212BIT1304 – Bioinformatics
Laboratory Record
Date: 24.01.2023
Reg. No: 9921001004
Name of the Student: Annie Magdaline
Experiment Number: 4
Experiment Title: Pairwise Sequence Alignment
Aim: To perform global and local alignment of pair of sequences using EMBOSS Needle and
EMBOSS water tools
Background Information:
Sequence Alignment or sequence comparison lies at heart of the bioinformatics, which describes
the way in which DNA/RNA or protein sequences are arranged, in order to identify the regions
of similarity among them. It is used to infer structural, functional and evolutionary relationship
between the sequences. Alignment finds similarity level between query sequence and different
database sequences. The algorithm works by dynamic programming approach which divides the
problem into smaller independent sub-problems. It finds the alignment more quantitatively by
assigning scores.
When a new sequence is found, the structure and function can be easily predicted by doing
sequence alignment. Since it is believed that, a sequence sharing common ancestor would exhibit
similar structure or function. Greater the sequence similarity, greater is the chance that they share
similar structure or function.
Methods of Sequence Alignment:
There are mainly two methods of Sequence Alignment:
Global Alignment: Closely related sequences which are of same length are very much
appropriate for global alignment. Here, the alignment is carried out from beginning till end of the
sequence to find out the best possible alignment.
The Needleman-Wunsch algorithm (A formula or set of steps to solve a problem) was developed
by Saul B. Needleman and Christian D. Wunsch in 1970, which is a dynamic programming
algorithm for sequence alignment. The dynamic programming solves the original problem by
dividing the problem into smaller independent sub problems. These techniques are used in many
different aspects of computer science. The algorithm explains global sequence alignment for
aligning nucleotide or protein sequences.
Local Alignment: Sequences which are suspected to have similarity or even dissimilar
sequences can be compared with local alignment method. It finds local regions with high level of
similarity.
These two methods of alignments are defined by different algorithms, which use scoring
matrices to align the two different series of characters or patterns (sequences). The two different
alignment methods are mostly defined by Dynamic programming approach for aligning two
different sequences.
Dynamic Programming:
Dynamic programming is used for optimal alignment of two sequences. It finds the alignment in
a more quantitative way by giving some scores for matches and mismatches (Scoring matrices),
rather than only applying dots. By searching the highest scores in the matrix, alignment can be
accurately obtained. The Dynamic Programming solves the original problem by dividing the
problem into smaller independent sub-problems. These techniques are used in many different
aspects of computer science. Needleman-Wunsch and Smith- Waterman algorithms for sequence
alignment are defined by dynamic programming approach.
Procedure:
The two sequences can be aligned globally using different algorithms. Needleman
Wunsch algorithm is one of the best algorithm for global alignment, which can be
performed using the online tool EMBOSS Needle (European Molecular Biology Open
Software Suite).
1. Get access to the tool EMBOSS Needle
([Link]
2. Copy and paste the FASTA formatted nucleotide sequence in the dialog box
3. One can also choose the file through “Choose File” option and can upload the
sequence file.
4. Similarly copy and paste or upload the second sequence for the alignment.
5. EMBOSS needle is predefined with the scoring matrices DNA full for
nucleotide sequence, BLOSUM62 for protein sequence
6. The gap open and gap extend penalty can be changed by user defined values. In
this example it kept as default values.
7. The user can be notified with the results through email, if the checkbox is checked
and the mail address is submitted.
8. The result will include % identity and % similarity between the two sequences,
the alignment score (based on BLOSUM 62 matrix) and also the alignment.
Local alignment of two sequences
Procedure
There are various algorithms to perform local alignment of two sequences, Smith-
Waterman algorithm is one of the best algorithms, which can be performed using the
online tool EMBOSS Water.
Steps to perform alignment
1. Get access to the tool EMBOSS Water ([Link]/Tools/psa/emboss_water)
2. Copy and paste the FASTA formatted nucleotide/protein sequence.
3. One can also choose the file through “Choose File” option and can upload
the sequence file.
4. Similarly copy and paste or upload the second sequence for the alignment.
5. EMBOSS Water is predefined with the scoring matrices DNA full for
nucleotide sequence, BLOSUM62 for protein sequence.
6. The gap open and gap extend penalty can be changed by user defined values. In
this example it kept as default values.
7. The user can be notified results through email, if the checkbox has been checked
and the mail address is submitted.
8. The result will include % identity and % similarity between the two sequences,
the alignment score (based on BLOSUM 62 matrix) and also the alignment.
Screenshots:
1. Perform pairwise sequence alignment for Two DNA sequences using Emboss Needle.
(NCBI GenBank)
Sequence 1 id: NM_001130527.3
Sequence 2 id: XM_045538358.1
Fig 4.1: Human sperm data.
Figure 4.2: Lemur Sperm Data
Figure 4.3: EMBOSS NEEDLE Home page with sequences.
Figure 4.4: Result page of EMBOSS NEEDLE.
2. Perform pairwise sequence alignment for Two Protein sequences using Emboss Needle.
(Uniprot)
Sequence 1 id: P06213
Sequence 2 id: P01317
Figure 4.5: Result page of EMBOSS Needle.
3. Perform pairwise sequence alignment for Two DNA sequences using Emboss Water. (NCBI
GenBank)
Sequence 1 id: AAB19865.1
Sequence 2 id: XP_045394314.1
Figure 4.6: Result page of pairwise sequence in EMBOSS Water.
4. Perform pairwise sequence alignment for Two Protein sequences using Emboss Water.
(Uniprot)
Sequence 1 id: P06213
Sequence 2 id: P01317
Figure 4.7: Result of pairwise sequences using Emboss Water (Uniprot).
Inference: This Emboss platform gives you an idea about how different sequences are related to
each other. It is of two types one will be the global alignment and local alignment.