Multiple sequence alignment is a method to arrange DNA, RNA, or protein sequences to identify regions of similarity that may indicate functional, structural, or evolutionary relationships. Computational approaches like dynamic programming are needed to align lengthy or numerous sequences. Global alignment finds the optimal alignment over the full length of sequences using the Needleman-Wunsch algorithm. Local alignment identifies similar regions within divergent sequences using the Smith-Waterman algorithm. Dot plot matrices can also show sequence similarities but have limitations like noise and difficulty extracting information.

1. Multiple Sequence Alignment
By- Gyanika Shukla

2. Previous Knowledge
• Genome?
• Phylogenetic analysis?
• DNA sequencing?
• Protein sequencing?
• Total number of amino acids?
• Letter ‘M’ denotes __________ amino acid?
• Bioinformatics
• DNA/ Protein sequence similarity search?

3. Multiple Sequence Alignment
• In bioinformatics, a sequence alignment is a way of arranging
the sequences of DNA, RNA, or protein to identify regions of
similarity that may be a consequence of functional, structural,
or evolutionary relationships between the sequences.
• Very short or very similar sequences can be aligned by hand.
However, most interesting problems require the alignment of
lengthy, highly variable or extremely numerous sequences
that cannot be aligned solely by human effort.
• Sequence similarity searches can only run by computational
approaches when the amino acid or DNA/ RNA sequences of
different organisms are parallel aligned.

5. • Aligned sequences of nucleotide or amino acid
residues are typically represented as rows within
a matrix
• Computational approaches to sequence
alignment generally fall into two categories:
global alignments and local alignments.
• Calculating a global alignment is a form of global
optimization that "forces" the alignment to span
the entire length of all query sequences (DNA or
Protein). By contrast, local alignments identify
regions of similarity within short sequences that
are often widely divergent overall.

6. Dynamic programming
• Dynamic programming is both a mathematical
optimization method and a computer
programming method. The technique of dynamic
programming can be applied to produce global
alignments via the Needleman-Wunsch
algorithm, and local alignments via the Smith-
Waterman algorithm. In typical usage,
a substitution matrix is used to assign scores to
amino-acid or DNA matches or mismatches, and
a gap penalty. In practice often simply assign a
positive match score, a negative mismatch score,
and a negative gap penalty.

7. GLOBAL ALIGNMENT
• Needleman-Wunsch algorithm: The algorithm
was developed by Saul B. Needleman and
Christian D. Wunsch and published in 1970.
• The alignment of sequences by this algorithm is
completed into three steps:
1) Scoring matrix (by taking a positive match
score, a negative mismatch score, and a
negative gap penalty)
2) Trace back
3) Alignment

8. Lets take an example that how the global alignment
software align the sequences by algorithm..
• Suppose the two sequences to be aligned are
• GCATG
• GATTA
• Lets take match value = +1 and miss-match value
is –1 (if two diagonally represented sequences
are same, then match value is assigned and if the
sequences are different miss-match value is
assigned)
• Gap penalty is – 1

9. Constructing the scoring matrix
• First construct a grid such as one shown in Figure. Start the first string in
the top of the third column and start the other string at the start of the
third row. Fill out the rest of the column and row headers as in Figure.
G C A T G
G
A
T
T
A

10. Filling in the matrix
• Start with a zero in the second row, second
column. Move through the cells of row and
column adjacent to the written sequences
calculating the score for each cell by adding
gap penalty to the next cell in the row and
column.

11. • The first case with existing scores in all 3 directions is the
intersection of our first letters. The surrounding cells are
below.
• This cell has three possible candidate sums:
• The diagonal top-left neighbor has score 0. The pairing of
G and G is a match, so add the score for match: 0+1 = 1
• The top neighbor has score −1 and moving from there
represents an box, so add the score for box: (−1) + (−1) =
(−2)
• The left neighbor also has score −1, represents a box and
also produces (−2).
• The highest candidate is 1 and is entered into the box.
• An arrow is marked from the box to the new box from
where the final value is assigned.

13. G C A T G
0 -1 -2 -3 -4 -5
G -1 1 0 -1 -2 -3
A -2 0 0 1 0 -1
T -3 -1 -1 0 2 1
T -4 -2 -2 -1 1 1
A -5 -3 -3 -1 0 0
G C A – T G
G – A T T A 50% similarity

14. Trace back and Alignment
The scoring matrix is traced back from the last box of the
matrix to the next boxes from where the highest values are
obtained. If the score of the box is obtained from the
diagonal box, both the sequences corresponding to that
row and column are written opposite to each other for
alignment. If score is from upper or beside box the
sequence corresponding to that column or row
respectively is taken as “gap” and only one sequence is
written.
If the highest score is from both diagonal and upper/beside
box, then diagonal value is preferred.
Once the sequences are aligned the similarity percentage can
be calculated.

15. LOCAL ALIGNMENT
• Smith–Waterman algorithm or zero-one algorithm
performs local sequence alignment; that is, for
determining similar regions between two strings of
nucleic acid sequences or protein sequences.
• Instead of looking at the entire sequence, the Smith–
Waterman algorithm compares segments of all
possible lengths and optimizes the similarity measure.
• The algorithm was first proposed by Temple F. Smith
and Michael S. Waterman in 1981
• The main difference to the Needleman–Wunsch
algorithm is that negative scoring matrix cells are set to
zero.

16. Lets take an example that how the global alignment
software align the sequences by algorithm..
• Suppose the two sequences to be aligned are
• GCATG
• GATTA
• Lets take match value = +1 and miss-match value
is –1 (if two diagonally represented sequences
are same, then match value is assigned and if the
sequences are different miss-match value is
assigned)
• Gap penalty is – 1

17. • The first case with existing scores in all 3 directions is the
intersection of our first letters. The surrounding cells are
below.
• This cell has three possible candidate sums:
• The diagonal top-left neighbor has score 0. The pairing of G
and G is a match, so add the score for match: 0+1 = 1
• The top neighbor has score −1 and moving from there
represents an box, so add the score for box: (−1) + (−1) = (−2)
• The left neighbor also has score −1, represents a box and
also produces (−2).
• The highest candidate is 1 and is entered into the box.
• An arrow is marked from the box to the new box from where
the final value is assigned.
• All the negative scores must be written as zero.

18. G C A T G
0 0 0 0 0 0
G 0 1 0 0 0 0
A 0 0 0 1 0 0
T 0 0 0 0 2 1
T 0 0 0 0 1 1
A 0 0 0 1 0 0
G C A T
G – A T
75% Similarity

19. Trace back and Alignment
The scoring matrix is traced back from the highest value
obtained in the matrix. If there are more than one highest
value, scoring can be done from any box.
If the score of the box is obtained from the diagonal box, both
the sequences corresponding to that row and column are
written opposite to each other for alignment. If score is
from upper or beside box the sequence corresponding to
that column or row respectively is taken as “gap” and only
one sequence is written.
If the highest score is from both diagonal and upper/beside
box, then diagonal value is preferred.
Once the sequences are aligned the similarity percentage can
be calculated.

20. Dot Plot Matrix
• The dot-matrix approach, which implicitly produces a family of
alignments for individual sequence regions, is qualitative and
conceptually simple, though time-consuming to analyze on a large
scale. In the absence of noise, it can be easy to visually identify
certain sequence features—such as insertions, deletions, repeats,
or inverted repeats—from a dot-matrix plot. To construct a dot-
matrix plot, the two sequences are written along the top row and
leftmost column of a two-dimensional matrix and a dot is placed at
any point where the characters in the appropriate columns match—
this is a typical recurrence plot. Some implementations vary the size
or intensity of the dot depending on the degree of similarity of the
two characters, to accommodate conservative substitutions. The
dot plots of very closely related sequences will appear as a single
line along the matrix's main diagonal.

21. Disadvantages of Dot Plot
• Problems with dot plots as an information
display technique include: noise, lack of
clarity, non-intuitiveness, difficulty extracting
match summary statistics and match positions
on the two sequences. There is also much
wasted space where the match data is
inherently duplicated across the diagonal and
most of the actual area of the plot is taken up
by either empty space or noise, and, finally,
dot-plots are limited to two sequences.

22. G C A T G
G
A
T
T
A
40% similarity
GCATG
GATTA