Download 14 Alignment 3(1)

How To Compare Two Sequences?
 pairwise
alignment includes precise tools
• Dot plots for graphical analysis
• Local or global alignments for
residue/residue analysis
In the alignment searching different models exist
which are designed to encapsulate a variety of
characteristics of biological sequences and/or
their evolutionary relatedness.
Two general model explore aligment:
Needleman e Wunsch (1970) considers similarity
across the full extent of the sequence (global
alignment);
Smith e Waterman (1981) focuses on regions of
similarity in parts of the sequences (local
alignment).
4 steps
1.
Definition of the substitution matrix and
gap penalties.
2. Alignment of the sequences through a dot
matrix.
3. Finding the network with the maximum
score also considering gaps.
4. Evaluation of the score derived by the
substitution matrix.
Basic Local Alignment Search Tool

Widely used similarity search tool
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based on Smith Waterman algorithm
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Finds best local alignments
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Provides statistical significance
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All combinations (DNA/Protein) query and database.
• DNA vs DNA
• DNA translation vs Protein
• Protein vs Protein
• Protein vs DNA translation
• DNA translation vs DNA translation
Many BLAST PROGRAMS . . .
BLASTing DNA Sequences
Blast
Pairwise comparison
Running a Blast program
NCBI http://blast.ncbi.nlm.nih.gov/Blast.cgi




Select the Blast program
Write the accession number or cut and paste
the query sequence
Write the accession number or cut and paste
the subject sequence
Run the program
Pairwise alignment using Blastp of nucleolin
between Homo Sapiens and Pan Troglodytes
NP_0053772
XP_516145
EXPECTATION VALUE (E-value)
IS THE MEASURE MOST COMMONLY USED FOR
ESTIMATING SEQUENCE SIMILARITY
IS ASSOCIATED WITH A SPECIFIC SCORE (“S”)
AND SEQUENCE
IS THE NUMBER OF DIFFERENT ALIGNMENTS
WITH SCORES EQUIVALENT TO OR BETTER
THAN “S”, THAT ARE EXPECTED TO OCCUR BY
CHANCE.
THE E-VALUE
THE LOWER THE E VALUE, THE MORE
SIGNIFICANT THE SCORE.
IF A MATCH IS HIGHLY UNEXPECTED, IT PROBABLY
RESULTS FROM SOMETHING OTHER THAN CHANCE
COMMON ORIGIN IS THE MOST LIKELY
EXPLANATION
E-value
An E-value of 1 assigned to a hit can mean that in the
database, one might expect to see one match with a
similar score simply by chance.
This means that the lower the E-value, or the closer it
is to “0”, the higher is the “significance” of the match.
However, it is important to note that searches with short
sequences can be virtually identical and have relatively high Evalue. This is because the calculation of the E-value also takes
into account the length of the query sequence. This is because
shorter sequences have a high probability of occurring in the
database purely by chance.
Meaning for E-Values
Low E-value  good hit
• 1 = bad e-Value
• 10-3 = borderline E-value
• 10-4 = good E-value
• 10-10 = very good E-value


E-values lower than 10-4 indicate possible homology
E-values higher than 10-4 require extra evidence to
support homology
BLAST Output: Alignments
Identical match
positive score
(conservative)
gap
Negative or zero
note
A pairwise sequence alignment from a BLAST report
The alignment is preceded by the sequence identifier, the full definition line,
and the length of the matched sequence, in amino acids. Next comes the bit
score (the raw score is in parentheses) and then the E-value. The following
line contains information on the number of identical residues in this
alignment (Identities), the number of conservative substitutions (Positives),
and if applicable, the number of gaps in the alignment. Finally, the actual
alignment is shown, with the query on top, and the database match is
labeled as Sbjct, below. The numbers at left and right refer to the position in
the amino acid sequence. One or more dashes (–) within a sequence
indicate insertions or deletions. Amino acid residues in the query sequence
that have been masked because of low complexity are replaced by Xs (see,
for example, the fourth and last blocks). The line between the two
sequences indicates the similarities between the sequences. If the query
and the subject have the same amino acid at a given location, the residue
itself is shown. Conservative substitutions, as judged by the substitution
matrix, are indicated with +.
The Alignments



Look for clusters
of identity
Gray residues are
low-complexity
regions
Grayed-out regions
have been removed
from your sequence
to avoid false hits
Pair-wise comparison between
Human nucleolin NP_005372.2
and
chimpanzee nucleolin XP_516145.3
Search for similarity of albumin
between
human and chimpanzee
human and Mus musculus
human and Gallus gallus
Link rete
Similarity searching
in the protein data base
BLAST MAKES PAIRWISE COMPARISON OF
THE QUERY SEQUENCE TO EACH
SEQUENCE IN THE DATABASE
PROCEDURE
• SEARCH FOR LOCAL ALIGNMENT
• EVALUATE THE SCORE
• SAVE THE HIGH SCORES
• REPEAT THE STEPS FOR EACH
SEQUENCE
Running blastp
http://blast.ncbi.nlm.nih.gov/Blast.cgi


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Select a database to search
Write the accession number or cut and paste the
query sequence
Click the BLAST button
Reading BLAST Output

Graphic Display
• Overview of the
alignments
 Hit List
• Gives the score of
each match
 Alignments
• Details of each
alignment
The Graphic Display


The Horizontal Axis (0-700)
corresponds to your protein
(query)
Color codes indicate match’s
quality
• Red: very good
• Green: acceptable
• Black: bad

Thin lines join independent
matches on the same sequence
The Hit List

Sequence accession number
• Depends on the database

Description
•

Taken from the database
Bit score
•
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High bit score = good match
E-Value
• Low E-value = good match
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Links
•
•
Genome
Uniref, database of
transcripts
BLAST Output: Descriptions
Sorted by e values
5 X 10-14
Link to entrez
Gene Linkout
NUCLEOLIN GENE IN HOMO SAPIENS
Search for homologous sequences

To human nucleolin
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In refseq protein database
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In mammals
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Exclude model proteins
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Use the default algorithm parameters
output results in Recen results window
Basic BLAST: Nucleotide
BLAST and BLAST-like programs

Megablast nucleotide only
• Contiguous megablast
• Nearly identical sequences
• Discontiguous megablast
• Cross-species comparison
Nucleotide Databases: Human and Mouse

Human and mouse genomic and transcript now default
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Separate sections in output for mRNA and genomic
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Direct links to Map Viewer for genomic sequences
Nucleotide Databases: Traditional
blastn
tblastn
tblastx
Universal Form: Nucleotide
Less
Sensitivity
More
More
Speed
Less
Search for homologous sequences
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To human nucleolin transcript
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In human genomic + transcript
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Exclude model proteins
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Select program for highly similar sequences

Use the default algorithm parameters
output results in Recen results window
Link to Genome View
Total Score: All Segments
Sortable Results
Separate Sections for
Transcript and
Genome
Link to Map Viewer
Genome BLAST
Genome BLAST pages
Map Viewer Homepage
Search for homologous sequences
to human nucleolin mRNA RefSeq
in Bos Taurus reference assembly