Department of

Microbiology

Unit no 3
Multiple Sequence Unit title Introduction
to databases
alignment Subject name and
code Bioinformatics
and Biostatistics and
02MB301

Dr. Purvi Rakhashiya

• Alignment of more than two DNA or Protein sequences of
similar length.

• A natural extension of pairwise alignment is multiple sequence

alignment.

• The dynamic programming algorithm used for optimal

alignment of pairs of sequences can be extended to three
sequences, but for more than three sequences, only a small
number of relatively short sequences may be analyzed.
 Uses
• Helps in identification of conserved regions in the sequences.

• An important step for phylogenetic analysis.

• Useful in designing experiments to test and modify the function of

specific proteins and also in predicting the function and structure of
proteins, and in identifying new members of protein families.
 Progressive algorithm
 Possible number of pairs is calculated first.

 Pair-wise alignment is done.

 Based on the scores, distance is calculated.

 Guide tree is built.

 Based on the guide tree, alignment is done again.

 T-Coffee
• Tree-based Consistency Objective Function For
alignment Evaluation.

• It is suitable for small alignments.

• Compare all the sequences two by two, producing a global alignment and a
series of local alignments (using lalign).

• Then combine all these alignments into a multiple alignment.

• T-Coffee is a consistency-based MSA tool that attempts to
mitigate the pitfalls of progressive alignment methods.

• It uses a progressive approach like ClustalW.

• But it has advanced features to evaluate the quality of the

alignments and some capacity for identifying occurrence of
motifs.
T-Coffee Multiple Sequence Alignment interface at EBI (
http://www.ebi.ac.uk/Tools/msa/tcoffee/)
1. Go to http://www.ebi.ac.uk/Tools/msa/tcoffee/ in your browser.
2. Enter your input sequence. (eg: iron superoxide dismutase (FeSODs) of Oryza sativa
subsp. indica (B8B2C9), Arabidopsis thaliana (P21276), Escherichia coli (P0AGD3),
Nostoc punctiforme (B2IZB2) and Synechococcus elongatus strain PCC 7942 (P18655))
3. Go to choose file and give your input sequence in any valid format (GCG, FASTA,
EMBL, GenBank, PIR, NBRF or UniProtKB/Swiss-Prot format).
4. Similarly give other input sequences (There is currently a limit of
500 sequences and 1MB of data).
5. Click on ‘more options’ button to set the alignment options. Matrix -used when
generating the MSA. Default value is: ‘None’, Other options: BLOSUM and PAM.
Order -in which the sequences appear in the final alignment. Default value is: ‘aligned’
Other option: ‘input’
6. Enter submit.
MSA in aln format
• ‘Alignment’ tab (default) -shows the alignment in aln format.

• By default an alignment will display the following symbols that denote the degree of
conservation observed in each column:
"*" -residues or nucleotides in that column are identical in all sequences in the
alignment.
":" -conserved substitutions have been observed. "." -semi-
conserved substitutions are observed.
• ‘Download Alignment File’ - to download the alignment in .aln format.

• ‘Show Colors’, -the alignment will be shown in colour.

• ‘ClustalW2_Phylogeny’- MSA can be directly parsed to ClustalW2 Phylogeny

program. This allows the user to control the method of tree construction.
Alignment displayed in colour based on their physicochemical properties
Result summary along with the JalView trigger button
• ‘Result Summary’ -displays the result files comprising the input
sequences for the alignment (.input), tool output (.output), which is a
log file created during the alignment, alignment in HTML format
(.html), alignment in PHYLIP format (.phylip), alignment in
CLUSTAL format (.clustalw), alignment in MSF format (.msf) and
guide tree (.dnd) that contains the information for building the
cladogram or phylogram.

• ‘Start JalView’ under JalView -triggers JalView, a Java based editor

in new window. This requires Java program to be preinstalled.
JalView editor
Guide tree generated during alignment process
• Phylogram
-Branching diagram (tree) assumed to be an
estimate of a phylogeny.
-Branch lengths are proportional to the amount of inferred
evolutionary change.

• Cladogram
-Branching diagram (tree) assumed to be an estimate of a
phylogeny where the branches are of equal length, thus cladograms show
common ancestry, but do not indicate the amount of evolutionary "time"
separating taxa.
Neighbour-joining tree without correcting the distances
submission details of the alignment
 MUSCLE
• MUltiple Sequence Comparison by Log-Expectation
• Better average accuracy and better speed than ClustalW2 or
T-Coffee
• An accurate MSA tool, especially good with proteins and suitable
for medium alignments.
• Aligns 5000 sequences with average length of 350.
• MUSLE algorithm includes
-fast distance estimation using kmer counting.
-progressive alignment using a new profile
function called the log‐expectation score.
-refinement using tree‐dependent partitioning. restricted
MUSCLE MSA interface at EBI (http://www.ebi.ac.uk/Tools/msa/muscle/)
1. Go to http://www.ebi.ac.uk/Tools/msa/muscle/ in your browser.
2. Enter your input sequence.
3. Go to choose file and give your input sequence in any valid format (GCG,
FASTA, EMBL, GenBank, PIR, NBRF or UniProtKB/Swiss-Prot format).
4. Similarly give other input sequences (There is currently a limit of 500
sequences and 1MB of data).
5. Click on ‘more options’ button to set the alignment options. Change output
format to clustalW to get results in aln format. Default value is
Pearson/FASTA [fasta]. Output Order -in which the sequences appear in
the final alignment. Default value is: ‘aligned’.
6. Enter submit.
MSA generated by MUSCLE MSA generated by T-Coffee

Comparison of MSA generated by MUSCLE and T-Coffee

 Both the programs use different algorithms, which is
clearly evident from the results generated by each program. It
has to be noted that, though alignments differ, conservation of
amino acids at the active site are still retained. Again a
phylogenetic tree construction is purely dependent on the
alignment. Hence one should utmost care in MSA.
• ‘Alignment’ tab (default) -shows the alignment in aln format.

• By default an alignment will display the following symbols that denote the degree of
conservation observed in each column:
"*" -residues or nucleotides in that column are identical in all sequences in the
alignment.
":" -conserved substitutions have been observed. "." -semi-
conserved substitutions are observed.
• ‘Download Alignment File’ - to download the alignment in .aln format.

• ‘Show Colors’ -the alignment will be shown in colour.

• ‘ClustalW2_Phylogeny’- MSA can be directly parsed to ClustalW2 Phylogeny

program. This allows the user to control the method of tree construction.
• ‘Result Summary’ -displays the result files comprising the input sequences for the
alignment (.input), tool output (.output), which is a log file created during the
alignment, alignment in HTML format (.html), alignment in PHYLIP format
(.phylip), alignment in CLUSTAL format (.clustalw), alignment in MSF format
(.msf) and guide tree (.dnd) that contains the information for building the
cladogram or phylogram.
• ‘Start JalView’ under JalView -triggers JalView, a Java based editor in new
window. This requires Java program to be preinstalled.
• ‘Phylogeny Tree’ -displays the phylogenetic tree of the sequences used. It is
actually a Neighbour-joining tree without correcting the distances
• Submission Details’ -displays the information regarding the program used, its
version, input parameters, etc
 MAFFT
• Multiple Alignment using Fast Fourier Transform.
• It uses FFT and is suitable for medium-large sequence
alignments.
• computational time is drastically reduced.
• 1st - homologous regions are identified by FFT, amino acid
sequence sequence composed of volume +
polarity values of each amino acid residue.
• 2nd -simplified scoring system is used for reducing computational
time and increasing the accuracy of alignments.
• Applicable for
-sequences having large insertions or
extensions
-distantly related sequences of similar length
• Methods
Progressive method (FFT-NS-2) - computational time is
drastically reduced with comparable accuracy
Iterative refinement method (FFT-NS-i) –is
100 times faster than T-COFFEE without sacrificing the
accuracy.
 Iterative method
• Problems in progressive alignment method
-errors in the initial alignments of the most
closely related sequences are propagated to the MSA.
-problem is more acute when the alignments are starting
between more distantly sequences. related

• Iterative methods
–rectify this problem by repeatedly realigning
subgroups of the sequences.
-then by aligning these subgroups into a global
alignment of all of the sequences.
-Major objective is to improve the overall
alignment score, such as a sum of pairs score.
• Selection of groups -based on the ordering of the sequences on
a phylogenetic tree.
MAFFT MSA interface at EBI (http://www.ebi.ac.uk/Tools/msa/mafft/)
•Click on ‘more options’ button to set the alignment options
Change the output format ‘clustalw’ (Default value is:
Pearson/FASTA [fasta]).
Matrix
Protein comparison matrix to be used when adding sequences to the alignment.
Matrix (Protein Only)
Default value is: BLOSUM 62 [bl62]
Gap Open
Penalty for first base/residue in a gap. Default
value is: 1.53
Gap Extension
Penalty for each additional base/residue in a gap.
Default value is: 0.123
Order
The order in which the sequences appear in the final alignment Default value is:
aligned
Tree Rebuilding Number
Default value is: 1 Guide Tree Output
Generate guide tree file
Default value is: ON [true]
Max Iterate
Maximum number of iterations to perform when refining the alignment.
Change the Max Iterate value to ‘2’ to change number of iterations for better
alignment.
Default value is: 0
Perform FFTS (Fast Fourier Transform) Default value is:
local pair
• Click ‘submit’
MSA generated by MAFFT
• The N terminal alignment in the result generated by the
MAFFT is similar to the one generated by the T-Coffee.
• The alignment is in the middle and C terminal is entirely
different from the T-Coffee or MUSCLE.
• Though alignments differ, conservation of amino acids at the
active site are retained.
 Formats
Gaps in sequences
• In all EMBOSS alignment formats, gaps indicated by ‘- 'character.
• Exception
-msf format which uses '.' as the gap character inside the sequences
-'~' as the gap character at the terminal ends of the alignment.
Head and tail of the format
• The majority of the alignment formats (except those that are also standard sequence formats, like fasta or MSF) have a block of information at the start of
the alignment describing the program, date, output filename, ID names of the sequences and some of the parameters and statistics of the alignment.
########################################
# Program: demoalign
# Rundate: Thu Jan 17 09:30:08 2002 # Report_file: stdout
########################################
#=====================================
Aligned_sequences: 4
# 1: IXI_234
# 2: IXI_235
# 3: IXI_236
# 4: IXI_237
# Matrix: EBLOSUM62

# Extend_penalty: ‐1 ##
# Gap_penalty: 9

Length: 131
# Identity: 95/131 (72.5%)
# Similarity: 127/131 (96.9%) # Gaps: 25/131 (19.1%)
#
9/27/2016 Alignment Formats http://emboss.sourceforge.net/docs/themes/AlignFormats.html 3/6 #
#====================================
There is also a block of information at the end of the alignment for summary information. This is used by a few programs e.g.
merger .
Length
The header block contains a line similar to:
# Length: 131
This is the length of the alignment, including any gaps that have been introduced to construct the alignment.
Identity
The header block contains a line similar to:
# Identity: 95/131 (72.5%)
This is a count of the number of positions over the length of the alignment where all of the residues or bases at that position are
identical. It is followed by '/131' the length of the alignment and '(72.5%)' the percentage of positions in the alignment where there are
identities.
Similarity
The header block contains a line similar to:
# Similarity: 127/131 (96.9%)
This is a count of the number of positions over the length of the alignment where >= 51% of the residues or bases at that position
are similar. Any two residues or bases are defined as similar when they have positive comparisons (as defined by the comparison
matrix being used in the alignment algorithm). It is followed by '/131' the length of the alignment and '(96.9%)' the percentage of
positions in the alignment where there are similarities. Note that the sum of identical and similar positions is greater than 100%.
This is because the count of similar positions includes the count of identical positions; if residues are identical,
they must also be similar.
Gaps
The header block contains a line similar to: # Gaps: 25/131
(19.1%)
This is a count of the number of positions over the length of the
alignment where there are one or more sequences with a gap.
9/27/2016 Alignment Formats
http://emboss.sourceforge.net/docs/themes/AlignFormat s.html4/6
It is followed by '/131' the length of the alignment and '(19.1%)' the
percentage of positions in the alignment where there are gaps.
Score
The header block may contain a line similar to:
# Score: 100.0
This is the score used by the program that calculated the alignment to determine which is the best
possible alignment to report. The algorithm that was used to derive the score is not part of the alignment
formatting routines. You should see documentation about the relevant algorithm to see how the score is
derived.
Alignment Formats (MSA)
 Alignment viewers/editors
Name Integrated with Can Align Can Calculate Other Features Formats License Link
Struct. Sequences Phylogenetic Supported
Prediction Tools Trees

AliView No Muscle is External Fast, very easy FASTA, GPL3 (http://www.ormb

2016 integrated. programs such navigation through PHYLIP, unkar.se/aliview)
Other as FastTree unlimited mouse Nexus, MSF
programs can be called wheel zoom in/out and Clustal
such as from within feature. Handles
MAFFT can unlimited file size
be alignments.
defined. Degenerate primer
design.

BioEdit No ClustalW rudimentary, plasmid drawing, Genbank, Free (http://www.mbio.

can read ABI Fasta, Phylip ncsu.edu/BioEdi
phylip chromatograms 3.2, Phylip 4, t/bioedit.html)
NBRF/PIR
CINEMA NO, but can ClustalW No Dotplot, 6 frame Nexus, MSF, Free (http://aig.cs.
read/show translation, Blast Clustal, man.ac.uk/researc
2D FASTA, h/utopia/cinema/c
structure PHYLIP, inema.php)
annotations PIR, PRINTS
DECIPHER Yes Yes UPGMA, NJ, Primer/Probe FASTA, GPL (http://deciphe
ML design, Chimera FASTQ, r.cee.wisc.edu/Do
finding GenBank wnload.html)
MEGA No Native UPGMA, NJ, extended support FASTA, Freeware, (http://www.m
ClustalW ME, MP, with to phylogenetics Clustal, registration egasoftware.net/),
bootstrap and analysis Nexus, requested table offeatures
confidence Mega, etc.. (http://www.mega
test software.net/feat
ures.html)
 Other Tools
• Clustal Omega
New MSA tool that uses seeded guide trees and HMM profile- profile techniques to
generate alignments (protein only). Suitable for medium-large alignments.
• DbClustal
Create a MSA from a protein BLAST result using the DbClustal program.
• MView
Transform a Sequence Similarity Search result into a MSA or
reformat a MSA using the MView program.
• WebPRANK
The EBI has a new phylogeny-aware MSA program which makes use of
evolutionary information to help place insertions and deletions.