Dr.

Sara Janiad (MMG)

BLAST (Basic Local Alignment Research Tool)

BLAST (Basic Local Alignment Search Tool) allows rapid sequence comparison of a query
sequence against a database.
The BLAST algorithm (method) is
 Fast
 Accurate
 Web-accessible

 It is a local alignment tool.

 It helps to find regions of local similarity between sequences.
 It is a programme compares nucleotide or protein sequences to sequence databases
and calculates the statistical significance of matches.
 It can be used to infer functional and evolutionary relationships between sequences
as well as help identify members of gene families.
 BLAST was developed by Stephen Altschul, Warren Gish, Webb Miller, Eugene
Myers and David J. Lipman at NCBI in 1990.
Why use BLAST?
BLAST searching is FUNDAMENTAL to understanding the relatedness of any favourite
query sequence to other known proteins or DNA sequences.
Applications of BLAST
• Identifying orthologs and paralogs
• Discovering new genes or proteins
• Discovering variants of genes or proteins
• Investigating expressed sequence tags (ESTs)
• Exploring protein structure and function
• Establishing Phylogeny
• DNA mapping
• Locating Domains
Four components to a BLAST search
(1) Choose the sequence (query)
(2) Select the BLAST program
(3) Choose the database to search
(4) Choose optional parameters
Then click “BLAST”
Major types of BLAST program
(1) Nucleotide BLAST
(2) Protein BLAST
(3) Translated BLAST
(4) Genome BLAST
Dr. Sara Janiad (MMG)

(5) Special BLAST

(6) Meta BLAST
Blastn is called (nucleotide BLAST)
blastp is called (protein BLAST)
blastx is called (translated BLAST)
tblastn is called (translated BLAST)
tblastx is called (translated BLAST)

DNA potentially encodes six proteins

Difference between Reading frame and open reading frame?

Reading Frame: In molecular biology, a reading frame is a way of dividing the sequence of
nucleotides in a nucleic acid (DNA or RNA) molecule into a set of consecutive, non-
overlapping triplets.
A DNA strand has three distinct reading frames. The double helix of a DNA molecule has
two anti-parallel strands; with the two strands having three reading frames each, there are
six possible frame translations.
Open Reading Frame: An open reading frame (ORF) contains a start codon (usually AUG)
and by definition cannot extend beyond a stop codon (usually UAA, UAG or UGA). That start
codon (not necessarily the first) indicates where translation may start.
Dr. Sara Janiad (MMG)

BLAST Overview
Three Steps: seeding, extension, and evaluation
• Seeding – identify where to start alignment
• Extension – extending alignment from seeds
• Evaluation – Determine which alignments are statistically significant
Dr. Sara Janiad (MMG)

It will take some time.

Dr. Sara Janiad (MMG)
Dr. Sara Janiad (MMG)
Dr. Sara Janiad (MMG)

BLAST result can be shown in 4 different formats.

1. Description
2. Graphical Summary
3. Alignment
4. Taxonomy
Dr. Sara Janiad (MMG)

Max/ Total Score (Bit score)

The higher the better.
Score constructed from length of total alignment of the high scoring pair.
Quality of the alignment is represented by the score.
E Value (Expectation value)
The lower the better.

Score measurement is determined by

 Match Award
 Mismatch Penalty
 Gap penalty
Dr. Sara Janiad (MMG)

The higher the score, then better the alignment.

If both penalties are set at Zero, it aims to find a maximum alignment.

What is Sequence Alignment?

Sequence alignment is a way of arranging sequences of DNA, RNA or protein to identify
regions of similarity is made to align the entire sequence.
 Alignment is the task of Locating Equivalent regions of two or more sequences to
maximize their similarity
 The Similarity may indicate the functional, structural and evolutionary significance
of the sequence.
 The sequence alignment is made between a known sequence and unknown
sequence or between 2 unknown sequences.
 The known sequence is called Reference sequence, however, the unknown
sequence is called Query sequence.

Sequence alignment is useful for discovering;

 Structural
 Functional &
 Evolutionary information

Methods of Sequence Alignment

 Dot Matrix method
 Word of k-tuple methods
 Dynamic Programming (DP) algorithm
Dr. Sara Janiad (MMG)

Points to Ponder
1. Alignment can reveal homology between sequences.
2. Similarity is descriptive term that tells about the degree of match between the two
sequences.
3. Sequence similarity does not always imply a common function.
4. Conserved function does not always imply similarity at the sequence level.
5. Convergent Evolution: Sequences are highly similar but are not homologous.
6. Alignment of related sequences is expected to give good scores compared with
alignments of randomly chosen sequences.
7. The correct alignment of 2 related sequences should ideally be the one that gives the
best score.
8. In practice, the correct alignment does not necessarily have the best score, since no
“perfect” scoring scheme has been devised.
9. Identity is the number of identical bases or amino acids matched between two aligned
sequences.
10. Percent identity is obtained by dividing this number by the total length of the aligned
sequences and multiplying by 100.

Terminologies for Sequence Comparison

 Sequence identity: Exactly the same Amino Acid or Nucleotide in the same
position.
 Sequence similarity: Substitutions with similar chemical properties.
 Sequence homology: General term that indicates evolutionary relatedness among
sequences; we usually measure of percentage identity of sequence homology.
 Pairwise alignment: It is used to find the best-matching piecewise (local) or global
alignments of two query sequences. Pairwise alignments can only be used between
two sequences at a time.
 Multiple sequence alignment: Try to align all of the sequences in a given query
set.
Similarity ≠ homology
Similarity = factual (% identity)
Homology = hypothesis supported by evidence
Dr. Sara Janiad (MMG)

Global vs Local Sequence Alignment

Global Alignment Local Alignment
Attempt is made to align the entire Finds local regions with the highest level
sequence (end to end align). of similarity between the two sequences.
Best if the query is very similar and about Works better for more divergent
the same length sequence
Should be closely related Can be distantly related
Often done for comparing homologous Used for finding conserved patterns in
genes, like 2 genes with same function. DNA or conserved domains or motifs in 2
proteins.
A general global alignment technique is A general local alignment method is
the Needleman-Wunsch algorithm. Smith-Waterman algorithm.
Tools Examples: Tools Examples:
EMBOSS Needle BLAST
EMBOSS Water
LALIGN
Dr. Sara Janiad (MMG)

Global Alignment

Local Alignment

Pairwise Sequence Alignment (PSA) vs Multiple Sequence Alignment (MSA)

Dr. Sara Janiad (MMG)