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The scoring scheme for sequence alignments consist of character
substitution scores (score for each possible character replacement)
and penalties for gaps.
The score is based on matches, mismatches and gaps in the
sequence alignment.
Thus, the alignment score is the sum of substitution scores and gap
penalties and indicates if the alignment is optimal.
There are different methods for scoring an alignment:
1. Dot matrix / Plot methods
2. Dynamic programming methods
Methods used in Scoring an alignment
Dot matrix method (Dot plot method)
This is a graphical way of comparing two sequences in a two dimensional matrix.
The two sequences to be compared are written in the horizontal and vertical axes
of the matrix.
Comparison is then done by scanning each residue of one sequence for similarity
with all residues in the other sequence.
If a residue match is found, a dot is placed within the graph.
When the two sequences have substantial regions of similarity, many dots line up
to form contiguous diagonal lines, which reveal the sequence alignment.
Interruptions in the middle of a diagonal line indicate insertions or deletions.
Parallel diagonal lines within the matrix represent repetitive regions of the
sequences
Dot matrix /plot analysis Online tools e.g NCBI BLAST
Example
>BAG32276.
MANLGCWMLVLFVATWSDLGLCKKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHG
GGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIH
FGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVV
EQMCITQYERESQAYYQRGSSMVLFSSPPVILLISFLIFLIVG
>ABP65297.
MVKSHIGSWLLVLFVATWSDIGFCKKWPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQ
PHGGGWGQPHGGGWGQPHGGGGWGQGGGSHGQWGKPSKPKTNMKHVAGAAAAGAVVGGLGGYMLGSAMSR
PLIHFGNDYEDRYYRENMYRYPNQVYYKPVDQYSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDMKIM
ERVVEQMCVTQYQRESEAYYQKGASAILFSPPPVILLISLLILLIVG
5
Procedure:
Step 1: Open the home page of NCBI BLAST through a web browser.
Step 2: Select “Nucleotide BLAST” for comparing two nucleotide
sequences, or “Protein BLAST” for comparing two protein sequences.
Step 3: Click the checkbox “Align two or more sequences” to change
the default layout to perform pairwise sequence alignment.
Step 4: Enter accession number(s), gi(s), or FASTA sequence(s) in
the text boxes (or upload FASTA format files) and click “BLAST”
button.
Step 5: Pairwise alignment result is displayed. Click the “Dot
Matrix /Plot View” link to expand the result of dot matrix.
Step 6: Click on MSA Viewer and zoom to sequence to view the
Dynamic programming methods
Dynamic programming methods is used to find the optimal alignment
between two proteins or nucleic acid sequences by comparing all
possible pairs of characters in the sequences.
Dynamic programming can be used to produce both global and local
alignments based on the two different algorithms: Needleman-Wunsch
algorithm and Smith-Waterman algorithm.
Needleman–Wunsch algorithm
This is used for global alignment and focuses on getting a maximum
score for the full-length sequence alignment.
This is however, risks missing the best local similarity thus it is used for
aligning two closely related sequences that are of the same length.
Smith–Waterman algorithm
This is is used for local alignment and it is considered a database search
algorithm.
This is because, the divergence level between the sequences to be aligned is not
easily known and sequence lengths are usually unequal.
Thus, identification of regional sequence similarity is of greater significance
than finding a match that includes all residues.
Positive scores are assigned for matching residues, zero for mismatches and
negative scores are not used.
Gaps are inserted where necessary.
The local alignment path may begin and end internally and gets the highest
score at the expense of the overall score for a full-length alignment.
Thus approach is used for aligning divergent sequences or sequences with
multiple domains of different origins.
Variants of BLAST
Nucleotide BLAST (BLASTN) queries nucleotide sequences with a nucleotide
sequence database.
Protein BLAST (BLASTP) uses protein sequences as queries to search against
a protein sequence database.
BLASTX uses nucleotide sequences as queries and translates them in all six
reading frames to produce translated protein sequences, which are used to query a
protein sequence database
TBLASTN queries protein sequences to a nucleotide sequence
database with the sequences translated in all six reading frames.
TBLASTX uses nucleotide sequences, which are translated in all
six frames, to search against a nucleotide sequence database that
has all the sequences translated in six frames.
Others types of BLAST for protein sequences:
PSI BLAST:‐ Allows the user to build a PSSM (position-specific
scoring matrix) using the results of the first BlastP run.
PHI BLAST:‐ Performs the search but limits alignments to those that
match a pattern in the query.
DELTA BLAST:‐ PSSM using the results of a Conserved Domain
Database search and searches a sequence database.
