Sequence alignment

Sequence alignment arranges two or more nucleotide or amino acid sequences to identify regions
of similarity between the sequences. These regions of similarity are helpful in understanding the
functional, structural, and evolutionary relationships between the sequences. It is considered the
most essential step in comparing biological sequences.
Types or Algorithms of sequence alignment
▪ Global alignment:
Global alignment is a method of comparing two sequences, which aligns the entire length of the
sequences by maximizing the overall similarity. This method is used when comparing sequences
that are of the same length.
▪ Local alignment: In local alignment, instead of attempting to align the entire length of the
sequences, only the regions with the highest density of matches are aligned. This is useful
for identifying short conserved regions in protein or nucleotide sequences.

Methods of Sequence alignment

❖ Pairwise Alignment
Pairwise sequence alignment is the type of sequence alignment that involves aligning two
sequences to identify the optimal pairing of the sequences.
It is based on a scoring system that assigns positive scores to matching characters and negative
scores to mismatching characters or gaps.
• The main objective of pairwise sequence alignment is to obtain the highest possible score,
which indicates the degree of similarity between the two sequences.
Methods of Pairwise sequence alignment
There are three main methods for generating pairwise alignments:
1. Dot-matrix method
It is also known as the dot plot method, is a graphical method of sequence alignment that involves
comparing two sequences by plotting them in a two-dimensional matrix.
• In a dot matrix, two sequences that must be compared are plotted along a matrix’s
horizontal and vertical axes. The method then scans each residue of one sequence to
identify similarities with all residues in the other sequence.
• If a residue in one sequence matches a residue in the other sequence, a dot is placed in the
corresponding position in the matrix. Otherwise, the matrix position is left blank.
• If the two sequences being compared are
highly similar, the dot plot will display as a
single line along the matrix’s main
diagonal. However, when the sequences
are less similar, the dot plot will show more
scattered dots with fewer diagonal lines,
indicating that the sequences share less
similarity.
• Dot plots can also find repeat elements in a
single sequence. Short parallel lines above
and below the main diagonal indicate the
presence of repeats.
2.Dynamic programming
The method 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. The
global pairwise alignment algorithm using dynamic programming is based on the
Needleman-Wunsch algorithm, while the dynamic programming in local alignment is
based on the Smith-Waterman algorithm.This method works in the following three steps.
i.Initialization of the scoring matrix: The first step is to create a two-dimensional matrix where
the two sequences to be aligned are written along the top and left sides. The matrix is initialized
with gap penalties and an initial score of zero at the top-left corner.
ii.Matrix filling with maximum scores: The next step involves filling the matrix with scores
based on a scoring matrix. Scoring matrices for nucleotide sequences are simple. A positive value
is given for a match, and a negative value for a mismatch. To calculate the alignment scores, the
algorithm starts at the upper left corner of the matrix and proceeds one row at a time toward the
lower right corner. The algorithm fills each cell in the matrix with the maximum score that can be
obtained by aligning the corresponding residues.
iii.Traceback to identify optimal alignment: After filling the matrix, the algorithm performs a
traceback to find the optimal alignment path. Starting from the bottom-right corner and moving
towards the top-left corner, adjacent cells are examined in reverse order to determine the best path
with the highest total score. The optimal alignment path is the one with the maximum score.
3. Word or k-tuple method
Word or k-tuple methods are heuristic methods best known for their use in the database search
tools FASTA and BLAST. The word method is a fast method for aligning two sequences. It begins
by identifying short identical sequences, also known as words or k-tuples, and then uses dynamic
programming to align the sequences based on these words.
❖ Multiple Sequence Alignment
Multiple Sequence Alignment involves aligning multiple (three or more) biological sequences to
achieve optimal sequence matching.
• Multiple sequence alignments are used to identify conserved sequence regions and to
construct phylogenetic trees, which help us understand the functional and evolutionary
relationships between different species or groups of organisms.
Methods of Multiple sequence alignment
Multiple Sequence Alignment involves aligning multiple (three or more) biological sequences to
achieve optimal sequence matching. Multiple sequence alignments are used to identify conserved
sequence regions and to construct phylogenetic trees, which help us understand the functional and
evolutionary relationships between different species or groups of organisms.
ultiple sequence alignment can be performed using either exhaustive or heuristic approaches.
1. Exhaustive algorithm
Exhaustive alignment involves examining all possible alignments at once. A multidimensional
search matrix is required to perform multiple sequence alignment using the exhaustive algorithm,
similar to the two-dimensional matrix used in dynamic programming for pairwise alignment. This
means that to align N sequences, an N-dimensional matrix is required.
• Dynamic programming is a powerful method for aligning sequences, but as the number of
sequences to be aligned increases, the amount of computational time and memory space
also increases. This means that the method becomes computationally impractical for large
data sets. As a result, dynamic programming is typically only used for small data sets with
fewer than ten short sequences.
2. Heuristic algorithm
i. Progressive method
This method, also known as the tree-based algorithm, is a step-
wise assembly of multiple alignments based on pairwise
similarity. This method is called progressive because it aligns
sequences in a step-wise manner.
i.First, it performs pairwise alignments of all the sequences
using the Needleman–Wunsch global alignment method and
records the similarity scores.
ii.Then, it converts the scores into evolutionary distances to
create a distance matrix.
iii.A guide tree is constructed from the distance matrix using
the neighbor-joining method.
iv.The guide tree is used to direct the realignment of sequences based
on their relative positions on the tree, starting with the two most closely
related sequences and adding more distant sequences one at a time
until all sequences are aligned.
v. Find consensus region within aligned sequences.
vi.Find the next most similar sequences have same consensus regions.
vii. Progressively add new sequences and results in final alignment.
ii. Iterative Method
The iterative method involves improving an initial suboptimal solution by repeatedly modifying it
until an optimal solution is reached. It is based on global alignment.
 • An initial pairwise alignment is conducted to create a tree that provides weights for creating
alignments. Aligned regions with gaps are identified and iteratively adjusted to enhance the
alignment score. The highest-scoring alignment is used in a new set of calculations to
predict a new tree, new weights, and new alignments. The procedure is repeated until there
is no more improvement in the alignment score.
iii. Block-based method
The progressive and iterative alignment methods are based on global alignment and may not be
effective in identifying conserved domains and motifs in highly divergent sequences of different
lengths.
• To align such divergent sequences, a local alignment-based approach is needed.
• The block-based method is one such method that identifies a block of ungapped alignment
that is shared by all sequences.
Tools for Multiple Sequence Alignment
1.Clustal Omega
This tool is used for high throughput analysis of a large number of sequences. It can align 100 to
1000s of sequences with fast speed. It is less sensitive.
2.Muscle
It is more accurate therefore it is best suitable for phylogenetic studies. It can only align 500
sequences (file of maximum 1 mb at a time.
Application/Significance of sequence alignment
• Sequence alignment can identify unknown sequences by comparing them with already
known sequences in databases.
• Sequence alignment is also used to identify conserved sequence patterns and motifs, which
helps to characterize the functions of the sequences.
• Sequence alignment can also produce phylogenetic trees and obtain information about the
evolutionary relationship between the sequences aligned.
• Sequence alignment can also predict proteins’ secondary and tertiary structures. It can also
predict gene locations and new members of gene families.
• Sequence alignment can also be used to develop degenerate PCR primers by analyzing
multiple related sequences.
• It can also be used in disease research and drug discovery.