Alignment Search Tool
• Algorithm first described in 1990
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman,
D.J. (1990) "Basic local alignment search tool." J. Mol. Biol.
215:403-410.
• And improved in 1997
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J.,
Zhang, Z., Miller, W. & Lipman, D.J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database
search programs. Nucleic Acids Res. 25: 3389-3402.
AG-ICB-USP
Blast search – four components
• Search purpose/goal
• Program
• Query sequence
• Database
AG-ICB-USP
BLAST – search purpose/goal
• What is the biological question? Examples:
• Which proteins of the database are similar to my protein sequence?
• Which proteins of the database are similar to the conceptual
translation of my DNA sequence?
• Which nucleotide sequences in the database are similar to my
nucleotide sequence?
• Which proteins coded by the conceptual translation of the database
sequences are similar to my protein sequence?
•Which proteins coded by the conceptual translation of the database
sequences are similar to the conceptual translation of my DNA
sequence?
AG-ICB-USP
BLAST – search purpose/goal
• Which proteins of the database are similar to my protein
sequence?
• I have sequenced a gene and derived the protein
sequence by concetpual translation. Alternatively, I
obtained the protein sequence directly. I am now interested
to find out its possible fnction.
• Using a similarity search, I can find protein sequences in
databases that are similar to mine: orthologs and paralogs.
• BLASTP – protein query x protein database
AG-ICB-USP
BLAST - search purpose/goal
• Which proteins of the database are similar to the conceptual
translation of my DNA sequence?
• I have sequenced an EST (expressed sequence tag) that
contains a protein coding region.
• I am interested to find out which proteins of the database
are similar to the conceptual translation of my nucleic acid
sequence.
• BLASTX – nucleotide (translated) query x protein
database
AG-ICB-USP
BLAST – search purpose/goal
• Which nucleotide sequences of the database are similar to
my DNA sequence?
• I have sequenced a DNA fragment.
• I am interested to find out which DNA sequences of the
database are similar to my nucleic acid sequence.
• BLASTN – nucleotide query x nucleotide database
AG-ICB-USP
BLAST - search purpose/goal
• Which proteins translated from a nucleic acid database are
similar to the conceptual translation of my DNA sequence?
• I have sequenced an EST (expressed sequence tag) that
contains a protein coding region.
• I am interested to find out which ESTs of other organisms
may be coding for homologous proteins.
• TBLASTX – nucleotide (translated) query x nucleotide
(translated) database
AG-ICB-USP
BLAST – search purpose/goal
• Which proteins coded by the conceptual translation of the
database sequences are similar to my protein sequence?
• I have a protein sequence on hands and am interested to
find out which genes of other organisms may be coding for
homologous proteins.
• TBLASTN – protein query x nucleotide (translated)
database
AG-ICB-USP
BLAST - programs
• BLASTP – protein query x protein database
• BLASTN – nucleotide query x nucleotide database
• BLASTX – nucleotide (translated) query x protein database
• TBLASTN
database
–
protein
query
x
nucleotide
(translated)
• TBLASTX – nucleotide query (translated) x nucleotide
(translated) database
AG-ICB-USP
BLAST – query sequence
FastA format
• The first line begins with the symbol '>' followed by the name
of the sequence
• The sequence is on the remaining lines.
• The sequence must not contain blanks.
• The sequence could be in upper or lower case.
• Below is an example sequence in FASTA format:\
>DNA sequence
GCCCCCGGCCCCGCCCCGGCCCCGCCCCCGGCCCCGCCCCGCAAGGGTC
ACAGGTCACGGGGCGGGGCCGAGGCGGAAGCGCCCGCAGCCCGGTACCG
GCTCCTCCTGGGCTCCCTCTAGCGCCTTCCCCCCGGCCCGACTCCGCTG
GTCAGCGCCAAGTGACTTACGCCCCCGACCTCTGAGCCCGGACCGCTAG
AG-ICB-USP
BLAST – database
• Nucleotide databases
• nr, refseq, est_human, est_mouse, est_others, wgs, etc.
• Protein databases – nr, Swiss-Prot, refseq, etc.
AG-ICB-USP
BLAST – web server
• BLAST web server address:
• http://blast.ncbi.nlm.nih.gov/Blast.cgi
AG-ICB-USP
AG-ICB-USP
AG-ICB-USP
AG-ICB-USP
AG-ICB-USP
AG-ICB-USP
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AG-ICB-USP
AG-ICB-USP
BLAST – some recommendations
• BLAST does not display ALL positive hits, only those with the
highest score. You may miss some important hits!
• Do not give up when getting “no hits” results. Try changing
some parameters:
– Substitution matrix
– Word size (lower values are more sensitive, but more computationally
intense)
– Gap penalty
AG-ICB-USP
BLAST – some recommendations
• If you have a large number of query sequences, you should
better run BLAST searches locally. WARNING NCBI limits the
number of simultaneous queries.
• Do not forget: protein sequences are more conserved than the
respective nucleotide sequences
– This is important if you are looking for distant orthologues
AG-ICB-USP