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Multiple sequence alignment

- Multiple sequence alignment (MSA) is the alignment of three or more biological sequences, such as DNA, RNA, or protein sequences. It involves identifying regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. - Several algorithms and tools exist for generating MSAs, including ClustalW, T-Coffee, MUSCLE, MAFFT, and MSA. They employ different methods like progressive alignment and iterative refinement. The choice of algorithm depends on factors like number of sequences and computational time. - MSAs aid in tasks like identifying conserved motifs and domains, phylogenetic analysis, and predicting protein structure and function. Formats like FASTA are used for representing MS

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Multiple Sequence Alignment
• 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 restricted partitioning.
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 starting alignments are between more distantly related sequences. • 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 # Gap_penalty: 9 # Extend_penalty: ‐1 ## 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. Markup Line The markup line is the line commonly placed between a pairwise alignment or at the bottom of alignments of 3 or more sequences that shows where sequences are mismatched, gapped, identical or similar. In general the markup line uses a space for a mismatch or a gap, '.' for any small positive score, ':' for a similarity which scores more than 1.0, and '|' for an identity where both sequences have the same residue regardless of its score ('W' matching 'W' scores much more than 'L' matching 'L' because a conserved tryptophan is more significant than a conserved leucine). The 'markx' set of alignment formats (produced by the FASTA suite of programs written by Bill Pearson) use '.' for similarity and ':' for an identity. The '|' character is not used. This was a design decision by Bill Pearson when he wrote the FASTA programs.
Alignment Formats (MSA)
Alignment viewers/editors Name Integrated with Struct. Prediction Tools Can Align Sequences Can Calculate Phylogenetic Trees Other Features Formats Supported License Link AliView 2016 No Muscle is integrated. Other programs such as MAFFT can be defined. External programs such as FastTree can be called from within Fast, very easy navigation through unlimited mouse wheel zoom in/out feature. Handles unlimited file size alignments. Degenerate primer design. FASTA, PHYLIP, Nexus, MSF and Clustal GPL3 (http://www.ormb unkar.se/aliview) BioEdit No ClustalW rudimentary, can read phylip plasmid drawing, ABI chromatograms Genbank, Fasta, Phylip 3.2, Phylip 4, NBRF/PIR Free (http://www.mbio. ncsu.edu/BioEdi t/bioedit.html) CINEMA NO, but can read/show 2D structure annotations ClustalW No Dotplot, 6 frame translation, Blast Nexus, MSF, Clustal, FASTA, PHYLIP, PIR, PRINTS Free (http://aig.cs. man.ac.uk/researc h/utopia/cinema/c inema.php) DECIPHER Yes Yes UPGMA, NJ, ML Primer/Probe design, Chimera finding FASTA, FASTQ, GenBank GPL (http://deciphe r.cee.wisc.edu/Do wnload.html) MEGA No Native ClustalW UPGMA, NJ, ME, MP, with bootstrap and confidence test extended support to phylogenetics analysis FASTA, Clustal, Nexus, Mega, etc.. Freeware, registration requested (http://www.m egasoftware.net/), table offeatures (http://www.mega 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.